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J Bacteriol. Aug 2004; 186(15): 5040–5051.
PMCID: PMC451633

Comparative Whole-Genome Hybridization Reveals Genomic Islands in Brucella Species

Abstract

Brucella species are responsible for brucellosis, a worldwide zoonotic disease causing abortion in domestic animals and Malta fever in humans. Based on host preference, the genus is divided into six species. Brucella abortus, B. melitensis, and B. suis are pathogenic to humans, whereas B. ovis and B. neotomae are nonpathogenic to humans and B. canis human infections are rare. Limited genome diversity exists among Brucella species. Comparison of Brucella species whole genomes is, therefore, likely to identify factors responsible for differences in host preference and virulence restriction. To facilitate such studies, we used the complete genome sequence of B. melitensis 16M, the species highly pathogenic to humans, to construct a genomic microarray. Hybridization of labeled genomic DNA from Brucella species to this microarray revealed a total of 217 open reading frames (ORFs) altered in five Brucella species analyzed. These ORFs are often found in clusters (islands) in the 16M genome. Examination of the genomic context of these islands suggests that many are horizontally acquired. Deletions of genetic content identified in Brucella species are conserved in multiple strains of the same species, and genomic islands missing in a given species are often restricted to that particular species. These findings suggest that, whereas the loss or gain of genetic material may be related to the host range and virulence restriction of certain Brucella species for humans, independent mechanisms involving gene inactivation or altered expression of virulence determinants may also contribute to these differences.

Brucellosis is a zoonotic disease endemic in many areas of the world and is characterized by chronic infections, abortion, and sterility (7). In humans, brucellosis is a systemic, febrile illness resulting in osteoarthritis, endocarditis, and several neurological disorders (7, 53). Brucellosis is caused by many species belonging to the genus Brucella that are aerobic facultative intracellular bacteria. Brucella species are closely related to intracellular symbionts and pathogens of plants and animals and are classified as α2-proteobacteria based on rRNA sequence comparison (36).

The genus Brucella consists of six species, designated on the basis of host preference, antigenic and biochemical characteristics as Brucella melitensis (goats and sheep), B. abortus (cattle), B. suis (pigs), B. canis (dogs), B. ovis (sheep), and B. neotomae (wood rats) (6). B. abortus, B. melitensis, and B. suis can all infect humans with similar serious disease consequences (7). B. melitensis, originally isolated as a pathogen of goats and sheep, is highly pathogenic and a frequent cause of human brucellosis. In contrast, human infections of B. ovis and B. neotomae have not been reported, and B. canis rarely causes infection in humans. Human brucellosis occurs via contact with infected animals or animal products and is common in countries where the disease is endemic in domestic animals (13). Brucella also can be readily dispersed through aerosol (13), making it a potential biowarfare threat (22, 26).

Although six species are recognized in the genus Brucella, DNA-DNA hybridization and multilocus enzyme electrophoresis studies have revealed very limited genetic diversity among Brucella species (16, 50). Further, Verger et al. have proposed, based on DNA-DNA hybridization studies, that B. melitensis is the only species in the genus Brucella, with other Brucella species being biovars of B. melitensis (50). Genomes of Brucella species are stable and have similar genomic organizations (23, 34). Comparison of the complete genome sequences of B. suis and B. melitensis has revealed extensive similarity in genetic content (>90% of the genes share 98 to 100% identity at the nucleotide level) and gene order (40). In addition, single-nucleotide polymorphisms between these two species are very low (40). These findings strengthen the notion of Brucella as a monospecific genus (16, 50) and suggest a relatively small number of differences are responsible for the host preference and virulence restriction of Brucella species.

