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The Brachyspira hyodysenteriae B204 genome sequence revealed three VSH-1 tail genes, hvp31, hvp60, and hvp37, in a 3.6-kb cluster. The location and transcription direction of these genes relative to those of the previously described VSH-1 16.3-kb gene operon indicate that the gene transfer agent VSH-1 has a noncontiguous, divided genome.

VSH-1 is a mitomycin C-inducible prophage of the anaerobic spirochete Brachyspira hyodysenteriae. Purified VSH-1 virions are noninfectious, contain random 7.5-kb fragments of the bacterial genome, and mediate generalized transduction of B. hyodysenteriae cells. In order to identify and sequence genes of this novel gene transfer agent (GTA), proteins associated either with VSH-1 capsids or with tails were purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal amino acid sequences of 11 proteins were determined. Degenerate PCR primers were designed from the amino acid sequences and used to amplify several VSH-1 genes from B. hyodysenteriae strain B204 DNA. A lambda clone library of B. hyodysenteriae B204 DNA was subsequently screened by Southern hybridization methods and used to identify and sequence overlapping DNA inserts containing additional VSH-1 genes. VSH-1 genes spanned 16.3 kb of the B. hyodysenteriae chromosome and were flanked by bacterial genes. VSH-1 identified genes and unidentified, intervening open reading frames were consecutively organized in head (seven genes), tail (seven genes), and lysis (four genes) clusters in the same transcriptional direction. Putative lysis genes encoding endolysin (Lys) and holin proteins were identified from sequence and structural similarities of their translated protein products with GenBank bacteriophage proteins. Recombinant Lys protein hydrolyzed peptidoglycan purified from B. hyodysenteriae cells. The identified VSH-1 genes exceed the DNA capacity of VSH-1 virions and do not encode traditional bacteriophage early functions involved in DNA replication. These genome properties explain the noninfectious nature of VSH-1 virions and further confirm its resemblance to known prophage-like, GTAs of other bacterial species, such as the GTA from Rhodobacter capsulatus. The identification of VSH-1 genes will enable analysis of the regulation of this GTA and should facilitate investigations of VSH-1-like prophages from other Brachyspira species.

Brucellosis is a worldwide disease of humans and livestock that is caused by a number of very closely related classical Brucella species in the alpha-2 subdivision of the Proteobacteria. We report the complete genome sequence of Brucella abortus field isolate 9-941 and compare it to those of Brucella suis 1330 and Brucella melitensis 16 M. The genomes of these Brucella species are strikingly similar, with nearly identical genetic content and gene organization. However, a number of insertion-deletion events and several polymorphic regions encoding putative outer membrane proteins were identified among the genomes. Several fragments previously identified as unique to either B. suis or B. melitensis were present in the B. abortus genome. Even though several fragments were shared between only B. abortus and B. suis, B. abortus shared more fragments and had fewer nucleotide polymorphisms with B. melitensis than B. suis. The complete genomic sequence of B. abortus provides an important resource for further investigations into determinants of the pathogenicity and virulence phenotypes of these bacteria.

The locus of enterocyte effacement (LEE), which includes five major operons (LEE1 through LEE4 and tir), enables enterohemorrhagic Escherichia coli (EHEC) O157:H7 to produce attaching and effacing lesions on host cells. Expression of LEE2, LEE3, and tir is positively regulated by ler, a gene located in LEE1. Transcriptional regulation of the esp operon (LEE4), however, is not well defined. Transposon mutagenesis was used to identify transcriptional regulators of the esp operon by screening for mutants with increased beta-galactosidase activity in an EHEC O157:H7 strain harboring an esp::lac transcriptional fusion. All mutants with significant increases in beta-galactosidase activity had transposon insertions in hha (hha::Tn). Specific complementation of the hha::Tn mutation with a plasmid-encoded copy of hha reduced beta-galactosidase activity to the level expressed in the parental esp::lac strain. Purified Hha, however, bound poorly to the esp promoter, suggesting that Hha might repress the transcription of a positive regulator of esp. Transposon mutagenesis of a Deltahha esp::lac strain expressing elevated levels of beta-galactosidase resulted in ler mutants with reduced beta-galactosidase activity. Purified Hha bound to the ler promoter with a higher affinity, and complementation of a Deltahha mutation in a Deltahha ler::lac strain repressed beta-galactosidase activity to the level expressed in a ler::lac strain. A positive regulatory role of ler in esp expression was demonstrated by specific binding of Ler to the esp promoter, reduced expression of beta-galactosidase in Deltaler esp::lac strains with and without hha, and severalfold-increased transcription of ler and espA in strains lacking hha. These results indicate that hha-mediated repression of ler causes reduced expression of the esp operon.

