Proc. Natl. Acad. Sci. USAVol. 93, pp. 10303-10308, September 1996Genetics

Highly plastic chromosomal organization in Salmonella typhiSHU-LIN Liu AND KENNETH E. SANDERSON*Salmonella Genetic Stock Centre, Department of Biological Sciences, University of Calgary, Calgary, AB, Canada T2N 1N4

Communicated by John R Roth, University of Utah, Salt Lake City, UT, June 20, 1996 (received for review March 8, 1996)

ABSTRACT Gene order in the chromosomes of Esche-richia coli K-12 and Salmonella typhimurium LT2, and in manyother species ofSalmonella, is strongly conserved, even thoughthe genera diverged about 160 million years ago. However,partial digestion ofchromosomal DNA ofSalmonella typhi, thecausal organism of typhoid fever, with the endonucleaseI-Ceul followed by separation of the DNA fragments bypulsed-field gel electrophoresis showed that the chromosomesof independent wild-type isolates of S. typhi are rearrangeddue to homologous recombination between the seven rrn genesthat code for ribosomal RNA. The order of genes within theI-Ceul fragments is largely conserved, but the order of thefragments on the chromosome is rearranged. Twenty-onedifferent orders of the I-CeuI fragments were detected amongthe 127 wild-type strains we examined. Duplications anddeletions were not found, but transpositions and inversionswere common. Transpositions of I-CeuI fragments into sitesthat do not change their distance from the origin ofreplication(oriC) are frequently detected among the wild-type strains, buttranspositions that move the fragments much further fromoriC were rare. This supports the gene dosage hypothesis thatgenes at different distances from oriC have different genedosages and, hence, different gene expression, and that duringevolution genes become adapted to their specific location;thus, cells with changes in gene location due to transpositionsmay be less fit. Therefore, gene dosage may be one of the forcesthat conserves gene order, although its effects seem less strongin S. typhi than in other enteric bacteria. However, both thegene dosage and the genomic balance hypotheses, the latter ofwhich states that the origin (oriC) and terminus (TER) ofreplication must be separated by 180°C, need further inves-tigation.

The general structure and the order of genes on the chromo-somes of different enteric bacteria are usually strongly con-served (1, 2). The genetic and physical genome maps ofSalmonella typhimurium LT2 (3-5) (Fig. 1), Escherichia coliK-12 (9, 10), Salmonella enteritidis (11), and Salmonella para-typhi B (12) are very similar in the order of genes, althoughthey sometimes differ by an inversion in the TER region (fortermination of replication), by insertions of nonhomologousDNA ("loops"), and by the presence or absence of a plasmid.In addition, 17 independent wild-type strains of S. typhimuriumhave very similar chromosomes (13). Although the chromo-somes of these species are very stable during evolution, thechromosomes frequently rearrange when the cells are grown inculture. Spontaneous duplications produced by recombinationbetween rrn (rRNA) operons occur at frequencies of 1O- to10-4 (14, 15), and deletions are also frequent (16). Inversionswith endpoints in the seven copies of the rrn operons arecommon in S. typhimurium (15) and E. coli (17); the frequencyof inversions at other sites varies from low to undetectable(18). This high frequency of rearrangements in culture, com-bined with the stability of the chromosome through evolution,indicates that cells with rearrangements are strongly selected

AL.

4.

at

0r,

\rrn)

CI,4,.

B271 1723 1775 3909Bini Xbal Blnl l-Ceul

01 y 6 kb

rrs(l rr 7(2 3 S

FIG. 1. (A) The location and orientation of transcription of theseven rrn genes for ribosomal RNA of S. typhimurium LT2 (3), eachof which has a cleavage site for the endonuclease I-CeuI (6, 7). Theseven I-Ceul fragments are in the order ABCDEFG. oriC is the siteof initiation of bidirectional replication of DNA; TER is the site oftermination of replication. Order of rn genes is the same in Esche-richia coli K-12 (1). (B) The structure of the rrnB operon for ribosomalRNA of E. coli K-12 (8). rrs, rrl, andf are the genes for 16S, 23S, and5S rRNA. The beginning and end of each gene in base pairs is shownunder the genes, and the positions in base pairs of sites for digestionby the endonucleases BlnI, XbaI, and I-Ceul are also shown (3).

against during evolution and, therefore, do not survive. Forexample, no rrn-mediated rearrangements have been detectedin wild-type strains of either S. typhimurium or E. coli, in spiteof the high frequency of rearrangements observed in culture.However, we found that the genome of Salmonella typhi is lessconservative, for rearrangements of the chromosome segmentsbetween the rrn genes were detected in strain Ty2 and in a fewother wild-type strains (19, 20). The order of genes on thechromosome segments between rrn genes is relatively con-served between S. typhi and S. typhimurium, but these seg-ments were rearranged in S. typhi, postulated due to homol-ogous recombination between the rrn operons.

