Purification and Characterization of Two Novel Antimicrobial Peptides Subpeptin JM4-A and Subpeptin JM4-B Produced by Bacillus subtilis JM4

An antimicrobial peptides-producing strain was isolated from soil and identified as Bacillus subtilis JM4 according to biochemical tests and 16S rDNA sequence analysis. The corresponding antimicrobial peptides were purified to homogeneity by ammonium sulfate precipitation, sequential SP-Sepharose Fast Flow, Sephadex G-25 and C18 reverse-phase chromatography, and in the final purification step, two active fractions were harvested, designated as Subpeptin JM4-A and Subpeptin JM4-B. The molecular weights, determined by mass spectrometry, were 1422.71 Da for Subpeptin JM4-A and 1422.65 Da for Subpeptin JM4-B, respectively. Amino acid sequencing showed that they differed from each other only at the seventh amino acid except for three unidentified residues, and the two peptides had no significant sequence homology to the known peptides in the database, indicating that they are two novel antimicrobial peptides. In addition, characteristic measurements indicated that both peptides had a relatively broad inhibitory spectrum and remained active over a wide pH and temperature range.     

Study on the production of chitin and chitosan from shrimp shell by using Bacillus subtilis fermentation

Fermentation of shrimp shell in jaggery broth using Bacillus subtilis for the production of chitin and chitosan was investigated. It was found that B. subtilis produced sufficient quantities of acid to remove the minerals from the shell and to prevent spoilage organisms. The protease enzyme in Bacillus species was responsible for the deprotenisation of the shell. The pH, proteolytic activity, extent of demineralization and deprotenisation were studied during fermentation. About 84% of the protein and 72% of the minerals were removed from the shrimp shell after fermentation. Mild acid and alkali treatments were given to produce characteristic chitin and their concentrations were standardized. Chitin was converted to chitosan by N-deacetylation and the properties of chitin and chitosan were studied. FTIR spectral analysis of chitin and chitosan prepared by the process was carried out and compared with spectra of commercially available samples.     

New inhibitors of colony spreading in Bacillus subtilis and Bacillus anthracis

We have recently characterized sliding motility in Bacillus subtilis strains that lack functional flagella, and here describe the discovery of inhibitors of colony spreading in these strains as well as the aflagellate pathogen, Bacillus anthracis. Aflagellate B. subtilis strains were used to screen for new types of antibacterials that might inhibit colony spreading on semi-solid media. From a diverse set of organic structures, p-nitrophenylglycerol (NPG), an agent used primarily in clinical laboratories to control Proteus swarming, was found to inhibit colony spreading. The four stereoisomers of NPG were synthesized and tested, and only the 1R,2S-(1R-anti) and 1R,2R-(1R-syn) NPG isomers had significant activity in a quantitative colony-spreading assay. Twenty-six NPG analogs and related structures were synthesized and tested to identify more active inhibitors. p-Methylsulfonylphenylglycerol (p-SPG), but not its ortho or meta analogs, was found to be the most effective of these compounds, and synthesis and testing of all four p-SPG stereoisomers showed that the 1R-anti-isomer was the most active with an average IC(50) of 16 μM (3-5 μg mL(-1)). For B. anthracis, the colony-spreading IC(50) values for 1R-anti-SPG and 1R-anti-NPG are 12 μM (2-4 μg mL(-1)) and >150 μM, respectively. For both Bacillus species tested, 1R-anti-SPG inhibits colony spreading of surface cultures on agar plates, but is not bacteriostatic or bacteriocidal in liquid cultures. Work is in progress to find the cellular target(s) of the NPG/SPG class of compounds, since this could lead to an understanding of the mechanism(s) of colony spreading as well as design and development of more potent inhibitors for the control of B. anthracis surface cultures.     

