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PubMed2002

Two novel psychrotolerant species, Bacillus psychrotolerans sp. nov. and Bacillus psychrodurans sp. nov., which contain ornithine in their cell walls.

Eleven psychrotolerant Bacillus strains with ornithine as diamino acid in position 3 of the peptide side chain of the cell wall and a G+C range of 35.7-38.4 mol% were characterized taxonomically. DNA-DNA hybridization studies confirmed previously physiologically established groups. High DNA-binding values (> 70%) were found within groups I A (consisting of the type strain of Bacillus insolitus DSM 5(T) and Bacillus insolitus DSM 2272), I B (consisting of isolates 3H1(T0, 71H1, 84E1, 87H2 and 4H2) and I C (consisting of isolates 68E39T), 61E1, 4E3 and 67E1). Low DNA-binding values (< 60%) were revealed between the three groups. Consequently, strains of groups I B and I C were considered as being representatives of new psychrotolerant species. For group I B strains the name Bacillus psychrotolerans sp. nov. is proposed with the type strain 3H1(T) (= DSM 11706(T) = NCIMB 13838(T)) and for group I C strains the name Bacillus psychrodurans sp. nov. is proposed with the type strain 68E3(T) (= DSM 11713(T) = NCIMB 13837(T)).

Bacillus↗

PubMed2003

Bacillus marisflavi sp. nov. and Bacillus aquimaris sp. nov., isolated from sea water of a tidal flat of the Yellow Sea in Korea.

Two Gram-positive or Gram-variable, endospore-forming, moderately halophilic rods (strains TF-11(T) and TF-12(T)) were isolated from a tidal flat of the Yellow Sea in Korea and were subjected to a polyphasic taxonomic study. Strains TF-11(T) and TF-12(T) had cell-wall peptidoglycan based on meso-diaminopimelic acid. The predominant menaquinone found in the two strains was MK-7. The cellular fatty acid profiles of both strains contained large amounts of branched and saturated fatty acids. The major fatty acids were anteiso-C(15 : 0) and iso-C(15 : 0). The DNA G+C contents of strains TF-11(T) and TF-12(T) were respectively 49 and 38 mol%. Phylogenetic analysis based on 16S rDNA sequences showed that strains TF-11(T) and TF-12(T) fell within the radiation of the cluster comprising Bacillus species. The level of 16S rDNA sequence similarity between strains TF-11(T) and TF-12(T) was 98.3 %. Strains TF-11(T) and TF-12(T) exhibited levels of 16S rDNA sequence similarity of less than 96.0 and 96.3 %, respectively, to Bacillus species. The mean level of DNA-DNA relatedness between the two strains was approximately 7 %. On the basis of phenotypic and phylogenetic data and genomic distinctiveness, strains TF-11(T) and TF-12(T) should be placed in the genus Bacillus as two distinct novel species, for which the names Bacillus marisflavi sp. nov. (type strain TF-11(T)=KCCM 41588(T)=JCM 11544(T)) and Bacillus aquimaris sp. nov. (type strain TF-12(T)=KCCM 41589(T)=JCM 11545(T)) are proposed.

Bacillus↗

PubMed2004

Bacillus odysseyi sp. nov., a round-spore-forming bacillus isolated from the Mars Odyssey spacecraft.

A round-spore-forming Bacillus species that produces an exosporium was isolated from the surface of the Mars Odyssey spacecraft. This novel species has been characterized on the basis of phenotypic traits, 16S rDNA sequence analysis and DNA-DNA hybridization. According to the results of these analyses, this strain belongs to the genus Bacillus and is a Gram-positive, aerobic, rod-shaped, endospore-forming eubacterium. Ultrathin sections of the spores showed the presence of an exosporium, spore coat, cortex and core. 16S rDNA sequence similarities between this strain, Bacillus fusiformis and Bacillus silvestris were approximately 96% and DNA-DNA reassociation values with these two bacilli were 23 and 17%, respectively. Spores of the novel species were resistant to desiccation, H2O2 and UV and gamma radiation. Of all strains tested, the spores of this strain were the most consistently resistant and survived all of the challenges posed, i.e. exposure to conditions of desiccation (100% survival), H2O2 (26% survival), UV radiation (10% survival at 660 J m(-2)) and gamma radiation (0.4% survival). The name proposed for this novel bacterium is Bacillus odysseyi sp. nov.; the type strain is 34hs-1T (=ATCC PTA-4993T=NRRL B-30641T=NBRC 100172T).

