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Transfer of Bacillus alginolyticus, Bacillus chondroitinus, Bacillus curdlanolyticus, Bacillus glucanolyticus, Bacillus kobensis, and Bacillus thiaminolyticus to the genus Paenibacillus and emended description of the genus Paenibacillus.

We determined the taxonomic status of six Bacillus species (Bacillus alginolyticus, Bacillus chondroitinus, Bacillus curdlanolyticus, Bacillus glucanolyticus, Bacillus kobensis, and Bacillus thiaminolyticus) by using the results of 16S rRNA gene sequence and cellular fatty acid composition analyses. Phylogenetic analysis clustered these species closely with the Paenibacillus species. Like the Paenibacillus species, the six Bacillus species contained anteiso-C15:0 fatty acid as a major cellular fatty acid. The use of a specific PCR primer designed for differentiating the genus Paenibacillus from other members of the Bacillaceae showed that the six Bacillus species had the same amplified 16S rRNA gene fragment as members of the genus Paenibacillus. Based on these observations and other taxonomic characteristics, the six Bacillus species were transferred to the genus Paenibacillus. In addition, we propose emendation of the genus Paenibacillus.

Bacillus↗

Study of mural painting isolates, leading to the transfer of 'Bacillus maroccanus' and 'Bacillus carotarum' to Bacillus simplex, emended description of Bacillus simplex, re-examination of the strains previously attributed to 'Bacillus macroides' and description of Bacillus muralis sp. nov.

A group of 24 strains was isolated from deteriorated mural paintings situated in Spain (necropolis of Carmona) and Germany (church of Greene-Kreiensen). (GTG)5-PCR genomic fingerprinting was performed on these strains to assess their genomic variability and the strains were delineated into four groups. Representatives were studied by 16S rRNA gene sequencing and were found to be closely related to Bacillus simplex and the species 'Bacillus macroides' (strain NCIMB 8796) and 'Bacillus maroccanus' (names not validly published) according to a fasta search. The close similarity between B. simplex, 'B. macroides' NCIMB 8796, 'B. maroccanus' and the mural painting isolates was confirmed by additional (GTG)5-PCR, ARDRA, FAME and SDS-PAGE analyses. Furthermore, these techniques revealed that strains of 'Bacillus carotarum', another name that has not been validly published, also showed high similarity to this group of organisms. On the other hand, it was shown that the strains labelled 'B. macroides' in different collections do not all belong to the same species. Strain NCIMB 8796 can be allocated to B. simplex, while strain DSM 54 (=ATCC 12905) shares the highest 16S rRNA gene sequence similarity with Bacillus sphaericus and Bacillus fusiformis (both around 98.6 %). On the basis of further DNA-DNA hybridization data and the study of phenotypic characteristics, one group of five mural painting strains was attributed to a novel species in the genus Bacillus, for which the name Bacillus muralis sp. nov. is proposed. Finally, the remaining mural painting strains, one (LMG 18508=NCIMB 8796) of two strains belonging to 'B. macroides' and strains belonging to 'B. maroccanus' and 'B. carotarum' are allocated to the species B. simplex and an emended description of B. simplex is given.

Bacillus↗

Taxonomic study of aerobic thermophilic bacilli: descriptions of Geobacillus subterraneus gen. nov., sp. nov. and Geobacillus uzenensis sp. nov. from petroleum reservoirs and transfer of Bacillus stearothermophilus, Bacillus thermocatenulatus, Bacillus thermoleovorans, Bacillus kaustophilus, Bacillus thermodenitrificans to Geobacillus as the new combinations G. stearothermophilus, G. th.

Five hydrocarbon-oxidizing strains were isolated from formation waters of oilfields in Russia, Kazakhstan and China. These strains were moderately thermophilic, neutrophilic, motile, spore-forming rods, aerobic or facultatively anaerobic. The G+C content of their DNA ranged from 49.7 to 52.3 mol%. The major isoprenoid quinone was menaquinone-7; cellular fatty acid profiles consisted of significant amounts of iso-15:0, iso-16:0 and iso-17:0 fatty acids (61.7-86.8% of the total). Based on data from 16S rDNA analysis and DNA-DNA hybridization, the subsurface isolates could be divided into two groups, one of which consisted of strains UT and X and the other of which consisted of strains K, Sam and 34T. The new strains exhibited a close phylogenetic relationship to thermophilic bacilli of 'Group 5' of Ash et al. [Ash, C., Farrow, J. A. E., Wallbanks, S. & Collins, M. D. (1991). Lett Appl Microbiol 13, 202-206] and a set of corresponding signature positions of 16S rRNA. Comparative analysis of the 16S rDNA sequences and fatty acid compositions of the novel isolates and established species of thermophilic bacilli indicated that the subsurface strains represent two new species within a new genus, for which the names Geobacillus subterraneus gen. nov., sp. nov., and Geobacillus uzenensis sp. nov. are proposed. It is also proposed that Bacillus stearothermophilus, Bacillus thermoleovorans, Bacillus thermocatenulatus, Bacillus kaustophilus, Bacillus thermoglucosidasius and Bacillus thermodenitrificans be transferred to this new genus, with Geobacillus stearothermophilus (formerly Bacillus stearothermophilus) as the type species.

