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The nucleotide sequence of formylmethionine tRNA from Mycoplasma mycoides sp. capri.

The nucleotide sequence of Mycoplasma mycoides sp. capri PG3 formylmethionine tRNA has been determined, using in vitro labeling techniques, to be pC-G-C-G-G-G-G-s4U-A-G-A-G-C-A-G-U-D (U)-G-G-D-A-G-C-U-C-G-C-C-G-G-G-C-U-C-A-U-A-A-C-C-C-G-G-A-G-G-C-C-G-C-A-G-G-U-psi- C-G-A-G-U-C-C-U-G-C-C-C-C-C-G-C-A-A-C-C-AOH. This tRNA contains only three modified nucleosides s4U, D and psi, all of which are derived from uridine. Both in the structural features which distinguish eukaryotic from prokaryotic initiator RNAs and in the overall sequence, this tRNA resembles a typical prokaryotic initiator tRNA. A comparison of the sequence of this tRNA with those of other prokaryotic initiator tRNAs suggests that taxonomically the Mycoplasma may be less related to the Cyanophyta (Anacystis nidulans) than to the bacteria and less related to the Enterobacteriaceae (Escherichia coli) than to the Bacillaceae (Bacillus subtilis).

Base Sequence↗

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↗

Phylogenetic position of the North American isolate of Pasteuria that parasitizes the soybean cyst nematode, Heterodera glycines, as inferred from 16S rDNA sequence analysis.

A 1341 bp sequence of the 16S rDNA of an undescribed species of Pasteuria that parasitizes the soybean cyst nematode, Heterodera glycines, was determined and then compared with a homologous sequence of Pasteuria ramosa, a parasite of cladoceran water fleas of the family Daphnidae. The two Pasteuria sequences, which diverged from each other by a dissimilarity index of 7%, also were compared with the 16S rDNA sequences of 30 other bacterial species to determine the phylogenetic position of the genus Pasteuria among the Gram-positive eubacteria. Phylogenetic analyses using maximum-likelihood, maximum-parsimony and neighbour-joining methods showed that the Heterodera glycines-infecting Pasteuria and its sister species, P. ramosa, form a distinct line of descent within the Alicyclobacillus group of the Bacillaceae. These results are consistent with the view that the genus Pasteuria is a deeply rooted member of the Clostridium-Bacillus-Streptococcus branch of the Gram-positive eubacteria, neither related to the actinomycetes nor closely related to true endospore-forming bacteria.

Animals↗

Phylogenetic analysis of the genus Thermoactinomyces based on 16S rDNA sequences.

The almost complete 16S rDNA sequences of the type strains of eight validly described species and two invalid species of the genus Thermoactinomyces were determined and phylogenetically analysed. The validly described Thermoactinomyces species formed phylogenetic lineages related to the family Bacillaceae, as shown previously. However, the available strains of 'Thermoactinomyces glaucus' and 'Thermoactinomyces monosporus' exhibited their closest phylogenetic affinities not to the genus Thermoactinomyces but to the genus Saccharomonospora. Some Thermoactinomyces species were shown to be closely related from 16S rDNA sequence analysis. Particularly, Thermoactinomyces vulgaris KCTC 9076T and Thermoactinomyces candidus KCTC 9557T had the same 16S rDNA sequences and Thermoactinomyces thalpophilus KCTC 9789T and Thermoactinomyces sacchari KCTC 9790T showed 16S rDNA similarity value of almost 100%. From phylogenetic analysis based on 16S rDNA sequences, it is suggested that the genus Thermoactinomyces should be taxonomically re-evaluated using other useful taxonomic markers.

DNA, Bacterial↗

Symbiobacterium thermophilum gen. nov., sp. nov., a symbiotic thermophile that depends on co-culture with a Bacillus strain for growth.

