Construction of in-frame gene deletions, point mutations, complementation constructs, and transcriptional reporter fusion plasmids.

Deletions and mutations were constructed via a splicing by overlap extension (SOE)-PCR protocol, or via a Gibson protocol (New England BioLabs [NEB]), and introduced into the V. cholerae chromosome via conjugation and selection for double recombination events, as described previously (21). SOE constructs were generated by designing two sets of primers that amplify ∼1,000 bp upstream and downstream of the region to be deleted. Primers 1 and 4 were the “exterior” primers, while the interior primers, primers 2 and 3, were designed to fall within a few amino acids of the start codon and stop codon of the gene of interest. The primers amplified a construct that generated a complete, in-frame deletion of the gene, leaving behind just the overlapping tag incorporated into primers 2 and 3, together with a small number of codons at the 5′ and 3′ ends of the gene. When mutations rather than deletions were constructed, primers 2 and 3 were designed to overlap the region that includes the mutation. The STAC domain deletion primers do not include an exogenous tag and completely overlapped one another without additional base pairs. The Gibson protocol was used to delete the pta-ackA operon from V. cholerae, and primers were designed according to the manufacturer's instructions (NEB). Following SOE-PCR with Q5 high-fidelity DNA polymerase (NEB), the PCR product was then digested with appropriate restriction enzymes and ligated into the pHC001B plasmid (49). The plasmid was transformed into DH5α λpir cells, and the construct was verified by sequencing and then moved into MFDpir cells (50). The plasmid was then conjugated into V. cholerae, and transconjugants carrying the plasmid was selected for by plating on kanamycin, as described previously (21). Sucrose selection was used to identify clones that had lost the plasmid, and integration of the correct construct was verified by PCR or by sequencing.

To generate complementation constructs, the gene of interest was amplified with Q5 high-fidelity DNA polymerase (NEB) using a forward primer that incorporated an NdeI restriction site into the start codon of the gene and a reverse primer that fell after the last stop codon and incorporated a second restriction site. The resulting fragment was digested, ligated into the pSRK-Km plasmid (51), and then transformed into E. coli for conjugation into V. cholerae.

To generate reporter plasmid constructs in the pBBRlux plasmid (52), fragments of the respective promoters were amplified using Q5 high-fidelity DNA polymerase (NEB) with primers incorporating restriction sites, digested, and ligated into pBBRlux. The resulting plasmid was then introduced into V. cholerae by conjugation, followed by selection on ampicillin and chloramphenicol (5 μg/ml).