Properties of motility in Bacillus subtilis powered by the H+-coupled MotAB flagellar stator, Na+-coupled MotPS or hybrid stators MotAS or MotPB

Bacillus subtilis has a single set of flagellar rotor proteins that interact with two distinct stator-force generators, the H+-coupled MotAB complex and the Na+-coupled MotPS complex, that energize rotation. Here, motility on soft agar plates and in liquid was assayed in wild-type B.subtilis and strains expressing only one stator, either MotAB, MotPS or hybrid MotAS or MotPB. The strains expressing MotAB or MotAS had an average of 11 flagella/cell while those expressing MotPS or MotPB had an average of seven flagella/cell, and a Mot-less double mutant had three to four flagella/cell. MotAB had a more dominant role in motility than MotPS under most conditions, but MotPS supported comparable motility to MotAB on malate-containing soft agar plating media at elevated pH and Na+. MotAB supported much faster swimming speeds in liquid than MotPS, MotAS or MotPB under all conditions, but a contribution of MotPS to wild-type swimming was discernible from differences in swimming speeds of wild-type and MotAB at elevated viscosity, pH and Na+. Swimming supported by MotPS and MotAS was stimulated by Na+ and elevated pH whereas the converse was true of MotAB and MotPB. This suggests that MotAS is Na+-coupled and MotPB is H+-coupled and that MotB and MotS are major determinants of ion-coupling. However, the swimming speed supported by MotPB, as well as MotPS and MotAS, was inhibited severely at Na+ concentrations above 300 mM whereas MotAB-dependent swimming was not. The presence of either the MotP or MotS component in the stator also conferred sensitivity to inhibition by an amiloride analogue. These observations suggest that MotP contributes to Na+-coupling and inhibition by Na+ channel inhibitors. Similarly, a role for MotA in H+-dependent stator properties is indicated by the larger effects of pH on the Na+-response of MotAS versus MotPS. Finally, optimal function at elevated viscosity was found only in MotPS and MotPB and is therefore conferred by MotP.     

The introduction of a phytase gene from Bacillus subtilis improved the growth performance of transgenic tobacco

Phytate, the main form of phosphorus storage in plant seeds, is well known to be an anti-nutrient and a major source of phosphorus pollution in animal manure. To improve phosphorus bio-availability, we introduced a recently characterized phytase from Bacillus subtilis into the cytoplasm of tobacco cells. Although the introduction of acid fungal phytase from Aspergillus niger in previous studies did not result in any phenotypic changes in tobacco, here we show that a tobacco line transformed with a neutral phytase exhibited phenotypic changes in flowering, seed development, and response to phosphate deficiency. The transgenic line showed an increase in flower and fruit numbers, small seed syndrome, lower seed IP6/IP5 ratio, and enhanced growth under phosphate-starvation conditions compared with the wildtype. The results suggest that the over-expression of Bacillus phytase in the cytoplasm of tobacco cells shifts the equilibrium of the inositol phosphate biosynthesis pathway, thereby making more phosphate available for primary metabolism. The approach presented here can be applied as a strategy for boosting productivity in agriculture and horticulture.     

Denitrification is a common feature among members of the genus Bacillus

Although several Gram-positive denitrifiers have been characterized in the past, there is still uncertainty about the occurrence of the denitrification trait among these bacteria. In an isolation campaign on luvisol soil, Bacillus spp. were among the most abundant retrieved cultured denitrifiers next to members of Rhizobiaceae family and genus Cupriavidus. Subsequent screening of 180 representatives of the genus Bacillus (encompassing more than half of the current validly described species diversity in Bacillus) was performed and demonstrated the potential for dissimilatory reduction of nitrogen compounds in 45 of the 87 investigated species, with 19 species containing denitrifying members. The influence of several electron donors and acceptors was tested. The use of more than one electron acceptor, e.g. both nitrate and nitrite, was crucial to detect the denitrification potential of reference strains. Complex electron donors, most suitable for aerobic growth, were ideal for denitrification testing, while retrieval of denitrifiers from the environment was facilitated by the use of defined electron donors, due to less interference of other anaerobic growers. The outcome of the isolation campaign and screening of reference strain set suggest that bacilli may be potential contributors to denitrification in terrestrial and possibly other ecosystems.     

Biochemical characterization of ribosome assembly GTPase RbgA in Bacillus subtilis

