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Genomics of secondary metabolite production by Pseudomonas spp


Harald Gross*a and Joyce E. Loper*b
Received 16th July 2009
First published as an Advance Article on the web 1st October 2009
DOI: 10.1039/b817075b

Covering: up to June 2009

Pseudomonas is a diverse genus of Gammaproteobacteria with more than 60 species exhibiting varied
life styles in a wide range of environments, including soil, water, plant surfaces, and animals. They are
well known for their ubiquity in the natural world, capacity to utilize a striking variety of organic
Published on 01 October 2009 on http://pubs.rsc.org | doi:10.1039/B817075B

compounds as energy sources, resistance to a wide range of medically- and agriculturally-important


antimicrobial compounds, and production of a remarkable array of secondary metabolites. Here, we
provide an overview of the astonishing metabolic capacity of the Pseudomonads, summarize the
knowledge of secondary metabolite biosynthesis in this group of organisms, and highlight the
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biological significance of these compounds to the diverse life styles exhibited by Pseudomonas spp. A
consistent theme throughout this discussion is the central role of genomics in natural product discovery,
characterization of metabolic gene clusters and patterns of their inheritance, and illuminaton of new
aspects of Pseudomonas biology.

1 Introduction: An overview of Pseudomonas spp. and 2.3.4 Pederin


their genomes 2.4 Other compounds
1.1 Pseudomonas aeruginosa 2.4.1 Phenazines
1.2 Pseudomonas entomophila 2.4.2 Quinolones
1.3 Pseudomonas fluorescens 2.4.3 Hydrogen cyanide
1.4 Pseudomonas syringae 3 New compounds discovered by genome-guided
2 Biosynthetic enzymes, genes and gene clusters of strategies in Pseudomonas spp.
Pseudomonads 3.1 Orfamides
2.1 Non-ribosomally produced peptides and other amino 3.2 Rhizoxins
acid-derived compounds 3.3 Enantio-pyochelin
2.1.1 Pyoverdines 3.4 Syringafactins AF
2.1.2 Pyochelin 4 Secondary metabolite gene clusters in the flexible
2.1.3 Pseudomonine genome of Pseudomonas spp.
2.1.4 Paerucumarin and pseudoverdine 5 Concluding remarks
2.1.5 Lipopeptides 6 Acknowledgements
2.1.6 Safracin 7 References
2.1.7 Tabtoxin
2.1.8 Phaseolotoxin
2.1.9 Pyrrolnitrin
2.1.10 Indole-3-acetic acid (IAA)
1 Introduction: An overview of Pseudomonas spp.
2.2 Polyketides and fatty acid derived compounds and their genomes
2.2.1 Mupirocin (pseudomonic acid A) Pseudomonas spp. entered the genomics era less than ten years
2.2.2 2,4-Diacetylphloroglucinol (DAPG) ago when the genome of P. aeruginosa PA01 became available,1
2.2.3 2,5-Dialkylresorcinols but genomics has since accelerated research in virtually all
2.3 Hybrid NRPS-PKS compounds aspects of Pseudomonas biology, including secondary metabo-
2.3.1 Syringolin lism. To date, the complete genomes of at least 22 strains rep-
2.3.2 Pyoluteorin resenting seven species have been sequenced, and many more
2.3.3 Coronatine genomic sequences of Pseudomonas spp. will soon become
available due, in part, to the application of new rapid and
a
Institute for Pharmaceutical Biology, Nussallee 6, 53115 Bonn, Germany. affordable sequencing technologies.2,3 This discussion will focus
E-mail: harald.gross@uni-bonn.de; Fax: +49 (0)228/73-3250; Tel: + 49 on new insights into secondary metabolism of the genus that has
(0)228/73-2676 been provided by genomics.
b
U.S. Department of Agriculture, Agricultural Research Service, 3420
N.W. Orchard Ave., Corvallis, OR, 97330, USA. E-mail: loperj@science. The remarkable ecological and metabolic diversity of Pseu-
oregonstate.edu; Fax: +1 541 738-4025; Tel: +1 541 738-4057 domonas spp. is reflected in the genomes of these bacteria.
This article is part of a themed issue on genomics. Genome size varies from 4.6 to 7.1 Mbases with 4237 to 6396

1408 | Nat. Prod. Rep., 2009, 26, 14081446 This journal is The Royal Society of Chemistry 2009
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predicted genes, and GC contents range from 57.8 to 66.6% four Pseudomonas species (Fig. 1). Therefore, the percentage of
(Table 1). Genomic diversity is particularly apparent from the the proteome shared among the four species varies from 40% for
relatively small size of the core genome that is shared among P. fluorescens Pf-5 (with 6137 predicted protein-encoding genes)
Pseudomonas species. For example, Mavrodi et al.4 determined to 46% for P. putida KT2440 (with 5,350 predicted protein-
that only 2468 genes are conserved among strains representing encoding genes). The core genome typically includes

Table 1 Selected strains of Pseudomonas spp. and their genomes

Strain Source of isolation Size [Mb] No. of genes GC content Ref.

P. aeruginosa 2192 Chronically-infected cystic fibrosis 6.9 6191 66.2 6


patient in Boston,
Massachusetts, USA
P. aeruginosa C3719 Manchester epidemic strain 6.2 5578 66.5 6
isolated from cystic fibrosis
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patient, UK
P. aeruginosa LESB58 Liverpool epidemic strain 6.6 6026 66.3 7
isolated from cystic fibrosis
patient, UK
P. aeruginosa PA14 Wound, from culture collection at 6.5 5905 66.3 411
University of California at
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Berkeley, USA
P. aeruginosa PAO1 Burn wound, Melbourne, Australia 6.3 5571 66.6 1
P. entomophila L48 Fruit or fruit fly, Guadeloupe 5.9 5293 64.2 13
P. fluorescens Pf-5 Soil, Texas, USA 7.1 6257 63.3 29
P. fluorescens PfO-1 Soil, Massachusetts, USA 6.4 5857 60.5 40
P. fluorescens SBW25 Leaf of sugar beet, Oxford, UK 6.7 6009 60.5 40
P. putida KT2440 Soil, Japan. Cured strain lacking 6.2 5481 61.5 412
the TOL plasmid
P. putida W619 Endophytic strain isolated from 5.8 5292 61.4 413
poplar, Washington, USA
P. stutzeri A1501 Rice paddy soils in China. 4.6 4237 63.9 414
Nitrogen-fixing strain used as
a crop inoculant in China
P. syringae pv. oryzae 1_6 Rice, China 6.7 4450 57.8 2
P. syringae pv. phaseolica 1448A Bean, Ethiopia 6.1 5436 57.9 48
P. syringae pv. syringae B728a Leaf of bean, Wisconsin, USA 6.1 5245 59.2 49
P. syringae pv. tomato DC3000 Tomato, Guernsey, UK 6.5 5721 58.3 47
P. syringae pv. tomato T1 Tomato 6.1 5771 58.6 3

Harald Gross studied pharmacy Joyce E. Loper studied biology


at the Friedrich-Alexander and plant pathology at the
University, Erlangen-Nurem- University of California (UC),
berg, Germany. In 2000, he receiving her BS and MS
started his PhD studies with degrees from UC Davis and her
Professor K onig at the Univer- PhD from UC Berkeley in 1983.
sity of Bonn, in which he focused She has been a Research Plant
on the chemical analysis of Pathologist in the US Depart-
bioactive metabolites from ment of Agricultures Agricul-
marine invertebrates with tural Research Service since
special emphasis on the elucida- 1985. She also holds the position
tion of their absolute configura- of Professor in the Department
tion. Harry then did of Botany and Plant Pathology
Harald Gross postdoctoral research, funded by Joyce E: Loper at Oregon State University. Her
the German Research Founda- research group studies the
tion (DFG), in the lab of Professor William Gerwick first at ecology, genetics and genomics of plant-associated Pseudomonas
Oregon State University and later at the Scripps Institution of spp., with a focus on secondary metabolite production by strains of
Oceanography, San Diego, CA, focusing on algal, cyanobacterial Pseudomonas fluorescens that suppress plant diseases.
and bacterial metabolites and their biosynthesis. In 2006, he
returned to the University of Bonn and started his Habilitation. His
current research is devoted to the mining of microbial genomes,
particularly of Pseudomonads and related bacteria for new natural
products.

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housekeeping genes and RNAs that are essential for the survival
of the organism, but most genes in individual Pseudomonas sp.
are either species-specific or shared by a subset of the species.
These genes comprise a flexible Pseudomonas genome, which
reflects adaptation of individual strains to a specific life style. The
flexible genome is thought to evolve through horizontal genetic
exchange mediated by a spectrum of mobile elements and sites
for recombination, which enable the acquisition and deletion of
genetic information. It is well known that horizontal gene
transfer (HGT) mediated by conjugation and site-directed
recombination play an important role in the evolution of
bacteria,5 and the core and flexible genomes of Pseudomonas spp.
exhibit a mosaic pattern of conserved and lineage-specific genes
as the remnants of these processes.69 Therefore, one can view
Published on 01 October 2009 on http://pubs.rsc.org | doi:10.1039/B817075B

a genomic sequence as a snapshot in the evolution of individual


strains, as they acquire and discard genomic fragments in the
process of developing a genetic repertoire customized to their
ecological niche. Genes conferring secondary metabolite
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biosynthesis are one component of this genetic repertoire, which


mediate the bacteriums interactions with plant or animal hosts,
its microbial co-inhabitants, or predators in the environment.
Secondary metabolites play important roles in the diverse life
styles of Pseudomonas spp., functioning in nutrient acquisition,
virulence, and defense against competitors and predators con-
fronted in natural habitats. In this section, we provide a brief
summary of the biology and genomics of P. aeruginosa,
P. entomophila, P. fluorescens and P. syringae. Taken together,
these four species produce an enormous spectrum of secondary
metabolites synthesized from gene clusters distributed between
the core and flexible genomes of each species.

1.1 Pseudomonas aeruginosa


Pseudomonas aeruginosa is a ubiquitous environmental bacte-
rium that is of increasing importance as an opportunistic human
pathogen. The bacterium causes respiratory infections, including
acute pneumonia, in patients breathing through mechanical
respirators or those with neutropenia or immunosuppression.10
P. aeruginosa can also cause persistent respiratory infections in
cystic fibrosis (CF) patients. As life expectancy of these patients
has increased with enhanced treatment, chronic pulmonary
infections, often caused by P. aeruginosa, have emerged as
a leading cause of morbidity and mortality.11 Over the course of
these infections, genetic changes in the bacterial population can
occur, reflected in phenotypes such as the overproduction of
extracellular alginate polysaccharides and loss of motility.12

Fig. 1 Venn diagrams showing the number of proteins shared or unique


among A) four Pseudomonas species, B) five strains of P. aeruginosa, and
C) three strains of P. fluorescens. Protein sequences of four Pseudomonas
genomes were compared, and bidirectional best matches that met speci-
fied criteria were scored as shared proteins.4,6 A and C) Criteria were: a
p value less than or equal to 105, identity of 35% or more, and match
lengths of at least 50% of the length of both query and subject sequence.
The number of shared genes is smaller than that reported earlier29 because
a more stringent standard was adopted in order to clearly identify shared
orthologues rather than homologues. B) Criteria included an alignment
over at least 60% of the query and subject sequence, and 90% of the
5021 genes in core P. aeruginosa having more than 98% identity.6
Diagrams were adapted from Mavrodi et al.4 and Mathee et al.6

1410 | Nat. Prod. Rep., 2009, 26, 14081446 This journal is The Royal Society of Chemistry 2009
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Factors contributing to virulence of P. aeruginosa are a subject of function as opportunistic human pathogens.19 Certain members
intense study, and the bacterium is known to infect numerous of P. fluorescens also exhibit toxicity against insects20 and
model host species in experimental settings, including the model nematodes21 in laboratory experiments.
plant Arabidopsis thaliana, the nematode Caenorhabditis elegans, The secondary metabolism of P. fluorescens is of particular
the insects Drosophila melanogaster and Galleria mellonella, and interest due its role in biological control of plant disease,22
the amoeba Dictyostelium discoideum.12 achieved by applying bacteria to plants or through cropping
Comparisons of the genomic sequences of P. aeruginosa have practices that increase populations of specific antibiotic-producing
revealed a core genome that is highly conserved within the strains of the species.2325 For example, soils that suppress take-all
species 5021 genes having at least 70% sequence identity are disease of wheat develop from agronomic practices that promote
conserved among the genomes of five strains of P. aeruginosa phenazine- or 2,4-diacetylphloroglucinol-producing populations
that were analysed by Mathee et al.6 (Fig. 1). 90% of these of P. fluorescens in the wheat rhizosphere.23 Wheat planted into
conserved genes share at least 98% sequence identity. This these disease suppressive soils does not develop the take-all disease
analysis conforms well to a study by Spencer et al.,8 who esti- even when the fungal pathogen Gaeumannomyces graminis var.
mated the nucleotide divergence in the core genomes of three tritici is present. Antibiotic production by rhizosphere-inhabiting
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other strains of P. aeruginosa at less than 0.5% (one in 200 strains of Pseudomonas spp. can also influence the fitness of the
nucleotides). To place this in perspective, this level of sequence producing strain26 especially in the presence of bacterial preda-
diversity is almost an order of magnitude lower than that found tors21 in soil or other natural substrates. Due to the importance of
in comparisons between E. coli O157 and K-12.8 Along with this antibiotics in biological control and microbial ecology, there has
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high level of conservation, each strain of P. aeruginosa has been great interest in determining the structure, biological activity,
unique components, which appear to arise primarily from and regulation of secondary metabolites produced by
insertions of gene sets acquired by horizontal gene transfer P. fluorescens.22,27,28
(HGT) as well as deletions. These insertions and deletions take The first genomic sequence for the species was obtained from
place at a limited number of sites in the genome of P. aeruginosa, P. fluorescens strain Pf-5,29 which was isolated from the soil in
representing hot spots for genetic recombination.6 Sets of genes College Station, Texas, USA.30 Pf-5 is a well characterized bio-
inserted in these sites compose the majority of the flexible logical control agent3035 that produces a wide spectrum of
genome of each strain, and contribute to the species capacity to secondary metabolites. Prior to genomic sequencing, Pf-5 was
occupy diverse environmental niches characterizing the species. known to produce pyrrolnitrin,30 pyoluteorin,31 2,4-diacetyl-
As detailed below, secondary metabolism is represented in both phloroglucinol,36 hydrogen cyanide,32 a pyoverdine siderophore
the core and flexible genomes of P. aeruginosa. of unknown structure, and a second siderophore related to
pyochelin. As described below, the spectrum of known secondary
metabolites produced by Pf-5 has been greatly expanded since
1.2 Pseudomonas entomophila
the genomic sequence of the strain became available.3739
Pseudomonas entomophila L48, which was isolated from fruit flies The genomic sequences of two additional strains of P. fluo-
or decaying fruits on the island of Guadeloupe, was first rescens, SBW25 and Pf0-1, were published recently.40 Strain
described for its capacity to induce a systemic immune response SBW25 was isolated from the leaf surface of a sugar beet plant
in larvae of the fruit fly Drosophila melanogaster.13,14 It is among grown in Oxfordshire, UK,41 and has become an important
the few isolates of Gram-negative bacteria that are pathogenic to model organism for studies on the ecology and evolution of
insects in both the larval and adult stages of development.13 environmental bacteria. Pf0-1 was isolated from soil in Sherborn,
P. entomophila L48 produces hydrogen cyanide15 and an extra- Massachusetts, USA,42 and has been the subject of studies
cellular protease, which contributes to insect pathogenesis.16 evaluating the molecular basis of bacterial attachment to soil
Insect virulence is thought to be conferred by multiple factors,16 particles and seeds, environmental fitness, and bacterial gene
and the genomic sequence has identified numerous secondary expression in natural habitats.
metabolite gene clusters that could also contribute to the insec- Comparisons between the three sequenced strains of P. fluo-
ticidal activity of P. entomophila.13 rescens indicate a high degree of genomic diversity at the species
level 3688 genes are conserved between the three strains, rep-
resenting 60% to 64% of the genome of each strain (Fig. 1).
1.3 Pseudomonas fluorescens
Although the percentage of the proteome shared among strains
Pseudomonas fluorescens is widely distributed on surfaces of of P. fluorescens is substantially greater than that shared among
virtually all plant tissues, and is also found in many other natural the Pseudomonas species, there is a large fraction of the proteome
habitats, including soil, organic matter, and water. The species is (1146 to 1574 genes) unique to each strain of P. fluorescens. This
extremely heterogeneous, and strains are differentiated into five genomic diversity was also noted by Silby et al.40 who provide
biovars defined by phenotypic and genotypic variation.17 Certain several lines of evidence indicating that P. fluorescens, as
plant-associated strains have the capacity to promote plant currently defined, is extremely heterogeneous and is likely to be
growth or function as biological control agents against plant a species complex.
disease, and some have been developed as commercial products
for management of plant diseases in agricultural settings.18 While
1.4 Pseudomonas syringae
the pathogenic potential of P. fluorescens has been considered
minimal, the species is found in clinical settings and concerns P. syringae is commonly found in water and on plant surfaces in
have been raised regarding the potential of some strains to natural and agricultural environments.43,44 The bacterium is

