Diversity in Natural Transformation Frequencies and Regulation across Vibrio Species

ABSTRACT In Vibrio species, chitin-induced natural transformation enables bacteria to take up DNA from the external environment and integrate it into their genome. Expression of the master competence regulator TfoX bypasses the need for chitin induction and drives expression of the genes required for competence in several Vibrio species. Here, we show that TfoX expression in Vibrio campbellii strains DS40M4 and NBRC 15631 enables high natural transformation frequencies. Conversely, transformation was not achieved in the model quorum-sensing strain V. campbellii BB120 (previously classified as Vibrio harveyi). Surprisingly, we find that quorum sensing is not required for transformation in V. campbellii DS40M4 or Vibrio parahaemolyticus in contrast to the established regulatory pathway in Vibrio cholerae in which quorum sensing is required to activate the competence regulator QstR. Similar to V. cholerae, expression of both QstR and TfoX is necessary for transformation in DS40M4. There is a wide disparity in transformation frequencies among even closely related Vibrio strains, with V. vulnificus having the lowest functional transformation frequency. Ectopic expression of both TfoX and QstR is sufficient to produce a significant increase in transformation frequency in Vibrio vulnificus. To explore differences in competence regulation, we used previously studied V. cholerae competence genes to inform a comparative genomics analysis coupled with transcriptomics. We find that transformation capability cannot necessarily be predicted by the level of gene conservation but rather correlates with competence gene expression following TfoX induction. Thus, we have uncovered notable species- and strain-level variations in the competence gene regulation pathway across the Vibrio genus. IMPORTANCE Naturally transformable, or competent, bacteria are able to take up DNA from their environment, a key method of horizontal gene transfer for acquisition of new DNA sequences. Our research shows that Vibrio species that inhabit marine environments exhibit a wide diversity in natural transformation capability ranging from nontransformability to high transformation rates in which 10% of cells measurably incorporate new DNA. We show that the role of regulatory systems controlling the expression of competence genes (e.g., quorum sensing) differs throughout both the species and strain levels. We explore natural transformation capabilities of Vibrio campbellii species which have been thus far uncharacterized and find novel regulation of competence. Expression of two key transcription factors, TfoX and QstR, is necessary to stimulate high levels of transformation in Vibrio campbellii and recover low rates of transformation in Vibrio vulnificus.

Unleashing Natural Competence in Lactococcus lactis by Induction of the Competence Regulator ComX

ABSTRACT In biotechnological workhorses like Streptococcus thermophilus and Bacillus subtilis, natural competence can be induced, which facilitates genetic manipulation of these microbes. However, in strains of the important dairy starter Lactococcus lactis, natural competence has not been established to date. However, in silico analysis of the complete genome sequences of 43 L. lactis strains revealed complete late competence gene sets in 2 L. lactis subsp. cremoris strains (KW2 and KW10) and at least 10 L. lactis subsp. lactis strains, including the model strain IL1403 and the plant-derived strain KF147. The remainder of the strains, including all dairy isolates, displayed genomic decay in one or more of the late competence genes. Nisin-controlled expression of the competence regulator comX in L. lactis subsp. lactis KF147 resulted in the induction of expression of the canonical competence regulon and elicited a state of natural competence in this strain. In contrast, comX expression in L. lactis NZ9000, which was predicted to encode an incomplete competence gene set, failed to induce natural competence. Moreover, mutagenesis of the comEA-EC operon in strain KF147 abolished the comX-driven natural competence, underlining the involvement of the competence machinery. Finally, introduction of nisin-inducible comX expression into nisRK-harboring derivatives of strains IL1403 and KW2 allowed the induction of natural competence in these strains also, expanding this phenotype to other L. lactis strains of both subspecies. IMPORTANCE Specific bacterial species are able to enter a state of natural competence in which DNA is taken up from the environment, allowing the introduction of novel traits. Strains of the species Lactococcus lactis are very important starter cultures for the fermentation of milk in the cheese production process, where these bacteria contribute to the flavor and texture of the end product. The activation of natural competence in this industrially relevant organism can accelerate research aiming to understand industrially relevant traits of these bacteria and can facilitate engineering strategies to harness the natural biodiversity of the species in optimized starter strains.

