CTX prophages in classical biotype Vibrio cholerae: functional phage genes but dysfunctional phage genomes

Abstract

CTXphi is a filamentous, lysogenic bacteriophage whose genome encodes cholera toxin, the primary virulence factor produced by Vibrio cholerae. CTX prophages in O1 El Tor and O139 strains of V. cholerae are found within arrays of genetically related elements integrated at a single locus within the V. cholerae large chromosome. The prophages of O1 El Tor and O139 strains generally yield infectious CTXphi. In contrast, O1 classical strains of V. cholerae do not produce CTXphi, although they produce cholera toxin and they contain CTX prophages integrated at two sites. We have identified the second site of CTX prophage integration in O1 classical strains and characterized the classical prophage arrays genetically and functionally. The genes of classical prophages encode functional forms of all of the proteins needed for production of CTXphi. Classical CTX prophages are present either as solitary prophages or as arrays of two truncated, fused prophages. RS1, a genetic element that is closely related to CTXphi and is often interspersed with CTX prophages in El Tor strains, was not detected in classical V. cholerae. Our model for CTXphi production predicts that the CTX prophage arrangements in classical strains will not yield extrachromosomal CTX DNA and thus will not yield virions, and our experimental results confirm this prediction. Thus, failure of O1 classical strains of V. cholerae to produce CTXphi is due to overall deficiencies in the structures of the arrays of classical prophages, rather than to mutations affecting individual CTX prophage genes.

FIG. 1

Derivatives of pCTX^ET that do not encode all of the phage proteins required for virion production are packaged into infectious virions by a classical strain of V. cholerae, O395. O395 transformed with wild-type pCTX^ET-Kn or a plasmid derivative containing an incomplete or mutant phage genome was used as a source of donor supernatant in transduction assays. At least one gene required for production of virions was inactive in or absent from each of the plasmids (except pCTX^ET-Kn), yet all of the plasmids yielded virions when maintained in O395. Phage titer per milliliter was normalized to the A₆₀₀ of the donor culture. Titers are average results based on at least three experiments. Sequences that no longer yield functional proteins are marked by diagonal grey lines. (S) and (M) denote SphI and MluI restriction sites that were filled in and religated to create frameshift mutations. It is not known whether the orfU mutation in pMW102 has a polar effect on downstream phage genes (marked with light stripes). Deletion of the PstI (P) fragment results in loss or inactivation of three phage genes. The Kn^r cassette has been inserted in place of most of ctxAB, which do not contribute to CTXφ production. pGP704 is an Ap^r suicide vector that cannot replicate independently in V. cholerae. N.A., not applicable.

FIG. 2

Comparison of the junctional sequences between CTX prophages and adjacent chromosomal DNA in classical and El Tor strains. pGP2 and pGP3 are plasmids derived from the prophages of the classical strain 569B (13). N16961 is the O1 El Tor strain sequenced by the V. cholerae genome project. The empty integration site in N16961 is the locus where the CTX prophage(s) are found on the small chromosome of classical V. cholerae. (A) Analysis of 3′ and 5′ ER sequences. Underlined bases differ from the typical El Tor junction sequences. Known genes and putative homologs that flank junction sequences are indicated. (B) Schematic alignment of sequences downstream of CTX prophage integration sites. Black triangles represent ER variants. Dark grey rectangles represent areas with essentially identical sequences. Light grey rectangles represent areas with similar sequences; the overlaid black lines and boxes represent differences and insertions. Adjacent genes are indicated. The diagram is not drawn to scale.

FIG. 3

Expected and actual structures of prophage arrays in classical strains of V. cholerae. Instead of an RS1 followed by a CTX prophage, CTX insertion sites that contain tandem elements in classical V. cholerae contain two truncated, fused prophages. The upstream prophage lacks the prophage genes downstream of cep (resulting in cep′), while the downstream prophage lacks ig-1 sequences starting with the ER (resulting in ′ig-1Δ1). The PCR primers used to amplify the sequences between rstB and rstA are shown as small black arrows. The DNA sequence of the unexpected 875-bp PCR product (grey line) was determined. The presence of genes not assayed by DNA sequencing or PCR has been inferred from restriction maps generated with SphI (S), NruI (N), and XbaI (X).

