Genome organization

in prokaryotes

Allan M Campbell
Stanford University, Stanford, USA

Most of the well-characterized prokaryotic genomes consist of doublestranded DNA organized as a single circular chromosome 0.6-10 Mb in length and one or more circular plasmid species of 2 kb-1.7 Mb. The past few years, however, have revealed some major variations in genome organization. In addition, a recent accumulation of data has shown that the location and orientation of the genes and repeated sequences (including prophages and transposons) on and among these elements is not always random. Some of the non-randomness is probably the result of unique historical events; in other cases it reflects selection for the optimization of function. Current Opinion in Genetics and Development 1993, 3:837-844

Introduction
The best studied prokaryotic genome, that of the K-12 strain of Escher-i&a coli, consists of a circular chromosome about 4.7 Mb in length, of which 1.6 Mb has been sequenced ll**j, and a plasmid (F) about 100 kb in length. F is a conjugative plasmid (able to transfer its DNA to other cells) of low copy number (l-2 per chromosome). As for the chromosome, the replication of F is stringently controlled (exactly one replication per division cycle) and daughter molecules are actively partitioned to sister cells at cell division 121. Many other bacteria harbor multicopy plasmids, usually smaller than F, which replicate randomly (relaxed control) during the cell cycle. Some of these can be introduced into E. coli K-12 by transformation. Stringently controlled plasmids may be likened to the accessory chromosomes of eukaryotes, whereas those under relaxed control act more like cytoplasmic elements. In addition, the K-12 chromosome contains several mobile elements: insertion sequences (ISI-IS5, and #>, most of which are present in several copies (IS2, IS.3 and fl being present in F as well), and active and defective prophages (h, its 4 defective relatives DLP12, rat, qin and e14, and a small remnant of 12 prophage, 131).Other bacterial strains and plasmids harbor transposons (insertible elements carrying genes that affect cell phenotype, such as antibiotic resistance), some of which can be transferred into E. coli K-12. Genes affecting cell phenotype are distributed among chromosomes, plasmids and mobile elements. We may ask why some of these genes are on chromosomes and others on plasmids, as well as why the chromosomal genes reside in their present locations along the chromosome. Two kinds of answers have been proposed: historical (attributing present location to a unique train of events in gene evolution or acquisition),

or functional (attributing present location to optimization of function and implying that other locations have been tried, but failed the test of natural selection). The dichotomy is useful only for classifying hypotheses; every real gene arrangement has arisen by some means and then survived natural selection.

Primarv

DNA structure

Chromosomal structure and the positioning of genes within it have been studied in numerous bacterial species 14,5*,6,7**,8,9,10-121. In E. coli K-12, the detailed ordering of loci by recombinational mapping 1131, has been supplemented by sequencing and restriction mapping, as well as by use of clone banks whose inserts are ordered along the chromosome 1141. For Bradyrhirobium japonicum, the circular topology of the chromosome was deduced from a map based on the pulsed-field gel electrophoresis (PFGE) of fragments generated by restriction enzymes that recognise few sites EA. Many genes were located on the map either by cloning them into a plasmid vector containing rare restriction sites followed by co-integration with the chromosome, or by inserting a kanamycin resistance cassette into the cloned DNA segment and recombining it into the chromosome. On the resulting map, the symbiosis genes (needed for nodulation and nitrogen fixation) all lie within a 380 kb segment of the 8.7 Mb chromosome. In the related species Rhirobium meliloti, the genome consists of one 3.4 Mb chromosome and two megaplasmids pSym1 (1.4 Mb) and pSym2 (1.7 Mb). These size estimates come from PFGE, using hybridization to markers that had previously been assigned to one of the three linkage groups by conjugational mapping

Abbreviations
Dam-DNA

adenine methylase; PFGE-pulsed 0 Current

field

gel electrophoresis;

REP-repetitive

extragenic

palindrome.