Of serious concern in Brucella research is the identification of genetic factors that permit these species to multiply within the host and cause disease. Well-characterized virulence factors of many pathogenic bacteria such as cytolysins, capsules, exotoxins, secreted proteases, pili and/or fimbriae, flagella, phage-encoded toxins, and virulence plasmids are absent in Brucella (8). Whole-genome comparison, at greater resolution, of closely related Brucella spp. expressing different pathogenicities may provide insights into their virulence determinants. DNA microarrays have been used to investigate genetic content among closely related bacteria (3, 9, 10, 12, 42, 45). The complete genome sequence of the Brucella species most pathogenic to humans, B. melitensis 16M, permits high-throughput whole-genome comparison of Brucella species by using microarrays. Here we have used the genomic sequence of B. melitensis 16M to assemble a whole-genome high-density oligonucleotide DNA microarray to compare 16M with other Brucella species genomes.

MATERIALS AND METHODS

Strains and culture conditions.

The Brucella strains used in the present study are listed in Table Table1.1. These strains were from diverse sources isolated over the last 40 to 50 years and include several strains from human infections. All Brucella strains were grown to stationary phase at 37°C in brucella broth or brain heart infusion broth (Becton Dickinson, Sparks, Md.) with or without 5% CO2. For microarray studies B. abortus S2308, B. canis RM6/66, B. melitensis 16M, B. neotomae 5K33, B. ovis REO198, and B. suis S100 were used.

TABLE 1.
Brucella strains used in this studya

B. melitensis 16M DNA microarray construction.

A high-density oligonucleotide array was designed by using the published DNA sequence of B. melitensis 16M genome (GenBank accession numbers AE008917 [chromosome I] and AE008918 [chromosome II]) (8). The genome has 3,294,931 total bases and encodes a predicted 3198 open reading frames (ORFs). We designed a high-density oligonucleotide array for the 16M genome by using several criteria for oligonucleotide probe selection. A general description of the array layout and methods for high-density in situ oligonucleotide microarray synthesis are previously described (46). Each oligonucleotide probe is 24 nucleotides, and probes are organized into probe pairs consisting of a perfect-match (PM) probe and a mismatch (MM) probe. The array has a total of 191,834 oligonucleotide probes that are organized into 95,917 probe pairs. The MM probe has two single-base substitutions relative to the corresponding PM probe sequence at positions 6 and 12 (bases were changed as follows: A→T, C→G, G→C, and T→A). Using the programs ProbeAnalyzer and CheckUnique (NimbleGen), potential probe sequences were scanned for high-quality probes and for minimal sequence redundancy by checking each probe against a database of the entire 16M genome and a collection of 8,500 mouse sequences from RefSeq (43). We screened probes against the mouse sequences so that the chip design could be used for in vivo expression profiling of Brucella in future experiments. A total of 17 probe pairs were selected from the coding strand of each of the 3,198 ORFs. For 3,113 ORFs, 17 probe pairs were selected that showed no redundancy within the 16M and mouse sequences. For the remaining 85 ORFs, probes were selected ignoring the fact that many probes for these ORFs had significant matches to mouse or 16M sequences. The array also consists of probes specific to non-ORF encoding regions of the genome, which include the untranslated RNA encoding genes. Probes for these regions were selected by using the same criteria as for the ORF probes. The probe pairs were synthesized at randomized locations on the chip by using a maskless array synthesizer (NimbleGen [46]).

Genomic DNA extraction and labeling.

The genomic DNA (gDNA) was prepared from Brucella strains grown to stationary phase in brucella broth by using MasterPure genomic DNA extraction kit (Epicentre, Madison, Wis.), and 10 to 15 μg of gDNA was fragmented to 50 to 150 bp by partial digestion with DNase I (0.015 U/μg of DNA; Roche, Indianapolis, Ind.) at 37°C for 10 min. DNase I was heat inactivated at 95°C for 15 min, and fragmentation was confirmed by agarose gel electrophoresis. Fragmented gDNA was purified through a Microcon YM-10 column (Millipore) and 3′ end labeled with 1 nM biotin-N6-ddATP (Perkin-Elmer, Boston, Mass.) by using 37.5 U of terminal transferase in 1× TdT buffer (0.2 M potassium cacodylate, 25 mM Tris-HCl, 0.25 mg/ml [pH 6.6]; Roche) and 2.5 mM CoCl2.

Array hybridization and washing.

