Profiling and comprehensive expression analysis of ABC transporter solute-binding proteins of Bacillus subtilis membrane based on a proteomic approach

We analyzed ABC transporter solute-binding proteins (SBPs) of the Bacillus subtilis membrane using a proteomic approach. We prepared a washed cell membrane fraction that was insoluble in 134 mM nondetergent sulfobetaine and then extracted proteins using mixtures of detergents in a stepwise manner. The membrane proteins were resolved by three two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) or two one-dimensional (1-D) PAGE procedures, electroblotted, and digested in the presence of 5% or 80% acetonitrile. Thereafter, matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF MS) identified 637 proteins corresponding to 15.9% of the total cellular proteins. We predicted that among these, 256 were membrane proteins, 101 were lipoproteins or secretory proteins and 280 were soluble proteins containing peripheral proteins that function in both the cytoplasm and the cell membrane such as SecA and FtsY. Among the 637 proteins, we identified 30 SBPs among 38 importers predicted by a bioinformatic search of the genome. We confirmed expression of the genes for the 30 SBPs using DNA microarray analysis. We compared the 2-D gel separation profiles of submembrane fractions solubilized by 1% n-dodecyl-beta-D-maltoside from cells cultured on Luria Bertani (LB), S7, and S7 medium without glutamate as well as DNA microarray data on LB and S7. The results suggested that YcdH, YtmK and YurO are binding proteins for Mn(++), glutamate and glucose, respectively, and that YqiX and YxeM are binding proteins for amino acids (tryptophan in S7 medium).     

Molecular cloning and expression of an endo-β-1,4-d-glucanase I (avicelase I) gene from Bacillus cellulyticus K-12 and characterization of the recombinant enzyme

Bacillus cellulyticus K-12 Avicelase (Avicelase I; EC 3.2.1.4) gene (ace A) has been cloned in Escherichia coli by using the vector pT7T3U19 and HindIII-HindIII libraries of the chromosomal inserts. The libraries were screened for the expression of avicelase by monitoring the immunoreaction of the antiavicelase (immunoscreening). Positive clones (Ac-3, Ac-5, and Ac-7) contained the identical 3.5-kb HindIII fragment as determined by restriction mapping and Southern hybridization, and expressed avicelase efficiently and constitutively using its own promoter in the heterologous host. From the immunoblotting analysis, a polypeptide that showed a carboxymethylcellulase (CMCase) activity with an M _r , of 64,000 was detected. The recombinant endo 1,4-β- _d -glucanase I was purified to homogeneity from an intracellular fraction of E. coli by DEAE-Toyopearl M650, Phenyl Toyoperal M650, and TSK gel HW50S chromatography. The enzyme had a monomeric structure, its relative molecular mass being 65 kDa by gel filtration and 64 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The pI was 5.3 and the optimal pH was 4.6, and the enzyme was stable at pH 4.0–10.5. The enzyme had a temperature optimum of 50°C and was stable at 55°C for 48 h, and retained approx 20% of its activity after 30 min at 70°C. It showed high activity toward carboxymethylcellulose (CMC) as well as p-nitrophenyl-β-d-cellobioside, 4-methylumbelliferyl cellobioside, Avicel, filter paper, and some cellooligosaccharides. K _m values for CMC and Avicel were 7.6 and 85.2 mg/mL, respectively, whereas V _max values were 201 and 9.2 μmol · min⁻¹ · mg⁻¹, respectively. Cellotetraose (G4) was preferentially cleaved into cellobiose (G2) and cellopentaose (G5) was cleaved into G2 + cellotriose (G3), whereas cellohexaose (G6) was cleaved into G4 + G2 and, to a lesser extent, into G3 + G3. G3 was not cleaved at all. G2 was the main product of Avicel hydrolysis. G2 inhibited whereas Mg⁺⁺ stimulated the activity of CMCase and Avicelase. Hydrolysis of CMC took place with a rapid decrease in viscosity but a slow liberation of reducing sugars. Based on these results, it appeared that the cellulase should be regarded as endo type, although it hydrolyzed Avicel.     

Continuous Production of Thermostable α-Amylase by Immobilized Bacillus Cells in a Fluidized-Bed Reactor

The exponential cells ofBacillus cereus immobilized in calcium alginate and spun into microcylindrical particles were used in a fluidized-bed reactor for continuous synthesis of thermostable α-amylase. The reactor was operated over a period of 30 d with a dilution rate of 0.33 h^-1, producing 1000–1200 U/mL of enzyme. The productivity of the reactor was in the range of 330–396 kU/h. A 20-fold increase in the productivity with respect to batch fermentation with free cells was attained.     

Capacity of Bacillus thuringiensis S-layer protein displaying polyhistidine peptides on the cell surface

S-layer protein of Bacillus thuringiensis strain CTC was used as the carrier protein to display polyhistidine (poly[6His]) peptides on the cell surface. Poly(6His) _n was fused with S-layer protein at two different sites, inserting just downstream of the S-layer protein homologous domain (slh) and replacing the non-slh region of S-layer protein, respectively. The two series chimeric proteins were both expressed by crystal negative B. thuringiensis strain 4Q7 and strain 171, respectively, as shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The recombinant B. thuringiensis cells gained Ni²⁺- and Cd²⁺-binding ability and had a capacity to display up to nine copies of poly(6His). The Cd²⁺ adsorption quantity of the recombinant strain with the strongest adsorption ability was twice that of the host strain.     

