Structure of the N-terminal oligomerization domain of DnaD reveals a unique tetramerization motif and provides insights into scaffold formation

DnaD is a primosomal protein that remodels supercoiled plasmids. It binds to supercoiled forms and converts them to open forms without nicking. During this remodeling process, all the writhe is converted to twist and the plasmids are held around the periphery of large scaffolds made up of DnaD molecules. This DNA-remodeling function is the sum of a scaffold-forming activity on the N-terminal domain and a DNA-dependent oligomerization activity on the C-terminal domain. We have determined the crystal structure of the scaffold-forming N-terminal domain, which reveals a winged-helix architecture, with additional structural elements extending from both N- and C-termini. Four monomers form dimers that join into a tetramer. The N-terminal extension mediates dimerization and tetramerization, with extensive interactions and distinct interfaces. The wings and helices of the winged-helix domains remain exposed on the surface of the tetramer. Structure-guided mutagenesis and atomic force microscopy imaging indicate that these elements, together with the C-terminal extension, are involved in scaffold formation. Based upon our data, we propose a model for the DnaD-mediated scaffold formation.     

Termination Structures in the Escherichia coli Chromosome Replication Fork Trap

The Escherichia coli chromosome contains two opposed sets of unidirectional DNA replication pause (Ter) sites that, according to the replication fork trap theory, control the termination of chromosome replication by restricting replication fork fusion to the terminus region. In contrast, a recent hypothesis suggested that termination occurs at the dif locus instead. Using two-dimensional agarose gel electrophoresis, we examined DNA replication intermediates at the Ter sites and at dif in wild-type cells. Two definitive signatures of site-specific termination—specific replication fork arrest and converging replication forks—were clearly detected at Ter sites, but not at dif. We also detected a significant pause during the latter stages of replication fork convergence at Ter sites. Quantification of fork pausing at the Ter sites in both their native chromosomal context and the plasmid context further supported the fork trap model.     

Structural analysis of a putative SAM-dependent methyltransferase,YtqB, from Bacillus subtilis

S-adenosyl-l-methionine (SAM)-dependent methyltransferases (MTases) methylate diverse biological molecules using a SAM cofactor. The ytqB gene of Bacillus subtilis encodes a putative MTase and its biological function has never been characterized. To reveal the structural features and the cofactor binding mode of YtqB, we have determined the crystal structures of YtqB alone and in complex with its cofactor, SAM, at 1.9 Å and 2.2 Å resolutions, respectively. YtqB folds into a β-sheet sandwiched by two α-helical layers, and assembles into a dimeric form. Each YtqB monomer contains one SAM binding site, which shapes SAM into a slightly curved conformation and exposes the reactive methyl group of SAM potentially to a substrate. Our comparative structural analysis of YtqB and its homologues indicates that YtqB is a SAM-dependent class I MTase, and provides insights into the substrate binding site of YtqB.     

Involvement of SpoVG in hemolysis caused by Bacillus subtilis

Bacillus subtilis is a facultative anaerobic Gram-positive non-pathogenic bacterium that includes members displaying hemolytic activity. To identify the genes responsible for hemolysis, a random mariner-based transposon insertion mutant library of B. subtilis 168 was constructed. More than 20,000 colonies were screened for the hypohemolytic phenotype on blood agar plates. One mutant showed significantly less pronounced hemolytic phenotype than the wild type. DNA sequencing and Southern blot analysis showed this mutant has a single transposable element inserted into the open reading frame (ORF) of the spoVG gene; complementation of the spoVG-disrupted mutant with a wild-type copy restored its hemolytic phenotype. It was therefore concluded that the spoVG gene, which plays a role in regulating asymmetric septation during sporulation in B. subtilis, is involved in hemolysis by B. subtilis.     

Bioconversion of shellfish chitin wastes for the production of Bacillus subtilis W-118 chitinase

Bacillus subtilis W-118, a strain that produces antifungal materials, excreted a chitinase when cultured in a medium containing shrimp- and crab-shell powder as the major carbon source. This chitinase, purified by sequential chromatography, had a molecular mass of 20,600 Da and a pI of 6. The optimum pH, optimum temperature, and pH stability of the chitinase were pH 6, 37 degrees C, and pH 5-7, respectively. The unique characteristics of the purified chitinase include low molecular mass and acidic pI. In the investigation of the inhibitory activity, it was found that the growth of Fusarium oxysporum was 100% inhibited after incubation for 1 day with sterilized W-118 chitinase solution (5.6 units/mL). The chitinase hydrolyzates of chitin with low degrees of polymerization (DP 1-6) were analyzed by HPLC. Longer reaction times led to the generation of chitin oligosaccharides with lower DP. The chitin oligosaccharides were examined for their inhibitory effects on F. oxysporum and human leukemia cell lines.     

