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Regulation of the FecI-type ECF sigma factor by transmembrane signalling

Volkmar Braun, Susanne Mahren and Monica Ogierman
Induction of the ferric citrate transport genes of Escherichia coli K-12 involves a signalling cascade that starts at the cell surface and proceeds to the cytoplasm. Three specic proteins are involved: FecA in the outer membrane, FecR in the cytoplasmic membrane, and FecI in the cytoplasm. The binding of dinuclear ferric citrate to FecA causes substantial structural changes in FecA, triggering the signal cascade. The amino-proximal end of FecA interacts with the carboxy-proximal end of FecR in the periplasm. FecR then transmits the signal across the cytoplasmic membrane into the cytoplasm and activates the FecI sigma factor, which binds to the RNA polymerase core enzyme and directs the RNA polymerase to the promoter upstream of the fecABCDE transport genes to initiate transcription. Transcription of the fecIR regulatory genes and the fec transport genes is repressed by the Fur protein loaded with Fe2. Therefore, transcription of the fec transport genes is subjected to double control: cells rst detect iron deciency and respond by synthesis of the regulatory proteins FecI and FecR, which initiate transcription of the fec transport genes, provided ferric citrate is available. FecI belongs to the extracytoplasmic function sigma factors, which are widespread among bacteria. With the recent sequencing of complete microbial genomes, it has become apparent that the FecIRA cascade is now a paradigm for the regulatory control of FecI family sigmas in Gram-negative bacteria.
Addresses Mikrobiologie/Membranphysiologie, Universtat Tuebingen, Auf der Morgenstelle 28, 72076 Tuebingen, Germany Correspondence: V Braun; e-mail: volkmar.braun@mikrobio.uni-tuebingen.de

plasmic function (ECF) s factors; Fec I was among this group. With genome sequencing has come the realisation that not all of the ECF sigma factors regulate extracytoplasmic functions. Hence, Helmann has proposed renaming these as the group-4 sigmas. The group-4 sigma FecI regulates the ferric citrate (Fec) transport system of Escherichia coli K-12 In turn, FecI is regulated by FecA and FecR. FecA is an outer membrane protein that receives the signal at the cell surface and transmits the signal across the outer membrane into the periplasm. The amino-terminal extension of FecA, which is not found in other active outer membrane transport proteins of E. coli K-12, is essential for signalling to FecR. FecR is a cytoplasmic membrane protein that transmits the signal across the cytoplasmic membrane and regulates the activity of FecI. The complete signalling cascade including the signal, the signal receptor, transfer of the signal across the outer membrane and the cytoplasmic membrane, and the receiver in the cytoplasm has only been fully elucidated in the E. coli Fec transport gene regulation system. The activity of outer membrane transport proteins requires energy that is derived from the proton motive force of the cytoplasmic membrane. The coupling device between the outer membrane and the cytoplasmic membrane consists of a protein complex composed of TonB, ExbB and ExbD. TonB contacts the transport proteins and converts them into active transporters. FecA requires TonB not only for transport of ferric citrate across the outer membrane but also for transmitting the regulatory signal across the outer membrane, contacting TonB through its TonB box, a heptapeptide at the carboxyterminal border of its amino-terminal external extension. A BLAST search reveals that FecI, together with other s factors, form a subgroup of ECF s factors with a score of more than 123 (see the lower branch in Figure 1). Only s factors of Gram-negative bacteria Pseudomonas species, Bordetella species and Xanthomonas campestris, all of which are potential pathogens for humans, animals and plants belong to the group. The upper branch of Figure 1 includes mainly Pseudomonas species but also Agrobacterium tumefaciens, Bordetella bronchiseptica and Caulobacter crescentus. ECF s factors of streptomycetes and mycobacteria (which are not included in Figure 1 and will not be discussed here) are also in the BLAST score window (5191). In this review, we discuss signal transduction from the cell surface to the cytoplasm which ultimately results in an active FecI ECF that initiates transcription of the ferric
Current Opinion in Microbiology 2003, 6:173180

Current Opinion in Microbiology 2003, 6:173180 This review comes from a themed issue on Cell regulation Edited by Andree Lazdunksi and Carol Gross 1369-5274/03/$ see front matter 2003 Elsevier Science Ltd. All rights reserved. DOI 10.1016/S1369-5274(03)00022-5

