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[Growth conditions and production of the Bacillus intermedius subtilisin-like serine proteinase by the recombinant Bacillus subtilis strain].

The effect of the components of the nutrient medium on growth and production of the Bacillus intermedius subtilisin-like serine proteinase by the recombinant strain Bacillus subtilis AJ73(pCS9) was studied. The production of proteinase was found to be dependent on the composition of the nutrient medium and showed two peaks, at the 28th and 48th h of growth. The concentrations of the main components of the nutrient medium (peptone and inorganic phosphate) optimal for the biosynthesis of subtilisin-like serine proteinase at the 28th and 48th h of growth were determined in factorial experiments. Complex organic substances, casein at concentrations of 0.5-1%, gelatin at concentrations of 0.5-1%, and yeast extract at a concentration of 0.5%, stimulated the production of subtilisin-like serine proteinase by the recombinant strain. The study of the sporulation dynamics in this strain showed that the proteinase peaks at the 28th and 48th h of growth correspond, respectively, to the initial stage of sporulation and to the terminal stages of endospore formation (V-VII stages of sporulation).

Bacillus↗

[Biosynthesis of the Bacillus intermedius subtilisin-like serine proteinase by the recombinant Bacillus subtilis strain].

The effect of certain nutrients on the growth and production of the Bacillus intermedius subtilisin-like serine proteinase by the recombinant strain Bacillus subtilis AJ73(pCS9) was studied. Glucose was found to inhibit the synthesis of proteinase in the early (28 h of growth) but not in the late stationary phase (48 h of growth). The inhibitory effect of the other mono- and disaccharides studied was less pronounced. Casamino acids added to the medium at concentrations of 0.1-1% as an additional carbon and nitrogen source stimulated enzyme biosynthesis. Individual amino acids (cysteine, asparagine, glutamine, tryptophan, histidine, and glutamate) also stimulated enzyme biosynthesis in the early stationary phase by 25-30%, whereas other amino acids (valine, leucine, alanine, and aspartate) were ineffective or even slightly inhibitory to enzyme production. The stimulatory effect of the first group of amino acids on the synthesis of proteinase in the late stationary phase was negligible. In contrast, the bivalent ions Ca2+, Mg2+, and Mn2+ stimulated biosynthesis of proteinase in the late stationary phase (by 20-60%) and not in the early stationary phase. The data indicate that there are differences in the biosyntheses of proteinase by the recombinant B. subtilis strain during the early and late periods of the stationary phases.

Amino Acids↗

Construction and characterization of the chimeric enzymes between the Bacillus subtilis cellulase and an alkalophilic Bacillus cellulase.

The amino acid sequences of cellulase from Bacillus subtilis (BSC) and that from an alkalophilic Bacillus sp. N-4 (NK1) show significant homology in most parts except for the C-terminal portions. Despite the high homology, the pH activity profiles of the two enzymes are quite different; BSC has its optimum pH at 6-6.5, whereas NK1 is active over a broad pH range from 6 to 10.5. In order to identify the structural features which determine such pH activity profiles, chimeric cellulases between BSC and NK1 were constructed using four restriction sites commonly present within the homologous coding sequences, and were produced in Escherichia coli. The chimeric cellulases showed various chromatographic behaviors, reflecting the origins of their C-terminal regions. The pH activity profiles of the chimeric enzymes in the alkaline range could be classified into either the BSC or NK1 type mainly depending on the origins of the fifth C-terminal regions. In the acidic range, the profile was determined only by the origin of the fourth enzyme region from the N terminus. Comparison of the kinetic parameters between pH 5 and 6 using p-nitrophenyl cellobioside as a substrate indicated that the fourth region is responsible for the pH-dependent change of the kcat value. Only a limited number of amino acids in the fourth region may affect on deprotonation of catalytic residues of the cellulases and modulate the catalytic activity in the acidic pH values.

Amino Acid Sequence↗

Relative potency of Bacillus thuringiensis var. israelensis and Bacillus sphaericus 2362 for Mansonia titillans and Mansonia dyari.

