Features of Bacillus subtilis IMB B-7023 and its streptomycin-resistant strain

Features of phosphate-mobilizing bacteria Bacillus subtilis IMB B-7023 and its streptomycin-resistant strain were investigated. While cultivated in medium with glucose and glycerophosphate, the growth rate of the antibiotic-marked strain was approximately similar to this parameter for Bacillus subtilis IMB B-7023 but cell sizes were 1.3-fold less. Both strains significantly stimulated the germinating of plant seeds, attached to their roots, and insignificantly differed in antagonistic activity toward phytopathogens and quantitative content of cell fatty acids and phosphatase activity. Streptomycin-resistant strain may be used for monitoring of Bacillus subtilis introduced to agroecosystem.     

Biological Properties of the Phosphorus-Mobilizing Bacillus subtilis Strain IMV V-7023

Biological characteristics of a new phosphate-mobilizing bacillus strain are reported. Species-level identification of the strain was performed according to morphological, cultural, and biochemical characteristics and the sequence of the 16S rRNA gene. The strain was identified as Bacillus subtilis IMV V-7023 and displayed a very high ability to mobilize phosphorus from its sparingly soluble inorganic and organic compounds and the capability of synthesizing biologically active substances; in addition, the strain essentially suppressed the growth of phytopathogenic bacteria, micromycetes, and agents causing various diseases of vegetable, cereal, leguminous, and other plants. The strain Bacillus subtilis IMV V-7023 is promising for developing bacterial preparations for crop production.     

Carbohydrate-containing cell wall polymers of some strains of the <Emphasis Type="Italic">Bacillus subtilis</Emphasis> group

A comparative study of the structures of carbohydrate-containing cell wall polymers isolated from the strains of the Bacillus subtilis group was performed by means of chemical and NMR spectroscopic meth ods. Polymers of different structure were revealed, namely, 1,3-poly(glycerol phosphates) with β-glucopyranose in Bacillus subtilis strains VKM B-520, VKM B-723, and VKM B-763 (= VKM B-911); 1,5-poly(ribitol phosphate) with α-glucopyranose in B. subtilis strains VKM B-722 and VKM B-922 (the structure is reported for the first time); and simultaneously two polymers in B. subtilis VKM B-761, 1,5-poly(ribitol phosphate) with β-glucopyranose and the disaccharide 1-phosphate polymer with the following repeating unit: -6)-α-D-Galp-(1-P-4)-gB-D-GlcpNAc-(1-, in which the hydroxyls at C3 and C6 of glucosamine residues are partially O-acetylated (the structure is reported for the first time). Heterogeneity of the B. subtilis group is con firmed by variations in the structure and composition of the cell wall polymers. The cell surface polymers are useful for discrimination of closely related bacilli strains and are cell wall marker components that may be an indispensable element of the Bacillus subtilis group taxonomy along with the genomosystematic methods.     

Phenotypic diversity and technological properties of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> species isolated from <Emphasis Type="Italic">okpehe</Emphasis>, a traditional fermented condiment

The Bacillus subtilis wild strains isolated from okpehe, a traditional fermented condiment used as seasoning in Nigeria, the reference and typed strains were investigated for their phenotypic diversity and their technological parameters with a view to obtain adequate data that would enable selection of appropriated starter cultures for vegetable protein fermentation in West Africa. All the 7 strains studied demonstrated diverse phenotypic characteristics and they were identified as Bacillus subtilis, based on the API 50 CHB combined with API 20E profile. Specific sugars that indicated a good hydrolytic potential of the wild strains were fermented. The highest proteinase activity of 90 AU/ml determined quantitatively was observed in the strain Bacillus subtilis BFE 5372, the proteinase was identified by the APIZYM gallery as chymotrypsin. Highest amylase activity of 13 AU/ml was noticed in strain Bacillus subtilis DSM 347 while only 4 strains produced polyglutamic acid with the strain Bacillus subtilis BFE 5359 producing the highest polyglutamate activity of 2.5 mm. Although strain Bacillus subtilis BFE 5301 did not release detectable polyglutamate, the strain demonstrated antagonism against different bacteria and the antimicrobial substance produced by strain Bacillus subtilis BFE 5301 was confirmed as a bacteriocin since its activities were lost after treatment with chymotrypsin and pepsin. The data generated showed the technological parameters that can aid selection of wild strains such as Bacillus subtilis BFE 5301, BFE 5359 and BFE 5372 for optimization of condiment production.     