Abbreviation: PFGE, pulsed-field gel electrophoresis.*To whom reprint requests should be addressed. e-mail: kesander@acs.ucalgary.ca.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

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S. typhi is the only 1 of the 2300 Salmonella serovars thatgrows exclusively in humans and causes typhoid enteric fever(21-23), a major public health problem in many parts of theworld (22). Serotyping data (24) and DNA reassociation data(25) clearly show that strains of S. typhi belong in the genusSalmonella. The following lines of evidence indicate that S.typhi is a very homogeneous species. All 26 S. typhi strainsstudied by Reeves et al. (26) using multi-locus enzyme elec-trophoresis fell into a single electrophoretic type; Selander etal. (27), studying over 300 strains, found two electrophoretictypes, but both groups found S. typhi to be well separated fromother species in subgenus I of Salmonella. In comparison withmany other species studied by Selander et al. (27), S. typhi wasone of the most homogeneous. Restriction enzyme digestionpatterns also show very little genetic variation among S. typhistrains (28).

Digestion of DNA by the intron-encoded endonucleaseI-Ceul, which cuts in the rrn gene for 23S rRNA (29) but at noother site so far detected, followed by separation of thefragments by pulsed-field gel electrophoresis (PFGE), hasbeen used to compare the genomes of related bacteria (13, 30).Partial digestion allows location of adjacent fragments (19). Inthis report, we extend the use of I-CeuI partial digestion andshow that the number of rrn genes, the lengths of the DNAfragments between these genes, and the order of all seven ofthese fragments on the chromosome of a strain (the "rrnskeleton" of the chromosome), can be determined from asingle lane of a gel. We determined the rrn skeleton of 127wild-type strains of S. typhi; these strains show a remarkabledegree of genomic plasticity with the seven rrn operon-flankedsegments arranged in different orders. This provides some cluesabout the forces that influence conservation ofgenome structure.

inger Mannheim (XbaI). Most other chemicals, includingagarose, were from Sigma.PFGE Methods. Preparation of high-molecular weight

genomic DNA, endonuclease cleaving of DNA in agaroseblocks, separation of the DNA fragments by PFGE, anddouble-digestion techniques were as reported (3, 4); analysiswith I-CeuI was as described (13). The analysis by PFGE wasdone with the Bio-Rad DRII, the Bio-Rad Mapper, or theHoefer Hula electrophoresis systems.

RESULTSAll 127 strains of S. typhi produced seven I-CeuI fragmentswhen DNA was completely digested with I-CeuI and separatedby PFGE, indicating that they all have seven rrn operons (datanot shown). In strain Ty2 these seven I-CeuI fragments havebeen identified as being equivalent to fragments I-CeuI-A toG in S. typhimurium (Fig. 1), based on their comparable sizeand their content of genes, using DNA probes (6) or TnlOinsertions into known genes (19, 20). All 127 strains have sevenfragments of indistinguishable size: I-CeuI-A (2400 kb), -B(724 kb), -C (502 kb), -D (136 kb), -E (146 kb), -F (44 kb), and-G (828 kb), except for a few that gave one or occasionally twofragments, which are slightly larger or smaller (data not shown).DNA of strains of S. typhi was partially digested with I-CeuI;

representative data from 7 of the 127 strains that wereanalyzed are shown in Fig. 24, and interpretations of thefragments are in Fig. 2B. For example, lane 3 shows sevenfragments (A-G) resulting from complete digestion, but in

