Genome Structure and Phylogeny in the Genus Brucella

Transcription

1 JOURNAL OF BACTERIOLOGY, May 1997, p Vol. 179, No /97/$ Copyright 1997, American Society for Microbiology Genome Structure and Phylogeny in the Genus Brucella SYLVIE MICHAUX-CHARACHON, GISELE BOURG, ESTELLE JUMAS-BILAK, PATRICIA GUIGUE-TALET, ANNICK ALLARDET-SERVENT, DAVID O CALLAGHAN, AND MICHEL RAMUZ* Unité 431, Faculté de Médecine, Institut National de la Santé et de la Recherche Médicale, Nîmes, France Received 21 October 1996/Accepted 7 March 1997 PacI and SpeI restriction maps were obtained for the two chromosomes of each of the six species of the genus Brucella: B. melitensis, B. abortus, B. suis, B. canis, B. ovis, and B. neotomae. Three complementary techniques were used: hybridization with the two replicons as probes, cross-hybridization of restriction fragments, and a new mapping method. For each type strain, a unique I-SceI site was introduced in each of the two replicons, and the location of SpeI sites was determined by linearization at the unique site, partial digestion, and end labeling of the fragments. The restriction and genetic maps of the six species were highly conserved. However, numerous small insertions or deletions, ranging from 1 to 34 kb, were observed by comparison with the map of the reference strain of the genus, B. melitensis 16M. A 21-kb SpeI fragment specific to B. ovis was found in the small chromosome of this species. A 640-kb inversion was demonstrated in the B. abortus small chromosome. All of these data allowed the construction of a phylogenetic tree, which reflects the traditional phenetic classification of the genus. Brucella is a small gram-negative bacterium pathogenic for animals and occasionally for humans. The genus is divided into six species on the basis of cultural, metabolic, and antigenic characteristics: B. melitensis, B. abortus, B. suis, B. ovis, B. canis, and B. neotomae (6). However, it was shown by DNA-DNA hybridization that the genus is more probably monospecific (42). In a previous work (1), we found that each of the socalled species exhibited a specific macrorestriction pattern by digestion of genomic DNA with XbaI, with only minor variations between biovars within a species. Recently, a physical map was proposed for the genus type strain, B. melitensis 16M, by a cross-hybridization method (30). We showed that the Brucella genome was constituted by two circular chromosomes with sizes of about 2.05 and 1.15 Mb. In this study, we established the PacI and SpeI restriction maps of the two chromosomes of the other reference strains in the genus Brucella: B. abortus 544, B. suis 1330, B. canis 62/290, B. ovis 63/290, and B. neotomae 5K33. Three different but complementary techniques were employed: hybridization with the two replicons as probes, cross-hybridization of restriction fragments, and a new mapping strategy using I-SceI. I-SceI is an endonuclease encoded by a group I intron of the Saccharomyces cerevisiae mitochondrial 21S rrna gene (31). Recently, by using artificially inserted I-SceI sites, a physical map of chromosome XI of S. cerevisiae (40) and a physical map of YAC inserts (5) were made. Since bacterial genomes lack natural I-SceI sites, we showed that the introduction of a unique restriction site recognized by I-SceI could be a useful tool for the study of the genome organization (20). In this study, we used an indirect end-labeling method to localize the SpeI sites, derived from a previously described procedure involving partial digestion of linearized DNA, followed by Southern blotting and hybridization with two end probes (37). Comparison of the six physical maps showed a rather high conservation of restriction sites among the six Brucella species genomes, even though a large inversion was revealed in the B. abortus 544 small chromosome. * Corresponding author. Phone: (33) Fax: (33) MATERIALS AND METHODS Bacterial strains. Bacterial strains used in the study are listed in Table 1. Brucella strains were grown on a low-salt 2YT medium (10 g of yeast extracts, 10 g of tryptone, 5gofNaCl per liter) and Escherichia coli on LB medium. Conjugation experiments. Introduction of a unique restriction I-SceI site in each replicon of the different Brucella species was described elsewhere (20). Briefly, the suicide vector pgf2, containing the Tn5Map with the 18-bp I-SceI recognition site (18), was transferred from E. coli SM10 pir into Brucella mutants resistant to either streptomycin or nalidixic acid. Transposition events were selected by kanamycin (25 