Whole-Genome Sequences of Variants of Bacillus anthracis Sterne and Their Toxin Gene Deletion Mutants.

Here, we report the draft genome sequences of three laboratory variants of Bacillus anthracis Sterne and their double (Δlef Δcya) and triple (Δpag Δlef Δcya) toxin gene deletion derivatives.

Complete Genome Assemblies for Two Single-Chromosome Vibrio cholerae Isolates, Strains 1154-74 (Serogroup O49) and 10432-62 (Serogroup O27).

Here, we report the completed genome sequences for two non-O1/non-O139 Vibrio cholerae isolates. Each isolate has only a single chromosome, as opposed to the normal paradigm of two chromosomes found in all other V. cholerae isolates.

Conformation of the hexasaccharide repeating subunit from the Vibrio cholerae O139 capsular polysaccharide.

In the past decade, several outbreaks of cholera have been reported to be caused by Vibrio cholerae O139, a strain which differs from the more common O1 strain in that the former is encapsulated. The hexasaccharide repeating subunit has been isolated from the V. cholerae O139 capsular polysaccharide by digestion with a recently discovered polysaccharide lyase derived from a bacteriophage specific for this serogroup. It specifically cleaves at a single position of the 4-linked galacturonic acid producing an unsaturated sugar product in quantities for conformational studies by (1)H and (13)C NMR spectroscopy. We report conformational studies on this oligosaccharide by molecular modeling and NMR spectroscopy including nuclear Overhauser effects and residual dipolar coupling of a sample weakly oriented in liquid crystalline solution. The structure contains a tetrasaccharide epitope homologous to the human Lewis(b) blood group antigen, which adopts a relatively well-defined single conformation. Comparison of these results with those of a previously published study of the intact capsular polysaccharide indicates that the conformations of the epitope in the two cases are identical or at least closely similar. Thus, this epitope, which may be essential for the pathogenicity of this V. cholerae strain, is not a "conformational epitope" requiring a certain critical size for antigenicity as has been reported for several other bacterial capsular antigens.

Sequence analysis of TnphoA insertion sites in Vibrio cholerae mutants defective in rugose polysaccharide production.

Vibrio cholerae can switch from a smooth to a wrinkled or rugose colony phenotype characterized by the secretion of a polysaccharide that enables the bacteria to survive harsh environmental conditions. In order to understand the genetic basis of rugosity, we isolated TnphoA-induced stable, smooth mutants of two O1 El Tor rugose strains and mapped the insertion sites in several of the mutants using a modified Y-adapter PCR technique. One of the TnphoA insertions was mapped to the first gene of the vps region that was previously shown to encode the rugose polysaccharide biosynthesis cluster. Three insertions were mapped to a previously unknown hlyA-like gene, also in the vps region. Five other insertions were found in loci unlinked to the vps region: (i) in the epsD gene (encodes the "secretin" of the extracellular protein secretion apparatus), (ii) in a hydG-like gene (encodes a sigma(54)-dependent transcriptional activator similar to HydG involved in labile hydrogenase production in Escherichia coli, (iii) in a gene encoding malic acid transport protein upstream of a gene similar to yeiE of E. coli (encodes a protein with similarities to LysR-type transcriptional activators), (iv) in dxr (encodes 1-deoxy-D-xylulose 5-phosphate reductoisomerase), and (v) in the intergenic region of lpd and odp (encode enzymes involved in the pyruvate dehydrogenase complex formation). These data suggest the involvement of a complex regulatory network in rugose polysaccharide production and highlight the general utility of the Y-adapter PCR technique described here for rapid mapping of transposon insertion sites.

Phylogeny of Vibrio cholerae based on recA sequence.

We sequenced a 705-bp fragment of the recA gene from 113 Vibrio cholerae strains and closely related species. One hundred eighty-seven nucleotides were phylogenetically informative, 55 were phylogenetically uninformative, and 463 were invariant. Not unexpectedly, Vibrio parahaemolyticus and Vibrio vulnificus strains formed out-groups; we also identified isolates which resembled V. cholerae biochemically but which did not cluster with V. cholerae. In many instances, V. cholerae serogroup designations did not correlate with phylogeny, as reflected by recA sequence divergence. This observation is consistent with the idea that there is horizontal transfer of O-antigen biosynthesis genes among V. cholerae strains.

Convergence of the secretory pathways for cholera toxin and the filamentous phage, CTXphi.

Virulence of Vibrio cholerae depends on secretion of cholera toxin (CT), which is encoded within the genome of a filamentous phage, CTXphi. Release of CT is mediated by the extracellular protein secretion (eps) type II secretion system. Here, the outer membrane component of this system, EpsD, was shown to be required for secretion of the phage as well. Thus, EpsD plays a role both in pathogenicity and in horizontal transfer of a key virulence gene. Genomic analysis suggests that additional filamentous phages also exploit chromosome-encoded outer membrane channels.

