Transient heterogeneity in extracellular protease production by Bacillus subtilis

The most sophisticated survival strategy Bacillus subtilis employs is the differentiation of a subpopulation of cells into highly resistant endospores. To examine the expression patterns of non-sporulating cells within heterogeneous populations, we used buoyant density centrifugation to separate vegetative cells from endospore-containing cells and compared the transcriptome profiles of both subpopulations. This demonstrated the differential expression of various regulons. Subsequent single-cell analyses using promoter-gfp fusions confirmed our microarray results. Surprisingly, only part of the vegetative subpopulation highly and transiently expresses genes encoding the extracellular proteases Bpr (bacillopeptidase) and AprE (subtilisin), both of which are under the control of the DegU transcriptional regulator. As these proteases and their degradation products freely diffuse within the liquid growth medium, all cells within the clonal population are expected to benefit from their activities, suggesting that B. subtilis employs cooperative or even altruistic behavior. To unravel the mechanisms by which protease production heterogeneity within the non-sporulating subpopulation is established, we performed a series of genetic experiments combined with mathematical modeling. Simulations with our model yield valuable insights into how population heterogeneity may arise by the relatively long and variable response times within the DegU autoactivating pathway

The Response of Lactococcus lactis to Membrane Protein Production

Background: The biogenesis of membrane proteins is more complex than that of water-soluble proteins, and recombinant expression of membrane proteins in functional form and in amounts high enough for structural and functional studies is often problematic. To better engineer cells towards efficient protein production, we set out to understand and compare the cellular consequences of the overproduction of both classes of proteins in Lactococcus lactis, employing a combined proteomics and transcriptomics approach. Methodology and Findings: Highly overproduced and poorly expressed membrane proteins both resulted in severe growth defects, whereas amplified levels of a soluble substrate receptor had no effect. In addition, membrane protein overproduction evoked a general stress response (upregulation of various chaperones and proteases), which is probably due to accumulation of misfolded protein. Notably, upon the expression of membrane proteins a cell envelope stress response, controlled by the two-component regulatory CesSR system, was observed. Conclusions: The physiological response of L. lactis to the overproduction of several membrane proteins was determined and compared to that of a soluble protein, thus offering better understanding of the bottlenecks related to membrane protein production and valuable knowledge for subsequent strain engineering.

Distinct Roles of ComK1 and ComK2 in Gene Regulation in Bacillus cereus

The B. subtilis transcriptional factor ComK regulates a set of genes coding for DNA uptake from the environment and for its integration into the genome. In previous work we showed that Bacillus cereus expressing the B. subtilis ComK protein is able to take up DNA and integrate it into its own genome. To extend our knowledge on the effect of B. subtilis ComK overexpression in B. cereus we first determined which genes are significantly altered. Transcriptome analysis showed that only part of the competence gene cluster is significantly upregulated. Two ComK homologues can be identified in B. cereus that differ in their respective homologies to other ComK proteins. ComK1 is most similar, while ComK2 lacks the C-terminal region previously shown to be important for transcription activation by B. subtilis ComK. comK1 and comK2 overexpression and deletion studies using transcriptomics techniques showed that ComK1 enhances and ComK2 decreases expression of the comG operon, when B. subtilis ComK was overexpressed simultaneously

Localized Plasticity in the Streamlined Genomes of Vinyl Chloride Respiring Dehalococcoides

Vinyl chloride (VC) is a human carcinogen and widespread priority pollutant. Here we report the first, to our knowledge, complete genome sequences of microorganisms able to respire VC, Dehalococcoides sp. strains VS and BAV1. Notably, the respective VC reductase encoding genes, vcrAB and bvcAB, were found embedded in distinct genomic islands (GEIs) with different predicted integration sites, suggesting that these genes were acquired horizontally and independently by distinct mechanisms. A comparative analysis that included two previously sequenced Dehalococcoides genomes revealed a contextually conserved core that is interrupted by two high plasticity regions (HPRs) near the Ori. These HPRs contain the majority of GEIs and strain-specific genes identified in the four Dehalococcoides genomes, an elevated number of repeated elements including insertion sequences (IS), as well as 91 of 96 rdhAB, genes that putatively encode terminal reductases in organohalide respiration. Only three core rdhA orthologous groups were identified, and only one of these groups is supported by synteny. The low number of core rdhAB, contrasted with the high rdhAB numbers per genome (up to 36 in strain VS), as well as their colocalization with GEIs and other signatures for horizontal transfer, suggests that niche adaptation via organohalide respiration is a fundamental ecological strategy in Dehalococccoides. This adaptation has been exacted through multiple mechanisms of recombination that are mainly confined within HPRs of an otherwise remarkably stable, syntenic, streamlined genome among the smallest of any free-living microorganism

Biology of Streptococcus mutans-Derived Glucosyltransferases: Role in Extracellular Matrix Formation of Cariogenic Biofilms

The importance of Streptococcus mutans in the etiology and pathogenesis of dental caries is certainly controversial, in part because excessive attention is paid to the numbers of S. mutans and acid production while the matrix within dental plaque has been neglected. S. mutans does not always dominate within plaque; many organisms are equally acidogenic and aciduric. It is also recognized that glucosyltransferases from S. mutans (Gtfs) play critical roles in the development of virulent dental plaque. Gtfs adsorb to enamel synthesizing glucans in situ, providing sites for avid colonization by microorganisms and an insoluble matrix for plaque. Gtfs also adsorb to surfaces of other oral microorganisms converting them to glucan producers. S. mutans expresses 3 genetically distinct Gtfs; each appears to play a different but overlapping role in the formation of virulent plaque. GtfC is adsorbed to enamel within pellicle whereas GtfB binds avidly to bacteria promoting tight cell clustering, and enhancing cohesion of plaque. GtfD forms a soluble, readily metabolizable polysaccharide and acts as a primer for GtfB. The behavior of soluble Gtfs does not mirror that observed with surface-adsorbed enzymes. Furthermore, the structure of polysaccharide matrix changes over time as a result of the action of mutanases and dextranases within plaque. Gtfs at distinct loci offer chemotherapeutic targets to prevent caries. Nevertheless, agents that inhibit Gtfs in solution frequently have a reduced or no effect on adsorbed enzymes. Clearly, conformational changes and reactions of Gtfs on surfaces are complex and modulate the pathogenesis of dental caries in situ, deserving further investigation

Role of asparagine 1134 in glucosidic bond and transglycosylation specificity of reuteransucrase from Lactobacillus reuteri 121

Glucansucrases from lactic acid bacteria convert sucrose into various α-glucans that differ greatly with respect to the glucosidic bonds present (e.g. dextran, mutan, alternan and reuteran). This study aimed to identify the structural features of the reuteransucrase from Lactobacillus reuteri 121 (GTFA) that determine its reaction specificity. We here report a detailed mutational analysis of a conserved region immediately next to the catalytic Asp1133 (putative transition-state stabilizing) residue in GTFA. The data show that Asn1134 is the main determinant of glucosidic bond product specificity in this reuteransucrase. Furthermore, mutations at this position greatly influenced the hydrolysis/transglycosylation ratio. Changes in this amino acid expands the range of glucan and gluco-oligosaccharide products synthesized from sucrose by mutant GTFA enzymes.