Involvement of luxS in Biofilm Formation by Capnocytophaga ochracea

Abstract

Capnocytophaga ochracea is present in the dental plaque biofilm of patients with periodontitis. Biofilm cells change their phenotype through quorum sensing in response to fluctuations in cell-population density. Quorum sensing is mediated by auto-inducers (AIs). AI-2 is involved in intercellular signaling, and production of its distant precursor is catalyzed by LuxS, an enzyme involved in the activated methyl cycle. Our aim was to clarify the role of LuxS in biofilm formation by C. ochracea. Two luxS-deficient mutants, TmAI2 and LKT7, were constructed from C. ochracea ATCC 27872 by homologous recombination. The mutants produced significantly less AI-2 than the wild type. The growth rates of these mutants were similar to that of the wild-type in both undiluted Tryptic soy broth and 0.5 × Tryptic soy broth. However, according to crystal violet staining, they produced significantly less biofilm than the wild type. Confocal laser scanning microscopy and scanning electron microscopy showed that the biofilm of the TmAI2 strain had a rougher structure than that of the wild type. Complementation of TmAI-2 with extrinsic AI-2 from the culture supernatant of wild-type strain did not restore biofilm formation by the TmAI2 strain, but complementation of LKT7 strain with luxS partially restored biofilm formation. These results indicate that LuxS is involved in biofilm formation by C. ochracea, and that the attenuation of biofilm formation by the mutants is likely caused by a defect in the activated methyl cycle rather than by a loss of AI-2.

A Human Pathogen <i>Capnocytophaga Ochracea</i> Exhibits Current Producing Capability

“…LKT7, lacking the luxS gene, is a QS-deficient strain, which produces significantly fewer auto-inducers (AI-2) and less biofilm formation than WT, but with a similar growth rate. 19 Current generation measurements using the same conditions (OD 600 of 0.5) revealed that LKT7 was also capable of producing significant anodic current, comparable to WT (Fig. 6A).…”

“…In the electrochemical experiments, C. ochracea at the late exponential growth phase or early stationary phase was used. The luxS gene (Coch_1216) deleted C. ochracea termed as LKT7 was previously constructed using a PCR-ligation-mutation strategy, 19 kindly provided by the group of Professor Kazuyuki Ishihara from Tokyo Dental College and grown with the same conditions as wild type for our experiments.…”

“…Four secondary-ion images of each sample were recorded with a raster size of 25 © 25 µm 2 and analyzed by Fiji (version 1.0) for the isotope assimilation calculation. Given C. ochracea cells tend to form web-like morphology, 19 to fully recover the isotope signals, the signals for each NanoSIMS image were averaged. Using the plugin, Open MIMS Image, all signal regions, including clusters of cells were selected from each image (regions of interest, ROI) and their pixel values were quantified for isotopic ratio analysis.…”

“…17,18 In the oral cavity, C. ochracea is prone to form biofilms with gliding motility and also known to possess a QS luxS system. 19 In addition, dehydrogenases-cytochromes in the cellular membrane of C. ochracea were identified four decades ago; 20 however, the biological function of these redox enzymes and the potential EET capability of this microbial strain have yet to be studied. Therefore, we conducted a whole-cell electrochemical assay, single-potential amperometry, and differential pulse voltammetry to study the EET capability and the effect of cell-density dependency on current production.…”

“…The EET mechanisms were further examined using a mutant strain of C. ochracea lacking the gene encoding the QS luxS system (termed as LKT7), which produced significantly fewer auto-inducers (AI-2) and substantially less biofilm formation than wild-type (WT). 19…”