Sporulation and regeneration efficiency of phosphobacteria (<Emphasis Type="Italic">Bacillus megaterium</Emphasis> var <Emphasis Type="Italic">phosphaticum</Emphasis>)

Sporulation in Bacillus megaterium var phosphaticum (PB — 1) was induced using modified nutrient media. This modified medium induced sporulation within 36 h. After spore induction the spores were kept under refrigerated (5°C) and room temperature (32°C) for five months and survival of spores was studied at 15 days intervals by plating them in nutrient agar medium. It was observed that there was not much variation in the storage temperature (5°C & 32°C). The spore cells of Bacillus megaterium var phosphaticum (PB — 1) were observed up to five months of storage under refrigerated (5°C) and room temperature (32°C). Regeneration of spore cells into vegetative cells was studied in tap water, rice gruel, nutrient broth, sterile lignite and sterile water at different concentrations of spore inoculum. The multiplication of sporulated Bacillus megaterium var phosphaticum culture was fast and reached its maximum (29.5 × 10⁸ cfu ml⁻¹) in nutrient broth containing 5 per cent inoculum level.     

Export,purification, and activities of affinity tagged <Emphasis Type="Italic">Lactobacillus reuteri</Emphasis> levansucrase produced by <Emphasis Type="Italic">Bacillus megaterium</Emphasis>

Fructosyltransferases, like the Lactobacillus reteri levansucrase, are important for the production of new fructosyloligosaccharides. Various His₆- and Strep-tagged variants of this enzyme were recombinantly produced and exported into the growth medium using the Gram-positive bacterium Bacillus megaterium. Nutrient-rich growth medium significantly enhanced levansucrase production and export. The B. megaterium signal peptide of the extracellular esterase LipA mediated better levansucrase export compared to the one of the penicillin amidase Pac. The combination of protein export via the LipA signal peptide with the coexpression of the signal peptidase gene sipM further increased the levansucrase secretion. Fused affinity tags allowed the efficient one-step purification of the recombinant proteins from the growth medium. However, fused peptide tags led to slightly decreased secretion of tested fusion proteins. After upscaling 2 to 3 mg affinity tagged levansucrase per liter culture medium was produced and exported. Up to 1 mg of His₆-tagged and 0.7 mg of Strep-tagged levansucrase per liter were recovered by affinity chromatography. Finally, the purified levansucrase was shown to synthesize new fructosyloligosaccharides from the novel donor substrates d-Gal-Fru, d-Xyl-Fru, d-Man-Fru, and d-Fuc-Fru. R. Biedendieck and R. Beine contributed equally to this work.     

Chromate reduction by <Emphasis Type="Italic">Bacillus megaterium</Emphasis> TKW3 isolated from marine sediments

Summary Bacillus megaterium strain TKW3 was isolated from multiple-metal-contaminated marine sediments of Tokwawan, Hong Kong SAR. This facultative aerobe utilized arabinose, mannitol, N-acetylglucosamine, maltose, caprate, citrate, butyrate or lactate as the sole source of carbon and energy for growth.B. megaterium TKW3 reduced highly toxic and soluble Cr⁶⁺ (as CrO₄²⁻) into almost non-toxic and insoluble Cr³⁺ under aerobic conditions. Complete reduction of 0.20 mM Cr⁶⁺ by B. megaterium TKW3 was achieved within 360 h. Initial Cr⁶⁺ concentration below 0.90 mM or inoculum less than 10⁷ cells ml⁻¹ did not have significant effect on ⁶⁺ reduction, while the residue Cr⁶⁺ concentration was the lowest at 10⁷ cells ml⁻¹. Cr⁶⁺ reduction by this strain was inhibited by high levels of NaCl (55%). B. megaterium TKW3 was also resistant to other oxyanions including 0.34 mM Cr₂O₇²⁻ 0.32 mM AsO₄³⁻, 0.58 mM SeO₃²⁻ and 0.53 mM SeO₄²⁻, and reduced soluble Se⁴⁺ (as SeO₃²⁻) to insoluble red amorphous Se⁰. B. megaterium TKW3 might have potential application in bioremediation of Cr-laden sediments associated with other oxyanions.     