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disseminated by water or in association with a plant host, and 2.1 Non-ribosomally produced peptides and other amino
typically establishes epiphytic populations on the plant surface acid-derived compounds
prior to infection of the host. Within the species, there are at least
Pseudomonas spp. produce a spectrum of structurally-diverse
50 pathovars, which cause a wide range of plant diseases exhib-
peptides, and many of these are synthesized by large, multi-
iting diverse symptoms, such as leaf or fruit lesions, cankers,
functional proteins called non-ribosomal peptide synthases
blasts, and galls. P. syringae produces several toxins (coronatine,
(NRPSs).45,55 These multienzyme complexes synthesize peptides
phaseolotoxin, syringomycin, and tabtoxin45) as well as phyto-
in a modular fashion, with each module catalyzing the addition
hormones that can function as virulence factors and contribute
and modification of a specific amino acid.56,57 The elongating
to disease symptomatology.
peptide moves from one module to the next in an assembly-line
The diverse pathovars of P. syringae are distributed among five
manner, and the number and sequence of amino acids in the
clades,46 and genomic sequences for strains representing four of
peptide product typically correspond to the number and order of
the five clades are now available. P. syringae pv. tomato DC3000 is
the modules in the NRPS, a phenomenon known as the colin-
a pathogen of Brassica spp., Arabidopsis thaliana, and tomato.47
earity rule. Each elongation module has a minimal set of three
The related strain P. syringae pv. tomato T1 causes bacterial speck
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domains. An adenylation domain (A) selects a specific amino


of tomato. P. syringae pv. phaseolicola 1448A causes halo blight
acid, activates it as an amino acyl adenylate, and transfers it to
disease of bean, characterized by a yellow halo surrounding leaf
the peptidyl carrier protein (PCP), which tethers the elongating
lesions that are associated with the pathogens production of
peptide chain to the module via a thioester bond. The conden-
phaseolotoxin, a chlorosis-inducing phytotoxin.48 P. syringae pv.
sation (C) domain catalyzes peptide bond formation between the
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syringae B728a49 causes brown spot of bean and also induces frost
amino acid present on the PCP of the same module and the
injury due to the presence of ice nucleation active proteins in the
peptidyl intermediate bound to the PCP of the preceding module.
bacterial outermembrane.50 P. syringae pv. oryzae 1_6, a path-
Because signature sequences within the A domain are specific to
ogen of rice,2 represents a fourth clade of P. syringae.
a given amino acid, the amino acid composition of the peptide
Comparison of four P. syringae genomes defined a core
product can be predicted bioinformatically.58 In addition to the
genome of 3594 genes, with 78107 variable regions representing
core domains (A, C, and PCP), other domains may be present,
2536% of the proteomes of P. syringae pv. tomato DC3000,
including epimerization (E), methyltransferase (MT), and cycli-
P. syringae pv. phaseolicola 1448A, and P. syringae pv. syringae
zation (Cy) domains, which are responsible for conversion of
B728a.9 Ninety-seven genes, representing 2.2% of the genome of
regular L-configured amino acids into the corresponding D-form,
P. syringae pv. oryzae 1_6, are unique to that strain.2 Gene
N-methylation, and formation of oxazole rings from Ser or
clusters for the biosynthesis of the phytotoxins coronatine,
thiazole rings from Cys, respectively. The latter ring systems can
phaseolotoxin, and syringomycin are present in the flexible
be further reduced by reductase (R) domains, leading to the
genomes of specific strains. These clusters are typically present in
corresponding oxazolidine and thiazolidine ring systems,
blocks of genes that appear to integrate into specific sites in the
respectively. A loading module, typically having only A and PCP
P. syringae core genome.9 Therefore, in P. syringae, as for other
domains, and a termination module, containing a thioesterase
species of Pseudomonas discussed herein, the flexible genome
(TE) domain required for release and optional cyclization of the
confers traits contributing to the specialized life style of indi-
peptide product, typically complete the NRPS assembly line. For
vidual strains.
more detailed information on NRPSs, we refer the reader to
several excellent reviews.56,57,5961
2 Biosynthetic enzymes, genes and gene clusters of
Pseudomonads
2.1.1 Pyoverdines. All fluorescent Pseudomonas spp. secrete
Pseudomonas species produce an enormous array of natural the yellow-green fluorescent, high-affinity strain-specific side-
products representing varied metabolic origins and exhibiting rophores termed pyoverdines (also called pyoverdins or pseu-
wide-ranging biological activities.45,5155 Although the biosyn- dobactins).62 They enable the acquisition of Fe(III) ions from the
thetic pathways for the Pseudomonas metabolites have much in environment63 and can serve as intracellular signalling
common with those of the well-studied Actinomycetes, they also compounds controlling gene expression.64,65 With the exception
exhibit unusual features. Consequently, the study of secondary of Azotobacter vinelandii, pyoverdines are exclusively produced
metabolism in Pseudomonas spp. has led to the discovery of novel by Pseudomonads. Chemically, pyoverdines are composed of
biosynthetic mechanisms, which are highlighted in the discussion a dihydroxy-quinoline chromophore attached to a variable
that follows. Table 2 summarizes the atypical features of Pseu- peptide chain that comprises 612 amino acids, and a dicarboxy-
domonas secondary metabolism, highlighting the ingenuity of lic acid or its amide. Typically, a given strain produces two to five
this genus regarding natural product biosynthesis. In this section, pyoverdines, differing only in the small dicarboxylic acid side
we provide an overview of selected secondary metabolism chain. Genes (pvd) responsible for the biosynthesis of pyo-
biosynthetic pathways and gene clusters in Pseudomonas spp., verdines are present in a single locus in some Pseudomonads,
omitting those compounds identified through genomic mining such as P. syringae, or up to five different loci in the genome of
approaches, which are featured in section 3. The characterized other species, such as P. fluorescens (Fig. 2).66
gene clusters and enzymes are presented according to their The pyoverdine biosynthesis gene cluster is best described in
metabolic origin. It is noteworthy that neither terpenoid P. aeruginosa PAO167 (Fig. 3). Both the chromophore68 and the
metabolites nor terpene synthases have been found to date in peptide chain69 are synthesized by NRPSs. From incorporation
Pseudomonas species. experiments, 2,4-diaminobutyrate70 and tyrosine71 have been

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Table 2 Features of secondary metabolism biosynthetic gene clusters identified from Pseudomonads

Compound Type Producer Unusual features

Pyochelin NRPS P. aeruginosa Unusual location of the E domain


Pseudomonine NRPS P. fluorescens AH2 Nonenzymatic rearrangement during biosynthesis
P. fluorescens WCS374
P. entomophila L48
Paerucumarin NRPS P. aeruginosa Isonitrile synthase, nonenzymatic conversion
Pseudoverdin
Syringomycin NRPS P. syringae pv. syringae Non-colinear, split modules, combined C/E domains,
non-heme FeII halogenase
Syringopeptin NRPS P. syringae pv. syringae Largest linear NRPS system described for
prokaryotes, combined C/E domains, tandem TE
domains
Arthrofactin NRPS Pseudomonas sp. MIS38 Combined C/E domains, tandem TE domains
Massetolides NRPS P. fluorescens SS101 Disconnected NRPS cluster, combined C/E domains,
Published on 01 October 2009 on http://pubs.rsc.org | doi:10.1039/B817075B

Pseudomonas sp. MF-30 tandem TE domains


Putisolvin NRPS P. putida PCL1445 Tandem TE domains
Orfamides NRPS P. fluorescens Pf-5 Tandem TE domains
Syringofactins NRPS P. syringae pv. syringae Tandem TE domains, linear lipopeptide
P. syringae pv. tomato
Safracin NRPS P. fluorescens A2-2 Iterative use of an NRPS module, reductive chain
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release
Tabtoxin AA-derived P. syringae pv. tabaci, Off-branching product from the lysine-pathway
pv. coronafaciens and pv. garcae
Pyrrolnitrin AA-derived P. fluorescens First example of the FADH2-dependent halogenases
P. aurantiaca BL915
Pseudomonas sp.
Indoleacetic acid AA-derived Pseudomonas spp. More than one pathway leading to the same molecule
Mupirocin PKS P. fluorescens NCIMB 10586 Trans-AT PKS, ACP doublets/triplets
DAPG PKS P. fluorescens Type III PKS chalcone synthase
2,5-Dialkylresorcinols PKS P. aurantiaca BL915 Modified fatty acid
Syringolin NRPS-PKS P. syringae pv. syringae Modified amino acids, unknown mechanism for the
incorporation of valine via a urea moiety
Pyoluteorin NRPS-PKS P. aeruginosa Inactive KR and DH domains, lack of loading
P. fluorescens Pf-5 modules, iteratively working FADH2-dependent
Pseudomonas sp. M18 halogenase, unusual location of TE domain
Coronatine NRPS-PKS P. syringae Unusual starter unit, L-allo-Ile specific A domain,
endotrig cyclization, cryptic halogenation with
a new type of halogenase, the non-heme-Fe2+-
ketoglutarate dependent halogenase
Pederin NRPS-PKS Pseudomonas sp. Split cluster, trans-AT PKS, unusual loading module,
tandem DH domains, MT domains for the
generation of geminal methyl groups, FAD-
dependent monooxygenase involved in the
production of the unusual oxidized single carbon
terminus of pederin, inactive modules (pedH)
Rhizoxins NRPS-PKS P. fluorescens Pf-5 Trans-AT PKS, inactive KS/ACP modules, tandem
ACP modules, polyketide-chain branching by an
enzymatic Michael addition
Phenazines P. chlororaphis Alkaloid derived from anthranilic acid
P. fluorescens
P. aeruginosa
Quinolone P. aeruginosa Alkaloid derived from anthranilic acid
HCN Pseudomonas spp. Toxin derived oxidatively from glycine

identified as precursors for biosynthesis of the chromophore via hydroxyaspartate.72 During the assembly or upon completion of
the NRPS PvdL, which consists of four modules. The deduced the peptide backbone, the peptide chain can be further modified,
amino acid sequence of the first domain in module 1 is similar to particularly to generate the above-mentioned unusual amino
acyl-CoA ligases (ACL) domains, but the function of this acids. The products of pvdA (ornithine hydroxylase)73 and pvdF
domain has not yet been elucidated. A domains of the remaining (transformylase)74 perform the respective hydroxylation and
NPRS modules activate and condense the amino acids gluta- formylation at N-5 of ornithine. For completion of the chro-
mate, tyrosine and 2,4-diamino-butyric acid. No TE is present at mophore, a second ring closure and several hydroxylation and
the C-terminus of PvdL, but a gene encoding a TE is clustered oxidation steps are required. Several mechanistic proposals for
with pvdL. The NRPSs PvdI, PvdJ, and PvdD, synthesize the these steps have been proposed.75 Stintzi et al. suggested that the
peptide chain of the pyoverdine, extending the tripeptide product products of a separate cluster of four genes (pvcABCD) perform
of PvdL by eight amino acids. The peptide chain comprises the corresponding reactions.76,77 At the time of discovery, the
L- and D-amino acids, some of which are unusual, such as proteins encoded by this operon showed similarity to proteins
N5-hydroxyornithine, N5-formyl-N5-hydroxyornithine and involved in spore wall maturation (PvcA), oxygenases (PvcB),

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Fig. 2 Varied organization of pyoverdine clusters in Pseudomonas spp. Homologous genes are shown in the same color, and genes are not drawn to
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scale. Revised from Swingle et al.415 and Ravel and Cornelis.66

Fig. 3 Biosynthesis model of pyoverdines in P. aeruginosa PAO1. ACL: acyl-CoA ligase, Dab 2,4-diaminobutyric acid.

hydroxylases (PvcC) and proteins of the cytochrome c protein production by P. aeruginosa.78 Therefore, although P. aeruginosa
family (PvcD). Chemically, it can be envisioned that PvcB creates may borrow the enzymatic Pvc machinery (PvcBCD) for the
a Da,b double bond in the tyrosine moiety, while PvcC and PvcD production of the pyoverdine chromophore under some
form a catechol structure at the aromatic ring of Tyr. The conditions, this is not always the case. Furthermore, alternative
resulting intermediate could undergo an intramolecular cycliza- pathways for completion of the chromophore apparently exist,
tion to give the characteristic pyoverdine chromophore. The because pyoverdines are produced by all fluorescent Pseudomo-
absence or malfunction of parts of the pvc cluster could explain nads whereas pvcABCD is found only in P. aeruginosa, and is not
the existence of ring-opened pyoverdines missing one double present in genomes of P. fluorescens, P. putida, or P. entomophila.
bond, termed ferribactins or dihydropyoverdines, which have Pyoverdines are a defining characteristic of the fluorescent
been found to accompany the pyoverdines.68 Recently, however, Pseudomonads, so it is no surprise that the pyoverdine gene
Brady and coworkers demonstrated that the pvc gene cluster clusters are a component of the core genome common to all
codes for the biosynthesis of paerucumarin and that pvcA, iden- species. Pseudomonas stutzeri, which does not produce pyo-
tified as an isonitrile synthase, is not required for pyoverdine verdine, poses an obvious exception to this generality. While the

1414 | Nat. Prod. Rep., 2009, 26, 14081446 This journal is The Royal Society of Chemistry 2009
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gene composition is generally conserved, the organization pch cluster.8587 As illustrated in Fig. 4, the cluster consists of the
of pyoverdine biosynthesis genes in the genomes of different two divergent operons pchDCBA and pchEFGHI, separated by
Pseudomonas spp. differ markedly (Fig. 2), as do the nucleotide the regulatory gene pchR. Salicylate is made from chorismate via
sequences of certain genes for pyoverdine biosynthesis and isochorismate by the action of the isochorismate synthase PchA
uptake. In landmark studies evaluating genome divergence in and isochorismate-pyruvate lyase PchB, respectively.88 PchD
strains of P. aeruginosa, the pyoverdine gene cluster was found to activates salicylate as salicyl-AMP in order to start the chain
be one of most highly divergent regions in the core genome.8 growth by a thiotemplate mechanism. Each of the two NRPS
Genes encoding the outer membrane pyoverdine receptor (fpvA) modules encoded by pchE and pchF performs an elongation with
and the NRPSs specifying the pyoverdine peptide chain (pvdI, cysteine, and the products are heterocyclized by cyclo-dehydra-
pvdJ, and pvdD) are the most divergent genes in the region,79 tion to thiazoline rings. During the formation of the N-terminal
indicating a co-evolution that maintains the mutual specificity of thiazoline ring, the E domain of PchE inverts the absolute
the siderophore for the receptor. While the pyoverdine gene configuration from L-Cys into D-Cys. PchE is noteworthy in the
cluster is a component of the core genome of P. aeruginosa, it unusual location of the E domain between the A and PCP
exhibits unusual codon usage and unusual oligonucleotide usage, domains, as E domains are typically located directly downstream
Published on 01 October 2009 on http://pubs.rsc.org | doi:10.1039/B817075B

both of which suggest a history of HGT, possibly from other of a PCP domain. The C-terminal thiazoline gets reduced by the
Pseudomonas spp.79 This HGT, coupled with extensive recom- NADPH-dependent reductase PchG87 (R-domain), thereby
bination and forces of diversifying selection, provides an expla- permitting N-methylation of the generated thiazolidine ring by
nation for the polymorphism of the pyoverdine gene cluster in the co-substrate S-adenosyl methionine (SAM) and the MT
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the Pseudomonas spp.79 Therefore, genomic analysis of the domain embedded in PchF.89 In a final step, the TE domain
pyoverdine regions of Pseudomonas spp. has revealed an extra- catalyzes the hydrolytic release to give the tandem bisheterocyclic
ordinary level of variation that is in line with the chemical compound pyochelin. The ABC transporter encoded by pchH
diversity of the pyoverdine structures themselves. and pchI exports the siderophore, whereas the pyochelin receptor
FtpA functions in uptake of the ferric-pyochelin complex.
2.1.2 Pyochelin. Since acquisition of iron is vitally important Pyochelin was first detected in iron-deficient cultures of
for microbes, Pseudomonads created by combinatorial evolution P. aeruginosa ATCC 15692 (strain PAO1) and has since been
and shuffling of bacterial assembly lines a range of siderophores. isolated from other Pseudomonads and Burkholderia species.9094
The iron-scavenging metabolite pyochelin has a very different Members of the Betaproteobacteria, the genus Burkholderia
scaffold than the pyoverdines. For acquisition of iron, pyochelin encompasses 40 species found in diverse environments, such as
forms a 2:1 complex with ferric iron and acts as a tetradentate soil, plant surfaces, and water, and associated with insect, fungal,
ligand.8082 Pyochelin is the condensation product of salicylic and mammalian hosts. Like P. aeruginosa, certain Burkholderia
acid and two molecules of cysteine.83 The structure contains three spp. are opportunistic human pathogens that cause respiratory
chiral centers (C-40 , C-200 , C-400 ) and exists as a mixture of the two infections in patients with cystic fibrosis. Prolific production of
interconvertible diastereomers pyochelin I (40 R, 200 R, 400 R) and secondary metabolites is a hallmark of Burkholderia spp., and
pyochelin II (40 R, 200 S, 400 R).84 pyochelin is one of many compounds produced by both
In P. aeruginosa, genes required for the biosynthesis of Pseudomonas and Burkholderia spp. The organization of the pch
pyochelin map to a ca. 21 kb genomic region designated as the gene cluster is identical in the genomes of Burkholderia spp. and

Fig. 4 Organization of the pch gene cluster in P. aeruginosa PAO1 and corresponding biosynthetic scheme for pyochelin formation. R: reductase
domain, responsible for the reduction of thiazoline to thiazolidine.

