Diversity of Genome Structure in Salmonella enterica Serovar Typhi Populations

JOURNAL OF BACTERIOLOGY, Apr. 2005, p. 2638–2650 Vol. 187, No. 80021-9193/05/$08.00�0 doi:10.1128/JB.187.8.2638–2650.2005Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Diversity of Genome Structure in Salmonella enterica SerovarTyphi Populations†

Sushma Kothapalli,1 Satheesh Nair,1 Suneetha Alokam,1 Tikki Pang,2 Rasik Khakhria,3‡David Woodward,3 Wendy Johnson,4 Bruce A. D. Stocker,5§

Kenneth E. Sanderson,1* and Shu-Lin Liu1,6,7

Department of Biological Sciences1 and Department of Microbiology and Infectious Diseases,6 University of Calgary,Calgary, and Bacteriology and Enteric Diseases Program, National Microbiology Laboratory, Health Canada,3

and Cangene Corporation,4 Winnipeg, Manitoba, Canada; Research Policy and Cooperation, WorldHealth Organization, Geneva, Switzerland2; Department of Medical Microbiology,

Stanford University, Stanford, California5; and Department of MicrobiologyPeking University School of Basic Medical Sciences, Beijing, China7

Received 24 September 2004/Accepted 6 January 2005

The genomes of most strains of Salmonella and Escherichia coli are highly conserved. In contrast, all 136wild-type strains of Salmonella enterica serovar Typhi analyzed by partial digestion with I-CeuI (an endonu-clease which cuts within the rrn operons) and pulsed-field gel electrophoresis and by PCR have rearrangementsdue to homologous recombination between the rrn operons leading to inversions and translocations. Recom-bination between rrn operons in culture is known to be equally frequent in S. enterica serovar Typhi and S.enterica serovar Typhimurium; thus, the recombinants in S. enterica serovar Typhi, but not those in S. entericaserovar Typhimurium, are able to survive in nature. However, even in S. enterica serovar Typhi the need forgenome balance and the need for gene dosage impose limits on rearrangements. Of 100 strains of genome types1 to 6, 72 were only 25.5 kb off genome balance (the relative lengths of the replichores during bidirectionalreplication from oriC to the termination of replication [Ter]), while 28 strains were less balanced (41 kb offbalance), indicating that the survival of the best-balanced strains was greater. In addition, the need forappropriate gene dosage apparently selected against rearrangements which moved genes from their accus-tomed distance from oriC. Although rearrangements involving the seven rrn operons are very common in S.enterica serovar Typhi, other duplicated regions, such as the 25 IS200 elements, are very rarely involved inrearrangements. Large deletions and insertions in the genome are uncommon, except for deletions of Salmo-nella pathogenicity island 7 (usually 134 kb) from fragment I-CeuI-G and 40-kb insertions, possibly a pro-phage, in fragment I-CeuI-E. The phage types were determined, and the origins of the phage types appearedto be independent of the origins of the genome types.

Salmonella enterica serovar Typhi is host restricted, for itgrows only in humans, where it causes typhoid enteric fever(13, 51). The annual global incidence of typhoid fever is esti-mated to be 21.6 million cases, with more than 220,000 deaths(10). The emergence of antibiotic-resistant strains (8) and theincreased incidence of typhoid fever in human immunodefi-ciency virus type 1-infected persons are causes for concern.The genus Salmonella is separated into two species and morethan 2,500 serovars (52) on the basis of the somatic and flagel-lar antigens. Many of the serovars, such as S. enterica serovarTyphimurium, are host generalists, growing in many differentanimal species and humans and causing gastroenteritis.

S. enterica serovar Typhi is more homogeneous than mostserovars of Salmonella. Using multilocus enzyme electrophore-sis, Reeves et al. (54) and Selander et al. (58) showed that S.enterica serovar Typhi strains constitute only one or two clonesthat are widely separated from the other serovars in subspeciesI. Membrane protein profiles (15, 17) and plasmids (42) showhomogeneity. Multilocus sequence typing of housekeepinggenes has suggested that S. enterica serovar Typhi evolved onlyabout 50,000 years ago from other Salmonella serovars (30).

The orders of orthologous genes in Escherichia coli K-12 andS. enterica serovar Typhimurium LT2 are almost identical,although the genera diverged about 100 to 160 million yearsago (38, 48, 59, 62). Within the genus Salmonella, the geneorder of S. enterica serovar Paratyphi B (33) and the geneorder of S. enterica serovar Enteritidis (36) are very similar tothe gene order of S. enterica serovar Typhimurium LT2, andchromosomes are also conserved in 17 independent strains ofS. enterica serovar Typhimurium (39). During growth in labo-ratory culture, duplications of segments of the chromosomeoccur at high frequencies (10�2 to 10�5) (4, 23), and someinversions, especially those with endpoints in the rrn operons,are common (22). Such remarkable conservation of the chro-mosome during evolution, in spite of the high frequency of

* Corresponding author. Mailing address for Kenneth E. Sanderson:Department of Biological Sciences, University of Calgary, CalgaryT2N 1N4, Canada. Phone: (403) 220-6792. Fax: (403) 289-9311.E-mail: [email protected]. Mailing address for Shu-Lin Liu: De-partment of Microbiology and Infectious Diseases, University of Cal-gary, Calgary T2N 1N4, Canada. Phone: (403) 220-3799. Fax: (403)270-2772. E-mail: [email protected].

† Supplemental material for this article may be found at http://jb.asm.org/.

‡ Present address: 32 Amberwood Crescent, Nepean, Ontario,Canada.

§ Deceased.

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rearrangements in culture, may have resulted from strong se-lective pressures that selectively removed rearranged genomes.

Pulsed-field gel electrophoresis (PFGE) permits rapid con-struction of genomic maps (16); partial digestion with the en-donuclease I-CeuI shows the number and locations of the rrlgenes for 23S rRNA and the order of adjacent fragments (34)(the rrn skeleton). I-CeuI, which is encoded by a class I mobileintron in the rrl gene for the large-subunit rRNA (23S-rRNA)in the chloroplast DNA of Chlamydomonas eugamatos (44),digests a 19-bp sequence in all seven rrl genes of enteric bac-teria (43). The rrn skeleton is highly conserved in enteric bac-teria, so related strains usually yield identical fingerprints (39).

Surprisingly, in view of the homogeneity in many properties,independent wild-type strains of S. enterica serovar Typhi showsignificant genomic rearrangements. The I-CeuI fragments ofS. enterica serovar Typhi strains were shown by PFGE to be inmany different orders, called genome types (40), that are me-diated through recombination between the seven rrn genes thatcode for rRNA. Partial I-CeuI digestion can determine theorder but not the orientation of the I-CeuI fragments.

Vi phage typing has high discriminatory power for subdivi-sion of strains of S. enterica serovar Typhi, and the method ofCraigie and Felix (9) has allowed over 100 different phagetypes to be recognized (49). Vi phage absorbs to the Vi (viru-lence) exopolysaccharide and adapts itself to the last strain inwhich it was propagated. In this study we determined thephage types of strains to determine if they are independent ofor correlated with genome types.