g/ml) and streptomycin resistance or kanamycin and nalidixic acid resistance according to the resistant Brucella mutant strain used. For mapping experiments, we used five insertion mutants for B. melitensis, two for B. abortus, five for B. suis, and eight for B. ovis. Endonuclease digestion of Brucella genomic DNA and pulsed-field gel electrophoresis (PFGE) conditions. Intact Brucella genomic DNA was prepared in agarose plugs and digested with rare-cutting endonucleases as previously described (30). For SpeI partial digestion, a 120-min diffusion period at 4 C followed by a 10-min digestion at 25 C gave optimal results with 3 U of endonuclease per plug (2 g of DNA). For double digestions, plugs were first partially digested with SpeI, incubated overnight at 37 C in 0.5 M EDTA (ph 8) containing proteinase K (1 mg/ml) and lauroyl sarcosine (1%), and then washed for 1 h in Tris-EDTA-phenylmethylsulfonyl fluoride (TE-PMSF) and three times in TE. Digestion with I-SceI (Boehringer Mannheim) was then performed as described elsewhere (20). PFGE of intact and digested DNA was performed in a contourclamped homogeneous electric field (CHEF) apparatus (CHEF-DRII; Bio-Rad) as previously described (30). Separation of double-digested DNA was carried out for 40 h in 1% agarose gels at 170 V by using two sets of pulse conditions, in separate runs, in order to estimate the fragment sizes more precisely: 100 to 12 s for separating the large bands and 50 to 10 s for separating the small bands. Hybridization experiments. Southern blots of PFGE agarose gels were prepared on Nytran membranes (Schleicher & Schuell) by using the VacuGene XL Blotting System (Pharmacia LKB) with 20 SSC (1 SSC is 0.15 M NaCl plus M sodium citrate) as the eluant. Preparation of probes for cross-hybridization of restriction fragments or bands separated from intact DNA was done as previously described (30). To obtain the 1.1- and 2.7-kb probes, corresponding to the ends of the I-SceI-linearized Tn5Map, plasmid ColE1::Tn5Map DNA (18) was successively digested by SphI, NotI, and then I-SceI. After separation on 1% low-melting-temperature agarose (SeaPlaque; FMC), the 1.1- and 2.7-kb restriction fragments were excised from the gel and labeled by a nonisotopic method (digoxigenin-dna labeling and detection kit; Boehringer). The Brucella DNA size markers were revealed with a total genomic DNA probe. Several genes were located on the maps by hybridizing SpeI restriction fragments with cloned or amplified (PCR) genes as described previously (30). Probes for pal, bfr, and P16.5 (8, 16, 41) were kindly provided by J. J. Letesson; for omp19 by A. Tibor; for brrp (phop homolog), brrn (ntrc homolog), htra (38), and reca (39) by B. Wren; for ery (36) by J. Aguerro; and for gale by L. Petrovska. A probe for dnaa was obtained by amplification using oligonucleotides calculated from the sequence of this gene in Rhizobium meliloti (29). brra is the gene of a response regulator amplified in our laboratory which is similar to ctra described in Caulobacter crescentus (unpublished data). 3244

2 VOL. 179, 1997 PHYSICAL MAPPING AND GENOME ORGANIZATION IN BRUCELLA 3245 TABLE 1. Brucella strains used in the study Species Strain a Host Phylogenetic analysis. Similarity between each pair of strains was calculated as the Dice coefficient as described by Grothues and Tümmler (13), with one modification: two fragments with the same size were considered as equivalent only if they cohybridized. Strains were clustered by using the Phylip program (10). RESULTS Geographic origin B. melitensis 16M (ATCC 23456) Goat United States B. abortus 544 (ATCC 23448) Cattle England B. suis 1330 (ATCC 23444) Swine United States B. canis RM/666 (ATCC 23365) Dog United States B. ovis 63/290 (ATCC 25840) Sheep Africa B. neotomae 5K33 (ATCC 23459) Desert wood rat a Type strains and biovar reference strains are listed by their FAO/OMS designations and their American Type Culture Collection numbers. Comparative PFGE-based macrorestriction analysis and determination of genome size. The genomic DNA from reference strains of the six Brucella species (Table 1) was digested with the rare-cutting endonucleases PacI (giving 9 to 10 fragments) and SpeI (26 to 28 fragments) and subjected to PFGE (Fig. 1). It could be seen that each species exhibited a specific restriction pattern with the two endonucleases, except for B. suis 1330 and B. canis, which had the same patterns. PFGE of intact genomic DNA revealed, as for B. melitensis 16M (30), the presence of two chromosomes in the other