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P. aeruginosa, but is different from that in P. fluorescens Pf-5, as pseudomonine. Very recently, it was demonstrated by in vitro
described below. The conservation and divergence of gene clus- reconstituation experiments that the pseudomonine NRPS
ters encoding for identical or similar metabolites in the Pseudo- (PmsDEG) shows relaxed substrate specificity.98 In vitro,
monads and Burkholderias is a central theme that emerges PmsDEG can utilize 2,3-dihydroxybenzoate instead of salicylate
repeatedly throughout this review. and Cys instead of Thr, yielding the siderophores acinetobactin
and anguibactin, which are produced by the Gram-negative
2.1.3 Pseudomonine. Pseudomonine, isolated from P. fluo- bacteria Acinetobacter baumannii and Vibrio anguillarum,
rescens AH2, is another Pseudomonas metabolite that functions respectively.
as a siderophore.95 Like pyochelin, the compound consists of
salicylic acid and two heterocyclic amino acid moieties. Despite 2.1.4 Paerucumarin and pseudoverdine. Paerucumarin is an
the chemical resemblance, the assembly lines of the two mole- isonitrile functionalized dihydroxycumarin that is produced by
cules exhibit intriguing differences at the genetic and biochemical the pvc gene cluster, the aforementioned four-gene operon that is
levels. The pseudomonine biosynthetic genes were localized by also reported to be involved in the biosynthesis of the pyoverdine
Bakker and coworkers in the biocontrol strain P. fluorescens chromophore. Recently, however, Brady and coworkers repor-
Published on 01 October 2009 on http://pubs.rsc.org | doi:10.1039/B817075B

WCS37496 and by Walsh and associates in P. entomophila L48.97 ted that the pvc cluster is responsible for the production of the
The 21 kb pms-cluster (Fig. 5) is divided into seven genes new bicyclic metabolite paerucumarin in P. aeruginosa.78 It is
encoding three NPRSs (PmsG,D,E) and four precursor biosyn- hypothesized that the isonitrile synthase PvcA generates an iso-
thetic enzymes (PmsA,B,C,F). The starting point in the biosyn- nitrile-functionalized tyrosine that is subsequently oxidized by
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thesis of pseudomonine is the formation of salicylic acid from the amino acid oxidizing enzyme PvcB (Fig. 6). The resulting
chorismate by the isochorismate synthase PmsC and iso- intermediate is then converted by the FAD-dependent mono-
chorismate-pyruvate lyase PmsB, respectively, followed by its oxygenases PvcC/D into the corresponding catechol structure,
activation by the A domain of PmsE. The Cy2 domain of PmsD which in turn undergoes an intramolecular cyclization to yield
transfers the salicylic acyl group to L-threonyl-S-pantetheinyl- paerucumarin. Due to the instability of isonitrile-containing
PmsG, while Cy1 of PmsD performs a cyclodehydration to metabolites, paerucumarin can decompose by the addition of
yield the salicylmethyloxazolinyl-PmsG covalent acylenzyme water to its N-formyl adduct pseudoverdine. Therefore, the
intermediate. The histidine decarboxylase PmsA and histamine- observed production ratio of paerucumarin to pseudoverdine
N-hydroxylase PmsF are involved in provision of N-hydroxy- varies with the purification procedure and also from strain to
histamine, which serves as a nucleophilic substrate for the C strain of P. aeruginosa. No biological functions of paerucumarin
domain of PmsG to release the corresponding oxazoline and pseudoverdine have been reported.
hydroxamate condensation product, termed pre-pseudomonine.
The latter compound is unstable and rearranges nonenzymati- 2.1.5 Lipopeptides. The cyclic lipopeptides (CLPs) are
cally to pseudomonine. An SN2 attack at the b-carbon of the a structurally-diverse class of compounds containing a fatty acyl
Thr-derived oxazoline results in the formation of an iso- residue ranging from C5C16 in length and chains of 725 amino
xazolidinone ring. This intramolecular reaction inverts the acids of which 414 form a lactone ring. Based on their structural
absolute configuration at the b-carbon of the Thr side chain, relationships, the CLPs of Pseudomonads can be generally
which explains the presence of L-allo-configured Thr in classified into six groups (Table 3).55 The combination of a polar

Fig. 5 Organization of the pms genes and corresponding biosynthetic scheme for pseudomonin formation.

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Fig. 6 The pvc gene cluster and corresponding biosynthetic scheme for paerucumarin and pseudoverdine formation.

peptide head with a lipophilic fatty acid tail is responsible for the structure of the final peptide. The incorporated serine and ami-
amphiphilic properties of these compounds, which can lower nobutyric acid are D-configured in the CLP product, yet no
Published on 01 October 2009 on http://pubs.rsc.org | doi:10.1039/B817075B

surface tension and interact with cellular membranes, thereby E domains were identified in their respective NRPS modules.
altering their integrity.55 The latter effect is assumed to Another mechanism by which D-configured amino acids can be
contribute to various interactions with other organisms, e.g. integrated into nonribosomal peptides is the direct selection of
plant pathogenicity,45 antifungal,99101 antibacterial,102 anti- the free D amino acid by a corresponding A domain, as is the case
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viral103 and phosphatidylinositol-specific phospholipase (PI- in for D-Ala1 of cyclosporine synthetase.117 Because SyrE2
PLC) inhibitory104 activity. Physiologically, this compound class recognizes only L-serine, however, the change of the configura-
may be produced by the bacterium for multiple reasons. CLPs tion is thought to occur at the peptidyl or at the aminoacyl stage
can first increase the bioavailability of water-insoluble and to be performed by an external racemase or epimerase.
substrates; second, promote cellular swarming, which enhances The mechanism for synthesis and attachment of the lipid
the colonization of surfaces;37,105 and third, enhance virulence or moiety to the peptide chain of syringomycin remains unclear. In
antagonism against other microorganisms.106108 other CLP-producing bacteria, such as Bacillus spp., genes
encoding these functions are commonly clustered with the NRPS
Syringomycin. Characterization of the biosynthesis pathway of structural genes. The itu gene cluster of Bacillus subtilis RB14,
the lipodepsinonapeptide syringomycin,109,110 a virulence deter- which codes for the biosynthesis of the CLP iturin A, contains
minant of the phytopathogenic strain P. syringae pv. syringae e.g. a fatty acid synthase,118 while in the srf operon of B. subtilis
B301D,111 provided the first insights into the synthesis of Pseu- ATCC21332, the hydroxy fatty acid moiety of the CLP surfactin,
domonas CLPs. Based on mutagenesis and feeding experiments is contributed by an acyl transferase.119 No evidence for the
on the one hand, and due to the presence of D-configured and presence of an internal or external synthase or transferase system
unusual amino acids on the other hand, it was hypothesized that has been reported to date for the syr cluster. However, the
an NRPS mechanism is involved in the biosynthesis pathway of presence of a C domain in the first module of SyrE provides
syringomycin. The syringomycin (syr) gene cluster of P. syringae a clue for a possible mechanism for incorporation of the lipid
pv. syringae strain B301D is about 37 kb in size and show an moiety. C domains preceding the first amino acid activation
unusual and nonlinear genetic organization. It comprises six domains are present in NRPSs having products with an acylated
genes with structural functions (syrB1, syrB2, syrC, syrE and amino acid in the first position, so C domains with this placement
syrP), three genes with regulatory functions (syrF, syrG and are thought to be involved in acylation.119122 Thus, it is likely
salA), and syrD with secretory functions112 (Fig. 7). The that this domain catalyzes the attachment of the 3-hydroxy fatty
biosynthesis of the syringomycin nonapeptide core is catalyzed acid moiety to the N-terminal serine, although a donor ACP
by two NRPSs that do not respect the colinearity rule. The syrB1 would still be necessary according to our current knowledge.
gene, responsible for the incorporation of the ninth amino acid, is Little is known about the origin of the incorporated 3-OH-fatty
located upstream of syrE, which encodes for the NRPS incor- acids, but they appear to be derived from 3-OH-alkanoates that
porating the first eight amino acids. Additionally, a split-module are accumulated in Pseudomonads and utilized as carbon and
phenomenon is present: the C-terminus of SyrE contains the C energy sources.123
and PCP domain of a ninth module but lacks an A domain,
which is present in SyrB1. SyrB1 activates and loads L-Thr Syringopeptin. Directly adjacent to the syr gene cluster in
which, while still tethered to the T-domain of SyrB1, is chlori- P. syringae pv. syringae strain B301D is a NRPS gene cluster
nated to 4-Cl-L-Thr by the non-heme FeII halogenase coding for the biosynthesis of the phytotoxic and necrosis-
SyrB2.113,114 The aminoacyltransferase SyrC shuttles the chloro- inducing CLP syringopeptin.124 The syringopeptins form a CLP
threonyl moiety in trans between the PCP domain of SyrB1 and class containing either 22 or 25 amino acids, depending on the
SyrE, thereby enabling the final elongation to the full-length bacterial strain,125127 and 3-hydroxydecanoic or 3-hydroxy-
nonapeptidyl.115 The TE domain located at the C-terminal end of dodecanoic acid as lipid moiety. At 74 kb and carrying
SyrE catalyzes the release and cyclization of the mature peptide 22 NRPS modules, the syringopeptide (syp) cluster represents the
chain. Recently, Walsh and coworkers demonstrated that SyrP is largest linear NRPS system described for prokaryotes.
involved in the b-hydroxylation of Asp into L-threo-3-OH-Asp The syp cluster consists of three large open reading frames,
while it is tethered to the T-domain of the eighth module.116 sypA, sypB and sypC.128 The order and number of the modules of
There is a discrepancy between the encoded genes and the stereo- the syp cluster are colinear to the amino acid sequence of the final

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Table 3 Primary structures of representatives of the six classes of cyclic lipopeptides (CLPs) produced by Pseudomonas spp.a

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Fig. 7 The syringomycin biosynthetic assembly line from Pseudomonas syringae pv. syringae B301D. Depicted are only the secrectory and structural
genes. The regulatory genes salA, syrF and syrG are not shown but are located downstream of syrE.

syringopeptin SP22. However, analogous to the syr gene cluster, each being responsible for both chain release and for the cycli-
no E domains are present, despite the presence of multiple zation reaction between Asp and the hydroxy group of the
D-configured amino acids in syringopeptin SP22; and no genes 3-hydroxy-decanoic acid, the latter being an unusual reaction in
governing the attachment of the lipid moiety have been identified CLP biosynthesis by Pseudomonas spp. Using site-directed
to date. In contrast to the syr gene cluster, two (instead of one) mutagenesis, Morikawa and coworkers demonstrated recently
internal TE domains (type I) were found at the end of SypC. that both TE domains are functional and work cooperatively:
Because a single C-terminal TE domain is commonly sufficient a mutation in the first TE domain abolished arthrofactin
for the hydrolytic cleavage of the peptide from its synthase and production completely, whereas a mutation in the second TE
subsequent cyclization, the function of the second TE domain is domain reduced the production of arthrofactin by 95%.133
unclear. Bioinformatic analysis indicated that the first TE Similar to the syr and syp gene cluster, the arf gene cluster
domain is intact whereas the second TE domain, which lacks contains no genes with putative functions in the fusion of the
a conserved histidine residue, may be non-functional.128 lipid moiety or the conversion of L- into D-configured amino
In P. syringae pv. syringae strains B301D and B728a, the syr acids. However, Walsh and associates shed light on latter
and syp clusters are located on large pathogenicity islands.129,130 phenomenon. Using several lines of evidence, they demonstrated
At more than 180 kb, the pathogenicity island of strain B728a that the epimerization is not catalyzed by external racemases but
accounts for nearly 3% of the genome, and includes genes for rather performed by dual condensation/epimerization (C/E)
biosynthesis of the siderophore achromobactin, antibiotic resis- domains, i.e. that the epimerase activity is cryptically embedded
tance, the modification of lipid A with arabinose, and a degen- with the C domain. These novel C/E domains, which are always
erate locus for phaseolotoxin biosynthesis.49 located downstream of the module having the amino acid that is
epimerized in the final product, can be recognized bio-
Arthrofactin. Tandem C-terminal TE domains are also present informatically by an elongated His motif conforming to the
in the arthrofactin synthase (arf) from Pseudomonas sp. MIS38, sequence HHI/LxxxxGD.134 The occurrence of this pattern was
the third well-investigated CLP biosynthetic pathway of the pre- also identified in the corresponding C-domains of the syringo-
genomic era. The colinear arf operon spans 39 kb and consists of mycin and syringopeptin gene cluster, which may account for the
the three genes arfA, arfB and arfC containing two, four and five formation of D-amino acids in these CLPs.
amino activating modules131 (Fig. 8), accounting for all eleven
amino acids found in the arthrofactin structure.132 The Massetolides. The massetolides consist of a nine amino
C-terminal end of ArfC contains two internal TE-type I domains, acid-containing cyclic peptide moiety linked to either

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Fig. 8 The arf biosynthesis gene cluster from Pseudomonas sp. MIS38 and resultant chemical structure of arthrofactin.
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a 3-hydroxy-decanoic, -undecanoic or -dodecanoic acid. Mas- D-configured amino acids in the molecule despite the lack of
setolides AH were first identified in Pseudomonas species iso- E domains. As described above for other Pseudomonas CLP
lated from a marine alga and a marine tubeworm, which were clusters, no genes involved in biosynthesis of the lipid side chain
collected near Masset Island, British Columbia, Canada.102 or in acyl transfer were identified. The predominant product of
Later, massetolides were isolated from terrestrial Pseudomo- the gene cluster is massetolide A, but the co-occurrence of the
nads, e.g. the biocontrol strains P. fluorescens SS101135 and minor analogs massetolides BH, differing in the amino acid and
Pseudomonas sp. MF-30.136 In addition to their anti- fatty acid composition, was ascribed to the relaxed substrate
mycobacterial properties,102 the massetolides exhibit antifungal specificity of the respective A and C domains. In particular, the
and potent surfactant activities, as well as destructive effects on flexibility in amino acid selection by A-domains was exploited in
zoospores of multiple Oomycete plant pathogens.137 In contrast a precursor-directed biosynthesis (mutasynthesis) study to
to the above-mentioned Pseudomonas CLP gene clusters, genes generate new derivatives, leading to the analogs massetolide IK,
for massetolide biosynthesis are not physically linked but are containing nonproteinogenic amino acids at position AA1, AA4
organized into two separate clusters in the genome of P. fluo- and AA9.102
rescens SS101.138 One gene cluster, spanning about 30 kb,
contains two NRPS genes, designated massBC, regulatory genes Putisolvin. The putisolvins I and II were the first known CLPs
of the luxR-type, and the efflux/resistance genes macA and macB, to have a peptide chain of 12 amino acids linked to a hexanoic
respectively (Fig. 9). A second gene cluster containing the NRPS lipid chain139,140 (Table 3). The two structures differ only at the
encoding massA is physically disconnected from the massBC second amino acid from the C-terminus, which is Val for puti-
cluster in the genome of strain SS101. In other respects, the solvin I and Leu/Ile for putisolvin II. In diverse bioassays,
NRPSs for massetolide biosynthesis are typical of other CLP putisolvins I and II were shown to reduce surface tension,
biosynthetic pathways: in agreement with the presence of nine stimulate swarming motility, inhibit biofilm formation, degrade
amino acids in the final product, MassABC comprise nine existing biofilms and micellate the organic solvent toluene. The
modules, each with a CAPCP domain structure, and MassC putisolvin-producing strain P. putida PCL1445 was isolated from
terminates with two TE domains. Phylogenetic analysis of the soil heavily polluted with polyaromatic hydrocarbons,141 so the
C-domains revealed that they group partially together with latter capability of putisolvins possibly represents an intriguing
established dual C/E domains, hence explaining the presence of survival strategy. Three NRPS genes, termed psoABC, were

Fig. 9 Organisation of the mass gene cluster in P. fluorescens SS101 encoding the CLPs massetolides AH.