Biology

Ltd ISSN 0959-437X

837

838

Gnomes

and evolution

[151. Such mapping is most conveniently performed by using the transposon TnS-Mob to insert oogins of transfer at random locations [6,161. The distinction between chromosome and plasmids is clear-cut for E. coli and most other bacteria that have been studied, first, because the chromosome is much larger than the plasmids, and second, because the plasmids are dispensable under optimal growth conditions. For Rhirobium, however, dispensability of the megaplasmids has not been demonstrated, and designation of the three Rhirobium DNAs as one chromosome and two plasmids is in part semantic. The megaplasmids contain the symbiosis genes (which occupy only a small fraction of their DNA length), but the only essential housekeeping genes that they contain are duplicated on the chromosome. Segments accounting for all but 200 kb of pSym1 can be deleted without loss of viability i171. A similar issue has arisen with Rhodobacter spberoides [lP*l and Bruce&a melitensis [l!W. B. melitensis has two independent DNA molecules (2.2 kb and 1.2 kb). DNA hybridization has mapped a dnaK gene to the larger molecule and a groE gene to the smaller one [lPl. The PFGE mapping of R. spberoides (recently confirmed by conjugation [18**1) shows two separate DNA molecules of 3Mb (chromosome 1) and 0.9Mb (chromosome 2), in addition to 450 kb of DNA in five small plasmids. The only genes essential for growth that have been localized to chromosome 2 are ribosomal RNA genes (rrnB and rm0. Chromosome 1 also carries a ribosomal RNA gene (&I. However, simultaneous inactivation of rrnB and rrnC results in very slow growth under all conditions tested. So, chromosome 2 is necessary for normal growth. Most characterized prokaryotic chromosomes and plasmids are circular. However, the chromosome of the spirochaete Borrelia burgdorferi, which causes Lyme disease, has a linear restriction map [7**,81. Spirochaetes of genera other than Borrelia (such as Leptospira and Treponema) have circular chromosomes [ill. All known chromosomal genes of B. burgdo@i have been located on the physical map, including ribosomal RNA genes [8] and recently identified analogs of dnaA [7**1, dnaJ dnaK, g?pE 1211 and rho 1201. Rbodococcus fascians also contains an otherwise uncharacterized 4 Mb DNA molecule, which could be the chromosome, that is presumed to be linear because it enters contour-clamped homogenous electric field (CHEF) gels f22.1. Linear plasmids (ranging in size from 5-420 kb 123,241) are found in many prokaryotes and are probably underrepresented in the published literature because they are lost in some extraction procedures designed to detect covalently closed circles.

ies known as nucleoids. After termination of replication, nucleoids divide to give daughter nucleoids, which are later partitioned at cell division. Nucleoids isolated from disrupted cells contain DNA, RNA, protein and cell membrane, and consist of about 50 domains whose superhelicity is independently relaxable by nicking. It is not known whether domain boundaries are fixed or vary from cell to cell. DNA extracted from the archaebacterium Halobactenum salinarium is found in two forms: free DNA (predominant in early exponential phase), and a chromatin-like DNA-protein complex (predominant in stationary phase) [25**1.

Replication

cycle

The primary foci of chromosomal organization are the origin and terminus of replication. The E. coli chromosome replicates bidirectionally from a unique origin (on0 at 84 minutes on the genetic map. Close by is the dnaA gene, which encodes the initiator protein for replication. Replication initiates at otiC exactly once during each division cycle; once initiation has occurred, some mechanism prevents re-initiation. The DNA close to oriC is rich in the tetranucleotide GATC, substrate of the Dam methylase (DNA adenine methylase). Following replication, DNA is transiently hemimethylated until the methylase can act on the newly synthesized strand. Hemimethylated origin DNA specifically binds to cell membrane 1261; Dam sites close to oriC and to the 5 end of dnaA (but not all intervening DNA) remain hemimethylated for 1&12minutes, much longer than sites elsewhere on the chromosome, suggesting that these sites are sequestered from the methylase and perhaps also from reinitiation f271. Antipodal to the replication origin is the terminus DNA, containing three sites so oriented as to stop counterclockwise fork movement (TerA at 28 minutes, TerD at 27 minutes, and TerE at 23 minutes) and two sites in the opposite orientation to stop clockwise fork movement (TerC at 34 minutes, and TerB at 36 minutes) 1281. Next to TerB is a gene, tus, whose product binds to Ter sites causing replication fork arrest 1291. Tus binding to TerB also autoregulates tus expression. The postulated function of terminus DNA is to deal with asynchrony in the rates of clockwise and counterclockwise replication, by forcing the first replication fork that arrives to wait for the other to complete the cycle. This ensures that any DNA segment outside the terminus region is always replicated in the same direction. Deletion of terminus DNA, leaving replication to stop wherever forks collide, is not lethal. The terminus of Bacillus subtilis resembles that of E. coli in having oppositely oriented sites that bind to a protein encoded by a gene adjacent to one of the sites [30*1. On completion of replication, partitioning of daughter chromosomes into sister cells requires several steps [2,311. First, the chromosomes must separate, and this requires topoisomerases to remove interlocks.