Bacillus↗

Bacillus novalis sp. nov., Bacillus vireti sp. nov., Bacillus soli sp. nov., Bacillus bataviensis sp. nov. and Bacillus drentensis sp. nov., from the Drentse A grasslands.

A group of 42 isolates were isolated from the soil of several disused hay fields, in the Drentse A agricultural research area (The Netherlands), that were taken out of production at different times. The group represents hitherto-uncultured Bacillus lineages that have previously been found, by a non-cultural method, to be predominant in soil. The strains were subjected to a polyphasic taxonomic study, including (GTG)5-PCR, 16S rDNA sequence analysis, DNA-DNA hybridizations, DNA base-ratio determination, fatty acid analysis and morphological and biochemical characterization. By comparing the groupings obtained by (GTG)5-PCR and 16S rDNA sequence analysis, six clusters of similar strains could be recognized. A DNA-DNA relatedness study showed that these clusters represented five novel genospecies. Further analysis supported the proposal of five novel species in the genus Bacillus, namely Bacillus novalis sp. nov. (type strain IDA3307T=R-15439T=LMG 21837T=DSM 15603T), Bacillus vireti sp. nov. (type strain IDA3632T=R-15447T=LMG 21834T=DSM 15602T), Bacillus soli sp. nov. (type strain IDA0086T=R-16300T=LMG 21838T=DSM 15604T), Bacillus bataviensis sp. nov. (type strain IDA1115T=R-16315T=LMG 21833T=DSM 15601T) and Bacillus drentensis sp. nov. (type strain IDA1967T=R-16337T=LMG 21831T=DSM 15600T).

Bacillus↗

Characterization of Bacillus brevis with descriptions of Bacillus migulanus sp. nov., Bacillus choshinensis sp. nov., Bacillus parabrevis sp. nov., and Bacillus galactophilus sp. nov.

Thirty-five Bacillus brevis strains obtained from culture collections, including protein-producing isolates, were taxonomically studied by using numerical analysis, DNA base composition, and DNA-DNA hybridization. Six DNA relatedness groups were represented, and these groups correlated well with clusters based on the numerical analysis. The B. brevis strains were separated into B. brevis sensu stricto, four new species, and an unidentified species of the genus Bacillus. Bacillus migulanus sp. nov., Bacillus choshinensis sp. nov., Bacillus parabrevis sp. nov., and Bacillus galactophilus sp. nov. are proposed.

Bacillus↗

Heat-stable toxin production by strains of Bacillus cereus, Bacillus firmus, Bacillus megaterium, Bacillus simplex and Bacillus licheniformis.

Strains of Bacillus cereus can produce a heat-stable toxin (cereulide). In this study, 101 Bacillus strains representing 7 Bacillus species were tested for production of heat-stable toxins. Strains of B. megaterium, B. firmus and B. simplex were found to produce novel heat-stable toxins, which showed varying levels of toxicity. B. cereus strains (18 out of 54) were positive for toxin production. Thirteen were of serovar H1, and it was of interest that some were of clinical origin. Two were of serovars 17B and 20, which are not usually implicated in the emetic syndrome. Partial purification of the novel B. megaterium, B. simplex and B. firmus toxins showed they had similar physical characteristics to the B. cereus emetic toxin, cereulide.

Bacillus↗

Bacillus aerius sp. nov., Bacillus aerophilus sp. nov., Bacillus stratosphericus sp. nov. and Bacillus altitudinis sp. nov., isolated from cryogenic tubes used for collecting air samples from high altitudes.

