Background: In recent years there has been an increasing problem with Staphylococcus aureus strains that are resistant to treatment with existing antibiotics. An important starting point for the development of new antimicrobial drugs is the identification of "essential" genes that are important for bacterial survival and growth.

dTDP-D-glucose 4,6-dehydratase (RmlB) was first identified in the L-rhamnose biosynthetic pathway, where it catalyzes the conversion of dTDP-D-glucose into dTDP-4-keto-6-deoxy-D-glucose. The structures of RmlB from Salmonella enterica serovar Typhimurium in complex with substrate deoxythymidine 5'-diphospho-D-glucose (dTDP-D-glucose) and deoxythymidine 5'-diphosphate (dTDP), and RmlB from Streptococcus suis serotype 2 in complex with dTDP-D-glucose, dTDP, and deoxythymidine 5'-diphospho-D-pyrano-xylose (dTDP-xylose) have all been solved at resolutions between 1.8 A and 2.4 A. The structures show that the active sites are highly conserved. Importantly, the structures show that the active site tyrosine functions directly as the active site base, and an aspartic and glutamic acid pairing accomplishes the dehydration step of the enzyme mechanism. We conclude that the substrate is required to move within the active site to complete the catalytic cycle and that this movement is driven by the elimination of water. The results provide insight into members of the SDR superfamily.

Bordetella pertussis lipopolysaccharide (LPS) is biologically active, being both toxic and immunogenic. Using transposon mutagenesis we have identified a genetic locus required for the biosynthesis of LPS in B. pertussis, which has been cloned and sequenced. We have also identified equivalent loci in Bordetella bronchiseptica and Bordetella parapertussis and cloned part of it from B. parapertussis. The amino acid sequences derived from most of the genes present in the sequenced B. pertussis locus are similar to proteins required for the biosynthesis of LPS and other complex polysaccharides from a variety of bacteria. The genes are in a unique arrangement in the locus. Several of the genes identified are similar to genes previously shown to play specific roles in LPS O-antigen biosynthesis. In particular, the amino acid sequence derived from one of the genes is similar to the enzyme encoded by rfbP from Salmonella enterica, which catalyses the transfer of galactose to the undecaprenol phosphate antigen carrier lipid as the first step in building oligosaccharide O-antigen units, which are subsequently assembled to form polymerized O-antigen structures. Defined mutation of this gene in the B. pertussis chromosome results in the inability to express band A LPS, possibly suggesting that the trisaccharide comprising band A is a single O-antigen-like structure and that B. pertussis LPS is similar to semi-rough LPS seen in some mutants of enteric bacteria.

Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica are closely related subspecies that cause respiratory tract infections in humans and other mammals and express many similar virulence factors. Their lipopolysaccharide (LPS) molecules differ, containing either a complex trisaccharide (B. pertussis), a trisaccharide plus an O-antigen-like repeat (B. bronchiseptica), or an altered trisaccharide plus an O-antigenlike repeat (B. parapertussis). Deletion of the wlb locus results in the loss of membrane-distal polysaccharide domains in the three subspecies of bordetellae, leaving LPS molecules consisting of lipid A and core oligosaccharide. We have used wlb deletion (⌬wlb) mutants to investigate the roles of distal LPS structures in respiratory tract infection by bordetellae. Each mutant was defective compared to its parent strain in colonization of the respiratory tracts of BALB/c mice, but the location in the respiratory tract and the time point at which defects were observed differed significantly. Although the ⌬wlb mutants were much more sensitive to complement-mediated killing in vitro, they displayed similar defects in respiratory tract colonization in C5 ؊/؊ mice compared with wild-type (wt) mice, indicating that increased sensitivity to complement-mediated lysis is not sufficient to explain the in vivo defects. B. pertussis and B. parapertussis ⌬wlb mutants were also defective compared to wt strains in colonization of SCID-beige mice, indicating that the defects were not limited to interactions with adaptive immunity. Interestingly, the B. bronchiseptica ⌬wlb strain was defective, compared to the wt strain, in colonization of the respiratory tracts of BALB/c mice beginning 1 week postinoculation but did not differ from the wt strain in its ability to colonize the respiratory tracts of B-cell-and T-cell-deficient mice, suggesting that wlb-dependent LPS modifications in B. bronchiseptica modulate interactions with adaptive immunity. These data show that biosynthesis of a full-length LPS molecule by these three bordetellae is essential for the expression of full virulence for mice. In addition, the data indicate that the different distal structures modifying the LPS molecules on these three closely related subspecies serve different purposes in respiratory tract infection, highlighting the diversity of functions attributable to LPS of gram-negative bacteria.Bordetellae are gram-negative bacteria that cause respiratory tract infections in mammals. Bordetella pertussis infects only humans, causing whooping cough (pertussis) in unvaccinated children and a milder coughing illness in adults. B. parapertussis also infects humans and causes a similar, albeit less severe, disease. B. bronchiseptica infects a variety of fourlegged mammals, causing atrophic rhinitis in pigs, kennel cough in dogs, and snuffles in rabbits. Most B. bronchiseptica infections, however, are asymptomatic (8). Very small numbers of B. bronchiseptica organisms are sufficient to establish persistent infection in laboratory...