Bacillus↗

PubMed1990

Presence of N6-methyladenine in GATC sequences of Bacillus popilliae and Bacillus lentimorbus KLN2.

Nine strains of Bacillus popilliae and Bacillus lentimorbus KLN2 contain N6-methyladenine in GATC sequences, as determined by using the restriction enzymes MboI and DpnI. Among eight other Bacillus species examined, all, except one strain of Bacillus brevis (ATCC 9999), lacked adenine methylation in GATC. A methylase with Escherichia coli dcm site specificity was not present in any of the Bacillus species studied.

Adenine↗

PubMed1984

Differentiation of Bacillus anthracis and other Bacillus species by lectins.

Bacillus anthracis was agglutinated by several lectins, including those from Griffonia simplicifolia, Glycine max, Abrus precatorius, and Ricinus communis. Some strains of Bacillus cereus var. mycoides (B. mycoides) were strongly reactive with the lectin from Helix pomatia and weakly reactive with the G. max lectin. The differential interactions between Bacillus species and lectins afforded a means of distinguishing B. anthracis from other bacilli. B. cereus strains exhibited heterogeneity with respect to agglutination patterns by lectins but could readily be differentiated from B. anthracis and the related B. mycoides. Spores of B. anthracis and B. mycoides retained lectin receptors, although the heating of spores or vegetative cells at 100 degrees C resulted in a decrease in their ability to be specifically agglutinated. Fluorescein-conjugated lectin of G. max stained vegetative cells of B. anthracis uniformly, suggesting that the distribution of lectin receptors was continuous over the entire cellular surface. B. anthracis cells grown under conditions to promote the production of capsular poly(D-glutamyl peptide) were also readily agglutinated by the lectins, suggesting that the lectin reactive sites penetrate the polypeptide layer. Trypsin, subtilisin, lysozyme, and mutanolysin did not modify the reactivity of B. anthracis with the G. max agglutinin, although the same enzymes markedly diminished the interaction between the lectin and B. mycoides. Because the lectins which interact with B. anthracis are specific for alpha-D-galactose or 2-acetamido-2-deoxy-alpha-D-galactose residues, it is likely that the bacteria possess cell surface polymers which contain these sugars. Lectins may prove useful in the laboratory identification of B. anthracis and possibly other pathogenic Bacillus species, such as B. cereus.

Agglutination Tests↗

PubMed2003

Detection of anthrax simulants with microcalorimetric spectroscopy: Bacillus subtilis and Bacillus cereus spores.

Recent advances in the development of ultrasensitive micromechnical thermal detectors have led to the advent of novel subfemtojoule microcalorimetric spectoscopy (CalSpec). On the basis of principles of photothermal IR spectroscopy combined with efficient thermomechanical transduction, CalSpec provides acquisition of vibrational spectra of microscopic samples and absorbates. We use CalSpec as a method of identifying nanogram quantities of biological micro-organisms. Our studies focus on Bacillus subtilis and Bacillus cereus spores as simulants for Bacillus anthracis spores. Using CalSpec, we measured IR spectra of B. subtilis and B. cereus spores present on surfaces in nanogram quantities (approximately 100-1,000 spores). The spectra acquired in the wavelength range of 690-4000 cm(-1) (2.5-14.5 microm) contain information-rich vibrational signatures that reflect the different ratios of biochemical makeup of the micro-organisms. The distinctive features in the spectra obtained for the two types of microorganism can be used to distinguish between the spores of the Bacillus family. As compared with conventional IR and Fourier-transform IR microscopic spectroscopy techniques, the advantages of the present technique include significantly improved sensitivity (at least a full order of magnitude), absence of expensive IR detectors, and excellent potential for miniaturization.