Chemotactic and adhesive properties of Azotobacter vinelandii and Bacillus subtilis

The effects of some factors on the chemotaxis of Azotobacter vinelandii IMV V-7076 and Bacillus subtilis IMV V-7023 and on their adhesion to cucumber roots have been studied. Glucose chemotaxis and adhesion to roots reach peak values in pH ranges characteristic of each strain. These ranges are 7.0–8.0 for A. vinelandii IMV V-7076 and 6.0–7.0 for B. subtilis IMV V-7023. The adhesion values of each species decrease significantly in their mixed suspension. The interaction of each of the strains with the clay mineral montmorillonite improves their adhesion to cucumber roots. The clay mineral palygorskite improves the adhesion of A. vinelandii but reduces that of B. subtilis.     

Peculiarities of luminescent response of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> recombinant strain bearing cloned <Emphasis Type="Italic">Vibro harveyi lux AB</Emphasis> genes to the action of thermostable blood serum compounds

A strain of Bacillus subtilis previously used as the test-organism in bacteriological and nephelometry methods for detection of thrombocytes cation protein (TCP) has been transformed by a plasmid pLFlux containing cloned luxAB genes of a sea luminescent bacterium Vibro harveyi. The designed luminescent biosensor B. subtilis of The All-Russia Collection of Industrial Microorganisms (Moscow) B-10191 demonstrated specific response to the thermostable fraction of guinea pig blood serum. Sensitivity to chromatographically purified TCP as part of the system has been analyzed and the correlation between luminescence inhibition and direct bacterial effect on the target cells has been demonstrated. The obtained results are considered as the first stage of the design of the bioluminescent technology for TCP detection in biological liquids with complicate composition of the components.     

Screening of <i>Bacillus subtilis</i> HAAS01 and Its Biocontrol Effect on <i>Fusarium</i> wilt in Sweet Potato

Fusarium wilt, a disease caused by Fusarium oxysporum f.sp batatas (Fob) is an important disease in sweet potato production. Using endophytic bacteria for biological control of sweet potato diseases is one of the important ways. A Bacillus subtilis with antagonistic effect on Fusarium wilt of sweet potato was isolated from soil by confrontation culture. According to the biological characteristics, 16S rDNA sequence analysis, and physiological and biochemical analysis, the Bacillus subtilis HAAS01 was named. A pot experiment was conducted for the biological control experiment of strain HAAS01, and the endogenous hormone content, antioxidant enzyme activity, soluble protein content, and related gene expressions of sweet potato plants were detected. The results showed that the HAAS01 strain could promote the production of endogenous hormones and resist the infection of plant diseases together with defensive enzymes and upregulation of related gene expressions. In summary, Bacillus subtilis HAAS01 was effective in controlling Fusarium wilt of sweet potato and has potential for application and development.     

Inhibitory Activity of Probiotic <Emphasis Type="Italic">Bacillus subtilis</Emphasis> UTM 126 Against <Emphasis Type="Italic">Vibrio</Emphasis> Species Confers Protection Against Vibriosis in Juvenile Shrimp (<Emphasis Type="Italic">Litopenaeus vannamei</Emphasis>)

The bacterial strain Bacillus subtilis UTM 126 produced antimicrobial activity against pathogenic Vibrio species, including V. alginolyticus, V. parahaemolyticus, and V. harveyi. The probiotic effect of B. subtilis was tested by feeding juvenile shrimp (Litopenaeus vannamei) food supplemented with B. subtilis (10⁵ CFU/g) for 28 days before an immersion challenge with V. harveyi at 10⁵ CFU/mL for 24 h. The treatment with B. subtilis UTM 126 decreased final mortality to 18.25%, compared with 51.75% in the control group. Bacillus subtilis UTM 126 has potential applications for controlling pathogenic V. harveyi in shrimp aquaculture.     

