Expression of the ςB-Dependent General Stress Regulon Confers Multiple Stress Resistance in Bacillus subtilis

The alternative sigma factor sigmaB of Bacillus subtilis is required for the induction of approximately 100 genes after the imposition of a whole range of stresses and energy limitation. In this study, we investigated the impact of a null mutation in sigB on the stress and starvation survival of B. subtilis. sigB mutants which failed to induce the regulon following stress displayed an at least 50- to 100-fold decrease in survival of severe heat (54 degrees C) or ethanol (9%) shock, salt (10%) stress, and acid (pH 4.3) stress, as well as freezing and desiccation, compared to the wild type. Preloading cells with sigmaB-dependent general stress proteins prior to growth-inhibiting stress conferred considerable protection against heat and salt. Exhaustion of glucose or phosphate induced the sigmaB response, but surprisingly, sigmaB did not seem to be required for starvation survival. Starved wild-type cells exhibited about 10-fold greater resistance to salt stress than exponentially growing cells. The data argue that the expression of sigmaB-dependent genes provides nonsporulated B. subtilis cells with a nonspecific multiple stress resistance that may be relevant for stress survival in the natural ecosystem.     

Dimethylglycine Provides Salt and Temperature Stress Protection to Bacillus subtilis

Glycine betaine is a potent osmotic and thermal stress protectant of many microorganisms. Its synthesis from glycine results in the formation of the intermediates monomethylglycine (sarcosine) and dimethylglycine (DMG), and these compounds are also produced when it is catabolized. Bacillus subtilis does not produce sarcosine or DMG, and it cannot metabolize these compounds. Here we have studied the potential of sarcosine and DMG to protect B. subtilis against osmotic, heat, and cold stress. Sarcosine, a compatible solute that possesses considerable protein-stabilizing properties, did not serve as a stress protectant of B. subtilis. DMG, on the other hand, proved to be only moderately effective as an osmotic stress protectant, but it exhibited good heat stress-relieving and excellent cold stress-relieving properties. DMG is imported into B. subtilis cells primarily under osmotic and temperature stress conditions via OpuA, a member of the ABC family of transporters. Ligand-binding studies with the extracellular solute receptor (OpuAC) of the OpuA system showed that OpuAC possesses a moderate affinity for DMG, with a K_d value of approximate 172 μM; its K_d for glycine betaine is about 26 μM. Docking studies using the crystal structures of the OpuAC protein with the sulfur analog of DMG, dimethylsulfonioacetate, as a template suggest a model of how the DMG molecule can be stably accommodated within the aromatic cage of the OpuAC ligand-binding pocket. Collectively, our data show that the ability to acquire DMG from exogenous sources under stressful environmental conditions helps the B. subtilis cell to cope with growth-restricting osmotic and temperature challenges.     

Purification and Characterization of Novel Transglutaminase from Bacillus subtilis Spores

Transglutaminase activity was detected in suspensions of purified spores prepared from lysozyme-treated sporulating cells of Bacillus subtilis AJ 1307. The enzyme was easily solubilized from the spores upon incubation at pH 10.5 at 37°C. The transglutaminase activity was separated into two fractions upon purification by hydrophobic interaction chromatography (TG1 and TG2). Each enzyme was purified to electrophoretic homogeneity (about 1,000-fold). Both enzymes had the same molecular weight of 29,000 as estimated by SDS-PAGE, had the same N-terminal 30 amino acid sequence, and also showed the same optimal temperature (60°C) and pH (8.2). The purified enzyme catalyzed formation of cross-linked ε-(γ-glutamyl)lysine isopeptides, resulting in the gel-formation of protein solutions such as α_s-casein and BSA.     

One of Two OsmC Homologs in Bacillus subtilis Is Part of the ςB-Dependent General Stress Regulon

In this report we present the identification and analysis of two Bacillus subtilis genes, yklA and ykzA, which are homologous to the partially RpoS-controlled osmC gene from Escherichia coli. The yklA gene is expressed at higher levels in minimal medium than in rich medium and is driven by a putative vegetative promoter. Expression of ykzA is not medium dependent but increases dramatically when cells are exposed to stress and starvation. This stress-induced increase in ykzA expression is absolutely dependent on the alternative sigma factor ς^B, which controls a large stationary-phase and stress regulon. ykzA is therefore another example of a gene common to the RpoS and ς^B stress regulons of E. coli and B. subtilis, respectively. The composite complex expression pattern of the two B. subtilis genes is very similar to the expression profile of osmC in E. coli.     

