Purification and characterization of a cell wall hydrolase encoded by the cw1A gene of Bacillus subtilis

A cell wall hydrolase of Bacillus subtilis was prepared from Escherichia coli cells harboring a plasmid containing the B. subtilis cwlA gene and purified by hydroxyapatite column chromatography and HPLC through TSK-gel G3000SWXL. In contrast to the molecular mass of 29,919 Da deduced from its nucleotide sequence, the purified CWLA is a 23 kDa protein. Characterization of the specific substrate bond cleaved by CWLA indicated the enzyme is an N-acetylmuramyl-L-alanine amidase. A 32-kDa precursor protein was detected on zymography of a crude cell homogenate. Some of the enzymatic properties of CWLA are also described.     

Sec-secretion and sortase-mediated anchoring of proteins in Gram-positive bacteria

Signal peptide-driven secretion of precursor proteins directs polypeptides across the plasma membrane of bacteria. Two pathways, Sec- and SRP-dependent, converge at the SecYEG translocon to thread unfolded precursor proteins across the membrane, whereas folded preproteins are routed via the Tat secretion pathway. Gram-positive bacteria lack an outer membrane and are surrounded by a rigid layer of peptidoglycan. Interactions with their environment are mediated by proteins that are retained in the cell wall, often through covalent attachment to the peptidoglycan. In this review, we describe the mechanisms for both Sec-dependent secretion and sortase-dependent assembly of proteins in the envelope of Gram-positive bacteria. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.     

Differences in effects on DNA gyrase activity between two glutamate racemases of Bacillus subtilis,the poly-gamma-glutamate synthesis-linking Glr enzyme and the YrpC (MurI) isozyme

Bacillus subtilis possesses two isogenes encoding glutamate racemases, the poly-gamma-glutamate synthesis-linking Glr enzyme and the YrpC isozyme, and produces abundant amounts of the Glr enzyme. The YrpC isozyme, but not the Glr enzyme, was found to influence the activity of DNA gyrase, as did the MurI-type glutamate racemase of Escherichia coli, which is involved in peptidoglycan synthesis during cell division.     

The effects of high-power microwaves on the ultrastructure of Bacillus subtilis

Aims:  To investigate the microbicidal mechanisms of high-power microwave (2·0 kW) irradiation on Bacillus subtilis and to determine the effect of this procedure on the ultrastructure of the cell wall.
Methods and Results:  We performed viability test, examined cells using transmission electron microscopy (TEM), and measured the release of intracellular proteins and nucleic acids. The inactivation rate of B. subtilis by 2·0-kW microwave irradiation was higher than that of a domestic microwave (0·5 kW). Few proteins were released from either microwaved or boiled cells. However, the leakage of nucleic acids from 2·0-kW-microwaved cells was significantly higher than that of 0·5-kW-microwaved or boiled cells. Therefore, we examined ultrastructural alterations of microwaved or boiled cells to analyse the pattern of release of cytoplasmic contents. Although boiled cells did not show any ultrastructural changes on TEM, 2·0-kW-microwaved cells showed disruption of the cell wall.
Conclusion:  The microbicidal mechanisms of 2·0-kW microwave irradiation include damage to the microbial cell wall, breakage of the genomic DNA, and thermal coagulation of cytoplasmic proteins.
Significance and Impact of the Study:  TEM images showed that the cytoplasmic protein aggregation and cell envelope damage by microwave irradiation were different from the ultrastructural changes observed after boiling.     

1株枯草芽胞杆菌体外拮抗6种肠道致病菌的研究

目的研究枯草杆菌BS-3株对大肠埃希菌等6种肠道致病菌的拮抗作用。方法通过在体外BS-3菌株分别与大肠埃希菌等6种致病菌混合培养后,观察不同时间内各菌的菌量变化。结果BS-3菌株与6种肠道致病菌混合培养24、48、72和96h,其菌量逐渐增加;6种致病菌的菌量随着培养时间延续逐渐减少,其中产毒性大肠埃希菌、致病性大肠埃希菌和宋内志贺菌与对照组比较差异更明显。结论BS3菌株在培养生长过程中,可抑制大肠埃希菌等6种肠道致病菌的生长。     

