Protein lysine acetylation is a post-translational modification that alters the charge, conformation, and stability of proteins. A number of genome-wide characterizations of lysine-acetylated proteins, or acetylomes, in bacteria have demonstrated that lysine acetylation occurs on proteins with a wide diversity of functions, including central metabolism, transcription, chemotaxis, and cell size regulation. Bacillus subtilis is a model organism for studies of sporulation, motility, cell signaling, and multicellular development (or biofilm formation). In this work, we investigated the role of global protein lysine acetylation in multicellular development in B. subtilis. We analyzed the B. subtilis acetylome under biofilm-inducing conditions and identified acetylated proteins involved in multicellularity, specifically, swarming and biofilm formation. We constructed various single and double mutants of genes known to encode enzymes involved in global protein lysine acetylation in B. subtilis. Some of those mutants showed a defect in swarming motility while others demonstrated altered biofilm phenotypes. Lastly, we picked two acetylated proteins known to be important for biofilm formation, YmcA (also known as RicA), a regulatory protein critical for biofilm induction, and GtaB, an UTP-glucose-1-phosphate uridylyltransferase that synthesizes a nucleotide sugar precursor for biosynthesis of exopolysaccharide, a key biofilm matrix component. We performed site-directed mutagenesis on the acetylated lysine codons in ymcA and gtaB, respectively, and assayed cells bearing those point mutants for biofilm formation. The mutant alleles of ymcA(K64R), gtaB(K89R), and gtaB(K191R) all demonstrated a severe biofilm defect. These results indicate the importance of acetylated lysine residues in both YmcA and GtaB. In summary, we propose that protein lysine acetylation plays a global regulatory role in B. subtilis multicellularity.

Environmental strains of the soil bacterium Bacillus subtilis have valuable applications in agriculture, industry, and biotechnology; however, environmental strains are genetically less accessible. This reduced accessibility is in sharp contrast to laboratory strains, which are well known for their natural competence, and a limitation in their applications. In this study, we observed that robust biofilm formation by environmental strains of B. subtilis greatly reduced the frequency of competent cells in the biofilm. By using model strain 3610, we revealed a cross-pathway regulation that allows biofilm matrix producers and competence-developing cells to undergo mutually exclusive cell differentiation. We further demonstrated that the competence activator ComK represses the key biofilm regulatory gene sinI by directly binding to the sinI promoter, thus blocking competent cells from simultaneously becoming matrix producers. In parallel, the biofilm activator SlrR represses competence through three distinct mechanisms involving both genetic regulation and cell morphological changes. Finally, we discuss the potential implications of limiting competence in a bacterial biofilm. IMPORTANCE The soil bacterium Bacillus subtilis can form robust biofilms, which are important for its survival in the environment. B. subtilis also exhibits natural competence. By investigating competence development in B. subtilis in situ during biofilm formation, we reveal that robust biofilm formation often greatly reduces the frequency of competent cells within the biofilm. We then characterize a cross-pathway regulation that allows cells in these two developmental events to undergo mutually exclusive cell differentiation during biofilm formation. Finally, we discuss potential biological implications of limiting competence in a bacterial biofilm.

12 13 14 15a. These two authors contributed equally to this work. 16 (She contributed to about 50% of the results in the initial submission and the 17 revision; Hunter initiated the project and contributed to about 30% of the 18 results in the initial submission.) Abstract 31 Environmental strains of the soil bacterium Bacillus subtilis have valuable 32 applications in agriculture, industry, and biotechnology. They are capable of forming 33 robust biofilms and demonstrate excellent biological control activities in plant 34 protection. However, environmental strains are genetically less accessible, a sharp 35 contrast to the laboratory strains well known for their natural competence and a 36 limitation toward their application. In this study, we observed that robust biofilm 37 formation of the environmental strains greatly reduces the rate of competent cells 38 within the biofilm. By using the model strain 3610, we reveal a cross-pathway 39 regulation that allows biofilm matrix producers and competence-developing cells to 40 undergo mutually exclusive cell differentiation. We show that the competence 41 activator ComK represses the key biofilm regulatory gene sinI by directly binding to 42 the sinI promoter, thus blocking competent cells from simultaneously becoming 43 matrix producers. In parallel, the biofilm activator SlrR represses competence 44 through three distinct mechanisms, involving both genetic regulation and cell 45 morphological changes. We discuss potential implications of limiting competence in 46 a bacterial biofilm. 47 48 3 Importance 49The soil bacterium Bacillus subtilis is capable of forming robust biofilms, a 50 multicellular community important for its survival in the environment. B. subtilis also 51 exhibits natural competence, the ability of cells to acquire genetic materials directly 52 from the environment. By investigating competence development in situ during B. 53 subtilis biofilm formation, we reveal that robust biofilm formation, an important 54 feature of the environmental strains of B. subtilis, often greatly reduces the rate of 55 competent cells within the biofilm. We characterize a cross-pathway regulation that 56 allows cells associated with these two developmental events to undergo mutually 57 exclusive cell differentiation during biofilm formation. Finally, we discuss potential 58 biological implications of limiting competence in a bacterial biofilm. 59 60 Bacillus subtilis is a soil-dwelling, spore-forming bacterium widely present in 61 nature, and plays important roles in environment, agriculture, and industry. B. subtilis 62 is also a plant growth-promoting rhizobacterium (PGPR), and a biological control 63 agent that shows various beneficial activities in plant protection (1). Biological control 64 by B. subtilis is attributed to a number of important abilities of the bacterium, 65 including antibiotic production, inhibition of pathogenic fungi and parasites, induction 66 of plant systemic resistance, and formation of plant root-associated biofilms (2-4).