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GLUCOSE CATABOLISM BY BACILLUS POPILLIAE AND BACILLUS LENTIMORBUS.

Pepper, Rollin E. (Michigan State University, East Lansing), and Ralph N. Costilow. Glucose catabolism by Bacillus popilliae and Bacillus lentimorbus. J. Bacteriol. 87:303-310. 1964.-Resting cells of Bacillus popilliae and B. lentimorbus catabolize glucose with the production of CO(2), lactic acid, acetic acid, glycerol, ethanol, and trace amounts of acetoin and acetaldehyde. The first three products are the major ones, and their ratios may be varied by controlling the availability of oxygen. Practically no lactic acid is produced when oxygen is not limiting, whereas it may comprise up to 80% of the total acid when oxygen is greatly limited. However, no glucose is catabolized by resting cells in the absence of molecular oxygen. Isotope and inhibitor studies and assays for key enzymes of the established metabolic routes all indicate that these organisms utilize both the Embden-Meyerhof and hexosemonophosphate pathways for glucose dissimilation. With a concentrated resting-cell suspension, the extent of participation of the latter route was estimated to be as high as 40% in an atmosphere of pure oxygen, and as low as 2% in air. Acetate was oxidized by only one of the cultures of B. popilliae tested, which is apparently a mutant. Cells of this strain from stationary phase cultures oxidized acetate at pH 7.0 or higher, but not at pH 6.0; however, they oxidized succinate, fumarate, and malate more rapidly at pH 6.0 than at 7.0. The oxidation of tricarboxylic acid cycle intermediates, the presence of condensing enzyme in extracts of cells capable of oxidizing acetate, and the complete inhibition of acetate oxidation by arsenite and partial inhibition by malonate all indicate that terminal oxidation of acetate by this strain of B. popilliae is via the tricarboxylic acid cycle.

Acetates↗

Cereulide synthetase gene cluster from emetic Bacillus cereus: structure and location on a mega virulence plasmid related to Bacillus anthracis toxin plasmid pXO1.

BACKGROUND: Cereulide, a depsipeptide structurally related to valinomycin, is responsible for the emetic type of gastrointestinal disease caused by Bacillus cereus. Recently, it has been shown that this toxin is produced by a nonribosomal peptide synthetase (NRPS), but its exact genetic organization and biochemical synthesis is unknown. RESULTS: The complete sequence of the cereulide synthetase (ces) gene cluster, which encodes the enzymatic machinery required for the biosynthesis of cereulide, was dissected. The 24 kb ces gene cluster comprises 7 CDSs and includes, besides the typical NRPS genes like a phosphopantetheinyl transferase and two CDSs encoding enzyme modules for the activation and incorporation of monomers in the growing peptide chain, a CDS encoding a putative hydrolase in the upstream region and an ABC transporter in the downstream part. The enzyme modules responsible for incorporation of the hydroxyl acids showed an unusual structure while the modules responsible for the activation of the amino acids Ala and Val showed the typical domain organization of NRPS. The ces gene locus is flanked by genetic regions with high homology to virulence plasmids of B. cereus, Bacillus thuringiensis and Bacillus anthracis. PFGE and Southern hybridization showed that the ces genes are restricted to emetic B. cereus and indeed located on a 208 kb megaplasmid, which has high similarities to pXO1-like plasmids. CONCLUSION: The ces gene cluster that is located on a pXO1-like virulence plasmid represents, beside the insecticidal and the anthrax toxins, a third type of B. cereus group toxins encoded on megaplasmids. The ces genes are restricted to emetic toxin producers, but pXO1-like plasmids are also present in emetic-like strains. These data might indicate the presence of an ancient plasmid in B. cereus which has acquired different virulence genes over time. Due to the unusual structure of the hydroxyl acid incorporating enzyme modules of Ces, substantial biochemical efforts will be required to dissect the complete biochemical pathway of cereulide synthesis.

Antigens, Bacterial↗

Complete genome sequence of the industrial bacterium Bacillus licheniformis and comparisons with closely related Bacillus species.