Microbial Treatment for Prevention and Removal of Paraffin Deposition on the Walls of Crude Pipelines

Two bacterial strains, paraffin removal strain and biosurfactant-producing strain, named BHJ-1 and QFL-1, were isolated from oil production wells in Daqing oilfield of China. They were subsequently identified as Bacillus cereus QAU68 and Bacillus subtilis XCCX, respectively. As an indicator of the degradation paraffin, the inoculum concentration of BHJ-1 and QFL-1 were added in different proportions, the optimum proportion was 5:2. In this proportion the degradation rate of paraffin could reach 64 %, the prevention rate of paraffin could reach 55 %.     

呼和浩特市大青山白桦根际土壤细菌群落结构研究

采用高通量测序技术对天然次生林生态系统演替过程中先锋树种白桦的根际土壤细菌多样性及群落结构进行了分析。研究结果表明:白桦根际土壤细菌隶属于28门、90纲、126目、213科、286属,在3个采样地中排名前8的优势细菌门的相对丰度均大于1%,分别为变形菌门(Proteobacteria)、酸杆菌门(Acidobacteria)、放线菌门(Actinobacteria)、芽单胞菌门(Gemmatimonadetes)、绿弯菌门(Chloroflexi)、硝化螺旋菌门(Nitrospirae)、疣微菌门(Verrucomicrobia)和拟杆菌门(Bacteroidetes)。各样地中前3个门的相对丰度之和均在60%以上。对白桦根际土壤细菌的α多样性指数、门水平的聚类热图以及PCoA聚类结果的分析表明,3个采样地中,小井沟(B2)和哈达门森林公园(C2)白桦根际土壤细菌的物种组成更为接近,与井儿梁(A2)的物种组成有一定差异;且小井沟和哈达门森林公园的物种多样性及丰度(ACE指数)显著高于井儿梁,表明细菌对不同环境的适应能力有明显差异。对细菌群落结构与土壤理化性质的RDA分析及相关性分析表明,环境因子对白桦根际土壤细菌的影响顺序为:全氮TN酸碱度pH含水量WC速效钾AK硝态氮NN铵态氮AN有机质OM有效磷EP,其中,TN、pH和WC是白桦根际土壤优势细菌的主要影响因子。研究结果为深入认识森林生态系统中根际土壤细菌的群落结构和影响因子提供了理论依据。     

Simultaneous cloning and expression of two cellulase genes from Bacillus subtilis newly isolated from Golden Takin (Budorcas taxicolor Bedfordi)

A bacterial strain with high cellulase activity was isolated of feces sample of Golden Takin (Budorcas taxicolor Bedfordi). The bacterium was classified and designated Bacillus subtilis LN by morphological and 16SrDNA gene sequence analysis. Two putative cellulase genes, CelL15 and CelL73, were simultaneously cloned from the isolated strain by PCR. The putative gene CelL15 consisted of an open reading frame (ORF) of 1470 nucleotides and encoded a protein of 490 amino acids with a molecular weight of 54 kDa. The CelL73 gene consisted of an open reading frame (ORF) of 741 nucleotides and encoded a protein of 247 amino acids with a molecular weight of 27 kDa. Both genes were purified and cloned into pET-28a for expression in Escherichia coli BL21 (DE3). The ability of E. coli to degrade cellulose was enhanced when the two recombinants were cultured together.     

Inhibition of the growth of Paenibacillus larvae, the causal agent of American foulbrood of honeybees, by selected strains of aerobic spore-forming bacteria isolated from apiarian sources

The bacterium Paenibacillus larvae, the causative agent of American foulbrood disease of honeybee larvae, occurs throughout the world and is found in many beekeeping areas of Argentina. The potential as biocontrol agents of antagonic aerobic spore-forming bacteria isolated from honey samples and other apiarian sources were evaluated. Each isolate was screened against one strain of Paenibacillus larvae (ATCC 9545) by using a perpendicular streak technique. Ten randomly selected bacterial strains from the group that showed the best antagonistic effect to P. larvae ATCC 9545 were selected for further study. These were identified as Bacillus subtilis (m351), B. pumilus (m350), B. licheniformis (m347), B. cereus (mv33), B. cereus (m387), B. cereus (m6c), B. megaterium (m404), Brevibacillus laterosporus (BLAT169), B. laterosporus (BLAT170), and B. laterosporus (BLAT171). The antagonistic strains were tested against 17 P. larvae strains from different geographical origins by means of a spot test in wells. The analysis of variance and posterior comparison of means by Tukey method (P < 0.01) showed that the best antagonists were B. megaterium (m404), B. licheniformis (m347), B. cereus (m6c), B. cereus (mv33), and B. cereus (m387).     

