Control of Competence in Vibrio fischeri

Abstract

Vibrio species, including the squid symbiont Vibrio fischeri, become competent to take up DNA under specific conditions. For example, V. fischeri becomes competent when grown in the presence of chitin oligosaccharides or upon overproduction of the competence regulatory factor TfoX. While little is known about the regulatory pathway(s) that control V. fischeri competence, this microbe encodes homologs of factors that control competence in the well-studied V. cholerae. To further develop V. fischeri as a genetically-tractable organism, we evaluated the roles of some of these competence homologs. Using TfoX-overproducing cells, we found that competence depends upon LitR, the homolog of V. cholerae master quorum sensing and competence regulator HapR, and on homologs of putative pili genes that in V. cholerae facilitate DNA uptake. Disruption of genes for negative regulators upstream of LitR, namely the LuxO protein and the sRNA Qrr1, resulted in increased transformation frequencies. Unlike LitR-controlled light production, however, competence did not vary with cell density under tfoX-overexpressing conditions. Analogous to V. cholerae, the requirement for LitR could be suppressed by loss of the Dns nuclease. We also found a role for the putative competence regulator CytR. Finally, we determined that transformation frequencies varied depending on the TfoX-encoding plasmid, and developed a new dual tfoX- and litR-overexpression construct that substantially increased the transformation frequency of a less genetically-tractable strain. By advancing the ease of genetic manipulation of V. fischeri, these findings will facilitate the rapid discovery of genes involved in physiologically-relevant processes, such as biofilm formation and host colonization. Importance The ability of bacteria to take up DNA (competence) and incorporate foreign DNA into their genomes (transformation) permits them to rapidly evolve and gain new traits and/or acquire antibiotic resistances. It also facilitates laboratory-based investigations into mechanisms of specific phenotypes, such as those involved in host colonization. Vibrio fischeri has long been a model for symbiotic bacteria-host interactions as well as for other aspects of its physiology such as bioluminescence and biofilm formation. Competence of V. fischeri can be readily induced upon overexpression of the competence factor TfoX. Relatively little is known about the V. fischeri competence pathway, although homologs of factors known to be important in V. cholerae competence exist. By probing the importance of putative competence factors that control transformation of V. fischeri, this work deepens our understanding of the competence process and advances our ability to genetically manipulate this important model organism.

Nocturnal Acidification: A Coordinating Cue in the Euprymna scolopes–Vibrio fischeri Symbiosis

“…Elucidation of the regulatory functions of these V. fischeri homologs began after experimental induction of competency was found through culturing in the presence of chitin oligosaccharides or by forcing overexpression of the putative competence regulatory factor, TfoX [ 76 ]. Recent investigations have found more corresponding regulatory homologs in V. fischeri , notably the HapR homolog LitR, several type IV pili structural proteins, and a putative cytidine responsive regulator, CytR [ 80 ]. V. fischeri litR knockout mutations were found to abrogate competence induction compared to wild type strains [ 80 ].…”

“…Recent investigations have found more corresponding regulatory homologs in V. fischeri , notably the HapR homolog LitR, several type IV pili structural proteins, and a putative cytidine responsive regulator, CytR [ 80 ]. V. fischeri litR knockout mutations were found to abrogate competence induction compared to wild type strains [ 80 ]. Knockouts of putative V. fischeri pilus genes pilA , pilB , pilC , pilQ , and comEA also abrogated competence induction, suggesting similar roles in transformation [ 80 ].…”

“…V. fischeri litR knockout mutations were found to abrogate competence induction compared to wild type strains [ 80 ]. Knockouts of putative V. fischeri pilus genes pilA , pilB , pilC , pilQ , and comEA also abrogated competence induction, suggesting similar roles in transformation [ 80 ]. Experiments in which knockouts of the V. fischeri putative cytidine-responsive regulator CytR abrogated natural transformation also suggest that environmental cytidine (from degraded DNA) availability is an inducer of competence [ 80 ].…”

“…Knowledge of several of the major environmental factors that induce in vitro competence in V. fischeri and of the signaling pathways involved has led to the development of increasingly powerful research tools for genetic manipulation studies [ 76 , 80 ]. As in V. cholera [ 78 ], the known cardinal regulators found to induce competence and transformation experimentally in V. fischeri (although not consistently) are the presence of environmental chitin or chitin derivatives (or over expression of the chitin-induced tfoX gene), low nutrient availability, the presence of environmental DNA or nucleosides (the repressor of CytR), and high cell density (which activates LitR through quorum-sensing pathways) [ 80 ]. Prior sections have highlighted the extensive research demonstrating that two of the cardinal inducers, high cell density (quorum), and environmental chitin, are found within the light organ and fluctuate rhythmically during the diel cycle in a coordinated interacting pattern with luminescence and acidification.…”

Quorum Sensing and Cyclic di-GMP Exert Control Over Motility of Vibrio fischeri KB2B1

“…Mutants of KB2B1 were generated as follows or as described in the Isolation of KB2B1 motility suppressors section: mutations generated in ES114 were transferred into KB2B1 using TfoX-mediated transformation ( Pollack-Berti et al, 2010 ; Brooks et al, 2014 ; Christensen et al, 2020b ; Cohen, Eichinger et al, 2021 ). The ES114-derived mutations were previously published as indicated in Table 1 , except Δ VF_1200 :FRT-Cm.…”