Bacillus subtilis transcriptional regulators interaction

Bacillus subtilis aprE gene codes for the extracellular protease subtilisin. Its expression is controlled by AbrB, DegU, Hpr, SinI, SinR and Spo0A transition state protein regulators. To determine in vivo the protein-protein interactions among these regulators, we used the LexA-based bacterial genetic two-hybrid system. Our results show homo-dimerization to all the analyzed proteins and hetero-dimerization between SinR-SinI and SinR-Hpr.     

Identification of Poly-N-acetylglucosamine as a Major Polysaccharide Component of the Bacillus subtilis Biofilm Matrix

Bacillus subtilis is intensively studied as a model organism for the development of bacterial biofilms or pellicles. A key component is currently undefined exopolysaccharides produced from proteins encoded by genes within the eps locus. Within this locus are four genes, epsHIJK, known to be essential for pellicle formation. We show they encode proteins synthesizing the broadly expressed microbial carbohydrate poly-N-acetylglucosamine (PNAG). PNAG was present in both pellicle and planktonic wild-type B. subtilis cells and in strains with deletions in the epsA–G and -L–O genes but not in strains deleted for epsH–K. Cloning of the B. subtilis epsH–K genes into Escherichia coli with in-frame deletions in the PNAG biosynthetic genes pgaA–D, respectively, restored PNAG production in E. coli. Cloning the entire B. subtilis epsHIJK locus into pga-deleted E. coli, Klebsiella pneumoniae, or alginate-negative Pseudomonas aeruginosa restored or conferred PNAG production. Bioinformatic and structural predictions of the EpsHIJK proteins suggest EpsH and EpsJ are glycosyltransferases (GT) with a GT-A fold; EpsI is a GT with a GT-B fold, and EpsK is an α-helical membrane transporter. B. subtilis, E. coli, and pga-deleted E. coli carrying the epsHIJK genes on a plasmid were all susceptible to opsonic killing by antibodies to PNAG. The immunochemical and genetic data identify the genes and proteins used by B. subtilis to produce PNAG as a significant carbohydrate factor essential for pellicle formation.     

Mechanism of Antimycin A Resistance in Bacillus subtilis

The site of action of antimycin A is known to lie between cytochrome b and c in the respiratory chain of mammalian cells. But in general, bacteria, even those which have cytochromes similar to those of mammalian cells such as Bacillus subtilis, are naturally resistant to this antibiotic.

The mechanism of this natural resistance is studied using a strain of B. subtilis. Succinoxidase activity of the intact cells of this bacterium showed very low sensitivity to the antibiotic, but on disruption of the cells, the sensitivity increased 7.5 times. Moreover, the activity of the intact cells could be sensitized by treatment with cationic detergent. In addition to the permeability barrier suggested by the above results, it was found that the electron transport system of this bacterium contained antimycin A insensitive by-path.     

Crystallization of NAD+ synthetase from Bacillus subtilis

NAD⁺-synthetase is a ubiquitous enzyme catalyzing the last step in the biosynthesis of NAD⁺. Mutants of NAD⁺ synthetase with impaired cellular functions have been isolated, indicating a key role for this enzyme in cellular metabolism. Crystals of the enzyme from Bacillus subtilis suitable for x-ray crystallographic investigation have been grown from polyethylene glycol solutions. Investigation on the structural organization of NAD⁺ synthetase, an enzyme fundamental for NAD⁺ biosynthesis, and belonging to the recently characterized amidotransferase enzymatic family, will provide more insight into the catalytic mechanism of deamido-NAD⁺ → NAD⁺ conversion, a biosynthetic process that is a potential target for the development of antibiotic compounds against Bacillus sp. and related bacteria. © 1996 Wiley-Liss, Inc.     