BACKGROUND: Bacillus licheniformis is a Gram-positive, spore-forming soil bacterium that is used in the biotechnology industry to manufacture enzymes, antibiotics, biochemicals and consumer products. This species is closely related to the well studied model organism Bacillus subtilis, and produces an assortment of extracellular enzymes that may contribute to nutrient cycling in nature. RESULTS: We determined the complete nucleotide sequence of the B. licheniformis ATCC 14580 genome which comprises a circular chromosome of 4,222,336 base-pairs (bp) containing 4,208 predicted protein-coding genes with an average size of 873 bp, seven rRNA operons, and 72 tRNA genes. The B. licheniformis chromosome contains large regions that are colinear with the genomes of B. subtilis and Bacillus halodurans, and approximately 80% of the predicted B. licheniformis coding sequences have B. subtilis orthologs. CONCLUSIONS: Despite the unmistakable organizational similarities between the B. licheniformis and B. subtilis genomes, there are notable differences in the numbers and locations of prophages, transposable elements and a number of extracellular enzymes and secondary metabolic pathway operons that distinguish these species. Differences include a region of more than 80 kilobases (kb) that comprises a cluster of polyketide synthase genes and a second operon of 38 kb encoding plipastatin synthase enzymes that are absent in the B. licheniformis genome. The availability of a completed genome sequence for B. licheniformis should facilitate the design and construction of improved industrial strains and allow for comparative genomics and evolutionary studies within this group of Bacillaceae.

Anti-Bacterial Agents↗

[Examination of toxin production from environmental Bacillus cereus and Bacillus thuringiensis].

Emetic toxin- and entero toxin-producing activities were examined for 524 strains of Bacillus cereus and 90 strains of Bacillus thuringiensis to determine the distribution and contamination in natural environment, including foods, vegetable, soil and etc. Emetic toxin was assayed by the production of acid from HEp-2 cells induced by the culture supernatants of Bacillus, which was developed as an improved method for the detection of B. cereus heat-stable emetic-toxin by us. In the 524 strains of B. cereus tested, 11 out of 60 strains obtained from bean paste and 5 out of 20 strains obtained from peach produced emetic toxin which was detected by the HEp-2 cell method. All these emetic toxin-producing isolate strains were classified into H1 serotype. The 90 strains of B. thuringiensis did not produce emetic toxin, but the 47 strains produced entero toxin.

Bacillus cereus↗

Lethality of chlorine, chlorine dioxide, and a commercial fruit and vegetable sanitizer to vegetative cells and spores of Bacillus cereus and spores of Bacillus thuringiensis.

Chlorine, ClO2, and a commercial raw fruit and vegetable sanitizer were evaluated for their effectiveness in killing vegetative cells and spores of Bacillus cereus and spores of Bacillus thuringiensis. The ultimate goal was to use one or both species as a potential surrogate(s) for Bacillus anthracis in studies that focus on determining the efficacy of sanitizers in killing the pathogen on food contact surfaces and foods. Treatment with alkaline (pH 10.5 to 11.0) ClO2 (200 microg/ml) produced by electrochemical technologies reduced populations of a five-strain mixture of vegetative cells and a five-strain mixture of spores of B. cereus by more than 5.4 and more than 6.4 log CFU/ml respectively, within 5 min. This finding compares with respective reductions of 4.5 and 1.8 log CFU/ml resulting from treatment with 200 microg/ml of chlorine. Treatment with a 1.5% acidified (pH 3.0) solution of Fit powder product was less effective, causing 2.5- and 0.4-log CFU/ml reductions in the number of B. cereus cells and spores, respectively. Treatment with alkaline ClO2 (85 microg/ml), acidified (pH 3.4) ClO2 (85 microg/ml), and a mixture of ClO2 (85 microg/ml) and Fit powder product (0.5%) (pH 3.5) caused reductions in vegetative cell/spore populations of more than 5.3/5.6, 5.3/5.7, and 5.3/6.0 log CFU/ml, respectively. Treatment of B. cereus and B. thuringiensis spores in a medium (3.4 mg/ml of organic and inorganic solids) in which cells had grown and produced spores with an equal volume of alkaline (pH 12.1) ClO2 (400 microg/ml) for 30 min reduced populations by 4.6 and 5.2 log CFU/ml, respectively, indicating high lethality in the presence of materials other than spores that would potentially react with and neutralize the sporicidal activity of ClO2.

Bacillus cereus↗

Partial purification and characterization of protease enzyme from Bacillus subtilis and Bacillus cereus.