Nucleoids
Intracellular DNA is folded and associated with protein (the major one in E. coli being HU) to form bod-

Genome

organization

in prokaryotes

Campbell

839

A site-specific recombinase acting near the terminus can resolve dimers that may have arisen through intramolecular recombination, and a myosin-related protein (MukB) moves separated nucleotides toward the poles. Stringently controlled low copy number plasmids (such as Pl, F and NR2) each have their own set of truns-acting partitioning factors, and at least one origin-linked &-acting site. No c&acting site has been identified for chromosomal-MukB interactions. Chromosomal partitioning is normal in dum mutants 1321, disproving the hypothesis that partitioning requires Dam-dependent membrane binding 1261, and leaving no evidence for a role of the origin in partitioning. F partitioning does not require MukB 1331. For linear plasmids and chromosomes, there is no direct information on the mechanism(s) of replication or partitioning. The structures of their molecular ends fall into two categories, similar to those of certain viruses whose replication has been studied. The 350 kb plasmid XI1 has a 80 kb inverted terminal repeat and a protein covalently bound to the 5 ends of DNA strands 1341,reminiscent of adenoviruses or the Bacillus phage +29. In these viruses, protein-primed synthesis initiates at the termini 135,3&l, and no circular or multimeric intermediates are formed. In the plasmid form of phage N15 1371and some linear plasmids of Borreliu 1381the two DNA strands are covalently joined to each other at each molecular end by a short DNA loop, as in poxviruses and iridoviruses. Three plasmids from the same Borrelia strain were shown to have the same terminal sequences and 4base loops, and these were most similar in sequence to the iridovirus African swine fever virus. In poxviruses, replication though a terminus generates an interrupted reverse repeat that is later resolved into new termini by site-specific exchange between two of the four strands in the subterminal DNA j391. If the Borrelia chromosome has similar termini, and if bidirectional replication starts from a unique origin (as suggested by the clustering of DNA replication genes [7**1), such resolution could convert the resulting head-to-head dimer into monomers (Fig. 1). If this hypothesis proves correct, we might expect to encounter some bacterium where it is used as an alternative to conventional topoisomerases for separating newly replicated circular chromosomes by transient linearization (as a reverse of the reaction could join ends to form daughter circles).

are readily lost from the genome, thus reducing the burden on the species of replicating every gene in every individual. This strategy has consequences that raise a number of questions 1401.One consequence is that the host range of the element must include diverse strains, either of the same or different species, one or more of which should serve as a reservoir from which the element can be transferred when needed. A major question is whether the element can survive, even in the reservoir strain, solely by its ability to outreplicate chromosomal DNA without conferring some selective advantage on the host. Mathematical modeling indicates that conjugative plasmids might perhaps be maintained without selection, but this is unlikely for non-conjugative plasmids or transposons 1411. Experimental studies have shown that a transposonderived antibiotic gene can confer a selective advantage, even in absence of antibiotic 1421. A complete understanding will require knowledge of why the chromosome has not captured the gene and jettisoned the rest of the element. The basic assumptions are that extrachromosomal genes are readily incorporated into the chromosome (an assumption supported by many demonstrated mechanisms) and that a natural equilibrium has been reached that optimizes the location of each gene. Whereas the distribution of genes between chromosomes and plasmids can be rationalized on the basis of gene function, simple explanations for non-random positioning of genes along chromosomes have been less convincing. Domain structure of nucleoids and the gradient of gene dosage from origin to terminus (about fourfold in rapidly growing E. coli) have been suggested, but historical explanations, such as chance rearrangements and chimeric construction of chromosomes, have also been considered. Several kinds of non-randomness are observed. Firstly, strong variations in the frequency of mapped loci are observable along the chromosomes of Sfrepfomyces coelicolorand E. coli 19,431. However, whether the segments of low gene density are truly poor in expressed genes, or simply include genes whose functions were not sampled, is unknown. In S. coelicolor, deletions of genetically silent DNA as large as 40 kb are innocuous. Secondly, genes or operons with related metabolic functions are located near, but not adjacent to, one another. For example, the chl genes required for molybdopterin synthesis in E. coli are in five contiguous clusters, three of which lie within 1 minute of each other on the chromosome 1131,and in Pseudomonas aeruginosa, most of the known auxotrophic mutations lie in one half of the chromosome, whereas catabolic genes are clustered but non-contiguous 14,441. Thirdly, some repeated sequences are distributed non-randomly. Although prokaryotic DNA is mostly comprised of unique sequence, all prokaxyotes contain some repeated sequences, including prophages, insertion sequences, ribosomal RNA genes, and sequences of oligonucleotide length 145*,46,471. Prokaryotic genomes have been described as streamlined compared to the genomes of higher eukaryotes, where selection to eliminate junk

location

of genes within

the genome

Plasmids and transposons frequently include genes that affect cell phenotype. Unlike chromosomal genes, the genes on plasmids and transposons are not essential for growth, but encode functions such as heavy metal resistance or catabolism of specific substrates that might be needed only occasionally in nature. This has been rationalized on the notion that the elements

840

Genomes

and evolution

(a)

(b)

(d)

(e)