枯草芽胞杆菌蛋白质表达分泌系统发展及展望

枯草芽胞杆菌作为革兰阳性模式菌株是基础研究和工业应用的常用宿主细胞。介绍了枯草芽胞杆菌中蛋白合成和分泌过程中的重要步骤及重要调控位点。在枯草芽胞杆菌蛋白表达及分泌系统中,可以针对目标基因在体内的转录、翻译、折叠、转运和菌株改造等方面对表达分泌系统进行优化改良,针对不同的目标蛋白,可进行不同优化模块的组装和拼搭,以达到针对目标蛋白产物定制化地提高产量和分泌量的目的。在未来,随着基因编辑和合成生物技术的发展,菌株改良策略的不断优化,枯草芽胞杆菌将会在工业生产蛋白质制品领域发挥更大的应用价值。     

Phosphate-mobilizing bacteria <Emphasis Type="Italic">Bacillus subtilis</Emphasis> as phenolic producers

Bacteria Bacillus subtilis IMVV-7023 accumulate biologically active phenolic substances in the culture liquid. They include significant amounts of phenylacetic (29.03%) and 4-hydroxyphenylacetic (10.49%) acids. These acids can induce root formation in plants. They also suppress fungal plant pathogens.     

Alkaline protease from <Emphasis Type="Italic">Bacillus sp.</Emphasis> isolated from coffee bean grown on cheese whey

Two strains of Bacillus, one from a culture collection (B. subtilis ATCC 6633) and a wild type (Bacillus sp. UFLA 817CF) isolated during coffee fermentation in the south of Minas Gerais, Brazil, were evaluated in relation to secretion of alkaline proteases. The strains were grown on nutrient broth, nutrient broth with sodium caseinate and nutrient broth with three different concentrations of cheese whey powder for 72 h. Samples were collected at 24-h intervals to evaluate the proteolytic activity, protein content and cell population. Maximum protease activity was observed after 24-h growth for both the microorganisms, a period that coincided with the end of the exponential phase. The specific activity values were, respectively, 839.8 U/mg for B. subtilis ATCC 6633 and 975.9 U/mg for Bacillus sp. UFLA 817CF. The 60% saturation presented the best results for specific protease activity in all the growth culture media tested with B. sp. UFLA 817CF. Bacillus sp. UFLA 817CF showed highest enzymatic activity at pH 9.0 and 40°C in the three culture media tested. The protease obtained from culture of the wild Bacillus strain presented stability at pH 7.0 and considerable heat stability at 40°C and 50°C, and could be an alternative for the industry to utilize cheese whey to produce proteolytic enzymes.     

Overexpression,purification and characterization of a recombinant secretary catalase from <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

A recombinant Bacillus subtilis strain (KN25) was generated for the large-scale preparation of catalase. The B. subtilis katA gene encoding for catalase was cloned into the shuttle vector PRB374, downstream of the constitutively active vegII promoter, followed by transformation of the B. subtilis strain WB600 with the plasmid. The transformant strain, KN25 secretes high levels (3,500 U/ml) of catalase, which facilitates its purification. Three simple purification steps yielded nearly homogeneous catalase, with ∼70% recovery. The purified recombinant catalase has a specific activity of 34,600 U/mg under optimal conditions, and is more resistant to acidic conditions than bovine liver catalase.     