FIG. 4

Southern blot of chromosomal DNA from classical V. cholerae strains demonstrating the heterogeneity of CTX prophage arrays. Chromosomal DNA was digested with SphI and probed with rstR^class, which detects only classical prophage DNA. No additional CTX prophage-related sequences were detected with a less specific probe (data not shown). Strains O395, GP12, CA401, C1, C14, C21, C33, and C34 (lanes 1 to 8, respectively) were compared. The single band in lanes 1, 2, and 7 was shown in additional analyses to be a doublet (data not shown).

FIG. 5

PCR amplification of prophage-prophage and chromosome-prophage junctions in classical V. cholerae. (A) Amplication of the junction between a CTX prophage and an upstream element, using primers RstBF2 and RstRproR (Fig. 3). An ∼875-bp product was generated from DNA of classical strains C34 (lane 1), CA401 (lane 2), and O395 (lane 3), indicating that all contain truncated, fused prophages. An ∼1,450-bp product was amplified from the RS1-CTX prophage junction in the O139 strain AS207 (lane 4), and no product was synthesized in the absence of template DNA (lane 5). (B) Detection of a junction between the chromosomal sequence of TLC and a CTX prophage, using primers TLCF1 and Ig1R1. A ∼600-bp product was amplified from the DNA of classical strains C34 (lane 1), CA401 (lane 2), and O395 (lane 3) as well as the O139 strain AS207 (lane 4); it was not synthesized if template DNA was omitted (lane 5). (C) Detection of a junction between CTX prophages in the classical strain-specific insertion site and upstream chromosomal sequence, using the primers PyrFF1 and Ig1R1. A ∼550-bp product was amplified from DNA of the classical strains O395 (lane 1), C34 (lane 2), and CA401 (lane 3); no product was amplified from AS207 DNA (lane 4), which lacks a CTX prophage at this insertion site, or in the absence of template DNA (lane 5).

FIG. 6

Restriction mapping of the CTX prophage insertions sites in classical biotype strains. (A) Summary of the restriction fragments detected on Southern blots of DNA from type I and type II classical strains digested with SphI (S), NruI (N), and XbaI (X) and probed with various prophage-derived probes. The precise sizes of restriction fragments (in kilobases) were derived from the V. cholerae genome database as well as laboratory sequence data. Band sizes on blots could not be determined as precisely, but all except one were consistent with sequence-derived sizes. X^★ denotes an XbaI site that is farther downstream than expected; classical biotypes apparently lack an XbaI site that is present in this region in El Tor strains. The location of the ctxA probe used for panel B is shown at the bottom. (B) ctxAB-spanning restriction fragments from diverse classical strains of V. cholerae are identical. A Southern blot of DNAs digested with SphI, XbaI, and NruI, as indicated, was probed with a ctxA probe. Each enzyme produced equal-size fragments from the classical strains C21 (lanes 1, 4, and 7) and O395 (lanes 2, 5, and 8). AS207 DNA (lanes 3, 6, and 9) had one fragment in common with the classical DNAs for each enzyme. Digests of classical DNA do not have the band with a constant size (∼7 kb), indicative of tandem prophages, that is produced from AS207 DNA. The AS207 prophage array was previously shown to contain an RS1 followed by three tandem CTX prophages. Faint bands in a few lanes resulted from incomplete digestion of DNA.

FIG. 6

Restriction mapping of the CTX prophage insertions sites in classical biotype strains. (A) Summary of the restriction fragments detected on Southern blots of DNA from type I and type II classical strains digested with SphI (S), NruI (N), and XbaI (X) and probed with various prophage-derived probes. The precise sizes of restriction fragments (in kilobases) were derived from the V. cholerae genome database as well as laboratory sequence data. Band sizes on blots could not be determined as precisely, but all except one were consistent with sequence-derived sizes. X^★ denotes an XbaI site that is farther downstream than expected; classical biotypes apparently lack an XbaI site that is present in this region in El Tor strains. The location of the ctxA probe used for panel B is shown at the bottom. (B) ctxAB-spanning restriction fragments from diverse classical strains of V. cholerae are identical. A Southern blot of DNAs digested with SphI, XbaI, and NruI, as indicated, was probed with a ctxA probe. Each enzyme produced equal-size fragments from the classical strains C21 (lanes 1, 4, and 7) and O395 (lanes 2, 5, and 8). AS207 DNA (lanes 3, 6, and 9) had one fragment in common with the classical DNAs for each enzyme. Digests of classical DNA do not have the band with a constant size (∼7 kb), indicative of tandem prophages, that is produced from AS207 DNA. The AS207 prophage array was previously shown to contain an RS1 followed by three tandem CTX prophages. Faint bands in a few lanes resulted from incomplete digestion of DNA.