Negative Interplay between Biofilm Formation and Competence in the Environmental Strains of Bacillus subtilis.

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.

Protein lysine acetylation plays a regulatory role in Bacillus subtilis multicellularity.

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.

The effects of elevated temperature and ocean acidification on the metabolic pathways of notothenioid fish.

The adaptations used by notothenioid fish to combat extreme cold may have left these fish poorly poised to deal with a changing environment. As such, the expected environmental perturbations brought on by global climate change have the potential to significantly affect the energetic demands and subsequent cellular processes necessary for survival. Despite recent lines of evidence demonstrating that notothenioid fish retain the ability to acclimate to elevated temperatures, the underlying mechanisms responsible for temperature acclimation in these fish remain largely unknown. Furthermore, little information exists on the capacity of Antarctic fish to respond to changes in multiple environmental variables. We have examined the effects of increased temperature and pCO2 on the rate of oxygen consumption in three notothenioid species, Trematomus bernacchii, Pagothenia borchgrevinki, and Trematomus newnesi. We combined these measurements with analysis of changes in aerobic and anaerobic capacity, lipid reserves, fish condition, and growth rates to gain insight into the metabolic cost associated with acclimation to this dual stress. Our findings indicated that temperature is the major driver of the metabolic responses observed in these fish and that increased pCO2 plays a small, contributing role to the energetic costs of the acclimation response. All three species displayed varying levels of energetic compensation in response to the combination of elevated temperature and pCO2. While P. borchgrevinki showed nearly complete compensation of whole animal oxygen consumption rates and aerobic capacity, T. newnesi and T. bernacchii displayed only partial compensation in these metrics, suggesting that at least some notothenioids may require physiological trade-offs to fully offset the energetic costs of long-term acclimation to climate change related stressors.

Characterization of nitrifying, denitrifying, and overall bacterial communities in permeable marine sediments of the northeastern Gulf of Mexico.

Sandy or permeable sediment deposits cover the majority of the shallow ocean seafloor, and yet the associated bacterial communities remain poorly described. The objective of this study was to expand the characterization of bacterial community diversity in permeable sediment impacted by advective pore water exchange and to assess effects of spatial, temporal, hydrodynamic, and geochemical gradients. Terminal restriction fragment length polymorphism (TRFLP) was used to analyze nearly 100 sediment samples collected from two northeastern Gulf of Mexico subtidal sites that primarily differed in their hydrodynamic conditions. Communities were described across multiple taxonomic levels using universal bacterial small subunit (SSU) rRNA targets (RNA- and DNA-based) and functional markers for nitrification (amoA) and denitrification (nosZ). Clonal analysis of SSU rRNA targets identified several taxa not previously detected in sandy sediments (i.e., Acidobacteria, Actinobacteria, Chloroflexi, Cyanobacteria, and Firmicutes). Sequence diversity was high among the overall bacterial and denitrifying communities, with members of the Alphaproteobacteria predominant in both. Diversity of bacterial nitrifiers (amoA) remained comparatively low and did not covary with the other gene targets. TRFLP fingerprinting revealed changes in sequence diversity from the family to species level across sediment depth and study site. The high diversity of facultative denitrifiers was consistent with the high permeability, deeper oxygen penetration, and high rates of aerobic respiration determined in these sediments. The high relative abundance of Gammaproteobacteria in RNA clone libraries suggests that this group may be poised to respond to short-term periodic pulses of growth substrates, and this observation warrants further investigation.

Microbial community diversity associated with carbon and nitrogen cycling in permeable shelf sediments.

Though a large fraction of primary production and organic matter cycling in the oceans occurs on continental shelves dominated by sandy deposits, the microbial communities associated with permeable shelf sediments remain poorly characterized. Therefore, in this study, we provide the first detailed characterization of microbial diversity in marine sands of the South Atlantic Bight through parallel analyses of small-subunit (SSU) rRNA gene (Bacteria), nosZ (denitrifying bacteria), and amoA (ammonia-oxidizing bacteria) sequences. Communities were analyzed by parallel DNA extractions and clone library construction from both sediment core material and manipulated sediment within column experiments designed for geochemical rate determinations. Rapid organic-matter degradation and coupled nitrification-denitrification were observed in column experiments at flow rates resembling in situ conditions over a range of oxygen concentrations. Numerous SSU rRNA phylotypes were affiliated with the phyla Proteobacteria (classes Alpha-, Delta-, and Gammaproteobacteria), Planctomycetes, Cyanobacteria, Chloroflexi, and Bacteroidetes. Detectable sequence diversity of nosZ and SSU rRNA genes increased in stratified redox-stabilized columns compared to in situ sediments, with the Alphaproteobacteria comprising the most frequently detected group. Alternatively, nitrifier communities showed a relatively low and stable diversity that did not covary with the other gene targets. Our results elucidate predominant phylotypes that are likely to catalyze carbon and nitrogen cycling in marine sands. Although overall diversity increased in response to redox stabilization and stratification in column experiments, the major phylotypes remained the same in all of our libraries, indicating that the columns sufficiently mimic in situ conditions.