Mutations in the extracellular protein secretion pathway genes (eps) interfere with rugose polysaccharide production in and motility of Vibrio cholerae.

Vibrio cholerae is the causal organism of the diarrheal disease cholera. The rugose variant of V. cholerae is associated with the secretion of an exopolysaccharide. The rugose polysaccharide has been shown to confer increased resistance to a variety of agents, such as chlorine, bioacids, and oxidative and osmotic stresses. It also promotes biofilm formation, thereby increasing the survival of the bacteria in the aquatic environments. Here we show that the extracellular protein secretion system (gene designated eps) is involved directly or indirectly in the production of rugose polysaccharide. A TnphoA insertion in epsD gene of the eps operon abolished the production of rugose polysaccharide, reduced the secretion of cholera toxin and hemolysin, and resulted in a nonmotile phenotype. We have constructed defined mutations of the epsD and epsE genes that affected these phenotypes and complemented these defects by plasmid clones of the respective wild-type genes. These results suggest a major role for the eps system in pathogenesis and environmental survival of V. cholerae.

Cloning and sequencing of the genes downstream of the wbf gene cluster of Vibrio cholerae serogroup O139 and analysis of the junction genes in other serogroups.

The DNA sequence of the O-antigen biosynthesis cluster (wbf) of a recently emergent pathogen, Vibrio cholerae serogroup O139, has been determined. Here we report the sequence of the genes downstream of the O139 wbfX gene and analysis of the genes flanking the wbf gene cluster in other serogroups. The gene downstream of wbfX, designated rjg (right junction gene), is predicted to be not required for O-antigen biosynthesis but appears to be a hot spot for DNA rearrangements. Several variants of the rjg gene (three different insertions and a deletion) have been found in other serogroups. DNA dot blot analysis of 106 V. cholerae strains showed the presence of the left and right junction genes, gmhD and rjg, respectively, in all strains. Further, these genes mapped to a single I-CeuI fragment in all 21 strains analyzed by pulsed-field gel electrophoresis, indicating a close linkage. The insertion sequence element IS1358, found in both O1 and O139 wb* regions, is present in 61% of the strains tested; interestingly, where present, it is predominantly linked to the wb* region. These results indicated a cassette-like organization of the wb* region, with the conserved genes (gmhD and rjg) flanking the divergent, serogroup-specific wb* genes and IS1358. A similar organization of the wb* region in other serogroups raises the possibility of the emergence of new pathogens by homologous recombination via the junction genes.

Instability of pUC19 in Escherichia coli transcription termination factor mutant, rho026.

The higher copy number of pUC19, compared to its parent plasmid pBR322, is known to be due to deletion of rop, also known as rom, and to an ori mutation that impedes RNAI:RNAII interaction. pUC19, unlike pBR322, fails to transform E. coli rho mutant rho026 cells. Here we identify two features of pUC19 that contribute to this transformation defect. (1) The pUCori mutation is involved because replacing the pUCori with that of pBR322 restored transformation. (2) Transcription from the lac promoter in pUC19 is important, since deletion or inversion of the promoter or insertion of a transcription terminator (lambdat0) downstream of it restored transformation. Host RNase E activity is responsible for the transformation defect because introduction of an rne-1 allele into rho026 cells suppressed this defect, indicating that RNAI instability due to RNase E is aggravated in the rho026 strain. We suggest that in rho026 cells pUC19 RNAI:RNAII interaction is more impeded than in rho+ cells and Rop/Rom may confer stability by protecting RNAI against RNase E activity because expression of a rom gene inserted into pUC19 restored transformation.

Effects on mRNA degradation by Escherichia coli transcription termination factor Rho and pBR322 copy number control protein Rop.

Mutants in Escherichia coli transcription termination factor Rho, termed rho(nusD), were previously isolated based on their ability to block the growth of bacteriophage T4. Here we show that rho(nusD) strains have decreased average half-lives for bulk cellular mRNA. Decreased E. coli message lifetimes could be because of increased ribonuclease activity in the rho mutant cells: if a Rho-dependent terminator precedes a ribonuclease gene, weaker termination in the rho mutants could lead to nuclease overexpression. However, inactivation of ribonuclease genes in rho026 cells did not relieve the defective phage growth. Unexpectedly, expression of the pBR322 Rop protein, a structure-specific, sequence-independent RNA-binding protein, in rho(nusD) cells restored the ability of T4 to grow and prolonged cellular message half-life in both the wild-type and the rho026 mutant. These results suggest that it is the RNA-binding ability of Rho rather than its transcription termination function that is important for the inhibition of bacteriophage growth and the shorter bulk mRNA lifetime. We propose that altered interaction of the mutant Rho with mRNA could make the RNA more susceptible to degradation. The inability of the RNA-binding proteins SrmB and DeaD to reverse the rho mutant phenotype when each is overexpressed implies that the required RNA interactions are specific. The results show novel roles for Rho and Rop in mRNA stability.