A ribosome–nascent chain sensor of membrane protein biogenesis in Bacillus subtilis

Proteins in the YidC/Oxa1/Alb3 family have essential functions in membrane protein insertion and folding. Bacillus subtilis encodes two YidC homologs, one that is constitutively expressed (spoIIIJ/yidC1) and a second (yqjG/yidC2) that is induced in spoIIIJ mutants. Regulated induction of yidC2 allows B. subtilis to maintain capacity of the membrane protein insertion pathway. We here show that a gene located upstream of yidC2 (mifM/yqzJ) serves as a sensor of SpoIIIJ activity that regulates yidC2 translation. Decreased SpoIIIJ levels or deletion of the MifM transmembrane domain arrests mifM translation and unfolds an mRNA hairpin that otherwise blocks initiation of yidC2 translation. This regulated translational arrest and yidC2 induction require a specific interaction between the MifM C‐terminus and the ribosomal polypeptide exit tunnel. MifM therefore acts as a ribosome–nascent chain complex rather than as a fully synthesized protein. B. subtilis MifM and the previously described secretion monitor SecM in Escherichia coli thereby provide examples of the parallel evolution of two regulatory nascent chains that monitor different protein export pathways by a shared molecular mechanism.     

Sporulation: an alternative way to recover recombinant proteins from Bacillus subtilis

A recombinant strain of Bacillus subtilis engineered for endocellular expression of human interleukin-1 receptor antagonist (IL-Ira) was subjected to sporulation. The recombinant protein was recovered from the sporulation supernatant in quantities, purity, and activity comparable with those obtained from a traditional cell lysate. Thus, exploitation of this natural mechanism of autolysis could overcome problems of intact protein recovery related to the cell disruption step. (c) 1995 John Wiley & Sons, Inc.     

The HPr proteins from the thermophile Bacillus stearothermophilus can form domain-swapped dimers

The study of proteins from extremophilic organisms continues to generate interest in the field of protein folding because paradigms explaining the enhanced stability of these proteins still elude us and such studies have the potential to further our knowledge of the forces stabilizing proteins. We have undertaken such a study with our model protein HPr from a mesophile, Bacillus subtilis, and a thermophile, Bacillus stearothermophilus. We report here the high-resolution structures of the wild-type HPr protein from the thermophile and a variant, F29W. The variant proved to crystallize in two forms: a monomeric form with a structure very similar to the wild-type protein as well as a domain-swapped dimer. Interestingly, the structure of the domain-swapped dimer for HPr is very different from that observed for a homologous protein, Crh, from B.subtilis. The existence of a domain-swapped dimer has implications for amyloid formation and is consistent with recent results showing that the HPr proteins can form amyloid fibrils. We also characterized the conformational stability of the thermophilic HPr proteins using thermal and solvent denaturation methods and have used the high-resolution structures in an attempt to explain the differences in stability between the different HPr proteins. Finally, we present a detailed analysis of the solution properties of the HPr proteins using a variety of biochemical and biophysical methods.     

Thermostability of In Vitro Evolved Bacillus subtilis Lipase A: A Network and Dynamics Perspective

Proteins in thermophilic organisms remain stable and function optimally at high temperatures. Owing to their important applicability in many industrial processes, such thermostable proteins have been studied extensively, and several structural factors attributed to their enhanced stability. How these factors render the emergent property of thermostability to proteins, even in situations where no significant changes occur in their three-dimensional structures in comparison to their mesophilic counter-parts, has remained an intriguing question. In this study we treat Lipase A from Bacillus subtilis and its six thermostable mutants in a unified manner and address the problem with a combined complex network-based analysis and molecular dynamic studies to find commonality in their properties. The Protein Contact Networks (PCN) of the wild-type and six mutant Lipase A structures developed at a mesoscopic scale were analyzed at global network and local node (residue) level using network parameters and community structure analysis. The comparative PCN analysis of all proteins pointed towards important role of specific residues in the enhanced thermostability. Network analysis results were corroborated with finer-scale molecular dynamics simulations at both room and high temperatures. Our results show that this combined approach at two scales can uncover small but important changes in the local conformations that add up to stabilize the protein structure in thermostable mutants, even when overall conformation differences among them are negligible. Our analysis not only supports the experimentally determined stabilizing factors, but also unveils the important role of contacts, distributed throughout the protein, that lead to thermostability. We propose that this combined mesoscopic-network and fine-grained molecular dynamics approach is a convenient and useful scheme not only to study allosteric changes leading to protein stability in the face of negligible over-all conformational changes due to mutations, but also in other molecular networks where change in function does not accompany significant change in the network structure.     