Across Genus Plasmid Transformation Between <Emphasis Type="Italic">Bacillus subtilis</Emphasis> and <Emphasis Type="Italic">Escherichia coli</Emphasis> and the Effect of <Emphasis Type="Italic">Escherichia coli</Emphasis> on the Transforming Ability of Free Plasmid DNA

The results presented in this article show that direct plasmid transfer from Escherichia coli carrying shuttle plasmid to Bacillus subtilis occurred when close contact between the two species was established by mixing E. coli and B. subtilis onto selective agar plates. The data demonstrate that the production of resistant colonies by plasmid transformation through cell contact was DNase I sensitive and dependent on transformable B. subtilis strains. Furthermore, another observation indicated that the E. coli strain is able to affect the transformation capability of B. subtilis. It is assumed that the donor strain is a momentous factor for taking up plasmid DNA. This conclusion is significant in the assessment of both the possibility of intercellular DNA transfer in natural habitats of micro-organisms and the risk of the application of genetically engineered micro-organisms.     

Isolation,identification and characterization of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> ZJB-063, a versatile nitrile-converting bacterium

Strain ZJB-063, a versatile nitrile-amide-degrading strain, was newly isolated from soil in this study. Based on morphology, physiological tests, Biolog and the 16S rDNA sequence, strain ZJB-063 was identified as Bacillus subtilis. ZJB-063 exhibited nitrilase activity without addition of inducers, indicating that the nitrilase in B. subtilis ZJB-063 is constitutive. Interestingly, the strain exhibited nitrile hydratase and amidase activity with the addition of ɛ-caprolactam. Moreover, the substrate spectrum altered with the alteration of enzyme systems due to the addition of ɛ-caprolactam. The constitutive nitrilase was highly specific for arylacetonitriles, while the nitrile hydratase/amidase in B. subtilis ZJB-063 could not only hydrolyze arylacetonitriles but also other nitriles including some aliphatic nitriles and heterocyclic nitriles. Despite comparatively low activity, the amidase of hydratase/amidase system was effective in converting amides to acids. The versatility of this strain in the hydrolysis of various nitriles and amides makes it a potential biocatalyst in organic synthesis.     

Potential Probiotics Bacillus subtilis KATMIRA1933 and Bacillus amyloliquefaciens B-1895 Co-Aggregate with Clinical Isolates of Proteus mirabilis and Prevent Biofilm Formation

A urinary tract infection (UTI) is a multi-factorial disease including cystitis, pyelonephritis, and pyelitis. After Escherichia coli, Proteus mirabilis is the most common UTI-associated opportunistic pathogen. Antibiotic resistance of bacteria and infection recurrence can be connected to biofilm formation by P. mirabilis. In this study, human and sheep isolates of P. mirabilis were investigated for antibiotic sensitivity using an antibiotic disk test. Co-aggregation of the tested potential probiotic bacilli, Bacillus amyloliquefaciens B-1895 and Bacillus subtilis KATMIRA1933, with the isolated pathogen was also evaluated. Then, the anti-biofilm activity of naturally derived metabolites, such as subtilin and subtilosin, in the bacilli-free supernatants was assessed against biofilms of P. mirabilis isolates. The isolated pathogens were sensitive to 30 μg of amikacin and 5 μg of ciprofloxacin but resistant to other tested antibiotics. After 24 h, auto-aggregation of B. amyloliquefaciens B-1895 was at 89.5% and higher than auto-aggregation of B. subtilis KATMIRA1933 (59.5%). B. amyloliquefaciens B-1895 strongly co-aggregated with P. mirabilis isolates from human UTIs. Cell-free supernatants of B. amyloliquefaciens B-1895 and B. subtilis KATMIRA1933 showed higher antimicrobial activity against biofilms of P. mirabilis isolated from humans as compared with biofilms of sheep isolates. According to our knowledge, this is the first report evaluating the anti-biofilm activity of probiotic spore-forming bacilli against clinical and animal UTI isolates of P. mirabilis. Further studies are recommended to investigate the anti-biofilm activity and the mode of action for the antimicrobial substances produced by these bacilli, subtilosin and subtilin.