Abbreviations ECF extracytoplasmic function Fec ferric citrate

Introduction
In 1994, Lonetto et al. [1] recognised that a group of transcription regulatory proteins were in fact sigma (s) factors which regulate genes that determine cell envelope functions and for this reason were designated extracytowww.current-opinion.com

174 Cell regulation

Figure 1

PA2896 P. aeruginosa

CC2707 C. crescentus PA3410 P. aeruginosa PAO149 P. aeruginosa PA1300 P. aeruginosa

ATU5309 A. tumefaciens

PA2387 P. aeruginosa ATU3692 A. tumefaciens

PvdS P. fluorescens PsbS P. B10 PbrA P. fluorescens PvdS P. aeruginosa PfrI P. aeruginosa

BupI B. bronchisepticum

CC0981 C. crescentus

PA4896 P. aeruginosa

PA2093 P. aeruginosa PA2050 P. aeruginosa PrhI R. solanacearum FiuI P. aeruginosa PupI P. putida PA0472 P. aeruginosa

FecI E. coli FecI X. campestris PA2468 P. aeruginosa PA1912 P. aeruginosa HurI B. pertussis RhuI B. avium PA3899 P. aeruginosa
Current Opinion in Microbiology

Phylogenetic tree demonstrating the degree of sequence identities among ECF sigma factors related to the E. coli K-12 fecI gene, as revealed by analysis with Clustal W.

citrate transport genes of E. coli K-12. This system has been characterised in the most detail and exhibits properties that will be typical for the whole class of FecI-type ECFs.

transported into the cytoplasm of cells clearly show that, for induction, ferric citrate is not taken up into the cytoplasm but acts from outside the cell.

Evidence for an extracytoplasmic regulation mechanism

Adding 1 mM of ferric citrate to growth medium induces the formation of the ferric citrate transport system [2]. Both the fact that citrate accumulated intracellularly (in an icd mutant devoid of isocitrate dehydrogenase activity) does not induce the Fec transport system [2] and that transport studies reveal it is iron, and not citrate, that is
Current Opinion in Microbiology 2003, 6:173180

The FecI system

FecI regulates transcription of the fecABCDE transport genes for ferric citrate in E. coli. The fecIR regulatory genes are located upstream of the fecABCDE genes and form a separate transcriptional unit (Figure 2) [2]. fecA encodes an outer-membrane transport protein and fecBCDE an ABC transport complex of the cytoplasmic membrane (Figure 2) [3]. Both fecIR and fecABCDE
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Regulation of the FecI-type ECF sigma factor by transmembrane signalling Braun, Mahren and Ogierman 175

Figure 2

FecA unloaded (Fe3+citrate)2

FecA loaded L8 L7

Outer membrane

N N C 160
c Fe B

TonB box TonB fragment C2 160

LLLV

C1 C N

TonB2

ExbD

Cytoplasmic membrane

FecE ADP+Pi ATP Fe+2

FecE ATP ADP+Pi FecI 4

ExbB TonB1

FecD

FecC

FecR

N 3

N C 2

RNAP core enzyme

PFur PecI
Fur
Fe2+

fecIR

PFur PfecA
Fur
Fe2+

fecABCDE
Fur
Fe2+
Current Opinion in Microbiology

Model of the ferric citrate transport and regulatory systems. The large structural changes in loops 7 (L7) and 8 (L8) that occur upon binding of dinuclear ferric citrate are shown. The movement of these loops closes off the cavity containing dinuclear ferric citrate from the external environment. The unknown conformation of the amino-proximal region of FecA, marked N, is shown to interact with region 160 of TonB. The figure shows the crystal structure of the TonB fragment. This is a dimer, although evidence exists to show that complete TonB forms a monomer [21]. The transmembrane topology of the energy transducing the TonBExbBExbD protein complex is shown, but the model does not reflect the unknown stoichiometry of the complex. A 7:2:1 ratio of ExbB : ExbD : TonB has been determined in cells but not in an isolated complex [22]. Interactions between FecA and the heptad leucine-zipper-like motif (marked LLLV) of FecR, between the amino-terminal region of FecR and region 4 of FecI [23], and between FecI and the RNA polymerase (RNAP) core enzyme (S Mahren and V Braun, unpublished data) are shown by arrows. The FecIRNAP (RNA polymerase) holoenzyme binds to the promoter of fecA. The Fur protein loaded with Fe2 (Fur Fe2) binds to the Fur boxes (PFur) upstream of fecI and fecA and represses fecIR and fecABCDE transcription. The ferric citrate transport system (ABC transporter) is shown on the left-hand side of the figure. www.current-opinion.com Current Opinion in Microbiology 2003, 6:173180