0 1993 Currenl Opinion in Genetics and lkvelopment

Fig. 1. Hypothetical model for chromosome replication in Borrelia. (a) Linear double-stranded DNA is joined at the ends by d-base loops. (b) Replication is initiated at the center of the chromosome and the replication bubble enlarges bidirectionally toward the termini. fc) When the replication reaches the termini, the 3 ends of the new DNA meet the 5 ends, and ligation occurs to produce a double-stranded circular DNA molecule. (d) Two-strand exchange (as in 1381) resolves the junctions: the top strands exchange on the left and the bottom strands on the right. Therefore, if exchange takes place within a subterminal inverted repeat, the sequence of the exchanged strands is the same in both cases. (e) Following exchange, separation produces two daughter molecules identical to the parental molecule in (a). The bold lines represent the terminal loops derived from the parental molecule. A, B and C (and their complementary sequences A, 6 and C) represent genes along the chromosome.

DNA is less intense, but in E.coli K-12, the active and defective lambdoid prophages alone comprise more than 3% of the genome, higher than the percentage of retroviral DNA in the mouse. A computer search through the K-12 sequence [ll recovered not only the previously known ribosomal RNA genes, IS elements, REP (repetitive extragenic palindrome) and Rhs (recombinational hot spot) sites, but also four new groups of sequences 30-46 bp in length, each repeated many times in identical or similar form. REP elements (which may represent gyrase-binding sites) are scattered in the genome [481, as are three of the four new groups. Ri-

bosomal RNA genes are not obviously clustered, either. On the other hand, the location of the eight identified group IV sites between 81.2 and 13.1 minutes is significantly non-random. Also, the insertion sites of natural lambdoid phages (which have little sequence similarity to one another) are clustered between 6 and 42 minutes
i491.

One caveat applies to the statistical significance of nonrandom location. In all cases cited, the probability that the observed distribution occurred by chance is less than 1%. However, these cases attract our attention pre-

Cenome

organization

in prokaryotes

Campbell

841

cisely because of that fact. The chl genes are clustered, but the operons for many other pathways are scattered. A skeptic might properly demand a universal survey. Where no functional explanation for non-randomness is evident, historical explanations have been entertained. Genetically silent regions may represent insertions of useless DNA. Because catabolic gene clusters are located in different positions on the chromosomes of Pseuciomonas aeruginosa and P. pritida (with respect to the auxotrophic markers, whose order is conserved in the two species), these clusters may have been inserted into the chromosome from plasmids, which carry similar genes in other pseudomonads. The E. coli K-12 chromosome might represent a recombinant of two lines, only one of which harbored lambdoid phages, an idea whose attractiveness is tempered by the fact that the same explanation cannot apply to both the phage attachment sites and the group IV sites, because their ranges are overlapping but not coextensive.

entation provides the best explanation for the observed conservation of gene order between bacteria such as E. coli and Salmonella typhimunum over a evolutionary time-span sufficient to allow 16% sequence divergence within genes 1561.

able 1. E. co/i K-l 2 elements

lements Orientation

with non-random

orientations.

Comments

References

;enes

Codirectional replication

with

This orientation avoids collision polymerase polymerase. documented transcription retards replication, that retards and suspected replication transcription. head-on of RNA with DNA It is that

147.48.50)

Orientation

of genes along the chromosome

Ihi sites

Preferred orientation with lo and respect replication transcription

Selective unknown

value

I481

Among 97 genes that encode rRNAs, tRNAs, ribosomal proteins and translation factors (all known to be rapidly transcribed in E. co/i K-121, 92 are oriented in the direction of replication fork movement [501, and in the 227 kb of DNA spanning the replication origin (of which 30% comprises open reading frames or structural RNA genes) 204 of the 272 genes are oriented in the direction of replication [51,52]). The obvious functional explanation is that collision of RNA and DNA polymerases would delay replication, transcription or both. Inhibition of replication fork movement by transcription has been demonstrated by the insertion of a plasmid replication origin into the chromosome, followed by electron microscopic determination of replication fork positions a few minutes after induction [531. The orientation of natural lambdoid prophages is highly non-random [491. A historical explanation can be contrived [541, but functional explanations, such as an effect of the direction of replication on prophage stability or cell growth, seem more likely. Within the 227 kb of sequenced DNA spanning the replication origin, 48 of the 56 copies of the recombination-promoting octanucleotide chi sites (GCTGGTGG) are oriented in the direction of replication. The orientation of chi is correlated both with direction of transcription/translation and with direction of replication. Chi sequences are among the more abundant octanucleotides in the 81.5-86.5 contig (as well as in the rest of the sequenced genome [1**,51**1). The same non-random orientations observed in the 81.5-86.5 contig are also observed in the second largest known contiguous sequence, at O-2.4 minutes [551. Present information on non-random orientation in

amboid rrophages

Preferred orientation chromosome on

Among lambdoid whose is known,

those prophages

[46,51**1

orientation only is ordered II one qin

(the defective prophage) oppositely

from the others. normally rather replicated

is also the only one counterclockwise than clockwise.