Labeled gDNA was denatured at 95°C for 5 min, incubated for 5 min at 45°C, and centrifuged at 14,000 × g for 5 min. Then 10 μg of labeled DNA was hybridized to the 16M microarray in 300 μl of hybridization solution containing 50 mM morpholineethanesulfonic acid (MES), 0.5 M NaCl, 10 mM EDTA, 0.005% (vol/vol) Tween 20, 1 nM CPK6 Oligo, 0.2 mg of acetylated bovine serum albumin, and 0.04 mg of herring sperm DNA for 16 h at 42°C. After hybridization, arrays were washed three times over a 5-min period at room temperature with nonstringent wash buffer (6× SSPE [1× SSPE is 0.18 M NaCl, 10 mM NaH2PO4, and 1 mM EDTA; pH 7.7], 0.01% [vol/vol] Tween 20), followed by stringent wash buffer (100 mM MES, 0.1 M NaCl, 0.01% Tween 20) four times over a 20-min period at 42°C. Arrays were stained with streptavidin-Cy3 conjugate (Amersham Pharmacia, Piscataway, N.J.) for 25 min at room temperature. After the staining step, arrays were washed with nonstringent wash buffer for 5 min, followed by a 30-s rinse in 1× final wash buffer (NimbleGen) and immediately dried with grade 5 argon.

Microarray scanning and data analysis.

The microarrays were scanned at 5-μm resolution by using an Axon 4000B scanner (Axon, Redwood City, Calif.), and the resulting image files were processed with NimbleScan Software (NimbleGen). For each species, two or three hybridizations were performed with separately labeled gDNA. The difference in intensity values for each probe pair was calculated by subtracting the MM intensity from PM intensity values. The average difference in intensity values (average difference values) for 17 probe pairs for each ORF was calculated by using the Tukey bi-weight mean algorithm (bi-weight constant = 6) (21). To normalize between arrays, array-specific scaling factors were calculated as the ratio of 1,000 divided by the average of the average difference values for all of the ORFs on the array. The average difference value for each ORF was multiplied by the array specific scaling factor. The scaling factors among replicate arrays ranged from 0.63 to 1.28. For each strain the scaled data were averaged across replicate arrays. For each ORF a ratio of the scaled average difference value relative to the value from 16M was calculated. For each strain, we calculated the average and standard deviation (SD) for this ratio for all ORFs, and potentially deleted ORFs were selected where the ratio was 4 SD below the average ratio.

PCR and sequencing.

Putative deleted regions identified by microarray experiments were examined by PCR and sequencing. PCR primers were designed within ORFs flanking the predicted edges of each deletion so that the amplified region spans the missing locus. The oligonucleotide primers used in the present study are listed in Table Table2.2. Primers were obtained from Integrated DNA Technologies (Coralville, Iowa). PCR was performed by using Taq PCR Master kit (Qiagen, Valencia, Calif.) or Herculase enhanced DNA polymerase (for long-range PCR; Stratagene, La Jolla, Calif.), and the products were analyzed by electrophoresis in 1% agarose gels. To determine the deletion boundaries, PCR products were purified by using the QIAquick PCR purification kit (Qiagen) and sequenced with PCR primers by using dye terminators at the DNA sequencing Core Facility, University of Wisconsin Biotechnology Center. Sequences were compared to the 16M genome sequence to determine the precise location of each deletion.

TABLE 2.
Oligonucleotide primers used to confirm the deletions identified by microarray hybridization

RESULTS

Microarray analysis.

To identify genetic determinants of 16M that contribute to its virulence and pathogenicity, we performed comparative genomic analysis of Brucella species. This microarray comparison only allows the detection of regions that are missing in other Brucella species relative to the 16M genome and will not detect regions that are unique to other Brucella strains. Hybridization results revealed that majority of the 16M ORFs are likely present across all five Brucella genomes examined as illustrated by the majority of ORFs with a signal intensity ratio close to 1 relative to 16M (Fig. (Fig.1).1). This finding is consistent with the notion that Brucella is a monospecific genus with limited genetic diversity (16, 50). Punctuating this pattern of conservation, the data suggest a number of regions likely to be deleted or duplicated in one or more of the Brucella species (Fig. (Fig.1).1). Many of the putative deleted regions were clustered within the 16M genome. We identified nine regions containing at least three contiguous ORFs, designated genomic islands (GIs), that were absent among five Brucella species. The GIs were numbered 1 through 9 based on the gene order in 16M.

