Natural Transformation in a Classical-Biotype Vibrio cholerae Strain

causes the gastrointestinal illness cholera, which spreads throughout the globe in large pandemics. The current pandemic is caused by O1 El Tor biotype strains, whereas previous pandemics were caused by O1 classical biotype strains. El Tor is noted for its ability to acquire exogenous DNA through chitin-induced natural transformation, which has been exploited for genetic manipulation of El Tor strains in the laboratory. In contrast, the prototypical classical strain O395 lacks this ability, which was suspected to be due to a mutation in the regulatory gene HapR and the regulator TfoX control expression of a third competence regulator, QstR. We found that artificial induction of both TfoX and QstR in the presence of HapR in O395 was required for efficient DNA uptake. However, natural transformation in the classical strain is still orders of magnitude below that of an El Tor strain. O395 expressing HapR could also undergo natural transformation after growth on chitin, which could be increased by artificial induction of TfoX and/or QstR. A plasmid that expresses both TfoX and QstR was created that allowed for consistent DNA uptake in O395 carrying a plasmid. This technique was also used to facilitate cotransformation into O395 of unmarked DNA (Δ , Δ , Δ ) for multiplex genome editing by natural transformation (MuGENT). These results demonstrate that the classical biotype O395 strain is functionally capable of DNA uptake, which allows for the rapid genetic manipulation of its genome. Natural transformation (uptake of exogenous DNA) in has contributed to the evolution of these human pathogens. Classical biotype strains were responsible for the first six cholera pandemics but were replaced by El Tor biotype in the current pandemic. This study demonstrates that classical is functionally capable of natural transformation, but inactivation of the transformation regulator HapR and inherent levels of transformation that are lower than those of El Tor suggest that the classical biotype may be less able to utilize natural transformation for horizontal gene transfer.