Bacillus thuringiensis var. israelensis (B.t.i.), HD-968-S-1983, and a primary powder of Bacillus sphaericus 2362 were assayed against Mansonia titillans and Ma. dyari. The susceptibility of the 2 species to the individual bacterial toxins was similar. The B.t.i. LC50 values were 50.5 micrograms/liter for Ma. dyari and 54.7 micrograms/liter for Ma. titillans; the B. sphaericus LC50 values were 339.0 micrograms/liter and 347.2 micrograms/liter, respectively. The log dose-probit mortality lines were parallel for both target species. The potency of B. sphaericus relative to B.t.i. was 0.15 for Ma. dyari and 0.16 for Ma. titillans.

Animals↗

Effect of metabisulphite on sporulation and alkaline phosphatase in Bacillus subtilis and Bacillus cereus.

The effect of metabisulphite on spore formation and alkaline phosphatase activity/production in Bacillus subtilis and Bacillus cereus was investigated both in liquid and semi-solid substrates. While supplementary nutrient broth (SNB) and sporulation medium (SM) were used as the liquid growth media, two brands of powdered milk were used as the food (semi-solid) substrates. Under both aerobic and anaerobic conditions, B. subtilis was more resistant to metabisulphite than B. cereus while the level of enzyme production and spores formed were generally higher under aerobic than anaerobic conditions. The metabisulphite concentrations required to inhibit spore production as well as alkaline phosphatase synthesis/activity were found to be relatively low and well within safety levels for human consumption. It is concluded that metabisulphite is an effective anti-sporulation agent and a recommendation for its general use in semi-solid and liquid foods is proposed.

Aerobiosis↗

[Survey of Bacillus thuringiensis and Bacillus sphaericus from soils of four provinces of China and their principal biological properties].

A number of isolates of Bacillus thuringiensis and Bacillus sphaericus were obtained from soils of Southwestern Area and Shaanxi Province of China. Among isolates of B. thuringiensis were under 13 sorts of serotype in total of 23 sorts of B. thuringiensis and about 20% of auto-agglutinate strains. Rules of ecologic distribution of two sorts of bacteria were analysed. Toxicities on six species of insects, morphology and crystal proteins of B. thuringiensis, as well as toxicities, morphology and crystal proteins of B. sphaericus, were investigated. 22 strains of more efficient of B. thuringiensis and 2 strains of more efficient of B. sphaericus were obtained. It was shown that B. thuringiensis is actually soil microorganism, and resource of B. thuringiensis is much fruitful in Southwestern Area of China.

Bacillus↗

[Expression of the Bacillus pumilus chloramphenicol acetyltransferase gene in Bacillus subtilis, achieved by the P-R-promotor of phage lambda].

The possibility of expression of the Bacillus pumilus chloramphenicol acetyltransferase gene (cat) in Bacillus subtilis from the pR promoter of phage lambda has been investigated in this work. For this purpose, the plasmid pPL703 carrying the B. pumilus DNA segment with the cat gene lacking promoter has been combined with the plasmid pBM21 containing the pR promoter. The recombinant plasmid pEL1 is capable of providing the 60 mkg/ml chloramphenicol resistance in Bac. subtilis cells.

Bacillus↗

Laboratory and field efficacy of Bacillus thuringiensis var. Israelensis and Bacillus sphaericus against Anopheles gambiae s.l. and Culex quinquefasciatus in Ouagadougou, Burkina Faso.

Two wettable powders (Bactimos and Vectobac) and one flowable concentrate (Teknar) of Bacillus thuringiensis var. israelensis (B.t.i.) and primary powders of Bacillus sphaericus isolates 1593 and 2362 were evaluated (laboratory) against field-collected larvae of Anopheles gambiae s.l. and Culex quinquefasciatus in Ouagadougou, Burkina Faso. Bactimos, Vectobac and a Corn-cob B.t.i. formulation (ABG-6138G) were field tested against Cx. quinquefasciatus and An. gambiae s.l. The isolates of B. sphaericus were also tested against An. gambiae s.l. in artificial ponds. Both wettable powders of B.t.i. showed superior activity than the flowable concentrate formulation against An. gambiae s.l. in the laboratory. Culex quinquefasciatus was more susceptible (3-4X) to B.t.i. (Bactimos) than An. gambiae s.l. The isolates of B. sphaericus were more effective (2-3X) against both mosquito species than Bactimos. In a ditch and two channels, Bactimos, Vectobac and ABG-6138G at 0.65, 1.5 and 5.6 kg/ha, respectively, gave 91-100% control of Cx. quinquefasciatus within 3 days of treatment. The same formulations at rates ranging from 0.25 to 5.6 kg/ha, produced 82-97% control of An. gambiae s.l. in rainwater pools 24 h after treatment. Isolates 1593 and 2362 at 0.12 and 0.24 kg/ha gave excellent control of An. gambiae s.l. in artificial ponds.

