Effect of Cultivation Conditions on Growth and Adhesion of Bacillus licheniformis

Adhesion to glass of actively growing cells of the thermophilic Bacillus licheniformis, isolated from the Medyaginskaya test borehole (Yaroslavl' oblast), was studied. The reversible adhesion (RA) manifests itself in a decline of cell density (without cell lysis) in the liquid culture over the first 20–40 min of growth followed by normal exponential growth. The RA is minimal under favorable growth conditions but increases when cells are transferred to a new medium, especially one with a pH, temperature, salinity, or concentration of Ca²⁺ ions nonoptimal for the given species. Under unfavorable growth conditions, the adhesion becomes irreversible. The obtained data suggest that RA represents an adaptation mechanism important for population survival.     

Development of low-cost media for the culture of mosquito larvicides, Bacillus sphaericus and Bacillus thuringiensis serovar. israelensis

Mosquito larvicides like Bacillus sphaericus and Bacillus thuringiensis serovar. israelensis have been widely and effectively used in mosquito control programs, but the industrial production of these bacilli is expensive. Here we have attempted to develop three cost-effective media, based on cheap sources, potato, common sugar and bengalgram. Growth and production of the insecticidal proteins from these bacteria were satisfactory. Bioassay studies with different mosquito larvae showed considerable toxicity. Therefore the investigation suggests that potato-based culture media are more economical for the industrial production of B. sphaericus and B. thuringiensis serovar. israelensis.     

Molecular typing of Bacillus thuringiensis serovars by RAPD-PCR

One hundred and twenty-six strains of Bacillus thuringiensis representing 57 serovars were allocated to 58 genomic types using random amplified polymorphic DNA (RAPD)-PCR patterns. Serovars darmstadiensis, israelensis, kenyae, kumamotoensis, kurstaki, morrisoni, pakistani, sotto, thuringiensis and tolworthi each encompassed identical or closely related strains. Despite this genomic homogeneity, most of these serovars also included at least one variant strain. Serovars aizawai, canadensis, entomocidus and sotto biotype dendrolimus, on the other hand, were genomically heterogeneous. Of the 57 serovars examined, 31 contained at least one strain with a closely related or identical RAPD pattern to a strain from a different serovar. We conclude that while the species is genomically diverse, the homogeneous serovars represent clonal lineages of successful insect pathogens.     

Hemolysin II is more characteristic of Bacillus thuringiensis than Bacillus cereus

To investigate the distribution of the hemolysin II determinant among strains of Bacillus cereus and Bacillus thuringiensis, thirteen strains of B. cereus and fourteen strains of B. thuringiensis strains were tested for hybridization of their chromosomal DNAs with a DNA probe containing the B. cereus hemolysin II gene. In addition, the production of hemolysin II, whose activity is not inhibited by cholesterol, was tested. The presence (absence) of the hybridization response in the microorganism's genome correlated with the presence (absence) of cholesterol-unaffected hemolysin production. Only four out of thirteen B. cereus strains were found to give a positive response in hybridization experiments, whereas thirteen out of fourteen B. thuringiensis strains responded positively. DNAs from ten B. thuringiensis strains contained a 3.5 kb EcoRV fragment, which hybridized with the B. cereus hemolysin II gene probe. The 3.5 kb EcoRV DNA fragment from one of these strains (B. thuringiensis VKM-B1555) was cloned and expressed in Escherichia coli cells. The hemolysin encoded by the cloned DNA fragment was not inhibited by cholesterol and possessed all other properties of B. cereus hemolysin II. The obtained data clearly show limited distribution of hemolysin II among B. cereus strains and demonstrate that hemolysin II is more characteristic of B. thuringiensis than B. cereus.     

Subspecies-specific distribution of intervening sequences in the Bacillus subtilis prophage ribonucleotide reductase genes

A collection of 212 gram-positive bacilli isolated from natural habitats was screened for the presence of intervening sequences (introns and intein-coding sequences) in the SPbeta prophage-related ribonucleotide reductase genes bnrdE and bnrdF. Three novel configurations were identified on the basis of the presence of (i) intervening sequences in bnrdE and bnrdF, and (ii) an ORF in the bnrdE-bnrdF spacer. Analysis of the cell wall genetic determinants as well as of the incorporation of radio-labelled glycerol into cell wall allowed newly and previously identified B. subtilis strains with different configurations of bnrdE/bnrdF intervening sequences to be assigned to one of two subspecies. Strains apparently belonging to the subsp. subtilis contain three intervening sequences many of which are associated with the putative homing endonuclease activity. Strains of the subsp. spizizenii contain only one or two ORF-less group I introns. Introns occupying bnrdF are confined to the subspecies subtilis.     