Comparative study on central metabolic fluxes of <Emphasis Type="Italic">Bacillus megaterium</Emphasis> strains in continuous culture using <Superscript>13</Superscript>C labelled substrates

Fluxes of central carbon metabolism [glycolysis, pentose phosphate pathway (PPP), tricarboxylic acid cycle (TCA cycle), biomass formation] were determined for several Bacillus megaterium strains (DSM319, WH320, WH323, MS941) in C- and N-limited chemostat cultures by ¹³C labelling experiments. The labelling patterns of proteinogenic amino acids were analysed by GC/MS and therefrom flux ratios at important nodes within the metabolic network could be calculated. On the basis of a stoichiometric metabolic model flux distributions were estimated for the different B. megaterium strains used at various cultivation conditions. Generally all strains exhibited similar metabolic flux distributions, however, several significant changes were found in (1) the glucose flux entering the PPP via the oxidative branch, (2) the reversibilities within the PPP, (3) the relative fluxes of pyruvate and acetyl-CoA fed to the TCA cycle, (4) the fluxes around the pyruvate node involving a futile cycle.     

Evidence of an association between poly(3-hydroxybutyrate) accumulation and phosphotransbutyrylase expression in<Emphasis Type="Italic"> Bacillus megaterium</Emphasis>

Molecular analysis of a genomic region of Bacillus megaterium, a polyhydroxybutyrate (PHB)-producing microorganism, revealed the presence of a gene coding for the enzyme phosphotransbutyrylase (Ptb). Enzyme activity was measured throughout the different growth phases of B. megaterium and was found to correlate with PHB accumulation during the late-exponential growth phase. Ptb expression was repressed by glucose and activated by the branched amino acids isoleucine and valine. Overexpression of Act_Bm, a ⁵⁴ regulator from B. megaterium whose gene is located upstream from ptb, caused an increase in Ptb activity and PHB accumulation in B. megaterium.     

Characterization of native <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> strains by PCR-RAPD based fingerprinting

Seventy isolates of Bacillus thuringiensis were isolated from soil samples collected from cotton fields. These isolates were characterized by randomly amplified poylmorphic DNA (RAPD) markers to determine their genetic diversity pattern based on their source of origin. Different random decamer primers were used for RAPD amplification, which generated a total of 1935 fragments; of these 1865 were polymorphic and 68 monomorphic. The primers OPA03, OPA08, OPD14, OPD19, OPD20, OPE17 and OPD19 produced 100% polymorphic fragments, whereas primers OPC06, OPC20 and OPD17 produced 20, 31 and 17 monomorphic fragments, respectively. When the RAPD banding pattern data was subjected to dendrogram construction, the 70 isolates fell into two separate clusters, cluster I and cluster II, which includes 26 and 44 B. thuringiensis isolates, respectively. These two main clusters were further divided into four subclusters at Eucledian distance of 150 and 80% similarity index. All primers showed amplification and indicated the good diversity of B. thuringiensis isolates. The RAPD pattern showed 4–10 bands per isolate, with MWt in the range of 0.4–3.5 Kb and an average of 193.5 fragments were produced per primer. The primer OPE17 was found to be the most discriminatory as it produced 286 polymorphic bands.     

Production of Exo-polysaccharide by <Emphasis Type="Italic">Rhizobium</Emphasis> sp.

Two fold increase in the yield of glucose and maltose containing exo-polysaccharide (EPS) by Rhizobium sp. was observed during its growth in modified YEMB. EPS production, plant growth promotion activity and root colonization of Rhizobium sp. studies showed enhanced EPS synthesis, more seed germination and over all improvement in plant growth over control and R. meliloti treatment. Groundnut seeds bacterized with Rhizobium sp. resulted in 69.75% more root length, 49.51% more shoot height, 13.75% more number of branches and 13.60% more number of pods over the control and R. meliloti treatment. Bacterization of wheat seeds increased the dry matter yield of roots (1.7-fold), and roots adhering soil (RAS) (1.5) and shoot mass (1.9-fold). Rhizobium sp. inoculation also increased the population density of EPS-producing bacteria on the rhizoplane. Roots of plants inoculated with Rhizobium sp. maintained a higher K⁺/Na⁺ ratio and K⁺–Na⁺ selectivity.     