Leptospires belong to a genus of parasitic bacterial spirochetes that have adapted to a broad range of mammalian hosts. Mechanisms of leptospiral molecular evolution were explored by sequence analysis of four genes shared by 38 strains belonging to the core group of pathogenic Leptospira species: L. interrogans, L. kirschneri, L. noguchii, L. borgpetersenii, L. santarosai, and L. weilii. The 16S rRNA and lipL32 genes were highly conserved, and the lipL41 and ompL1 genes were significantly more variable. Synonymous substitutions are distributed throughout the ompL1 gene, whereas nonsynonymous substitutions are clustered in four variable regions encoding surface loops. While phylogenetic trees for the 16S, lipL32, and lipL41 genes were relatively stable, 8 of 38 (20%) ompL1 sequences had mosaic compositions consistent with horizontal transfer of DNA between related bacterial species. A novel Bayesian multiple change point model was used to identify the most likely sites of recombination and to determine the phylogenetic relatedness of the segments of the mosaic ompL1 genes. Segments of the mosaic ompL1 genes encoding two of the surface-exposed loops were likely acquired by horizontal transfer from a peregrine allele of unknown ancestry. Identification of the most likely sites of recombination with the Bayesian multiple change point model, an approach which has not previously been applied to prokaryotic gene sequence analysis, serves as a model for future studies of recombination in molecular evolution of genes.

Serpulina hyodysenteriae B204 cells treated with mitomycin (20 microg of mitomycin/ml of culture broth) lysed and released bacteriophages. Bacteriophage particles, precipitated by using polyethylene glycol and purified by CsC1 density gradient ultracentrifugation, had a buoyant density of 1.375 g/cm3 and consisted of a head (45-nm diameter) and an ultrastructurally simple (noncontractile) tail (64 by 9 nm) composed of at least 13 proteins with molecular masses ranging between 13 and 101 kDa. The purified bacteriophage has been designated VSH-1 (VSH for virus of S. hyodysenteriae). VSH-1 was incapable of lytic growth on any of five intestinal spirochete strains, representing three Serpulina species. VSH-1 nucleic acid was determined to be approximately 7.5 kb in size and to be linear, double-stranded DNA based on differential staining with acridine orange, DNase I sensitivity, electrophoretic mobility, and contour length as measured by electron microscopy. Phage DNA digested by the restriction enzymes SspI, AseI, EcoRV, and AflII gave electrophoretic banding patterns nearly identical to those of digested chromosomal DNA from S. hyodysenteriae. Additionally, VSH-1 DNA fragments hybridized with probes complementary to S. hyodysenteriae chromosomal genes nox and flaA1. When purified bacteriophages induced from cultures of S. hyodysenteriae A203 (deltaflaA1 593-762::cat) were added to growing cells of strain A216 (deltanox 438-760::kan), transductants (Cmr Kmr) were obtained at a frequency of 1.5 x l0(-6) per phage particle (enumerated by electron microscopy). These findings indicate that induced VSH-1 virions package DNA of S. hyodysenteriae and are capable of transferring host genes between cells of that spirochete. To our knowledge, this is the first report of genetic transduction of a spirochete.

A combined physical and genetic map of the Serpulina hyodysenteriae B78T genome was constructed by using pulsed-field gel electrophoresis and DNA blot hybridizations. The S. hyodysenteriae genome is a single circular chromosome about 3.2 Mb in size. The physical map of the chromosome was constructed with the restriction enzymes BssHII, EclXI, NotI, SalI, and SmaI. The physical map was used to constructed a linkage map for genes encoding rRNA, flagellum subunit proteins, DNA gyrase, NADH oxidase, and three distinct hemolysins. Several flaB2-related loci, encoding core flagellum subunit proteins, were detected and are dispersed around the chromosome. The rRNA gene organization in S. hyodysenteriae is unusual. S. hyodysenteriae has one gene each for 5S (rrf), 16S (rrs), and 23S (rrl) rRNAs. The rrf and rrl genes are closely linked (within 5 kb), while the rrs gene is about 860 kb from the other two rRNA genes. Using a probe for the S. hyodysenteriae gyrA gene, we identified a possible location for the chromosomal replication origin. The size and genetic organization of the S. hyodysenteriae chromosome are different from those of previously characterized spirochetes.