     

A <Emphasis Type="Italic">Bacillus subtilis</Emphasis> strain HPC248 from an effluent treatment plant with antimicrobial activity

This study reports the identification and demonstration of an organism with antimicrobial activity isolated from activated biomass of an effluent treatment plant (ETP) treating wastewater containing pesticides. While assessing the heterotrophic diversity of biomass collected from ETP, clear zones were observed on Luria Bertani plates. The bacterial isolate producing the zone as well as the bacterial cells surrounding the zone were isolated and purified by sub-culturing. Both isolates were identified by partial sequencing of the 16S rDNA clone. Presence of antimicrobial activity was demonstrated against various laboratory strains, isolated from different treatment plants and also against waterborne pathogens. The isolate that produced antimicrobial activity was identified as Bacillus subtilis strain HPC248 and the sensitive strain was identified as Bacillus sphaericus strain HPC249.     

Purification and characterization of an extracellular keratinase from a hornmeal-degrading <Emphasis Type="Italic">Bacillus subtilis</Emphasis> MTCC (9102)

Native proteolytic microorganisms were isolated from the hornmeal, which is a product obtained by treatment of horns and hoofs with steam under high pressure. Keratinolytic activities of these organisms were screened in mineral salt medium with 1% hornmeal. Bacillus subtilis MTCC (9102), a keratinase-producing organism causing extensive degradation of hornmeal has been identified. Keratinase was purified (45-fold) by ion exchange, and gel filtration chromatography. Among the keratinases produced by the various organisms, keratinase from the Bacillus subtilis strain reported by us was found to have a molecular weight range between 64 and 69 kDa and high activity in the pH range between 5 and 7, with maximum activity at pH 6.0 and at an optimum temperature of 40°C. It remained stable up to 70°C. The keratinase activity was completely inhibited by ethylenediamine tetraacetic acid (EDTA), and 1 10-phenanthroline, and remained unaffected by phenylmethanesulfonyl fluoride (PMSF, relative activity: 93%), whereas iodoacetamide inhibited considerably. Zinc, magnesium, calcium, manganese, and nickel were found to enhance the enzyme activity, whereas mercury and copper inhibited its activity completely. The keratinolytic metalloprotease from native Bacillus subtilis differed from the other serine proteases. It may have potential applications in the bioconversion of keratinous wastes and eco-friendly dehairing in the leather industry.     

Effects of nematodes,fungi and bacteria on the growth of young apple trees grown in apple replant disease soil

Growth, dry root weight of seedlings and root score of apple seedlings cv. McIntosh were reduced when soils were inoculated with Pratylenchus penetrans, Penicillium janthinellum, Constantinella terrestris, Trichoderma sp., and 4 strains of Bacillus subtilis. Trichoderma sp., and B-1 and B-26 strains of B. subtilis alone reduced plant growth but the combination of Trichoderma sp. + B. subtilis (B-1) and Trichoderma sp. + B. subtilis (B-26) increased plant height. Plant height, root weight and root score were significantly reduced when P. penetrans plus B. subtilis or P. penetrans plus fungi plus bacteria were present in the soil. It is suggested that fungi, bacteria, nematodes alone or their combinations such as nematodes plus bacteria or nematodes plus fungi plus bacteria may contribute towards the occurrence of apple replant disease.Contribution number 700.Contribution number 700.     

Biocontrol efficacy of protoplast fusants between Bacillus thuringiensis and Bacillus subtilis against Spodoptera litura Fabr.

Bacillus thuringiensis (Bt) is a microbial pesticide widely used to control crop pests. Its strains have good biocontrol activity against crop insect pest, but lack some desirable characteristics that are found in Bacillus subtilis. An attempt has been made to combine those desirable characteristics; we used a highly effective biocontrol strain of B. thuringiensis in protoplast fusions with a strain of B. subtilis. The fusants were identified through cell culture and stained with crystal violet. The Bt and B. subtilis protoplasts were induced to fuse by PEG 6000. The fusants were produced almost 95% mortality in first instar larvae of Spodoptera litura. The lethal doses (The LC₅₀ and LC₉₀) for mortality of S. litura values were significantly in lower level in the fusant-treated larvae, when compared with Bt and B. subtilis individual treatment. The consumption and digestion of S. litura significantly decreased after treatment with fusant. Also the approximate digestibility of S. litura increased significantly.