Four novel bacterial strains were isolated from cryogenic tubes used to collect air samples at altitudes of 24, 28 and 41 km. The four strains, 24K(T), 28K(T), 41KF2a(T) and 41KF2b(T), were identified as members of the genus Bacillus. Phylogenetic analysis based on 16S rRNA gene sequences indicated that three of the strains, 24K(T), 28K(T) and 41KF2a(T), are very similar to one another (>98 % sequence similarity) and show a similarity of 98-99 % with Bacillus licheniformis and 98 % with Bacillus sonorensis. DNA-DNA hybridization studies showed that strains 24K(T), 28K(T) and 41KF2a(T) exhibit <70 % similarity with each other and with B. licheniformis and B. sonorensis. Differences in phenotypic and chemotaxonomic characteristics between the novel strains and B. licheniformis and B. sonorensis further confirmed that these three isolates are representatives of three separate novel species. Strain 41KF2b(T) showed 100 % 16S rRNA gene sequence similarity to Bacillus pumilus, but differed from its nearest phylogenetic neighbour in a number of phenotypic and chemotaxonomic characteristics and showed only 55 % DNA-DNA relatedness. Therefore, the four isolates represent four novel species for which the names Bacillus aerius sp. nov. (type strain, 24K(T)=MTCC 7303(T)=JCM 13348(T)), Bacillus aerophilus sp. nov. (type strain, 28K(T)=MTCC 7304(T)=JCM 13347(T)), Bacillus stratosphericus sp. nov. (type strain, 41KF2a(T)=MTCC 7305(T)=JCM 13349(T)) and Bacillus altitudinis sp. nov. (type strain, 41KF2b(T)=MTCC 7306(T)=JCM 13350(T)) are proposed.

Air Microbiology↗

Fatty acids in the genus Bacillus. II. Similarity in the fatty acid compositions of Bacillus thuringiensis, Bacillus anthracis, and Bacillus cereus.

The nature and relative abundance of fatty acids produced by two strains each of Bacillus thuringiensis and of B. anthracis were studied by gas-liquid chromatography on a 12,000 theoretical plate polyester column capable of partially resolving iso- and anteiso-fatty acids with the same number of carbon atoms. Unsaturated fatty acids as the bromo derivatives were separated from the saturated acids and resolved in a short SE-30 column by use of programmed-temperature gas chromatography. All four strains produced 16 major fatty acids: 9 branched (i-C(12), i-C(13), i-C(14), i-C(15), i-C(16), i-C(17), a-C(13), a-C(15), and a-C(17)), 3 normal (n-C(14), n-C(15), and n-C(16)), and 4 monounsaturated (i-C(16) (1=), i-C(17) (1=), a-C(17) (1=), and n-C(16) (1=)), in addition to some minor fatty acids. In all cases, 12 branched acids, including saturated and monounsaturated, made up over 70% of the total fatty acids, and iso-C(15) acid was most abundant. These fatty acid distribution patterns were very similar to those of B. cereus and B. cereus var. mycoides. There were, however, minor but clear differences between the fatty acid distribution patterns of B. thuringiensis and B. anthracis. B. thuringiensis, like B. cereus, produced higher proportions of i-C(13), a-C(13), and i-C(14) fatty acids than did B. anthracis. This difference between these two species could be useful as a supplemental criterion in their differentiation. Indications are that the enzyme systems for monounsaturated fatty acid synthesis in B. thuringiensis and B. anthracis prefer normal fatty acids as substrates rather than branched-chain fatty acids.

Bacillus↗

MICs of selected antibiotics for Bacillus anthracis, Bacillus cereus, Bacillus thuringiensis, and Bacillus mycoides from a range of clinical and environmental sources as determined by the Etest.

This paper presents Etest determinations of MICs of selected antimicrobial agents for 76 isolates of Bacillus anthracis chosen for their diverse histories and 67, 12, and 4 cultures, respectively, of its close relatives B. cereus, B. thuringiensis, and B. mycoides derived from a range of clinical and environmental sources. NCCLS breakpoints are now available for B. anthracis and ciprofloxacin, penicillin, and tetracycline; based on these breakpoints, the B. anthracis isolates were all fully susceptible to ciprofloxacin and tetracycline, and all except four cultures, three of which had a known history of penicillin resistance and were thought to originate from the same original parent, were susceptible to penicillin. Based on NCCLS interpretive standards for gram-positive and/or aerobic bacteria, all cultures were susceptible to amoxicillin-clavulanic acid and gentamicin and 99% (one with intermediate sensitivity) of cultures were susceptible to vancomycin. No group trends were apparent among the different categories of B. cereus (isolates from food poisoning incidents and nongastrointestinal infections and food and environmental specimens not associated with illness). Differences between B. anthracis and the other species were as expected for amoxicillin and penicillin, with all B. anthracis cultures, apart from the four referred to above, being susceptible versus high proportions of resistant isolates for the other three species. Four of the B. cereus and one of the B. thuringiensis cultures were resistant to tetracycline and a further six B. cereus and one B. thuringiensis cultures fell into the intermediate category. There was a slightly higher resistance to azithromycin among the B. anthracis strains than for the other species. The proportion of B. anthracis strains fully susceptible to erythromycin was also substantially lower than for the other species, although just a single B. cereus strain was fully resistant. The Etest compared favorably with agar dilution in a subsidiary test set up to test the readings, and it compared with other published studies utilizing a variety of test methods.