In this study we used PFGE with I-CeuI to determine thegenome types of a set of strains of S. enterica serovar Typhiwhich had been assembled from a variety of sources; we thendetermined the orientation of the I-CeuI fragments usingPCR. In addition, we determined the Vi phage types and theflagellar antigens of the strains. The sizes of the seven frag-ments (the I-CeuI fingerprint) are indistinguishable in moststrains, so they are characteristic of the species, but the sizes ofa few fragments are increased or decreased due to insertions ordeletions. We show here that the phage type is largely inde-pendent of the genome type. The need for chromosome bal-ance between the two replichores and for maintaining appro-priate gene dosage appears to restrict the range of genomicrearrangements.

MATERIALS AND METHODS

Bacterial strains and cultivation conditions. The S. enterica serovar Typhistrains and their sources are shown in Table 1. All strains were maintained in15% glycerol at �70°C in the collection of the Salmonella Genetic Stock Center(www.ucalgary.ca/�kesander), and single-colony isolates were isolated prior touse. The strains were grown at 37°C in Luria-Bertani medium; solid mediacontained 1.5% agar.

Enzymes and chemicals. Endonucleases were obtained from New EnglandBiolabs (AvrII [� BlnI], I-CeuI, and SpeI) and Boehringer-Mannheim (XbaI).Taq polymerase and deoxynucleoside triphosphates were obtained from Amer-sham. Other chemicals, including Luria-Bertani medium and agarose, were ob-tained from Sigma Chemicals.

Endonuclease digestion and PFGE methods. Preparation of high-molecular-weight genomic DNA, endonuclease cleavage of DNA in agarose blocks, sepa-ration of the DNA fragments by PFGE, and double-digestion techniques wereperformed as described previously (32, 35, 37). For digestion by I-CeuI, includingpartial digestion, we used the methods described previously (39).

Primers. The primers were designed based upon the sequence of DNA flank-ing each of the seven rrn operons in S. enterica serovar Typhi CT18 (50) (GenBank accession no. NC_003198) by using the Primer3 program (http:

//frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi/), and they were synthesizedby the University Core DNA Services (Health Sciences Centre, University ofCalgary). The sequences of the primers used in this study and their locationsrelative to rrn operons are shown in Table S1 in the supplemental material. Thelocations of the primers in the genomes of S. enterica serovar Typhimurium LT2and S. enterica serovar Typhi CT18 are shown in Fig. 1. These primers were usedin different combinations to amplify the rrn operons.

PCR amplification and agarose gel electrophoresis. Chromosomal DNA usedin PCR was isolated with a Wizard genomic DNA purification kit (Promega)used in accordance with the manufacturer’s instructions.

Each PCR was carried out by using a HotStart storage and reaction tube(Gordon Technologies Inc.) in an Eppendorf gradient thermal cycler. In each50-�l (total volume) PCR mixture, 20 �l was the lower mixture and 30 �l was theupper mixture. The lower mixture contained 250 ng of template DNA, 1 �l ofeach primer (0.4 �M), and 2 �l of deoxynucleoside triphosphates (0.4 mM) (theconcentrations of the components of the lower mixture were calculated based onthe 50-�l reaction mixture), and the final volume was adjusted with double-distilled water. Denaturation was done at 90°C for 30 s to melt the wax pellet.After the mixtures were cooled to room temperature, the reactions were initiatedby addition of 5 �l of 1� PCR buffer, 3 �l of MgCl2 (1.5 mM), and 2.5 U of TaqDNA polymerase (0.25 �l), and 21.75 �l of double-distilled water was added(upper mixture) (the concentrations of the components of the upper mixturewere calculated based on the 50-�l reaction mixture). PCR amplification wasperformed with 30 cycles of denaturation at 96°C for 1 min, annealing at 57°C for1 min, and extension at 72°C for 10 min, followed by a final extension at 72°C for10 min.

The PCR product was electrophoresed at 65 V on a 1% agarose gel in 0.5�Tris-borate-EDTA buffer (45 mM Tris, 45 mM boric acid, 10 mM EDTA [pH 8])with 1 �g of ethidium bromide per ml. Following electrophoresis the gel wasphotographed under UV light.

Computer methods. The individual fragment sizes were estimated by using theS. enterica serovar Typhi CT18 genome sequence (50) (GenBank accession no.NC_003198) and the BLAST Program produced by National Center for Bio-technology Information, Bethesda, Md. (www.ncbi.nlm.nih.gov/BLAST).

Phage typing and serotyping. Bacteriophage typing was performed at theNational Microbiology Laboratory (formerly the Laboratory Centre for DiseaseControl), Health Canada, as reported previously by Khakhria et al. (29), by usingthe methods and scheme described by Anderson and Williams (3) and the Viphage of S. enterica serovar Typhi. Serotyping was performed at the NationalMicrobiology Laboratory, Health Canada, by the methods described in a reporton the Kauffmann-White scheme (53).

RESULTS

Partial digestion by I-CeuI in S. enterica serovar Typhi.Partial digestion yielded the seven bands expected from com-plete digestion plus other bands resulting from a failure tocleave between adjacent fragments; representative data areshown in Fig. 2. All strains produced seven fragments (frag-ments A to G), which ranged from 44 to about 2,400 kb long,and usually the lengths were indistinguishable in differentstrains (with the exception of fragment G in strain SARB64[Fig. 2, lane 2], which was about 130 kb smaller than thenormal fragment G). However, the fragments resulting frompartial digestion were different in different strains, indicatingthat the fragments in the chromosome are in different orders.For example, the following partial digestion bands were ob-served in Fig. 2, lanes 1 and 6: DF, EF, and DEF. These dataindicate that the order is EFD. The order of these three frag-ments is different in other strains. For example, the partialdigestion bands DF, DE, and DEF were observed in Fig. 2,lane 4; these data indicate that the order is fragment FDE. Theorder of many of the other fragments could be determinedfrom the same gel. Some of the expected partial digestionfragments could not be recognized in other lanes because thegel was overloaded; in order to determine the order of all

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TABLE 1. Phenotypic and genotypic characteristics of S. enterica serovar Typhi strains used in this study

Straina SGSC no.b Source(reference)c

Locality ofisolation

Year ofisolation

Flagellarantigend Phage

typeeFragment order

in genomef PCR orderg Genometypeh

Altered 1-Ceu-Ifragment(s)i

d J

26.001 3124 NML Manitoba 1994 � � E1 BCFEDG A�C� 226.003 3126 NML Alberta 1993 � � UT(Vi-ve) BCDEFG A�C� 1 G, �130 (SP17�)26.004 3127 NML British Columbia

(India)1993 � � I�IV BCEFDG A�C� 3

26.005 3128 NML British Columbia(India)

