Genomic analysis of the Mozambique strain of Vibrio cholerae O1 reveals the origin of El Tor strains carrying classical CTX prophage

Abstract

Cholera outbreaks in subSaharan African countries are caused by strains of the El Tor biotype of toxigenic Vibrio cholerae O1. The El Tor biotype is the causative agent of the current seventh cholera pandemic, whereas the classical biotype, which was associated with the sixth pandemic, is now extinct. Besides other genetic differences the CTX prophages encoding cholera toxin in the two biotypes of V. cholerae O1 have distinct repressor (rstR) genes. However, recent incidences of cholera in Mozambique were caused by an El Tor biotype V. cholerae O1 strain that, unusually, carries a classical type (CTX(class)) prophage. We conducted genomic analysis of the Mozambique strain and its CTX prophage together with chromosomal phage integration sites to understand the origin of this atypical strain and its evolutionary relationship with the true seventh pandemic strain. These analyses showed that the Mozambique strain carries two copies of CTX(class) prophage located on the small chromosome in a tandem array that allows excision of the prophage, but the excised phage genome was deficient in replication and did not produce CTX(class) virion. Comparative genomic microarray analysis revealed that the strain shares most of its genes with the typical El Tor strain N16961 but did not carry the TLC gene cluster, and RS1 sequence, adjacent to the CTX prophage. Our data are consistent with the Mozambique strain's having evolved from a progenitor similar to the seventh pandemic strain, involving multiple recombination events and suggest a model for origination of El Tor strains carrying the classical CTX prophage.

Fig. 1.

Results of microarray-based comparative genomic analyses of the Mozambique strain of V. cholerae O1 with diverse V. cholerae O1 and non-O1 strains. The identity of strains shown in different lanes are A–E: WBDV-101E, WBDH697, WBDH712, FY2G, and NWBD40–1F1; F–K: WBDH552, WBDH336, AM15622, AM19226, MZO2, and MZO3; L–N: 569B, NIH41, and O395; O–R: MAK757, C6709, HK1, and N16961; S: Mozambique strain B-33; T and U: 2470–80 and NCTC; and V: MO10. The serogroups of different strains are shown above the figure.

Fig. 2.

Sequence comparison and alignment of chromosome I dif sites in various V. cholerae strains. The attRS1 site is underlined; B33 is the 2004 Mozambique isolate; N16961 seventh pandemic El Tor; O395 classical biotype; 2740–80 is an environmental isolate that is clonal with TCP⁺CTX⁺ isolates but lacks CTX; AM19226 and 1587 are non-O1, non-O139 clinical isolates of V. cholerae; and RC385 is a non-O1, non-O139 environmental isolate of V. cholerae.

Fig. 3.

Arrangement of the CTX prophage, RS1, TLC, and their adjacent genes as well as various attachment sites and related sequences on both chromosomes of a typical El Tor strain N16961, a classical strain O395, and the recently emerged Mozambique strain B-33. N16961 Chromosome I, dif core1 and dif core2 sites are separated by CTX^ET. Both dif core sites are deviated from consensus, and they contain either an attRS1 or end repeat (ER, similar 17-bp sequence, instead of 18 bp). N16961 chromosome II, no phage insertion, therefore dif core1 and dif core2 are intact (overlapping). O395 chromosome I, similar to N16961 dif core1 and dif core2, they have either overlapping attRS1 or ER site, but dif cor1 and dif core2 are separated only by CTX^Class. O395 Chromosome II, CTX^Class is inserted into dif core1 and dif core2 sites with the exception that dif core1 does not contain attRS1 or ER site. B33 Chromosome I, contains a dif core2 site with ER but does not have a dif core1 site and lacks TLC. B33 Chromosome II, as in O395 chromosome II, CTX^Class is integrated into dif core1 and dif core2 sites and dif core1 does not include attRS1 or ER.

Fig. 4.

Proposed recombination event in the deletion of the entire region containing CTX prophage, RS1, and TLC from the large chromosome of a putative precursor strain in the pathway to the emergence of the Mozambique strain B-33.