176 Cell regulation

transcription is repressed by the Fur protein loaded with ferrous iron. Under iron-limiting growth conditions resulting in low intracellular iron concentrations, Fur is unloaded and transcription of fecIR occurs. By contrast, iron limitation is not sufcient to induce transcription of the fecABCDE transport genes. Rather, both iron limitation and the presence of ferric citrate are required for fecABCDE transcription. FecA transports iron citrate and interacts with FecR to allow FecI activation (see below). High expression of fecA guarantees that a minimal level of FecA is present in uninduced cells to respond to ferric citrate and initiate the regulatory cascade. When an intracellular iron surplus is reached, ferrous iron binds to Fur, which represses transcription of the fecIR and fecABCDE genes [2]. FecI incubated with puried RNA polymerase is sufcient to bind to the fecA promoter and initiate transcription [2]. The exclusive use of FecI in fec transport gene regulation was shown in a study in which the seven E. coli s-factors, s70, sN, sH, sF, sE, sS and puried FecI, were incubated with the RNA polymerase core enzyme. Only FecI RNA polymerase transcribed a DNA fragment containing the fecA promoter [4].

membrane. It is assumed that additional structural changes occur through interactions with TonB, or that the structural changes induced by (Fe3 citrate)2 binding are modied by TonB. Because the signal initiated in FecA needs to reach the cytoplasm where transcription of the fec genes takes place, the signal must cross the periplasmic space and the cytoplasmic membrane. Crossing the periplasm is achieved by interaction of the amino-terminal end of FecA with the carboxy-terminal end of FecR; both of which are localised in the periplasm (Figure 2) [6,7]. The appealing concept that the amino-terminal end of FecA interacts in the periplasm with the carboxy-proximal end of FecR was demonstrated by a bacterial two-hybrid system that revealed specic interaction of FecA179 (residues 179 of mature FecA) with FecR101317 [8]. FecR contains a motif composed of repeating heptapeptides (residues 247268) that are anked by three leucine residues and one valine residue (Figure 3). It resembles leucine zipper motifs. The motif is highly conserved in FecI-like ECF anti-s factors (Figure 3). Replacement of the leucine and valine residues by proline results in derivatives with a strongly decreased interaction with FecA and a low fecA transcription in response to ferric citrate (S Enz and V Braun, unpublished data). Either the conformation of the repeat sequence and/or the leucine residues are important for interaction with FecA. Analogous in vivo experiments using the LexA system demonstrate binding of FecI to FecR185, FecR158 and FecR985 [8]. Residues 958 seem to be sufcient for the binding of FecR to FecI. In vitro, FecR(His)6 (six histidine residues fused to the carboxy-terminal end of FecR) bound to a Ni-agarose column binds FecI [8]. Signal transfer across the outer membrane by FecA, interactions between FecA and FecR in the periplasm, signal transfer across the cytoplasmic membrane by FecR, and interactions between FecR and FecI in the cytoplasm form a complete signalling cascade from the cell surface to the cytoplasmic site of transcription initiation. Sequence analysis and biochemical studies reveal that s factors, including ECF s factors, are divided into structurally and functionally conserved subregions (Figure 4). A FecI deletion analysis using the LexA two-hybrid system reveals that regions 4.1 and 4.2 of FecI interact with FecR185 [9]. Additional mutagenesis and overexpression studies support this conclusion.