Growth,sporulation and toxin production byBacillus thuringiensis subsp.israelensis andB. sphaericus in media based on mustard-seed meal

Bacillus thuringiensis subsp.israelensis andB. sphaericus strains 2362 and 1593 were grown in media based on defatted mustard-seed meal (MSM). The meal contains 40% (w/w) protein, with glutamic acid and arginine as the major amino acids. The toxic potencies of the final bacterial powders towardsCulex pipens quinquefasciatus Say, compared with those of the respective international reference standards, were 46% forB. thuringiensis subsp.israelensis, 62% forB. sphaericus 2362 and 88% forB. sphaericus 1593 when 2% (w/v) MSM was used for growth. With 4% (w/v) MSM,B. thuringiensis subsp.israelensis grew better but had undetectable larvicidal activity, whereas theB. sphaericus strains not only grew better but gave a higher degree of sporulation and toxicity. The potencies ofB. sphaericus in medium with 4% MSM were comparable with those of international reference standards.The authors are with the Department of Life Sciences, University of Bombay, Bombay 400 098, India.     

FT-IR spectroscopic characteristics of differently cultivated Bacillus subtilis

Bacillus subtilis is an aerobic endospore forming bacterium widely spread in different environments. Because it represents a biological agent of some health relevance, its rapid detection and identification is highly desirable. By using FT-IR spectroscopy for this purpose slightly different characteristics were obtained from cell mass grown in differently composed cultural media, and harvested in different phases of bacterial growth. If cultivated uniformly, i.e., 24h at 30 degrees C in a minimum-strength nutrient broth, cell mass of B. subtilis delivered a well differentiated spectrum with major absorption bands of nucleic acid structures at 3300cm(-1), cell wall constituents at 3000-2800cm(-1), proteinaceous structures at 1660, 1544 and 1235cm(-1), and some aliphatic structural units at 1080cm(-1). Attenuated total reflectance, and absorption/transmission scanning techniques, delivered structurally identical spectra but those obtained by the former technique were more expressed.     

BpaI and BpnI: novel type II restriction endonucleases from Bacillus pasteurii and Bacillus pantothenticus

Two novel type II restriction endonucleases, designated as BpaI and BpnI, were isolated from Bacillus pasteurii strain1761 and Bacillus pantothenticus strain1639, respectively. They were partially purified and SDS-PAGE indicated Mr values of 28 and 67 kDa for BpaI, 28 and 48 kDa for BpnI. The partially purified endonucleases hydrolyzed DNA into discrete fragments: pUC18 (2.6 kb for BpaI; 1.8 and 0.8 kb for BpnI), pBR322 (2.5 and 1.8 kb for BpaI; 2.6 and 1.7 kb for BpnI) and phix174 DNA (3.2 and 2.1 kb for BpaI; 4 and 1.3 kb for BpnI).     

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.     

Occurrence and pathogenic potential of Bacillus cereus group bacteria in a sandy loam

The major part (94%) of the Bacillus cereus-like isolates from a Danish sandy loam are psychrotolerant Bacillus weihenstephanensis according to their ability to grow at temperatures below 7 °C and/or two PCR-based methods, while the remaining 6% are B. cereus. The Bacillus mycoides-like isolates could also be␣divided into psychrotolerant and mesophilic isolates. The psychrotolerant isolates of B. mycoides could␣be discriminated from the mesophilic by the two PCR-based methods used to characterize B.␣weihenstephanensis. It is likely that the mesophilic B. mycoides strains are synonymous with Bacillus pseudomycoides, while psychrotolerant B. weihenstephanensis, like B. mycoides, are B. mycoides senso stricto. B. cereus is known to produce a number of factors, which are involved in its ability to cause gastrointestinal and somatic diseases. All the B. cereus-like and B. mycoides like isolates from the sandy loam were investigated by PCR for the presence of 12 genes encoding toxins. Genes for the enterotoxins (hemolysin BL and nonhemolytic enterotoxin) and the two of the enzymes (cereolysin AB) were present in the major part of the isolates, while genes for phospolipase C and hemolysin III were present in fewer isolates, especially among B. mycoides like isolates. Genes for cytotoxin K and the hemolysin II were only present in isolates affiliated to B. cereus. Most of the mesophilic B. mycoides isolates did not possess the genes for the nonhemolytic enterotoxin and the cereolysin AB. The presence of multiple genes coding for virulence factors in all the isolates from the B. cereus group suggests that all the isolates from the sandy loam are potential pathogens.     

Production and purification of a calcium-dependent protease from Bacillus cereus BG1

The production and purification of a calcium-dependent protease by Bacillus cereus BG1 were studied. The production of the protease was found to depend specifically on the calcium concentration in the culture medium. This suggests that this metal ion is essential for the induction of protease production and/or stabilisation of the enzyme after synthesis. The calcium requirement is highly specific since other metal ions (such as Mg²⁺ and Ba²⁺, which both activate the enzyme) are not able to induce protease production. The most appropriate medium for growth and protease production comprises (g L^–1) starch 5, CaCl₂ 2, yeast extract 2, K₂HPO₄ 0.2 and KH₂PO₄ 0.2. The protease of BG1 strain was purified to homogeneity by ultrafiltration, heat treatment, gel filtration on Sephacryl S-200, ion exchange chromatography on DEAE-cellulose and, finally, a second gel filtration on Sephacryl S-200, with a 39-fold increase in specific activity and 23% recovery. The molecular weight was estimated to be 34 kDa on SDS-PAGE. The optimum temperature and pH of the purified enzyme were determined to be 60°C and 8.0, respectively, in 100 mM Tris-HCl buffer + 2 mM CaCl₂.     