Characterization of Bacillus subtilis bacteriophages

Brodetsky, Anna M. (University of California, Los Angeles), and W. R. Romig. Characterization of Bacillus subtilis bacteriophages. J. Bacteriol. 90:1655-1663. 1965.-A group of six phages, SP5, SP6, SP7, SP8, SP9, and SP13, which use the Marburg strain of Bacillus subtilis as host was characterized. These phages, referred to as group 1, were examined for the following properties: host range, plaque morphology, stability, adsorption kinetics, one-step growth characteristics, calcium requirements, serum neutralization, thermal inactivation, and inactivation by ultraviolet irradiation. Five unrelated B. subtilis phages, SP3, SP10, PBS1, SP alpha, and SP beta, were included in the studies. When first isolated, none of the group 1 phages was able to replicate efficiently on B. subtilis SB19, a mutant of the "transforming" B. subtilis 168. Host range mutants capable of growth in SB19 were isolated for all of the group 1 phages except SP13, and are designated the "star" phages (SP5* through SP9*). For characterization, SB19 was used as host for the star phages, and another B. subtilis mutant, 168B, was host for SP13.     

Growth and Metabolism of Senna as Affected by Salt Stress

Pot culture experiments were conducted using different NaCl concentrations to assess their impact on the growth and metabolic changes in senna (Cassia angustifolia Vahl.). Five treatments (0, 40, 80, 120, and 160 mM NaCl) were given to the plants at three phenological stages, i.e. at pre-flowering, (45 days after sowing, DAS); flowering (75 DAS) and post-flowering (90 DAS) stages. A significant reduction in the biomass and length of the roots and shoots, photosynthetic rate, stomatal conductance, the total chlorophyll content, protein content, nitrate reductase activity, and reduced nitrogen content of the leaves was observed at each phenological stage with each salt concentration applied. Contrary to this, proline and nitrate contents of the leaves increased markedly. The post-flowering stage was most sensitive to NaCl treatment.     

枯草芽孢杆菌中一种新型双向启动子的功能鉴定

本研究旨在通过转录组分析预测的方法,由地衣芽孢杆菌中筛选获得一种新型双向启动子,鉴定其启动强度。以已知强组成型启动子pShuttle-09为对照,检测其对克劳氏芽孢杆菌碱性蛋白酶基因的表达活性。成功构建了3种重组碱性蛋白酶表达载体及对应的工程菌株。在新型启动子pLA和其反向启动子pLB调控转录下,克劳氏芽孢杆菌碱性蛋白酶表达活性达到164 U/mL和111 U/mL。结果表明,pLA的启动强度明显高于pShuttle-09和pLB,pLA启动子与pLB启动子均可表达碱性蛋白酶。从而为枯草芽孢杆菌表达系统中异源基因的表达提供一个新的方向,也为原核生物中共同表达两种基因提供了新的思路。     

Characterization of the Bacillus subtilis W23 genome by sedimentation

Sedimentation measurements of DNA gently extracted from stationary-phase cells of Bacillus subtilis demonstrated molecules large enough to account for the total genome. The observed sedimentation coefficient of the putative genome showed a strong dependence on centrifuge speed and was, therefore, measured at very low speeds. The value for the sedimentation coefficient extrapolated to zero speed was 159 ± 19 s, from which was calculated molecular weights of (2.5 ± ^1.2_0.8) × 10⁹ a.m.u. for linear molecules and (1.8±^0.8_0.7) × 10⁹ a.m.u. for circular molecules (relative to the following values for T2 DNA: 57 s and 132±12 × 10 daltons; Leighton &; Rubenstein, 1969). The genome also showed extreme shear sensitivity.     

Characterization of highly purified metalloprotease produced by Bacillus subtilis

Proteolytic enzyme produced by Bacillus subtilis is characterized as typical metalloprotease with a molecular weight of 27.9 kD; the enzyme shows its highest activity at pH 7.0-9.0, possesses substrate specificity with respect to different proteins, its temperature optimum is 52 degrees C and its specific activity exceeds that of all known commercial analogues. At 37 degrees C the enzyme is half inactivated in 72 hours.     

Characterization of recF suppressors in Bacillus subtilis

A recF mutation renders Bacillus subtilis cells very sensitive to DNA-damaging agents. Such a recF defect is partially suppressed either by the presence of the recA73 mutation or by the presence of a plasmid-borne, heterologous, single-stranded DNA-binding (ssb) protein gene. Plasmids carrying ssb genes also suppressed the recR and recL defects. Our results suggest that suppression occurs by increasing recombinational repair. The effect of the suppressors may be at the level of induction of the SOS response.