Anti-Bacterial Agents↗

Comparative sequence analyses on the 16S rRNA (rDNA) of Bacillus acidocaldarius, Bacillus acidoterrestris, and Bacillus cycloheptanicus and proposal for creation of a new genus, Alicyclobacillus gen. nov.

Comparative 16S rRNA (rDNA) sequence analyses performed on the thermophilic Bacillus species Bacillus acidocaldarius, Bacillus acidoterrestris, and Bacillus cycloheptanicus revealed that these organisms are sufficiently different from the traditional Bacillus species to warrant reclassification in a new genus, Alicyclobacillus gen. nov. An analysis of 16S rRNA sequences established that these three thermoacidophiles cluster in a group that differs markedly from both the obligately thermophilic organisms Bacillus stearothermophilus and the facultatively thermophilic organism Bacillus coagulans, as well as many other common mesophilic and thermophilic Bacillus species. The thermoacidophilic Bacillus species B. acidocaldarius, B. acidoterrestris, and B. cycloheptanicus also are unique in that they possess omega-alicylic fatty acid as the major natural membranous lipid component, which is a rare phenotype that has not been found in any other Bacillus species characterized to date. This phenotype, along with the 16S rRNA sequence data, suggests that these thermoacidophiles are biochemically and genetically unique and supports the proposal that they should be reclassified in the new genus Alicyclobacillus.

Bacillaceae↗

beta-Amylase production by some Bacillus cereus, Bacillus megaterium and Bacillus polymyxa [correction of polymaxa] strains.

The production of extracellular beta-amylase by some Bacillus cereus, Bacillus megaterium and Bacillus polymyxa [corrected] strains was investigated, and the maximal yields of the enzyme were 3.6; 9.3 and 20.4 U/mL of the culture fluid, respectively (U, 1 mumol of maltose equivalent per min at 30 degrees C). Several cultivation media were used for beta-amylase production. Bacillus cereus and some strains of Bacillus megaterium gave good yields of beta-amylase only in medium with the addition of nutrient broth. However, beta-amylase produced during growth in protein rich medium (nutrient broth) was highly unstable, probably due to inactivation by proteolytic enzymes co-existing in the culture fluid. Bacillus polymyxa [corrected] strains can produce good yields of beta-amylase on a semi-synthetic medium consisting of inorganic salts, potato starch and inexpensive soybean extract instead of costly peptone and meat extract. The most potential beta-amylase producer was the strain Bacillus polymyxa [corrected] NCIB 8524. The tested Bacillus megaterium and Bacillus polymyxa [corrected] strains were apparently differentiated by temperature cultivation (30 and 37 degrees C) suitable for beta-amylase amylase yield.

Bacillus↗

Bacillus arenosi sp. nov., Bacillus arvi sp. nov. and Bacillus humi sp. nov., isolated from soil.

A group of nine Gram-positive endospore-forming bacteria was isolated from soil of the Drentse A agricultural research area in The Netherlands. Using (GTG)5-PCR genomic fingerprinting and fatty acid analysis, the nine isolates were divided into three consistent groups. On the basis of 16S rRNA gene sequence similarity of representative strains, the nine isolates were shown to belong to the genus Bacillus. The first group of four isolates was most closely related to Bacillus carboniphilus (95.5 %) and Bacillus sporothermodurans (95.5 %). The second and third groups of three and two isolates, respectively, showed highest sequence similarity to Bacillus neidei (97.0 and 97.1 %, respectively) and Bacillus pycnus (both 96.7 %). A DNA-DNA relatedness study confirmed the consistency of the three groups delineated by (GTG)5-PCR and fatty acid analysis. A small number of phenotypic characters allowed differentiation of the three groups of isolates. The three groups therefore represent novel species, for which the names Bacillus humi, Bacillus arenosi and Bacillus arvi are proposed, with LMG 22167T (=DSM 16318T), LMG 22166T (=DSM 16319T) and LMG 22165T (=DSM 16317T) as the respective type strains.

Bacillus↗

Bacillus hwajinpoensis sp. nov. and an unnamed Bacillus genomospecies, novel members of Bacillus rRNA group 6 isolated from sea water of the East Sea and the Yellow Sea in Korea.