The ribosome biogenesis GTPase A protein RbgA is involved in the assembly of the large ribosomal subunit in Bacillus subtilis, and homologs of RbgA are implicated in the biogenesis of mitochondrial, chloroplast, and cytoplasmic ribosomes in archaea and eukaryotes. The precise function of how RbgA contributes to ribosome assembly is not understood. Defects in RbgA give rise to a large ribosomal subunit that is immature and migrates at 45 S in sucrose density gradients. Here, we report a detailed biochemical analysis of RbgA and its interaction with the ribosome. We found that RbgA, like most other GTPases, exhibits a very slow k(cat) (14 h(-1)) and has a high K(m) (90 μM). Homology modeling of the RbgA switch I region using the K-loop GTPase MnmE as a template suggested that RbgA requires K(+) ions for GTPase activity, which was confirmed experimentally. Interaction with 50 S subunits, but not 45 S intermediates, increased GTPase activity by ～55-fold. Stable association with 50 S subunits and 45 S intermediates was nucleotide-dependent, and GDP did not support strong interaction with either of the subunits. GTP and guanosine 5'-(β,γ-imido)triphosphate (GMPPNP) were sufficient to promote association with the 45 S intermediate, whereas only GMPPNP was able to support binding to the 50 S subunit, presumably due to the stimulation of GTP hydrolysis. These results support a model in which RbgA promotes a late step in ribosome biogenesis and that one role of GTP hydrolysis is to stimulate dissociation of RbgA from the ribosome.     

Cost-effective production of Bacillus thuringiensis by solid-state fermentation

Production of Bacillus thuringiensis (Bt) was standardized on wheat bran based media in 250 ml Erlenmeyer flasks. Scale-up of Bt production on the best medium in plastic tubs with aeration at 8 h intervals starting 16 h after incubation yielded a significant increase in spore count and toxin content of the product. Maximum lysis of Bt cells was obtained by 60 h of incubation at 30 degrees C. This protocol was suitable for production of Bt strains and local isolates. The Bt produced proved highly effective at 0.1% concentration against larvae of castor semilooper, Achaea janata L, resulting in complete mortality by three days in laboratory bioassays. In field trials, the population of castor semilooper larvae on the castor bean crop was reduced significantly by three days after application. The cost for material production of 1 kg of Bt was approximately US dollars 0.70.     

The Bacillus subtilis Chemoreceptor McpC Senses Multiple Ligands Using Two Discrete Mechanisms

Bacillus subtilis can perform chemotaxis toward all 20 l-amino acids normally found in proteins. Loss of a single chemoreceptor, McpC, was previously found to reduce chemotaxis to 19 of these amino acids. In this study, we investigated the amino acid-sensing mechanism of McpC. We show that McpC alone can support chemotaxis to 17 of these amino acids to varying degrees. Eleven amino acids were found to directly bind the amino-terminal sensing domain of McpC in vitro. Sequence analysis indicates that the McpC sensing domain exhibits a dual Per-Arnt-Sim (PAS) domain structure. Using this structure as a guide, we were able to isolate mutants that suggest that four amino acids (arginine, glutamine, lysine, and methionine) are sensed by an indirect mechanism. We identified four candidate binding lipoproteins associated with amino acid transporters that may function in indirect sensing: ArtP, GlnH, MetQ, and YckB. ArtP was found to bind arginine and lysine; GlnH, glutamine; MetQ, methionine; and YckB, tryptophan. In addition, we found that ArtP, MetQ, and YckB bind the sensing domain of McpC, suggesting that the three participate in the indirect sensing of arginine, lysine, methionine, and possibly tryptophan as well. Taken together, these results further our understanding of amino acid chemotaxis in B. subtilis and gain insight into how a single chemoreceptor is able to sense many amino acids.     

Bioconversion of shellfish chitin wastes for the production of Bacillus subtilis W-118 chitinase

Bacillus subtilis W-118, a strain that produces antifungal materials, excreted a chitinase when cultured in a medium containing shrimp- and crab-shell powder as the major carbon source. This chitinase, purified by sequential chromatography, had a molecular mass of 20,600 Da and a pI of 6. The optimum pH, optimum temperature, and pH stability of the chitinase were pH 6, 37 degrees C, and pH 5-7, respectively. The unique characteristics of the purified chitinase include low molecular mass and acidic pI. In the investigation of the inhibitory activity, it was found that the growth of Fusarium oxysporum was 100% inhibited after incubation for 1 day with sterilized W-118 chitinase solution (5.6 units/mL). The chitinase hydrolyzates of chitin with low degrees of polymerization (DP 1-6) were analyzed by HPLC. Longer reaction times led to the generation of chitin oligosaccharides with lower DP. The chitin oligosaccharides were examined for their inhibitory effects on F. oxysporum and human leukemia cell lines.     