Overproduction of alkaline polygalacturonate lyase in recombinant Escherichia coli by a two-stage glycerol feeding approach

This work aims to achieve the overproduction of alkaline polygalacturonate lyase (PGL) with recombinant Escherichia coli by a two-stage glycerol feeding approach. First, the PGL coding gene from Bacillus subtilis WSHB04-02 was expressed in E. coli BL21 (DE3) under the strong inducible T7 promoter of the pET20b (+) vector. And then the influence of media composition, induction temperature, and inducer isopropyl β-D-1-thiogalactopyranoside (IPTG) concentration on cell growth and PGL production was investigated. Finally, a two-stage glycerol feeding strategy was proposed and applied in a 3-L fermenter, where cultivation was conducted at a controlled specific growth rate (μset=0.2) during pre-induction phase, followed by a constant glycerol feeding rate of 12 ml h(-1) at post-induction phase. The total PGL yield reached 371.86 U mL(-1), which is the highest PGL production by recombinant E. coli expression system.     

Study on the production of chitin and chitosan from shrimp shell by using Bacillus subtilis fermentation

Fermentation of shrimp shell in jaggery broth using Bacillus subtilis for the production of chitin and chitosan was investigated. It was found that B. subtilis produced sufficient quantities of acid to remove the minerals from the shell and to prevent spoilage organisms. The protease enzyme in Bacillus species was responsible for the deprotenisation of the shell. The pH, proteolytic activity, extent of demineralization and deprotenisation were studied during fermentation. About 84% of the protein and 72% of the minerals were removed from the shrimp shell after fermentation. Mild acid and alkali treatments were given to produce characteristic chitin and their concentrations were standardized. Chitin was converted to chitosan by N-deacetylation and the properties of chitin and chitosan were studied. FTIR spectral analysis of chitin and chitosan prepared by the process was carried out and compared with spectra of commercially available samples.     

一株促生秸秆降解菌的分离与鉴定

针对秸秆处理不当影响全世界环境污染的问题，筛选多功能秸秆降解菌，旨在得到高效降解秸秆且具有促生作用的微生物菌种。结合纤维素钠-刚果红(CMC-Na)平板筛选，通过16S rRNA基因分析，进行菌株鉴定，得到一株具有纤维素降解效果的菌株XJ-132,经16S rRNA基因鉴定为枯草芽胞杆菌(Bacillus subtilis)。与单独施用秸秆处理相比，加入菌株XJ-132 60 d后，秸秆降解率提高21.0%,且对水稻生长促进作用显著，地上、下部鲜重分别增加17.8%和9.6%。水稻种子喷施菌株XJ-132发酵液，低浓度发酵液对种子萌发具有一定促进作用。结果表明，菌株XJ-132可能通过产吲哚乙酸(IAA)、产铁载体、产氨等多种有益物质，降解秸秆的同时促进水稻生长。筛选具有促生作用的秸秆降解菌能够更好地加速秸秆降解，具有广泛的开发利用前景。     

The crystal structure of YloQ, a circularly permuted GTPase essential for Bacillus subtilis viability