FIG. 1.
Comparison of five Brucella species genomes to B. melitensis 16M by microarray. 16M arrays were hybridized with labeled gDNA from Brucella species (two or three hybridizations from gDNA labeled in separate reactions for each species). For each ORF, the ...

Putative deletions identified by microarray-based comparisons were examined by PCR amplification and sequencing. Table Table33 lists the ORFs altered and the precise boundaries of deleted regions with respect to the 16M genome. The predicted product of each altered ORF and its distribution across Brucella species can be seen in Fig. Fig.2.2. Amplification and sequencing of the predicted deletion junction confirmed nearly all of the predicted differences. ORFs that were present in multiple copies in the 16M genome or partially missing were scored as present by microarray hybridization and later confirmed to be absent from a GI by PCR and sequencing. Although the number of probe pairs for each ORF was constant (17 probe pairs), their spacing within ORFs was variable. Since we averaged probe pair values across ORFs, detection of partially deleted ORF was dependent on probe location in an ORF and on ORF length. Only two ORFs, BMEI0728 and BMEI1314, although called deleted by the microarray, were present by PCR and sequencing. ORF BMEII1071 was identified as absent by microarray analysis in B. canis, B. neotomae, and B. suis, even though a DNA fragment of size similar to 16M was amplified from each species. Sequencing revealed that the BMEII1071 region from these three species was highly divergent compared to 16M.

FIG. 2.FIG. 2.
Genomic comparison of five Brucella species. ORFs, deleted either partially or completely in five Brucella species relative to 16M, are shown. Putative deletions were selected if the average hybridization signal ratio was 4 SD below the mean. Each putative ...
TABLE 3.
ORFs deleted in five Brucella spp. compared to B. melitensis 16Ma

16M ORFs absent in Brucella species: comparison with B. ovis REO198.

B. ovis causes contagious epididymitis in rams and, rarely, abortion in ewes. B. melitensis, although originally isolated as a pathogen of goats and sheep, is pathogenic to humans, whereas B. ovis does not infect humans and the ovine brucellosis due to B. ovis is not zoonotic. Comparison of B. ovis and B. melitensis genomes may identify the factors responsible for these changes in host specificity. Array hybridization, followed by PCR and sequencing, revealed 84 ORFs distributed in nine chromosomal loci were either partially or completely absent (Table (Table33 and Fig. Fig.2).2). All but four deleted ORFs were clustered in five islands relative to the 16M genome (GI-1, GI-2, GI-5, GI-7, and GI-9).

GI-1 (~8.1 kb; nine ORFs) contains mostly ORFs encoding hypothetical proteins (HPs) and phage-related genes, including a resolvase similar to phage Mu DNA invertase.

GI-2 (~15.1 kb; 20 ORFs) includes ORFs encoding HPs, transposases, and a phage family integrase. Interestingly, this island also contains two ORFs (BMEI0997 and BMEI0998) involved in lipopolysaccharide (LPS) biosynthesis and their absence may contribute to the rough phenotype associated with B. ovis. In addition, an ORF encoding a 25-kDa outer membrane protein precursor, Omp25, is also included in this cluster.

GI-5 (~44.1 kb; 42 ORFs) includes ORFs encoding peptide ABC transporters such as spermidine-putrescine (Pot), oligopeptide (Opp), and dipeptide (Dpp); transcriptional regulators similar to an AtrA regulator that is required for attachment and virulence of Agrobacterium tumefaciens (2); and two ORFs similar to cephalosporin acylases. B. suis and B. abortus genome sequences contain these two cephalosporin acylases encoding ORFs from 16M (317 and 466 amino acids) fused as a single ORF encoding a 761-amino-acid protein 47% identical to 774-amino-acid protein from Pseudomonas species (30). Cephalosporin acylases are involved in the activation of cephalosporin antibiotics, and the eightfold-higher MIC observed for B. ovis relative to B. abortus (49) may be due to lack of acylase from B. ovis.

