pIII CTX , a Predicted CTXφ Minor Coat Protein, Can Expand the Host Range of Coliphage fd To Include Vibrio cholerae

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Abstract
CTXφ is a filamentous bacteriophage that encodes cholera toxin. CTXφ infection of its host bacterium, Vibrio cholerae , requires the toxin-coregulated pilus (TCP) and the products of the V. cholerae tolQRA genes. Here, we have explored the role of OrfU, a predicted CTXφ minor coat protein, in CTXφ infection. Prior to the discovery that it was part of a prophage, orfU was initially described as an open reading frame of unknown function that lacked similarity to known protein sequences. Based on its size and position in the CTXφ genome, we hypothesized that OrfU may function in a manner similar to that of the coliphage fd protein pIII and mediate CTXφ infection as well as playing a role in CTXφ assembly and release. Deletion of orfU from CTXφ dramatically reduced the number of CTXφ virions detected in supernatants from CTXφ-bearing cells. This defect was complemented by expression of orfU in trans , thereby confirming a role for this gene in CTXφ assembly and/or release. To evaluate the requirement for OrfU in CTXφ infection, we introduced fragments of orfU into gIII in an fd derivative to create OrfU-pIII fusions. While fd is ordinarily unable to infect V. cholerae , an fd phage displaying the N-terminal 274 amino acids of OrfU could infect V. cholerae in a TCP- and TolA-dependent fashion. Since our findings indicate that OrfU functions as the CTXφ pIII, we propose to rename OrfU as pIII CTX . Our data also provide new evidence for a conserved pathway for filamentous phage infection.