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Fig. 10 The pso biosynthesis gene cluster from Pseudomonas putida PCL1445 and resultant chemical structures of putisolvin I (R1 CH3, R2 R3 H)
and II (R1 R2 CH3, R3 H or R1 H, R2 R3 CH3).
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identified in P. putida PCL1445 and shown to be responsible for incorporated by three modules was revealed in the homologous
the production of both putisolvins (Fig. 10). The structural saframycin A gene cluster (sfm) from Streptomyces lavandulae
NRPS genes are flanked downstream by the genes macA and NRRL 11002, where SacA and SacB recognize and activate Ala
macB, which are likely to function in putisolvin export, and and Gly, respectively, and SacC acts in an iterative fashion,
upstream by the luxR-type regulatory gene psoR. PsoABC codes activating the tyrosine derivative 3h5mOmTyr twice to produce
for 12 typical NRPS modules which obey the co-linearity rule. the required tetrapeptide intermediate.149 In addition to the
Bioinformatic predictions for the substrate specificity of the A iterative use of one module, the NRPS assembly line composed
domain of module 11 indicates a preference of Val over Leu or of SacABC is also conspicuous due to substitution of the TE
Ile, which correlates well with the commonly observed ratios of domain, typically found as the C-terminal domain of NRPSs, with
putisolvin I/II (3:2).140,142 In addition, relative production of the a terminal reductase (R) domain, suggesting a reductive chain
two putisolvins is influenced by the availability of Val, Leu or Ile release similar to that in saframycin,150 myxochelin151 or nosto-
in the culture medium.143 cyclopeptide152 biosynthesis. In nostocyclopeptide biosynthesis,
the R domain performs dual functions, catalyzing the reductive
2.1.6 Safracin. Safracins, which exhibit antitumor activity, release of the matured peptide chain as an aldehyde and triggering
have been isolated from P. fluorescens A2-2144 and SC 12695145 the spontaneous formation of an imino head-to-tail linkage.
and exhibit an extraordinary three-dimensional molecular Similarly, the R domain in SacC may catalyze the chain release of
architecture based on a tetrahydroisoquinoline skeleton. The the matured peptide chain as an aldehyde and then trigger the
discovery of the safracin biosynthetic pathway was driven by the spontaneous formation of the imino head-to-tail linkage, leading
structural similarity of safracin to the potent anti-tumor agent to the closure of ring C of safracin. Before or during the subse-
ecteinascidin (ET-743, trabectedin, Yondelis), isolated from quent cyclization reactions, one of the two 3h5mOmTyr moieties
the tunicate Ecteinascidia turbinata. Neither the harvest of is additionally hydroxylated by the monoxygenase SacJ, which
natural populations nor aquaculture could provide sufficient leads to a quinone ring structure, while the methyltransferase SacI
quantities of ecteinascidin to continue clinical development. performs N-methylation at the amine of the remaining
Furthermore, manufacturing or total synthesis was not 3h5mOmTyr residue. In a final step, the resulting safracin B is
economical at an industrial scale. Therefore, a partial synthesis converted by SacH into safracin A, which lacks a hydroxyl-group
of ecteinascidin starting from safracin B, produced by Pseudo- at C-21. Because safracin A exhibits lower antibiotic and anti-
monads, was envisioned.146,147 tumoral activity than safracin B, sacH is considered to be a resis-
The safracin gene cluster (sac) in P. fluorescens A2-2 comprises tance gene functioning in detoxification.
ten open reading frames coding for three NPRSs (SacA, SacB,
SacC), three precursor biosynthetic enzymes (SacD, SacF, 2.1.7 Tabtoxin. Tabtoxin represents another example of
SacG), two tailoring enzymes (SacI, SacJ), one resistance protein phytotoxic peptides from P. syringae. The compound consists of
(SacH) and one protein of unknown function148 (Fig. 11). The tabtoxine-b-lactam (TbL) and threonine. The small dipeptide is
unusual amino acid derivative 3-hydroxy-5-methyl-O-methyl- known to be secreted by three pathovars of P. syringae: tabaci,
tyrosine (3h5mOmTyr) is synthesized from L-Tyr by two methyl- coronafaciens and garcae.153 The phytotoxin is associated with
transferases, SacF and SacG, and the hydroxylase SacD. Two the symptoms of wildfire disease on tobacco and halo blight of
molecules of 3h5mOmTyr, together with Ala and Gly, are oats. However, the chlorosis-inducing effects occur only after
selected and loaded on specific NPRS modules to form the hydrolysis of the dipeptide with aminopeptidases present in
safracin tetrapeptide basic skeleton. The NRPS pathway exhibits either the bacteria or the plant, yielding the actual toxin TbL.
several unusual features. While the peptidic backbone of safracin The mode of action of TbL is the irreversible inhibition of
contains a stretch of four amino acids, sacABC encode only three glutamine synthase, which catalyzes the synthesis from glutamic
NRPS modules. The mechanism by which four amino acids are acid to glutamine in amino acid metabolism. This inhibition

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Fig. 11 Structural organization of the sac gene cluster of P. fluorescens A2-2 and proposed safracin biosynthesis mechanism. R: reductase domain,
responsible for reductive chain release.

results in reduced availability of Gln, which blocks the only acyltransferase), and TblE in conjunction with TblF (putative
efficient ammonia detoxification pathway in plants, leading to membrane protein, forming a functional pair with a D-Ala D-Ala
ammonia toxicity.154,155 ligase). It has been shown that these enzymes are essential for
The biosynthetic enzymes of the virulent isolate P. syringae BR2 tabtoxin biosynthesis, but details about the enzymatic mechanisms
are encoded by the 15 kb tab/tbl gene cluster156 (Fig. 12). Several remain to be elucidated.160,161 Upon assembly, the completed
genes of the cluster (e.g. tabA and tabB) show a high resemblance tabtoxin can be converted by the metallopeptidase TabP into the
to genes of the bacterial lysine biosynthesis pathway. toxin TbL, which is subsequently exported by the transporter
Labeling157,158 and knockout159 studies demonstrated that tabtoxin TblR. The cleavage of tabtoxin occurs only in the presence of zinc,
biosynthesis proceeds along the lysine pathway (dabABCDE), which is required for peptidase activity.162,163
branching off after tetrahydropicolineate and before dia- Evidence for HGT is found in sequences surrounding the tbl/
minopimelate formation. From this branch point, tabtoxin is tab cluster in P. syringae pv. tabaci.156 The cluster is bordered by
assembled by the action of TblS (putative b-lactam synthetase), a lysC tRNA gene, which is a common site for integration of
TblC (putative clavaminic acid synthase), TblD (putative GNAT genomic islands in bacteria including Pseudomonas spp.,164166

Fig. 12 Structural organization of the tab/tbl gene cluster and proposed biosynthetic pathway to tabtoxin.

1422 | Nat. Prod. Rep., 2009, 26, 14081446 This journal is The Royal Society of Chemistry 2009
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Fig. 13 Pht gene cluster of Pseudomonas syringae pv. phaseolicola NPS3121 and its resulting structure phaseolotoxin. In planta, phaseolotoxin is
processed by a peptidase to create the active toxin PSOrn, also known as octicidine.
Published on 01 October 2009 on http://pubs.rsc.org | doi:10.1039/B817075B

and a tyrosine recombinase, which is also associated with HGT and Orn, which are synthesized by transamination from Arg and
of the phaseolotoxin gene cluster, described below. The tabtoxin Lys, respectively.181 Regarding the assembly of the tripeptidic
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biosynthetic gene cluster has been reported to excise readily from backbone of phaseolotoxin, the involvement of a non-ribosomal
the chromosome of P. syringae pv. tabaci,167 and the locus thiotemplate mechanism was always assumed, but corresponding
exhibits a patchy distribution among the major groups of NRPS genes have not yet been identified. Very recently, experi-
P. syringae.46 Certain genes in the region are also present in the mental work by Kino and Arai gave indications of another
PAPI-1 pathogenicity island of P. aeruginosa PA14.156 Together, possible biosynthesis strategy for the tripeptide moiety. In the
these data provide convincing evidence for HGT in the evolution genome of P. syringae pv. phaseolicola 1448A, Kino et al. iden-
of tabtoxin-producing strains of P. syringae. tified phtU as a novel L-amino acid ligase-encoding gene. The
protein encoded by phtU (PSPPH_4299) was proven to synthe-
2.1.8 Phaseolotoxin. Phaseolotoxin is a phytotoxic tripeptide size various hetero-dipeptides, but not a tripeptide, in an ATP-
consisting of ornithine, alanine and homoarginine, linked to an dependent manner.182 Therefore, it can be envisioned that the
inorganic sulfodiaminophosphinyl moiety,168,169 that is produced peptide backbone might be constructed by PhtU and an addi-
by P. syringae pvs. phaseolicola and actinidiae,170 pathogens tional, currently unidentified L-amino-acid ligase instead of the
causing halo blight of bean and canker of kiwi fruit, respectively, NRPS machinery. However, a growing body of evidence is
as well as by P. syringae pv. syringae CFBP3388.171 Phaseo- pointing to the fact that at least one gene outside the argKtox
lotoxin exerts its phytotoxic effects by competitive inhibition of gene cluster, possibly encoding an NRPS, is partly involved in
ornithine carbamoyl transferase (OCTase), a key enzyme in the the synthesis and assembly of the tripeptide moiety (S. Aguilera
urea cycle that converts ornithine and carbamoyl phosphate to and A. Alvarez-Morales, personal communication).
citrulline. As a result, ornithine accumulates and a deficiency in In phaseolotoxin-producing strains of P. syringae pv. actini-
intracellular pools of arginine occurs. Although phaseolotoxin is diae and P. syringae pv. phaseolicola, argKtox clusters have
a reversible inhibitor of OCTase, it is hydrolyzed in planta by been associated with genomic islands integrated into the genomes
peptidases to Nd-(N0 -sulfodiamino-phosphinyl)ornithine (syn. in a site-specific manner.183 These clusters are flanked by inser-
PSorn or octicidine), which is a strong irreversible inhibitor of tion sequences or genes encoding for members of the tyrosine
OCTase.172 The 28 kb phaseolotoxin gene cluster, termed the recombinase family, which facilitate integration of horizontally-
argKtox cluster, is composed of 23 genes (Fig. 13).173175 At acquired DNA into the genome.183 The genomic sequence of the
different stages of the discovery process, the designation of the phaseolotoxin-producing strain P. syringae pv. phaseolicola
genes changed, and the following text conforms to a recently 1448A contains an argKtox cluster, as expected, as well as
proposed nomenclature.175 Despite long-standing efforts reach- a second cluster containing homologs (4080% similarity) to
ing back more than two decades, the identities and roles of most a subset of the argKtox genes.48 The same degenerate cluster
of the gene products of the phaseolotoxin cluster remain to be was found in the genome of P. syringae pv. syringae B728a,
determined. The unambiguous genes identified so far are argK, which does not produce phaseolotoxin, where it exists on the
amtA and desI. By the production of ArgK, a resistant OCTase large genomic island also containing genes for syringomycin,
isoform, P. syringae pv. phaseolicola, protects itself against an syringopeptin and achromobactin biosynthesis.49 In both B728a
impairment of its own prokaryotic urea cycle.176178 The product and 1448A, the degenerate cluster appears to define a lateral-
of the desI gene shows similarity to a fatty acid desaturase, a class transfer hotspot that exhibits evidence of multiple HGT
of enzymes that commonly generate unsaturated fatty acids for events.48 Thus, the phaseolotoxin gene cluster provides another
the synthesis of phospholipids in the cytoplasmic membrane.179 It example of secondary metabolite gene cluster acquisition
has been hypothesized that DesI may facilitate the export of through HGT events.
phaseolotoxin across the bacterial membrane at the low
temperatures (1820  C) conducive to phaseolotoxin produc- 2.1.9 Pyrrolnitrin. Pyrrolnitrin is a chlorinated phenylpyrrole
tion.180 AmtA encodes an amidinotransferase and is demon- compound and its production by Pseudomonas pyrocinia was first
strably involved in the formation of the amino acids homo-Arg discovered in 1964.184 Since that time, production of pyrrolnitrin

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Fig. 14 The genetic organization of the prnABCD gene cluster from P. fluorescens BL915 and biosynthetic route to pyrrolnitrin.

has been reported from other Pseudomonas spp., as well as the genome lacking REP regions, as described in Section 4. In
Enterobacter agglomerans, Myxococcus fulvus, Burkholderia three B. pseudomallei genomes, prnABCD is flanked by putative
spp., and Serratia spp.185187 Pyrrolnitrin is an inhibitor of fungal transposases, which could mediate insertion of transferred DNA
respiratory chains188 and, due to its strong antifungal activity, sequences into the genomes. A closely-related strain of
pyrrolnitrin was developed as a topical antimycotic for human B. pseudomallei lacks only prnABCD but retains the transposases
Published on 01 October 2009 on http://pubs.rsc.org | doi:10.1039/B817075B

use.189,190 A derivative of pyrrolnitrin exhibiting greater envi- and flanking genes, perhaps representing a predecessor of a HGT
ronmental stability is used as an agricultural fungicide.191,192 A event or a successor of an excision event. Notably, however, the
four-gene cluster (prnABCD) for the biosynthesis of pyrrolnitrin regions lack certain hallmarks of genomic islands, exhibiting no
was first described in Pseudomonas aurantiaca (previously iden- abrupt alterations in G + C content or dinucleotide or trinucleo-
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tified as P. fluorescens) BL915.193,194 The order of the four prn tide skew from the surrounding genomic regions.186
genes corresponds to the order of their protein products in the
biosynthetic pathway for pyrrolnitrin (Fig. 14). L-Tryptophan 2.1.10 Indole-3-acetic acid (IAA). Indole-3-acetic acid is
serves as the basic precursor,195199 which is chlorinated by the a naturally occuring indole-based phytohormone that influences
tryptophan halogenase PrnA to form 7-chloro-Trp. PrnB cata- virtually every aspect of plant development and physiology. IAA
lyzes the rearrangement from the indole to the phenylpyrrole is produced by many microorganisms, including plant pathogens
skeleton including a decarboxylation reaction to give mono- such as P. syringae203 and non-pathogenic strains of Pseudo-
dechloro-pyrrolnitrin, which is in turn halogenated at position 3 monas spp.204207 IAA production is a well established virulence
of the pyrrol ring by the halogenase PrnC. In the last step, the factor of plant pathogens that cause hyperplasia, such as Pseu-
amino group of the resulting intermediate metabolite amino- domonas savastanoi, which causes gall formation on olive and
pyrrolnitrin is converted into a nitro group by the action of the oleander.208 P. savastanoi, many pathovars of P. syringae203 and
oxidase PrnD to yield pyrrolnitrin. Especially notable are the two certain strains of other plant-associated Pseudomonas spp.204
halogenases PrnA and PrnB, which exhibit high substrate spec- produce high concentrations of IAA, whereas low levels are
ificity and regioselectivity and provided the first examples of the produced by rhizosphere-inhabiting strains that are beneficial to
FADH2-dependent halogenases.200 plants.206 The effects of exogenous IAA on plant growth and
The four pyrrolnitrin biosynthetic genes (prnABCD) are highly development are concentration-dependent, so the concentrations
conserved among strains of P. fluorescens that produce pyrrol- of IAA produced by bacteria on plant surfaces are thought to be
nitrin.187,193,194,201 To date, P. fluorescens Pf-5 is the only pyrrol- key to the plant response.206 The relationship between IAA
nitrin-producing Pseudomonad whose complete genomic production and plant response is further complicated, however,
sequence is available, but the highly-conserved prnABCD cluster by the fact that certain IAA-producing Pseudomonas sp. also
is also present in the completed genomes of several Burkholderia catabolize IAA.209 Auxin signaling pathways are important
spp.39,186 Whereas a syntenic prnABCD cluster is conserved, the components of plant defense strategies, and recent evidence
composition and organization of flanking genes differ markedly indicates that pathogens such as P. syringae can interfere with
between the two genera, and even among certain of the Bur- these pathways to overwhelm host defense.210212 Although it is
kholderia spp. Flanking the four known biosynthetic genes in the commonly assumed that IAA production evolved in bacteria due
Pf-5 genome are genes with regulatory, transport, and biosyn- to its importance in plantbacteria interactions, IAA can also
thetic functions that could play a role in pyrrolnitrin production. function as a signaling molecule in prokaryotes, effecting many
Especially notable is the flavin reductase PrnF that, when aspects of bacterial physiology206 including secondary metabo-
expressed in Escherichia coli, provides reduced FADH2 to the lism in P. syringae.213
PrnD oxygenase.202 Genes having putative regulatory, transport In bacteria, the biosynthesis of IAA proceeds from L-trypto-
and flavin reductase functions are also found near the prnABCD phan to IAA via at least five possible biosynthetic routes, clas-
operon in several (but not all) of the Burkholderia genomes.186 sified in terms of their intermediates.206,214,215 Pseudomonads
Because pyrrolnitrin is produced by heterologous host bacteria utilize the indole-3-acetamide (IAM) pathway and the indole-3-
such as Escherichia coli harboring the prnABCD operon alone,193 pyruvic acid (IPyA) pathway, including its shortcut, the trypto-
the flanking genes are not strictly required for pyrrolnitrin phan side chain pathway (Fig. 15).214,215 A single strain of
biosynthesis. Pseudomonas spp. may utilize two of the three pathways for IAA
Burkholderia spp. and Pseudomonas spp. are coinhabitants of biosynthesis.213,216 The plant pathogen P. savastanoi and certain
soil and other natural habitats, and it is likely that the presence of strains of P. syringae assemble IAA primarily via the IAM
the highly conserved prnABCD cluster in these distantly related pathway,217 but may also use the IPyA pathway. In general, the
bacteria is a function of HGT. In P. fluorescens Pf-5, the IAM pathway is constitutively expressed whereas IPyA path-
prnABCD cluster is in a large (ca. 70 kb) lineage-specific region of ways are expressed only in the presence of tryptophan.218220