GI-7 (~4.4 kb; five ORFs) and GI-9 (~4.9 kb; four ORFs) consist of AraC and GntR family transcriptional regulators, a hydrolase, and an ORF involved in amino acid metabolism.

Comparison with B. neotomae 5K33.

B. neotomae only infects desert wood rats under natural conditions and is not associated with disease. Whole-genome comparison of B. neotomae might reveal a defined set of genes critical for 16M pathogenesis. Hybridization of B. neotomae gDNA to the 16M array revealed a very similar gene content, a finding consistent with previous work (34), suggesting a close phylogenetic relationship between these two species compared to other Brucella species. Only 17 ORFs were identified as altered, including a region containing 10 contiguous ORFs (~7.5 kb, GI-6) (Table (Table33 and Fig. Fig.2).2). ORFs in GI-6 include those encoding transposases with significant similarity to ORFs on plasmid pNGR234a from Rhizobium species (15). A 2.2-kb region in chromosome II containing ORFs involved in denitrification, including a transcriptional regulator of the CRP/FNR family (BMEII0986), was also missing.

Comparison with B. canis RM6/66.

B. canis is a pathogen of dogs that causes epididymo-orchitis in males and abortion in females. Although B. canis infects humans, clinical disease is rarely reported. Comparison of B. canis genome to 16M may reveal determinants responsible for pathogenicity in humans. A total of 38 ORFs distributed in seven chromosomal loci were found either partially or completely deleted (Table (Table33 and Fig. Fig.2).2). Most B. canis deletions were identical to B. suis deletions except for three ORFs, a finding consistent with the presumed close phylogenetic relationship between these two species (16, 34). Thirty ORFs clustered within a 21-kb region in chromosome I (GI-3), mostly encoding proteins of unknown functions were missing in B. canis as well as B. suis. There are three ORFs absent from B. canis but present in all other Brucella species, including B. suis. Particularly interesting is the absence of a polysaccharide deacetylase that is similar to chitooligosaccharide deacetylase NodB of Rhizobium species, since this factor is involved in establishing a symbiotic interaction between bacteria and host (44).

Comparison with B. suis S100.

A total of 39 ORFs were absent either completely or partially (Table (Table33 and Fig. Fig.2),2), and these alterations were nearly identical to the set identified in B. canis. A B. suis genome has been sequenced, and the differences that are described by whole-genome sequence comparison (40) are almost entirely consistent with the microarray results except for three small regions (<200 bp) that were not identified by microarray analysis. Closer analysis of these ORFs suggested that there were very few probes representing the deleted regions on the array and hence were not identified as deleted. Interestingly, we also identified a 2-kb region involving two ORFs missing in B. suis 100 (BMEI1746 and BMEI1747) but present in the sequenced B. suis 1330 (biotype 1). B. suis 100 is also biotype 1, isolated from swine with abortion, and is virulent in the IRF-1−/− mouse model similar to B. suis 1330 and B. melitensis 16M (G. Rajashekara and G. Splitter, unpublished data), suggesting that this 2-kb region is dispensable for virulence and pathogenicity.

Comparison with B. abortus S2308.

Forty ORFs were absent from the B. abortus genome, including two clusters involving more than three contiguous ORFs, one in chromosome I (5 ORFs, ~3.8 kb, GI-4) and another in chromosome II (25 ORFs, ~25.1, GI-8) (Table (Table33 and Fig. Fig.2).2). GI-8 primarily contains ORFs encoding proteins involved in sugar metabolism and LPS biosynthesis, whereas GI-4 has ORFs encoding butanoate metabolism. Other ORFs include a response regulator (BMEII0292) and a diguanylate cyclase (BMEI0929). Most of these regions were absent from the genome sequence of B. abortus (University of Minnesota [http://www.cbc.umn.edu/ResearchProjects/AGAC/Pub_Brucella/Brucellahome.html]), except for GI-4, suggesting that the strain we used, similar to B. suis, was not identical to the sequenced strain.