The aim of this experimental study was to isolate and partially purify protease enzyme from Bacillus cereus and Bacillus subtilis. Protease enzyme is obtained by inducing spore genesis of bacteria from Bacillus species in suitable nutrient plates. The partial purification was realized by applying, respectively, ammonium sulfate precipitation, dialysis, and DEAE-cellulose ion-exchange chromatography to the supernatant that was produced later. Optimum pH, optimum temperature, pH stability, and temperature stability were determined, as well as the effects of pH, temperature, substrate concentration, reaction time, and inhibitors and activators on enzyme activity. In addition, the molecular mass of the obtained enzyme was investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The specific activity of partially purified enzyme from B. subtilis was determined to be 84 U/mg. The final enzyme preparation was eight-fold more pure than the crude homogenate. The molecular mass of the partially purified enzyme was found to be 45 kDa by using SDS-PAGE. The protease enzyme that was partially purified from B. cereus was purified 1.2-fold after ammonium sulfate precipitation. The molecular mass of the partially purified enzyme was determined to be 37 kDa by using SDS-PAGE.

Bacillus cereus↗

[Structural-functional characteristics of Bacillus mesentericus promoters cloned in Bacillus subtilis cells].

Bacillus mesentericus DNA fragments carrying promoters were cloned in Bacillus subtilis. The nucleotide sequences of four promoters, the sites of transcription initiation and the levels of promoter-mediated expression of pPL603 CAT gene at different stages of cell growth were determined. It was identified, that the two promoters (P435 and P442) are the typical sigma 43-RNA polymerase promoters. Promoter P462 possessed the unusual nucleotide sequence and induced the expression of indicator CAT gene at the middle of the stationary phase of cell growth. The next P428 promoter was found to be a complex promoter composed of three overlapping ones: P428-1, P428-2 and P428-3. P428-3 is the sigma 43-specific promoter which revealed the high degree of homology with Bacillus subtilis spoOB gene promoter. We suggest the promoters P428-1 and P428-2 to be sigma 43- and sigma 37-RNA polymerase specific, respectively.

Bacillus↗

Efficacy of Bacillus sphaericus and Bacillus thuringiensis var. israelensis for control of Culex pipiens and floodwater Aedes larvae in Iowa.

Granular and flowable concentrate formulations of Bacillus thuringiensis var. israelensis provided a 90-100% reduction in Aedes vexans and Culex spp. larvae in natural larval habitats. A briquet formulation of B. thuringiensis was less effective, providing a 12-76% reduction. No residual activity occurred in sites treated with B. thuringiensis. Granular formulations of Bacillus sphaericus (2.78-8.42 kg/ha) caused a 100% reduction in Culex pipiens larvae in natural sites and artificial pools. Bacillus sphaericus also controlled (84-98% reduction) a mixed population of Aedes trivittatus and Cx. pipiens in subplots of a retention pond. In field sites, B. sphaericus continued to control Cx. pipiens larvae for 96 hr.

Aedes↗

Processing of Bacillus cereus 569/H beta-lactamase I in Escherichia coli and Bacillus subtilis.

The gene for Bacillus cereus 569/H beta-lactamase I, penPC, has recently been cloned and sequenced (Mézes, P. S. F., Yang, Y. Q., Hussain, M., and Lampen, J. O. (1983) FEBS Lett. 161, 195-200). A typical prokaryotic signal peptide but with no lipoprotein modification site, as present in the Bacillus licheniformis 749/C beta-lactamase, was indicated by the DNA sequence for this secretory protein. We have here purified the beta-lactamase I products found in Escherichia coli and Bacillus subtilis carrying penPC and have determined the first 20 NH2-terminal amino acids of each of the forms. Processing of the beta-lactamase I in E. coli occurs at a single site which is characteristic for cleavage by a signal peptidase. B. subtilis secreted two distinct products to the culture medium which were both smaller than the single product formed in E. coli. Sequencing of [35S]Met-labeled pre-beta-lactamase I from phenylethyl alcohol-treated cells of B. cereus 569/H indicated that UUG is being utilized as the initiation codon for penPC. The same result was obtained for the pre-beta-lactamase I from similarly treated cells of the closely related B. cereus 5/B strain.

Amino Acid Sequence↗

Integrated management of waste tire mosquitoes utilizing Mesocyclops longisetus (Copepoda: Cyclopidae), Bacillus thuringiensis var. israelensis, Bacillus sphaericus, and methoprene.