A BMATERIALS AND METHODS

Bacterial Strains and Cultivation Conditions. The S. typhistrains were obtained from the following sources: WendyJohnson and Rasik Khakhria (Laboratory Centre for DiseaseControl, Ottawa, Canada), Tikki Pang (Centre for AdvancedStudies, University of Malaysia), Bruce Stocker (Departmentof Medical Microbiology, Stanford University), Centers forDisease Control (Atlanta), Chandar Anand (Provincial Lab-oratory, Calgary, Canada), and Robert Selander (Institute ofMolecular Evolutionary Genetics, Pennsylvania State Univer-sity). All of these strains were identified as members of thefamily Enterobacteriaceae, because they are facultatively an-aerobic Gram-negative rods, and as Salmonella based onbiochemical reactions. They were identified as S. typhi basedon biochemical and antigenic characterizations, which weredetermined by the laboratories of origin, and confirmed by theLaboratory Centre for Disease Control (Ottawa). The anti-genic formula of S. typhi is 9,12 for somatic (0) antigen, Vi forthe virulence antigen, d for phase 1 flagellar (H) antigen, and(usually) negative for phase 2 antigen. Almost all strains hadthis pattern, but a few are Vi-negative, i.e., devoid of Vi antigenand untypeable with the Vi-phagetyping system for S. typhi. Asan independent test Vi-negative strains were identified as S.typhi by typical biochemical characterization (31, 32). A fewstrains, originating from Indonesia, have the flagellar antigenj in phase 1. Some strains, either d or j in phase 1, have theflagellar antigen z66, presumably in phase 2 (33).

Luria-Bertani (LB) medium (10 g tryptone/5 g yeast ex-tract/10 g NaCl/3.5 ml of 1 M NaOH) was used for cultivationof all strains; solid medium also contained 1.5% agar. Theminimal medium (MM) used is a modified Davis medium,which has been previously described (34). Tetracycline wasused at 20 ,ug/ml. Strains were maintained in 15% glycerol at-70°C, and a single colony was isolated prior to use.Enzymes and Chemicals. Endonucleases were from New

England Biolabs [AvrII (=BlnI), I-CeuI, and SpeI], and Boehr-

1 2 3 4 56 7 Frags. kbCHR 4800 -A 2400 -

G+C 1330 -B+C 1226 -

D+F+G 1008E+G 974>D+G 964G+F 872-G 828-

C+E+D 784 -B+F 768 -B 724-

C+E 648 -

12 34567

C+F 546 _C 502-1_

E+D+F 326 _ _ _E+D 282-._

E+F 190-D+F 1 80 -

E 146-D 136 -

F 44 I-

FIG. 2. (A) Partial digestion of DNA of strains of S. typhi with theendonuclease I-CeuI and separation by PFGE. Lanes: 1, strain 26T4;2, strain 26T9; 3, strain 26T12; 4, strain 26T19; 5, strain 26T38; 6, strain26T48; 7, strain 26T49. (B) The fragments observed inA are indicatedby bars, and their size and the fragments they are inferred to includeare labeled on the left. The fragments labeled 502, 724, and 828 kb areconsidered indistinguishable in size; the apparent variations are dueonly to loading differences.

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addition the following fragments result from partial digestion:D + F (180 kb), D + E (282 kb), E + D + F (326 kb), indicatingthe order EDF. Similarly, C + F (546 kb), G + E (974 kb), andB + C (1226 kb) taken together indicate the order GEDFCB.In selected cases, the identity of these multiple fragments wasconfirmed by excising the fragments from the gel, redigestingwith I-CeuI, and reelectrophoresing.The order of I-CeuI fragments, inferred from partial I-CeuI

digestion data, is shown in Fig. 3 as a linear map in whichfragments B, C, D, E, F, and G are illustrated as a contiguousblock arranged in different orders in different genome types;this linear unit is joined at both ends to fragment A to produce

a circular chromosome as in Fig. 1. Type 1 includes strains inwhich the fragment order is BCDEFG, the same as in E. coliK-12, S. enteritidis (11), S. paratyphi B (12), and 17 independentstrains of S. typhimurium (13) (the order of some of thesefragments, and their lengths, are shown at the bottom of Fig.3). The order of the strain in Fig. 2, lane 3, is shown (in reverseorder) as genome type 6 in Fig. 3. Similarly, the sevenfragments in Fig. 2, lane 4, resulting from complete digestion,are the same size as in lane 3, but the fragments resulting frompartial digestion indicate the fragment order BCEDFG (type5 in Fig. 3). The order of I-Ceul fragments in other strains wasdetermined in the same way. Digestions of strains were run two