枯草芽胞杆菌孢子表面展示外源蛋白的研究

枯草芽胞杆菌孢子表面展示技术是最近十几年新兴的一种外源蛋白固定方法,已在酶学、疫苗学、靶向药物制备、金属污染治理等领域获得了广泛应用。以孢子衣壳蛋白为载体蛋白,已经成功地把许多抗原、酶和其他蛋白展示在孢子外表面。枯草芽胞杆菌孢子衣壳由多种衣壳蛋白组成,但可用做载体蛋白的并不多,且它们的特性不同。综合介绍了枯草芽胞杆菌孢子表面展示外源蛋白这种新型技术的具体机理,及其在国内外各领域应用的研究进展。     

Molecular insights into the initiation of sporulation in Gram-positive bacteria: new technologies for an old phenomenon

The last decade has witnessed extensive, and widespread, changes in scientific technologies that have impacted significantly upon the study of the life sciences. Arguably, the biggest advances in our comprehension of simple and complex biological processes have come as a consequence of obtaining the complete DNA sequence of organisms. It is likely that we will become accustomed to hearing of quantum leaps in the study and understanding of the biology of higher eukaryotes in the coming years, now that (near) complete genome sequences are available for man, mouse and rat. In this review, we will discuss the impact of genome sequence data, and the use of new scientific technologies that have emerged largely as consequence of the availability of this information, on the study of the master regulator of sporulation, Spo0A, in low G+C Gram-positive endospore-forming bacteria.     

Deciphering distinct biological control and growth promoting potential of multi-stress tolerant Bacillus subtilis PM32 for potato stem canker

Plant growth-promoting rhizobacteria (PGPR) represent a set of microorganisms that play significant role in improving plant growth and controlling the phytopathogens. Unpredictable performance after the application of PGPR has been observed when these were shifted from in-vitro to in-vivo conditions due to the prevalence of various abiotic stress conditions. During growing period, the potato crop is subjected to a combination of biotic and abiotic stresses. Rhizoctonia solani, a soil-borne plant pathogen, causes reduced vigor and yield of potato crop worldwide. In the current study, multi-stress-tolerant rhizobacterial strain, Bacillus subtilis PM32, was isolated from field-grown potato with various plant growth promoting (PGP) traits including zinc and potassium solubilization, biological nitrogen fixation, ammonia and siderophore, as well as extracellular enzyme productions (cellulase, catalase, amylase, protease, pectinase, and chitinase). The strain PM32 exhibited a distinct potential to support plant growth by demonstrating production of indole-3-acetic acid (102.6 μM/mL), ACC-deaminase activity (1.63 μM of α-ketobutyrate/h/mg protein), and exopolysaccharides (2.27 mg/mL). By retarding mycelial growth of R. solani the strain PM32 drastically reduced pathogenicity of R. solani. The strain PM32 also suppressed the pathogenic activity significantly by impeding mycelial expansion of R. solani with inhibition co-efficient of 49.87. The B. subtilis PM32 also depicted significant tolerance towards salt, heavy metal (Pb), heat and drought stress. PCR based amplification of ituC and acds genes coding for iturin and ACC-deaminase activity respectively indicated potential of strain PM32 for lipopeptides production and ACC deaminase enzyme activity. Results of both in-vitro and pot experiments under greenhouse conditions depicted the efficiency of B. subtilis PM32 as a promising bio-control agent for R. solani infection together with enhanced growth of potato plants as deciphered from biomass accumulation, chlorophyll a, b, and carotenoid contents. Therefore, it was envisioned that application of indigenous multi-stress tolerant PGPR may serve to induce biotic and abiotic stress tolerance in crops/plants for pathogen control and sustainable global food supply.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12298-021-01067-2.     

