The Cyclic AMP (cAMP)-cAMP Receptor Protein Signaling System Mediates Resistance of Vibrio cholerae O1 Strains to Multiple Environmental Bacteriophages

Toxigenic Vibrio cholerae, the causative agent of the epidemic diarrheal disease cholera, interacts with diverse environmental bacteriophages. These interactions promote genetic diversity or cause selective enrichment of phage-resistant bacterial clones. To identify bacterial genes involved in mediating the phage-resistant phenotype, we screened a transposon insertion library of V. cholerae O1 El Tor biotype strain C6706 to identify mutants showing altered susceptibility to a panel of phages isolated from surface waters in Bangladesh. Mutants with insertion in cyaA or crp genes encoding adenylate cyclase or cyclic AMP (cAMP) receptor protein (CRP), respectively, were susceptible to a phage designated JSF9 to which the parent strain was completely resistant. Application of the cyaA mutant as an indicator strain in environmental phage monitoring enhanced phage detection, and we identified 3 additional phages to which the parent strain was resistant. Incorporation of the cyaA or crp mutations into other V. cholerae O1 strains caused similar alterations in their phage susceptibility patterns, and the susceptibility correlated with the ability of the bacteria to adsorb these phages. Our results suggest that cAMP-CRP-mediated downregulation of phage adsorption may contribute to a mechanism for the V. cholerae O1 strains to survive predation by multiple environmental phages. Furthermore, the cyaA or crp mutant strains may be used as suitable indicators in monitoring cholera phages in the water.Bacteriophages contribute to the evolution of bacteria by mediating horizontal gene transfer and genomic rearrangements, as well as by bactericidal selection, in which bacterial strains that are able to resist phage predation thrive over competing phage-susceptible strains (5, 10, 11). Toxigenic Vibrio cholerae, the causative agent of the epidemic diarrheal disease cholera, interacts with diverse phages, both in the aquatic environment and in the host milieu, and these interactions may promote genetic diversity and/or cause selective enrichment of particular bacterial clones (10, 11, 26, 27).Historically, cholera is an ancient disease with the occurrence of seven distinct pandemics since the first pandemic of cholera began in 1817, but the disease still affects millions of people (9, 16). The current seventh pandemic of cholera, which originated in Indonesia in 1961, is the most extensive in geographic spread and duration, and the causative agent is V. cholerae O1 of the El Tor biotype. The sixth pandemic and presumably the earlier pandemics were caused by the classical biotype, which now seems to be extinct.Molecular epidemiological surveillance has revealed continually changing relative prevalences of different clones of pathogenic V. cholerae (9), and the emergence of new clones has been attributed to possible horizontal transfer of clusters of genes associated with virulence or environmental fitness as well as resistance to different antibiotics (9, 20). The recent recognition that phage predation may play a role in the natural control of cholera epidemics (10, 11, 14) reinforces predictions that changes in this pathogen and the prevalences of different clones may also be driven by environmental phages. The emergence of certain strains is likely to be enhanced by phages through the bactericidal mechanism in which phage-sensitive strains are killed while providing a selective advantage to phage-resistant strains. Therefore, the ability to evade phage predation constitutes an important factor in attaining increased evolutionary fitness.In the present study we screened a transposon insertion library of V. cholerae O1 El Tor biotype strain C6706, to identify genes whose inactivation would enhance the susceptibility of the bacteria to environmental phages. Presumably, these genes contribute in mediating resistance to the relevant phages and thus allow the bacteria to survive phage predation. Bacteria with increased phage susceptibility due to mutations in the appropriate genes may also have application as improved indicator strains to monitor the prevalence of relevant phages in the environment.