MstX and a Putative Potassium Channel Facilitate Biofilm Formation in Bacillus subtilis

Citation: Lundberg ME, Becker EC, Choe S ( MstX and a Putative Potassium Channel Facilitate Biofilm Formation in Bacillus subtilis Matthew E. Lundberg 0 Eric C. Becker 0 Senyon Choe 0 Adam Driks, Loyola University Medical Center, United States of America 0 1 Structural Biology Laboratory, The Salk Institute, La Jolla, California, United States of America, 2 Division of Biology, University of California San Diego , La Jolla, California , United States of America Biofilms constitute the predominant form of microbial life and a potent reservoir for innate antibiotic resistance in systemic infections. In the spore-forming bacterium Bacillus subtilis, the transition from a planktonic to sessile state is mediated by mutually exclusive regulatory pathways controlling the expression of genes required for flagellum or biofilm formation. Here, we identify mstX and yugO as novel regulators of biofilm formation in B. subtilis. We show that expression of mstX and the downstream putative K+ efflux channel, yugO, is necessary for biofilm development in B. subtilis, and that overexpression of mstX induces biofilm assembly. Transcription of the mstX-yugO operon is under the negative regulation of SinR, a transcription factor that governs the switch between planktonic and sessile states. Furthermore, mstX regulates the activity of Spo0A through a positive autoregulatory loop involving KinC, a histidine kinase that is activated by potassium leakage. The addition of potassium abrogated mstX-mediated biofilm formation. Our findings expand the role of Spo0A and potassium homeostasis in the regulation of bacterial development. - Funding: Funding provided by the National Institutes of Health and the Chapman foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Nearly all bacteria are capable of forming multicellular communities through complex signaling events that lead to differentiation into a myriad of cell types. These sessile, surfaceattached bacterial populations, referred to as biofilms, create an elaborate extracellular matrix comprised of protein and exopolysaccharides that enhance survival in a nutrient-depleted state and mediate attachment to surfaces [1,2]. Cells lacking either component of the extracellular matrix form flat, featureless colonies devoid of complex architecture and they fail to adhere to surfaces [3]. A signature feature of biofilm communities is their increased resistance to antibiotics and environmental stresses; both features make them particularly problematic in clinical and industrial settings. For instance, biofilms constitute over 65% of bacterial infections and represent a formidable source of innate multidrug resistance [4]. A small percentage of bacteria in a biofilm give rise to dormant, persistent cells that are recalcitrant to antibiotic treatment. The molecular mechanisms by which biofilms acquire antibiotic resistance have only recently begun to emerge [57]. The Gram-positive bacterium B. subtilis is capable of forming thin, floating biofilms at the liquid-air interface (pellicles) with sporulation at apical tips that project into the air [8]. Previous studies have identified multiple transcriptional regulatory networks that give rise to multicellular communities in B. subtilis. These pathways include the regulatory proteins Spo0A, sH, the transition state regulator AbrB, the master regulator for biofilm formation SinR, and Slr [812]. Biofilm assembly requires expression of the 15-gene operon epsA-O, which encodes enzymes that synthesize the exopolysaccharide layer, the tapA-sipW-tasA operon, which encodes the protein component, and BslA, a protein that forms a hydrophobic layer on the surface of biofilms. SinR is a transcription factor that directly represses exopolysaccharide production and the flagellar motor inhibitor EpsE during exponential growth [13]. It also inhibits Slr, a transcriptional factor that activates biofilm genes while repressing motility [14]. The balance between SinR and Slr activity depends on Spo0A-P accumulation, which allows production of SinI, an inhibitor of SinR, which therefore turns on matrix production and turns off motility [12]. The switch between motility and biofilm formation therefore critically depends on the phosphorylation state of Spo0A, which is controlled by a variety of kinases and phosphatases that respond to different stimuli including oxidative stress, K+ leakage, osmotic pressure, and malic acid ([1517]. These kinases (KinA, KinB, KinC, and KinD) help facilitate biofilm formation through spatial regulation but can be partially redundant through signaling overlap [18]. Mistic (MstX) is a unique protein found in a small number of Bacillus species, including B. subtilis, that enables high-level, heterologous expression and targeting of integral membrane protein sequences to cell membranes when fused to the Nterminus of a cargo protein construct [19]. In spite of its small and highly acidic nature, MstX associates with the membrane, presumably through autonomous association with the phospholipid bilayer, thereby bypassing or facilitating the traditional secretory apparatus [19]. The MstX homologues in Bacillus atrophaeus, Bacillus mojavensis, and Bacillus licheniformis, like B. subtilis, facilitate heterologous integral membrane protein expression when used as part of a fusion construct [20]. Furthermore, in all cases, mstX homologues precede a putative potassium ion channel yugO, suggesting that the MstX protein might be involved in membrane insertion of YugO (Figure 1). No similar sequence with a known function exists, raising the question as to what function MstX might serve in Bacillus subtilis. The initial goal of the present work was to elucidate the function of mstX in the Gram-positive bacterium, Bacillus subtilis. During the course of this investigation, we discovered novel roles for mstX during biofilm development. We show that mstX is necessary for robust biofilm formation. The mstX promoter is regulated by SinR, the master regulator for biofilm formation, and induces biofilm formation at least partially through KinC mediated phosphorylation of Spo0A, and correspondent increases in expression of the regulators abbA and sinI. A mutation in SinR proved epistatic to the mstX biofilm film defect, restoring both colony morphology and pellicle formation in a double mutant. Supplementation of media with potassium or disruption of the downstream putative potassium ion channel abrogated mstX-mediated biofilm formation, illustrating the importance of KinC activation and potassium in biofilm development. These data suggest that mstX operates through a potassium efflux-driven positive feedback loop that enhances biofilm formation in B. subtilis. Methods and Materials Strains, media, and culture conditions The parent strains for al (...truncated)