Many critical bacterial virulence factors and determinants of resistance to antimicrobial agents are encoded on mobile genetic elements such as plasmids and bacteriophages. My laboratory studies the mechanisms that regulate transfer of these mobile elements and the ways in which these elements influence production of the virulence factors they encode. We primarily investigate these issues in Vibrio cholerae, the Gram-negative bacterium that causes the severe diarrheal disease cholera.

CTXφ

Vibrio cholerae colonizes the mucosal surface of the human small intestine and secretes cholera toxin. This toxin stimulates secretion of water and electrolytes by the cells of the small intestine, leading to the severe watery diarrhea that is characteristic of cholera. The genes encoding cholera toxin, ctxA and ctxB, are located on the V. cholerae chromosome and were not initially recognized to be part of a self-transmissible genetic element. As a postdoctoral fellow, I discovered that ctxA and ctxB are part of the genome of a filamentous bacteriophage, CTXφ. Thus, the evolution of toxigenic V. cholerae depended upon infection of an ancestral V. cholerae by CTXφ. The CTXφ receptor is TCP, a cell surface organelle that is also a key virulence factor, since it is essential for the organism’s colonization of the intestine.

Compared to the well-characterized filamentous coliphages, such as M13, CTXφ has several unusual features. Unlike M13, CTXφ integrates site-specifically into the V. cholerae genome. Other phages that integrate in a site-specific fashion encode an integrase: an enzyme that catalyzes recombination between the phage and host genomes. CTXφ does not encode an integrase; instead, we found that two chromosome-encoded recombinases (XerC and XerD) that ordinarily function to resolve chromosome dimers are essential for CTXφ integration into the V. cholerae genome. Examination of the sequences of the integration sites of other integrating filamentous phages suggests that the XerCD recombinases also mediate the integration of these phage genomes into their respective hosts’ chromosomes.

CTXφ also differs from M13 in that its genome lacks a homolog of M13’s gene IV, which encodes the outer membrane pore (‘secretin’), pIV, through which M13 exits from its host. We found that V. cholerae produces a pIV-like protein from a chromosomal locus, epsD, that serves as the CTXφ secretin. EpsD is the putative outer membrane pore for the eps (extracellular protein secretion) secretion apparatus that is essential for secretion of cholera toxin and several other virulence factors from V. cholerae. Thus, EpsD plays a role both in pathogenicity and in horizontal transfer of a key virulence gene. Phage exploitation of a host secretin has not been demonstrated previously; however, analyses of the genomes of additional filamentous phages suggest that reliance upon a chromosome-encoded secretin may be a common strategy for phage secretion.

In all characterized isolates of V. cholerae from the ongoing seventh pandemic of cholera, CTXφ DNA is maintained as part of a chromosomal array that contains one or more prophages plus one or more copies of a related genetic element known as RS1. We have discovered that these arrays routinely yield hybrid virions, composed of DNA from two adjacent prophages or from a prophage and a downstream RS1. The presence of tandem elements is required for production of virions. Generation of the replicative (plasmid) form of CTXφ, pCTX, does not depend on reversal of the process for site-specific integration of CTXφ DNA into the chromosome or RecA-mediated homologous recombination between adjacent prophages. Our work suggests that the CTXφ-specific proteins required for replication of pCTX can also function on a chromosomal substrate, and that, unlike the processes used by other integrating phages, production of CTXφ does not require excision of the prophage from the chromosome. Use of this replication strategy maximizes vertical transmission of prophage DNA while still enabling dissemination of CTXφ to new hosts.

The CTXφ related element RS1 contains three genes that are also found in CTXφ, but it lacks the genes encoding cholera toxin and the proteins required for secretion and packaging of CTXφ, such as the phage’s major coat protein. RS1 contains a single gene, rstC, that is not found in the CTXφ genome and that bears no significant homology to any other gene in GenBank. We found that RS1 is a satellite phage whose transmission depends upon proteins produced from a CTX prophage (its helper phage). However, unlike other satellite phages and satellite animal viruses, RS1 can aid the CTX prophage as well as exploit it, due to the function of RstC. RstC is an antirepressor that counteracts the activity of the CTXφ repressor RstR. RstC promotes transcription of genes required for phage production and thereby promotes transmission of both RS1 and CTXφ. Furthermore, antirepression by RstC also induces expression of the cholera toxin genes and thus may contribute to the virulence of V. cholerae.

Transfer of antibiotic resistance by the SXT constin

Vibrio cholerae O139, the first non-O1 serogroup of V. cholerae to give rise to epidemic cholera, is characteristically resistant to several antibiotics. We found that the genes encoding these resistances are present in a 100kb self-transmissible, conjugative, chromosomally integrating element designated SXT. SXT integrates site-specifically into the 5’ end of prfC, the gene encoding peptide chain release factor 3. The mechanism of integration and excision of SXT shares several features with site-specific recombination by lambdoid phages. However, unlike λ and conjugal broad host range plasmids, the extrachromosomal form of SXT is not replicative. Although some of the properties of the SXT element are similar to the so-called conjugative transposons, certain features of SXT are distinctive. We have therefore proposed to classify this element as a constin, an acronym for conjugal, self-transmissible, integrating element.

Constins very similar to SXT have been found in all recent V. cholerae clinical isolates from the Indian subcontinent. We have also identified SXT-like elements in other diarrheal pathogens, suggesting that constins disseminate antibiotic resistances and probably other properties to many bacterial pathogens. Additionally, SXT can mobilize certain plasmids in trans and chromosomal DNA in cis. This finding raises the possibility that SXT and other constins play a general role in horizontal gene transfer among Gram-negative bacteria.

We sequenced the ~100kb SXT genome and found that this element appears to be a chimera composed of plasmid, phage and transposon-associated antibiotic resistance genes. Functional studies revealed that the element’s plasmid-related genes are required for SXT conjugation and its phage-related genes are required for regulation of its transfer, chromosomal integration and excision. The bacterial response to DNA damage (SOS) promotes SXT transfer by diminishing repression by the SXT repressor, SetR, of the SXT transcriptional activators, setC and setD. The discovery of this novel stimulus of conjugative transfer suggests that the use of antimicrobial agents that induce SOS may promote the dissemination of resistance genes.

Phage Control of Virulence of Enterohemorrhagic Escherichia coli

The phage encoded Shiga toxins play a critical role in the pathogenesis of the hemolytic uremic syndrome that can result from infection with E. coli O157:H7 and other Enterohemorrhagic E. coli. In collaboration with David Friedman (Michigan), we have determined that production of Shiga toxin 2 in E. coli O157:H7 depends upon the phage late promoter, pR’. Since transcription initiating at pR’ requires activation of the phage lytic cascade, expression of the Shiga toxin (Stx) genes in E. coli O157:H7 primarily depends on prophage induction. By showing this central role for the prophage in stx gene expression, our findings contradict the prevailing notion that phages merely serve as agents for virulence gene transfer. Since many commonly used antibiotics induce Stx-encoding bacteriophages, our studies provide a plausible explanation for the recent epidemiologic observation that antibiotic treatment is associated with increased morbidity in patients with STEC infections. Furthermore, if toxin production is a consequence of prophage induction, then new therapeutic strategies can be targeted toward preventing prophage induction