TypeA B C D E F G

2400 724 502 136 146 44 828 240(

2 l,, : - -77773 :I4

6

7

8

1 2

13

- :---,:- :--,;-----; :,;::- - z

N~~~~~~~~1'M//.~;.,. ,.-.,.:.,,-

rmnG trnD-4-

S

...---*

I'l / N

rrnC rrnA rrnB rrnE-* r-bo -O

No. of Representativestrains strain

A

2 26T322 26T157 26T45 26T82 26T1 9

8 26T123 CC62 26T504 Ty20

2 26T490

2 CDC382-822 26T380

1 26T91 In4

2 25T354 26T40

00

2 4l7Ty2 26T562 26T32

1 SARB631 PL27566

rtinH

FIG. 3. The order of I-CeuI fragments in 127 strains of S. typhi and in other enteric bacteria. The sizes in kilobases of the fragments are shownat the top, and these sizes are to scale in the bars. The order of I-CeuI fragments B-G was determined from the representative data in Fig. 2. All127 strains were digested and electrophoresed, many several times. The order of I-CeuI fragments for strain Ty2 (genomic type 9) had been previouslydetermined by analysis with TnlO insertions (19); the same order was confirmed by partial I-CeuI digestion. The I-CeuI-A fragment (2400 kb) isinferred to join the left end to the right end of the fragments shown, forming a circle, but its orientation is not known. The orientation of I-CeuIfragments B, D, E, F, and G can be inferred from the polarity of the rrn genes. The solid dot in the I-CeuI-C fragment is the location of oriC; thisis known in E. coli K-12, S. typhimurium LT2, and S. typhi Ty2. Because I-CeuI-C is flanked by inverted rrn operons, the fragment could be inverted,so the location of oriC for most S. typhi strains is not known. The number of strains of each genome type is shown; some of the theoretical genometypes shown here were not detected. The order and size of the fragments between rrn genes were previously determined for E. coli K-12 (6, 9, 10),S. enteritidis (11), S. paratyphi B (12), and S. typhimurium LT2 (3); these are illustrated at the bottom, drawn to scale, and all have the same orderof fragments as genome type 1 of S. typhi (*). Genome type 9 (**) was previously determined for S. typhi Ty2 (19, 20).

14

15

16171819

2021

22

2324

2526

E. coliK12S. en

S. p8S. tmLT2

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or more times under a variety of pulsing conditions to assureseparation of complete and partial digestion products.The 127 strains of S. typhi fall into 21 different orders for the

7 I-CeuI fragments (see Fig. 3). In types 1-6, the small I-CeuIfragments D, E, and F are present as a contiguous block,adjacent to I-CeuI-C, arranged in all six possible orders withrespect to fragment C. Although wild-type strains carrying allsix of these genomic types were found, their frequencies varygreatly: 60 strains are of genome type 3, but only 2 are genometype 1, the type invariably found in wild-type strains of S.typhimurium (13) and in E. coli K-12. Genomic types 7-12resemble types 1-6, except that the order of fragments I-CeuI-G and -B is rearranged; the frequencies of these types arelower, and only four of the six possible types are detected.Strain Ty2, which is used as a wild-type strain and for which wedetermined a detailed chromosome map (20), is of genometype 9, a relatively rare type. Genomic types 13-16, 17-20, and21-24 represent types in which I-CeuI fragments F, D, and E,respectively, are detected on the opposite side of fragment Cfrom the other two. The frequencies of these types are low, andnot all types are detected, although these other genomic typesmight be found in a larger sample of strains. Types 25 and 26,each detected in only a single strain, are exceptional, for onlyin these strains are the large fragments I-CeuI-B and -G foundon the same side of fragment C.

DISCUSSIONWe believe that the rearrangements that we detect in S. typhiare present in wild-type strains at the time of their isolationfrom humans and that they did not simply occur duringsubsequent storage. This conclusion is based on two types ofdata: first, many of the strains in our sample of 127 indepen-dent wild-type strains were very recently isolated from patients

A. Duplication

A B C_ D-EFJ G A

~~ _5

1

.-"- -- -

.. :::................

with typhoid fever and, therefore, had very limited opportunityfor rearrangement in culture, yet these strains show the samespectrum of rearrangements as the strains we obtained fromculture collections that have been stored for many years;second, rearrangements occur infrequently in culture, forseveral independent lines of Ty2, a wild-type strain kept inculture for many years in many different laboratories, all havethe same genomic type (type 9).We feel confident that the genetic events that have rear-

ranged the chromosome must be due to recombination be-tween the rrn genes and not at other sites, because the lengthsof the I-CeuI fragments remain unaltered in almost all of thestrains shown here. Inversions or transpositions involvingother sites would drastically change the lengths of thesefragments; we have observed such changes rarely in S. typhi-murium and in S. paratyphi C, but not in S. typhi.Four classes of chromosome rearrangements might result