species (data not shown). To recognize to which replicon belong the PacI and SpeI restriction fragments, the bands corresponding to the two chromosomes of each Brucella species undigested genomic DNA were excised from the PFGE gel, labeled, and used as probes for hybridizing Southern blots of the same DNA digested with PacI or SpeI. The addition of restriction fragment sizes allowed a good estimation of the two replicon sizes for each Brucella species, and these were always close to 2.05 and 1.15 Mb, except for the small chromosomes of B. suis 1330, B. canis, and B. neotomae, which were 50 kb longer. Then, for each species, the PacI restriction fragments were used as linking probes on Southern blots of genomic DNA digested with SpeI. As the large PacI restriction fragments, ranging from 900 to 260 kb, hybridized with more than two SpeI restriction fragments, another technique was needed to localize all the SpeI sites within each PacI fragment. SpeI physical mapping by introducing a unique I-SceI site in each replicon. We used a Tn5 derivative, Tn5Map, to introduce a unique I-SceI site into both replicons of each of the six Brucella species, as described previously (20). For each species, Tn5Map insertion mutant clones were selected and the genomic DNA was digested with SpeI, separated by PFGE, and tested by Southern hybridization for the presence of Tn5Map using labeled ColE1::Tn5Map as a probe (data not shown). For mapping experiments, agarose plugs of genomic DNA from each Tn5Map insertion mutant were first partially digested with SpeI and then I-SceI digestion was performed. DNA products of double digestion were separated by PFGE in duplicate, on both sides of the Brucella DNA size markers. After Southern blotting of the gel, each half of the nylon membrane, containing a sample of I-SceI-SpeI digested DNA, was hybridized with one of the two probes containing the ends of Tn5Map. The SpeI restriction sites were directly localized on each side of the insertion of the I-SceI site, so that the labeled fragments read from the bottom to the top of the membrane, allowing the construction of the SpeI physical map. We show an example of the two hybridization patterns in Fig. 2 for the B. ovis clone 63/290 7, with the I-SceI site insertion in the 130-kb SpeI fragment on the small chromosome. The sizes of the DNA fragments labeled with the 1.1-kb probe (Fig. 2, lane 1) were, from the bottom to the top, 80, 85, 105, 250, 300, 480, and 500 kb, and with the 2.7-kb probe (lane 2) they were 45, 65, 80, (85), (105), (250), (300), 325 and 440 kb (parentheses indicate fragments with fainter signals). In fact, all the restriction fragments labeled with the 1.1-kb probe were also found to hybridize with the 2.7-kb probe, although some had fainter signals. The 2.7-kb probe hybridized weakly with bands labeled by the 1.1-kb probe. We therefore needed to ignore the bands which also hybridized with the 1.1-kb probe. Starting from this 130-kb fragment, the order of the SpeI restriction fragments was 5, 20, 145, 50, 180, and 20 kb on the 1.1-kb end probe side and 20, 15, 245, and 115 kb on the 2.7-kb end probe side. From these results, when constructing the SpeI restriction map of the B. ovis 63/290 small chromosome, the main difficulty encountered in aligning the fragments was the existence of fragments of slightly different sizes. To separate by PFGE the fragments obtained by partial digestion, large pulses were needed. In these conditions, the determination of the sizes of the restriction fragments from the hybridization patterns was not sufficiently accurate to identify the fragments precisely, particularly in the higher part of the run. To overcome this problem, we used Brucella genomic DNA digested with SpeIor PacI instead of the usual markers such as lambda concatemer DNA or S. cerevisiae chromosomes. This gave a ladder ranging from 900 to 3 kb (Fig. 2, lanes S and P). Furthermore, when necessary, we used two different pulse ramps in separate runs for a better size calculation. Data from cross-hybridization experiments between SpeI and PacI restriction fragments and the use of several mutants with different Tn5Map insertion sites further confirmed the map. Using these methods, we constructed physical maps of the FIG. 1. PFGE. SpeI digestion of genomic DNA from six Brucella species reference strains. Lane 1, B. melitensis 16M; lane 2, B. abortus 544; lane 3, B. ovis 63/290; lane 4, B. suis 1330; lane 5, B. canis RM/666; lane 6, B. neotomae 5K33. Size markers in kilobases are at the left of the figure.