Animals↗

[Cloning in Bacillus subtilis cells of the Bacillus mesentericus genes that determine the enzyme synthesis of tryptophan metabolism].

In this work, we tried to clone some Bacillus mesentericus genes coding for tryptophan synthesis in Bacillus subtilis cells. We succeeded in obtaining two identical plasmids carrying some genes of Bac. mesentericus and complementing the trpC2 and trpF mutations of Bac. subtilis. The cloned genes of Bac. mesentericus completely replaced the functions of trpC2 and trpF genes.

Aldose-Ketose Isomerases↗

Effect of temperature on toxicity of two bioinsecticides spherix (Bacillus sphaericus) and bactoculicide (Bacillus thuringiensis) against larvae of four vector mosquitoes.

Two bioinsecticide preparations, viz. Spherix (Bacillus sphaericus) and Bactoculicide (Bacillus thuringiensis H-14), were tested in the laboratory against larvae of Anopheles culicifacies, Anopheles stephensi, Culex quinquefasciatus and Aedes aegypti at different temperatures. The LCs50 of Spherix against III instar larvae of these species at 27 +/- 2 degrees C were 2.0, 0.19, 0.05 and > 40 mg/litre, respectively and those of Bactoculicide were 0.32, 0.16, 0.06 and 0.03 mg/litre, respectively. The toxicity of two bioinsecticides, especially Spherix, varied to a great extent when the tests were repeated at 21 +/- 2 degrees C and 31 +/- 2 degrees C. At 21 +/- 2 degrees C, Spherix was almost non-toxic against larvae of An. culicifacies and An. stephensi (LC50 > 10 mg/l) but at 31 +/- 2 degrees C the bioinsecticide was highly toxic against An. culicifacies (LC50 = 0.48 mg/litre) and An. stephensi (LC50 = 0.04 mg/litre). A similar effect of the temperature was also observed with Bactoculicide.

Animals↗

[Effect of carbon and nitrogen sources and complex B vitamins on the synthesis of alkaline protease by different strains of Bacillus mesentericus and Bacillus subtilis].

The effect of different sources of carbon, nitrogen, amino acids and vitamins on the synthesis of alkaline proteases by the stock and mutant strains of Bacillus mesentericus and by the natural strain of Bacillus subtilis-12 has been investigated. The maximum synthesis of alkaline protease has been obtained in the media containing starch or its hydrolysates--dextrine and maltose as the carbon source. Ammonium phosphate and casein as the nitrogen source prove to be optimal for Bac. mesentericus and Bac. subtilis, respectively. Complex B vitamins added to the nutrient medium accelerate the enzyme synthesis 2.5-4-fold.

Ammonia↗

Efficacy of selected hand hygiene agents used to remove Bacillus atrophaeus (a surrogate of Bacillus anthracis) from contaminated hands.