Simultaneous detection of pathogenic <Emphasis Type="Italic">B. cereus,S. aureus</Emphasis> and <Emphasis Type="Italic">L. monocytogenes</Emphasis> by multiplex PCR

Three important foodborne pathogens, Bacillus cereus, Listeria monocytogenes and Staphylococcus aureus are of major concern for food safety in terms of frequency and seriousness of the disease. The occurrence these three important pathogens and their coexistence in food matrices are predominant. Moreover, symptoms associated with B. cereus and S. aureus food poisoning not only closely resembles each other but can also be overlapping with other foodborne infections. In this context, detection of these three pathogens simultaneously in food samples by a single multiplex PCR (mPCR) would have advantages in terms of rapidity and cost saving, when compared with single organism specific PCRs. mPCR has been standardized by targeting three major diarrheal enterotoxin genes hbl A, cyt K and nhe A of B. cereus, virulence associated nuc and Ent B genes of S. aureus and virulence associated hly and iap genes of L. monocytogenes along with internal amplification control (IAC). The results showed that mPCR accurately identified all the three organisms individually or in combination without non-specificity. The mPCR was able to detect as low as 10 to 100 organisms per ml of growth following overnight enrichment of spiked food samples (vegetable biriyani and milk) and their presence in naturally contaminated samples also. The high throughput and cost effective multiplex PCR method developed in this study could provide a powerful tool for simultaneous, rapid and reliable detection of B. cereus, S. aureus and L. monocytogenes in food samples.     

Cyclodextrin production and genetic characterization of cyclodextrin glucanotranferase of <Emphasis Type="Italic">Paenibacillus graminis</Emphasis>

Paenibacillus graminis strains were described recently as cyclodextrin (CD) producers. Cyclodextrins are produced by cyclodextrin glucanotransferase (CGTase) which has not been characterized in P. graminis. Similar amounts of α- and β-CDs were produced by P. graminis (MC22.13) and P. macerans (LMD24.10^T). Primers were designed to sequence the gene encoding CGTase from P. graminis. A phylogenetic tree was constructed and P. graminis CGTase protein showed to be closer (79.4% protein identity) to P. macerans |P31835|. Hybridization studies suggested that the gene encoding CGTase is located in different positions in the genomes of P. macerans and P. graminis.     

Effect of <Emphasis Type="Italic">Glomus</Emphasis> species on physiology and biochemistry of <Emphasis Type="Italic">Catharanthus roseus</Emphasis>

The present study on efficacy of different Glomus species, an arbuscular mycorrhizal (AM) fungus (G. aggregatum, G. fasciculatum, G. mosseae, G. intraradices) on various growth parameters such as biomass, macro and micronutrients, chlorophyll, protein, cytokinin and alkaloid content and phosphatase activity of pink flowered Catharanthus roseus plants showed that all Glomus species except G. intraradices enhanced the chlorophyll, protein, crude alkaloid, phosphorus, sulphur, manganese and copper contents of C. roseus plants along with phosphatase activity significantly over uninoculated plants. However only G. mosseae and G. fasciculatum exhibited superior symbiotic relationship with the plant. G. mosseae was found to be the best for increasing the crude alkaloid content (8.19%) in leaf and also in increasing the quantity of important alkaloids vincristine and vinblastine.     

Rapid Affinity Purification Processes for Cyclodextrin Glycosyltransferase from <Emphasis Type="Italic">Bacillus</Emphasis><Emphasis Type="Italic">circulans</Emphasis>

Two rapid and easy-to-scale-up methods for the purification of cyclodextrin glycosyltransferase (CGTase) from Bacillus circulans were developed: affinity precipitation with starch and aqueous two-phase partition. The first method, optimised by a factorial design, gave an 80% CGTase adsorption at 11% starch and 1.6% ammonium sulphate, and a 65% recovery after elution with 10 mM α-cyclodextrin. The purification factor was 17. Aqueous two-phase partition yielded a 72% CGTase recovery in a two-step procedure; CGTase was obtained in the bottom phase with a purification factor of 37.     

Cloning and expression of <Emphasis Type="Italic">mel</Emphasis> gene from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Previous work from our laboratory has shown that most of Bacillus thuringiensis strains possess the ability to produce melanin in the presence of l-tyrosine at elevated temperatures (42 °C). Furthermore, it was shown that the melanin produced by B. thuringiensis was synthesized by the action of tyrosinase, which catalyzed the conversion of l-tyrosine, via l-DOPA, to melanin. In this study, the tyrosinase-encoding gene (mel) from B. thuringiensis 4D11 was cloned using PCR techniques and expressed in Escherichia coli DH5 . A DNA fragment with 1179 bp which contained the intact mel gene in the recombinant plasmid pGEM1179 imparted the ability to synthesize melanin to the E. coli recipient strain. The nucleotide sequence of this DNA fragment revealed an open reading frame of 744 bp, encoding a protein of 248 amino acids. The novel mel gene from B.thuringiensis expressed in E. coli DH5 conferred UV protection on the recipient strain.     