Conclusions
Only within the last few years have major variations in genome organization (such as linear chromosomes or multiple chromosomes) come to light, and additional studies will doubtless broaden the range still further. There is no great virtue in the mere demonstration of diversity unless underlying rules can be identified. Where the location of genes within genomes has a functional basis, its study may provide clues to higher order chromosome structure, subtle effects of gene dosage, and the selective conditions affecting mobile elements in cell lineages and in natural populations. Aspects of gene organization resulting from recent historical accidents, such as the chance occurrence of a

E. coli K-12 is summarized in Table 1. Selection for ori-

842

Genomes

and evolution closely linked clusters in a 380 kb segment. This contrasts to Rhinoblum melfloti, where the symbiosis genes are on megaplasmids. 6. OSTEI& M, STANLEYJ, BHOUCHTON WJ, DOWLING DN: A Chromosomal Gcnctic Map of Rbfzoblum sp NGR234 GCIP crated with Tn5-Mob. Mol Gen Gener 1989, 220:157-160.

chromosomal rearrangement or insertion of a transposon, may provide no special insights beyond their collective impact on variation in natural populations. More ancient events, which have defined the structure of a whole group of descendants, can be useful not only in tracing phylogenies but also in interpreting the array of secondary events that have later been selected. For example, the orientation of highly transcribed genes with respect to direction of replication must have developed over time and must create a strong pressure against any rearrangements that disturb it, as well as pressure for maintaining a specific terminus region. Intuition may prove a poor guide as to which events fall into that category. Both linear chromosomes and multiple chromosomes seem like fundamental deviations from the E. coli paradigm, yet the linear chromosomes of BorreIiu are not found in other spirochetes, and Rhodobacter capsulatus and Bradybirobium japonicum store in one chromosome information that is distributed among two or three replicons in Rbodobacter spberoides and Rbixobium meliloti, respectively. Transition to these variant forms may be relatively recent and facile, in which case efforts to uncover a selective advantage specific to the natural habitat seem appropriate.

7. ..

CA~JENSS, HUANG WM: Linear Chromosomal Physical and Gcnctic Map of Bortvlia butgdorferr. Mol Mlcrobiol 1993, 8967-980. All known genes of Borrelfa brqdo@ri are localized on the 952 kb linear chromosome, except for two genes encoding outer surface proteins which map to a 53 kb linear plasmid. From the location of dna genes it is proposed that replication is initiated near the center of the chromosome. 8. DAVIIXON BE, MACDOUGALL J, SAINT GIHONS I: Physical Map of the Linear Chromosome of the Bacterium Borre&a burgdotferf 212, a Causative Agent of Lyme Discasc, and Le cahzation of rRNA Gcncs. J Backrlol 1992, 174:376&3774.

9. .

KIFXR HM, KI~XR T, HOPWOOD DA: A Combined Genetic and Physical Map of the Stmpromyces coellcolor k?(2) Chre mosomc. J Baclerfol 1992, 174:5496-5507. Physical mapping of the Streptomyccs coekolor chromosome confirms the presence of long segments with few if any genes. Some long deletions in these segments have no observed phenotypic effects. 10. FONSXIN M, ZHENG S, HASELKORNH: Physical Map of the Gcnomc of RbodobacterXapsulahs SB1003. J Baclerfol 1992, 174:407O-i077. BARIL C, HERMANN JL, RICHAUD C, MARGARITA D, SAINI GIHONS I: Scattering of the rItNA Genes on the Physical Map of the Circular Chromosome of Lcptospira-Irr tcrrogans Scrovar Ictcrhaemorrhagiac. J BackHo 1992, 174:7566-7571. KIM NW, BINGHAM, H. KHAWAJA H, Lovt~ H, HANI E, NEOIF K, CHAN VL: Physical Map of CampylobacterJejunf TGH9011 And Localization of 10 Gcnctic Markers by USC of Pulsed-Field Gel Electrophorcsis. J Bacletiol 1992, 113494-3498. BACHMANNBJ: Linkage Map of Escbericbla calf K-12. Edition 8. Microbfol Rev 1990, 54:130-197. NISHIMURAA, AKIYAMA K, KOHARA Y, HOIUUCHI K: Corrclation of the pLC Plasmids to the Physical Map of Escberlcbfa toll K-12. Microbial Rev 1992, 56:137-151. SOI~RALBWS, HONEYCLIT~AJ, AIHI;RLY AG, MCCLELLANDM: Electrophorctic Separation of the Three Rblzoblum mellIoN Rcplicons. J Bacwiol 1991. 173:5173-5180. KLEIN S, LOHMAN K, CLOVI;.RR, WALKER GC. SIGNERER: A Directional, High-Frequency Chromosomal Mobilization Sys tcm for Genetic Mapping of Rbizobium mellIoN. J Bactetiol 1992, 1743324326. CHAHLFS.TC, FINAN TM: Analysis of a 1600-Kilobase Rbfzc+ bium melflotf Mcgaplasmid Using Dchncd Dclctions Gcncrated in viva. Genello- 1991, 127:F20.