from recombination between the rrn genes: duplications,deletions, transpositions, and inversions (Fig. 4). Two of theclasses, duplications and deletions, were not found, althoughboth would be efficiently detected using I-CeuI digestion.Deletions could be detected through complete loss of singleI-CeuI fragments, and duplications through the detection ofdoubled intensity of the duplicated new fragments and alsothrough new combinations following partial digestion. Dele-tions of the I-CeuI-F fragment due to rrnB and rrnE rear-rangement occur at frequencies of about i0-4 in E. coli K-12,but result in lethality unless the segment is duplicated else-where (35); our failure to find deletions among wild-typestrains is not surprising. Duplications of the segments betweenrrn genes (14, 15) or between the insertion sequences IS200(36) occur commonly in culture and can be maintained usingselective systems, but we did not detect any in the wild-typestrains of S. typhi we studied.

A_ B _ C DEFEF_ G A

B. Deletion.

A- B C -D

C. Transposition

Q9F

A - B 4- C _DE

F

--->A_ B

QF

CD5 E_G _A

A-

B _,C -F_D_E -G -A4- 4-- p-4-4I-4-4--G -A

D. Inversion

A_ B -C- -D EF -G -A4-----4-@ -4-4- 44 4

rrnD rrnE

A_. G _ C_ D_E_F B -.A

FIG. 4. Postulated patterns of homologous recombination between the rrn genes (modified from ref. 14). The arrows show the rn genes andthe orientation of their transcription. The letters refer to the I-CeuI fragments between the rrn genes. The dot in fragment C is the inferred positionof oriC; DNA replication proceeds in both directions from oriC and meets at TER in the A fragment. In each case the initial order is type 1 inFig. 3, as found in S. typhimurium LT2 and E. coli K-12. (A) Duplications can occur by interchromosomal recombination. (B) Deletion can resultfrom intrachromosomal recombination. (C) Transposition results if the deleted segment (as in B) reinserts into the chromosome by homologousrecombination at a different rrn gene, forming a new arrangement (the combination shown here is type 6 in Fig. 3). (D) Inversion results froma recombination between genes that are oriented in different directions, as shown here by the dotted line between rrnD and rrnE. This rearrangementresults in types 7-12 (Fig. 3).

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However, the chromosome of almost all wild-type strains ofS. typhi has at least one change when compared with the(presumed) ancestral order of I-CeuI fragments in S. typhi-murium LT2. The rrn operons are oriented in the samedirection as DNA replication, thus, the rrnC, rrnA, rrnB, rrnE,and rrnH genes on one arm are all oriented toward the TER,and the rrnD and rrnG operons on the opposite arm areoriented in opposite direction, also toward TER (Fig. 1). Thus,segments that are transposed within one arm are expected toremain oriented in the same direction, because of the polarityof the recombination of the rrn genes (Fig. 4C), whereastranspositions to another arm are expected to be reversed inorientation (as in transposition of I-CeuI-F into rrnD ingenomic types 13 and 14) (Fig. 3). The orientation of all of theI-Ceul fragments of strain Ty2, where genes were mappedpermitting determination of orientation, were found to fitthese above predictions (20).

Transpositions could result from the reinsertion of a deletedsegment, as indicated in Fig. 4C, but could be formed by othermechanisms. Though strains with duplications are not de-tected, they could be formed, perhaps during growth in thehuman hosts, and later resolved by deletions. Their resolutionmight result in a transposition, e.g., deletions from the dupli-cation DEFEF (Fig. 4A) could produce DFEF and then DFE.

Inversions are also observed, e.g., types 7-12 (Fig. 3) mightresult from inversion of the CDEF fragments due to recom-bination between rrnD and rrnE (Fig. 4D). Inversions resultfrom recombination between rrn operons on different arms ofthe chromosome. The orientation of the fragments can beinferred for most inversions based on the orientation of the rrnoperons.We can estimate the minimum number of recombination