1994 � � E1 BCFEDG A�C� 2

26.006 3129 NML British Columbia 1994 � � UT(Vi-ve) GFCEDB A�C� 1626.007 3130 NML British Columbia 1994 � � A BCEFDG A�C� 326.008 3131 NML British Columbia 1994 � � D2 BCDFEG A�C� 426.009 3132 NML Manitoba 1994 � � B1 GFCEDB A�C� 1626.010 3133 NML Manitoba 1994 � � E1 BCFEDG A�C� 226.011 3134 NML Alberta (Kenya) 1994 � � B2 BCEFDG A�C� 326.012 3135 NML Manitoba (India) 1994 � � O BCFDEG A�C� 626.015 3138 NML British Columbia 1994 � � J1 BCEFDG A�C� 326.016 3139 NML British Columbia 1994 � � DVS BCFDEG A�C� 626.017 3140 NML British Columbia 1994 � � B1 BCDFEG A�C� 426.018 3141 NML British Columbia 1994 � � B2 BCEFDG A�C� 326.019 3142 NML Alberta 1994 � � A BCEDFG A�C� 526.020 3143 NML British Columbia 1994 � � Atypical BCEDFG A�C� 526.021 3144 NML British Columbia 1994 � � O BCFDEG A�C� 626.022 3145 NML Alberta 1994 � � I�IV BCEFDG A�C� 326.023 3146 NML Alberta 1994 � � UT(Vi-ve) BCEFDG A�C� 326.024 3147 NML Ontario 1994 � � E1 BCFEDG A�C� 226.027 3150 NML Quebec 1994 � � 46 BCFEDG A�C� 226.028 3151 NML Quebec 1994 � � DVS BCFDEG A�C� 6 B, �8826.029 3152 NML Quebec 1994 � � UT(Vi-ve) BCEFDG A�C� 326.03 3153 NML Quebec 1994 � � I�IV BCEFDG A�C� 326.031 3154 NML Quebec 1994 � � Atypical GDCEFB A�C� 1926.032 3155 NML Quebec 1994 � � I�IV GECFDB A�C� 2426.033 3156 NML Quebec 1994 � � Atypical BCEFDG A�C� 3 B, �8026.034 3157 NML Quebec 1994 � � I�IV BCEFDG A�C� 326.035 3158 NML Quebec 1994 � � I�IV BCEFDG A�C� 326.037 3160 NML British Columbia 1994 � � I�IV BCFEDG A�C� 226.038 3161 NML British Columbia 1994 � � E1 BFCEDG A�C� 1426.04 3163 NML British Columbia 1994 � � M3 GDCEFB A�C� 19 G, �5026.041 3164 NML Alberta (El

Salvador)1994 � � B3 BCEFDG A�C� 3 B, �80

26.042 3165 NML Alberta(California)

1994 � � B2 BCEFDG A�C� 3 E, �40

26.043 3166 NML Alberta 1994 � � DVS BCEFDG A�C� 3 B, �8026.044 3167 NML British Columbia 1994 � � F4 BCFEDG A�C� 2 B, �9026.045 3168 NML British Columbia 1994 � � D8 BCEFDG A�C� 326.047 3170 NML British Columbia 1994 � � A variant BCEFDG A�C� 326.048 3171 NML British Columbia 1994 � � B1 BCFEDG A�C� 226.049 3172 NML British Columbia 1994 � � B1 GCEDFB A�C� 1126.05 3173 NML Alberta 1994 � � I�IV GCFEDG A�C� 2 B, �8026.051 3174 NML British Columbia 1994 � � DVS BCDEFG A�C� 126.054 3177 NML Quebec 1994 � � E1 BCFEDG A�C� 2 B, �1526.055 3178 NML Quebec 1994 � � Atypical BCFEDG A�C� 226.056 3179 NML Quebec 1994 � � F1 GECDFB A�C� 2325.035 2667 NML Manitoba 1994 � � F1 BDCFEG A�C� 18 G, �8025.036 2668 NML Alberta 1993 � � E1 BCEFDG A�C� 325.037 2669 NML British Columbia 1993 � � D5 BCEFDG A�C� 325.039 2671 NML British Columbia 1993 � � O BCFDEG A�C� 225.04 2672 NML British Columbia

(Guatamala)1993 � � E1 BCDFEG A�C� 4

25.041 2673 NML Alberta 1993 � � B2 BCEFDG A�C� 3 E, �40; B, �2025.042 2674 NML Ontario 1993 � � E1 BCFEDG A�C� 2ST60 2770 Pang Malaysia 1986 � � C4 BCFEDG A�C� 2 G, �20ST24A 2771 Pang Malaysia 1986 � � DVS BCEFDG A�C� 3ST143 2773 Pang Malaysia 1994 � � D2 BCDFEG A�C� 4ST145 2774 Pang Malaysia 1994 � � I�IV BCEFDG A�C� 3 E, �30ST308 2775 Pang Malaysia 1987 � � E1 BCEFDG A�C� 3ST1002 2776 Pang Malaysia 1987 � � E1 GCEFDB A�C� 9ST168 2777 Pang Malaysia 1987 � � UT(Vi-ve) BCEFDG A�C� 3 E, �40; G, �70ST309 2779 Pang Malaysia 1987 � � E1 BCEFDG A�C� 3 B, �15ST1106 2780 Pang Malaysia 1987 D1 BCDFEG A�C� 4 E, �40ST495 2781 Pang Malaysia 1987 � � B1 BCEFDG A�C� 3In14 2782 Pang Indonesia 1994 � � Atypical BCEFDG A�C� 3In15 2783 Pang Indonesia 1994 � � D2 BCEFDG A�C� 33123 3184 Pang Chile 1983 rough BCEFDG A�C�3125 3185 Pang Chile 1983 � � 46 BCEFDG A�C� 3T189 3187 Pang Thailand 1990 � � N GCEDFB A�C� 11T202 3189 Pang Thailand 1990 � � UT(Vi-ve) BCEFDG A�C� 3 G, �130 (SP17�)

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TABLE 1—Continued

Straina SGSC no.b Source(reference)c

Locality ofisolation

Year ofisolation

Flagellarantigend

Phage typee Fragment orderin genomef PCR orderg Genome

typehAltered 1-Ceu-I

fragment(s)i

d J

T104 3188 Pang Thailand 1990 � � UT(Vi-ve) BCDEFG A�C� 1 G, �130 (SP17�)In4 3190 Pang Indonesia 1992 � � 53 BDCEFG A�C� 17 B, �80In20 3191 Pang Indonesia 1992 � � A GCEFDB A�C� 9 E, �40In24 3192 Pang Indonesia 1992 � � C3 BCEFDG A�C� 3PNG30 3193 Pang Papua New