Interaction of regulatory proteins in the signalling cascade from cell surface to cytoplasm
Transcription regulation of the fec transport genes is accomplished by three regulatory proteins: FecA, FecR and FecI. FecA serves two functions: induction of fec transport gene transcription, and transport of ferric citrate. Various mutants indicate that these two functions can be uncoupled. Binding of ferric citrate to FecA without transport is sufcient to initiate the signalling cascade that nally leads to transcription of the fec transport genes. The crystal structure of the FecA protein reveals that the ferric citrate form that binds to FecA is dinuclear ferric citrate (Fe3 citrate)2 [5], thus denitely identifying the inducing species. (Fe3 citrate)2 binds to ten residues of FecA, which are located in a cavity that lies well above the outer boundary of the outer-membrane lipid bilayer. Binding of (Fe3 citrate)2 induces strong long-range structural transitions in FecA. Surface loops 7 and 8 are translated 11 A and 15 A, respectively, and cover the entry of the surface cavity, presumably preventing the escape of (Fe3 citrate)2 back to the external milieu. In the FecA region exposed to the periplasm, a short helix is unwound. Smaller translations of 2 A and less are observed in several other regions of FecA. The massive structural changes in FecA presumably generate a signal that is transmitted across the outer membrane. However, the structural changes that occur upon binding of (Fe3 citrate)2 to FecA are not sufcient to initiate the transcription initiating signalling cascade. Signalling is dependent on TonB activity, as is (Fe3 citrate)2 transport across the outer
Current Opinion in Microbiology 2003, 6:173180

Mechanism of ferric citrate transcription regulation

FecI recruits the RNA polymerase core enzyme and directs it to the promoter of the fecA gene, which is the major or only promoter of the fecABCDE transport gene transcription. The activity of most ECF s factors seems to be controlled by anti-s factors. In the absence of anti-s
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Regulation of the FecI-type ECF sigma factor by transmembrane signalling Braun, Mahren and Ogierman 177

Figure 3

1 N

84 101
TM

246 269
LLLV

317 C FecR

Cytoplasmic domain

Periplasmic domain

FecR E.coli FecR X. campestris PupR P. putida FiuR P. aeruginosa PA0471 P. aeruginosa PA1911 P. aeruginosa PA2051 P. aeruginosa PA3409 P. aeruginosa Contig312 P. fluorescens Contig259 P. fluorescens Frag10708 P. putida Frag10749 P. putida Frag10715 P. putida Frag3802 P. syringae Contig812 B. pertussis Contig1034 B. pertussis RhuR B. avium

247 254 261 268 WTKDILSFSDKPLGEVIATLTRYRNGVLRCDP WERGQLIADELRLDAFVAELERYRPGLLRCDP WSQGMLVAQGQPLAAFIEDLARYRRGHLACDP WAQGMLVVENARLADLVAELGRYSPALLQVDP WAQGMLVVENARLADLVAELGRYSPALLQVDP WTRGMLMADRMPLAEVLAELARYRRGVLRCDP WLDGRLEVRDRPLGEVLEALRAYRRGIISVAD WRRGLLVFDEQPLGEVVARLNRYRPGHLLVAP WDKGMLLASNMRLDELLGELSRYRRGVLRCHP WVQGRLEVRDRPLSEVIDSLRSYRRGILHLSP WTEGVLSVQQMPLAEFASELGRYRPGLLRCAP WEHGMLLARDMRLADLLQELARYRPGVLRCHP WREGALRLDDRPLGELLHELRRYRPGVLRWAP WTRGVLKVDDQPLSEVLQTLATYRHGLLRYDT WEDGLLVVHGWRLDRLAAQLARYRLGVIRVDP WEDGLLVVHGWRLDRLAAQLARYRLGVIRVDP WLRGVLHVNAMPLAAFAAELGRYRRGLVRCAQ * . * .*. * *. ...
Current Opinion in Microbiology

Sequence comparison of predicted FecR-like proteins in the region LLLV through which FecR interacts with FecA (S Enz and V Braun, unpublished data). The conserved leucine residues (L), which form the flanking residues of four heptad repeats, are shown in grey. The asterisks represent identical amino acids in all listed proteins; full stops (.) represent identical proteins in most of the listed proteins.

factors, the s factors initiate transcription without extracytoplasmic signals. There is no evidence that FecR acts as an anti-s factor. In the absence of FecR, there is virtually no fecABCDE transcription. By contrast, cells containing the cytoplasmic FecR185 and even fragments of FecR185 display a high constitutive transcription of the fecABCDE genes. Cells containing longer FecR derivatives which extend from residue 1 to 273 (out of a total of 317 residues) [2] that do not interact with FecA [8] also transcribe fecABCDE constitutively, but to a lower level. Although FecR is necessary for FecI activity, it cannot be ruled out that FecR acts as an anti-s factor. If FecI is unstable, spontaneously denatures, precipitates or is degraded by
Figure 4

proteases, binding to FecR could maintain FecI in a stable conformation. When the signal from FecA occupied by (Fe3 citrate)2 arrives through FecR, FecR undergoes a conformational change that may result in the dissociation of FecI from FecR and immediate binding of FecI to the RNA polymerase core enzyme. In this model, FecR acts as both a chaperone for FecI and as an anti-s factor, given that FecI is kept in an active conformation or assumes an active conformation with FecR but cannot exert activity while it is bound to FecR.