Synthesis of silver nanoparticles by solar irradiation of cell-free Bacillus amyloliquefaciens extracts and AgNO3

Silver nanoparticles (AgNPs) were obtained by solar irradiation of cell-free extracts of Bacillus amyloliquefaciens and AgNO₃. Light intensity, extract concentration, and NaCl addition influenced the synthesis of AgNPs. Under optimized conditions (solar intensity 70,000 lx, extract concentration 3 mg/mL, and NaCl content 2 mM), 98.23 ± 0.06% of the Ag⁺ (1 mM) was reduced to AgNPs within 80 min, and the ζ-potential of AgNPs reached −70.84 ± 0.66 mV. TEM (Transmission electron microscopy) and XRD (X-ray diffraction) analysis confirmed that circular and triangular crystalline AgNPs with mean diameter of 14.6 nm were synthesized. Since heat-inactivated extracts also mediated the formation of AgNPs, enzymatic reactions are likely not involved in AgNPs formation. A high absolute ζ-potential value of the AgNPs, possibly caused by interaction with proteins likely explains the high stability of AgNPs suspensions. AgNPs showed antimicrobial activity against Bacillus subtilis and Escherichia coli in liquid and solid medium.     

Extracellular production of human cystatin S and cystatin SA by Bacillus subtilis

We herein describe the development of a Bacillus subtilis system that can be used to produce large quantities of recombinant (r-) human salivary cystatins, a cysteine protease inhibitor of family 2 in the cystatin superfamily. The B. subtilis that lacked the alkaline protease E gene (DeltaaprE type mutant strain) was prepared by homologous recombination. The cDNA fragments coding for mature cystatins (S and SA) were ligated in frame to the DNA segment for the signal peptide of endoglucanase in the pHSP-US plasmid vector that was then use to transform the DeltaaprE type mutant strain of B. subtilis. The transformants carrying the expression vectors were cultivated in 5-L jar fermenters for 3 days at 30 degrees C. Both r-cystatin S and r-cystatin SA were successfully expressed and secreted into the culture broth, and were purified using a fast performance liquid chromatography system. The first use of DeltaaprE type mutant strain of B. subtilis made it possible to obtain a high yield of secreted protein, which makes this system an improvement over expression in Escherichia coli. We conclude that this system has high utility for expression of commercial quantities of secreted proteins.     

A novel type of teichoic acid from the cell wall of Bacillus subtilis VKM B-762

The cell wall of Bacillus subtilis VKM B-762 contains, along with 1,5-poly[4-O-(2-acetamido-2-deoxy-β-d-glucopyranosyl)ribitol phosphate], a novel type of glycopolymer involving three types of inter-monomeric bonds in the repeating unit, viz., amide, glycosidic and phosphodiester:Such a structural pattern of natural glycopolymers has been hitherto unknown. This polymer represents a novel type of teichoic acids.     

Bioreduction of Cr(VI) by alkaliphilic Bacillus subtilis and interaction of the membrane groups

Detoxification of Cr(VI) under alkaline pH requires attention due to the alkaline nature of many effluents. An alkaliphilic gram-positive Bacillus subtilis isolated from tannery effluent contaminated soil was found to grow and reduce Cr(VI) up to 100% at an alkaline pH 9. Decrease in pH to acidic range with growth of the bacterium signified the role played by metabolites (organic acids) in chromium resistance and reduction mechanism. The XPS and FT-IR spectra confirmed the reduction of Cr(VI) by bacteria into +3 oxidation state. Chromate reductase assay indicated that the reduction was mediated by constitutive membrane bound enzymes. The kinetics of Cr(VI) reduction activity derived using the monod equation proved (K_s = 0.00032) high affinity of the organism to the metal. This study thus helped to localize the reduction activity at subcellular level in a chromium resistant alkaliphilic Bacillus sp.     

Identification and characterization of a novel spermidine/spermine acetyltransferase encoded by gene ste26 from Streptomyces sp. 139

Streptomyces sp. 139 produces a novel exopolysaccharide (EPS) designated Ebosin which can bind IL-1R specifically and exhibits anti-rheumatic arthritis activity in vivo. With the Ebosin biosynthesis gene cluster (ste) consisting of 27 ORFs identified previously the focus of this study was to characterize the protein encoded by ste26 gene. After cloning and expressing ste26 in Escherichia coli BL21, we purified the recombinant Ste26 protein and revealed its ability of transferring the acetyl group from AcCoA to spermidine and spermine, with spermine being the preferred substrate. Therefore Ste26 has been determined to be a spermidine/spermine acetyltransferase which can use spermine (Km of 72.1 ± 7.4 μM), spermidine (Km of 147.2 ± 11 μM), AcCoA (Km of 45.7 ± 2.5 μM) and poly-l-lysine (Km of 99.7 ± 11 μM) as substrates. The optimum pH, temperature and time for the activity have been shown to be 7.5, 37°C and 10 min, respectively. This is the first spermidine/spermine acetyltransferase characterized in Streptomyces and its function in Ebosin biosynthesis is discussed.     