Analysis of the open and closed conformations of the GTP-binding protein YsxC from Bacillus subtilis

Genetic analysis has suggested that the product of the Bacillus subtilis ysxC gene is essential for survival of the microorganism and hence may represent a target for the development of a novel anti-infective agent. B.subtilis YsxC is a member of the translation factor related class of GTPases and its crystal structure has been determined in an apo form and in complex with GDP and GMPPNP/Mg2+. Analysis of these structures has allowed us to examine the conformational changes that occur during the process of nucleotide binding and GTP hydrolysis. These structural changes particularly affect parts of the switch I and switch II region of YsxC, which become ordered and disordered, respectively in the "closed" or "on" GTP-bound state and disordered and ordered, respectively, in the "open" or "off" GDP-bound conformation. Finally, the binding of the magnesium cation results in subtle shifts of residues in the G3 region, at the start of switch II, which serve to optimize the interaction with a key aspartic acid residue. The structural flexibility observed in YsxC is likely to contribute to the role of the protein, possibly allowing transduction of an essential intracellular signal, which may be mediated via interactions with a conserved patch of surface-exposed, basic residues that lies adjacent to the GTP-binding site.     

枯草芽孢杆菌MH25 Iturin A操纵子的克隆与分析

根据已知非核糖体肽合成抗生素操纵子的保守序列设计引物,从对棉花立枯病有很好拮抗作用的枯草芽孢杆菌(Bacillus subtilis)MH25菌株中克隆相关操纵子.获得了枯草芽孢杆菌MH25的一个非核糖体肽合成抗生素操纵子序列,其包括4个ORF(ORF1,ORF2,OKF3,ORF4),与枯草芽孢杆菌RB14的ituD,ituA,tiuB和ituC的同源性分别为99%,98.70%,98.99%和99.48%,4个ORF编码的氨基酸序列与ItuD,ItuA,ItuB,ItuC的相似性分别为98%,98.54%,98.69%和98%.然后将4个ORF分别进行结构域分析,ORF3的14 779～14 963序列与ituB相对应区域的相似性为86.24%.该操纵子的启动子区为TATACACA-16bp-TAGGAT,与σA-10和-35(TTGACA-17bp-TATAAAT)不同.枯草芽孢杆菌MH25的Iturin A操纵子序列已在GenBank中注册,登陆号为EU263005.     

Glutamine synthetase gene of Bacillus subtilis

The glutamine synthetase gene (glnA) of Bacillus subtilis was purified from a library of B. subtilis DNA cloned in phage lambda. By mapping the locations of previously identified mutations in the glnA locus it was possible to correlate the genetic and physical maps. Mutations known to affect expression of the glnA gene and other genes were mapped within the coding region for glutamine synthetase, as determined by measuring the sizes of truncated, immunologically cross-reacting polypeptides coded for by various sub-cloned regions of the glnA gene. When the entire B. subtilis glnA gene was present on a plasmid it was capable of directing synthesis in Escherichia coli of B. subtilis glutamine synthetase as judged by enzymatic activity, antigenicity, and ability to allow growth of a glutamine auxotroph. By use of the cloned B. subtilis glnA gene as a hybridization probe, it was shown that the known variability of glutamine synthetase specific activity during growth in various nitrogen sources is fully accounted for by changes in glnA mRNA levels.     

Characterization of signal-sequence-coding regions selected from the Bacillus subtilis chromosome

Signal-sequence-coding regions for protein export were selected from chromosomal Bacillus subtilis DNA. The number of different signals obtained was higher than expected on the basis of known exported proteins in B. subtilis.

Most of the selected regions showed the characteristics of typical signal sequences, including a basic N-terminal region followed by a hydrophobic core and a potential signal-peptidase cleavage site.

The signal-coding regions were functionally interchangeable between the β. licheniformis -amylase and Escherichia coli TEM β-lactamase genes. In addition to the signal-sequence-coding regions, the nature of the host cells, and the mature parts of the reporter proteins contributed to the amounts of protein secreted.     

Single-stranded DNA binding of the cold-shock protein CspB from Bacillus subtilis: NMR mapping and mutational characterization