yloQ is one of 11 essential genes in Bacillus subtilis with unknown roles in the physiology of the cell. It encodes a polypeptide of 298 residues with motifs characteristic of GTPases. As a contribution to elucidating its indispensable cellular function, we have solved the crystal structure of YloQ to 1.6 A spacing, revealing a three-domain organisation. At the heart of the molecule is the putative GTPase domain, which exhibits a classical alpha/beta nucleotide-binding fold with a topology very similar to that of Ras and Era. However, as anticipated from the order in which the conserved G protein motifs appear in the sequence, the GTPase domain fold in YloQ is circularly permuted with respect to the classical GTPases. The nucleotide-binding pocket in YloQ is unoccupied, and analysis of the phosphate-binding (P) loop indicates that conformational changes in this region would be needed to accommodate GTP. The GTPase domain is flanked at its N terminus by a beta-barrel domain with an oligonucleotide/oligosaccharide-binding (OB) fold, and at its C terminus by an alpha-helical domain containing a coordinated zinc ion. This combination of protein modules is unique to YloQ and its orthologues. Sequence comparisons reveal a clustering of conserved basic and aromatic residues on one face of the OB domain, perhaps pointing to a role for YloQ in nucleic acid binding. The zinc ion in the alpha-helical domain is coordinated by three cysteine residues and a histidine residue in a novel ligand organisation. The juxtaposition of the switch I and switch II regions of the G domain and the OB and zinc-binding domains suggests that chemical events at the GTPase active site may be transduced into relative movements of these domains. The pattern of conserved residues and electrostatic surface potential calculations suggest that the OB and/or Zn-binding domains participate in nucleic acid binding consistent with a possible role for YloQ at some stage during mRNA translation.     

Subtle interplay of stochasticity and deterministic dynamics pervades an evolutionary plausible genetic circuit for Bacillus subtilis competence

Here we study the interplay of stochastic and deterministic dynamics in an evolutionary plausible candidate core genetic circuit for Bacillus subtilis competence. We find that high noise would not necessarily be detrimental to the circuit’s ability to deliver the phenotype, due to an unexpected built-in robustness that we further investigate. Also, we find that seemingly subtle deterministic dynamical features of the regulation, unstable and stable limit cycles, while in the presence of biochemical noise, would result in a distinctive new observable in the phenotype. We conduct mathematical analyses of the system’s stability at the fixed points and derive some general model-independent consequences. We also show how imperfect time-scale separation in the system would result in observables detrimental to the phenotype, that nature could have harnessed for selection.     

The Replication System of Plasmids from Bacillus subtilis Environmental Isolates

Restriction enzyme analysis, cloning, and sequencing showed that large (more than 90 kb) plasmids isolated from different Bacillus subtilisstrains are identical in structure of the region ensuring stable inheritance of plasmid replicons and are widespread in Belarussian environmental strains of B. subtilis.     

Surfactin production enhances the level of cardiolipin in the cytoplasmic membrane of Bacillus subtilis

Surfactin is a cyclic lipopeptide antibiotic that disturbs the integrity of the cytoplasmic membrane. In this study, the role of membrane lipids in the adaptation and possible surfactin tolerance of the surfactin producer Bacillus subtilis ATCC 21332 was investigated. During a 1-day cultivation, the phospholipids of the cell membrane were analyzed at the selected time points, which covered both the early and late stationary phases of growth, when surfactin concentration in the medium gradually rose from 2 to 84 μmol·l^− 1. During this time period, the phospholipid composition of the surfactin producer's membrane (Sf⁺) was compared to that of its non-producing mutant (Sf⁻). Substantial modifications of the polar head group region in response to the presence of surfactin were found, while the fatty acid content remained unaffected. Simultaneously with surfactin production, a progressive accumulation up to 22% of the stress phospholipid cardiolipin was determined in the Sf⁺ membrane, whereas the proportion of phosphatidylethanolamine remained constant. At 24 h, cardiolipin was found to be the second major phospholipid of the membrane. In parallel, the Laurdan generalized polarization reported an increasing rigidity of the lipid bilayer. We concluded that an enhanced level of cardiolipin is responsible for the membrane rigidification that hinders the fluidizing effect of surfactin. At the same time cardiolipin, due to its negative charge, may also prevent the surfactin-membrane interaction or surfactin pore formation activity.     

Optimizing Bacillus subtilis spore isolation and quantifying spore harvest purity

Investigating the biochemistry, resilience and environmental interactions of bacterial endospores often requires a pure endospore biomass free of vegetative cells. Numerous endospore isolation methods, however, neglect to quantify the purity of the final endospore biomass. To ensure low vegetative cell contamination we developed a quality control technique that enables rapid quantification of endospore harvest purity. This method quantifies spore purity using bright-field and fluorescence microscopy imaging in conjunction with automated cell counting software. We applied this method to Bacillus subtilis endospore harvests isolated using a two-phase separation method that utilizes mild chemicals. The average spore purity of twenty-two harvests was 88 ± 11% (error is 1σ) with a median value of 93%. A spearman coefficient of 0.97 correlating automated and manual bacterial counts confirms the accuracy of software generated data.