11.

Acknowledgments
12. I thank Joe Campbell, Nyles Charon, Sam Karlin and Sharon Long for useful discussions and information. This work was supported by grant AlO8573, National Institutes of Allergy and Infectious Diseases. 13.

References

and recommended

reading

14.

Papers of particular interest, published within the annual period OF review, have been highlighted as: . of special interest .. of outstanding interest BWSDELL BE, HUDD KE, MAI~N A, KAHLIN S: Significant Dis pcrscd Recurrent DNA Sequences in the Ekcberfcbia coli Gcnomc. J Mol Btol 1993, 229:833-t%% New computer methods are used in a global survey of short dispersed repeats in the 1.6 Mb of sequenced B calf genome. .Severdl new classes of repeats are reported, and their positional relationship to genes of known Function is analyzed. 2. 3. HIRAGA S: Chromosome and Plasmid Partitioning in Escbericbla cvll. Anntr Rev Blochem 1992, 62:283306. BARREIRO,V, HAGG~RDLJUNCQUISIE: Attachment Sites for Bactcriophagc P2 on the &cbericbti coli Chromosome: DNA Scqucnccs, Localization on the Physical Map, and De tcction of a PZliic Remnant in E. coli K-12 Dcrivativcs. J BaCle?iOl 1992, 174:4086--4093. KRAWIECS, RILEY M: Organization of the Bacterial Chrome some. Mlcrobiol Rev 1990, 54:130-197. 1. ..

15.

16.

17.

SUWANTOA. KAPLAN S: Chromosome Transfer in Rbodobacter spbervfdes: Hfr Formation and Genetic Evidence for Two Unique Circular Chromosomes. J Bactetiol 1992, 174:1135-1145. Insertion of origins of conjugative transfer confirms the conclusion (from restriction mapping) that Rbodohacter spherokies has two chromosomes. Evidence that both chromosomes are needed for normal growth is mentioned. MICHAUX S, PAILLISSONJ, CARLX~ NURIT M-J, BOURG G, ALLARDI-X-SERVANT F&MUX M: Prcscncc of Two IndcA, pcndcnt Chromosomes in the Brucella melltens&s 16M Gcnomc. J Backrio 1993, 175:701-705. The B. melfk?uY.s genome consists of two circular DNA molecules (2.2 kb and 1.2 kb). A dnaKgene was mapped to the larger molecule and the gtoE gene to the smaller one, by DNA hybridization. 19. .

18. ..

4. 5. .

KUNDIG C. HENNECKEH, GOTIFERI M: Corrclatcd Physical and Genetic Map of the BradyrbLzoblum japonicum 110 Gcnomc. J Bacterlol 1993, 175:613-622. 63 genes arc located on a physical map of the 8700 kb chromosome of Bmdynbfzobium japonfcum. Symbiosis genes are in two

Cenome 20. K. HAUSER R, CAMPBELLJ, O.STHEIMER Isolation of J: dnaj dnaK aml ppE Homologues from Bone&a butgdop feri and Complcmcntation of Eschricbia coli Mutants. Mol Microbial 1993, 7:359-369. TILLY K, CAMPI~IXL J: A Bormlia burgdo@ri Homolog of the Escberichia coli rbo Gene. Nucleic Acids Res 1993. 21:2140.
TILLY,

organization

in prokaryotes

Campbell

843

21. 22. .