Relaxation of replication control in chaperone-independent initiator mutants of plasmid P1.

Escherichia coli chaperones DnaJ, DnaK and GrpE increase P1 plasmid initiator binding to the origin by promoting initiator folding. The binding allows initiation and also promotes pairing of origins which is believed to control initiation frequency. Chaperone-independent DNA binding mutants are often defective in replication control. We show here that these mutants have increased rates of association for DNA binding and defects in origin pairing. The increases in association rates were found to be due either to increased protein folding into active forms or to increases in the association rate constant, kon. Since the dissociation rate constants for DNA release with these mutants are not changed, it is unlikely that the DNA binding domain is affected. The pairing domain may thus control replication and modulate DNA binding. The role of the pairing domain in DNA binding can be significant in vivo as the selection for chaperone-independent binding favors pairing-defective mutants.

Heat shock proteins DnaJ, DnaK, and GrpE stimulate P1 plasmid replication by promoting initiator binding to the origin.

Binding of the P1-encoded protein RepA to the origin of P1 plasmid replication is essential for initiation of DNA replication and for autoregulatory repression of the repA promoter. Previous studies have shown defects in both initiation and repression in hosts lacking heat shock proteins DnaJ, DnaK, and GrpE and have suggested that these proteins play a role in the RepA-DNA binding required for initiation and repression. In this study, using in vivo dimethyl sulfate footprinting, we have confirmed the roles of the three heat shock proteins in promoting RepA binding to the origin. The defects in both activities could be suppressed by increasing the concentration of wild-type RepA over the physiological level. We also isolated RepA mutants that were effective initiators and repressors without requiring the heat shock proteins. These data suggest that the heat shock proteins facilitate both repression and initiation by promoting only the DNA-binding activity of RepA. In a similar plasmid, F, initiator mutants that confer heat shock protein independence for replication were also found, but they were defective for repression. We propose that the initiator binding involved in repression and the initiator binding involved in initiation are similar in P1 but different in F.

Participation of the Escherichia coli heat shock proteins DnaJ, DnaK, and GrpE in autorepression of the P1 plasmid repA promoter.

The replicon of the low copy number plasmid P1 uses the three Escherichia coli heat shock proteins DnaJ, DnaK, and GrpE for the efficient initiation of its DNA replication. The only P1-encoded protein required for plasmid replication is the initiator, RepA. Binding of RepA to the origin also represses the promoter for the repA gene, which is located within the origin. We found that repression is incomplete in E. coli strains with mutations in the dnaJ, dnaK, or grpE genes. Since there is no decrease in RepA concentration in the mutant strains, the mutations are likely to affect the protein-DNA or protein-protein reactions required for repression, thereby decreasing RepA binding at its promoter. We also showed that the deficit in repression can be overcome by providing excess RepA, implying that the mechanism of repression is not altered in the mutant strains. Since repression requires RepA binding to the origin, a binding deficit might account for the replication defect in the heat shock mutants.

Plus-origin mapping of single-stranded DNA plasmid pE194 and nick site homologies with other plasmids.

Staphylococcus aureus plasmid pE194 manifests a natural thermosensitivity for replication and can be established in several species, both gram positive and gram negative, thus making it attractive for use as a delivery vector. Like most characterized plasmids of gram-positive bacteria, pE194 generates single-stranded DNA. The direction of pE194 replication is clockwise, as determined by the strandedness of free single-stranded DNA. Significant homology exists between a 50-base-pair sequence in the origin of pE194 and sequences present in plasmids pMV158 (Streptococcus agalactiae), pADB201 (Mycoplasma mycoides), and pSH71 (Lactococcus lactis). We used an initiation-termination reaction, in which pE194 initiates replication at its own origin and is induced to terminate at the related pMV158 sequence, to demonstrate that pE194 replicates by a rolling-circle mechanism; the initiation nick site was localized to an 8-base-pair sequence.

Transposon mutagenesis and genetic mapping of the rglA and rglB loci of Escherichia coli.

The rglA and rglB genes code for two different proteins which cleave the hydroxymethylated cytosine residues of T-even phages. We isolated Tn10 and Tn5 insertion mutants of the above genes and of the genes in and around the rglA and rglB loci. These insertions were used to construct a detailed genetic map. Our results show that the rglA gene maps at 25.24 min and the rglB gene at 98.39 min on the standard Escherichia coli K12 genetic map.