Unleashing natural competence inLactococcus lactisby induction of the competence regulator ComX

In biotechnological work horses likeStreptococcus thermophilusandBacillus subtilisnatural competence can be induced, which facilitates genetic manipulation of these microbes. However, in strains of the important dairy starterLactococcus lactisnatural competence has not been established to date. However,in silicoanalysis of complete genome sequences of 43L. lactisstrains revealed complete late-competence gene-sets in 2L. lactissubspeciescremorisstrains (KW2 and KW10) and 8L. lactissubspecieslactisstrains, including the model strain IL1403 and the plant-derived strain KF147. The remainder of the strains, including all dairy isolates, displayed genomic decay in one or more of the late competence genes. Nisin-controlled expression of the competence regulatorcomXinL. lactissubsp.lactisKF147 resulted in the induction of expression of the canonical competence regulon, and elicited a state of natural competence in this strain. By contrast,comXexpression inL. lactisNZ9000, predicted to encode an incomplete competence gene-set, failed to induce natural competence. Moreover, mutagenesis of thecomEA-ECoperon in strain KF147, abolished thecomXdriven natural competence, underpinning the involvement of the competence machinery. Finally, introduction of nisin-induciblecomXexpression intonisRK-harboring derivatives of strains IL1403 and KW2 allowed the induction of natural competence also in these strains, expanding this phenotype to otherL. lactisstrains of both subspecies.Significance statementSpecific bacterial species are able to enter a state of natural competence in which DNA is taken up from the environment, allowing the introduction of novel traits. Strains of the speciesLactococcus lactisare very important starter cultures for the fermentation of milk in the cheese production process, where these bacteria contribute to the flavor and texture of the end-product. The activation of natural competence in this industrially relevant organism can accelerate research aiming to understand industrially relevant traits of these bacteria, and can facilitate engineering strategies to harness the natural biodiversity of the species in optimized starter strains.

Important Contribution of the Novel LocuscomEBto Extracellular DNA-Dependent Staphylococcus lugdunensis Biofilm Formation

The coagulase-negative speciesStaphylococcus lugdunensisis an emerging cause of serious and potentially life-threatening infections, such as infective endocarditis. The pathogenesis of these infections is characterized by the ability ofS. lugdunensisto form biofilms on either biotic or abiotic surfaces. To elucidate the genetic basis of biofilm formation inS. lugdunensis, we performed transposon (Tn917) mutagenesis. One mutant had a significantly reduced biofilm-forming capacity and carried a Tn917insertion within the competence genecomEB. Site-directed mutagenesis and subsequent complementation with a functional copy ofcomEBverified the importance ofcomEBin biofilm formation. In several bacterial species, natural competence stimulates DNA release via lysis-dependent or -independent mechanisms. Extracellular DNA (eDNA) has been demonstrated to be an important structural component of many bacterial biofilms. Therefore, we quantified the eDNA in the biofilms and found diminished eDNA amounts in thecomEBmutant biofilm. High-resolution images and three-dimensional data obtained via confocal laser scanning microscopy (CSLM) visualized the impact of thecomEBmutation on biofilm integrity. ThecomEBmutant did not show reduced expression of autolysin genes, decreased autolytic activities, or increased cell viability, suggesting a cell lysis-independent mechanism of DNA release. Furthermore, reduced amounts of eDNA in thecomEBmutant biofilms did not result from elevated levels or activity of theS. lugdunensisthermonuclease NucI. In conclusion, we defined here, for the first time, a role for the competence genecomEBin staphylococcal biofilm formation. Our findings indicate thatcomEBstimulates biofilm formation via a lysis-independent mechanism of DNA release.