CTXphi infection of Vibrio cholerae requires the tolQRA gene products

Abstract

CTXphi is a lysogenic filamentous bacteriophage that encodes cholera toxin. Filamentous phages that infect Escherichia coli require both a pilus and the products of tolQRA in order to enter host cells. We have previously shown that toxin-coregulated pilus (TCP), a type IV pilus that is an essential Vibrio cholerae intestinal colonization factor, serves as a receptor for CTXphi. To test whether CTXphi also depends upon tol gene products to infect V. cholerae, we identified and inactivated the V. cholerae tolQRAB orthologues. The predicted amino acid sequences of V. cholerae TolQ, TolR, TolA, and TolB showed significant similarity to the corresponding E. coli sequences. V. cholerae strains with insertion mutations in tolQ, tolR, or tolA were reduced in their efficiency of CTXphi uptake by 4 orders of magnitude, whereas a strain with an insertion mutation in tolB showed no reduction in CTXphi entry. We could detect CTXphi infection of TCP(-) V. cholerae, albeit at very low frequencies. However, strains with mutations in both tcpA and either tolQ, tolR, or tolA were completely resistant to CTXphi infection. Thus, CTXphi, like the E. coli filamentous phages, uses both a pilus and TolQRA to enter its host. This suggests that the pathway for filamentous phage entry into cells is conserved between host bacterial species.

FIG. 1

The organization of the tol gene clusters in V. cholerae and E. coli is identical. The predicted lengths of the V. cholerae Tol proteins were derived from an ORF map of the V. cholerae DNA sequence with MacVector. Percent identity and similarity were determined by comparing the predicted amino acid sequences of V. cholerae and E. coli Tol proteins with MacVector. The solid lines flanked by vertical bars represent the positions of the fragments of each tol gene that were used to construct the insertion mutations. The solid lines flanked by arrows represent the sequences cloned into pBAD33 used for the complementation studies.

FIG. 2

V. cholerae tolQRAB mutants (DH1, DH2, DH3, and DH4) produce amounts of TcpA similar to those produced by O395. All strains are derivatives of O395. The DH1 revertant was made by growing a small colony of DH1 in LB broth overnight at 37°C in the absence of ampicillin. The excision of pDH235 from tolQ in this revertant strain was confirmed by Southern analysis. All strains were grown in LB broth at 30°C. Whole-cell lysates were prepared in sample buffer as previously described (23) and run on an 4 to 12% Tris-Bis gradient gel (Novex, San Diego, Calif.). The proteins were then transferred to nitrocellulose and probed with anti-TcpA polyclonal antiserum.

FIG. 3

Kinetics of growth of O395 and O395-derived tol mutant strains. All strains were grown in LB broth with the appropriate antibiotics at 37°C and their OD₆₀₀ (A) and CFU (B) were determined over time.

FIG. 4

Filamentous morphology of V. cholerae tol mutant strain DH4 (A) compared with O395 (B). O395 derivatives with insertions in tolQ, tolR, and tolA also exhibited filamentation.