events required to change the presumed ancestral type (ge-nome type 1) to each of the other types. Types 2-6 showchanges in fragment DEF. All of these changes require trans-positions; for example, the order FED (genome type 2) isunlikely to be due to inversion, because of the polarity of therrn operons, so it must be due to two transpositions, perhapsfirst producing the order DFE and then FED. Type 3 (EFD)requires only one transposition, moving D between F and G.In type 4, F transposes between D and E. In type 5, Dtransposes between E and F. In type 6, F transposes betweenC and D. Genome types 7-12 have the same arrangements ofDEF as types 1-6, respectively, but in addition, have aninversion due to recombination between rrnD and rrnE (asillustrated in Fig. 4D). Genome types 13-16 all have a trans-position of fragment F into a position to the left of C; types 15and 16 also have the inversion due to rrnD-rrnE recombina-tion, and types 14 and 16 have a transposition ofD with respectto E. Genome types 17-20 are equivalent to types 13-16,except that D (rather than F) is inserted to the left of fragmentC, and genome types 21-24 are also equivalent, with E insertedto the left of fragment C. Type 25 (CBEFDG) resembles type3, the most common type, except that fragment B has trans-posed between C and E. Type 26 (ECBFDG) resembles type22, except that fragment B has transposed between C and F.Of course, there is no assurance that the rearrangementsresulting in these strains actually occurred in this order.Two of the hypotheses for conservation of gene order are as

follows. (i) The gene dosage hypothesis states that duringreplication, genes close to oriC are present in extra copies,causing increased gene expression (37); this may result inadaptation of genes to the position they occupy and, thus,during evolution selection against cells with rearrangementsthat result in inappropriate levels of gene expression. Our datasupport this hypothesis, for in spite of the many rearrange-ments, there are few that dramatically alter the distance ofgenes from oriC. For example, fragments I-CeuI-D, -E, or -Ftranspose readily; types 1-12 (Fig. 3) include 115 strains inwhich these 3 fragments shuffle positions but remain adjacent

to each other, close to oriC. They also transpose infrequentlyso that one of the three fragments is on the opposite side ofI-CeuI-C, which leaves them still about the same distance fromoriC (types 13-24, only 18 strains). However, the three frag-ments are never detected next to I-CeuI-A (at the left or rightend of the segments shown in Fig. 3), which would move themfar from oriC. We assume that such transposition occurs, butthat location far from oriC results in reduced gene expressionand failure to survive in nature during evolution. (ii) Thegenomic balance hypothesis states that the origin (oriC) andterminus (TER) of replication must be separated by 180° (19,38). Our data in this report neither confirm nor deny thishypothesis, for although the position of oriC, which is onI-CeuI-C, has changed dramatically in some strains (Fig. 3), wedo not know the location of TER; it has been shown that TERcan be displaced by inversions within the I-Ceu-A fragment,and such an inversion of 500 kb has been observed in S. typhiTy2 (19, 20), and might be present in other strains. In addition,we cannot exclude the possibility of inversions of fragment Cwhich would change the position of oriC, or of fragment A,which would change the position of TER (20, 30).

Bacteria have been divided into two groups in terms ofgenomic stability (39): in one group, which includes Salmonellaspp. andE. coli, the genomes are highly conserved. E. coli K-12,S. typhimurium LT2, and 16 other independent wild-typestrains of S. typhimurium all have an rrn skeleton of genometype 1. Approximately 50 other Salmonella strains, which arepart of the SAlmonella Reference A (SARA) set of strains (40)representing several different species are also of genome type1 (S.-L.L. and K.E.S., unpublished data). In the second group,which includes Bacillus cereus and Leptospira interrogans, ahigh level of intraspecies heterogeneity is found. We assumethat genomic rearrangements have occurred commonly duringdivergence of E. coli and S. typhimurium, but that constraintson gene order, due to genomic balance or gene dosage or someother cause, have prevented survival of strains with rearrange-ments [except for an inversion over the terminus (3)]. How-ever, these constraints appear to have been partially relaxedduring evolution of S. typhi. The order of I-CeuI fragments isgreatly rearranged, although the order of genes within thesefragments (based on data from S. typhi Ty2) is substantiallyconserved.

We thank Wendy Johnson and Rasik Khakhria (Laboratory Centrefor Disease Control, Ottawa), Tikki Pang (Centre for AdvancedStudies, University of Malaysia), and Bruce Stocker (Stanford Uni-versity) for strains received; Rasik Khakria and David Woodword(Laboratory Centre for Disease Control, Ottawa) for identification ofS. typhi strains using biochemical and antigenic analysis; and Lili Leifor technical assistance. The work was supported by grants from theNatural Sciences and Engineering Research Council of Canada andGrant R01AI34829 from the National Institute of Allergy andInfectious Diseases of the National Institutes of Health (Bethesda,MD).

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