Anthrax↗

PubMed2000

Prevalence and expression of enterotoxins in Bacillus cereus and other Bacillus spp., a literature review.

Members of the Bacillus genus are ubiquitous soil microorganisms and are generally considered harmless contaminants. However, a few species are known toxin producers, including the foodborne pathogen, B. cereus. This species produces two distinct types of foodborne illness, the emetic (vomit-inducing) syndrome, associated with consumption of toxin in cooked rice dishes, and the diarrheal illness seen occasionally following consumption of contaminated meats, sauces, and certain dairy products. In the latter case, illness results from the production of enterotoxins by vegetative cells in the small intestine of the host. In dairy products, the occurrence of Bacillus spp. is inevitable, and the spore-forming ability of this organism allows it to easily survive pasteurization. Many strains have been shown to grow and produce enterotoxin in dairy products at refrigeration temperatures. Evaluation of toxin gene presence and toxin expression in Bacillus spp. other than B. cereus has not been thoroughly investigated. However, the presence of natural isolates of Bacillus spp. harboring one or more enterotoxin gene(s) and subsequent demonstration of conditions which may support toxin expression holds crucial importance in the food safety arena.

Bacillus↗

PubMed1994

Cloning and expression in Bacillus subtilis of the npr gene from Bacillus thermoproteolyticus Rokko coding for the thermostable metalloprotease thermolysin.

We report the isolation, cloning and expression, in Bacillus subtilis, of the gene coding for thermolysin, a thermostable metalloprotease which is produced by Bacillus thermoproteolyticus Rokko. The nucleotide sequence has revealed that, like neutral proteases produced by other members of the Bacillus species, thermolysin is probably produced as a preproenzyme carrying a typical N-terminal membrane signal sequence. Further, the thermolysin gene shares a strong homology with two other previously cloned genes from two different strains of Bacillus stearothermophilus. The sequence of the mature secreted protease, inferred from the DNA sequence, is, with two exceptions, identical with the previously published protein sequence of thermolysin [Titani, Hermodson, Ericsson, Walsh and Neurath (1972) Nature (London) 238, 35-37]. The exceptions are Asn37 and Gln119, originally reported to be Asp and Glu respectively. The biochemical characterization of the secreted recombinant protein shows that it is indistinguishable from the wild-type thermolysin.

Amino Acid Sequence↗

PubMed2003

Degradation of proteins during the fermentation of African locust bean (Parkia biglobosa) by strains of Bacillus subtilis and Bacillus pumilus for production of Soumbala.

AIMS: To examine isolates of Bacillus subtilis and B. pumilus predominant in Soumbala for their ability to degrade African locust bean proteins (ALBP). METHODS AND RESULTS: Agar diffusion test in casein and ALBP agar was used for screening of isolates. The profiles of water-soluble proteins and free amino acids (FAA) during the fermentation of ALBP by the Bacillus isolates were studied by SDS-PAGE and cation exchange chromatography. The profile of soluble proteins changed with the fermentation time and varied depending on the isolate. The quantity of total FAA and essential FAA such as lysine was increased sharply between 24 and 48 h of fermentation and differed among the isolates. Simultaneously, a pH increase was observed. Cysteine, methionine, leucine, isoleucine, tyrosine and phenylalaline appeared during fermentation. CONCLUSION: The Bacillus isolates studied degraded ALBP leading to a profile of soluble proteins and FAA specific for each isolate. SIGNIFICANCE AND IMPACT OF THE STUDY: This study contributes to the selection of Bacillus strains to be used as starter cultures for controlled production of Soumbala.

Amino Acids↗

PubMed2005

Peptidoglycan N-acetylglucosamine deacetylases from Bacillus cereus, highly conserved proteins in Bacillus anthracis.

