Vibrio cholerae causes the gastrointestinal illness cholera, which spreadsthroughout the globe in large pandemics. The current pandemic is caused by O1 ElTor biotype strains, whereas previous pandemics were caused by O1 classical biotypestrains. El Tor V. cholerae is noted for its ability to acquire exogenous DNA throughchitin-induced natural transformation, which has been exploited for genetic manipulationof El Tor strains in the laboratory. In contrast, the prototypical classical strain O395lacks this ability, which was suspected to be due to a mutation in the regulatory genehapR. 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 HapRin O395 was required for efficient DNA uptake. However, natural transformation in theclassical strain is still orders of magnitude below that of an El Tor strain. O395 expressingHapR could also undergo natural transformation after growth on chitin, which couldbe increased by artificial induction of TfoX and/or QstR. A plasmid that expresses bothTfoX and QstR was created that allowed for consistent DNA uptake in O395 carrying ahapR plasmid. This technique was also used to facilitate cotransformation into O395 ofunmarked DNA (DlacZ, DflaA, DflgG) for multiplex genome editing by natural transformation(MuGENT). These results demonstrate that the classical biotype O395 strain isfunctionally capable of DNA uptake, which allows for the rapid genetic manipulation ofits genome.
- DNA uptake
- genetic manipulation
- natural transformation systems
ASJC Scopus subject areas
- Food Science
- Applied Microbiology and Biotechnology