3 3246 MICHAUX-CHARACHON ET AL. J. BACTERIOL. FIG. 2. Hybridization of I-SceI-linearized and partially SpeI-digested genomic DNA of B. ovis clone 63/290 7 with the end probes. The electrophoretic conditions were optimized for the fragments below 450 kb. Lane 1, hybridization with the 1.1-kb probe; lane 2, hybridization with the 2.7-kb probe; lanes S and P, SpeI digestion and PacI digestion, respectively, of B. ovis DNA labeled with a total genomic DNA probe. The sizes of the PacI restriction fragments are given in kilobases. small and large chromosomes for the different Brucella species: B. abortus 544, B. suis 1330, B. canis 62/290, and B. ovis 63/290. The B. neotomae 5K33 map was deduced from cross-hybridization of restriction fragments (Fig. 3). The construction of the B. melitensis 16M physical map by this method allowed us to test the validity of the new mapping technique using I-SceI and to confirm the existing map (30). The restriction map of B. suis 1330 was also confirmed by hybridization of SpeI fragments with linking clones from a B. suis 1330 DNA cosmid library: one to three of 68 clones linked two adjacent SpeI fragments, except for three sites. Comparison of the physical maps. Since a physical map has been established for the genus type strain, B. melitensis 16M (biovar 1) (M1), we arbitrarily decided it was the reference map and we compared the three other maps to it. The six physical maps are shown in Fig. 3. The most frequent differences are small insertions or deletions (indels) ranging from 1 to 34 kb. These events are most noticeable in the small chromosome. We cannot, however, exclude the possibility that small indels exist in the large SpeI fragments, which are predominantly found in the large chromosome, since size determination is less accurate for the large fragments. A 21-kb fragment was found between two SpeI sites in the small chromosome of B. ovis which did not hybridize to DNAs from the other Brucella species (data not shown). Although other strain-specific nucleotide sequences were not found, their existence cannot be ruled out. For B. abortus 544, a 640-kb inversion with respect to M1 was found in the small chromosome between two SpeI restriction sites. The comparison of cross-hybridization results between SpeI and PacI restriction fragments of M1 DNA and those of these B. abortus strains confirmed this large inversion. However, this inversion was found only in biovars 1, 2, 3, and 4, the small chromosome of the other biovars being very similar to that of M1 (unpublished results). The map of B. suis 1330 was again related to that of M1. However, modifications of restriction fragment length and the creation or loss of restriction sites were more often observed in this species than in the other. The small chromosome is 50 kb longer than that of M1. B. canis 62/690 had a physical map identical to that of B. suis B. neotomae 5K33 has a small chromosome that is very similar to that of B. suis 1330, while its large chromosome is more similar to that of M1. Genomic organization. In order to compare the genomic organization of the two chromosomes of the four Brucella species, each of the SpeI restriction fragments of B. melitensis 16M was taken as a probe and hybridized with those of the other species. At this macrorestriction level, the genetic information seemed to be in the same order in the four genomes except for the B. abortus 544 small chromosome, showing a large inversion (see below). Only a few genes have been cloned in Brucella, and some of them were located on the maps by hybridization with SpeI restriction fragments (Fig. 3). The localization of rrn loci, omp1 and omp2, BCSP 31, dnak, groe, and IS6501 copies has already been published for B. melitensis (30) and was confirmed for the other species. The localization of the other genes was obtained as described in Materials and Methods. All the genes were found in the corresponding SpeI restriction fragments in the different species. We also identified the SpeI restriction fragments containing at least one copy of the insertion sequence IS6501 described in the Brucella genome (33). We found 16 of them in B. ovis 63/290 and only 5 in the three other strains (Fig. 4). But, by hybridization with EcoRI-digested DNA of