Diversity in natural transformation frequencies and regulation acrossVibriospecies

Abstract

24In marine Vibrio species, chitin-induced natural transformation enables bacteria to take up DNA 25 from the external environment and integrate it into their genome via homologous recombination. 26Expression of the master competence regulator TfoX bypasses the need for chitin induction and 27 drives expression of the genes required for competence in several Vibrio species. Here, we 28 show that TfoX expression in two Vibrio campbellii strains, DS40M4 and NBRC 15631, enables 29 high frequencies of natural transformation. Conversely, transformation was not achieved in the 30 model quorum-sensing strain V. campbellii BB120 (previously classified as Vibrio harveyi).31 Surprisingly, we find that quorum sensing is not required for transformation in V. campbellii 55 Natural transformation is a physiological state in which bacteria are able to take up 56 extracellular DNA from the environment, transport it across the cell envelope, and integrate it 57 into their genome via homologous recombination. In marine Vibrio species, competence is 58 induced by growth on the chitinous exoskeletons of crustaceans (1, 2). This process has been 59 best studied in Vibrio cholerae (reviewed in (2)). Insoluble polysaccharide chitin is broken down 60 by secreted extracellular chitinases (3). Soluble chitin oligosaccharides ultimately induce 61 expression of the master competence regulator TfoX (4), which activates expression of 62 numerous components of the competence machinery required to take up extracellular DNA. In 63 addition to the chitin-sensing system, V. cholerae cells must also have a functional quorum- 64sensing system for natural transformation to be successful (1). Quorum sensing, the process of 65 cell-cell communication, allows bacterial cells to respond to changes in population density and 66 alter gene expression (5). The quorum-sensing systems in Vibrio species rely on detection of 67 extracellular autoinducers that are sensed by membrane-bound sensor kinases, which shuttle 68 phosphate to or away from the core response regulator LuxO at low cell density or high cell 69 density, respectively (5). At the end of this phosphorylation cascade at high cell density, the 70 master transcription factor called LuxR is activated, which controls expression of hundreds of 71 genes (6). LuxR is the name of the master transcription factor in Vibrio campbellii (previously 72 called Vibrio harveyi), whereas the homologs in other Vibrio species have different names:73 HapR (V. cholerae), SmcR (Vibrio vulnificus), and OpaR (Vibrio parahaemolyticus) (7). In V. 74 cholerae, DhapR mutants are not competent, and this is due to HapR regulation of various 75 competence genes, including comEA, comEC, qstR, and dns (1, 8, 9). HapR directly activates 76 qstR (encoding a transcriptional regulator) and represses dns (encoding an extracellular DNase) 77 (8). QstR subsequently activates downstream genes required for DNA uptake and integration, 78 such as comEA, comEC, and comM (10). The requirement for HapR for competence can be79 circumvented if qs...

The quorum-sensing systems ofVibrio campbelliiDS40M4 and BB120 are genetically and functionally distinct

“…To determine whether homologs of previously tested BB120 quorum-sensing sRNAs and proteins have similar functions in DS40M4, we generated numerous mutant strains, including Δ cqsA , Δ luxS , Δ cqsA Δ luxS , Δ luxO , Δ luxR, luxO D47E (a LuxO phosphomimic allele), in addition to every combination of qrr deletion (single or combined). We previously showed, using a P luxCDABE -gfp reporter plasmid, that wild-type DS40M4 activates the luxCDABE promoter from BB120, and this is dependent on DS40M4 LuxR (15). We have since constructed a plasmid encoding the BB120 luxCDABE operon under control of its endogenous promoter (pCS38) for use in DS40M4, which enables us to monitor bioluminescence of various DS40M4 strains and assess LuxR regulation as a function of upstream quorum-sensing components.…”

“…We previously assessed the molecules sensed by DS40M4 receptors CqsS and LuxPQ (15). Our data showed that DS40M4 senses the AI-2 and CAI-1 molecules produced by BB120 but not BB120 HAI-1, even though DS40M4 encodes a LuxN homolog.…”

“…Thus, making mutations in BB120 is laborious and requires construction of suicide plasmids and the use of counter-selection and colony screening. Here we expanded upon our previous study of natural transformation in DS40M4 (15) to show the power of natural transformation for constructing up to four unmarked mutations simultaneously. The Qrr genes in the quorum-sensing circuit are only one example of a gene set for which MuGENT is useful in studying combinatorial mutant phenotypes.…”

“…Other Vibrio species, such as Vibrio natriegens , can successfully undergo chitin-independent transformation via overexpression of V. cholerae TfoX. Notably, the field has been unable to achieve natural transformation in BB120 with either method of transformation (15). The inability to rapidly generate defined mutations in BB120 has been a significant disadvantage, especially in comparison to other Vibrio species like V. cholerae that have more genetic tractability.…”

“…Recently, we showed that natural transformation is achievable in two wild isolates of V. campbellii , DS40M4 and NBRC 15631, by inducing expression of TfoX (15). Furthermore, DS40M4 can transform at frequencies similar to V. natriegens , which has been reported as the strain with the highest transformation frequency thus far.…”