The genomes of Bacillus cereus and its closest relative Bacillus anthracis contain 10 polysaccharide deacetylase homologues. Six of these homologues have been proposed to be peptidoglycan N-acetylglucosamine deacetylases. Two of these genes, namely bc1960 and bc3618, have been cloned and expressed in Escherichia coli, and the recombinant enzymes have been purified to homogeneity and further characterized. Both enzymes were effective in deacetylating cell wall peptidoglycan from the Gram(+) Bacillus cereus and Bacillus subtilis and the Gram(-) Helicobacter pylori as well as soluble chitin substrates and N-acetylchitooligomers. However, the enzymes were not active on acetylated xylan. These results provide insight into the substrate specificity of carbohydrate esterase family 4 enzymes. It was revealed that both enzymes deacetylated only the GlcNAc residue of the synthetic muropeptide N-acetyl-D-glucosamine-(beta-1,4)-N-acetylmuramyl-L-alanine-D-isoglutamine. Analysis of the constituent muropeptides of peptidoglycan from B. subtilis and H. pylori resulting from incubation of the enzymes BC1960 and BC3618 with these polymers and subsequent hydrolysis by Cellosyl and mutanolysin, respectively, similarly revealed that both enzymes deacetylate GlcNAc residues of peptidoglycan. Kinetic analysis toward GlcNAc(2-6) revealed that GlcNAc4 was the favorable substrate for both enzymes. Identification of the sequence of N-acetychitooligosaccharides (GlcNAc(2-4)) following enzymatic deacetylation by using 1H NMR revealed that both enzymes deacetylate all GlcNAc residues of the oligomers except the reducing end ones. Enzymatic deacetylation of chemically acetylated vegetative peptidoglycan from B. cereus by BC1960 and BC3618 resulted in increased resistance to lysozyme digestion. This is the first biochemical study of bacterial peptidoglycan N-acetylglucosamine deacetylases.

Acetylglucosamine↗

PubMed2005

Synthesis of additional endotoxins in Bacillus thuringiensis subsp. morrisoni PG-14 and Bacillus thuringiensis subsp. jegathesan significantly improves their mosquitocidal efficacy.

A fundamental principal of resistance management is that the more complex and potent a toxin mixture, the slower resistance will develop to the mixture in an insect population. Thus, to develop more complex and potent mosquitocidal bacteria, we genetically engineered Bacillus thuringiensis subsp. morrisoni PG-14 and Bacillus thuringiensis subsp. jegathesan, to synthesize, respectively, the binary (Bin) toxin of Bacillus sphaericus or a combination of Bin and the CytlA protein of Bacillus thuringiensis subsp. israelensis. Engineering these two larvicidal bacteria in general significantly improved their efficacy against fourth instars in comparison with their wild-type parental strains. For B. thuringiensis subsp. morrisoni PG-14, which naturally synthesizes Cyt1A, synthesis of Bin improved efficacy nine-fold (LC50 from 4.5 to 0.5 ng/ml) against Culex quinquefasciatus Say, although no improvement was observed (LC50 of 2 ng/ml for both strains) against Aedes aegypti L. For B. thuringiensis subsp. jegathesan, cosynthesis of Bin plus Cyt1A in combination with its normal complement of endotoxins improved efficacy 17-fold (LC50 from 34 to 2 ng/ml) against Cx. quinquefasciatus and 3.2-fold (LC50 from 68 to 21 ng/ml) against Ae. aegypti. Addition of Bin alone to B. thuringiensis subsp. jegathesan did not improve toxicity (LC50 from 68 to 65 ng/ml) against Ae. aegypti, indicating that CytlA synergized the activity of the endotoxins in this strain against Ae. aegypti. These results demonstrate that mosquitocidal efficacy of these strains and likely their resistance management properties can be improved significantly by increasing their toxin complexity and the amount of toxin they synthesize.

Animals↗

PubMed1991

Comparative analysis of Bacillus anthracis, Bacillus cereus, and related species on the basis of reverse transcriptase sequencing of 16S rRNA.