Genetic maps were constructed for Leptospira interrogans serovars icterohaemorrhagiae and pomona. Previously we independently constructed physical maps of the genomes for these two serovars. The genomes of both serovars consist of a large replicon (4.4 to 4.6 Mb) and a small replicon (350 kb). Genes were localized on the physical maps by using Southern blot analysis with specific probes. Among the probes used were genes encoding a variety of essential enzymes and genes usually found near bacterial chromosomal replication origins. Most of the essential genes are on the larger replicon of each serovar. However, the smaller replicons of both serovars contain the asd gene. The asd gene encodes aspartate beta-semialdehyde dehydrogenase, an enzyme essential in amino acid and cell wall biosyntheses. The finding that both L. interrogans replicons contain essential genes suggests that both replicons are chromosomes. Comparison of the genetic maps of the larger replicons of the two serovars showed evidence of large rearrangements. These data show that there is considerable intraspecies heterogeneity in L. interrogans.

The structure and composition of periplasmic flagella (PF) from Leptospira interrogans serovar pomona type kennewicki were characterized. Electron microscopic observations showed that leptospiral PF were complex structures composed of an 11.3-nm-diameter core surrounded by two sheath layers with 21.5- and 42-nm diameters. Two-dimensional gel electrophoresis of isolated PF showed the presence of seven different proteins ranging in mass from 31.5 to 36 kDa. Rabbit polyclonal and mouse monoclonal antibodies against PF proteins were prepared and were used to localize specific proteins to portions of the PF structure by immunoelectron microscopy. A 34-kDa protein was associated with the 11.3-nm-diameter core filament, while a 36-kDa protein was associated with a PF sheath (21.5-nm-diameter filament). The amino termini of the 34- and 35.5-kDa proteins were homologous to PF core proteins of other spirochetes. The experimental data suggested that L. interrogans PF contains 2 proteins (34 and 35.5 kDa) in the PF core.

The Leptospira biflexa rpsL and rpsG genes were sequenced. Although similar in many respects, proteins encoded by these L. biflexa genes had several unusual features when compared with homologous proteins of other organisms. Unlike the rpsL genes of other eubacteria, the L. biflexa rpsL gene is adjacent to a rpoC-like gene.

Coxiella burnetii was isolated from persistently infected fibroblast host cells by a rapid mechanical lysis technique. Macromolecular synthesis was initiated in these otherwise dormant cells by incubation at pH 4.5. The synthesis of protein proceeded for as long as 24 h. Initiation of protein synthesis in C. burnetii was dependent upon RNA synthesis. Approximately 24 species of polypeptides were synthesized, and some of these appeared to be major synthetic products. Increases in protein biomass of 15 to 30% were calculated to occur during incubation. Inhibition of DNA synthesis affected protein synthesis after 12 h of incubation. The results suggest that although these parasitic bacteria did not grow in the axenic media devised, significant biosynthetic processes occurred.

The genus Leptospira, as a member of the order Spirochaetales, forms one of the most ancient evolutionary branches of the eubacteria. These spirochetes are morphologically and physiologically different from most eubacteria, and little is known about Leptospira genetics. In this communication, we report the first nucleotide sequence of a Leptospira gene. A gene which complements an argE mutation in Escherichia coli was isolated from a plasmid-based genomic library composed of Leptospira biflexa serovar patoc DNA. The functional region for the complementing activity was localized by transposon mutagenesis and restriction enzyme mapping and by subcloning. Nucleotide sequence analysis indicated a single open reading frame within the region containing argE complementing activity. The size of the predicted protein, 31,071 daltons, was in excellent agreement with data obtained from coupled transcription-translation reactions primed with cloned L. biflexa DNA. One surprising result was that the predicted amino acid sequence of this protein closely resembles portions of the beta' subunits of RNA polymerases from bacteria and chloroplasts.