Interaction of <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> and <Emphasis Type="Italic">Glomus intrarradix</Emphasis> in Sugar Cane Roots

Fifteen-day-old variety NA 56-79 sugar cane seedlings were inoculated with Azospirillum brasilense and Glomus intrarradix. This article aims at examining changes in sugar cane root seedlings inoculated with Glomus intrarradix and Azospirillum brasilense, the increase in microbial biomass and the acetylene reduction process as well. The internal root colonization was studied 20 days after inoculation using scanning and a transmission electron microscope. Both microorganisms entered the sugar cane root through the emergent lateral roots. The microorganisms were capable of coexisting both intra and intercellularly, producing changes in the cell wall, thus allowing colonization and interaction between the organisms. These changes increased the number of microorganisms inside the root as well as acetylene nitrogen reduction. Sugar cane plant biomass increased with joint-inoculation. The number of endophytic microorganisms and nitrogen fixing activity increased when they were colonized by Azospirillum and Glomus together.     

Co-inoculation of Urea and DAP Tolerant <Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis> and <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> as Integrated Approach for Growth Enhancement of <Emphasis Type="Italic">Brassica juncea</Emphasis>

Two plant growth promoting rhizobacteria––Sinorhizobium meliloti RMP1 and Pseudomonas aeruginosa GRC₂ were studied for integrated nutrient management to obtain improved yield of Brassica juncea. Low concentrations of urea and diammonium phosphate (DAP) stimulated the growth of both S. meliloti RMP1 and P. aeruginosa GRC₂. 1 M of urea and 0.35 M of DAP was found lethal for RMP1, while 1.3 M and 0.37 M concentrations of urea and DAP proved to be toxic for GRC₂. Lc₅₀ was observed as 0.49 M of urea and 0.15 M of DAP for RMP1, and 0.66 M urea and 0.18 M of DAP for GRC₂. Urea and DAP adaptive variants of RMP1 and GRC₂ was isolated. Adaptive bacterial variants had better growth rates at sub-lethal (Lc₅₀) concentrations of urea and DAP as compared to non-adaptive variants. They also retained plant growth promoting attributes similar to non adaptive variants. GRC₂ and RMP1 did not affect the growth of each other and were chemotactically active for DAP, urea as well as root exudates of B. juncea. Both the isolates colonized well in the rhizosphere of B. juncea, as their populations were recorded ≈5 log₁₀ cfu g⁻¹ after 120 days. Interestingly, the colonization ability was found even better when both strains were co-inoculated, as their population was recorded in the range of ≈6 log₁₀ cfu g⁻¹ after 120 days. In field trials, application of RMP1 and GRC₂ resulted in significant increase in biomass and yield of B. juncea as compared to control. However, yield was better with application of half dose and full dose of recommended fertilizers. Interestingly, the biomass as well as yield improved further when both isolates were applied together along with half dose of recommended fertilizers.     

High level expression of a recombinant phospholipase C from <Emphasis Type="Italic">Bacillus cereus</Emphasis> in <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Twenty-two Bacillus cereus strains were screened for phospholipase C (PLC, EC 3.1.4.3) activity using p-nitrophenyl phosphorylcholine as a substrate. Two strains (B. cereus SBUG 318 and SBUG 516) showed high activity at elevated temperatures (>70°C) at acidic pH (pH 3.5–6) and were selected for cloning and functional expression using Bacillus subtilis. The genes were amplified from B. cereus DNA using primers based on a known PLC sequence and cloned into the expression vector pMSE3 followed by transformation into B. subtilis WB800. On the amino acid level, one protein (PLC318) was identical to a PLC described from B. cereus, whereas PLC516 contained an amino acid substitution (E173D). PLC production using the recombinant strains was performed by an acetoin-controlled expression system. For PLC516, 13.7 U g⁻¹ wet cell weight was determined in the culture supernatant after 30 h cultivation time. Three purification steps resulted in pure PLC516 with a specific activity of 13,190 U mg⁻¹ protein.     