The primary structures of the 16S rRNAs of Bacillus anthracis, Bacillus cereus, Bacillus mycoides, and Bacillus thuringiensis were determined by using the reverse transcription-dideoxy sequencing method. All of the strains exhibited very high levels of sequence similarity (greater than 99%) that were consistent with the close relationships shown by previous DNA hybridization studies. The sequences of B. anthracis Sterne and B. cereus emetic strain NCTC 11143 were found to be identical for a continuous stretch of 1,446 bases and differed from the sequence of B. cereus NCDO 1771T (T = type strain) by only a single nucleotide. The 16S rRNA sequences of B. mycoides and B. thuringiensis differed from each other and from the sequences of B. anthracis and B. cereus by four to nine nucleotides.

Bacillus anthracis↗

PubMed1994

Differentiation of Bacillus anthracis from other Bacillus cereus group bacteria with the PCR.

Variation among isolates of Bacillus anthracis was examined by using restriction fragmentation patterns and the PCR performed with arbitrary and sequence-specific oligonucleotide primers. The patterns were compared with the patterns generated from strains of closely related species belonging to the "Bacillus cereus group" of bacteria, including B. cereus, Bacillus thuringiensis, and Bacillus mycoides. All B. anthracis profiles were identical for each of 18 restriction enzymes, each of 10 arbitrary PCR primers, and a repetitive extragenic palindrome-specific PCR primer. The PCR profiles generated with a coliphage M13-based primer exhibited slight pattern variation in a 400- to 500-bp band region. The B. anthracis profiles were unique compared with the profiles of the other species examined. In these other species, strain-to-strain variations were observed. Our results showed that isolates of B. anthracis are almost completely homogeneous, indicating a clonal lineage, and are distinct from other members of the B. cereus group and that B. anthracis, as a species in its own right, may have evolved only relatively recently.

Bacillus anthracis↗

PubMed1983

Comparative studies on extracellular penicillinases of the same structural gene, penP, expressed in Bacillus licheniformis and Bacillus subtilis.

Extracellular penicillinases produced by Bacillus licheniformis ATCC 9945A and Bacillus subtilis from the same structural gene, penP, were compared. The two strains secreted the same exo-large penicillinase (mol. wt, 305000; isoelectric point, pI = 5.00-5.04; NH2-terminal amino acid, Ser). In contrast, the exo-small enzyme from Bacillus subtilis (mol. wt, 29500; pI = 5.00-5.04; NH2-terminal amino acid, Glu or Asn) was slightly different from that of Bacillus licheniformis (mol. wt, 29500; pI = 5.13; NH2-terminal amino acid, Lys). The difference in the NH2-terminal residue is most probably due to differences in degradation by host-specific proteolytic enzymes.

Amino Acid Sequence↗

PubMed2025

Fast-growing Bacillus sensu lato rhizosphere populations are constrained by antagonistic Pseudomonadota, Actinomycetota and other Bacillus sensu lato.

Copiotrophic Bacillus and related taxa grow rapidly and are commonly isolated from soil. Despite their growth rate, Bacillus sensu lato (BSL) constitute less than one percent of soil bacterial communities, and the nutrient-enriched rhizosphere contains even fewer. Amendment of bulk soil with synthetic root exudate did not lead to increase in Bacillus culturable counts. We hypothesized that BSL populations in soil enriched with growth-supporting carbon are suppressed by various soil microbes. A screen using B. pseudomycoides as tester strain yielded 124 growth inhibiting isolates, aligning by 16S rRNA genes to 3 Alphaproteobacteria, 6 Betaproteobacteria, 5 Gammaproteobacteria, 3 Streptomyces, and 19 Bacillaceae. Most antagonists also suppressed four other BSL, and over 70% of the BSL isolates suppressed each other. The 11 sequenced BSL genomes encoded between 2 and 10 antibiotic biosynthetic gene clusters. Incubation of multiple isolates in artificial soil microcosms resulted in population growth restraint through a high percentage of endospores formed. This indicated that growth suppression by antagonists was due primarily to induction of sporulation. These results support our hypothesis that Bacillus populations in soil enriched with growth-supporting carbon are suppressed by various soil microbes.

