Identification of genes induced in Vibrio cholerae in a dynamic biofilm system

Abstract

The facultative human pathogen Vibrio cholerae, the causative agent of the severe secretory diarrheal disease cholera, persists in its aquatic reservoirs in biofilms during interepidemic periods. Biofilm is a likely form in which clinically relevant V. cholerae is taken up by humans, providing an infective dose. Thus, a better understanding of biofilm formation of V. cholerae is relevant for the ecology and epidemiology of cholera as well as a target to control the disease. Most previous studies have investigated static biofilms of V. cholerae and elucidated structural prerequisites like flagella, pili and a biofilm matrix including extracellular DNA, numerous matrix proteins and exopolysaccharide, as well as the involvement of regulatory pathways like two-component systems, quorum sensing and c-di-GMP signaling. However, aquatic environments are more likely to reflect an open, dynamic system. Hence, we used a biofilm system with constant medium flow and a temporal controlled reporter-system of transcription to identify genes induced during dynamic biofilm formation. We identified genes known or predicted to be involved in c-di-GMP signaling, motility and chemotaxis, metabolism, and transport. Subsequent phenotypic characterization of mutants with independent mutations in candidate dynamic biofilm-induced genes revealed novel insights into the physiology of static and dynamic biofilm conditions. The results of this study also reinforce the hypotheses that distinct differences in regulatory mechanisms governing biofilm development are present under dynamic conditions compared to static conditions.

Extracellular DNA builds and interacts with vibrio polysaccharide in the biofilm matrix formed by Vibrio cholerae

“…As stated above, the biofilm formed in the late logarithmic phase showed the highest amount of eDNA that might allow efficient attachment of bacterial cells to surfaces and among themselves to develop a healthy biofilm. Similar to this finding, several workers also reported the presence of eDNA in the biofilm matrix of many bacteria, B. cereus, P. aeruginosa, S. epidermidis, E. faecalis including V. cholerae (O'Gara, 2007;Thomas et al, 2008;Tang et al, 2013;Seper et al, 2014;Panda and Singh, 2018). eDNA acts as an essential element in the initiation of biofilm formation and the maintenance of biofilm.…”

“…Both nucleases are induced at the late stage of mature biofilm, where nucleases are involved in the detachment process triggering biofilm dissolution. After that, utilizing degraded eDNA as a source of phosphate under nutrient limitation (Seper et al, 2014). In contrast, eDNA is secreted independent of cell lysis in the early stage of biofilm.…”

“…The Vibrio can survive in adverse conditions through the selfaggregation of micro-colonies in the biofilm matrix, showing higher tolerance/resistance to antibiotics than planktonic cells (Faruque et al, 2006;Alam et al, 2007). The formation of biofilm in V. cholerae requires flagella, pili and exopolysaccharide biosynthesis Chiavelli et al, 2001;Lauriano et al, 2004;Meibom et al, 2004;Moorthy and Watnick, 2004;Houot and Watnick, 2008;Yang et al, 2009;Houot et al, 2010;Krasteva et al, 2010), and two-component system, quorum sensing and c-di-GMP signalling pathways for its regulation (Watnick et al, 2001;Haugo and Watnick, 2002;Hammer and Bassler, 2003;Tischler and Camilli, 2004;Reguera and Kolter, 2005;Fong et al, 2006;Fong and Yildiz, 2007;Absalon et al, 2011;Seper et al, 2011;Berk et al, 2012;Seper et al, 2014). Vibrio polysaccharide (VPS) genes are arranged into two clusters vpsA-K and vpsL-Q.…”

Silence is golden: gene silencing of V. cholerae during intestinal colonization delivers new aspects to the acid tolerance response

“…For example, we recently adapted RIVET to identify genes induced during biofilm formation of V. cholerae, representing its aquatic lifestyle. 40 Amongst other interesting candidates, extracellular nucleases were found to be induced during biofilm formation, which resulted in the characterization of extracellular DNA as a novel matrix component of V. cholerae biofilms. 41 Similarly, identification of Figure 3.…”

Characterization of Biofilm Extracts from Two Marine Bacteria

“…Flagella involved in swimming and swarming play a role during the adhesion initial phase of biofilm formation, whereas type IV pili participate in the biofilm maturation [74]. Flagella and pili are often present in Vibrio cells [75,76]. Raguenes et al [40] have shown that V. diabolicus cells display one single flagellum in liquid medium and many lateral pili on a solid medium, which can contribute to swarming.…”