Harvard Catalyst Profiles

The focus of my research has been on the plasticity and maintenance of the Vibrio cholerae genome. Lateral gene transfer has played a major role in the evolution of pathogenic bacteria because most virulence factors are encoded on mobile genetic elements. My work has focused on two virulence-linked mobile elements in Vibrio cholerae, the agent of cholera. My laboratory has defined several of the molecular steps in the life-cycles of two novel mobile genetic elements: 1) CTXƒÖ, an integrating filamentous phage that encodes cholera toxin, the principal virulence factor of Vibrio cholerae, and 2) SXT, a V. cholerae-derived integrating conjugative element (ICE) that encodes multiple antibiotic resistance genes.

CTXƒÖ infection of non-toxigenic V. cholerae strains can render them fully pathogenic. My laboratory has dissected many of the events in the CTXƒÖ lifecycle and demonstrated the profound dependence of CTXƒÖ on its host. Cellular factors directly mediate integration of the CTXƒÖ genome into the V. cholerae chromosome, secretion of viral particles, and regulation of phage gene transcription. Currently, we are defining the molecular features of the ¡¥genetic switch¡¦ that controls CTXƒÖ prophage induction to further knowledge of the molecular controls that govern cholera toxin gene transfer.

We are studying SXT, a V. cholerae-derived integrative conjugative element (ICE) that encodes resistance to multiple antibiotics, to learn about the environmental and genetic factors that control dissemination of antibiotic resistance genes and to define the key properties of this poorly understood but pervasive class of mobile elements. Like conjugative plasmids, ICEs are transferred between cells in a cell-contact dependent fashion; unlike plasmids, ICEs do not autonomously replicate but integrate into the chromosome of the new host. We have carried out genomic and functional analyses of the ~100kb SXT and identified many of the genes that mediate its integration, excision, and conjugation. My laboratory discovered that SXT is part of a family of closely related ICEs and, most importantly, defined some of the key components of a regulatory circuit that controls SXT transfer. Currently we are exploring the molecular mechanisms that mediate the generation of novel ICEs and investigating the mechanisms that limit SXT transfer.

Another focus of my research has been the study of the mechanisms that control and coordinate the replication and segregation of the two V. cholerae chromosomes. Studies of prokaryotic chromosome replication and segregation have focused almost exclusively on organisms with one chromosome. We defined and characterized the origins of replication of the two V. cholerae chromosomes, oriCIvc and oriCIIvc. OriCIIvc-based replication requires a hypothetical gene (designated rctB) that flanks oriCIIvc. RctB is conserved among diverse genera of the family Vibrionaceae and encodes an origin binding protein. Currently we are defining the biochemical activities of RctB and conducting high-throughput screens to identify small molecules inhibitors of this essential Vibrionaceae-specific replication factor.

We found that in all stages of the cell cycle, the two origins localize to distinct subcellular locations. The differences in localization and timing of segregation of oriCIvc and oriCIIvc suggest that distinct mechanisms govern the segregation of the two V. cholerae chromosomes. Currently, we are exploring the mechanisms that mediate the segregation of the two chromosomes.

Another area of my work has been the study of Vibrio cholerae sRNAs. Recently it has become clear from studies in E. coli that small untranslated RNAs (sRNAs) regulate many cellular processes. We have evidence that sRNAs regulate V. cholerae virulence. We developed a computer program, sRNAPredict, which enables the rapid identification of putative sRNAs in intergenic regions of bacterial genomes. Currently, we are investigating the targets and mechanisms of action of several of the V. cholerae sRNAs that were identified using this software and exploring the use of new high density sequencing technologies in sRNA discovery.

In addition, we are studying the pathogenicity of enterohemorrhagic Escherichia coli (EHEC). EHEC (E. coli O157), are important foodborne pathogens. In collaboration with David Friedman (U. Mich.), we showed that transcription of phage-borne genes encoding Shiga toxin (Stx), the principal EHEC virulence factor, is largely dependent on a phage promoter and that toxin release depends on phage mediated cell lysis. Our current goals are to explore Stx prophage induction within the intestine and to identify previously uncharacterized horizontally transmitted genes that influence EHEC intestinal colonization using an infant rabbit model of EHEC pathogenesis that we developed.