The oxidative stress response in Bacillus subtilis

Abstract Bacillus subtilis undergoes a typical bacterial stress response when exposed to low concentrations (0.1 mM) of hydrogen peroxide. Protection is thereby induced against otherwise lethal, challenge concentrations (10 mM) of this oxidant and a number of proteins are induced including the scavenging enzymes, catalase and alkyl hydroperoxide reductase, and a putative DNA binding and protecting protein. Induced protection against higher concentrations (10–30 mM) of hydrogen peroxide is eliminated in a catalase-deficient mutant. Both RecA and Spo0A influence the basal but not the induced resistance to hydrogen peroxide. A regulatory mutation has been characterized that affects the inducible phenotype and is constitutively resistant to high concentrations of hydrogen peroxide. This mutant constitutively overexpresses the proteins induced by hydrogen peroxide in the wild-type. The resistance of spores to hydrogen peroxide is partly attributable to binding of small acid soluble proteins by the spore DNA and partly to a second step which coincides with the depletion of the NADH pool, which may inhibit the generation of hydroxyl radicals from hydrogen peroxide.     

枯草芽孢杆菌(Bacillus subtilis)发酵生产乙偶姻的pH调控策略

【目的】为了提高Bacillus subtilis CCTCC M 208157发酵生产乙偶姻的效率。【方法】在7 L发酵罐水平上考察不同pH条件对菌株生长及乙偶姻合成的影响。【结果】pH对菌株合成乙偶姻有显著影响,pH 4.5有利于细胞合成乙偶姻,但是延迟期较长;pH 5.5时菌株生长较快,但乙偶姻的产量偏低。因此提出了两阶段pH控制策略:发酵前期(0 16 h),控制pH 5.5;发酵中后期(16 72 h),控制pH 4.5。【结论】通过此策略,菌株合成乙偶姻的能力得到进一步提高,乙偶姻的产量、产率和生产强度分别为32.7 g/L、0.41 g/g和0.91 g/(L.h),分别比初始发酵条件下提高了41%、42%和69%。     

Spherical and rod shaped protoplasts from β-lactam antibiotic treated cultures of Bacillus subtilis

Abstract Addition of β-lactams to exponentially growing cultures of an autolytically deficient Bacillus subtilis met c3 lyt -2 strain FJ6 caused increase in optical density to stop after 1 h when it had about doubled, and thereafter to remain constant for at least 6 h. The number of protoplasts to be derived per unit dry weight of bacteria started to fall when the antibiotic was added and after 1 h had reached 50% of the initial value. Also during the first hour but after a lag of 20–30 min an increasing number of membrane bound rod shaped protoplasts were seen among the normal spherical ones. These swelled to spheres and ultimately burst if the concentration of sucrose in the suspending fluid was gradually reduced. These results would be best explained by rapid inhibition of initiation of cell division followed by membrane damage to an increasing proportion of cells.     

Diversity of the growth patterns of Bacillus subtilis colonies on agar plates

Abstract A Bacillus subtilis strain showed a variety of colony growth patterns on agar plates. The bacterium grew to a fractal colony through the diffusion-limited aggregation process, a round colony reminiscent of the Eden model, a colony with a straight and densely branched structure similar to the dence branching, morphology, a colony spreading without any openings, and a colony with concentric rings, on plates with various agar and nutrient concentrations. The microstructures of these colonies were also characteristic and dynamic. The patterns of these bacterial colonies were thought to grow in relation to the diffusion of nutrient in the agar plate.     

Inhibitory effects of Bacillus probionts on growth and toxin production of Vibrio harveyi pathogens of shrimp

Aim:  To investigate the effects of Bacillus subtilis , Bacillus licheniformis and Bacillus megaterium in terms of toxin and growth of pathogenic Vibrio harveyi .
Methods and Results:  Three Bacillus probionts were isolated from probiotic BZT aquaculture and identified using a 16S rDNA sequence. Growth inhibition assay showed that supernatants from the 24-h culture of three Bacillus species were able to inhibit the growth of V. harveyi (LMG 4044); B. subtilis was the most effective based on the well diffusion method. Results of a liquid culture model showed that B. subtilis was also widely effective in inhibiting three strains of V. harveyi (isolated from Thailand, the Philippines and LMG 4044), and that both B. licheniformis and B. megaterium inhibit the growth of V. harveyi isolated from the Philippines. Moreover, a haemolytic activity assay demonstrated that V. harveyi (IFO 15634) was significantly decreased by the addition of B. licheniformis or B. megaterium supernatant.
Conclusions:  Bacillus subtilis inhibited Vibrio growth, and both B. licheniformis and B. megaterium suppressed haemolytic activity in Vibrio .
Significance and Impact of the Study:  The cell-free supernatants produced by Bacillus probionts inhibit Vibrio disease, and Bacillus probionts might have an influence on Vibrio cell-to-cell communications.     