Genes required for infection of mice by Salmonella Typhimurium can be identified by the interrogation of random transposon mutant libraries for mutants that cannot survive in vivo. Inactivation of such genes produces attenuated S. Typhimurium strains that have potential for use as live attenuated vaccines. A quantitative screen, Transposon Mediated Differential Hybridisation (TMDH), has been developed that identifies those members of a large library of transposon mutants that are attenuated. TMDH employs custom transposons with outward-facing T7 and SP6 promoters. Fluorescently-labelled transcripts from the promoters are hybridised to whole-genome tiling microarrays, to allow the position of the transposon insertions to be determined. Comparison of microarray data from the mutant library grown in vitro (input) with equivalent data produced after passage of the library through mice (output) enables an attenuation score to be determined for each transposon mutant. These scores are significantly correlated with bacterial counts obtained during infection of mice using mutants with individual defined deletions of the same genes. Defined deletion mutants of several novel targets identified in the TMDH screen are effective live vaccines.

Nature achieves the epimerization of carbohydrates by a variety of chemical routes. One common route is that performed by the class of enzyme defined by dTDP-6-deoxy-D-xylo-4-hexulose 3,5-epimerase (RmlC) from the rhamnose pathway. Earlier studies failed to identify the key residues in catalysis. We report the 1.3 A structure of RmlC from Streptococcus suis type 2 and its complexes with dTDP-D-glucose and dTDP-D-xylose. The streptococcal RmlC enzymes belong to a separate subgroup, sharing only 25% identity with RmlC from other bacteria, yet the S. suis enzyme has similar kinetic properties and structure to other RmlC enzymes. Structure, sequence alignment, and mutational analysis have now allowed reliable identification of the catalytic residues and their roles.

A defined allelic-replacement mutant of the sly gene, encoding a thiol-activated cytolysin, from a European isolate of Streptococcus suis serotype 2 was generated and characterized. Unlike the parental strain, it is nonhemolytic, noncytotoxic for cultured macrophage-like cells, avirulent in a mouse infection model, yet only slightly attenuated in a porcine model of systemic infection. Streptococcus suis has been described as the etiological agent for a number of infectious disease syndromes in pigs, including arthritis, septicemia, meningitis, and pneumonia. S. suis produces a secreted hemolysin (suilysin) which has been suggested as playing a role in virulence (2,(4)(5)(6)(7)10). In order to investigate the role played by suilysin in the pathogenesis of a European serotype 2 isolate of S. suis, we generated a defined allelic-replacement mutant of the sly gene and compared the wild-type organism with the sly mutant in a number of assays.Bacterial strains and media. S. suis type 2 strain P1/7 was grown on Columbia agar (Oxoid) containing 10% defibrinated horse blood or in liquid cultures of Todd-Hewitt broth (Oxoid) supplemented with 7% fetal calf serum (FCS) (Gibco). Allelicreplacement mutants of S. suis were maintained on 1 g of erythromycin (Sigma) per ml.Mutagenesis of the suilysin gene from S. suis type 2. An erythromycin resistance gene cassette was introduced into an EcoRV site within a 1,278-bp fragment of the sly gene, contained in the vector pT7-Blue (Stratagene), amplified by PCR using primers suis1 (5Ј-AGCTTGACTTACGAGCCACAAG AG-3Ј) and suis2 (5Ј-CCACCATTCCCAAGCTAATCCTGT-3Ј) with chromosomal DNA from P1/7 as a template. The resulting plasmid, pSUI-erm, contains the erm gene in the same orientation as the sly gene. Plasmid pSUI-erm was introduced into P1/7 by electroporation (22.5 V/cm, 25 F, and 1,000 ⍀). A transformant which had undergone a doublecrossover event, confirmed by Southern hybridization and PCR, with concomitant insertion mutation of the sly gene, was isolated and named S7c.Phenotypic analysis of S7c. Overnight growth of suilysin mutant S7c on Columbia blood agar plates revealed no ␤-hemolysis. Secreted proteins from anaerobically grown overnight cultures of P1/7 and S7c were concentrated 100-fold by ammonium sulfate (50%, wt/vol) precipitation; then 10 l each of these preparations was spotted onto a Columbia horse blood agar plate and incubated at 37°C for 30 min. The proteins from P1/7 show clear zones of hemolysis, whereas there is a complete absence of hemolytic activity in the proteins obtained from S7c (Fig. 1a). The hemolytic activity from the wild-type parental bacteria was enhanced by the addition of ␤-mercaptoethanol, as previously reported (7) (Fig. 1a).The lack of expression of suilysin was confirmed by Western blotting. Supernatants from aerobically grown cultures of both P1/7 and S7C (sterilized using a 0.22-m-pore-size filter and concentrated 25-fold using Amicon filters) were probed with a monoclonal antibody (INT-STS-28-02; A. C. Jacobs, Intervet) raised against purified s...

Medicine, John Radcliffe Hospita't Oxford OX3 9DUtCopies of a multi-gene family, named PRTl (protease 3, encoding a subtilisinlike serine protease were cloned from the opportunistic fungal pathogen Pneumocystis carinii. Comparison of the nucleotide sequence of a genomic clone and a cDNA clone of PRTl from P. carinii f. sp. carinii revealed the presence of seven short introns. Several different domains were predicted from the deduced amino acid sequence: an N-terminal hydrophobic signal sequence, a pro-domain, a subtilisin-like catalytic domain, a P-domain (essential for proteolytic activity), a proline-rich domain, a serinerthreoninerich domain and a C-terminal hydrophobic domain. The catalytic domain showed high homology to other eukaryotic subtilisin-like serine proteases and possessed the three essential residues of the catalytic active site. Karyotypic analysis showed that PRTl was a multi-gene family, copies of which were present on all but one of the P. carinii f. sp. carinii chromosomes. The different copies of the PRTl genes showed nucleotide sequence heterogeneity, the highest level of divergence being in the proline-rich domain, which varied in both length and composition. Some copies of PRTl were contiguous with genes encoding the P. carinii major surface glycoprotein. UK