Guinea1994 � � D2 BCFEDG A�C� 2

PNG31 3194 Pang Papua NewGuinea

1994 � � D2 BCFEDG A�C� 2

PNG32 3195 Pang Papua NewGuinea

1994 � � D2 BCEFDG A�C� 2

ST1 2728 Pang � � I�IV BDCFEG A�C� 18 G, �130 (SP17�)CC6 3198 Ho Thailand � � A GCDEFB A�C� 7CC7 3199 Ho Thailand � � A GCDEFB A�C� 7382-82 2664 CDC Marshall Islands � � M1 BFCDEG A�C� 139032-85 2663 CDC Taiwan � � UT(Vi-ve) BFDCEG A�C� 23 G, �151707-81 2661 CDC Liberia � � UT(Vi-ve) BCEFDG A�C� 31196-74 2662 CDC Mexico � � A BCFDEG A�C� 6 B, �803434-73 2658 CDC Peru � � G1 BECFDG A�C� 223137-73 2660 CDC India � � K1 BCFDEG A�C� 63815-73 2659 CDC Unknown � � T BCEFDG A�C� 3SARB63 2520 Selander Dakar, Senegal 1988 � � A CBEFDG A�C� 25SARB64 2521 Selander Dakar, Senegal 1988 � � UT(Vi-ve) BDCEFG A�C� 19 G, �130 (SP17�)SA4825 2655 ProvLab Calgary, Canada 1993 � � B2 BCEFDG A�C� 3 E, �40PL27566 2990 ProvLab � � M1 ECBFDG A�C� 26 B, �20PL45838 2991 ProvLab 1994 � � DVS BCEFDG A�C� 3 B, �80PL73203 2992 ProvLab 1995 � � A BCFEDG A�C� 257639-199 2682 1990 � � O BCFDEG A�C� 6Lysin�SA4 2683 � � I�IV BCFDEG A�C� 6ISP1820

(ATCC55047)

2406 Hone (25) Chile 1983 � � 46 BFECDG A�C� 19

Ty2 2408 Hone (25) USSR 1918 � � E1 GCEFDB A�C� 9200Ty 2272 Stocker � � E1 GCEFDB A�C� 9403Ty 2273 Stocker BCEFDG A�C� 3 G, �130541Ty 2758 Stocker GCDEFB A�C� 7R1637 2692 ProvLab � � E2 BFCEDG A�C� 14R1962 2693 ProvLab UT(Vi-ve) BCDEFG A�C� 1 G, �130 (SP17�)R1167 2694 ProvLab � � A GDCEFB A�C� 19R136 2695 ProvLab � � D9 BCEFDG A�C� 3 E, �40R2101 2696 ProvLab � � M1 BCEFDG A�C� 3R70 2697 ProvLab � � 46 BCFEDG A�C� 2R196 2698 ProvLab � � E1 BCFEDG A�C� 2414Ty 3212 Stocker Australia 1981 � � I-IV BCEFDG A�C� 3 E, �40415Ty 3213 Stocker The Netherlands 1982 � � UT(Vi-ve) BCEFDG A�C� 3 E, �40; G, �130

(SP17�)416Ty 3214 Stocker Japan 1982 � � UT(Vi-ve) BCEFDG A�C� 3 E, �40417Ty 3215 Stocker New Caledonia 1982 � � HIV BECFDG A�C� 22 E, �40; G, �130418Ty 3216 Stocker The Netherlands 1988 � � I�IV BCEFDG A�C� 3 E, �40419Ty 3217 Stocker The Netherlands 1988 � � I�IV BCEFDG A�C� 3 E, �40420Ty 3218 Stocker Japan 1982 � � UT(Vi-ve) BCEFDG A�C� 3 E, �40421Ty 3219 Stocker France 1984 � � UT(Vi-ve) BCEFDG A�C� 3 E, �40; G, �130

(SP17�)422Ty 3220 Stocker The Netherlands 1988 � � I�IV BCEFDG A�C� 3 E, �40423Ty 3221 Stocker Australia 1981 � � I�IV BCEFDG A�C� 3 E, �40424Ty 3222 Stocker The Netherlands 1988 � � I�IV BCEFDG A�C� 3 E, �40425Ty 3223 Stocker � � I�IV BCEFDG A�C� 3 E, �40444Ty 3224 Stocker � � I�IV BCEFDG A�C� 3 E, �40445Ty 3225 Stocker BCEFDG A�C� 3 E, �40446Ty 3226 Stocker � � I�IV BCEFDG A�C� 3 E, �40447Ty 3227 Stocker � � I�IV BCEFDG A�C� 3 E, �40701Ty 3485 Stocker CEFBDG A�C� 27 E, �40702Ty 3486 Stocker BCEFDG A�C� 3 E, �40TYT1668 3487 Mora Chile � � M1 BECDFG A�C� 21TYT1669 3488 Mora Chile � � UT(Vi-ve) BCFDEG A�C� 6 G, �170 (part of

SP17 deleted)TYT1670 3489 Mora Chile � � 46 BCEFDG A�C� 3TYT1671 3490 Mora Chile BCEFDG A�C� 3TYT1672 3491 Mora Chile � � E1 GECDFB A�C� 23TYT1673 3492 Mora Chile � � F8 BCEFDG A�C� 3TYT1674 3493 Mora Chile � � E1 GECDFB A�C� 23TYT1675 3494 Mora Chile � � E1 GECDFB A�C� 23TYT1676 3495 Mora Chile � � E1 GECDFB A�C� 23TYT1677 3496 Mora Chile � � F8 BCEDFG A�C� 5 G, �80ST318 2778 Pang Malaysia � � Atypical BCEFDG A�C� 3CT18 4072 Sanger Centre

(50)Vietnam A�C� 9

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fragments, several gels with different loading and electrophore-sis conditions were run if necessary.

A set of 136 strains of S. enterica serovar Typhi which wereassembled from a variety of sources (Table 1) was analyzed bypartial digestion with I-CeuI, as shown in Fig. 2. Many differentarrangements of I-CeuI fragments were detected; these ar-rangements apparently resulted from inversions and translo-cations following recombination between rrn operons, as illus-trated in Fig. 3. Figure 4 shows I-CeuI fragments A, B, C, D,E, F, and G as a contiguous block arranged in different orders;27 different genome types are shown, some of which were notdetected in the 136 strains. This linear unit is joined at bothends through fragment A to produce a circular chromosome,as shown in Fig. 1.

Genome types 1 to 6 had all possible rearrangements of thethree small fragments, fragments D, E, and F; all of theserearrangements were detected, although some were muchmore frequent than others. Genome type 1 (I-CeuI-ABCDEFG) is most common in the enteric bacteria; e.g., it has beenobserved for S. enterica serovar Typhimurium LT2 (39), for 17wild-type strains of S. enterica serovar Typhimurium (41), for S.enterica serovar Enteritidis (36), and for S. enterica serovarParatyphi B (33), as well as for the following strains of E. coliwhose complete sequences have been deposited in the Gen-Bank database: E. coli K-12, E. coli CFT073, and E. coliO157:H7 (strains EDL933 and Sakai). However, only 4 of 136strains of S. enterica serovar Typhi are genome type 1. Genometype 3 (I-CeuI-BCEFDG) is by far the most common genometype, represented by 59 strains. Translocation of the fragmentsto new locations could result from deletion of a fragment dueto homologous recombination between rrn operons, followedby insertion of the fragment in a different rrn operon (Fig. 3).Genome types 7 to 12 involve the same arrangements of the D,E, and F fragments, but fragments B and G are inverted,presumably due to a crossover between rrnD and rrnE; thesetypes are less common than genome types 1 to 6. Strain Ty2,which is widely used as a wild-type strain, is a genome type 9organism; the detailed genomic cleavage map for the enzymesXbaI, BlnI, SpeI, and I-CeuI for Ty2 (38), the partial I-CeuIdigestion data (this study), and the complete nucleotide se-quence (12) all confirm the same order. Genome types 13 to 16are types in which I-CeuI-F has been translocated to a position

to the left of I-CeuI-C; genome types 17 to 20 and 21 to 24represent equivalent translocations of I-CeuI-D, and I-CeuI-E,respectively. Genome types 13 to 24 are uncommon, and someof these types were not detected in the sample studied, al-though they might be found in a larger sample. All combina-tions in which I-CeuI-B and -G are adjacent to fragment A arerepresented in genome types 1 to 24; genome types 25 to 27 arethree of the rarely encountered types in which a differentfragment is adjacent to fragment A.