Transcription regulation of the Fec type in species other than E. coli

Pseudomonas putida WCS358 expresses an iron transport system via the siderophore pseudobactin BN8, which

1 1.2 N 1.2

9 2.1 2.2 2.3 2.4

84 3.1 3.2

113 4.1 4.2 -35 recognition Binding site of FecR9-59

173 C FecI

-10 recognition

Current Opinion in Microbiology

Domain structure of the FecI s factor. Fecl lacks the 1.1 region of s70 factors. The regions through which Fecl binds to the 10 and 35 promoter regions and to FecR959 (residues 959 of FecR) are shown. www.current-opinion.com Current Opinion in Microbiology 2003, 6:173180

178 Cell regulation

Figure 5

fecI fecR hurI hurR rhuI rhuR bupI bupR prhI prhR pupI pupR fpvI fpvR

fecA bhuR bhuR bfrZ prhA pupB fpvA

fecB bhuS

fecC bhuT

fecD bhuU

fecE bhuV

pvdS
Current Opinion in Microbiology

to pyoverdin in the growth medium but retains pyoverdin transport [12]. PvdS binds to the RNA polymerase core enzyme in a 1:1 ratio and this complex binds to a DNA promoter fragment of the pvdA pyoverdin biosynthesis gene [13]. The proposed transcription model is similar to the Fec transcription model. Fe3 pyoverdin binds to the FpvA outer-membrane transport protein that interacts with FpvR. The signal is transmitted across the cytoplasmic membrane via the predicted FecR transmembrane segment, and then PvdS dissociates from FpvR and functions as an ECF s factor. A second fecI homologue, fpvI, maps adjacent to fpvR but is transcribed in the opposite direction. FpvI also receives a transcription initiation signal from FpvR. In a study demonstrating interactions of FecI and FecR in E. coli, two pairs of FecIR homologues of P. aeruginosa were included [9]. As demonstrated by the LexA twohybrid system, the FecI homologue PA2468 (Figure 1) and its truncated form PA2468110172 dimerise with the related FecR185 homologue PA2467190 but not with the unrelated FecR185 homologue PA3900185 [9]. PA3900185 only dimerises with the related FecI homologue PA3899 (Figure 1) and the truncated PA3899105170. The truncated FecI-like fragments cover region 4, demonstrating their involvement in the interaction with the FecR homologues of P. aeruginosa. As with fecIR, the fecIR-like Pseudomonas genes are preceded by Fur boxes. In addition, genome analysis of P. aeruginosa reveals 14 ECF s factors of the fecI type, which are adjacent to fecR-type regulatory genes. Ten of these fecIR-like genes are adjacent to fecA-like genes that encode proteins with aminoterminal extensions like FecA [14]. Regulatory systems analogous to FecAIR have been identied experimentally in Bordetella pertussis [15], Bordetella bronchiseptica [16] and in Bordetella avium [17] (Figure 5). They all regulate iron transport systems for which the iron ligand responsible for induction or regulation by iron has been shown. B. pertussis and B. bronchiseptica encode the two regulatory genes hurI and hurR upstream of the heam transport gene cluster bhuRSTUV. Synthesis of the BhuR outer-membrane transport protein for haem is enhanced when cells are grown in a medium supplemented with hemin [15]. In addition, synthesis of a putative ferric siderophore outer-membrane transport protein (BfrZ) of B. bronchiseptica is regulated by two proteins, BupI and BupR, which are homologous to the fecI and fecR gene products, respectively [16]. Overexpression of bupI induces bfrZ transcription and BfrZ is observed in the outer-membrane fraction. B. avium contains a haem utilisation system in which the synthesis of the related outer-membrane transport protein BhuR is induced by haem and requires RhuI (which is homologous to FecI). BhuR synthesis is enhanced in cells that overexpress RhuI. Overexpression of RhuI reduces transcription of a ss-dependent gene, suggesting competition
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Comparison of the arrangement of the fecIR and fecABCDE genes of E. coli K-12 with similar transport and signalling systems. The arrows indicate the transcription polarity of the genes. (See text for designation of the genes.)