1424 | Nat. Prod. Rep., 2009, 26, 14081446 This journal is The Royal Society of Chemistry 2009
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In the IPyA pathway, tryptophan is converted by a trans-


aminase to the unstable intermediate indole-3-pyruvic acid,
which is decarboxylated to yield indole-3-acetaldehyde (IAAld).
In a final step, IAAld dehydrogenase transforms IAAld into
IAA.214 The tryptophan side chain pathway, in which tryptophan
side chain oxidase (TSO) converts tryptophan directly to IAAld,
represents a shortcut to the IPyA pathway (Fig. 15)232 that is
preferred for IAA production in at least some strains of P. flu-
orescens.216 Surprisingly, almost nothing is known about the
genetics that underlie the IPyA/trytophan side chain pathways.
An indolepyruvate decarboxylase gene (ipdC) sequence was
reported for P. putida GR12-2,233 but has since been shown to
originate from a strain of Enterobacter cloacae.
Published on 01 October 2009 on http://pubs.rsc.org | doi:10.1039/B817075B

2.2 Polyketides and fatty acid derived compounds


Polyketides are a large class of structurally-diverse natural
products that include compounds with antibiotic, chemothera-
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peutic, and phytotoxic activities.51,234,235 The formation of poly-


ketides can be envisioned as a series of decarboxylative Claisen
thioester condensations of acetic acid units.236 In this way, poly-
Fig. 15 A) Pathways leading from Trp to IAA in Pseudomonads: [I] ketide and fatty acid biosynthesis are quite similar, but important
Indolacetamide pathway, catalyzed by Trp-2-mono-oxygenase and IAM distinctions differentiate the polyketide synthases (PKSs) from
hydrolase. [II] Tryptophan side chain pathway, utilizing Trp side chain fatty acid synthases (FASs).237 PKSs use a broader range of
oxidase (TSO) and IAAld-dehydrogenase. [III], indolepyruvic acid biosynthetic building blocks, exhibit wider variation in chain
pathway [III], initiated by Trp transaminase and followed by decarboxy- length of the final product, and do not fully reduce each acyl unit
lation to produce IAAld and subsequently IAA. B) Organization of the
following every round of chain elongation as done by FASs.
iaaMH and iaaL genes in the plasmid plIAA1 of P. syringae subsp.
Historically, PKSs have been categorized in three major types:
savastanoi (P. savastanoi).
type I PKSs are large, modular proteins;237 type II PKSs are
composed of complexes of monofunctional proteins;236 and type
The beneficial plant growth-promoting Pseudomonas strains, III PKSs238 are most commonly associated with chalcone and
which produce lower amounts of IAA, use primarily the IpyA stilbene synthases in plants, enzymes containing a single active-
pathway or its shortcut, the side chain pathway.216,221,222 site cysteine and lacking acyl carrier protein (ACP) functional-
The gene cluster directing IAA biosynthesis via the IAM ities found in type I and type II PKSs. Collectively, polyketide
pathway, which was first identified in P. savastanoi,223 comprise biosynthesis in Pseudomonas spp. involves each of the three types
two genes organized in one operon, iaaH and iaaM.224 The latter of PKSs.
gene encodes a tryptophan 2-monooxygenase, catalyzing the Like the NRPSs described above, Type I PKSs are large multi-
oxidative decarboxylation of tryptophan to indole-3-acetamide functional proteins organized into modules, each of which
(Fig. 15).225,226 iaaH, coding for indoleacetamide hydrolase, functions in the addition and modification of an extender unit
subsequently hydrolyzes the intermediate indole-3-acetamide to (usually acetyl-CoA or malonyl-CoA).234,237 PKS modules are
IAA and ammonia. In P. savastanoi, the iaaMH operon is composed of three core domains: An acyltransferase (AT)
commonly accompanied by iaaL, which is located upstream and domain, which selects the extender unit and transfers it to an acyl
in the opposite orientation. This gene encodes N-(indole-3- carrier protein (ACP) domain, having a phosphopantetheine
acetyl)-L-lysine synthetase, which is responsible for the produc- prosthetic group that is linked to the extender unit via a covalent
tion of biologically inactive IAAlysine conjugates227229 with thioester bond, and a ketosynthase (KS) domain, which catalyzes
proposed roles in regulation of the IAA pool size and viru- the condensation reaction between the extender unit and the
lence.229 In strains of P. savastanoi, the iaaLiaaHM cluster can polyketide intermediate on the preceding module. The three core
be located on the chromosome or on conjugative plasmids,230 domains are present in all elongating modules, whereas the
such as those in the pPT23A family,231 which are widely loading module lacks a KS domain, and the terminal module
distributed in phytopathogenic Pseudomonas spp. and are known contains a TE domain, responsible for the release of the poly-
to carry genes required for virulence and ecological fitness. ketide product from the PKS. Modules may also contain
Although IAA production is common in many pathovars of domains for tailoring reactions that contribute to polyketide
P. syringae, the iaaMH operon is found in only a subset of IAA- diversity. For background and perspectives on PKS biosynthesis,
producing strains,203 indicating that other IAA biosynthetic we refer the reader to excellent reviews on the subject.236240
pathways (such as IPyA) are more common. The iaaMH operon
is present in only one (B728a) of the strains of P. syringae with 2.2.1 Mupirocin (pseudomonic acid A). Pseudomonic acids
published genomic sequences. Although iaaL appears to be more are a mixture of antimicrobial compounds isolated from P. flu-
widely distributed in the species,203 it is also present in only one orescens consisting of four components: pseudomonic acids A,239
(DC3000) of the strains of P. syringae sequenced to date. B,240 C241 and D.242 The four compounds share certain features,

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including a dihydroxylated tetrahydropyran ring bearing func- codes for six multi-domain enzymes (MmpAF) with sequence
tionalized side chains in position 2 and 5. Pseudomonic acid A, similarity to type I PKSs or fatty acid synthases and 29 addi-
the major metabolite in the pseudomonic acid mixture, exhibits tional proteins (MupAX and MacpAE), resembling type II
the highest level of antibacterial activity towards Gram-posi- PKSs, auxiliary, tailoring and resistance enzymes. The cluster
tives243 and has been developed as a topical antibiotic (Bac- exhibits two extraordinary features. First, the mupirocin cluster
troban, Centany or Eismycin, INN: mupirocin). It is used was one of the early examples of trans-AT PKS gene clus-
today clinically for dermal staphylococcal infections, particularly ters:254,255 these PKS modules lack AT domains, but genes
those caused by methicillin-resistant Staphylococcus aureus encoding ATs are present in the cluster, acting iteratively
(MRSA).244 The use of mupirocin is limited to topical application in trans.255 Second, the pathway contains several tandem ACP
because it is inactivated rapidly by esterases in the human serum doublets or triplets, which are hypothesized to increase the
and bound with high affinity to serum proteins, resulting in poor throughput rate or to bind multiple substrates simultaneously.
bioavailability. Mupirocin has been shown to interfere with The biosynthesis of monic acid starts with the production of
RNA, proteins and, to a lesser extent, with cell wall biosyn- a pentaketide moiety by MmpD (Fig. 16). Starter units could be
thesis.245,246 Mupirocin is a competitive inhibitor of the bacterial activated by MupU or MupQ, which show similarity to acyl-
Published on 01 October 2009 on http://pubs.rsc.org | doi:10.1039/B817075B

isoleucyl-tRNA synthetase, and this inhibition leads to reduced CoA synthases, and one of the AT domains from MmpC
protein biosynthesis. The methyl end of mupirocin mimics an Ile transfers an activated acetyl group from acetyl-CoA to the first
moiety and interacts with the amino acid binding site of Ile- ACP domain. Due to a mutation in the conserved active site
tRNA synthase, while the remaining molecule interacts with the region, the dehydration domain of module 1 of MmpD is inac-
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respective ATP binding site.247 Pseudomonic acid-producing tive, but all other domains are functional and catalyze the first
Pseudomonads protect themselves from the inhibitory effects of four condensations. The assembly of monic acid is continued by
the antibiotic by altering their own isoleucyl-tRNA synthetase.248 the transfer of the pentaketide product of MmpD to MmpA,
Biosynthetically, mupirocin is the product of an esterification of which performs further extension with two malonyl-CoA units.
the polyketide monic acid and the fatty acid 9-hydroxy-nonanoic Gene knockout studies indicated that the first KS domain of
acid (Fig. 16). Feeding studies indicated that the molecule is MmpA is essential for mupirocin production, but does not act as
derived from acetate units, except for C-16 and C-17, which are a classic ketosynthase; instead, it facilitates the transfer of the
introduced by SAM biomethylation, and C-15, which has been pentaketide intermediate between MmpD and MmpA. The
proposed to be derived from acetate via 3-hydroxy-3-methyl- incorporation at C-15 has been proposed to be performed by the
glutarate (HMG).249251 action of the putative HMG-CoA-synthase MupH. In order to
In 1995, Thomas et al. identified the genomic region of complete the structure of monic acid, several oxidation and
P. fluorescens NCIMB 10586 that is required for mupirocin reduction post-PKS tailoring reactions are required to create the
production.252 Eight years later, the same group provided the epoxide group and the tetrahydropyran ring system. Candidate
nucleotide sequence of the complete 74 kb cluster253 (Fig. 16). It enzymes for these steps are MupC (NADH oxidoreductase),

Fig. 16 The mupirocin gene cluster and biosynthesis model. Asterisks indicate an inactive domain.

1426 | Nat. Prod. Rep., 2009, 26, 14081446 This journal is The Royal Society of Chemistry 2009
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MupO (a cytochrome P450), MupT (ferredoxin dioxygenase) to DAPG has been experimentally demonstrated to date.274 The
and MupW (dioxygenase). Further gene knockout experiments level of produced DAPG is regulated mainly by degradation
indicated that MupW is actually involved in the formation of the through the specific hydrolase PhlG, which converts DAPG into
tetrahydrapyran ring and that MupO,Q,S,T,U,V and MacpE are MAPG, and by the regulatory proteins PhlF and PhlH. PhlF
essential for the production of pseudomonic acid A.256 In represses the expression of the phlABCD operon by binding to
contrast, MupO, MupU, MupV and MacpE are not essential for two conserved sites in the phlA leader region. DAPG itself is able
production of pseudomonic acid B, which bears an additional to dissociate the repressor PhlF from the phlA promotor, hence
hydroxyl group at C-18. These results indicate that pseudomonic acting as an autoinducer of it own biosynthesis.267 Since DAPG is
acid B is not simply produced by hydroxylation of pseudomonic exported and diffusible, it can function as a signaling
acid A, but represents either a precursor of pseudomonic acid A compound.275 PhlH, the second pathway-associated transcrip-
or a shunt product. Upon completion of the tailoring reactions, if tional regulator, is hypothesized to antagonize the repressive
not earlier, monic acid is fused to the lipid 9-hydroxy-nonanoic effect of PhlF.28
acid, which is proposed to be synthesized by MmpB with DAPG production is characteristic of a subset of strains of
a 3-hydroxy-propionate starter unit and three malonyl-CoA P. fluorescens, which include the fully-sequenced strain Pf-5.
Published on 01 October 2009 on http://pubs.rsc.org | doi:10.1039/B817075B

extender units. Alternatively, since MmpB possesses an enoyl Moynihan et al.276 recently reported that the cluster is ancestral
reductase domain, the putative enoyl reductase MupE could in P. fluorescens, based upon its presence in a single phyloge-
possibly catalyze the final reduction step in the 9-hydroxy-non- netically-defined branch of the species. To date, the phl cluster
anoic acid biosynthesis. Apart from the discussed structural has been found in three distinct locations, defined by flanking
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genes, mupM has been identified as a resistance gene, encoding genes, in the genomes of different strains of P. fluorescens. Along
the modified mupirocin-resistant Ile-tRNA-synthase, which is with the known phl biosynthetic genes, an additional gene was
essential for survival. The expression of mupirocin is controlled found adjacent to the cluster only in the DAPG-producing
by MupR and MupI, which are members of the LuxR and LuxI strains.276 While the role of this gene in DAPG production
family of quorum sensing regulators.253 Several genes of the remains to be explored, it provides an excellent example for the
mupirocin cluster have not yet been characterized in detail and value of genomics in delimiting biosynthetic gene clusters. The
their functions warrant future investigation. phl cluster is flanked by conserved genes of the core P. fluorescens
genome in Pf-5, whereas the cluster is present in a large lineage-
2.2.2 2,4-Diacetylphloroglucinol (DAPG). DAPG is a specific region in the genome of another DAPG-producing
primary factor contributing to biological control of plant disease strain, P. fluorescens F113.
by many plant-associated Pseudomonas strains.36,257260
The phenolic molecule is toxic to a wide range of plant patho- 2.2.3 2,5-Dialkylresorcinols. A series of three 2-n-hexyl-
genic fungi259262 and also exhibits antibacterial,262 anti- resorcinols bearing in position 5 either an n-propyl, n-pentyl or
helmintic263 and, in high concentrations, phytotoxic n-heptyl side chain are produced by specific strains of Pseudo-
properties.262 DAPG also triggers systemic resistance of plants monas spp.277280 The 2,5-dialkylresorcinols, like the structurally-
against disease,264 and is a primary determinant of the disease- related acetylated phloroglucinols, also exhibit antifungal and
suppressive properties of certain soils against the take-all patho- antibacterial activity. Despite the structural resemblance,
gen of wheat.265 The 6.5 kb DAPG biosynthetic locus (Fig. 17) however, the 2,5-dialkylresorcinols and acetylated phlor-
has been identified and analyzed in P. fluorescens strains oglucinols have completely different biosynthetic pathways.
Q2-87,266 CHA0,267 and F113.268 The gene cluster is highly Several labeling experiments and genomic analysis suggested that
conserved among DAPG producers258 and comprises genes for dialkylresorcinol biosynthesis represents a unique offshoot of
biosynthesis (phlACBD),266 efflux (phlE),269 degradation (phlG)270 fatty acid metabolism in which medium-chain-length fatty acid-
and regulation (phlF and phlH).267,268,271 Quite rare and unusual derived precursors are further modified. The 4-kb dar gene
for microorganisms,272 the biosynthesis of DAPG is mediated by cluster was identified in Pseudomonas aurantiaca (previously
PhlD, a type III PKS chalcone synthase. Frost and coworkers identified as P. fluorescens) BL915, which produces 2-hexyl-5-
proved that PhlD first condenses three molecules of malonyl- propyl-alkylresorcinol as the predominant analog of several
CoA to form an activated 3,5-diketoheptanedioate, which is in dialkylresorcinols.281 As Fig. 18 illustrates, the dar cluster
turn cyclized via a Claisen condensation upon decarboxylation to consists of five genes; darABC are sufficient for 2,5-di-
yield phloroglucinol272,273 (Fig. 17). Stepwise acetylation per- alkylresorcinol biosynthesis, whereas darS and darR encode
formed by PhlABC leads to monoacetylphloroglucinol (MAPG) regulatory proteins. DarA catalyzes an unusual head-to-head
and 2,4-DAPG, respectively, but only the conversion of MAPG condensation between two b-ketoacyl thioester precursors

Fig. 17 The phl gene cluster and proposed biosynthesis pathway of 2,4-diacylphloroglucinol (2,4-DAPG).

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Fig. 18 The dar gene cluster of P. aurantiaca and proposed biosynthesis pathway of 2,5-dialkylresorcinols, exemplified with 2-hexyl-5-propylresorcinol.
Published on 01 October 2009 on http://pubs.rsc.org | doi:10.1039/B817075B

R ACP or acyl-CoA.

(3-ketohexanoate and 3-ketodecanoate) to form, after subse- proteins cannot be degraded, which effects the regulation of the
quent dehydration, a dioxocyclohexene intermediate. The two cell cycle, gene expression and responses to oxidative stress,
b-ketoacyl thioesters originate from the medium-chain-length leading ultimately to cell death. In human cancer cells, the
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fatty acid octanoate, which is partially degraded by b-oxidation interaction of syringolins with the proteasome leads to the inhi-
to give the shorter-chain precursor 3-ketohexanoate. The longer bition of cell proliferation and apoptosis.286 The major deriva-
chain precursor, 3-ketodecanoate, is generated via adol conden- tive, syringolin A, consists of a 12-membered ring structure,
sation by DarB, a b-ketoacyl-acyl carrier protein synthase III, formed by the two non-proteinogenic amino acids 5-methyl-4-
and DarC, an acyl carrier protein. Upon thioester hydrolysis of amino-2-hexenoic acid and 3,4-dehydrolysine. The a-amino
the dioxocyclohexene intermediate, a decarboxylation reaction group of the latter is joined by a peptide bond to a valine residue
at C-6 is thought to occur. Aromatization of this decarboxylated that, in turn, is linked to a second valine via a urea moiety. Other
structure through ketoenol tautomerism leads to the formation members of the syringolin family differ from syringolin A by the
of 2-hexyl-5-propyl-alkylresorcinol. substitution of 3,4-dehydrolysine by lysine or of one or both
valine residues by isoleucine residues.
The biosynthetic genes for syringolin A have been described by
2.3 Hybrid NRPS-PKS compounds
Dudler and coworkers.287 This biosynthetic locus is also assumed
2.3.1 Syringolin. Syringolins constitute a family of structur- to be responsible for the biosynthesis of the minor structural
ally-related phytotoxic cyclic peptide-polyketides produced by variants syringolins BF, whose appearance can be attributed to
certain strains of P. syringae pv. syringae.282,283 Syringolins can the relaxed substrate specificity of A domains and to the alterable
activate defense-related genes in plants, thereby reducing the efficacy of an involved desaturase. The syringolin (syl) gene
severity of plant disease282,284 and their production is a virulence cluster spans about 28 kb and consists of the five open reading
factor in P. syringae pv. syringae.285 The mode-of-action was frames sylABCDE (Fig. 19). SylA is a transcriptional regulator
recently attributed to irreversible inhibition of the eukaryotic and SylE, which shows similarity to an efflux pump, may be
proteasome.285 As a consequence, unneeded and damaged involved in the efflux of syringolins. Together, sylC and sylD

Fig. 19 The syl biosynthesis gene cluster encoding syringolins.