Deletions in Brucella species are present in multiple strains.

Since microarray hybridizations were performed with only one strain of each species, it is possible that the deletions observed are specific to the strain that was used in the microarray experiment. To confirm that the microarray data comparing single strains is in fact representative of the species, we examined 47 strains representing B. abortus, B. canis, B. ovis, and B. suis by PCR. PCR was performed with primers specific to ORFs flanking the predicted edges of each deletion. As expected, the deletions observed in these four species were present in multiple strains of the same species (Table (Table4).4). Only two regions in B. abortus (BMEI0929 and BMEI1919-1923; GI-4), one region in B. ovis (BMEII0405), and two regions in B. suis (BMEI0899-0900 and BMEI1746-1747) were not deleted in some strains (see Tables S1A to D in the supplemental material for the PCR results for each strain). For B. neotomae, however, more strains could not be tested since only two strains have been described, including the one used here. Since deletions identified by microarray are conserved in multiple strains of other Brucella species, it is more likely that the changes in B. neotomae are species specific. In contrast, except for the two regions (BMEI0899-0900 and BMEI0926), none of the 15 B. melitensis isolates had alterations in regions that were identified by microarray as deleted in other Brucella species (Table (Table4)4) (see Tables S2A and B in the supplemental material for the PCR results for each strain). Interestingly, the GIs missing in Brucella species that are nonpathogenic to humans are present in all other Brucella species, including 15 B. melitensis strains examined (Table (Table4)4) and in at least 3 B. abortus and 2 B. suis strains (data not shown), suggesting that these GIs may contribute to human infection.

TABLE 4.
PCR analysis of Brucella strains to detect deletions identified by microarraya

16M GI acquisition.

Analysis of sequences surrounding the GIs in 16M suggests that many of the GIs were acquired through horizontal transfer. Figure Figure33 shows the schematic representation of five GIs with hallmarks of horizontal transfer. Typical of pathogenicity islands (PIs) of many pathogenic bacteria (17, 19), these islands were integrated adjacent to or within tRNA genes with an integrase or insertion sequence (IS) flanking the ends. The presence of direct or inverted repeats at the island boundaries may indicate the sites of recombination (Fig. (Fig.3).3). In addition, these regions have variable GC content compared to the remaining genome and most ORFs present in GI-1, -2, and -3 have no significant similar sequence in GenBank. ORFs on GI-5 have an atypical trinucleotide composition, whereas ORFs in GI-6 are similar to ORFs on Rhizobium plasmid pNGR234a (15). These features suggest that these GIs were acquired by lateral transfer of DNA.

FIG. 3.
Schematic representation of the genomic regions of the five B. melitensis 16M GIs (only relevant features are shown; the figure is not drawn to scale). Identical repeats at the deletion boundaries are shown in hatched boxes, the orientations of the repeats ...

Duplication of a GI in B. ovis.

Microarray analysis revealed 30 ORFs with ratios significantly >1, suggesting that these ORFs are present in multiple copies (Table (Table5).5). In the B. ovis genome, a cluster of 22 contiguous ORFs relative to 16M (~25.5 kb; duplicated region [DR]) mostly encoding HPs and l-amino acid transport genes, had ratios of ~2, suggesting duplication of the entire region. One ORF (BMEI1214) in the cluster, however, had a ratio of ~3, suggesting a higher copy number of this ORF. In addition, seven ORFs, five encoding ISs were duplicated across four species (Table (Table5),5), including IS6501 ORFs that are known to be present in more copies in B. ovis than B. melitensis (37).

TABLE 5.
Duplication of ORFs in Brucella spp.

DISCUSSION

Microarray analysis revealed extensive similarities among Brucella species in their gene content. Only 217 ORFs were absent either completely or partially compared to 16M. We also discovered that >3,110 ORFs of the 3,198 ORFs represented on the microarray were present in any given Brucella species. Our results suggest that genomes of Brucella species are similar and imply that a relatively small number of genetic changes may be responsible for differences in host preference and virulence among Brucella species. In nature, brucellae are found predominantly associated with macrophages and are not known to harbor extragenetic material such as plasmids, and there is no evidence of natural transfer of genetic material. Therefore, stability of Brucella genomes may be largely due to a lifestyle that keeps these bacteria genetically isolated similar to obligate intracellular symbionts of Buchnera species (48).