induces synthesis of the PupB outer-membrane transporter [10]. Two genes, pupI and pupR, are located upstream of pupB (Figure 5). Transcription induction of pupB by pseudobactin BN8 requires pupI and pupR. pupI shows 42.8% and pupR 36.6% sequence identity to fecI and fecR, respectively. Although regulation of pupB transcription resembles regulation of fecABCDE transcription, PupR might function only as an anti-s factor, given that PupI in the absence of PupR induces pupB transcription constitutively. In light of the data collected with the Fec system, one may conclude that the structural change in PupA upon binding of pseudobactin 358 is mediated through the amino-terminal extension of PupB to PupR, which then dissociates from PupI, releases PupI and acts as a pupB-specic s factor. In Pseudomonas aeruginosa, synthesis of the siderophore pyoverdin and transport of ferric pyoverdin is controlled by a FecIR-like regulatory device [11]. Pyoverdin (presumably after loading with Fe3) functions as an inducer of the pyoverdin synthesis and Fe3 pyoverdin transport genes and induces formation of exotoxin A and an extracellular endoproteinase. In this system, the FecR homologue fpvR and the fpvA gene (equivalent to fecA) map close to the pyoverdin synthesis operon, whereas the fecI homologue pvdS lies some distance away (Figure 5). PvdS is an ECF s factor and is required for the synthesis of pyoverdin, exotoxin A and endoproteinase. Synthesis is high in fpvR mutants; overexpressed fpvR inhibits synthesis, indicating that FpvR functions as an anti-PvdS s factor. Furthermore, FpvA is required for the induction by pyoverdin. Analogous to FecA, FpvA contains an amino-terminal extension; deletion of the extension abolishes induction of pyoverdin synthesis in response
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Regulation of the FecI-type ECF sigma factor by transmembrane signalling Braun, Mahren and Ogierman 179

between RhuI and ss for the RNA polymerase core enzyme [17]. Ralstonia solanacearum elicits a hypersensitive response on non-host plants. At the beginning of the regulatory cascade that eventually induces the hrp hypersensitivity genes stands a regulatory device of the Fec type [18,19,20]. In the R. solanacearum system, it is highly interesting that the initial signal is generated by physical contact between the bacteria and the plant cells, without involvement of a diffusible substance. A signalling system that starts at the bacterial cell surface is perfectly suited to respond to cellcell contact, as was previously predicted [2]. For the expression of the prhJ regulatory gene, the PrhA outer-membrane protein, PrhI (homologous to FecI) and PrhR (homologous to FecR) are required. As with the fecIR genes, the prhIR genes form a separate regulatory unit (Figure 5) that does not require PrhA for expression and is not autoregulated. In addition, cells synthesising a truncated PrhR protein are fully pathogenic on host plants and this is reminiscent of the constitutive expression of the fec transport genes in cells synthesising truncated FecR proteins.

transmission of the signal through the periplasm and across the cytoplasmic membrane to convert FecI to an active s factor. As FecI shows virtually no activity in the absence of FecR, FecR cannot function purely as an anti-s factor. If FecR is an anti-s factor, which is likely in the light of the regulation of other ECF s factors, FecR must display an additional function for example, to maintain FecI in an active conformation. The conformational change in FecR that results from the signal received from FecA occupied with (Fe3 citrate)2 might cause dissociation of FecI from FecR, which then immediately binds to the RNA polymerase core enzyme to avoid inactivation by proteolysis, denaturation or precipitation. FecR might also activate FecI by changing the FecI conformation. There is no evidence for a chemical modication of FecI, however. It is also difcult to envisage that FecR catalyses chemical modication of FecI, because the smallest fragment of FecR that activates FecI is FecR168 [2], which is unlikely to display enzymatic activity. Without a signal, the cytoplasmic fragment of FecR, which shows constitutive expression of the fec transport genes in the absence of inducer, might activate FecI by spontaneous dissociation from FecI or by spontaneous induction of the active conformation in FecI. The few studies on other ECF s factors of the FecI type suggest a mechanism similar to the FecAIR signalling cascade. Bacterial species living in environments of poor and frequently changing nutrients such as soil and water are particularly rich in ECF s factors. They are also rich in TonB-dependent outer-membrane transport proteins that indicate that scarce nutrients, other than iron, are taken up by active-transport systems across the outer membrane. These transport proteins might not only function as transporters but also as signalling proteins for ECF s factors. All the systems discussed in this review concern regulation of virulence factors of pathogenic or potentially pathogenic bacteria. Regulation of the genes that control the hypersensitivity response in plants by R. solanacearum, and exoproteinase and exotoxin synthesis in P. aeruginosa, extends the signals and the functions that are controlled by the FecIRA-type regulation beyond regulation of iron-transport systems. Following the discovery of signal generation by cellcell contact in the R. solanacearumplant system, it will be of interest to see if a similar regulatory device functions during infection of animals or humans for example, in the regulation of the formation of type III and type IV secretion systems that are induced upon contact of bacteria with eukaryotic cells of their hosts.