Cold-shock proteins (CSPs) bind to single-stranded nucleic acids, thereby acting as a "RNA chaperone." To gain deeper insights into the rather unspecific nature of ssDNA/RNA binding, we characterized the binding interface of CspB from Bacillus subtilis to a 25-mer of ssDNA (Y-Box25) using heteronuclear 2D NMR spectroscopy. Seventeen residues, including eight out of nine aromatic amino acids, are directly involved in the Y-Box25 interaction and were identified by extreme line broadening of their cross-peaks. Eight residues belong to the earlier proposed RNP binding motifs. A second set of seven backbone amides becomes evident by major chemical shift perturbations reporting remote conformational rearrangements upon binding. These residues are located in loop beta3-beta4 and loopbeta4-beta5, and include Ile18. The individual contributions of the so-identified residues were examined by fluorescence titration experiments of 15 CspB variants. Phenylalanine substitutions in- and outside the RNP motifs significantly reduce the binding affinity. Unrestricted possible backbone conformations of loop beta3-beta4 also markedly contribute to binding. Stopped-flow fluorescence kinetics revealed that the different binding affinities of CspB variants are determined by the dissociation rate, whereas the association rate remains unchanged. This might be of importance for the "RNA chaperone" activity of CspB.     

Methylmalonate-semialdehyde dehydrogenase from Bacillus subtilis: substrate specificity and coenzyme A binding

Methylmalonate-semialdehyde dehydrogenase (MSDH) belongs to the CoA-dependent aldehyde dehydrogenase subfamily. It catalyzes the NAD-dependent oxidation of methylmalonate semialdehyde (MMSA) to propionyl-CoA via the acylation and deacylation steps. MSDH is the only member of the aldehyde dehydrogenase superfamily that catalyzes a β-decarboxylation process in the deacylation step. Recently, we demonstrated that the β-decarboxylation is rate-limiting and occurs before CoA attack on the thiopropionyl enzyme intermediate. Thus, this prevented determination of the transthioesterification kinetic parameters. Here, we have addressed two key aspects of the mechanism as follows: 1) the molecular basis for recognition of the carboxylate of MMSA; and 2) how CoA binding modulates its reactivity. We substituted two invariant arginines, Arg-124 and Arg-301, by Leu. The second-order rate constant for the acylation step for both mutants was decreased by at least 50-fold, indicating that both arginines are essential for efficient MMSA binding through interactions with the carboxylate group. To gain insight into the transthioesterification, we substituted MMSA with propionaldehyde, as both substrates lead to the same thiopropionyl enzyme intermediate. This allowed us to show the following: 1) the pK(app) of CoA decreases by ～3 units upon binding to MSDH in the deacylation step; and 2) the catalytic efficiency of the transthioesterification is increased by at least 10(4)-fold relative to a chemical model. Moreover, we observed binding of CoA to the acylation complex, supporting a CoA-binding site distinct from that of NAD(H).     

A novel cold-inducible expression system for Bacillus subtilis

Production of recombinant proteins at low temperatures is one strategy to prevent formation of protein aggregates and the use of an expensive inducer such as IPTG. We report on the construction of two expression vectors both containing the cold-inducible des promoter of Bacillus subtilis, where one allows intra- and the other extracellular synthesis of recombinant proteins. Production of recombinant proteins started within the first 30min after temperature downshock to 25 degrees C and continued for about 5h.     

The Regulation of Bacillus intermedius Glutamyl Endopeptidase Biosynthesis in the Recombinant Bacillus subtilis Strain AJ73 during Sporulation

The growth of the recombinant Bacillus subtilis strain AJ73 carrying the Bacillus intermedius 3-19 glutamyl endopeptidase gene on a multicopy plasmid and the effect of some nutrients on the efficiency of extracellular glutamyl endopeptidase production in the stationary growth phase were studied. In this phase, the concentration of glutamyl endopeptidase in the culture liquid peaked at the 48th and 78th hours of cultivation and depended on the composition of the cultivation medium. Unlike the synthesis of glutamyl endopeptidase in the trophophase (i.e., during vegetative growth), which was suppressed by glucose, the synthesis of this enzyme during sporulation was resistant to glucose present in the cultivation medium. A multifactorial experimental design allowed optimal proportions between the concentrations of major nutrients (peptone and inorganic phosphate) to be determined. Inorganic phosphate and ammonium ions augmented the production of glutamyl endopeptidase by 30–150%, and complex organic substrates, such as casein and gelatin, enhanced the production of glutamyl endopeptidase by 50–100%. During sporulation, the production of glutamyl endopeptidase was stimulated by some bivalent cations (Ca²⁺, Mg²⁺, and Co²⁺) and inhibited by others (Zn²⁺, Fe²⁺, and Cu²⁺). The inference is drawn that the regulatory mechanisms of glutamyl endopeptidase synthesis during vegetative growth and sporulation are different.