Analysis of the minor autolysins of Bacillus subtilis during vegetative growth by zymography

Abstract SDS-PAGE and zymographic analysis of protein extracts from Bacillus subtilis AN8, which is deficient in the major 50-kDa amidase (CwlB[LytC]), revealed another distinct but relatively weak 50-kDa protein and its strong activity band. As well as the 50-kDa protein (designated as CwIE), a 35-kDa protein (designated as CwlF) and its activity were also found. In contrast to CwlE production, CwlF production was unaffected by a flaDl ( sinR ) point mutation which represses other vegetative phase autolysins. These newly identified autolysin activities quickly disappeared when cell growth entered stationary phase. The introduction of a sigD -null mutation caused the disappearance of Cw1E activity but Cw1F activity was unaffected by the mutation, as judged on zymography. The possible roles of CwlE and CwlF during vegetative growth are discussed.     

PspA外源表达对枯草芽孢杆菌168蛋白质分泌的影响

PspA同源物广泛存在于细菌和高等生物的组织中.在本研究中克隆了来源于地衣芽孢杆菌的PspA基因,并将其克隆于用于大肠-芽孢穿梭诱导表达载体pDG-StuI中构建重组质粒pDG-PspA.将构建的诱导表达型的重组质粒转化到Bacillus subtilis 168中,研究PspA的外源表达对该菌的生长,总蛋白分泌,以及Sec分泌途径中α-淀粉酶分泌的影响,结果表明,PspA基因的外源表达,在发酵过程后期能在一定程度上提高总蛋白的分泌量,在发酵过程后期能在一定程度上提高分泌的α-淀粉酶浓度.     

Cloning and expression of two autolysin genes, cwIU and cwIV, which are tandemly arranged on the chromosome of Bacillus polymyxa var. colistinus

The cwlV gene, which encodes Bacillus polymyxa var. colistinus autolysin was cloned and sequenced. cwlV comprises a 1497-bp ORF and encodes a polypeptide of 499 amino acid (aa) residues (M_r of 53,707 Da). The N-terminal sequence of the mature 23-kDa CwlV protein is NSXGKKVVVIDAGXGAKD(X, undetermined aa); this processed form corresponds to the C-terminal portion (183 aa, M_r of 20,050 Da) of the cwlV ORF. Sequencing of the flanking region revealed that another putative autolysin gene, cwlU, is located upstream of cwlV. cwlU encodes a polypeptide of 524 aa and its deduced sequence is 34.9% identical to the full-length sequence of CwlV. Downstream of cwlV, the genes for a deduced lipoprotein (OrfW), an endonuclease III homolog (Nth), a non-homologous OrfX, a glutathione peroxidase homolog (Gpx), and the N-terminal region of OrfZ containing a ATP/GTP-binding site motif were found. Northern blotting and primer-extension analyses revealed that cwlU is transcribed as a single cistron, but cwlV is transcribed with orfW. The unprocessed forms of CwlV and CwlU (VΔS and UΔS, respectively) and their predicted mature forms (Vcat and Ucat, respectively) were expressed in, and purified from, Escherichia coli. Enzyme analysis indicated that VΔS and Vcat exhibit low and high cell wall hydrolase activities toward B. polymyxa cell wall, respectively, but UΔS and Ucat exhibit almost no and low cell wall hydrolase activities, respectively. Received: 14 April 1999 / Accepted: 26 July 1999     