PCR to detect chromosomal rearrangements in independentwild-type strains of S. enterica serovar Typhi. Partial digestionwith I-CeuI determines the order of fragments, but not theirorientation. The orientation of rrn operons limits the types ofrearrangements which can be formed. The chromosome iscomposed of two replichores, and replication begins at oriCand proceeds bidirectionally to the termination of replication(Ter). For example, in S. enterica serovar Typhimurium LT2(Fig. 1A) replichore 1 contains rrnCABEH and replichore 2contains rrnDG, and all of these genes are oriented so that theyare transcribed from oriC toward Ter. Homologous recombi-nation between rrn operons to produce inversions or translo-cations can occur only between rrn operons in the same orien-tation; thus, the orientation of fragments which have twosimilarly oriented rrn operons at their ends, such as I-CeuI-B,-D, -E, -F, and -G, can be predicted. However, I-CeuI-C(which contains oriC) has two rrn operons at its ends tran-scribed away from oriC, and I-CeuI A (which contains Ter) hasthe two rrn operons at its ends transcribed toward Ter. Thus,both fragments can be inverted by recombination, which canoccur between rrn operons at their ends (Fig. 1 and 3); there-fore, the orientation of these fragments cannot be predicted byPFGE methods. Recombination between other rrn operons indifferent replichores results in inversions (Fig. 3).

Therefore, we used PCR analysis (as first described by Helmand Maloy [20]) to confirm the order of the seven I-CeuIfragments analyzed by PFGE and to determine their orienta-tions. All 14 primers were designed from the S. enterica serovarTyphi CT18 sequence (Fig. 1B; see Table S1 in the supple-mental material) and are located inside the genes which areadjacent to each of the seven rrn operons on either side. Theseprimers were used in different pairwise combinations, based onthe order of I-CeuI fragments predicted from PFGE analysis

TABLE 1—Continueda Designation used in the laboratory which provided the strain.b Strain number used at the Salmonella Genetic Stock Centre.c NML, National Microbiology Laboratory, Winnipeg, Canada (formerly Laboratory Centre for Disease Control) (R. Khakhria and David Woodward); Pang, T.

Pang, Research Policy and Cooperation, World Health Organization, Geneva, Switzerland; Ho, M. Ho, Department of Microbiology and Infectious Diseases,University of Calgary; CDC, Centers for Disease Control (J. J. Farmer) (54); Selander, Robert K. Selander, Pennsylvania State University (part of SARB set,Salmonella Reference B) (6); ProvLab, Southern Alberta Provincial Lab (C. Anand); Hone, D. Hone, Center for Vaccine Development, Baltimore, Md. (25) (H238.2is ISP1820 with aroD1013, and H251.1 is Ty2 with aroC101); Stocker, B. A. D. Stocker, Stanford University; Mora, G. Mora, University of Chile, Santiago, Chile.

d Flagellar antigens d and j are two alternative states of the phase 1 antigen.e Phage typing with Vi typing phage II of Craigie and Felix (9) was done at the Laboratory Centre for Disease Control in Ottawa, Canada.f Fragment order was determined by partial digestion of DNA with endonuclease I-CeuI and separation of the fragments by PFGE; the data are the order of I-CeuI

fragments.g Order and orientation of the A and C fragments as determined by PCR. A plus sign indicates that the fragment is in the normal (uninverted) orientation, and a

minus sign indicates that the fragment is in the inverted orientation.h Specific order of I-CeuI fragments, as shown in Fig. 4.i Altered I-CeuI fragments and their sizes (in kilobases) are indicated. All of the strains of S. enterica serovar Typhi yielded seven fragments following complete

digestion with I-CeuI, and in most strains the sizes of these fragments were indistinguishable from the sizes observed in strain Ty2 (12) and CT18 (50). In certain strainsthe sizes of the fragments differed by the amounts indicated; larger sizes are indicated by a plus sign, and smaller sizes are indicated by a minus sign. Deletion of theI-Ceul-G fragment resulted in loss of all or part of Salmonella pathogenicity island 7 (SPI7), a 134-kb island which has the viaB operon for Vi exopolysaccharide (47).The sizes of all of the fragments not listed are not distinguishable from the sizes of the normal fragments.


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(Fig. 2); appropriate pairwise combinations resulted in ca. 6-kbamplicons containing the rrn operons. Since the orientations offragments A and C were unknown, two primer combinationsmight work for these two fragments. PFGE indicated that theorder of I-CeuI fragments for S. enterica serovar Typhi 26.047was ABCEFDG (genome type 3) (Table 1). Template DNAfrom this strain gave successful amplification with the followingprimer pairs, indicating that specific I-CeuI fragments are ad-jacent: primers 3 and 4 (AB), primers 15 and 12 (BC), primers14 and 13 (CE), primers 6 and 5 (EF), primers 10 and 9 (FD),primers 16 and 11 (DG), and primers 8 and 7 (GA) (Fig. 5A).Primer pairs 7-4 and 3-8, as well as primer pairs 15-14 and12-13, would have produced an amplicon if the A and C frag-ments, respectively, were inverted; all these combinationsfailed. This indicates that both fragment A and fragment C arein the orientation present in most strains of Salmonella and E.coli. This was illustrated by the locations of the pro and hisgenes in I-CeuI-A and the location of oriC in fragment C at theclockwise end; we called these orientations A�C� (Fig. 5A).

When the same primer pairs were used with genomic DNAof S. enterica serovar Typhi 425Ty as the template (genometype 3) (Table 1), seven PCR amplicons were again produced(Fig. 5B). Primer pairs for amplification of the rrn operonsbetween the following fragments produced the same patternthat was observed with S. enterica serovar Typhi 26.047 (Fig.5A): B and C, C and E, E and F, F and D, and D and G.However, primer pairs 7-4 and 3-8 yielded PCR amplicons,whereas primer pairs 3-4 and 7-8 did not, indicating that the Afragment was inverted. The lack of amplicons for primer pairs15-14 and 12-13 indicated that fragment C was in the normalorientation (uninverted); this structure is represented byA�C� (Fig. 5B).