The large number of changes seen in the B. ovis genome (five GIs) may have resulted from more active ISs in this species compared to other Brucella spp. B. ovis has more IS6501 copies (~30) than other Brucella species (4 to 10 copies), and IS6501 hybridization could distinguish strains of B. ovis but not strains of B. melitensis (37), suggesting a more active IS element in B. ovis. ORFs in GI-2 and GI-5 encode factors involved in Brucella virulence. Smooth LPS is one of the key virulence factors in Brucella pathogenesis (41); rough LPS mutants of Brucella are attenuated (35). Smooth LPS O chain is involved in inhibition of early fusion between Brucella containing phagosomes and lysosomes in murine macrophages and mutations that inactivate the LPS biosynthetic gene, wbdA (BMEI0997), in B. suis attenuate its ability to multiply within macrophages (25). Likewise, Omp25 is implicated in virulence of B. melitensis and B. abortus, and inactivation of Omp25 in Brucella results in attenuation in BALB/c mice (11). Omp25 is an Omp3 homologue found in many pathogens or symbionts of α-proteobacteria, where it plays a role in bacterial surface control and host cell interactions (18). GI-5 has 19 ORFs encoding peptide ABC-type transporters such as Dpp, Opp, and Pot systems. Homologs of these transporters in other bacteria are important for root colonization, intracellular survival, attachment to host cell, and virulence (4, 27, 32). B. ovis is highly sensitive to cationic antimicrobial peptides in contrast to other Brucella species, including the rough mutants of B. abortus (14). Similarly, Salmonella enterica serovar Typhimurium Opp mutants exhibit increased susceptibility to antimicrobial peptides and reduced virulence (39). Absence of the Opp system in B. ovis may cause increased uptake of peptides due to disregulation of rate of peptide uptake, influencing their intracellular survival. In fact, B. ovis is more readily destroyed within nonprofessional phagocytes compared to other Brucella (14). Further studies are required to determine whether GI-2 and GI-5 play important roles in Brucella pathogenesis and may constitute novel PIs. Despite lacking these potential virulence factors, B. ovis is virulent in sheep, suggesting that unknown genes in B. ovis that are absent in 16M may contribute to pathogenicity in sheep.

In B. neotomae, denitrification genes were altered. BMEII0986 encodes a CRP/FNR family transcriptional regulator with significant homology to the denitrification regulator nnrR of other α-proteobacteria that is required for anaerobic growth in the presence of nitrite or nitrate and regulates nitrite reductase activity (1, 28, 33, 52). In fact, nnrR (BMEII0986) of B. melitensis 16M restores denitrification in NnrR-deficient A. tumefaciens, suggesting that denitrification is regulated similarly in α-proteobacteria (Seung-Hun Baek, G. Rajashekara, G. A. Splitter, and J. P. Shapleigh, unpublished data). The nitrate, nitrous oxide, and nitric oxide reductase genes of B. melitensis are predicted to be highly expressed (24), suggesting that Brucella can efficiently use alternative energy sources under anaerobic conditions. The lack of nitrite reductase activity suggests that B. neotomae, unlike other Brucella spp., cannot reduce nitrite and is likely not able to use nitrogen as an alternative energy source in oxygen deprivation and be more sensitive to toxic effects of NO encountered within the macrophage.

Our results do not clearly identify deletions that might explain the phenotypic differences, such as oxidase activity, CO2 requirement, H2S production, growth on basic fuchsin- or thionin-containing medium, that are conventionally used to distinguish Brucella species. These phenotypic differences may be related to some of the genes whose functions are unknown or due to mutations such as single base changes that are unlikely to be detected by using microarray hybridization.