Prevalence of Very Low Numbers of Potential Pathogenic Isolates of <Emphasis Type="Italic">Yersinia</Emphasis><Emphasis Type="Italic">enterocolitica</Emphasis> and <Emphasis Type="Italic">Yersinia intermedia</Emphasis> in Traditional Fast Foods of India

In this study, an incidence pattern of 1.7% for Yersinia enterocolitica and 2.5% for Y. intermedia were observed in an analysis of 120 diversified food samples collected from the local market of Mysore, Southern India. Two native isolates characterized as Y. enterocolitica belonged to biotype 1B and revealed the presence of major virulence related traits such as regulator of virulence, mucoid Yersinia factor regulator, attachment invasion locus, heat stable enterotoxin, Yersinia type II secretory system and phospholipase A in PCR. Force type neighbor-joining phylograms generated for Y. enterocolitica based on PCR amplicons of rovA and ypl showed 100% homology with two to three strains of Y. enterocolitica and about 75% homology with several strains of Y. pestis.     

The expression of <Emphasis Type="Italic">Bacillus intermedius</Emphasis> glutamyl endopeptidase gene in <Emphasis Type="Italic">Bacillus subtilis</Emphasis> recombinant strains

The gene encoding for B. intermedius glutamyl endopeptidase (gseBi) has previously been cloned and its nucleotide sequence analyzed. In this study, the expression of this gene was explored in protease-deficient strain B. subtilis AJ73 during stationary phase of bacterial growth. We found that catabolite repression usually involved in control of endopeptidase expression during vegetative growth was not efficient at the late stationary phase. Testing of B. intermedius glutamyl endopeptidase gene expression with B. subtilis spo0-mutants revealed slight effect of these mutations on endopeptidase expression. Activity of glutamyl endopeptidase was partly left in B. subtilis ger-mutants. Probably, gseBi expression was not connected with sporulation. This enzyme might be involved in outgrowth of the spore, when germinating endospore converts into the vegetative cell. These data suggest complex regulation of B. intermedius glutamyl endopeptidase gene expression with contribution of several regulatory systems and demonstrate changes in control of enzyme biosynthesis at different stages of growth.     

Negative regulation of pathogenesis in <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">tabaci</Emphasis> 11528 by ATP-dependent lon protease

Pseudomonas syringae pv. tabaci causes wildfire disease in tobacco plants. The hrp pathogenicity island (hrp PAI) of P. syringae pv. tabaci encodes a type III secretion system (TTSS) and its regulatory system, which are required for pathogenesis in plants. Three important regulatory proteins-HrpR, HrpS, and HrpL-have been identified to activate hrp PAI gene expression. The bacterial Lon protease regulates the expression of various genes. To investigate the regulatory mechanism of the Lon protease in P. syringae pv. tabaci 11528, we cloned the lon gene, and then a Δlon mutant was generated by allelic exchange. lon mutants showed increased UV sensitivity, which is a typical feature of such mutants. The Δlon mutant produced higher levels of tabtoxin than the wild-type. The lacZ gene was fused with hrpA promoter and activity of β-galactosidase was measured in hrp-repressing and hrp-inducing media. The Lon protease functioned as a negative regulator of hrp PAI under hrp-repressing conditions. We found that strains with lon disruption elicited the host defense system more rapidly and strongly than the wild-type strain, suggesting that the Lon protease is essential for systemic pathogenesis.     

Characterization of the <Emphasis Type="Italic">codY</Emphasis> gene and its influence on biofilm formation in <Emphasis Type="Italic">Bacillus </Emphasis><Emphasis Type="Italic">cereus</Emphasis>

The foodborne pathogen Bacillus cereus can form biofilms on various food contact surfaces, leading to contamination of food products. To study the mechanisms of biofilm formation by B. cereus, a Tn5401 library was generated from strain UW101C. Eight thousand mutants were screened in EPS, a low nutrient medium. One mutant (M124), with a disruption in codY, developed fourfold less biofilm than the wild-type, and its defective biofilm phenotype was rescued by complementation. Addition of 0.1% casamino acids to EPS prolonged the duration of biofilms in the wild-type but not codY mutant. When decoyinine, a GTP synthesis inhibitor, was added to EPS, biofilm formation was decreased in the wild-type but not the mutant. The codY mutant produced three times higher protease activity than the wild-type. Zymogram and SDS-PAGE data showed that production of the protease (∼130 kDa) was repressed by CodY. Addition of proteinase K to EPS decreased biofilm formation by the wild-type. Using a dpp-lacZ fusion reporter system, it was shown that that the B. cereus CodY can sense amino acids and GTP levels. These data suggest that by responding to amino acids and intracellular GTP levels CodY represses production of an unknown protease and is involved in biofilm formation.