Conclusions
Since its identication in 1994, the ECF s factor family has rapidly grown. In the FecIR subgroup, all studied systems except one regulate the synthesis of Fe3 transport systems. The reason might be found in the very low Fe3 availability, which requires intricate transport systems across the outer membrane, through the periplasm, and across the cytoplasmic membrane. Control that starts from the cell surface enables transcription initiation without formation of the transport system in the cytoplasmic membrane. It is economic to synthesise the iron-import systems only when they are needed. Iron starvation alone is not a sufcient signal for the formation of the transport systems, since a single strain may express several iron transport systems. For example, E. coli has up to eight such systems, and only one system transports the source of iron that may be available at a given time. An additional regulatory system that confers specicity to the available iron source limits the metabolic activity of the cells to what is required. This is the case for the regulation of the ferric citrate transport system. Iron starvation induces synthesis of the regulatory proteins FecI and FecR, of which only a few molecules are synthesised. When ferric citrate is in the medium, synthesis of FecA is strongly increased from a basal level to around 80,000 molecules per cell, making FecA one of the most highly synthesised proteins of E. coli. The high levels of FecA facilitate capture of the scarce (Fe3 citrate)2 molecules in the medium. Binding of (Fe3 citrate)2 to FecA at the cell surface initiates a signal that eventually ends in the cytoplasm, where transcription of the fec transport genes is initiated. FecR is required for
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Acknowledgements
The authors work was supported by the Deutsche Forschungsgemeinschaft (Br 330/19-1), the Alexander von Humboldt Foundation (M Ogierman) and the Fonds der Chemischen Industrie. Current Opinion in Microbiology 2003, 6:173180

180 Cell regulation

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:  of special interest  of outstanding interest 1. Lonetto MA, Brown KL, Rudd KE, Buttner MJ: Analysis of the Streptomyces coelicolor sigE gene reveals the existence of a subfamily of eubacterial RNA polymerase r factors involved in the regulation of extracytoplasmic functions. Proc Natl Acad Sci USA 1994, 91:7573-7577. Braun V: Surface signaling: novel transcription initiation mechanism starting from the cell surface. Arch Microbiol 1997, 167:325-331. Luck SN, Turner SA, Rajakumar K, Sakellaris H, Adler B: Ferric dicitrate transport system (FEC) of Shigella exneri 2a YSH6000 is encoded on a novel pathogenicity island carrying multiple antibiotic resistance genes. Infect Immun 2001, 69:6012-6021. Maeda H, Jishage M, Nomura T, Fujita N, Ishihama A: Two extracytoplasmic function sigma subunits rE and rFecI of Escherichia coli: promoter selectivity and intracellular levels. J Bacteriol 2000, 182:1181-1184.