these Brucella strains, it was shown that the number of IS6501 copies was around 30 in the B. ovis genome and between 5 and 10 in the three others (33). Finally, three rrn loci were found in all the strains, located in the corresponding SpeI fragments. Phylogeny within the Brucella genus. Restriction fragment length polymorphism has been used to study the phylogeny of Pseudomonas aeruginosa (13). We employed a related method to study the phylogeny within the genus Brucella, using 16 type strains representing all the species and biovars. The dendrogram is shown in Fig. 4. This tree is unrooted, since macrorestriction patterns are very specific for Brucella and definition of a related extragroup to place the root is artificial. The isolates were scattered in several groups. The two B. abortus groups (biovars 1 to 4 and biovars 5 to 9) form a tightly related branch. The three B. melitensis biovars are regrouped to the latter but are more remote from one another. B. ovis is more or less related to B. melitensis biovar 1 by the method used for the construction of the tree. The four B. suis biovars are localized at the opposite extremities of the tree and are relatively separated from one another. B. canis is not represented in a separate branch, since its physical map is the same as that of B. suis biovar 1. Finally, B. neotomae is an intermediate between the B. melitensis-b. abortus group and the B. suis biovar group.

4 VOL. 179, 1997 PHYSICAL MAPPING AND GENOME ORGANIZATION IN BRUCELLA 3247 Downloaded from FIG. 3. Physical maps of the two chromosomes of B. melitensis 16M (B. m), B. abortus 544 (B. a), B. suis 1330 (B. s), B. canis RM/666, B. ovis 63/290 (B. o), and B. neotomae (B. n). The two circular chromosomes are represented in linear form, each one starting from a conserved SpeI fragment. (A) large chromosomes; (B) small chromosomes. For each chromosome map, SpeI sites are located above and PacI sites are located below. Sizes are given in kilobases for all the restriction fragments of the B. melitensis chromosomes, and only for those different from B. melitensis for the other species. Several genes are located (see Materials and Methods). rrn loci are represented by open squares, IS6501 copies by stars, and Tn5Map insertions by open circles. The inversion in the small chromosome of B. abortus is shown by two lines. on August 16, 2018 by guest DISCUSSION The first striking observation concerning our results is that the restriction maps of the six Brucella species are very similar. Genome mapping of several isolates belonging to the same bacterial species or genus has been performed for few bacteria, and comparison showed different patterns of genome conservation. First, chromosomal mapping could reveal a high conservation of the restriction site or gene order (2, 22, 24, 34). The second pattern associated the conservation of gene order with a high restriction polymorphism (4, 25, 32). In a third pattern, comparison of genome mapping revealed a mosaic structure showing the presence of conserved and modified regions (3, 11, 23). In the most extreme case, the restriction site or gene order differed markedly (19, 43). The close relationship found among the six physical maps in our study allows us to assign the Brucella genus to the first group. Small indels and creation or loss of restriction sites are frequently observed, most often in the small chromosome. Variability is localized to certain regions, similar to the polymorphic hot spots observed in Clostridium perfringens (2). A 640-kb inversion was found in the small chromosome of B. abortus 544 (biovar 1). PacI restriction polymorphism showed that this inversion is also present in biovars 2, 3, and 4 of the same species but not in biovars 5, 6, and 9. It is unlikely that the phenotypic differences between the two groups of biovars can be explained by this chromosomal rearrangement. Similar inversions with a size of several hundred kilobases have also been described in other bacterial species (21, 22, 25 28, 35, 43). It has been demonstrated that large genomic rearrangements in E. coli and in Salmonella serovars occur by homologous recombination at the rrn loci (17, 27, 28) or repeated nucleotide sequences, such as insertion sequences (7, 12, 21).