Use of a continuous culture fermentation system to investigate the effect of GanedenBC30 (Bacillus coagulans GBI-30, 6086) supplementation on pathogen survival in the human gut microbiota

Single-stage continuous fermentation systems were employed to examine the effects of GanedenBC(30) supplementation on the human gastrointestinal microbiota in relation to pathogen challenge in vitro. Denaturing gradient gel electrophoresis analysis demonstrated that GanedenBC(30) supplementation modified the microbial profiles in the fermentation systems compared with controls, with profiles clustering according to treatment. Overall, GanedenBC(30) supplementation did not elicit major changes in bacterial population counts in vitro, although notably higher Bcoa191 counts were seen following probiotic supplementation (compared to the controls). Pathogen challenge did not elicit significant modification of the microbial counts in vitro, although notably higher Clit135 counts were seen in the control system post-Clostridium difficile challenge than in the corresponding GanedenBC(30)-supplemented systems. Sporulation appears to be associated with the anti-microbial activity of GanedenBC(30), suggesting that a bi-modal lifecycle of GanedenBC(30)in vivo may lead to anti-microbial activity in distal regions of the gastrointestinal tract.     

Tannase production by Bacillus licheniformis

Bacillus licheniformis KBR 6 produced maximum extracellular tannase activity at 0.21 U ml^–1 with 1.5% (w/v) tannic acid either in the absence or presence of glucose (1 g l^–1) after 18–21 h growth though the organism did not attain maximum growth until 36 h.     

Siderophores of Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis

Three Bacillus anthracis Sterne strains (USAMRIID, 7702, and 34F2) and Bacillus cereus ATCC 14579 excrete two catecholate siderophores, petrobactin (which contains 3,4-dihydroxybenzoyl moieties) and bacillibactin (which contains 2,3-dihydroxybenzoyl moieties). However, the insecticidal organism Bacillus thuringiensis ATCC 33679 makes only bacillibactin. Analyses of siderophore production by previously isolated [Cendrowski et al., Mol. Microbiol. 52 (2004) 407-417] B. anthracis mutant strains revealed that the B. anthracis bacACEBF operon codes for bacillibactin production and the asbAB gene region is required for petrobactin assembly. The two catecholate moieties also were synthesized by separate routes. PCR amplification identified both asbA and asbB genes in the petrobactin producing strains whereas B. thuringiensis ATCC 33679 retained only asbA. Petrobactin synthesis is not limited to the cluster of B. anthracis strains within the B. cereus sensu lato group (in which B. cereus, B. anthracis, and B. thuringiensis are classified), although petrobactin might be prevalent in strains with pathogenic potential for vertebrates.     

Melanin pigment formation and increased UV resistance in Bacillus thuringiensis following high temperature induction

The pigment melanin is well known to protect against the damaging effects of UV radiation. In this study, we show that thirty-five of thirty-seven tested Bacillus thuringiensis strains possess the potential to produce melanin in the presence of L-tyrosin at elevated temperature (42 degrees C). These findings offer a method of protecting insecticidal toxins produced by B. thuringiensis from UV degredation and may therefore have important applications in the field of crop protection. Toxicity assays on Heliothis armigera suggested that the insecticidal activity of B. thuringiensis that produced melanin was significantly higher after UV irradiation than when melanin was not produced.     

Bacillus algicola sp. nov., a novel filamentous organism isolated from brown alga Fucus evanescens

A slightly yellowish, Gram-positive, filamentous with 'cross-like' branching, aerobic, spore-forming bacterium was isolated from enrichment culture during degradation of the thallus of the brown alga Fucus evanescens. The bacterium studied was chemoorganotrophic, tolerant to 3% NaCl, alkalitolerant, and alginolytic. The predominant cellular fatty acid was ai15:0 which accounted more than 65% of total fatty acids, while i14:0, il5:0 i16:0, and ai17:0 made up 25%. DNA base composition was 37 mol% GC. Phylogenetic analysis of 16S rDNA gene revealed that this isolate was a member of the genus Bacillus, with no close relatives at the species level (16S rRNA gene sequence similarity less 97%). On the basis of the significant differences demonstrated in the phenotypic and chemotaxonomic characteristics, it is suggested that the bacterium be classified as a novel species; the name Bacillus algicola sp. nov. is proposed. The type strain is KMM 3737T (= CIP 107850T).