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encode three typical NRPS modules that account for the resorcinol ring is derived from polyketide biosynthesis292,293 and
assembly of the tripeptide Val-Lys-Val. The latter valine residue that L-proline is the primary precursor of the dichloropyrrole
represents an activated precursor, which gets further extended by moiety.294 Initially, L-proline is activated to L-prolyl-AMP by the
two carbon atoms through a classical type I PKS. Subsequent aminoadenylation domain of PltF and is then transfered to the
release and ring closure lead to a mixed peptide-polyketide phosphopantetheinyl arm of carrier protein PltL295 (Fig. 20).
intermediate. SylB exhibits similarity to a fatty acid desaturase While tethered to PltL, the prolyl moiety is desaturated by the
domain and is hypothesized to be responsible for the desatura- dehydrogenase PltE and subsequently dichlorinated first at
tion of lysine. However, it is not known at which stage of the position 5 and then at position 4 by the FADH2-dependent
biosynthesis this reaction may occur. Furthermore, the mecha- halogenase PltA.296 The dichloropyrrolyl residue is most likely
nism by which the second valine is linked via the ureido group to passed on to the type I polyketide synthases PltB and PltC,
the core structure is unknown. which add three malonyl-CoA monomers. Subsequent cycliza-
The syringolins are structurally related to the glidobactins, tion to resorcinol and release by the thioesterase PltG yields
a group of antifungal, cytotoxic, acylated tripeptides produced pyoluteorin.
by Burkholderia spp.288 Homologs of sylB, sylC, and sylD are The biosynthetic gene cluster (plt) for pyoluteorin production,
Published on 01 October 2009 on http://pubs.rsc.org | doi:10.1039/B817075B

present in the genomes of several Burkholderia spp. and Photo- regulation and efflux was discovered in P. fluorescens
rhabdus luminescens, an endosymbiont of entomopathogenic Pf-5,293,294,297 where it encompasses 17 genes spanning a total of
nematodes in the genus Heterorhabditis.288 Structural differences about 30 kb (Fig. 20). The plt cluster shows several unusual
between the syringolins and glidobactins have been attributed to characteristics. The PKSs PltB and PltC lack a loading module
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variations in the A domains of the three modules of the NRPSs and a terminal TE; pltG, which encodes a TE, is located three
present in all three bacterial genera, as well as the activities ORFs downstream of pltC. In addition, PltB contains a non-
conferred by auxiliary genes specific to each cluster. The syrin- functional KR and PltC a defective DH domain. The formation
golins and glidobactins provide additional examples of gene of the dichloropyrrolyl moiety in pyoluteorin is unique in several
clusters that are likely to have been acquired via HGT between aspects. A single halogenase (PltA) appears to be solely respon-
Pseudomonas spp. and Burkholderia spp. sible for the chlorination at both positions of the pyrrole ring,
despite the presence of two additional genes encoding putative
2.3.2 Pyoluteorin. Pyoluteorin was first isolated in the late halogenases in the plt cluster, i.e. pltD and pltM. Mutations in
1950s from cultures of the P. aeruginosa strains T359 and IFO pltD and pltM abolish pyoluteorin production by P. fluorescens
3455,289 and later from many other strains of Pseudomo- Pf-5,294 indicating an essential role of these genes in pyoluteorin
nads.51,290 This hybrid NRPS/PKS natural product is toxic biosynthesis. On the other hand, PltD lacks a conserved NADH
against Oomycetes,31 certain bacteria and fungi289 and, at high binding site, which is essential to the function of other halo-
concentrations, exhibits phytotoxicity against certain plants.291 genases. Therefore, the functions of PltM and PltD in pyolu-
The compound is best known for its toxicity against the Oomy- teorin biosynthesis remain unclear. Furthermore, it is unusual
cete Pythium ultimum,31 an important plant pathogen causing that the pyrrolyl substrate is recognized and accepted only when
broad-scale economic losses to agriculture. Chemically, pyolu- tethered to the carrier protein scaffold, whereas the free pyrrole-
teorin is composed of a resorcinol ring attached to a bichlori- 2-carboxylate is not accepted for halogenation by PltA. Apart
nated pyrrole moiety. Feeding experiments revealed that the from the structural genes pltABCDEFGLM, genes functioning in

Fig. 20 The plt gene cluster and model of pyoluteorin biosynthesis. Asterisks indicate an inactive domain.

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efflux of pyoluteorin (pltIJKNOP) and regulation (pltZ and pltR) which the 32.8 kb cor cluster is borne on the plasmid p4180.312
are present in the biosynthetic gene cluster.294,297299 The pyolu- The intriguing biosynthesis of coronatine proceeds by formation
teorin biosynthetic locus of Pseudomonas sp. M18 is identical to of CMA and CFA, which are then linked to produce corona-
that of P. fluorescens Pf-5, although the gene nomenclature tine.313,314 The structural genes for CFA and CMA biosynthesis
deviates slightly from that used in Pf-5.298 A pyoluteorin are located at opposite ends of the gene cluster (Fig. 21), sepa-
biosynthesis cluster of identical synteny is also present in rated by three regulatory genes (corPSR) involved in the tran-
a genomic island of P. aeruginosa LESB58.7 Intriguingly, this scriptional control of the two regions.315317
cluster is adjacent to a putative chalcone synthase most closely The nonproteinogenic character of CMA suggested the
related to phlD of P. fluorescens Pf-5. involvement of a thiotemplate mechanism and the work of Parry
et al. demonstrated that CMA was derived from L-allo-isoleu-
2.3.3 Coronatine. The chlorosis-inducing non-host-specific cine.318,319 Initially, a set of four genes, cmaABTU was identi-
phytotoxin coronatine is produced by several pathovars of fied.314 Three genes thereof, cmaABT, were proven to be essential
P. syringae, including atropurpurea, glycinea, maculicola, for coronatine production, while the role of cmaU remained
morsprunorum, and tomato.300302 Xanthomonas campestris pv. unknown. Later on, the genes cmaC, D and E were recognized in
Published on 01 October 2009 on http://pubs.rsc.org | doi:10.1039/B817075B

phormiicola also produces analogs of coronatine.303,304 Corona- the cma cluster and added by the Walsh group. The biosynthesis
tine acts as a virulence factor,305307 promotes entry of the of CMA starts with the didomain protein CmaA which catalyzes
bacteria into the plant host by stimulating the opening of the adenylation of L-allo-isoleucine and the attachment of this
stomata308 and suppresses salicylic acid-dependent host activated amino acid to the CmaA PCP domain.320 The A
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defenses.309,310 The toxin is composed of the ethylcyclopropyl domain was proven to prefer L-allo-Ile over other branched
amino acid moiety coronamic acid (CMA), which is linked to amino acids and represents the first example of an A domain
a bicyclic hydrindane ring-based polyketide moiety termed specific for this amino acid. How L-allo-Ile is synthesized remains
coronafacic acid (CFA). Due to the close resemblance of CMA unknown. The aminoacyltransferase CmaE transfers the ami-
and CFA to precursors of the endogenous plant hormones noacyl group from the PCP-domain of CmaA to a second PCP
ethylene and jasmonic acid, respectively, coronatine is thought to domain, CmaD.321 While tethered to CmaD, the formation of the
impact signaling in host plants via the ethylene and jasmonic cyclopropyl ring system occurs by cryptic halogenation. CmaB,
acid311 pathways. a member of the non-heme Fe2+a-ketoglutarate-dependent
Genes required for coronatine production were first identified enzyme superfamily, chlorinates the g-position of L-allo-Ile113 in
by Bender and coworkers in P. syringae pv. glycinea PG4180, in a regiospecific manner, thereby activating this position for

Fig. 21 Biosynthesis of coronatine. ACL: acyl-CoA ligase, CCR: crotonyl-CoA reductase.

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further chemical reactions. CmaC then catalyzes the formation a genomic island in the genome of another plant pathogen,
of a kinetically stabilized a-C carbanion, which displaces the Pectobacterium carotovora pv. atroceptica, provides further
g-Cl group in an intramolecular cyclization reaction to form evidence for HGT of the locus. As in P. syringae, the cfa genes
a cyclopropyl ring.322 CmaB was the founding member of a new contribute to virulence of P. carotovora pv. atroceptica,332
type of halogenases, the non-heme Fe2+ketoglutarate-depen- providing intriguing insights into the role of HGT in the evolu-
dent halogenases. Along with PrnA and B, CmaB provides tion of pathogenesis in these bacteria.
another example in which novel mechanisms of biohalogenation
were discovered through the analysis of secondary metabolite 2.3.4 Pederin. Pederin is a highly active cytotoxic agent,333
biosynthesis pathways of Pseudomonas spp. isolated originally from rove beetles in the species Paederus and
The polyketide portion of coronatine, CFA, is synthesized by Paederidus, which use this structurally unique compound as
the cfa gene cluster consisting of nine open reading frames, cfa1 a chemical weapon against predators. Intriguingly, the occur-
through cfa9323 (Fig. 21). The translation products of the first rence of pederin was correlated to the presence of a bacterial
three genes, cfa123, are related to ACPs, fatty acid dehydratases, symbiont of the beetle, so the biosynthesis of pederin was
and b-ketoacyl synthetases, respectively. The role of cfa4 could ascribed to a non-cultivated Pseudomonas species.334,335 Using
Published on 01 October 2009 on http://pubs.rsc.org | doi:10.1039/B817075B

not be predicted, while Cfa5 showed sequence similarity to acyl- a metagenomic approach, Piel and co-workers confirmed this
CoA ligases (ACL). Cfa6 and Cfa7 represent a two modular type hypothesis by cloning the pederin biosynthesis cluster from total
I PKS system responsible for CFA production. Cfa8 is also DNA isolated from the beetle Peaderus fuscipes.336,337 The ped
required for CFA biosynthesis and represents a crotonyl-CoA cluster and the pederin biosynthetic pathway have several
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reductase (CCR) thought to be involved in the formation of unusual and fascinating features. Whereas genes for the
butyryl-CoA, which is in turn commonly used as a four-carbon- biosynthesis, efflux, and regulation of secondary metabolites are
containing extender unit in polyketide synthesis. Cfa9, which commonly clustered in a single site in Pseudomonas genomes, the
exhibits similarity to TE domains, has proven to be dispensable ped cluster is distributed on three distinct genomic regions,
for CFA production.324 With the exception of Cfa6 and Cfa7, designated pedABCDEFGH, pedIJK, and pedLMNOPQR
little is known about the interplay of the cfa gene products and (Fig. 22 and Fig. 23). Due to the presence of transposase gene
their role in CFA biosynthesis. The current proposed route to fragments flanking each ped region, it has been hypothesized that
CFA starts with a cyclopentenone compound, commonly translocation of DNA to different genome regions may have
synthesized by coronatine producers,325 which is shuttled to the caused the observed fragmentation of a formerly intact gene
first modular PKS, Cfa6 (Fig. 21). The starter unit is at first cluster. Furthermore, the modules are devoid of AT domains.
elongated with ethyl malonate via the action of Cfa6. The Instead, PedCD encode two acyl transferases, which act in trans
resulting intermediate is then transferred to the downstream PKS to acylate each PKS module in the cluster.
Cfa7, where it is condensed with a malonate unit, yielding Pederin biosynthesis starts with the assembly of the pederin
a latent reactive product. Upon the latter elongation step, a tetraketide performed by the tetramodular PKS PedI (Fig. 22).
reactive b-keto thioester intermediate is formed that undergoes An acetyl starter unit is first selected and incorporated by
a rapid conformationally controlled 6-endo-trig cyclization a GCN5-related N-acetyltransferase (GNAT). The three
before the tethered intermediate is reduced and dehydrated on
the assembly line by the remaining DH and KR domains of
Cfa7.326 These domains act subsequently on the enzyme-bound
bicyclic hydrindane skeleton to complete the structure of CFA.
The final step in the coronatine pathway is presumed to be the
ligation of CFA and CMA via formation of an amide bond.327,328
Small quantities of coronatine congeners were isolated from
P. syringae pv. glycinea when CMA was replaced by norcoro-
namic acid, Leu, Val, Ile, allo-Ile, Ser or Thr, indicating that
the responsible enzyme(s) catalyzing this reaction lacks rigid
specificity.329331
In many strains of P. syringae, the cor biosynthetic gene cluster
is carried on large (80110 kb) indigenous plasmids in the
pPT23A family that are transferred readily via conjugation
between strains of the pathogen.231 In other strains, such as
P. syringae pv. tomato DC3000, the cor genes are borne on the
chromosome where they are co-localized with other genes
involved in virulence, including two clusters of effector genes.47
Plasmid-encoded cor genes are typically in a single cluster
whereas chromosomal cor genes can be dispersed, as in the
genome of DC3000 where 26 kb separates the cfa and cma loci.
The region of the DC3000 genome containing the cfa and cma Fig. 22 Organization of the pedIJK and pedLMNOPQR fragments of
gene clusters is enriched in mobile genetic elements of several the ped cluster. CR: crotonase domain, EST: esterase, GNAT: GCN5-
classes, which could provide a mechanism for HGT of the related N-acetyltransferase, HMGS: 3-hydroxy-3-methylglutaryl-CoA
region.47 The presence of a full complement of cfa genes on synthase, OR: oxidoreductase.

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Fig. 23 Organization of the pedABCDEFGH fragement of the ped cluster and proposed biosynthetic scheme for pederin formation. OR: oxidore-
ductase.

remaining PKS modules of PedI catalyze the extension of the like intermediate, which is then cleaved by PedG to form the
backbone by three acetate units and trigger the cyclization of the proposed intermediate 7,10-didehydroxy-6,10,17,18-tetrademe-
resultant tetraketide intermediate. The formation of the unusual thoxypederin. Upon the assembly of the basic carbon skeleton of
exomethylene group at the C-4 position in pederin is assumed to pederin, several hydroxylations and methylations occur as
be generated by the two crotonase domains (CR) in the third tailoring reactions. Recently, PedO was shown to selectively
module of PedI. As a mechanism, a nucleophilic attack of acetyl- methylate the terminal 18-OH group.339 The additional methoxy
CoA on a keto group, followed by a decarboxylative Grob-type groups are presumably provided by the O-methyltransferases
fragmentation of the resulting b-hydroxy-acid, has been PedA or PedE. Since pederin carries four methoxy groups, but
proposed.338 Since this reaction requires the involvement of an only three MT genes were identified, PedA or PedE could act
HMG-CoA synthase, a KS, an ACP, and an additional CR repeatedly. Alternatively, the fourth methylation could be per-
domain, it was hypothesized that pedLMNPQ, which encode formed by the beetle host. Bioinformatic analyses indicate that
proteins possessing these catalytic properties, might participate in PedR has regulatory functions, PedQ functions as an esterase,
exomethylene bond formation. The tetraketide intermediate and PedB and PedJ show similarity to oxidoreductases.
assembled by PedI is then transferred to mixed PKS/NPRS
megaenzyme PedF (Fig. 23). In a canonical fashion, the tetrake-
2.4 Other compounds
tide is at first elongated with glycine and subsequently in four
cycles with acetate units. Noteworthy are the methyltransferase 2.4.1 Phenazines. Phenazines are a large family of colorful
domain in module four, presumably attaching the geminal nitrogen-containing tricyclic molecules with antibiotic,
dimethyl group, and the tandem dehydratase domain in the antitumor, and antiparasitic activity.54,340 The unusually broad-
subsequent module, which is most likely responsible for pyran spectrum activity of phenazines relies on interactions with poly-
ring formation. Finally, pedG, which encodes a FAD-dependent nucleotides, topoisomerase inhibition, and the generation of free
monooxygenase, catalyzes oxidative cleavage of a two-carbon radicals.54 Among the Pseudomonads, strains of P. aeruginosa,
extension unit in a BaeyerVilliger fashion in order to produce the P. chlororaphis, and P. fluorescens are the most prominent producers
unusual and rare oxidized single-carbon terminus of pederin. The of phenazines.54,341 Pyocyanin (5-N-methyl-1-hydroxyphenazine)
additional PKS/NRPS gene pedH has no structural counterpart, is a pathogenicity factor produced by P. aeruginosa,342,343 whereas
but the corresponding enzyme would hypothetically extend the phenazine-1-carboxylic acid, 2-hydroxyphenazine-1-carboxylic
pederin structure to the skeleton of the sponge-derived compound acid, and phenazine-1-carboxamide are antifungal compounds
onnamide A. In addition to the previously-described biosynthetic produced by rhizosphere isolates of P. fluorescens and
route, it can be also envisioned that PedH produces an onnamide- P. chlororaphis.54 These redox-active compounds function as