nism for Protein-Primed DNA Replication. Proc Natl Auad Sci USA 1992, 8939579-9583. In ultra study of the protein-primed initiation of phage $29 DNA synthesis defines the sequence requirements for template DNA and shows that replication initiates with the second nucleotide, then slides back to copy the terminal nucleotide. The result may be relevant to the replication mechanism of one class of linear plasmids. 37. TILLY K: Independence of Bacteriophage N15 Lytic and Liar ear Plasmid Replication from the Heat Shock Proteins DnaJ DnaK and GrpE. J Bacte?iol 1991, 173663-2. HINNEBUSCHJ, BARBOUR AG: Linear Plasmids of Bormlia butgdo@ri Have a Telomeric Structure and Sequence Similar to Those of a Eukaryotic Virus. J Bacterial 1991, 173:72337239. MERCHLINSKY Resolution of Poxvirus Telomem: Process M: ing Vaccinia Vi Concatemer Insettions by Conservative Strand Exchange. J Vtrol 1990, 643437-3446. CAMPl3eLLA: Evolutionary Significance of Accessory DNA Elements. Annu Rev Mkxobiol 1981, 35:55-84. CONDIT R, STEWART FM, LEVIN BR: The Population Biology of Bacterial Transposons: a Priori Conditions for Maintenance as Parasitic DNA. Amer Nat 1988, 132:12%147.
BLOT M, MEYER J, ARBER W: Bleomycin-Resistance Gene De rived from the Transposon TnS Confers Selective Advantage to Escbericbia coli K-12. Proc Nat1 Acad Sci USA 1991, 88:9112-9116,

CRESPI MFSSENS CAYLANAI), VAN MON~AGU M, D~:SOMER M, E, J: Fasciation Induction by the Phytopathogen Rbodococcus fkscians Depends Upon a Linear Plasmid Encoding a Cytokinin Synthase Gene. EMBO J 1992, 11:795-804. A bacterial-plant symbiosis requires a linear conjugative plasmid in the bacterium. HINNEBU.scHJ, I3ARI3OURA: Linear- and Circular-Plasmid 23. Copy Numbers in Bormlia burgdotferi. J Bacterial 1992, 1745251-5257. Hydrogen Autotrophy of Nocatrlla opaca Strains is Encoded by Linear Mcgaplasmids. J Gen Mfcrobfol 1990, 136:114%1151. TAKAYANACI S, MORIMURA KUSAOKE H. YOKOYAMA Y, KANO S, 25. .. K. SHIOI)A M: Chromosomal Structure of the Halophilic Archacbactcriwn Halobacterium-Solinarium. J Bacterial 1992. 174:7207-7216. Halobactetium DNA can be isolated as a chromatin-like complex more stable than protein-DNA complexes from most bacteria. In early exponential phase mo.st DNA is free, but it is converted to the chromatin-like complex by stationary phase.
KALKUS J, hH M, SCHI.ECEL H:

38.

39.

24.

40. 41.

42.

26.

OGDEN GB, PRAI-I MJ, SCHAECHTER The Replication OriM: gin of the E. coli Chromosome Binds to Cell IMembranes Only when Hcmimethylated. Cell 1988, 54:127-135.

43.

27.

CAMPU~LL, KLECKN~RN: E. coli Replication Origin (orlc) JL, and the dnuA Gene Promoter are Sequestered from Dam Methyltransfcrase Following Passage of the Chromosomal Replication Fork. Cell 1990. 62967-979. HIDAKA M. KOI~AYASHI T, HORIUCHI T: A Newly Identified 28. DNA Replication Terminus Site, TcrE, on the Escbericbia coli Chromosome. J Bactetfol 1991, 173:391-393. ROECKLEINBA, KUEM~EL PL: In Vfuo Characterization of fus 29. . Gene Expression in Escbericbia coli. Moi Microhiol 1992. 6:1655-1661. Binding of the termination protein Tus to the TerB terminator site of Escberichia calf causes both replication fork arrest and autoreguhtion of its own synthesis. SMITH MT, WAKE RG: Definition and Polarity of Action of 30. . DNA Replication Terminators in Bacillus suMIlk J Moi Biol 1992. 227648-667. Bacllius subtfk has two oppositely oriented terminators and a terminator protein Rtp which binds to them. Each terminator contains two Rtp-binding sites, both of which are needed for fork arrest. L@UNER-OLESEN KUEMPELPL: Chromosomal Partitioning in A. 31. .. Escberkbia coli. J Bacterial 1992. 174:7887889. The separation of E. calf chromosomes at the end of the replication cycle and the segregation of daughter nucleoids are described, and models for nucleoid formation are proposed and evaluated. VINELLA D, JAFPEA, DARI R, KOHIYAMA M, H~J~;Ho P: Chrc+ 32. mosomc Partitioning in Escbericbia coli in the Absence of Dam-Directed Methylation. J Bacteriol 1992, 174:2380-2390. 33. EZAKI B, OGURA T, NIKI H. HIRAGA S: Partitioning of a MiniF Plasmid into Anucleate Cells of the m&B Null Mutant. J Bacterial 1991. 1736643-6646. KINASHI H. SHIMAJI-MURAYAMA Physical Characterization M: of SCPl, a Giant Linear Plasmid from Smppromyces coelicolor. J Bacterial 1991, 173:1523-1531. SALAS M: Protein Primiig of DNA Replication. Anntr Rev Blochem 1991, 60:39-71. MOUNDED!BLANCO L, ESTHSAN JA, BERNARDA, SALAFM: IniJ, tiation of $29 DNA Replication Occurs at the Second 3 Nuclcotidc of the Linear Template: a Slidiig-Back Mecha-

44.