12. Leoni L, Orsi N, De Lorenzo V, Visca P: Functional analysis of PvdS, an iron starvation sigma factor of Pseudomonas aeruginosa. J Bacteriol 2000, 182:1481-1491. 13. Shen J, Meldrum A, Poole K: FpvA receptor involvement in  pyoverdine biosynthesis in Pseudomonas aeruginosa. J Bacteriol 2002, 184:3268-3275. This paper provides biochemical evidence that FpvA acts as a transcription regulatory protein and as an outer-membrane transporter. 14. Visca P, Leoni L, Wilson MJ, Lamont IL: Iron transport and  regulation, cell signalling and genomics: lessons from Escherichia coli and Pseudomonas. Mol Microbiol 2002, 45:1177-1190. This paper provides a comprehensive overview of regulatory devices of the FecIR type in P. aeruginosa. 15. Vanderpool CK, Armstrong SK: The Bordetella bhu locus is required for heme iron utilization. J Bacteriol 2001, 183:4278-4287. 16. Pradel E, Locht C: Expression of the siderophore receptor gene bfrZ is controlled by the extracytoplasmic-function sigma factor BupI in Bordetella bronchiseptica. J Bacteriol 2001, 183:2910-2917. 17. Kirby AE, Metzger DJ, Murphy ER, Connell TD: Heme utilization in Bordetella avium is regulated by RhuI, a heme-responsive extracytoplasmic function sigma factor. Infect Immun 2001, 69:6951-6961. 18. Marenda M, Brito B, Callard D, Genin S, Barberis P, Boucher C, Ariat M: PrhA controls a novel regulatory pathway required for the specic induction of Ralstonia solanacearum hrp genes in the presence of plant cells. Mol Microbiol 1998, 27:437-453. 19. Aldon D, Brito B, Boucher C, Genin S: A bacterial sensor of plant cell contact controls the transcriptional induction of Ralstonia solanacearum pathogenicity genes. EMBO J 2000, 19:2304-2314. 20. Brito B, Aldon D, Barberis P, Boucher C, Genin S: A signal transfer  system through three compartments transduces the plant cell contact-dependent signal controlling Ralstonia solanacearum hrp genes. Mol Plant Microbiol 2002, 15:109-119. This paper shows that ECF sigma-factor-controlled transcription initiation occurs at the start of a signal cascade that regulates the hypersensitivity response. 21. Moeck GS, Letellier L: Characterization of in vitro interactions between a truncated TonB protein from Escherichia coli and the outer membrane receptors FhuA and FepA. J Bacteriol 2001, 183:2755-2764. 22. Higgs PI, Larsen RA, Postle K: Quantication of known components of the Escherichia coli TonB energy transduction system: TonB, ExbB, ExbD and FepA. Mol Micobiol 2002, 44:271-281. 23. Stiefel A, Mahren S, Ochs M, Schindler PT, Enz S, Braun V: Control of the ferric citrate transport system of Escherichia coli: mutations in region 2.1 of the FecI extracytoplasmic-function sigma factor suppress mutations in the FecR transmembrane regulatory protein. J Bacteriol 2001, 183:162-170.

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Ferguson AD, Chakraborty R, Smith BS, Esser L, van der Helm D, Deisenhofer J: Structural basis of gating by the outer membrane transporter FecA. Science 2002, 295:1715-1719. This paper describes the crystal structure of FecA in the unloaded and ferric-citrate-loaded conformation. It also reveals substrate gating, and forms the basis for the interpretation of the properties of FecA mutant proteins. 6. Kim I, Stiefel A, Plantor S, Angerer A, Braun V: Transcription induction of the ferric citrate transport genes via the Nterminus of the FecA outer membrane protein, the Ton system and the electrochemical potential of the cytoplasmic membrane. Mol Microbiol 1997, 23:333-344. Welz D, Braun V: Ferric citrate transport of Escherichia coli: functional regions of the FecR transmembrane regulatory protein. J Bacteriol 1998, 180:2387-2394. Enz S, Mahren S, Stroeher UH, Braun V: Surface signaling in ferric citrate transport gene induction: interaction of the FecA, FecR and FecI regulatory proteins. J Bacteriol 2000, 182:637-646. Mahren S, Enz S, Braun V: Functional interaction of region 4 of the extracytoplasmic function sigma factor FecI with the cytoplasmic portion of the FecR transmembrane protein of the Escherichia coli ferric citrate transport system. J Bacteriol 2002, 184:3704-3711.

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10. Koster M, van Klompenburg W, Bitter W, Leong J, Weisbeek P: Role for the outer membrane ferric siderophore receptor PupB in signal transduction across the bacterial cell envelope. EMBO J 1994, 13:2805-2813. 11. Lamont IL, Beare PA, Ochsner U, Vasl AI, Vasil ML: Siderophoremediated signaling regulates virulence factor production in Pseudomonas aeruginosa. Proc Natl Acad Sci USA 2002, 99:7072-7077.

Current Opinion in Microbiology 2003, 6:173180

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