The order and orientation of the seven rrn operons weredetermined in the same way by using template DNA from all136 wild-type strains of S. enterica serovar Typhi previouslytested by PFGE. In all cases the template DNA yielded sevenPCR amplicons, and the order agreed with the order of frag-ments determined by PFGE and with the orientation of frag-ments B, D, E, F, and G (inferred from the polarity of the rrnoperons). The genome type and orientation of fragments Aand C are indicated for each strain in Table 1 and are sum-marized in Fig. 4, which shows the number of strains for each

FIG. 1. Location and order of the seven I-CeuI fragments on thechromosome (the rrn skeleton). oriC is the site of initiation of bidirec-tional replication; Ter is the termination site. The numbers with arrowsrepresent the different primer combinations used to amplify the sevenrrn operons (indicated by arrows outside the circles). The numbersoutside the circles indicate the sizes of the I-CeuI fragments (in kilo-bases) based on the previously published sequences (45, 50). (A) S.enterica serovar Typhimurium LT2. (B) S. enterica serovar Typhi CT18.

FIG. 2. Partial digestion of DNA of strains of S. enterica serovarTyphi with endonuclease I-CeuI, separation by PFGE, and stainingwith ethidium bromide. The gel is shown on the left. The fragments areshown on the right, and the inferred composition and sizes (in kilo-bases) are indicated. Lane 1, strain SARB63 (fragment order, CBE-FDG; genome type 25); lane 2, SARB64 (fragment order, BDCEFG;genome type 19); lane 3, 27566 (fragment order, ECBFDG; genometype 26); lane 4, SA4864 (fragment order, BCFDEG; genome type 6);lane 5, SA4665 (fragment order, GFCEDB; genome type 16); lane 6,Ty2 (fragment order, GCEFDB; genome type 9).


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genome type and for each of the four orientations of fragmentsA and C. Fragment I-CeuI-A was frequently inverted, andI-CeuI-C was rarely inverted. Thus, strains of S. enterica sero-var Typhi showed many different rearrangements of the genesegments between rrn operons (the I-CeuI fragments).

Normally, we did not use all possible pairwise combinationsof primers for testing each of the strains; we used only theprimer pairs predicted by PFGE data to be effective. However,in a few cases we tested all possible combinations, and onlythose primer pairs predicted by the PFGE results were effec-tive (data not shown).

Changes in I-CeuI fragment lengths. PFGE data indicatedthat the lengths of I-CeuI fragments are highly conserved, forall seven sizes were indistinguishable from the sizes observed instrain Ty2 (38) for 86 of the 136 strains, as shown in Table 1.PFGE did not detect changes in fragment I-CeuI-C (517 kb),I-CeuI-D (134 kb), or I-CeuI-F (42 kb) in any of the strains(Table 2), although changes of a few kilobases should have

been detectable by PFGE. Our methods could not detectchanges in the large I-CeuI-A fragment (2,422 kb). The 57strains with detectable changes included many types (Table 2).The following numbers of insertions were detected: 13 strainshad 20- to 90-kb insertions in I-CeuI-B; 25 strains had 20- to40-kb insertions in I-CeuI-E; and six strains had 15- to 80-kbinsertions in I-CeuI-G. The only strains with a fragment withdeletions were 12 strains with deletions in I-CeuI-G. Thesedeletions were shown previously to result from a loss of all orpart of Salmonella pathogenicity island 7, a 134-kb island whichhas the viaB operon for Vi exopolysaccharide, due to recom-bination between genes for tRNAPhe. All these strains areuntypeable by the phages used for Vi typing and are not ag-glutinated by Vi antiserum (7, 47). Thus, deletions were veryrare. Insertions were more common, including the insertions in25 strains with a 40-kb insertion in I-CeuI-E and 19 otherinsertions of various sizes in I-CeuI-B, -E, and -G. However,small changes in fragment sizes (1 to 2 kb in small fragmentsand about 10 to 20 kb in larger fragments), which could bedetected by sequencing, were not observed by PFGE.

Determination of phage type by using Vi phage and of flagel-lar antigens. The phage types of most of the wild-type strainsare shown in Table 1. The data show that many different phagetypes are associated with specific genome types (for example,the 59 genome type 3 strains have many different phage types).This indicates that the phage type and the genome type arelargely independent of each other. In a few cases, the fre-quency of occurrence of a specific genome type in a phage typeis greater than that expected by chance. For example, of 19phage type E1 strains, 7 are genome type 2, although genometype 2 is found in only 20 of 136 strains; this may representisolation of similar strains from a clonal population. Flagellarantigens were determined for almost all of the strains; theseantigens were usually the d antigen, although a few strains hadthe alternative j antigen.

DISCUSSION

Serovars of Salmonella which are pathogens for a wide rangeof hosts (generalists, such as S. enterica serovar Typhimurium)have very conserved genomes, while serovars which have verylimited host ranges (specialists), such as S. enterica serovarTyphi, S. enterica serovar Paratyphi C, S. enterica serovar Galli-narum, and S. enterica serovar Pullorum, show a high fre-quency of rearrangements among wild-type strains due to ho-mologous recombination between rrn operons (40). Helm et al.(19) showed in laboratory experiments that the frequency ofrecombination between rrn operons is not distinguishable instrains of S. enterica serovar Typhi and S. enterica serovarTyphimurium. This result suggests that differences in selectivevalue rather than differences in rearrangement frequency arelikely to be responsible for the higher frequency of rearrange-ments found in S. enterica serovar Typhi than in S. entericaserovar Typhimurium. Thus, greater survival of a recombinantmight be due to the different lifestyles of generalist and hostspecialist species.

The following four classes of chromosome rearrangementsmight be formed due to recombination between rrn operons:deletions, duplications, translocations, and inversions (Fig. 3)(40) (56). Deletions of entire I-CeuI fragments would be

FIG. 3. Proposed model of genomic rearrangements due to homol-ogous recombination between rrn operons resulting in inversions ortranslocations. oriC is indicated by a shaded circle in fragment C(which corresponds to I-CeuI-C), and Ter is indicated by a shadedsquare in fragment A (I-CeuI-A). pro (proline requirement) and his(histidine requirement) indicate the positions of standard genes.(A) Both the A and C fragments are in the normal, uninverted orien-tation (A�C�), and the fragment order is I-CeuI-ABCDEFG. (B andC) Inversion. Recombination between rrnH and rrnG results in inver-sion of fragment A. (D to F) Translocation. Recombination betweenrrnC and rrnA deletes fragment D, which is reinserted by recombina-tion with rrnE; this results in translocation to produce the fragmentorder ABCEFDG.