WILLIAMSONKM. HPIHERINCTON JACKSONJH: Detection of J, Fundamental Principles and a Level of Order for -Scale Gene Clustering on the E.scberic&ia co/i Chromosome. J Mol Euol 1993, 36:347-360. RAINANINGSHAK E, DHAHMSTITS, KR~SHNAPILWV, MORGAN A, SINCIAIR M, HOLLOWAY BW: A Combined Physical and Genetic Map of Pseudomonas aeruginosa PAO. J Gen Mtcrobiol 1990, 136:2351-2357.

45. .

LU~SKI JR, WEINST~CK GM: Short, Interspersed Repetitive DNA Sequence in Prokaryotic Genomes. J Bacrertol 1992, 174:4525-4529. A good summary of the literature on short dispersed repeats in prokaryotes. 46. HERMANSPWM, WANSOOWNGEND, VANEMUDENJDA: Characterization of a Major Polymorphic Tandem Repeat in Mycobacterfum-Tuberculosis and Its Potential Use in the Epidemiology of Mycobacterium-Kansasii and Mycobacterium-Gonlonae. J BackvIol 1992, 174:41574465. MARTIN B, HUMMERS 0. CAMARA M, GUENZI E, WALKER J, MITCHELL T, ANDREW P. PRUDHOMME M, ALLOING G, HAKENI3ECKR, ET AL.: A Highly Conserved Repeated DNA Elcmcnt Located in the Chromosome of Sheprococuspneumonlae. Nucleic Acids Res 1992, 20~3479-3483. DIMR~ GP, RUDD KE, MORGAN MK, BAYAL H, FERROLuw AMES G: Physical Mapping of Repetitive Palindrome Sequcnccs in Escbericbia coli and Phyiogenetic Distribution among Escbericbia coli Strains and Other Emetic Bacteria. J Bacterial 1992, 174:458s593. CAMPUELLAM: Chromosomal Insertion sites for Phagcs and Plasmids. J Bacteriol 1992, 174:749+7499. BREWERBJ: Replication and the Transcriptional Organiz+ tion of the Escberkbia coli Chromosome. In ne Bactertal Chromosome. Edited by Drlca K, Riley M. Washington DC; Amer Sot Microbial; 1990:6143.

47.

48.

49. 50.

34.

35. 36. .

BURLANDV, PLUNKS G III, DANIELSDL, B~AT~NERFR: DNA Sequence and Analysis of 136 Kilobases of the I&berfcbfu co/i Genomc. Organizational Symmetry around the Origin of Replication. Cenomics 1993, 16:551-561. Previous work showed that highly expressed genes are generally oriented in the direction of replication fork movement. From the complete sequence of 227 kb of E coil DNA around the replication ori-

51. ..

844

Genomes

and evolution
54.

gin, this rule is shown to apply to all ORFs, the switch in preferred direction lying at the origin site. Furthermore. the recombination-promoting oligonucleotide chi sites are also preferentially oriented with respect to the direction of replication and transcriptionbarxskdtion. 52. DANIELS DL, PLUNKWI G III, BURLAND V. BLA~INER FR: Analysis of the Es&ericbka toll Genome. DNA Sequence of the Region from 84.5 to 86.5 Minutes. Scbzce 1992, 2573771-778.

55.

56.

53. ..

FHENCH S: Consequences of Replication Fork Movement through Transcription Units In Ww. Sctence 1992, 250:1362-1365. , Insertion of a plasmid replication origin into the E. cdl chromosome and electron microscopy of the DNA after induction of initiation indicates that replication fork advance is strongly retarded by opposing trdnscription.

CAMPUEI.LA, SCHNEIDEHSJ. SONG 1% Lambdoid Phags as Elements of Bacterial Genomes. Gmerluc 1992. B&259-267. YURA T, MW H, NAGA~A T, ISHIHAMA A, FUJITA N, ISUNO K, MIZOBUCHI K, NAGAIA A: Systematic Sequencing of the Escbericbla coli Genome: Analysis of the O-2.4 Min Region. Nucleic Acti Res 1992, 20:330>33OB. SHARP PM: Determinants of DNA Sequence Divergence Between Escbericbia co/i and Salmonella @pblmurfum: Codon Usage, Map Position. and Concerted Evolution. /Mel Ewl 1991, 33:23-33.

AM Campbell, Department of I3iOlOgicdl sity, Stanford, California 94305, USA.

Sciences, Stanford Univer-