Structure and organisation of SinR, the master regulator of biofilm formation in Bacillus subtilis

sinR encodes a tetrameric repressor of genes required for biofilm formation in Bacillus subtilis. sinI, which is transcribed under Spo0A control, encodes a dimeric protein that binds to SinR to form a SinR-SinI heterodimer in which the DNA-binding functions of SinR are abrogated and repression of biofilm genes is relieved. The heterodimer-forming surface comprises residues conserved between SinR and SinI. Each forms a pair of α-helices that hook together to form an intermolecular four-helix bundle. Here, we are interested in the assembly of the SinR tetramer and its binding to DNA. Size-exclusion chromatography with multi-angle laser light scattering and crystallographic analysis reveal that a DNA-binding fragment of SinR (residues 1-69) is a monomer, while a SinI-binding fragment (residues 74-111) is a tetramer arranged as a dimer of dimers. The SinR(74-111) chain forms two α-helices with the organisation of the dimer similar to that observed in the SinR-SinI complex. The tetramer is formed through interactions of residues at the C-termini of the four chains. A model of the intact SinR tetramer in which the DNA binding domains surround the tetramerisation core was built. Fluorescence anisotropy and surface plasmon resonance experiments showed that SinR binds to an oligonucleotide duplex, 5′-TTTGTTCTCTAAAGAGAACTTA-3′, containing a pair of SinR consensus sequences in inverted orientation with a K_d of 300 nM. The implications of these data for promoter binding and the curious quaternary structural transitions of SinR upon binding to (i) SinI and (ii) the SinR-like protein SlrR, which “repurposes” SinR as a repressor of autolysin and motility genes, are discussed.     

ChIP‐seq and transcriptome analysis of the OmpR regulon of Salmonella enterica serovars Typhi and Typhimurium reveals accessory genes implicated in host colonization

OmpR is a multifunctional DNA binding regulator with orthologues in many enteric bacteria that exhibits classical regulator activity as well as nucleoid‐associated protein‐like characteristics. In the enteric pathogen Salmonella enterica, using chromatin immunoprecipitation of OmpR:FLAG and nucleotide sequencing, 43 putative OmpR binding sites were identified in S. enterica serovar Typhi, 22 of which were associated with OmpR‐regulated genes. Mutation of a sequence motif (TGTWACAW) that was associated with the putative OmpR binding sites abrogated binding of OmpR:6×His to the tviA upstream region. A core set of 31 orthologous genes were found to exhibit OmpR‐dependent expression in both S. Typhi and S. Typhimurium. S. Typhimurium‐encoded orthologues of two divergently transcribed OmpR‐regulated operons (SL1068–71 and SL1066–67) had a putative OmpR binding site in the inter‐operon region in S. Typhi, and were characterized using in vitro and in vivo assays. These operons are widely distributed within S. enterica but absent from the closely related Escherichia coli. SL1066 and SL1067 were required for growth on N‐acetylmuramic acid as a sole carbon source. SL1068–71 exhibited sequence similarity to sialic acid uptake systems and contributed to colonization of the ileum and caecum in the streptomycin‐pretreated mouse model of colitis.     

Importance ofeps genes fromBacillus subtilis in biofilm formation and swarming

Unicellular organisms naturally form multicellular communities, differentiate into specialized cells, and synchronize their behaviour under certain conditions. Swarming, defined as a movement of a large mass of bacteria on solid surfaces, is recognized as a preliminary step in the formation of biofilms. The main aim of this work was to study the role of a group of genes involved in exopolysaccharide biosynthesis during pellicle formation and swarming inBacillus subtilis strain 168. To assess the role of particular proteins encoded by the group ofepsI-epsO genes that form theeps operon, we constructed a series of insertional mutants. The results obtained showed that mutations inepsJ-epsN, but not in the last gene of theeps operon (epsO), have a severe effect on pellicle formation under all tested conditions. Moreover, the inactivation of 5 out of the 6 genes analysed caused total inhibition of swarming in strain 168 (that does not produce surfactin) on LB medium. Following restoration of thesfp gene (required for production of surfactin, which is essential for swarming of the wild-type bacteria), thesfp ⁺ strains defective ineps genes (exceptepsO) generated significantly different patterns during swarming on synthetic B medium, as compared to the parental strain 168sfp ⁺.     

Bistability and biofilm formation in Bacillus subtilis

Biofilms of Bacillus subtilis consist of long chains of cells that are held together in bundles by an extracellular matrix of exopolysaccharide and the protein TasA. The exopolysaccharide is produced by enzymes encoded by the epsA-O operon and the gene encoding TasA is located in the yqxM-sipW-tasA operon. Both operons are under the control of the repressor SinR. Derepression is mediated by the antirepressor SinI, which binds to SinR with a 1:1 stoichiometry. Paradoxically, in medium promoting derepression of the matrix operons, the overall concentration of SinR in the culture greatly exceeded that of SinI. We show that under biofilm-promoting conditions sinI, which is under the control of the response regulator Spo0A, was expressed only in a small subpopulation of cells, whereas sinR was expressed in almost all cells. Activation of Spo0A is known to be subject to a bistable switch, and we infer that SinI reaches levels sufficient to trigger matrix production only in the subpopulation of cells in which Spo0A is active. Additionally, evidence suggests that sinI is expressed at intermediate, but not low or high, levels of Spo0A activity, which may explain why certain nutritional conditions are more effective in promoting biofilm formation than others.