Structural characterization of gentiobiosyl diglycerides fromBacillus pumilus associated with ascidiaHalocynthia aurantium

A mixture of 1(3),2-di-O-acyl-3(1)-O-β-gentiobiosylglycerols was isolated from a sea isolate ofBacillus pumilus. The components of the mixture were structurally characterized by mass spectrometry and¹H and¹³C NMR spectroscopy data for the native compounds and their derivatives. The predominant component contains two C₁₅ acyl groups, while the second component contains C₁₅ and C₁₇ fatty acids. Six minor components differ in residues of fatty acids and/or their combinations. Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 1, pp. 166–170, January, 2000.     

Electrochemical Studies on De-Emulsification: Effect of a Biosurfactant Produced by Bacillus subtilis MO-1

Model emulsions were de-emulsified using a biosurfactant produced by Bacillus subtilis MO-1 mixed with chemical de-emulsifiers. The speed and efficiency of de-emulsification by polyether type de-emulsifier (G-17) were enhanced by combined use of a biosurfactant produced by B. subtilis MO-1 (BS); this effect was more apparent at low concentrations. Polyether/biosurfactant synergy was confirmed by electrochemical measurement of the interfacial film electrical resistance (R _m ) and capacitance (C _m ) during de-emulsification. These values were closely related to the rate of water removal, demonstrating that electrical techniques are suitable for studying de-emulsification phenomena.     

He-Ne激光在异种间原生质体融合中的应用

用632.8nm 12mW的氦氖激光及聚乙二醇（PEG）复合诱导融合枯草芽孢杆菌与黑曲霉原生质体.为了提高枯草芽孢杆菌的糖化酶产酶能力,对两亲本在融合中采用不同的激光照射时间,选育出的融合子与枯草芽孢杆菌亲本相比,糖化酶酶活提高了2倍,并通过酯酶同工酶谱分析对融合子进行了鉴定,结果表明:融合子遗传性状与亲本相比发生了显著变化,通过传代培养,融合子具有良好的遗传稳定性.     

Investigation of the effect of berberines alkaloids in <Emphasis Type="Italic">Coptis chinensis</Emphasis> Franch on <Emphasis Type="Italic">Bacillus shigae</Emphasis> growthby microcalorimetry

The inhibitory effects of five berberines alkaloids (BAs) from rhizoma of Coptis chinensis Franch, a traditional Chinese medicinal (TCM) herb, on Bacillus shigae (B. shigae) growth were investigated by microcalorimetry. The power-time curves of B. shigae with and without BAs were acquired; meanwhile, the extent and duration of inhibitory effects on the metabolism were evaluated by growth rate constants (k1, k2), half inhibitory ratio (IC50), maximum heat output (Pmax), and peak time (tp). The values of k1 and k2 of B. shigae in the presence of the five BAs decreased with the increasing concentrations of BAs. Moreover, Pmax was reduced, and the value of tp increased with increasing concentrations of the five drugs. The inhibitory activity varied with different drugs. IC50 of the five BAs was respectively 75 μg/mL for berberine, 90 μg/mL for coptisine, 115 μg/mL for palmatine, 220 μg/mL for epiberberine, and 400 μg/mL for jatrorrhizine. The sequence of antimicrobial activity of the five BAs berberine ＞ coptisine ＞ palmatine ＞ epiberberine ＞ jatrorrhizine. The functional groups methylenedioxy at C2 and C3 on phenyl ring improve antimicrobial activity more strongly than methoxyl at C2 and C3 on phenyl ring. However, the effect of bacteriostasis is not significant with methylenedioxy or methoxyl at C9 and C10 on phenyl ring.     

Silver nanoparticles formed in bio- and chemical syntheses with biosurfactant as the stabilizing agent

In this work, the comparison of the physical properties of silver nanoparticles (AgNPs) obtained via the reduction of silver nitrate (AgNO₃) in biological and chemical (model) syntheses supplemented with the biosurfactant surfactin is described. In the studies, two strains of Bacillus subtilis (denoted T’1 and I’1a) were used. The biological synthesis of AgNPs was performed using supernatants obtained from cultures of bacteria growing on brewery effluents, molasses, and Luria–Bretani (LB) medium. In model experiments, ascorbic acid served as the reductant; surfactin acted as the stabilizing agent. The surfactin concentrations were adjusted to 5 and 30?mg/L, which corresponded to minimum and maximum surfactin concentrations as measured in the supernatants obtained from the B. subtilis cultures. The chemical synthesis was carried out at acidic as well as alkaline pH. Dynamic light scattering (DLS) revealed that in model and biological samples, single AgNPs were accompanied by aggregated structures. Transmission electron microscopy showed that the contribution of the aggregates in bacterial supernatants and in chemical synthesis is negligible under acidic conditions. However, in the alkaline environment, this contribution predominates. In the model experiments, smaller nanoparticles were formed with higher concentrations of surfactant. The presence of surfactin significantly increased the stability of AgNPs in both bio- and chemical syntheses.