Optimizing Bacillus subtilis spore isolation and quantifying spore harvest purity

Investigating the biochemistry, resilience and environmental interactions of bacterial endospores often requires a pure endospore biomass free of vegetative cells. Numerous endospore isolation methods, however, neglect to quantify the purity of the final endospore biomass. To ensure low vegetative cell contamination we developed a quality control technique that enables rapid quantification of endospore harvest purity. This method quantifies spore purity using bright-field and fluorescence microscopy imaging in conjunction with automated cell counting software. We applied this method to Bacillus subtilis endospore harvests isolated using a two-phase separation method that utilizes mild chemicals. The average spore purity of twenty-two harvests was 88 ± 11% (error is 1σ) with a median value of 93%. A spearman coefficient of 0.97 correlating automated and manual bacterial counts confirms the accuracy of software generated data.     

Effects of terbium chelate structure on dipicolinate ligation and the detection of Bacillus spores

Terbium-sensitized luminescence and its applicability towards the detection of Bacillus spores such as anthrax are of significant interest to research in biodefense and medical diagnostics. Accordingly, we have measured the effects of terbium chelation upon the parameters associated with dipicolinate ligation and spore detection. Namely, the dissociation constants, intrinsic brightness, luminescent lifetimes, and biological stabilities for several Tb(chelate)(dipicolinate)_x complexes were determined using linear, cyclic, and aromatic chelators of differing structure and coordination number. This included the chelator array of NTA, BisTris, EGTA, EDTA, BAPTA, DO2A, DTPA, DO3A, and DOTA (respectively, 2,2′,2″-nitrilotriacetic acid; 2,2-bis(hydroxymethyl)-2,2′,2″-nitrilotriethanol; ethylene glycol-bis(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid; ethylenediamine-N,N,N′,N′-tetraacetic acid; 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; 1,4,7,10-tetraazacyclododecane-1,7-diacetic acid; diethylenetriamine-N,N,N′,N″,N″-pentaacetic acid; 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid; and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid). Our study has revealed that the thermodynamic and temporal emission stabilities of the Tb(chelate)(dipicolinate)_x complexes are directly related to chelate rigidity and a ligand stoichiometry of x = 1, and that chelators possessing either aromaticity or low coordination numbers are destabilizing to the complexes when in extracts of an extremotolerant Bacillus spore. Together, our results demonstrate that both Tb(EDTA) and Tb(DO2A) are chemically and biochemically stable and thus applicable as respectively low and high-cost luminescent reporters for spore detection, and thereby of significance to institutions with developing biodefense programs.     

Improved secretory production of glucoamylase in Pichia pastoris by combination of genetic manipulations

Rhizopus oryzae glucoamylase (GA) has been genetically engineered with modified signal peptide (MSP), increased copy number of the gene, and coexpression of SEC4, a gene encoding a Rab protein associated with secretory vesicles, and its secretion level has been successfully raised up to 100-fold in Pichia pastoris. The MSP was designed to contain the signal peptide of mouse salivary alpha-amylase (S8L) fused to the pro-region of the signal peptide of Saccharomyces cerevisiae alpha-mating factor to replace the wild type signal peptide (WTSP) of GA. The P. pastoris transformant MSPGA-1 containing a single copy of MSPGA gene showed a 3.6-fold increase in GA secretion as compared to that of WTSPGA-1. Moreover, the P. pastoris transformant MSPGA-7 harboring seven copies of the MSPGA inserts was identified and showed 56-fold higher secreted GA than WTSPGA-1. In addition, we found that overexpression of SEC4 further doubled the secretion level of GA in each MSPGA/P. pastoris transformant. Taken together, the MSPGA-7-SEC4 clone showed as much as 100-fold secretion level of GA when compared to WTSPGA-1. In summary, we have demonstrated that combination of the aforementioned genetic manipulations resulted in high level secretion of R. oryzae GA in P. pastoris.