Examination of sequences within or flanking several 16M GIs revealed striking similarities in genomic organization to PIs from other bacteria. PIs are mostly found adjacent to or integrated into tRNA genes and flanked by ISs. Integrase genes encoded on phages or plasmids often mediate insertion at tRNA genes, suggesting that PI acquisition is a phage- or plasmid-mediated process (5). Five of the 16M GIs were found either within or adjacent to tRNA genes with ISs flanking one or both ends and have direct or inverted repeats at their ends (Fig. (Fig.3),3), suggesting that these GIs were acquired by lateral gene transfer. Insertions have resulted in either duplication of the entire tRNA gene (GI-1) or reconstitution with a portion of tRNA genes present as a repeat at the opposite end (GI-2 and GI-3). Strikingly, many of the sites of GI integration in Brucella are known to be sites of the integration of islands in other bacteria. Insertion of GI-2 into a Gly-tRNA gene is similar to island insertion in Pseudomonas and Xanthomonas species (29). A symbiosis island in one other member of the α2-proteobacteria, Mesorhizobium loti, also integrates into a Phe-tRNA gene (47) similar to GI-3. Integration into Gly- or Phe-tRNA genes is likely phage mediated, particularly, insertion of GI-3 seems to be mediated by a P4 family integrase by using the terminal CCA of the tRNA gene as an attachment site similar to the symbiosis island of M. loti. Although mechanisms of GI-3 insertion appear similar, the gene contents are different as ORFs in GI-3 have no similar sequence in M. loti or other members of the α-proteobacteria.

Ser-tRNA genes are also frequent targets for horizontal transfer of genetic materials (20, 51), and GI-5 and GI-6 are associated with this locus. Unlike GI-1, -2, or -3, incorporation of GI-5 and GI-6 is likely not phage mediated. GI-5 is present upstream of a Ser-tRNA gene and is flanked by identical copies of an IS similar to S. sonnei IS600 (31). ORFs on GI-5 are conserved in broad range of species and have an atypical trinucleotide composition, suggesting a foreign origin. Whereas, GI-6, is located downstream of a second Ser-tRNA gene and encodes several ISs similar to ORFs on a plasmid pNGR234a of Rhizobium species (15).

In conclusion, our study has identified a defined set of genes missing from Brucella species that are not pathogenic to humans. Our findings now facilitate additional studies to understand the relevance of these genes or gene clusters in host adaptation and virulence restriction of certain Brucella species to humans. Furthermore, our results revealed several genomic islands are likely acquired in Brucella, and many islands have large numbers of ORFs encoding proteins with unknown functions. Brucella lacks well-characterized virulence factors of many pathogenic bacteria such as cytolysins, capsules, exotoxins, secreted proteases, pili and/or fimbriae, flagella, phage-encoded toxins, and virulence plasmids (8, 40). The GIs missing in B. ovis are present in other Brucella species that are pathogenic to humans. However, B. neotomae, a species that is not pathogenic to humans and domestic animals, also possesses these islands. In addition, B. canis and B. suis, although differing in virulence to humans, are genetically very similar. These findings imply that, in addition to the loss or gain of genetic content in Brucella species, mechanisms involving gene inactivation or altered expression of virulence traits as seen in Bordetella species (38) may contribute to differences in host range and virulence of Brucella species for humans. However, we cannot rule out the possibility of genetic contents that are unique to Brucella species other than 16M contributing to these differences. Future studies are necessary to understand the relevance of these GIs to the host adaptation and virulence of Brucella species.

Supplementary Material

[Supplemental material]

Acknowledgments

This study was supported by National Institutes of Health grant R01AI048490, NIH/NIAID Regional Center of Excellence for Biodefense and Emerging Infectious Diseases Research (RCE) Program grant 1-U54-AI-057153, and grants BARD-US 2968-98C (to G.A.S.) and R44HG002193 to Roland Green, NimbleGen Systems.

We thank David Warshauer and Tim Monson at the Wisconsin State Laboratory of Hygiene for providing Brucella strains and Phil Elzer and Sue Hagius at Louisiana State University for providing B. ovis strains. We thank John Luecke, and Irene Ong for assistance with array hybridization and analysis. We also thank Nicole Perna for invaluable discussions.

Footnotes

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

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