化感物质对枯草芽孢杆菌(Bacillus subtilis)在厌氧条件下的生长及反硝化作用的影响

研究由秸秆腐解产生的化感物质 :阿魏酸 ( t-FA)、对羟基苯甲酸 ( p-HA)和苯甲酸 ( BA)在不同浓度下对厌氧培养的枯草芽孢杆菌 ( Bacillussubtilis)的生长及其反硝化活性的影响。结果表明 ,3种浓度的阿魏酸 ( 5.1 5、2 .58、0 .2 6mmol/L)均表现出对枯草芽孢杆菌的生长有抑制作用。对羟基苯甲酸 ( 0 .3 6、3 .62、7.2 4 mmol/L )对生长影响不明显。 8.1 9mmol/L和 4 .0 9mmol/L的苯甲酸有一定的刺激作用 ,而 0 .4 1 mmol/L的苯甲酸与对照无差别。实验表明枯草芽孢秆菌不仅能转化 NO- 3生成 NO- 2 ,而且还能转化 NO- 2 生成 N2 O。 3种化感物质对 NO- 3的转化均表现抑制作用 ,其抑制作用强弱依次为阿魏酸 >对羟基苯甲酸 >苯甲酸。高浓度的抑制作用强于低浓度。阿魏酸在 5.1 5mmol/L和 2 .58mmol/L浓度下 ,其抑制作用的差异显著性分别为 P<0 .0 1 ,P<0 .0 5。 NO- 2 的生成与 NO- 3的减少相互有关联 ,第 3天测定时 ,各处理中NO- 3急剧减少 ,而 NO- 2 急剧增加。在阿魏酸、苯甲酸处理中的 NO- 2 积累高峰在第 3天、第 4天 ,然后下降。而在对羟基苯甲酸的处理中 NO- 2 的积累一直上升 ,在第 6天的观察中仍未出现下降趋势。 3种化感物质均能抑制 N2 O的生成 ,至于在田间的抑制效果尚需进一步试验     

枯草芽孢杆菌活菌体外拮抗6种肠道致病菌的研究

对枯草芽孢杆菌BS 3株在体外对 6种常见肠道致病菌 (肠产毒性大肠杆菌、肠侵袭性大肠杆菌、肠致病性大肠杆菌、鼠伤寒沙门菌、福氏志贺菌和宋内志贺菌 )的拮抗作用进行了研究。结果表明 ,枯草芽孢杆菌BS 3株对宋内志贺菌、肠致病性大肠杆菌及肠产毒性大肠杆菌拮抗作用较为明显。     

Associated roles of hemolysin and p60 protein for the intracellular growth of Bacillus subtilis

Hemolysin expressing Bacillus subtilis strain (B. subtilis ble/hlA) was used as a carrier for listerial protein p60 to study the impact of this protein on bacterial virulence independent of other gene products of Listeria monocytogenes. Bacillus subtilis ble/hlyA exhibited longer cell chains than B. subtilis ble/hlyA/iap. Recombinant Bacillus strains are able to adhere to the mouse macrophage-like J774 and human epithelial-like Int407 cell lines. The bacterial number of B. subtilis ble/hlyA/iap strain that adhered to the Int407 cell lines was 2.52-fold higher, and its invasion level strain was 2.66-fold higher than that observed for the hemolytic strain. Microscopy analysis of infected monolayers showed that recombinant B. subtilis cells were localized inside the cytoplasm of epithelial cells, near to the nuclei, in cellular compartments with low internal pH. Furthermore, in cells infected with bacteria, the actin structures rapidly changed and accumulation of a fat, wide actin layer around the nucleus zone was observed.     

A new strategy to apply Bacillus subtilis MA139 for the production of solid-state fermentation feed

Aim:  The study investigated the potential of using Bacillus subtilis MA139 in combination with Lactobacillus fermentum and Saccharomyces cerevisae to produce solid-state fermentation feed.
Methods and Results:  In a pure fermentation, B. subtilis MA139 was able to grow and synthesize antimicrobial substances at temperatures from 25 to 37°C and at a pH from 5·0 to 9·0. Subsequently, B. subtilis MA139, Lact. fermentum and S. cerevisae were used as starter strains co-inoculated in unsterilized substrate (feed-grade soybean meal and wheat bran). Following 10 days of fermentation in a newly developed plastic bag equipped with a one-way valve, lactic acid bacteria and Bacillus became the predominant strains while S. cerevisae cells decreased slightly. Enterobacteriaceae ( Escherichia coli K88 and Salmonella typhimurium ) were not detected.
Conclusions:  Use of B. subtilis MA139 as a starter strain co-inoculated with S. cerevisae and Lact. fermentum successfully controlled the growth of enterobacteriaceae.
Significance and Impact of the Study:  This study provided a facile and low-cost way to produce solid-state fermentation feed.