Kenneth M. Peterson

Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes

Four new antibiotic-resistant derivatives of the broad-host-range (bhr) cloning vector pBBR1MCS have been constructed. These new plasmids have several advantages over many of the currently available bhr vectors in that: (i) they are relatively small (< 5.3 kb), (ii) they possess an extended multiple cloning site (MCS), (iii) they allow direct selection of recombinant plasmid molecules in Escherichia coli via disruption of the LacZ alpha peptide, (iv) they are mobilizable when the RK2 transfer functions are provided in trans and (v) they are compatible with IncP, IncQ and IncW group plasmids, as well as with ColE1- and P15a-based replicons.

A putative integrase gene defines the distal end of a large cluster of ToxR-regulated colonization genes in Vibrio cholerae

A large cluster of virulence genes encoding proteins involved in Vibrio cholerae accessory colonization factor (ACF) expression and toxin-coregulated pilus (TCP) biogenesis is flanked by sequences that resemble bacteriophage attachment (att) half-sites. Adjacent to the attL-like site is a gene (int) that encodes a protein related to the integrase family of site-specific recombinases. The putative vibrio integrase appears to be most closely related to the Escherichia coli cryptic prophage (CP4-57) integrase protein (52% identity, 73% similarity). Genomic analysis of numerous V. cholerae strains (O1, non-O1 and O139) revealed that only vibrios capable of causing epidemic Asiatic cholera possess the TCP-ACF colonization gene cluster in association with the integrase. The fact that the integrase gene is absent in avirulent strains suggests that epidemic strains of V. cholerae obtained the TCP-ACF colonization gene cluster via horizontal transfer.

The accessory colonization factor and toxin-coregulated pilus gene clusters are physically linked on the Vibrio cholerae 0395 chromosome

A partial nucleotide sequence of the first gene in the Vibrio cholerae accessory colonization factor gene cluster was obtained by sequencing a cloned acfB::TnphoA fusion junction. Analysis of the sequence data revealed that the ATG start codon of the acfB structural gene is located 6 bp downstream of the tcpJ TGA stop codon. This establishes a large region in excess of 20 kbp of the Vibrio cholerae chromosome as a cluster of coordinately regulated virulence genes involved in intestinal colonization. The absence of a characteristic transcription termination site in the tcpJ-acfB intergenic region indicates that acfB may be encoded on the same messenger RNA as tcpJ. The deduced amino acid sequence for the N-terminal 143 amino acids of AcfB reveals a potential leader peptide, that lacks a consensus signal peptidase I recognition sequence. Computer algorithms fail to detect any significant similarities between the partial AcfB amino acid sequence and any entries in the SWISS-PROT database.

ISOLATION AND CHARACTERIZATION OF A VIBRIO CHOLERAE GENE (TAGA) THAT ENCODES A TOXR-REGULATED LIPOPROTEIN

The Vibrio cholerae (Vc) gene (tagA) coding for the TagA lipoprotein has been isolated. Sequencing of tagA revealed the presence of an open reading frame (ORF) of 568 amino acids with a characteristic signal peptidase II cleavage site at the N terminus. Electrophoretic analysis of proteins synthesized by Escherichia coli (Ec) cells following T7 promoter/RNA polymerase-directed expression of tagA, revealed a closely migrating doublet of proteins corresponding to two species of TagA. Computer-generated alignment algorithms predict that a homology exists between Vc TagA and Ec K99 fimbriae biogenesis determinant FanD.

Identification of a Vibrio cholerae ToxR-activated gene (tagD) that is physically linked to the toxin-coregulated pilus (tcp) gene cluster.

The toxin-coregulated pilus (TCP)-encoding gene cluster (tcp) specifies a type-IV pilus that is a major colonization determinant of Vibrio cholerae. We have identified a gene 200 bp upstream from the tcp cluster that requires ToxR for expression. We have designated this gene tagD (ToxR-activated gene) and have shown that tagD is encoded on a 600-nt transcript. The deduced tagD product is a 164-amino-acid polypeptide (20 kDa). Interestingly, TagD shares a high degree of similarity to a protein of Streptococcus sanguis 12 that is thought to play a role in fimbriae synthesis or assembly. The high degree of similarity between tagD and the Ss 12 protein provides preliminary evidence that tagD represents an additional member of the tcp cluster.

Identification of a ToxR-activated gene, tagE, that lies within the accessory colonization factor gene cluster of Vibrio cholerae O395

The nucleotide (nt) sequence has been determined for a Vibrio cholerae ToxR-activated gene designated tagE that is located within a cluster of genes required for efficient intestinal colonization. The tagE gene encompasses 909 nt and is predicted to encode a 303-amino-acid (aa) protein with an estimated molecular mass of 34,468 Da. Computer-assisted similarity searches revealed that TagE possesses aa sequence similarity with Escherichia coli OrfU and Staphylococcus simulans lysostaphin, two proteins that are involved in cell-wall biosynthesis and peptidoglycan degradation, respectively. The role, if any, that TagE plays in the accessory colonization factor phenotype is currently under investigation.

Isolation and characterization of the Vibrio cholerae acfA gene, required for efficient intestinal colonization

The nucleotide sequence of the Vibrio cholerae acfA gene (encoding an accessory colonization factor) has been determined. Sequence analysis revealed the presence of an open reading frame of 215 amino acids with a characteristic signal peptidase I (SPI) cleavage site at the N terminus. Electrophoretic analysis of proteins synthesized by Escherichia coli cells, following T7 promoter/RNA polymerase-directed expression of acfA, revealed a 23-kDa protein corresponding to the mature form of AcfA. The T7 expression system also showed that, in the presence of known SPI inhibitors, a 25-kDa unprocessed form of AcfA is produced.

The Vibrio cholerae hlyC gene encodes a protein that is related to lipases of Pseudomonas species

The nucleotide sequence of the Vibrio cholerae N16961 hlyC gene was determined. The hlyC gene encompasses 513 nucleotides that are predicted to encode a 171-amino acid protein with a calculated molecular weight of 18.2 kDa. The predicted HlyC protein contains a region that is 93.5% similar to the substrate-binding/catalytic domain of the Pseudomonas species triacylglycerol acylhydrolase (lipase). The proposed catalytic serine residue is also conserved in the HlyC protein. The contribution of the putative HlyC lipase to the physiology of V. cholerae is currently under investigation.

Multiple intraintestinal signals coordinate the regulation of Vibrio cholerae virulence determinants

Vibrio cholerae is a Gram-negative motile bacterium capable of causing fatal pandemic disease in humans via oral ingestion of contaminated water or food. Within the human intestine, the motile vibrios must evade the innate host defense mechanisms, penetrate the mucus layer covering the small intestine, adhere to and multiply on the surface of the microvilli and cause disease via the action of cholera toxin. The explosive diarrhea associated with V. cholerae intestinal colonization leads to dissemination of the vibrios back into the environment to complete this phase of the life cycle. The host phase of the vibrio life cycle is made possible via the concerted action of a signaling cascade that controls the synthesis of V. cholerae colonization determinants. These virulence proteins are coordinately synthesized in response to specific host signals that are still largely undefined. A more complete understanding of the molecular events involved in the V. cholerae recognition of intraintestinal signals and the subsequent transcriptional response will provide important information regarding how pathogenic bacteria establish infection and provide novel methods for treating and/or preventing bacterial infections such as Asiatic cholera. This review will summarize what is currently known in regard to host intraintestinal signals that inform the complex ToxR regulatory cascade in order to coordinate in a spatial and temporal fashion virulence protein synthesis within the human small intestine.