This study evaluated the compatibility and efficacy of using a predatory copepod, Mesocyclops longisetus in concert with 3 "biorational" compounds for mosquito control in waste tires. The toxicity of Bacillus thuringiensis var. israelensis (B.t.i), Bacillus sphaericus, and methoprene to Mesocyclops longisetus was assessed in the laboratory using concentrations 10 times the maximum labeled or suggested rate and based on a water depth of 7.6 cm. Microbials were tested using mature copepods exposed for durations of 24, 48, and 72 h. Methoprene bioassays consisted of individually exposing newly hatched copepods (i.e., nauplius larvae) and monitoring their development to maturity. The toxicity tests indicated B.t.i., B. sphaericus, and methoprene were not deleterious to copepods at concentrations exceeding those expected in the field. Copepods exposed to methoprene matured normally, and when mated, 50% developed egg sacs. A 5-month field test, integrating the copepod and B.t.i., B. sphaericus, and methoprene provided better mosquito reduction together than either copepods or control agents alone. When copepods were combined with B.t.i. or methoprene, overall reduction of 3rd- and 4th-instar larvae during the 5-month interval was equal to or greater than 90%. Bacillus thuringiensis var. israelensis alone temporarily produced a high degree of larval reduction (up to 100%), however reapplications were necessary to maintain that level of control. Of all the treatments, B. sphaericus alone produced the lowest degree of mosquito suppression due to lack of toxicity to Aedes albopictus, the predominant species during the study. It is recommended that mosquito control managers consider integrating M. longisetus and B.t.i. or methoprene against mosquitoes in waste tires.

Aedes↗

[Extrachromosomal DNA spectrum of Bacillus thuringiensis Berliner and Bacillus sphaericus Meyer et Neide].

Analysis of the pattern of extrachromosomal DNA in different cultures of Bacillus thuringiensis and Bacillus sphaericus demonstrated a higher content of extrachromosomal DNA in B. thuringiensis than in B. sphaericus. The quantity and approximate molecular weights of the plasmids were determined. The assumption that the plasmid DNA content in B. thuringiensis strains is higher than in the other representatives of the genus Bacillus was confirmed.

Bacillus↗

Differentiation of species in the Bacillus brevis group and the Bacillus aneurinolyticus group based on the electrophoretic whole-cell protein pattern.

Ninety strains of eleven Bacillus species in the Bacillus brevis group and the Bacillus aneurinolyticus group were compared with the electrophoretic whole-cell protein pattern. The strains were separated into two clusters at the similarity of 55%. One cluster (cluster 1) was consisted of strains from the B. brevis group, and another cluster (cluster 2) was composed of strains from the B. aneurinolyticus group. The cluster 1 was separated into eight subclusters. Out of eight subclusters, seven subclusters contain strains from B. brevis, B. laterosporus, B. agri, B. reuszeri, B. choshinensis, B. formosus, and B. borstelensis. Another subcluster was further separated into two related clusters, which corresponded to B. centrosporus and B. parabrevis, and they were fused at the similarity of 76%. Cluster 2 was separated into two subclusters, which corresponded to B. aneurinolyticus and B. migulanus. The above eleven species showed characteristic patterns distinct from one another, and this correlated well with the published DNA relatedness data. The comparison of the electrophoretic whole-cell protein pattern proved to be useful for evaluation of taxonomic relationships among these taxa.

Bacillus↗

On the appearance of Bacillus subtilis intracellular serine protease in the cell membrane and culture medium. Comparison of the enzyme and other Bacillus subtilis serine proteases.

While about 80% of the cell-bound intracellular serine protease of Bacillus subtilis A-50 have been recovered in the soluble fraction upon disruption of cells, the rest of the enzyme was found to be associated with the membrane fraction. Soluble cytoplasmic intracellular serine protease, as well as membrane-bound serine protease liberated by non-ionic detergent treatment, have been isolated in a pure state and shown to be identical. The same protease might also be found extracellularly, due presumably to cell lysis or altered membrane permeability. Intracellular serine protease of Bacillus subtilis A-50 was clearly related to Bacillus subtilis serine proteases W1 and bacillopeptidase F described as extracellular enzymes.

Amino Acids↗

Bacillus arsenicoselenatis, sp. nov., and Bacillus selenitireducens, sp. nov.: two haloalkaliphiles from Mono Lake, California that respire oxyanions of selenium and arsenic.