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readily detectable but were not observed, which is not surpris-ing since all the fragments have essential genes. Deletions of a9-kb segment between two rrn operons were detected in Ba-cillus subtilis (27), indicating that this short segment has noessential genes. Duplications would be detected by doubled

intensity of the duplicated I-CeuI fragments and in partialdigestion data, but these were not seen in these strains. Roth etal. (56) showed that 3% of the cells in cultures of S. entericaserovar Typhimurium LT2 had duplications of the segmentbetween the closest rrn operons (the I-CeuI-F fragment) and

FIG. 4. Order and orientation of I-CeuI fragments in 136 independent wild-type strains of S. enterica serovar Typhi. The sizes (in kilobases)of the fragments based on the sizes in CT18 (50) are indicated at the top, shown approximately to scale. The order of I-CeuI fragments B to Gwas determined by PFGE (Fig. 2) and was confirmed by PCR (Fig. 5). The I-CeuI-A fragment (2,422 kb) is inferred to join the left end to the rightend of a fragment to form a circle. The orientation of I-CeuI fragments B, D, E, F, and G was inferred from the polarity of the rrn genes and wasconfirmed by PCR. The order and sizes of fragments for E. coli K-12 and S. enterica serovar Typhimurium LT2 (STM LT2) and the orientationof rrn operons are indicated at the bottom. The chromosomes of the different genome types are shown in the A�C� orientation (with both theA and C fragments uninverted); the open square in fragment A indicates pro (proline utilization), and the open triangle indicates his (histidinerequirement). Since both I-CeuI-C and I-CeuI-A are flanked by inverted rrn operons, these fragments could be inverted. The number of strainsof each genome type that fall into each of the four sets of orientation of A and C fragments was determined from the PCR data (see Fig. 5). Thedot in the I-CeuI-C fragment indicates the location of oriC; T indicates the terminus. The sizes of the fragments (in kilobases) were calculated frompreviously published sequences of S. enterica serovar Typhimurium LT2 (45) (GenBank accession no. NC_003197) and E. coli K-12 (5) (GenBankaccession no. NC_000913); these fragments are shown at the bottom.


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found that these duplications are unstable since they revert tothe haploid state; our failure to find strains with duplicationsconfirms that duplications of this type are unstable.

Inversions and translocations occur frequently, for not oneof the 136 strains tested had genome type 1 A�C� (Fig. 4)(the genome order normally found in Salmonella and E. coli);even the four genome type 1 strains were A�C�. Thus, allstrains tested had at least one translocation or inversion com-

pared with the standard type. Two separate mechanisms couldexplain translocation. First, there could be deletion of a seg-ment due to recombination between two rrn operons in thesame replichore, thus forming a circular fragment, followed byreinsertion of the circle into another rrn operon (Fig. 3D andE). Hill and colleagues observed circles of a size appropriatefor the interval from rrnB to rrnE (about 42 kb) (24); thiscorresponds to the 42-kb I-CeuI-F fragment. Second, translo-

FIG. 5. PCR analysis of the rrn skeleton of S. enterica serovar Typhi genome type 3 strains. The primer pairs are indicated above the gel showingthe PCR products. Lane M contained the marker (HindIII-digested lambda). The inferred rrn skeleton is shown on the left. (Upper set) TemplateDNA of strain 26.047 (genome type 3, A�C�). (Lower set) Template DNA of strain 425Ty (genome type 3, A�C�).

TABLE 2. Changes in lengths of I-CeuI fragments in 136 strains of S. enterica serovar Typhi, measured by PFGE

I-CeuIfragment

Fragmentsize (kb)a Deletion size (kb) (no. of strains)b Insertion size (kb) (no. of strains)b No. of fragments

altered

A 2,422 NDc

B 706 20 (4), 80 (7), 90 (2) 13C 517 0D 134 0E 149 30 (1), 40 (25) 26F 42 0G 839 70 (1), 134 (10), 170 (1) 15 (1), 20 (2), 50 (1), 80 (2) 18

a Normal fragment sizes were determined from the sequence of strain CT18 (50); these sizes are very similar to those of strain Ty2 (12). The sizes are slightly correctedfrom those determined previously by PFGE (38).

b Deletions and insertions represent decreases or increases in the size of a fragment relative to the normal size; the number of strains with each size class is indicatedin parentheses. The specific strains are shown in Table 1.

c ND, not determined, because an accurate size of the I-CeuI-A fragments could not be determined by PFGE.


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cations could be formed by duplications (e.g., to form DEFEF)followed by two independent deletions of fragments E and F(to form DFE). Genome types 1 to 6 are postulated to resultfrom translocations of the small fragments I-CeuI-D, -E, and-F to form all six combinations. Genome types 13 to 16 involvetranslocation of I-CeuI-F into rrnD to the left of I-CeuI-C.Inversions are also commonly detected; e.g., genome types 7 to12 are due to an inversion resulting from recombination be-tween rrnD and rrnE. In addition, these strains have the trans-locations found in genome types 1 to 6; i.e., genome types 1and 7 and genome types 2 and 8 have the same translocation,etc.

Several hypotheses were devised by Roth et al. (56) to ex-plain the highly conserved genomes usually found in entericbacteria. At first glance, it seems that rearrangements in S.enterica serovar Typhi have resulted in total reshuffling of thegenome. However, in spite of the many genome rearrange-ments that we have detected in S. enterica serovar Typhi (Fig.4), there is still considerable conservation (although not asmuch as in S. enterica serovar Typhimurium); our data supportthe gene balance and gene dosage hypotheses for genomeconservation in S. enterica serovar Typhi.

Genome balance. Lengths of the replichores between theoriC and Ter sites on a circular bacterial chromosome must bemaintained for balanced bidirectional replication (23). Hill andGray (22) showed that moving oriC relative to Ter reduced thegrowth rate of E. coli K-12. S. enterica serovar Typhi CT18fragment sizes determined from the sequence (50) were usedas the standards to calculate the genome balance for all wild-type S. enterica serovar Typhi strains (the sizes calculated fromthe sequence of strain Ty2 [12] are very similar). The positionsof the origin of replication (oriC) and the termination of rep-lication were determined from the E. coli K-12 oriC sequence(46) (GenBank accession no. K01789) and the position of thedif (deletion-induced filamentation) sequence of E. coli (31)(GenBank accession no. S62735), respectively; locations on thechromosome of S. enterica serovar Typhi CT18 were detectedwith BlastN (2).

Rearrangements only in fragments D to F, which result ingenome types 1 to 6 (Fig. 4), do not change the genomebalance because they are all in the same replichore. Whenthese genome types are A�C�, the size of replichore 1, fromoriC in I-CeuI-C through fragments E, F, D, G, and A to Ter,is 2,362 kb; the size of replichore 2, from fragment C throughfragments B and A to Ter, is 2,447 kb. Genome balance wascalculated by dividing the size of each replichore by the totalgenome size; the off-balance value, which was one-half thedifference between the replichore sizes, was 42.5 kb (Fig. 6 andTable 3). The genome balance was calculated for genome types1 to 6 for the four different orientations in fragments A and C,which changed the lengths of the replichores, and the numbersof strains of each type were summarized from Fig. 4, as shownin Table 3. Of the 100 strains with genome types 1 to 6, 28 wereA�C� (42.5 kb off-balance), 72 were A�C� (25.5 kb off-balance), and none were A�C� or A�C� (over 400 kb off-balance). Thus, the fragment orientations which gave the bestchromosome balance were the most frequently detected ori-entations; among independent wild-type strains, strains withinversions of I-CeuI-C, which would have been highly unbal-anced, were not detected. It has been noticed previously by

Eisen et al. (14) that chromosomal inversions around the ori-gin and termination of replication are usually symmetrical,thus retaining chromosome balance, even when comparisonsare done between groups as widely separated as E. coli andVibrio cholerae.