CONTEXT: The intentional use of Bacillus anthracis transmitted via the US mail in October-November 2001 resulted in 22 people developing inhalation or cutaneous anthrax. Glove use with handwashing prior to and after contact with potential contaminated environmental surfaces and cutaneous lesions has been recommended. However, only limited data are available on the susceptibility of B anthracis to antiseptics. OBJECTIVE: To evaluate the efficacy of several hand antiseptics (interventions) and soap and water (control) against Bacillus atrophaeus, a surrogate of B anthracis. DESIGN, SETTING, AND PARTICIPANTS: Challenge study conducted among healthy adult volunteers, using the Standard Test Method for Evaluation of the Effectiveness of Health Care Professional Handwash Formulations (American Society for Testing and Materials E 1174-94) to determine the efficacy of various hand hygiene products at wash times of 10, 30, and 60 seconds. Volunteers were excluded if they had eczema, psoriasis, or other chronic skin conditions; nonintact skin; or allergies to any study agent. Study agents were a waterless rub containing 61% ethyl alcohol, a 2% chlorhexidine gluconate preparation, and an antibacterial microfiber towel that releases hypochlorite. A nonantimicrobial soap was used as a control. MAIN OUTCOME MEASURE: Reduction of B atrophaeus spores (log10 CFU/mL) on contaminated hands. RESULTS: Washes of 10, 30, and 60 seconds with either soap and water or 2% chlorhexidine gluconate eliminated 1.5 to 2.0 log10 CFUs/mL of B atrophaeus spores at wash 3. Mean reductions (95% confidence intervals) with 10-, 30-, and 60-second washes with soap and water were 2.4 (2.2-2.5), 2.3 (2.2-2.4), and 2.1 (1.9-2.4) log(10) CFUs/mL, respectively; and with 2% chlorhexidine gluconate, 2.1 (2.0-2.3), 1.8 (1.5-2.0), and 1.7 (1.5-1.9) log10 CFUs/mL, respectively. Handwashing with chlorine-containing towels was increasingly effective as the wipe time increased; reductions at 10, 30, and 60 seconds were 1.3 (1.1-1.5), 1.6 (1.2-2.0), and 2.2 (2.1-2.2) log10 CFUs/mL, respectively. A waterless rub containing 61% ethyl alcohol was ineffective in eliminating B atrophaeus spores at all times tested (0 [-0.1 to 0.1], -0.2 [-0.3 to -0.1], and 0 [-0.2 to 0.2] log10 CFUs/mL). CONCLUSIONS: In this evaluation of hand hygiene agents, handwashing with soap and water, 2% chlorhexidine gluconate, or chlorine-containing towels reduced the amount of B atrophaeus spore contamination, whereas use of a waterless rub containing ethyl alcohol was not effective in removing spores.

Adult↗

Engineering of baker's yeasts, E. coli and Bacillus hosts for the production of Bacillus subtilis Lipase A.

Lipases are versatile biocatalists showing multiple applications in a wide range of biotechnological processes. The gene lipA coding for Lipase A from Bacillus subtilis was isolated by PCR amplification, cloned and expressed in Escherichia coli, Saccharomyces cerevisiae and Bacillus subtilis strains, using pBR322, YEplac112 and pUB110-derived vectors, respectively. Lipase activity analysis of the recombinant strains showed that the gene can be properly expressed in all hosts assayed, this being the first time a lipase from bacterial origin can be expressed in baker's S. cerevisiae strains. An important increase of lipase production was obtained in heterologous hosts with respect to that of parental strains, indicating that the described systems can represent a useful tool to enhance productivity of the enzyme for biotechnological applications, including the use of the lipase in bread making, or as a technological additive.

Bacillus subtilis↗

Complete sequence of Bacillus subtilis plasmid p1414 and comparison with seven other plasmid types found in Russian soil isolates of Bacillus subtilis.

We determined the complete sequence of a cryptic 7949-bp plasmid isolated from naturally occurring Bacillus subtilis found in Russian soil from Moscow. We found 15 putative open reading frames (ORFs), all of which were preceded by a ribosome binding site. One encodes the gene (rep) which should be essential for vegetative rolling circle replication (RCR). The putative double-stranded origin as well as a palT1-like single-stranded origin was also identified. The predicted product of another ORF showed similarity to a moblization protein while a third showed similarity to a ubiquitous family of small proteins whose members have so far been associated with stress response. We used fragments with these latter ORFs to probe representatives of seven other groups of cryptic RCR plasmids from geographically related B. subtilis isolates. All plasmids carried the mob function, suggesting a common ancestor for the rep/mob region but the putative hsp was present only on some of the plasmids. This suggests that the putative hsp gene is not an essential plasmid component and may therefore be present as a phenotypic marker-perhaps providing response to stress. This adds weight to the growing evidence that these small Bacillus plasmids may not be cryptic but may provide an adaptive advantage for the host in its natural environment.

Amino Acid Sequence↗

Safety evaluation of pullulanase enzyme preparation derived from Bacillus licheniformis containing the pullulanase gene from Bacillus deramificans.

