Intracellular reactive oxygen species mediate suppression of sporulation in Bacillus subtilis under shear stress

Sporulation is an important cellular response to stress that is also significant from a bioreactor operation viewpoint. While sporulating organisms are known to show an enhanced sporulation response under several stress situations, the sporulation response to shear stress has not been investigated thus far. Such a study could be of interest since shear stress, to a greater or lesser degree, is always present in bioreactor operation. In this article, we investigate the sporulation extents of the Gram-positive bacteria Bacillus subtilis at various defined shear levels. We show that, contrary to expectations, shear inhibits sporulation. We found an inverse correlation between the shear rate-dependent specific intracellular reactive oxygen species level (siROS), and the sporulation extent. A 10-fold increase in siROS resulted in about 17-fold decrease in sporulation extent. The involvement of reactive oxygen species (ROS) in sporulation was unknown thus far. Further, through experiments that specifically increased and reduced intracellular ROS (iROS), we established that siROS is responsible for the inhibition of sporulation under shear stress. In addition, we found that shear induced siROS regulated the expression levels of the general stress proteins Ctc and sigma(B). Based on the above, we hypothesize that siROS may regulate suppression of sporulation under high shear by altering sigma(B) and Ctc expression levels, and a model for the same is presented.     

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.     

c-di-AMP reports DNA integrity during sporulation in Bacillus subtilis

The bacterium Bacillus subtilis produces the DNA integrity scanning protein (DisA), a checkpoint protein that delays sporulation in response to DNA damage. DisA scans the chromosome and pauses at sites of DNA lesions. Structural analysis showed that DisA synthesizes the small molecule cyclic diadenosine monophosphate (c-di-AMP). Here, we demonstrate that the intracellular concentration of c-di-AMP rises markedly at the onset of sporulation in a DisA-dependent manner. Furthermore, exposing sporulating cells to DNA-damaging agents leads to a global decrease in the level of this molecule. This drop was associated with stalled DisA complexes that halt c-di-AMP production and with increased levels of the c-di-AMP-degrading enzyme YybT. Reduced c-di-AMP levels cause a delay in sporulation that can be reversed by external supplementation of the molecule. Thus, c-di-AMP acts as a secondary messenger, coupling DNA integrity with progression of sporulation.     

Cold shock response in sporulating Bacillus subtilis and its effect on spore heat resistance

Cold shock and ethanol and puromycin stress responses in sporulating Bacillus subtilis cells have been investigated. We show that a total of 13 proteins are strongly induced after a short cold shock treatment of sporulating cells. The cold shock pretreatment affected the heat resistance of the spores formed subsequently, with spores heat killed at 85 or 90 degrees C being more heat resistant than the control spores while they were more heat sensitive than controls that were heat treated at 95 or 100 degrees C. However, B. subtilis spores with mutations in the main cold shock proteins, CspB, -C, and -D, did not display decreased heat resistance compared to controls, indicating that these proteins are not directly responsible for the increased heat resistance of the spores. The disappearance of the stress proteins later in sporulation suggests that they cannot be involved in repairing heat damage during spore germination and outgrowth but must alter spore structure in a way which increases or decreases heat resistance. Since heat, ethanol, and puromycin stress produce similar proteins and similar changes in spore heat resistance while cold shock is different in both respects, these alterations appear to be very specific.     

Comparative genome analysis of Bacillus cereus group genomes with Bacillus subtilis

Genome features of the Bacillus cereus group genomes (representative strains of Bacillus cereus, Bacillus anthracis and Bacillus thuringiensis sub spp. israelensis) were analyzed and compared with the Bacillus subtilis genome. A core set of 1381 protein families among the four Bacillus genomes, with an additional set of 933 families common to the B. cereus group, was identified. Differences in signal transduction pathways, membrane transporters, cell surface structures, cell wall, and S-layer proteins suggesting differences in their phenotype were identified. The B. cereus group has signal transduction systems including a tyrosine kinase related to two-component system histidine kinases from B. subtilis. A model for regulation of the stress responsive sigma factor sigmaB in the B. cereus group different from the well studied regulation in B. subtilis has been proposed. Despite a high degree of chromosomal synteny among these genomes, significant differences in cell wall and spore coat proteins that contribute to the survival and adaptation in specific hosts has been identified.     