Two gram-positive anaerobic bacteria (strains E1H and MLS10) were isolated from the anoxic muds of Mono Lake, California, an alkaline, hypersaline, arsenic-rich water body. Both grew by dissimilatory reduction of As(V) to As(III) with the concomitant oxidation of lactate to acetate plus CO2. Bacillus arsenicoselenatis (strain E1H) is a spore-forming rod that also grew by dissimilatory reduction of Se(VI) to Se(IV). Bacillus selenitireducens (strain MLS10) is a short, non-spore-forming rod that grew by dissimilatory reduction of Se(IV) to Se(0). When the two isolates were cocultured, a complete reduction of Se(VI) to Se(0) was achieved. Both isolates are alkaliphiles and had optimal specific growth rates in the pH range of 8.5-10. Strain E1H had a salinity optimum at 60 g l-1 NaCl, while strain MLS10 had optimal growth at lower salinities (24-60 g l-1 NaCl). Both strains have limited abilities to grow with electron donors and acceptors other than those given above. Strain MLS10 demonstrated weak growth as a microaerophile and was also capable of fermentative growth on glucose, while strain E1H is a strict anaerobe. Comparative 16S rRNA gene sequence analysis placed the two isolates with other Bacillus spp. in the low G+C gram-positive group of bacteria.

Anaerobiosis↗

Revision of the taxonomic position of the xylanolytic Bacillus sp. MIR32 reidentified as Bacillus halodurans and plasmid-mediated transformation of B. halodurans.

Bacillus sp. MIR32 has been isolated using xylan as the only carbon source, and one of its xylanolytic enzymes has been extensively studied. Biochemical analysis first related this strain to Bacillus amyloliquefaciens, but further studies based on a comparison of 16S rDNA sequences, G+C content, and DNA-DNA hybridization showed that strain MIR32 should be classified as a member of the species Bacillus halodurans. This change is also supported by the typical phenotype observed and by the results of PCR amplification directed toward spacers in rDNA and tDNA genes, which were assayed and compared with those of B. halodurans DSM 497(T). Although among alkaliphilic bacilli competence development has not been experimentally demonstrated, in this work both B. halodurans MIR32 and DSM 497(T) were transformed according to a simple procedure developed in our laboratory, reaching 10(2)-10(3) stable transformants per microgram of plasmid DNA.

Bacillus↗

Reidentification of the keratinase-producing facultatively alkaliphilic Bacillus sp. AH-101 as Bacillus halodurans.

Alkaliphilic Bacillus sp. AH-101 was characterized in terms of physiological and biochemical characteristics, and 16S rDNA sequence homology and DNA-DNA hybridization analyses were performed. Phylogenetic analysis of strain AH-101 based on comparison of 16S rDNA sequences revealed that this strain is closely related to Bacillus halodurans. DNA-DNA hybridization of AH-101 and related Bacillus reference strains showed that the highest level of DNA-DNA relatedness (88%) was found between strain AH-101 and the B. halodurans type strain (DSM497). Our findings demonstrate that strain AH-101 is a member of the species B. halodurans.

Bacillus↗

Analyses of a Bacillus subtilis homologue of the Na+/H+ antiporter gene which is important for pH homeostasis of alkaliphilic Bacillus sp. C-125.

Bacillus subtilis was revealed to have a homologous region to the DNA fragment responsible for alkaliphily of alkaliphilic Bacillus sp. C-125 on the genome, as reported previously [1]. The yufT gene on the B. subtilis genome showed a significant similarity with ORF1 of Bacillus sp. C-125, which is related to membrane potential (DeltaPsi)-driven Na+/H+ antiport activity and is important for pH homeostasis in an alkaline condition. Disruption of the yufT gene resulted in the decrease of Na+/H+ antiport activity, and the growth of the yufT disrupted strain was impaired with an increase in the external Na+ concentration. We conclude that the yufT gene encodes a Na+/H+ antiporter, which has a dominant role in the extrusion of cytotoxic Na+.

Bacillus subtilis↗

Bacillus aeolius sp. nov. a novel thermophilic, halophilic marine Bacillus species from Eolian Islands (Italy).

Phylogenetic relationships of a thermophilic, halophilic, aerobic spore-forming strain 4-1(T), isolated from the water of a shallow sea hot spring at Vulcano Island (Italy), revealed its relatedness to members of the genus Bacillus. Chemotaxonomic and phenotypic properties of strain 4-1(T) are sufficiently different from related moderately thermophilic species, e.g., B. smithii, B. fumarioli, B. oleronius, B. sporothermodurans and B. infernus to describe strain 4-1(T) as a new Bacillus species, for which the name Bacillus aeolius sp. nov. is proposed. Strain 4-1(T) is characterised by the potential biotechnological important properties such as exopolysaccharide production, surfactant activity, and utilisation of hydrocarbons.

Aerobiosis↗