The remaining 36 strains were also grouped into sets, such asgenome types 7 to 12, etc., and were analyzed to determinechromosome balance and frequency; these strains are shown inTable S2 in the supplemental material. Most of the genometypes occur in small numbers; many of them are off-balance by100 to 300 kb. The most striking are genome types 25 and 27(731.5 kb off-balance), represented by two strains, and onestrain of genome type 26 (582.5 kb off-balance).

It was surprising to find such a high degree of genomicrearrangement in S. enterica serovar Typhi (and in S. entericaserovar Paratyphi C [21], which causes paratyphoid fever) sincethe genomes of most enteric bacteria are highly conserved. Wepropose that the insertion of a large block of foreign DNA intothese two organisms (134 kb in Salmonella pathogenicity island7 containing the viaB genes inserted into I-CeuI-G) (38) mayhave resulted in chromosome imbalance which triggered aseries of rearrangements (32).

Gene dosage. Due to bidirectional replication, there areextra copies of genes close to oriC, resulting in increased geneexpression (57), and since dosage differences may cause thestrengths of promoters to be evolutionarily optimized for theirspecific positions, cells in which genes are a different distancefrom oriC are at a selective disadvantage (56). Although rear-rangements were seen in all 136 strains, some classes of rear-rangements were rare or never detected. Three small frag-ments, I-CeuI-D, -E and -F, are frequently translocated fromthe normal order DEF into all possible orders in genome types1 to 6 and also into new sites on both sides of I-CeuI-C ingenome types 12 to 24. However, there is not a single case oftranslocation of any of these three fragments into rrnH be-tween I-CeuI-A and I-CeuI-G, although this translocationwithin the same replichore would not change the chromosomebalance (Fig. 4). We postulate that translocations involving rrnoperons occur at random at these positions, as well as at otherlocations, but that the cells are at a selective disadvantagebecause, according to the gene dosage hypothesis, such trans-locations move the genes far from oriC such that the copynumber and thus the rate of gene expression are less adaptive;thus, rearrangements of these types occur, but strains withthese genome types do not survive in nature.

Gene position might also be conserved because promotersare tuned to the degree of local supercoiling of the DNA, sothat rearrangements would be disadvantageous (55). This maypartially explain the conservation that we detected. The direc-tion of transcription and replication is normally the same inhighly expressed genes, such as those for rRNA, and this maybe an additional basis for conservation of the genome (55, 56).It must be emphasized that in the many inversions and trans-locations observed in strains of S. enterica serovar Typhi, in-cluding those between replichores, I-CeuI fragments retain thesame orientation with respect to replication, because homolo-gous recombination between rRNA operons, all of which havetranscription oriented from oriC to Ter, enforces this.

Genomic rearrangements other than those involving rrnoperons are rare in S. enterica serovar Typhi. This was revealed


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by the fact that the lengths of the I-CeuI fragments in wild-typestrains seldom varied, except due to rare insertions or deletionsin individual fragments (Table 3), which indicates that the vastmajority of genome rearrangements which occur are due torecombination between rrn operons. Recombination might oc-cur between the 25 IS200 elements of S. enterica serovar TyphiCT18 or between the six IS200 elements in S. enterica serovarTyphimurium LT2, for IS200 is 700 bp long and should be agood target for homologous recombination. Recombinationbetween IS200 elements can occur; PCR methods detected aninversion in S. enterica serovar Typhi between two IS200 ele-ments (1) (these were in the same I-CeuI fragment and thuswere undetectable by PFGE methods in the present study); in

addition, unstable duplications with IS200 endpoints were de-tected in S. enterica serovar Typhimurium (18). However, thereis no evidence that rearrangements involving IS200 or otherduplicated regions occurred among the 136 strains in thisstudy, for such events should cause simultaneous changes inthe lengths of two I-CeuI fragments if their ends flank an rrnoperon.

The genomes of most strains of E. coli and Shigella appear tobe stable, like the genomes of most strains of Salmonella, butstudies with I-CeuI digestion indicated that rearrangementsoccur frequently in Shigella dysenteriae and Shigella flexneristrains; originally, this was postulated to be due to rrn-medi-ated rearrangements (60). However, the complete genome se-

FIG. 6. Analysis of genome balance. The chromosome structure of genome type 3 A�C� illustrates the genome balance. Since the chromo-some is bidirectionally replicated from oriC, two replichores are shown. The dot in fragment C represents oriC. (A) Linear form. The cross-hatchedline represents calculation of the total fragment sizes to determine the length of replichore 1 and replichore 2. (B) Circular form. Rep1, replichore1; Rep2, replichore 2.

TABLE 3. Genome balance analysis of S. enterica serovar Typhi strains belonging to genome types 1 to 6a

ParameterA�C�b A�C� A�C� A�C�

Replichore 1 Replichore 2 Replichore 1 Replichore 2 Replichore 1 Replichore 2 Replichore 1 Replichore 2

Genome balancec 0.491 0.508 0.505 0.495 0.589 0.410 0.604 0.395Amt off-balance (kb)d �42.5 42.5 25.5 �25.5 432.5 �432.5 500.5 �500.5No. of isolatese 28 72 0 0

a Genome types 1 to 6 have the fragment order I-CeuI-ABC(DEF)G; DEF can be in any order.b The orientation of fragments A and C as shown in Fig. 4.c Genome balance was determined from fragment sizes (Fig. 6).d See the Discussion for the method used to calculate the amount off-balance.e The number of strains in each type of orientation, as shown in Fig. 4.


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quences show that although both S. flexneri 2a strain 301 (28)and strain 2457T (61) have large symmetrical chromosomalinversions spanning the replication origin and terminus, mostof these rearrangements are due to recombination betweeninsertion sequences, as was also seen in two strains of Yersiniapestis (11). This is quite unlike S. enterica serovar Typhi, inwhich recombination occurred between rrn operons.

The 136 strains of S. enterica serovar Typhi belong to manydifferent phage types (Table 1). Phage types are very stable andthus have been used a great deal in bacterial typing (49).Genome types are relatively stable but show occasionalchanges. For example, during 10 years, as we have worked withisolates of S. enterica serovar Typhi strain Ty2 (genome type 9A�C�) (38), we have detected only rare changes in genometype (less than 1 per 100 single-colony isolates). Hughes (26)summarized the frequency of genomic rearrangements formembers of many different genera and within the same speciesand genus. He noted that rearrangements occurred most fre-quently in clinical isolates of pathogens of humans and ani-mals. He concluded that orderly and efficient replication of thegenome and global regulation of gene expression are bothcritically important; our data support these conclusions, for weshow the importance of genome balance (for genome replica-tion) and gene dosage (for gene expression).

ACKNOWLEDGMENTS

The work reported here was supported by grant RO1AI34829 fromthe National Institute of Allergy and Infectious Diseases and by adiscovery grant from the Natural Sciences and Engineering ResearchCouncil to K.E.S. and by a discovery grant from the Natural Sciencesand Engineering Research Council to S.L.L.

We thank Barney Truong and Martin Papez for assistance with theexperiments.

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Diversity of Genome Structure in Salmonella enterica Serovar Typhi Populations

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