Two Gram-positive or -variable, endospore-forming, slightly halophilic strains (SW-72(T) and SW-93) were isolated from sea water of the East Sea and the Yellow Sea in Korea, respectively, and subjected to polyphasic taxonomic study. Both strains had cell-wall peptidoglycan that was based on meso-diaminopimelic acid and MK-7 as the predominant menaquinone. The two strains contained large amounts of saturated and branched fatty acids, with anteiso-C(15 : 0) as the major fatty acid. The DNA G+C contents of strains SW-72(T) and SW-93 were 40.9 and 41.0 mol%, respectively. Phylogenetic analysis based on 16S rDNA sequences showed that strains SW-72(T) and SW-93 fall within the radiation of the cluster that comprises members of the genus Bacillus, particularly Bacillus rRNA group 6. There were five nucleotide differences between the 16S rDNA sequences of strains SW-72(T) and SW-93. The mean level of DNA-DNA relatedness between strains SW-72(T) and SW-93 was 21.5 %. Strains SW-72(T) and SW-93 showed 93.1-95.2 % 16S rDNA sequence similarity to the type strains of Bacillus species that are assigned to rRNA group 6. Strains SW-72(T) and SW-93 could not be differentiated clearly by using their phenotypic properties. On the basis of phenotypic properties, phylogeny and genomic data, it is proposed that strain SW-72(T) (=KCCM 41641(T)=JCM 11807(T)) should be placed in the genus Bacillus as the type strain of a novel species, Bacillus hwajinpoensis sp. nov., and that strain SW-93 (=KCCM 41640=JCM 11806) should be placed in the genus Bacillus as an unnamed Bacillus genomospecies.

Bacillus↗

Bacillus farraginis sp. nov., Bacillus fortis sp. nov. and Bacillus fordii sp. nov., isolated at dairy farms.

Forty-eight bacterial strains were isolated at dairy farms from raw milk, the milking apparatus, green fodder or feed concentrate after a heat treatment of 30 min at 100 degrees C. In this way, spore-forming bacteria with a very high intrinsic heat resistance were selected for. The aerobic spore-forming isolates were subjected to a polyphasic taxonomical study, including repetitive element sequence-based PCR typing, whole-cell protein profiling, 16S rDNA sequence analysis, DNA-DNA hybridizations, DNA base composition, fatty acid analysis, and morphological and biochemical characteristics. A comparison of the REP- and (GTG)5-PCR and whole-cell protein SDS-PAGE profiles resulted in three clusters of similar strains. Analysis of the 16S rDNA sequences and DNA-DNA relatedness data showed that these clusters represented three novel species. The highest 16S rDNA similarity to a recognized species found for the three groups was around 94% with Bacillus lentus and Bacillus sporothermodurans. Further phenotypic characterization supported the proposal of three novel species in the genus Bacillus, Bacillus farraginis, Bacillus fortis and Bacillus fordii. The respective type strains are R-6540T (=LMG 22081T=DSM 16013T), R-6514T (=LMG 22079T=DSM 16012T) and R-7190T (=LMG 22080T=DSM 16014T); their G+C DNA base contents are 43.7, 44.3 and 41.9 mol%, respectively. Although in variable amounts, a predominance of the branched fatty acids iso-C(15 : 0) and anteiso-C(15 : 0) was observed in all three novel species.

Aerobiosis↗

Expression of Bacillus protease (Protease BYA) from Bacillus sp. Y in Bacillus subtilis and enhancement of its specific activity by site-directed mutagenesis-improvement in productivity of detergent enzyme-.

An attempt was made to express protease BYA produced by an alkalophilic Bacillus sp. Y in Bacillus subtilis by gene engineering methods. The gene encoding protease BYA was cloned from Bacillus sp. Y, and expression vector pTA71 was constructed from the amylase promoter of Bacillus licheniformis, DNA fragments encoding the open reading frame of protease BYA, and pUB110. Protease BYA was secreted at an activity level of 5100 APU/ml in the common industrial culture medium of Bacillus subtilis transformed with pTA71. We then attempted to increase the specific activity of protease BYA by site-directed mutagenesis. Amino acid residue Ala29 next to catalytic Asp30 was replaced by one of three uncharged amino acid residues (Val29, Leu29, Ile29), and each mutant enzyme was expressed and isolated from the culture medium. Val29 mutant enzyme was secreted at an activity level of greater than 7000 APU/ml in culture medium, and its specific activity was 1.5-fold higher than that of the wild-type enzyme. Other mutant enzymes had specific activity similar to that of the original one and were less stabile than the wild-type enzyme. It can be thought that the substitution at amino acid residue 29 affects the level of activity and stability of protease BYA.

Amylases↗