Characterization of the major control region of Vibrio cholerae bacteriophage K139: immunity, exclusion, and integration

Abstract

The temperate bacteriophage K139 is highly associated with pathogenic O1 Vibrio cholerae strains. The nucleotide sequence of the major control region of K139 was determined. The sequences of four (cox, cII, cI, and int) of the six deduced open reading frames and their gene order indicated that K139 is related to the P2 bacteriophage family. Two genes of the lysogenic transcript from the mapped promoter PL encode homologs to the proteins CI and Int, with deduced functions in prophage formation and maintenance. Between the cI and int genes, two additional genes were identified: orf2, which has no significant similarity to any other gene, and the formerly characterized gene glo. Further analysis revealed that Orf2 is involved in preventing superinfection. In a previous report, we described that mutations in glo cause an attenuation effect in the cholera mouse model (J. Reidl and J. J. Mekalanos, Mol. Microbiol. 18:685-701, 1995). In this report, we present strong evidence that Glo participates in phage exclusion. Glo was characterized to encode a 13.6-kDa periplasmic protein which inhibits phage infection at an early step, hence preventing reinfection of vibriophage K139 into K139 lysogenic cells. Immediately downstream of gene int, the attP site was identified. Upon analysis of the corresponding attB site within the V. cholerae chromosome, it became evident that phage K139 is integrated between the flagellin genes flaA and flaC of O1 El Tor and O139 V. cholerae lysogenic strains.

FIG. 1

DNA sequence of the phage K139 control region. The deduced amino acid sequences are shown below the DNA sequence. Oligonucleotides used for sequencing (seq 1 to 18) are indicated by solid-line arrows, and those used for the construction of the Glo expression system are shown as dashed-line arrows. Transcriptional and translational relevant sites are indicated; ribosomal binding sites are marked as S.D., and putative promoters (P_L and P_R) are marked by arrows. The attP site is underlined. Tn10d-cat insertions are indicated at bp 2031 and 2863. The mapped start site of transcription is indicated at position 1222. Putative helix-turn-helix motifs are marked by underlining of the amino acids comprising CII and Cox.

FIG. 1

DNA sequence of the phage K139 control region. The deduced amino acid sequences are shown below the DNA sequence. Oligonucleotides used for sequencing (seq 1 to 18) are indicated by solid-line arrows, and those used for the construction of the Glo expression system are shown as dashed-line arrows. Transcriptional and translational relevant sites are indicated; ribosomal binding sites are marked as S.D., and putative promoters (P_L and P_R) are marked by arrows. The attP site is underlined. Tn10d-cat insertions are indicated at bp 2031 and 2863. The mapped start site of transcription is indicated at position 1222. Putative helix-turn-helix motifs are marked by underlining of the amino acids comprising CII and Cox.

FIG. 1

DNA sequence of the phage K139 control region. The deduced amino acid sequences are shown below the DNA sequence. Oligonucleotides used for sequencing (seq 1 to 18) are indicated by solid-line arrows, and those used for the construction of the Glo expression system are shown as dashed-line arrows. Transcriptional and translational relevant sites are indicated; ribosomal binding sites are marked as S.D., and putative promoters (P_L and P_R) are marked by arrows. The attP site is underlined. Tn10d-cat insertions are indicated at bp 2031 and 2863. The mapped start site of transcription is indicated at position 1222. Putative helix-turn-helix motifs are marked by underlining of the amino acids comprising CII and Cox.

FIG. 1

DNA sequence of the phage K139 control region. The deduced amino acid sequences are shown below the DNA sequence. Oligonucleotides used for sequencing (seq 1 to 18) are indicated by solid-line arrows, and those used for the construction of the Glo expression system are shown as dashed-line arrows. Transcriptional and translational relevant sites are indicated; ribosomal binding sites are marked as S.D., and putative promoters (P_L and P_R) are marked by arrows. The attP site is underlined. Tn10d-cat insertions are indicated at bp 2031 and 2863. The mapped start site of transcription is indicated at position 1222. Putative helix-turn-helix motifs are marked by underlining of the amino acids comprising CII and Cox.

FIG. 2

Construction of the control region encoding plasmid pJBcII-int and its derivatives. (A) Orientation of the subcloned StuI-XmnI phage fragment in the FspI site of pBR322. (B) Deduced ORFs of the 5,031-bp DNA phage fragment along with the putative intergenic region and promoters P_L and P_R. Filled bars indicate the corresponding DNA fragments, subcloned on pBR322 (pJBcII-orf2, pJBcII-glo, and pJBcII-int) and pTrckan (pTrcglokan). cm9 and cm2 indicate the locations and direction of Tn10d-cat insertions.

FIG. 3

Cellular localization of Glo. V. cholerae MAK757 and MAK757 K139 were cell fractionated. (A) SDS-polyacrylamide gels (15%) stained with Coomassie brilliant blue. (B) Corresponding Western blots after reaction with a Glo-specific polyclonal rabbit antiserum. Lanes 1 to 4, cytoplasmic, inner membrane, periplasmic, and outer membrane extracts, respectively; lane 5, purified Glo protein; lane S, 10-kDa molecular mass ladder (Gibco-Life Technologies); lane M, Kaleidoscope polypeptide standard (Bio-Rad). Sizes are indicated in kilodaltons.

FIG. 4

Identification of the phage and bacterial attachment site. (A) Chromosomal organization of integrated phage K139 between flaA and flaC and locations of the primers. Also indicated are the orientations of phage-harbored int and glo genes, attL and attR, and the PCR products expected from the primer combinations a (JN-flaA/JN-flaC) and b (JN-flaA/JN-int). (B) A 0.7% agarose gel shows the PCR products generated with primer combinations a and b. Lane S, 1-kb ladder DNA standard (Gibco Life Technologies); lane 1, classical O1 V. cholerae strain O395; lanes 2 to 7, O1 El Tor V. cholerae strains MAK757, MAK757 K139, M799, M804, M807, and M817, respectively; lanes 8 and 9, O139 V. cholerae strains AI1838 and MO10, respectively. PCR was performed for 30 cycles of 94°C for 1 min, 60°C for 1 min, and 72°C for 2 min.

FIG. 5

Comparison of the major control regions of different phages. Sequence similarity between phages K139 of V. cholerae, HP1 of H. influenzae, and P186 of E. coli are indicated for the ORFs CII, Cox, CI, and Int as black arrows; light arrows show the corresponding genes for TP-J34 and HP1 without detectable sequence similarity. SS (black boxes), signal sequence which can also be found in Orf142 of phage TP-J34, encoding a putative lipoprotein (43); attP, phage attachment site; P_L and P_R, locations of promoter structures.