金属离子对枯草芽孢杆菌转酮酶缺失突变株合成D-核糖的影响

研究了金属离子Mn2 +、Fe2 +、Zn2 +对枯草芽孢杆菌 (Bacillussubtilis)转酮酶 (EC 2 .2 .1 .1 )缺失突变株FBL0 4 531D 核糖合成的影响。发现Mn2 +对该突变株合成D 核糖和形成芽孢具有非常显著的影响。     

AbrB is a regulator of the sigma(W) regulon in Bacillus subtilis

The Bacillus subtilis global regulator AbrB was found to negatively control expression of sigW and genes of the sigma(W) regulon. AbrB bound to DNA regions in the autoregulatory sigW promoter and to some, but not all, of the other sigma(W)-dependent promoters in B. subtilis. Defects in antibiotic resistance properties caused by spo0A mutations are at least partially correlated with AbrB repression of the sigma(W) regulon.     

Sterilization effect of atmospheric plasma on Escherichia coli and Bacillus subtilis endospores

Aims:  Escherichia coli and Bacillus subtilis spores were treated with an atmospheric plasma mixture created by the ionization of helium and oxygen to investigate the inactivation efficiency of a low-temperature plasma below 70°C.
Methods and results:  An electrical discharge plasma was produced at a radio frequency (RF) of 13·56 MHz, connected to a perforated circular electrode with a discharge spacing of 1–15 mm. The discharge gas was helium with 0–2% oxygen. For the plasma treatment, a dried E. coli cell or B. subtilis endospore suspension on a cover-glass was exposed to oxygen downstream of the plasma from holes in an RF-powered electrode. The sterilization effect of the RF plasma was highest with 0·2% oxygen, corresponding to the maximum production of oxygen radicals.
Conclusions:  Oxygen radicals generated by RF plasma are effective for the destruction of bacterial cells and endospores.
Significance and Impact of the study:  Low-temperature atmospheric plasma can be used for the disinfection of diverse objects, especially for the inactivation of bacterial endospores.     

枯草芽胞杆菌168不对称转化产生磷霉素的蛋白质组学分析

旨在用蛋白质组学方法揭示枯草芽胞杆菌Bacillus subtilis 168将顺丙烯磷酸转化成磷霉素的机理.B.subtilis 168能够将顺丙烯磷酸不对称转化成磷霉素.气相色谱分析发现在转化培养基发酵液中的磷霉素的含量达816.6 tg/mL,转化率为36.05％.将分别培养在含有底物和不含底物的培养基中的B.subtilis 168的胞质蛋白进行双向凝胶电泳.对两种条件下的电泳图谱进行比较,发现有98个差异表达蛋白.其中在有底物存在时,表达量下调的点有20个,表达量上调的点52个,底物特异性表达的点有26个.对差异表达蛋白进行质谱鉴定,共鉴定到80个蛋白点,其中下调的点17个,上调的点45个,底物特异性表达的点18个.这些蛋白分别参与胁迫反应、氧化还原反应、物质转运、核苷酸代谢、糖代谢、氨基酸和蛋白质代谢等.根据上述对B.subtilis 168蛋白质组学分析结果,推测菌株是通过两步将顺丙烯磷酸转化成磷霉素的.第一步是水化反应,第二步是脱氢反应.     

The effect of Bacillus subtilis producing Surfactin in ROS production and transformation efficiency of tobacco cells

Surfactin secreted by bacilli has biological functions in plant. Surfactin C14 and C15 have the highest effect on inducing hydrogen peroxide species release in the plant. Surfactin production in the two Bacillus strains ACCT21332 and FKR3 were analysed by HPLC and the phytotoxicity of the Bacilli-derived surfactins was determined in Tobacco cell culture. Surfactin C14 and C15 were detected in ACCT21332 but not in FKR3 strain. Extracellular hydrogen peroxide produced by tobacco cell culture cells exposed to ATCC21332 and FKR3 strains increased compared to untreated ones. The Agrobacterium mediated transformation rate of tobacco cells drops from 4% transformed cells to 0.8 and 1.2% when pretreated with ATCC21332 or FKR3 strain, respectively. The strong drop in transformation rate of plant cell culture after FKR3 strain pre-treatment indicates that Surfactin C14 and C15 are not the major or the only cause in protecting plant cells from Agrobacterial infection and transformation.     