5 3248 MICHAUX-CHARACHON ET AL. J. BACTERIOL. An insertion sequence, IS711 (15) or IS6501 (33), was described in Brucella species. These mechanisms cannot explain the rearrangement in the small chromosome of B. abortus, since rrn loci or IS copies were not located at the edges of the inversion. It is also noteworthy that the B. ovis genome contains three to four times more copies of IS6501 than the other Brucella species, but its restriction map is very similar to that of these species, without evidence of major chromosomal rearrangements. Other repeated sequences exist in the Brucella genome which may also have played a role in the genomic rearrangements observed (14). The dendrogram deduced from the maps grossly reflects the traditional phenetic classification of the genus Brucella, confirming a previous study in which we found that Brucella species exhibited different XbaI restriction patterns, that biovars within a species are less polymorphic, and that, except for B. ovis isolates (33), field strains within a biovar have exactly the same pattern (1). This result was not expected since this division of the genus into species and biovars is based solely upon a small number of metabolic traits, two lipopolysaccharide epitopes, and sensitivity to a set of phages which are host-range variants of the same ancestor phage (6). A recent study, using sequence data from the omp2 locus from different Brucella species, confirms the existence of species-specific lineages (9). As in our study, a close relationship was found between the same strains (between B. suis and B. canis or between B. melitensis and B. abortus, the latter being divided into the same two subgroups), but an extreme divergence of B. ovis and B. neotomae from the other species was also observed. However, the authors suggested the existence of a gene conversion for this locus in these two species, explaining the divergence with the other species. The nucleotide sequence similarity between all the Brucella strains, measured by DNA-DNA hybridization, is so high that Verger et al. (42), following the international recommendations for bacterial classification, proposed that the genus is monospecific. However, although virulence is not recognized FIG. 4. Phylogenetic tree of the six Brucella species. as a taxonomic criterion, the division of Brucella into several species has been always more or less influenced by the restriction of the virulence of each so-called species to one or a small number of mammalian hosts. With view to a phylogenetic classification, such a proposal can be made only if the different species are evolutive lineages restricted to a narrow niche. This is the case for Brucella for three reasons. First, because of their fastidious growth requirements, Brucella organisms cannot actively multiply in the environment but only in infected animals which harbor huge numbers of bacteria in their tissues. Second, since mechanisms of genetic exchange, such as plasmid, temperate bacteriophage, or transformation, have never been demonstrated to occur naturally in Brucella, each species is genetically isolated, despite the frequent coexistence of different strains infecting different animals in the same farm. Third, virulence of the different species is restricted to a small number of hosts. For example, B. melitensis infects goats and sheep while B. abortus infects cattle. While both species are also pathogenic for humans and B. melitensis occasionally infects cattle, the abnormal host is always contaminated via the natural host and the infection is an epidemiological dead end. A narrower restriction of the virulence is observed for B. canis, B. ovis, and B. neotomae, which are, respectively, pathogenic for dogs, sheep, and desert wood rats. Finally, B. suis is essentially virulent for swine. The similarity of the maps argues for a monospecific genus, as suggested by Verger et al., and the tree for the existence of subspecies corresponding to evolutive lineages (pathovars) adapted to a particular host. Liu and Sanderson have recently compared the physical and genetic maps of several different Salmonella serovars (27). The maps of the different serovars of Salmonella enterica (e.g., S. typhimurium and S. enteritidis) are closely related. S. enterica infections in humans range from gastroenteritis to fatal septicemia, but the organism also infects a wide range of warm- and cold-blooded animals. The genomic organization of the ubiquitous E. coli was also similar (but not identical) to that of S. enterica. However, the physical map of S. typhi, the agent of

6 VOL. 179, 1997 PHYSICAL MAPPING AND GENOME ORGANIZATION IN BRUCELLA 3249 typhoid fever in humans, is very different in both its organization and content. Moreover, when four wild-type S. typhi isolates were tested, all were found to have identical physical maps. Since S. typhi is pathogenic only for humans and has no other animal hosts, the authors proposed that the restricted host range has exerted specific selective constraints, conducive to the selection of a particular clone. The very close homology between the different species, shown by the comparison of macrorestriction maps, requires a still more precise analysis of the Brucella genome in order to understand the differences in the virulence restriction of the different species. High-resolution maps are now being constructed in our laboratory. REFERENCES 1. Allardet-Servent, A., G. Bourg, M. Ramuz, M. Pages, M. Bellis, and G. Roizes DNA polymorphism in strains of the genus Brucella. J. Bacteriol. 170: Canard, B., B. 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