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Fig. 24 Comparison of phenazine biosynthetic loci from different Pseudomonads and current biosynthesis scheme.
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intercellular signals influencing transcriptional regulation of the responsible for the generation of the various known congeners of
producing cell and having broad effects on bacterial physiology PCA are either linked to the core operon (e.g. phzM encoding
and fitness, including biofilm formation.344346 In addition to a SAM-dependent N-methyltransferase in P. aeruginosa PAO1) or
Pseudomonas spp., strains of Burkholderia, Brevibacterium, reside elsewhere in the genome (e.g. phzH performing the conver-
Streptomyces, Pectobacterium, and Pantoea produce phena- sion of carboxylic acids into carboxamides in P. aeruginosa
zines.54,340,347 More than 50 naturally-occurring phenazine PAO1).351 A linked pair of regulatory genes in the luxR/luxI family
compounds have been described and a few strains produce ten control phenazine production in a density-dependent manner, and
different congeners at the same time. The basic skeleton is usually this system serves as a model for the role of quorum sensing in the
extended by hydroxyl or carboxylic acids groups on the benzene ecology of bacteria in natural environments.352
ring moiety and by oxides and methyl groups on the nitrogen atoms. The phenazine gene cluster provides an excellent example of the
The core phenazine biosynthetic locus (phzABCDF) is highly modification of secondary metabolites to a range of diverse
conserved among phenazine-producing strains of Pseudomonas functions by auxiliary genes that complement a common set of
spp., with some some species (such as P. aeruginosa) having two core genes. Across diverse bacterial genera, the core biosynthetic
copies (Fig. 24). Chorismic acid is converted to 2-amino-4-deoxy- genes (phzB, phzD, phzE, phzF and phzG) are conserved among all
chorismic acid (ADIC) by the anthranilate synthase homologue phenazine-producing strains, indicating that they are essential for
PhzE. PhzD, a member of the isochorismatase enzyme family, synthesis of the phenazine scaffold.353,354 Variations in the core
cleaves ADIC to generate trans-2,3-dihydro-3-hydroxyanthranilic phenazine cluster include the absence of the DAHP synthase-
acid (DHHA). The isomerase PhzF performs a 1,5-prototropic encoding gene phzC in some bacterial genera and presence of
shift, yielding an enol that converts to the corresponding ketone redundant phzA/B genes in the Pseudomonads and Streptomyces
6-amino-5-oxocyclohex-2-ene-1-carboxylic acid. Subsequently, cinnamonensis.353,354 In P. aeruginosa PA01, the phenazine
two of these amino-cyclohexenone molecules undergo a symmet- biosynthesis genes are in a region of the chromosome having GC
rical head-to-tail double condensation reaction, yielding a double content, codon usage patterns, and dinucleotide frequencies
imine. While this bimolecular key step is spontaneous and does not typical of the genome at large. The phz gene clusters in other
require enzyme catalysis in vitro,348 it is catalyzed by the dimeric Pseudomonas spp. are also located on the chromosome,54 but phz
protein PhzA/B in vivo.349 Together, PhzA and its duplicate PhzB genes are plasmid-borne in at least one bacterium, Pantoea
form a small dimeric protein of the D5-3-ketosteroid isomerase/ agglomerans Eh1087.355 In other species, such as the plant path-
nuclear transport factor 2 family, which acts as an acid/base cata- ogen Pectobacterium carotovora subsp. atroseptica, phz genes are
lyst and significantly increases the reaction rate by orienting two located within a defined genomic island.332 In Mycobacterium
substrate molecules and by neutralizing the negative charge of abscessus and Burkholderia cepacia, transposases and insertion
tetrahedral intermediates through protonation. Possibly catalyzed regions adjacent to the phz gene cluster could have played a role in
also by PhzA/B, the condensation product rearranges further to HGT of the region.353,354 Analysis of the fully sequenced genomes
prevent back-hydrolysis. The resultant rearranged condensation available to date indicates that the phenazine locus has a complex
product is prone to oxidation and undergoes oxidative decarbox- evolutionary history that includes HGT.353,354
ylation in a nonenzymatic reaction. The final aromatization of the
ring system is performed by the FMN-dependent oxidase PhzG, 2.4.2 Quinolones. Quinolone derivatives are small antibac-
leading to PCA.350 PhzC, the remaining gene of the core operon, terial alkaloids, isolated from several P. aeruginosa strains.356
encodes a 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) The basic skeleton of 4-quinolone is typically substituted with an
synthase, which catalyzes the first step of the shikimate pathway. It alkane or alkene chain at position 2. Occasionally, the structures
is hypothesized that PhzC acts to redirect intermediates from are also formulated in the tautomeric forms and referred to as
primary metabolism into phenazine biosynthesis. The genes 4-hydroxy-2-alkylquinolines (HAQs). Additionally, N-hydroxy

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derivatives can be observed.341 The antibacterial activity of However, the enzymatic functions of PqsABCD remain to be
P. aeruginosa or its preparations was discovered in the late 19th elucidated. HHQ represents an interim end-product of the
century,357359 but it was not until 1945 that the bioactivity was biosynthesis gene cluster because, as a messenger of cell-to-cell
attributed to the presence of the quinolones Pyo I-IV.360 In communication, it is released into the extracellular environment
addition to their antibacterial roles, 4-quinolones also serve as immediately upon synthesis. HHQ can then be taken up by
signal molecules for cell-to-cell communication. The model neighboring cells und converted into PQS by the action of the
compound 2-heptyl-3-hydroxy-4-quinolone was therefore FAD-dependent monoxygenase PqsH.366 Subsequently, PQS is
termed the Pseudomonas quinolone signal (PQS). PQS regulates exported and functions as an intercellular signal molecule.367
diverse target genes including those coding for elastase, rham- Homologs of the pqsABCDE cluster are present in the fully-
nolipid, the PA-IL lectin and pyocyanin, as well as diverse sequenced genomes of several Burkholderia spp.368,369 As in
phenotypes such as biofilm development and cellular fitness. P. aeruginosa, certain HAQs function as intercellar signaling
Chemical investigations and labeling experiments indicate that molecules in Burkholderia spp. The predominant HAQs
PQS and other 4-quinolones originate from a head-to-head produced by Burkholderia spp. are methylated and saturated; the
condensation of anthranilate and b-keto-(do)decanoic acids.361363 less common unsaturated forms have a double bond at the
Published on 01 October 2009 on http://pubs.rsc.org | doi:10.1039/B817075B

The genes directing the biosynthesis of PQS were found by 2-position, in contrast to the 1-position found in the unsaturated
serendipity during a random transposon mutagenesis screen in HAQs produced by P. aeruginosa. Differences in the prevalence
P. aeruginosa PAO1 for genes involved in the regulation of the of unsaturated vs. saturated forms of HAQs may reflect differ-
phenazine pyocyanin synthesis. The screening approach hit the ences in the pool of fatty acid intermediates between Bur-
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phnAB region, which was thought previously to be involved in kholderia spp. and P. aeruginosa. In addition to the core genes
phenazine biosynthesis, and led to the identification of the 9 kb hmqABCDE (also called hhqABCDE), the clusters in the Bur-
cluster364 (Fig. 25). It comprises seven structural genes encoding kholderia genomes contain an additional two genes: hmqG, which
PqsA, PqsB, PqcC, PqsD, PqsH, PhnA, and PhnB, and two genes encodes a putative SAM-dependent methyltransferase, and
encoding regulatory functions, PqsE and PqsR. PhnA and PhnB hmqF. HmqF, which is predicted to contain two acyl-CoA
presumably synthesize the anthranilate precursor of PQS from dehydrogenase domains and an attachment site for 3-phospho-
chorismate. Alternatively, in other P. aeruginosa strains, anthra- pantetheine, could be responsible for the double bond at the
nilic acid can also be provided by the kynurenine pathway via 2 position in the unsaturated HAQs produced by Burkholderia
tryptophan degradation (Fig. 25).365 In the next steps, anthranilic spp.,369 a possibility that has not yet been verified experimentally.
acid is condensed with b-ketodecanoic acid and subsequent Alternatively, differences in the biosynthesis of the 3-keto fatty
decarboxylative cyclization yields the PQS precursor 4-hydroxy-2- acid intermediates carried out by pqsBCD vs. hmqBCD may
heptylquinoline (HHQ). PqsABCD have been shown to be account for the altered position of the double bond in the
essential for these biosynthetic steps. PqsA exhibits similarity to respective HAQ products. HmqG appears to be responsible for
benzoate A ligases, possibly involved in anthranilate activation, the presence of the methyl group on the HAQs produced by
whereas PqsB,C and D may be b-keto-acyl-ACP synthetases. Burkholderia spp.369 Therefore, the HAQ gene cluster, like the
phenazine gene cluster described above, provides an additional
example of the modification of a secondary metabolite structure
by auxiliary genes that complement core genes of the biosyn-
thetic gene cluster. Further chemical diversity may be afforded
by variations in the availability of primary precursors for
secondary metabolism, represented by pool of saturated vs.
unsaturated fatty acids in the case of HAQ.

2.4.3 Hydrogen cyanide. Hydrogen cyanide (HCN, syn.


prussic acid) is extremely poisonous to most organisms due to its
effective inhibition of cytochrome c oxidase and other metal-
loproteins. At physiological pH and in the absence of complexing
ions, HCN is largely undissociated, volatile (boiling point:
25.6  C) and colorless. Biological HCN production has been
demonstrated in many insects,370 higher plants371 and fungi,372
but in only a few species of bacteria in the genera Chromo-
bacterium,373 Bacillus,374377 Burkholderia,378 and particularly
Pseudomonas.374,379383 HCN production by Pseudomonads
inhabiting the rhizosphere can be beneficial to the host plant
because it contributes to the suppression of various plant
diseases.384,385 In cystic fibrosis patients infected by P. aeruginosa,
elevated HCN concentrations were detected in the sputum, thus
suggesting that HCN production is a virulence factor.386
Early experiments indicated that Pseudomonads synthesize
Fig. 25 The PQS biosynthetic operon of P. aeruginosa and biosynthetic HCN from glycine,387,388 which is converted by oxidative reactions
route to PQS. into HCN and CO2. Several reaction mechanisms have been

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unstable nitrile derivative which undergoes in a third step a rapid


CC bond cleavage, yielding HCN and CO2.383,389 Because HcnA
showed similarity to formate dehydrogenases, LaVille et al.
proposed that HCN and formic acid are formed from the inter-
mediate iminoacetic acid, whereby formic acid is further oxidized
to CO2 by HcnA.390 However, the exact functions and the interplay
of the HcnABC subunits remain to be elucidated.
Fig. 26 The hcn gene cluster and current working hypotheses for
The hcnABC operon is highly conserved in sequence and
cyanide formation from glycine by Pseudomonads.
organization among the cyanogenic strains of Pseudomonas
spp.,15,39,384 although the genomic context of the operon differs
proposed for bacterial HCN biosynthesis,389 but the instability of among species. hcnABC is also present in the genomes of Chro-
the produced intermediates and of the HCN synthase itself mobacterium violaceum and many species of Burkholderia,378
impeded early efforts to define the biosynthetic pathway. It was not providing another example of shared metabolic capabilities of
until the HCN biosynthetic gene cluster of P. fluorescens CHA0 the Pseudomonads and Burkholderias.
Published on 01 October 2009 on http://pubs.rsc.org | doi:10.1039/B817075B

was described that the biochemistry of cyanogenesis was illumi-


nated.390,391 Three contiguous structural genes, hcnABC, which 3 New compounds discovered by genome-guided
together encode a membrane-bound HCN synthase complex, were
strategies in Pseudomonas spp.
shown to be sufficient for cyanogenesis. The amino acid sequences
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of HcnB and HcnC are similar to amino acid oxidases (HcnB and From a natural product chemistry point of view, Pseudomonads
HcnC), an observation that supports the dehydrogenase biosyn- were known for decades only for the production of siderophores,
thesis model proposed by Wissing.383 According to this mechanism, phenazines and small molecular weight antibiotics or phytox-
glycine is oxidized in the first dehydrogenase reaction to imino- ins.341 This picture changed fundamentally as it became evident
acetic acid (Fig. 26). Subsequently, with the help of a second that the genus has a tremendous capacity to assemble exciting
dehydrogenase reaction, iminoacetic acid is converted into an metabolites using intriguing biochemistry. Appreciation for the

Fig. 27 Circular representation of the genome of Pseudomonas fluorescens Pf-5. The outer scale designates coordinates in base pairs (bp), with the origin
of replication at 1 bp. The first circle (outermost circle) shows predicted coding regions on the plus strand, and the second circle shows predicted coding
regions on the minus strand, color-coded by role categories. The third circle shows the set of 1489 P. fluorescens Pf-5 genes that are not found in the
genomes of P. aeruginosa PAO1, P. syringae pv. tomato DC3000, and P. putida KT2440 (see Fig. 1A). The fourth circle shows the set of 1472 genes that
are not found in the genomes of P. fluorescens SBW25 or PfO-1 (See Fig. 1B). The fifth circle shows nine secondary metabolite gene clusters. The sixth
circle shows REP repeat elements. The seventh circle shows the PFGI-1 mobile island in olive, and phage regions. The eighth circle shows trinucleotide
composition. The ninth circle shows percentage G + C in relation to the mean G + C in a 2000-bp window. The tenth circle shows rRNA genes in green,
tRNA genes in blue.

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metabolic potential of the Pseudomonads increased further once 3.1 Orfamides


the prevalence of secondary metabolite gene clusters in their
The first new compounds to be identified from mining of Pseu-
genomes was discovered. In addition to illuminating the genomic
domonas genomes were the orfamides discovered by the groups
context and the comparative analysis of known metabolic clus-
of Gerwick and Loper37 from P. fluorescens Pf-5. Following
ters, as described above, genomic sequence data highlighted the
a bioinformatic analysis of the Pf-5 genome for genes encoding
presence of many new genetic loci with the hallmark sequences
NRPSs and PKSs, three orphan gene clusters were identified.29
typical of NRPSs or PKSs in Pseudomonas genomes.9,29,49,61 It
One of the orphan gene clusters contained three contiguous genes
quickly became evident that most Pseudomonads are capable of
termed ofaABC, whose deduced amino acid sequences are similar
producing, in addition to pyoverdines, an additional siderophore
to NRPSs. Together, ofaABC comprise ten modules with the
(i.e. pyochelin, pseudomonine, achromobactin, or yersinia-
typical CAPCP architecture of NRPSs synthesizing a decap-
bactin),47,1,29,49 lipopeptides,392 and numerous non-ribosomally
eptide (Fig. 28). With OfaA lacking a typical initiation module
derived peptides, polyketides, or hybrids thereof, many of
and OfaC having two TE domains near the C-terminus, the
unknown structure.9,29,61 Indeed, bioinformatic analyses revealed
cluster exhibits characteristics of NRPSs synthesizing CLPs in
the presence of orphan biosynthetic gene clusters, i.e. loci
Published on 01 October 2009 on http://pubs.rsc.org | doi:10.1039/B817075B

other Pseudomonas spp. (see section 2.1.5). In silico analysis of


encoding secondary metabolites whose product is yet unknown,
the substrate specificity of the A domains allowed the prediction
in the genomes of almost all Pseudomonas spp. In P. fluorescens
of the amino acid sequence of the resulting peptide product,
Pf-5, for example, secondary metabolic gene clusters, including
which indeed resembled that of CLPs of the viscosin group
those with known and unknown products, were estimated to
(Table 3). The product of the ofa gene cluster was isolated using
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occupy approximately 6% of the genome29 (Fig. 27). Therefore,


a traditional bioassay-guided as well as a new genomisotopic
the identified orphan gene clusters in Pseudomonas spp. are
approach, in parallel. The latter approach uses an isotopically-
comparable in number, novelty and diversity, to the prolific
labeled amino acid, predicted to be a precursor of the considered
Actinomycetes.
peptide, to guide the fractionation and purification process. In
One of the most exciting outcomes of Pseudomonas genomics
the case of the ofa cluster, leucine was predicted to be present
is the discovery of novel traits, including secondary metabolite
four times in the final peptide and not to be present in other
production, exhibited by these bacteria. Here, we highlight the
metabolites. Hence, 15N-labeled L-leucine was chosen as the
novel compounds discovered through genome mining strategies
labeled precursor and a 1H15N HMBC NMR experiment for its
applied to Pseudomonas genomes.
detection. Both the genomisotopic and the bioassay-guided

Fig. 28 Organization of the ofa gene cluster and corresponding biosynthetic scheme for orfamide A formation.