Unraveling the molecular basis for ligand binding in truncated hemoglobins: The trHbO Bacillus subtilis case

Truncated hemoglobins (trHbs) are heme proteins present in bacteria, unicellular eukaryotes, and higher plants. Their tertiary structure consists in a 2‐over‐2 helical sandwich, which display typically an inner tunnel/cavity system for ligand migration and/or storage. The microorganism Bacillus subtilis contains a peculiar trHb, which does not show an evident tunnel/cavity system connecting the protein active site with the solvent, and exhibits anyway a very high oxygen association rate. Moreover, resonant Raman results of CO bound protein, showed that a complex hydrogen bond network exists in the distal cavity, making it difficult to assign unambiguously the residues involved in the stabilization of the bound ligand. To understand these experimental results with atomistic detail, we performed classical molecular dynamics simulations of the oxy, carboxy, and deoxy proteins. The free energy profiles for ligand migration suggest that there is a key residue, GlnE11, that presents an alternate conformation, in which a wide ligand migration tunnel is formed, consistently with the kinetic data. This tunnel is topologically related to the one found in group I trHbs. On the other hand, the results for the CO and O₂ bound protein show that GlnE11 is directly involved in the stabilization of the cordinated ligand, playing a similar role as TyrB10 and TrpG8 in other trHbs. Our results not only reconcile the structural data with the kinetic information, but also provide additional insight into the general behaviour of trHbs. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

四溴甘脲对枯草杆菌黑色变种芽胞损伤作用的电镜观察

为探索四溴甘脲消毒剂杀灭细菌的机理,采用透射电镜技术对四溴甘脲消毒剂处理过的枯草杆菌黑色变种芽胞的超微结构进行了分析和比较.结果显示,以含有效溴274mg/L的四溴甘脲消毒剂作用30min,可使枯草杆菌黑色变种芽胞杀灭率达到100%.在透射电镜下观察到,经该消毒剂作用的枯草杆菌黑色变种芽胞壳质破损断裂明显,壳内结构模糊,核心溶解,有的芽胞近似空壳.结果显示,四溴甘脲消毒剂杀灭芽胞效果优于普通含氯消毒剂,对细菌芽胞超微结构破坏明显.     

Characterization of Bacillus subtilis mutants resistant to cold shock-induced autolysis

Bacillus subtilis vegetative cells undergo autolysis when exposed to cold shock treatment. A mutant (CA1) resistant to cold shock was isolated, and its DNA was used for the transformation of B. subtilis 168AR. The transformant (TR1) and CA1 had almost completely lost major vegetative autolysins (CwlB and CwlG) and motility, and showed a filamentous cell morphology during the exponential phase. Expression of the sigD-lacZ fusion was reduced in TR1. But the introduction of a SigD overproducing plasmid, pHYSigD, into TR1 led to a considerable increase in the amount of autolysin, a normal cell morphology (short rod), and the cold shock-sensitive phenotype. However, motility was not restored in the transformant. The roles of pleiotropic genes in cold shock-induced autolysis are discussed.     

Evidence that dipicolinic acid is covalently bound to specific macromolecules in spores of Bacillus subtilis

Abstract Two dipicolinic acid (DPA)-binding macromolecules with molecular masses of about 440 kDa and 230 kDa were detected in the soluble fractions of dormant and germinated spores of Bacillus subtilis using native PAGE and an immunological technique. In SDS-PAGE, only one band with the molecular mass of about 50 kDa was found. Proteinase K partially digested the 440-kDa macromolecule of dormant spores to convert it into a 230-kDa one, and completely digested both the 440-kDa and 230-kDa bands of germinated spores. DNase I did not affect either DPA-binding macromolecules. This suggests that the two DPA-binding macromolecules are of similar origin, their main component is protein and a conformational change may occur during germination. DPA was not dissociated from the DPA-binding macromolecules by extensive dialysis and SDS treatment, suggesting the presence of a covalent bonding.