Bacillus↗

PubMed2006

Structure-activity relationships of Bacillus cereus and Bacillus anthracis dihydrofolate reductase: toward the identification of new potent drug leads.

New and improved therapeutics are needed for Bacillus anthracis, the etiological agent of anthrax. To date, antimicrobial agents have not been developed against the well-validated target dihydrofolate reductase (DHFR). In order to address whether DHFR inhibitors could have potential use as clinical agents against Bacillus, 27 compounds were screened against this enzyme from Bacillus cereus, which is identical to the enzyme from B. anthracis at the active site. Several 2,4-diamino-5-deazapteridine compounds exhibit submicromolar 50% inhibitory concentrations (IC(50)s). Four of the inhibitors displaying potency in vitro were tested in vivo and showed a marked growth inhibition of B. cereus; the most potent of these has MIC(50) and minimum bactericidal concentrations at which 50% are killed of 1.6 mug/ml and 0.09 mug/ml, respectively. In order to illustrate structure-activity relationships for the classes of inhibitors tested, each of the 27 inhibitors was docked into homology models of the B. cereus and B. anthracis DHFR proteins, allowing the development of a rationale for the inhibition profiles. A combination of favorable interactions with the diaminopyrimidine and substituted phenyl rings explains the low IC(50) values of potent inhibitors; steric interactions explain higher IC(50) values. These experiments show that DHFR is a reasonable antimicrobial target for Bacillus anthracis and that there is a class of inhibitors that possess sufficient potency and antibacterial activity to suggest further development.

4-Aminopyridine↗

PubMed2002

Genetic differentiation between sympatric populations of Bacillus cereus and Bacillus thuringiensis.

Little is known about genetic exchanges in natural populations of bacteria of the spore-forming Bacillus cereus group, because no population genetics studies have been performed with local sympatric populations. We isolated strains of Bacillus thuringiensis and B. cereus from small samples of soil collected at the same time from two separate geographical sites, one within the forest and the other at the edge of the forest. A total of 100 B. cereus and 98 B. thuringiensis strains were isolated and characterized by electrophoresis to determine allelic composition at nine enzymatic loci. We observed genetic differentiation between populations of B. cereus and B. thuringiensis. Populations of a given Bacillus species--B. thuringiensis or B. cereus--were genetically more similar to each other than to populations of the other Bacillus species. Hemolytic activity provided further evidence of this genetic divergence, which remained evident even if putative clones were removed from the data set. Our results suggest that the rate of gene flow was higher between strains of the same species, but that exchanges between B. cereus and B. thuringiensis were nonetheless possible. Linkage disequilibrium analysis revealed sufficient recombination for B. cereus populations to be considered panmictic units. In B. thuringiensis, the balance between clonal proliferation and recombination seemed to depend on location. Overall, our data indicate that it is not important for risk assessment purposes to determine whether B. cereus and B. thuringiensis belong to a single or two species. Assessment of the biosafety of pest control based on B. thuringiensis requires evaluation of the extent of genetic exchange between strains in realistic natural conditions.

Alleles↗

PubMed2000

Expression of the neutral protease gene from a thermophilic Bacillus sp. BT1 strain in Bacillus subtilis and its natural host: identification of a functional promoter.

The expression of the neutral protease gene (npr) from the thermophilic Bacillus sp. BT1 strain was studied in its natural host and in mesophilic Bacillus subtilis. In the thermophilic BT1 strain, the transcription of the protease gene is initiated from its own promoter, just 5' to the gene. In contrast, in heterologous B. subtilis this thermophilic npr promoter does not function, and expression of the npr gene results from transcription originating upstream of an adjacent gene, open reading frame X (ORF X). A functional promoter was identified 5' to ORF X that is required for efficient expression of the npr gene in Bacillus subtilis as verified by primer extension, reverse transcription-PCR, and 5' rapid amplification of cDNA ends experiments. These data suggest that transcriptional signals used in thermophilic Bacillus sp. BT1 strain are different from those used in B. subtilis.

Bacillus↗