1436 | Nat. Prod. Rep., 2009, 26, 14081446 This journal is The Royal Society of Chemistry 2009
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strategies led to the isolation of orfamides A, B and C, which mutagenesis and subsequent metabolite profiling.38,393,394 In this
represent founding members of a new class of CLPs character- way, five analogs of rhizoxin, one of which had not been
ized by a 3-hydroxy-dodecanoic or tetradecanoic (myristic) acid described before,38 were correlated with this cluster (Fig. 30).
connected to the N-terminus of a 10 amino acid containing cyclic Rhizoxins are 16-membered polyketide macrolides that exhibit
depsipeptide. The order and identity of the amino acids forming significant phytotoxic,395 antifungal396 and antitumoral397 prop-
the peptide chain of orfamide A, the dominant CLP produced by erties by binding to b-tubulin,398 thereby interfering with
Pf-5, conforms exactly to that predicted in silico. Due to flexi- microtubule dynamics during mitosis. They were originally iso-
bility in amino acid and fatty acid selection by the NRPS system, lated from the fungus Rhizopus microsporus,396 but it is now
Pf-5 produces orfamides B and C that differ in the amino acid recognized that the compounds are not produced by the
composition and in the length of the lipid side chain. As observed fungus, but by the endosymbiotic bacterium Burkholderia
for many CLPs produced by Pseudomonas spp., the stereo- rhizoxinica.399,400
chemistry of the amino acids and the generation of the fatty acid Except for some inactive KS-ACP modules and one twin ACP,
moiety are not reflected in the ofa gene cluster. In the orfamides, the order and number of the modules in the rzx cluster are
the occurrence of D-configured amino acids can not be fully colinear with the backbone of the isolated structures. Intrigu-
Published on 01 October 2009 on http://pubs.rsc.org | doi:10.1039/B817075B

explained by the presence of C/E domains134 because the six of ingly, the polyketide chain is branched at C-5 by an enzymatic
the ten C domains that contain the characteristic C/E sequence Michael addition involving the addition of an acetyl building
motif are not strictly associated with D-amino acids in the block to an acryloyl precursor.401 In general, the rzx cluster
resulting peptide. Regarding its biological significance, orfamide resembles the rhi cluster in Burkholderia rhizoxinica, encom-
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A was shown to function as an antifungal agent and a bio- passing genes for the biosynthesis rhizoxin itself (Fig. 30).402
surfactant, influencing swarming motility of Pf-5 and lysing Noteable differences between the two clusters are the gene order
zoospores produced by an Oomycete plant pathogen.37 and the absence of a rhiJ equivalent, coding for an oxygenase, in
the rzx cluster. This oxygenase could be responsible for the
epoxide functionality at position C-2 and C-3 of rhizoxin, which
3.2 Rhizoxins
is absent in the rhizoxin analogs produced by P. fluorescens Pf-5.
An orphan NRPS/PKS gene cluster was the second subject of Compound WF-1360 F, the predominant rhizoxin derivative
genomic mining approaches employed to identify novel metabo- produced by Pf-5, and the new compound 22Z-WF-1360 F are
lites produced by P. fluorescens Pf-5. Six of the nine genes in the highly antifungal, anti-oomycetal, differentially phytotoxic and
79 kb cluster (Fig. 29) have predicted functions as PKS or mixed cytotoxic.
NPRS/PKS (rzxA-F), whereas the remaining genes have putative In Pf-5, the rhx cluster is located in a very large lineage-specific
functions as a tandem acyl transferase (rzxG), a methyltransfer- region of the genome that also includes fitD, a gene encoding
ase (rzxI) and a P450 monooxygenase (rzxH). All PKS modules a functional insect toxin related to the Mcf toxin of Photorhabdus
lack integral AT domains and the tandem AT encoded by rzxG luminescens.20 Rhizoxin provides another example of a bio-
provides a third example of ATs acting in trans in the Pseudo- synthetic locus shared among specific strains of Pseudomonas and
monads. The products synthesized from this pathway were Burkholderia spp., and the fit cluster is the second example (along
identified using a genomics-guided strategy involving insertional with syringolin) of a locus shared with P. luminescens. Again,

Fig. 29 Biosynthetic gene cluster for rhizoxin analogs (rzx) including putative functions of the genes in P. fluorescens Pf-5 and biosynthetic pathway for
rhizoxin (rhi) in B. rhizoxinica. OXY: oxygenase, HC: condensation-heterocyclization, P450: cytochrome P-450 monooxygenase, B: domain involved in
b-branching, GNAT: N-acetyltransferase. Asterisks indicate domains with motifs deviating from consensus sequences of functional domains and
indicate therefore possibly inactive domains.

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Fig. 30 Rhizoxin and rhizoxin analogs obtained from P. fluorescens Pf-5.

HGT is a likely mechanism for the presence of these closely related pchPAO1, all genes of pchPf-5 are organized in one operon. Second,
loci in diverse bacterial genera. PchGPf-5 showed limited sequence similarity to its counterpart in
PAO1 and was therefore renamed to PchK. However, a motif
search indicated that PchK could have a reductase and/or
3.3 Enantio-pyochelin
epimerase function and therefore could fulfill the reduction task
The sequenced genome of P. fluorescens Pf-5 continued to be an of PchGPAO1. Third and most strikingly, pchE of Pf-5 lacked, in
inspiration for the isolation of new compounds from Pseudo- comparison with pchEPAO1, the E domain required to generate
monads by genome-guided approaches. In the case of enantio- the R configuration at the asymmetric center, C-40 (see section
pyochelin, Reimmann and coworkers discovered that the pch 2.1.4 and Fig. 31).403 According to the colinearity rules, the new
cluster of Pf-5 (pchPf-5) deviated from the cluster in P. aeruginosa 40 S enantiomers enantio-pyochelin I (40 S, 200 S, 400 S) and II (40 S,
PAO1 (pchPAO1).403 Three major differences were noticed 200 R, 400 S) were predicted products of this biosynthetic pathway.
(Fig. 31). First, in contrast to the two operon-structure of Enantio-pyochelin was isolated from P. fluorescens strain CHA0,

Fig. 31 Comparison of the two divergent pch gene cluster of P. aeruginosa and P. fluorescens Pf-5/CHA0 and the resultant stereoisomers of pyochelin.

1438 | Nat. Prod. Rep., 2009, 26, 14081446 This journal is The Royal Society of Chemistry 2009
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a strain closely related to Pf-5 that contains the same modified conditions, six related octalipopetides, syringafactins AF, were
pch cluster, and its chemical structure and absolute configuration obtained.393 Their planar chemical structures were proposed
were determined using a combination of spectroscopy and based on mass-spectrometry data and amino-acid analysis. With
chemical synthesis.403 The modification of PchE and therefore the exception of one amino acid, the peptide sequence correlated
the production of the optical antipode of pyochelin represent well with the bioinformatic prediction. The analogues syringa-
a physiological advantage for the bacterium. Strain CHA0 factin AF vary either in the length of the attached 3-hydroxy
utilizes ferric-complexes of enantio-pyochelin, but not pyochelin fatty acid (C10 vs. C12) or in the amino acid residue integrated
itself, whereas P. aeruginosa utilizes pyochelin, not enantio- into the molecule at position AA6 (Val, Leu, Ile). Both variations
pyochelin, for iron nutrition.403 By producing enantio-pyochelin, can be readily attributed to a recognition flexbility of the A
strains Pf-5 and CHA0 have evolved a mechanism for seques- domain of the NRPS module 6 towards branched amino acids
tering iron in a form that is available to themselves, but not to and of the initiating C domain of SyfA, respectively.
competing bacteria. It is hypothesized that this strategy provides Surprisingly, in contrast to the CLPs produced by many
a competitive advantage for Pf-5 and CHAO in natural envi- Pseudomonads, all isolated syringafactins are linear rather than
ronments where concentrations of biologically-available iron are cyclic. The linear octalipopeptide corrugatin is produced by
Published on 01 October 2009 on http://pubs.rsc.org | doi:10.1039/B817075B

limited. Pseudomonas corrugata,405 but corrugatin possesses a non-func-


tionalized lipid side chain (n-octanoic acid) and a peptide
(OH-His/Dab/Dab/Ser/Ser/OH-Asp/Dab/OH-Asp) containing
3.4 Syringafactins AF
a high number of polar amino acids due to its physiological role
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In silico mining of the genomes of P. syringae pv. tomato as a siderophore. In contrast, the syringafactins are not side-
DC300047 and P. syringae pv. syringae B728a49 by several rophores, but function, like CLPs, in swarming motility. There-
research groups led to the identification of an orphan biosyn- fore, the syringafactins are related to the classical Pseudomonas
thetic gene cluster involved in the production of an octalipo- CLPs, forming a new group within this compound family.
peptide.61,392,404 The cluster, dubbed syf, consisted of two NRPS
genes (syfA and syfB) that are flanked by syfR, encoding a LuxR- 4 Secondary metabolite gene clusters in the flexible
like transcriptional regulator, and syfC and syfD, whose prod-
genome of Pseudomonas spp.
ucts may be involved in the export of the resultant lipopeptide.
Together, the two structural genes syfA and syfB encode eight In the Pseudomonas spp., most secondary metabolites are
NRPS modules that are terminated by tandem TE domains produced only by specific lineages, contributing to their
(Fig. 32). Inspection of the A domains of the NRPS for their specialized associations with plant or animal hosts or altering
amino acid substrate specificities, as well as the bioinformatics- their competitive interactions with other organisms in the envi-
based investigation of the corresponding C domains for hidden ronment. Accordingly, the genetic loci for the biosynthesis of
epimerase activity, allowed the prediction of the resulting peptide these metabolites are, by definition, in the flexible genome of
sequence: lipid/L-Leu/D-Leu/D-Gln/D-Leu/L-Thr/D-Val/L-Leu/ Pseudomonas spp. Biosynthetic gene clusters are considered to be
404
D-Leu (Fig. 32). Typical of the NRPSs for lipopeptides, the among lifes most diverse and rapidly evolving genetic
N-terminal module of SyfA includes a C domain, which was elements,406 and their diversity is thought to be mediated
expected to recognize and condense a thioesterified fatty acid primarily via HGT followed by mutation, recombination, and
from primary metabolism to the first amino acid of the peptide. duplication or excision events.407,408 Gene clusters for secondary
To test the hypothetical structure developed by bioinformatics, metabolism are therefore considered to be prototypic members
Thomas and coworkers proceeded with metabolite analysis of the prokaryotic mobilome, which consists of bacteriophages,
applying the prediction and screening approach393 to P. syrin- plasmids, transposable elements and associated genes.5 Indeed,
gae pv. syringae DC3000. After identification of optimal culture secondary metabolite biosynthetic clusters (coronatine or

Fig. 32 The syf biosynthesis gene cluster from P. syringae pv. tomato DC3000 and resultant chemical structures of syringafactins. The indicated
stereochemistry is deduced only by bioinformatics but not experimentally proven, and was therefore not included in the resultant structure.

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phytohormones such as indole acetic acid) have long been skew, and the lack of REP sequences (Fig. 27).29 Therefore,
associated with plasmids in pathovars of P. syringae,409 analysis of the genome of Pf-5 provides strong evidence for HGT
providing an obvious mechanism for HGT. In the vast majority as a fundamental mechanism for the inheritance of metabolic
of strains, however, secondary metabolite gene clusters are gene clusters, which is consistent with the conclusions from
located on the chromosome, in regions that vary from species to studies focused on many of the individual compounds discussed
species and from strain to strain. previously in this review.
As described above, bacterial genomes are mosaics composed
of relatively stable core regions interspersed with more variable
5 Concluding remarks
regions that diverge from even closely-related strains with respect
to both the composition and organization of genes.5 Variable Pseudomonas spp. exhibit enormous metabolic capabilities and
regions are commonly described as genomic islands, defined as versatile biochemistry through their production of structurally
horizontally acquired genomic regions. The sequence composi- diverse, bioactive chemical structures. The metabolic pathways
tion of genomic islands often deviates from that of the core for the biosynthesis of these compounds often reveal surprising
genome, and this deviation can sometimes be quantified from sophistication, as well as divergence from the model systems
Published on 01 October 2009 on http://pubs.rsc.org | doi:10.1039/B817075B

sequence properties. Features reported to be associated with studied in the Gram-positive actinomycetes. Some Pseudomonas
genomic islands include the presence of flanking repeats, biosynthetic pathways have functionally combined modular,
mobility genes (e.g., integrases and transposases), proximal dissociated, or chalcone synthase-like polyketide synthases with
transfer RNAs (tRNAs), atypical guanine and cytosine content, adenylating enzymes or with components of fatty acid synthases
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and skews in dinucleotide or trinucleotide composition. Some (2,4-diacetylphloroglucinol, pyoluteorin, mupirocin, corona-
genomic islands are associated with mobile genetic elements or tine). The common occurance of trans-AT polyketide synthases
integrate preferentially into regions flanked by tRNAs. The in PKS biosynthetic gene clusters (mupirocin, pederin, rhizoxins)
structures and sequences of genomic islands or their flanking is striking in the genus. Intriguingly, Pseudomonads also employ
regions do not always clearly reflect a mechanism for their cryptic halogenation in order to activate alipathic groups (cor-
transmission or integration into the genome, however, because onatine). Many Pseudomonas pathways are composed of
variable regions continue to be shaped by a range of processes, enzymes that rearrange and modify primary metabolic products
including excision, recombination, duplication, or mutation,5 to produce secondary metabolites (pyrrolnitrin, phenazine
and these obscure the evidence of HGT. Over time, the compo- 1-carboxylic acid). Genomic analysis has highlighted major
sitional variation between the core genome and GIs lessens, and themes contributing to chemical diversity in the Pseudomonads,
consequently, more ancient HGT events are detected with less including variations in the numbers and composition of struc-
sensitivity. tural domains in the modular NRPSs and PKSs, the comple-
Comparative genomic analysis of Pseudomonas spp. provides mentation of core genes by auxiliary genes (phenazines,
convincing evidence that the remarkable diversity of secondary syringolins, and rhizoxin), and relaxed substrate specificities of
metabolism in Pseudomonas spp. is commonly a consequence of biosynthetic enzymes. Through genomic comparisions between
the acquisition of genomic islands containing biosynthetic loci. Pseudomonas and other Proteobacteria, notably Burkholderia
By comparing the genomic sequences of different Pseudomonas spp., it is clear that HGT is a major process leading to chemical
spp. or strains, genes unique to a given strain can be identified diversity operate across the phylum.
(Fig. 1), and the distribution of these unique genes provides One of the contributions of genomics to prokaryotic biology is
a pictoral view of the mosaic nature of the bacterial genome. a deepened recognition for the prevalence of HGT and its
Using P. fluorescens Pf-5 as an example, the non-random consequences as a driving force enhancing microbial diversity.5
distribution of the unique genes is striking (Fig. 27): blocks of Comparative genomics provides ample evidence for the move-
genes unique to Pf-5, whether defined at the strain or species ment of biosynthetic gene clusters among microorganisms,
level, coincide on the genomic map. Therefore, the distribution of which, along with subsequent alterations, can translate into
lineage specific genes, whether defined at the strain or the species novel chemical structures. This is certainly the case for Pseudo-
level, provided one criterion used to identify genomic islands in monas spp., and many of the structures discussed herein provide
the Pf-5 genome.29 Other criteria were atypical trinucleotide clear examples of this phenomenon. In addition to the discovery
composition and the distribution of 1052 copies of a 34-bp REP of novel structures, the identification of new biosynthetic loci in
sequence. REP elements are repetitive and palindromic Pseudomonas spp. has provided new direction for studies
sequences, varying in length between 21 and 65 bases, that are exploring the biological significance of compounds, and in some
found the in the extragenic regions of some bacterial genomes.410 cases has revealed new aspects of the biology of the producing
The REP elements are clustered in the Pf-5 genome, with distinct strain. We predict that future exploration of novel gene clusters
gaps that often correspond to regions of atypical nucleotide in this heterogeneous genus will greatly expand the already-
content, the presence of prophages, and genes unique to Pf-5.29 remarkable spectrum of secondary metabolites known to be
In short, they segregate preferentially into the core genome produced by Pseudomonas species.
shared with other species of Pseudomonas and other strains of
P. fluorescens.
6 Acknowledgements
Strain Pf-5 produces at least nine secondary metabolites, and
biosynthetic gene clusters for many of these are located in We are grateful to Marcella Henkels, Rachel Blumhagen,
genomic islands identified using the criteria described above: Edward Davis, and Kedy Shen for assistance in preparation of
distribution of genes unique to the strain, atypical trinucleotide this article. We express sincere thanks to D. Mavrodi,

1440 | Nat. Prod. Rep., 2009, 26, 14081446 This journal is The Royal Society of Chemistry 2009
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L. Thomashow, S. Aguilera and A. Alvarez-Morales for sharing 22 D. Haas and G. Defago, Nat. Rev. Microbiol., 2005, 3, 307319.
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L. S. Thomashow, Annu. Rev. Phytopathol., 2002, 40, 309.
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1446 | Nat. Prod. Rep., 2009, 26, 14081446 This journal is The Royal Society of Chemistry 2009

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