The Vibrio cholerae O139 Calcutta bacteriophage CTXphi is infectious and encodes a novel repressor

Abstract

CTXphi is a lysogenic, filamentous bacteriophage. Its genome includes the genes encoding cholera toxin (ctxAB), one of the principal virulence factors of Vibrio cholerae; consequently, nonpathogenic strains of V. cholerae can be converted into toxigenic strains by CTXphi infection. O139 Calcutta strains of V. cholerae, which were linked to cholera outbreaks in Calcutta, India, in 1996, are novel pathogenic strains that carry two distinct CTX prophages integrated in tandem: CTX(ET), the prophage previously characterized within El Tor strains, and a new CTX Calcutta prophage (CTX(calc)). We found that the CTX(calc) prophage gives rise to infectious virions; thus, CTX(ET)phi is no longer the only known vector for transmission of ctxAB. The most functionally significant differences between the nucleotide sequences of CTX(calc)phi and CTX(ET)phi are located within the phages' repressor genes (rstR(calc) and rstR(ET), respectively) and their RstR operators. RstR(calc) is a novel, allele-specific repressor that regulates replication of CTX(calc)phi by inhibiting the activity of the rstA(calc) promoter. RstR(calc) has no inhibitory effect upon the classical and El Tor rstA promoters, which are instead regulated by their cognate RstRs. Consequently, production of RstR(calc) renders a CTX(calc) lysogen immune to superinfection by CTX(calc)phi but susceptible (heteroimmune) to infection by CTX(ET)phi. Analysis of the prophage arrays generated by sequentially integrated CTX phages revealed that pathogenic V. cholerae O139 Calcutta probably arose via infection of an O139 CTX(ET)phi lysogen by CTX(calc)phi.

FIG. 1

Structure and sequence of CTX prophages within AS207, an O139 Calcutta strain of V. cholerae. (A) Within the AS207 chromosome, an RS1 element and a CTX^ET prophage are followed by two CTX^calc prophages (shown in light grey). The Calcutta prophage is structurally similar to the previously described CTX prophages within El Tor and classical strains, which contain two major domains known as RS2 and core. RS2 contains three genes (rstA, rstB, and rstR) whose transcriptional orientation is indicated by the arrows. The black triangles represent attRS-ER sequences. (B) Alignment of the nucleotide sequences of rstR, ig-2, and parts of rstA and ig-1 from Calcutta, El Tor, and classical prophages. The arrows underneath the sequences depict the ORFs that encode the three variants of RstR.

FIG. 2

Detection of transfer of CTX^calcφ and CTX^ETφ from supernatants of AS207 to O395 using Southern blot analysis of plasmid DNA. Undigested plasmid DNAs were run on agarose gels, transferred to a nylon membrane, and sequentially hybridized with probes for rstR^calc (left panel) and rstR^ET (right panel). Plasmid DNA was prepared from O395 (lanes 1), AS207 (lanes 2), 2740-80 (CTX^ET-Kn) (lanes 3), O395 cultured with AS207 supernatant (lanes 4), and O395 cultured with 2740-80 (CTX^ET-Kn) supernatant (lanes 5). The Kn^r cassette is slightly larger than the ctxAB genes it replaces in CTX^ET-Knφ, so pCTX^ET-Knφ migrates more slowly than pCTX^ETφ.

FIG. 3

Relative efficiency of transformation of pCTX^calc-Ap versus pCTX^ET-Ap into 2740-80, 2740-80 (CTX^ET-Kn), and 2740-80 (CTX^calc-Kn). Percentage of total Ap^r transformants = (pCTX^calcAp or pCTX^ETAp transformants)/(pCTX^calcAp + pCTX^ETAp transformants).

FIG. 4

Integration site selection by sequentially integrated plasmids. (A) PCR followed by restriction digestion was used to determine the order of prophages within the chromosome following sequential integration of CTX^ET and CTX^calc antibiotic-marked plasmids. When the CTX^calc prophage is the furthest 5′, the TLCF1-RstR Rev PCR product contains a HindIII site but not a BglII site. Conversely, when CTX^ET is upstream, the PCR product contains a BglII site but not a HindIII site. (B) A representative agarose gel containing PCR products digested with HindIII. Template DNA is as follows: 2740-80(CTX^ET-Kn) transformed with pCTX^calc-Ap (lanes 1 to 6), 2740-80 (CTX^calc-Kn) transformed with pCTX^ET-Ap (lanes 7 to 12), and 2740-80 (CTX^calc-Kn) (lane 13). (C) Summary and model of integration site selection following sequential CTXφ integration. Phage DNA does not integrate into the 5′ ER (black triangle) if the chromosome already contains a CTX prophage. Instead, the new phage DNA inserts into the 3′ ER.