Pullulanase enzyme is an amylopectin debranching enzyme used in starch hydrolysis. This article describes studies conducted to investigate the safety of a pullulanase enzyme preparation produced by a strain of Bacillus licheniformis that has been transformed by introduction of genetic material from another Bacillus species, B. deramificans. A 4-week dietary toxicity study in rats was conducted in which test animals received pullulanase in the feed at concentrations of 0.2, 1.0, and 5.0%. No adverse treatment-related effects were observed. Lack of genetic toxicity potential was demonstrated by the results of a bacterial mutation assay in Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, and TA1538, in an in vitro histidine forward mutation study in mouse lymphoma cells, and in in vivo mouse bone marrow chromosome aberration and micronucleus assays. The enzyme preparation also has been shown to be a nonirritant in eye and primary dermal irritation tests in rabbits and is nontoxic by inhalation exposure. Finally, the genetically altered B. licheniformis has been demonstrated to be nonpathogenic upon single intraperitoneal injection to rats of both live and killed cells at doses up to 10(11) cells/kg. The results of these studies demonstrate that the enzyme preparation may be considered safe when employed in starch processing.

Animal Feed↗

Molecular cloning and nucleotide sequence of the glycogen branching enzyme gene (glgB) from Bacillus stearothermophilus and expression in Escherichia coli and Bacillus subtilis.

The structural gene for the Bacillus stearothermophilus glycogen branching enzyme (glgB) was cloned in Escherichia coli. Nucleotide sequence analysis revealed a 1917 nucleotide open reading frame (ORF) encoding a protein with an Mr of 74787 showing extensive similarity to other bacterial branching enzymes, but with a shorter N-terminal region. A second ORF of 951 nucleotides encoding a 36971 Da protein started upstream of the glgB gene. The N-terminus of the ORF2 gene product had similarity to the Alcaligenes eutrophus czcD gene, which is involved in cobalt-zinc-cadmium resistance. The B. stearothermophilus glgB gene was preceded by a sequence with extensive similarity to promoters recognized by Bacillus subtilis RNA polymerase containing sigma factor H (E - sigma H). The glgB promoter was utilized in B. subtilis exclusively in the stationary phase, and only transcribed at low levels in B. subtilis spoOH, indicating that sigma factor H was essential for the expression of the glgB gene in B. subtilis. In an expression vector, the B. stearothermophilus glgB gene directed the synthesis of a thermostable branching enzyme in E. coli as well as in B. subtilis, with optimal branching activity at 53 degrees C.

1,4-alpha-Glucan Branching Enzyme↗

ssp genes and spore osmotolerance in Bacillus thuringiensis israelensis and Bacillus sphaericus.

It was shown previously that spores and vegetative cells of Bacillus sphaericus (Bf) and Bacillus thuringiensis israelensis (Bti) are very sensitive to osmotic variations. Since spore osmotolerance has been associated with their SASP (small acid soluble spore proteins) content coded by ssp genes, hybridization assays were performed with sspE and sspA genes from B. subtilis as probes and showed that Bti and Bf strains could lack an sspE-like gene. The B. subtilis sspE gene was then introduced into Bti 4Q2 strain; spores were obtained and showed a 65 to 650 times higher level of osmotolerance to NaCl, without affecting other important properties: hypoosmotic resistance in vegetative cells, spore UV resistance, and larvicidal activity against diptera larvae.

Animals↗

Synthesis and secretion of a heat-stable carboxymethylcellulose from Clostridium thermocellum in Bacillus subtilis and Bacillus stearothermophilus.

The cellulase gene celA of Clostridium thermocellum coding for the thermostable endoglucanase A was transferred from Escherichia coli to Bacillus subtilis 168 and B. stearothermophilus CU21 using plasmids derived from the Bacillus vector pUB110. When the structural part of the gene was joined to a pUB110 promoter the recombinant plasmids (pSE102, pSE105) were stably maintained and expressed carboxymethylcellulose (CMCase) activity. In B. stearothermophilus CU21 (pSE105) the clostridial CMCase was produced over a wide temperature range up to the maximal growth temperature (68 degrees C). In contrast to E. coli, all of the CMCase synthesized in bacilli was released into the culture medium. About 50% of the extracellular protein secreted by B. subtilis 168 (pSE102) carrying the celA gene consisted of endoglucanase A. These findings demonstrate the feasibility of producing cellulolytic enzymes from thermophilic anaerobes in bacilli.

Bacillus subtilis↗