A Gram-negative and tryptophanase-positive thermophile, whose growth is dependent on co-culture with an associating Bacillus strain, had been reported and tentatively named Symbiobacterium thermophilum strain T(T). Axenic culture of strain T(T) was recently established by dialysing cultures with the supporting bacterial strains or adding their culture broth. Phylogenetic analysis of strain T(T), based on the 16S rDNA sequence, was conducted for the validation of S. thermophilum. The sequence of strain T(T) was located at the outermost position in the high-G+C Gram-positive group distinctly isolated from any other branches hitherto known. Ten sequences identical to that of strain T(T), and one sequence closely related to it, were identified for the first time from soil and compost samples. The outer membrane of strain T(T) had a three-layered structure, outside the cytoplasmic membrane, which is similar to the S-layer in the cells of members of the Bacillaceae. Chemical analysis of the cells revealed that menaquinone-6 is a major component of the quinone system. According to these results, along with several previous observations (i.e. a G+C DNA content of 65 mol% and the identification of iso-C15:0 and iso-C17:0 acids as major cellular fatty acids), the new taxon Symbiobacterium thermophilum gen. nov., sp. nov. is proposed. The type strain is S. thermophilum strain T(T) (= IAM 14863T).

Bacillus↗

Bacillus siralis sp. nov., a novel species from silage with a higher order structural attribute in the 16S rRNA genes.

A novel bacterial strain (171544T) was recently isolated from silage and was classified in the genus Bacillus by 16S rDNA sequence analysis. Additional silage samples have been investigated in the present study and four organisms resembling strain 171544T were isolated. Phenotypic and genotypic characterization of these bacteria showed that they constitute a new species of the genus Bacillus. This taxon was positioned in the family Bacillaceae on the basis of evolutionary distance trees using 16S rDNA sequences. Bacillus circulans, Bacillus firmus and Bacillus benzoevorans were the most closely related species with 165 rDNA similarities of 97.2, 96.3 and 95.9%, respectively. All five silage isolates shared a higher order structural feature in the 3' region of the 16S rRNA gene comprising an extension to helix 49 of 24 bp and highly similar random amplified polymorphic DNA patterns that distinguished them from the type strains of B. circulans and B. firmus. Moreover, they possessed a unique pattern of phenotypic features including subterminally or terminally located endospores which distinctly swelled the sporangium, strictly aerobic metabolism but with the ability to utilize nitrate as a terminal electron acceptor under anaerobic conditions, and hydrolysis of casein but not starch. The name Bacillus siralis is therefore proposed for this new taxon. The type strain of B. siralis is strain 171544T (= NCIMB 13601T = CIP 106295T).

Bacillus↗

Bacillus subterraneus sp. nov., an iron- and manganese-reducing bacterium from a deep subsurface Australian thermal aquifer.

A facultatively anaerobic bacterium, designated strain COOI3B(T) (= ATCC BAA 136T = DSM 13966T), was isolated from the waters emitted by a bore well tapping the deep subterranean thermal waters of the Great Artesian Basin of Australia. The cells were straight to slightly curved rods (0.5-0.8 x 2-25 microm) that occurred singly and rarely in pairs or in chains. Strain COOI3B(T) was motile by peritrichous flagella. It stained gram-negative, but electron micrographs showed a gram-positive-type cell wall. Spores were never observed and cells were heat-sensitive. Yeast extract at 0.02% (w/v) was required for growth and could also be used as a sole carbon and energy source at concentrations higher than 0.1% (w/v). The strain utilized amorphous iron(III), manganese(IV), nitrate, nitrite and fumarate as electron acceptors in the presence of yeast extract, glucose, sucrose, fructose, maltose, xylose, starch, glycerol, ethanol or lactate. Electron acceptors were not obligately required and growth was better in the presence of nitrate than in its absence. Acid was not produced from growth on carbohydrates. Tryptophan deaminase, H2S, arginine dihydrolase, lysine decarboxylase, beta-galactosidase, arabinosidase, glucuronidase, glucosaminidase, nitroanilidase, xylosidase and ornithine decarboxylase were not produced. Starch and gelatin, but not casein, were hydrolysed. Aesculin and catalase, but not oxidase and urease, were produced. Strain COOI3B(T) grew optimally at temperatures between 37 and 40 degrees C (the temperature growth range was 25-45 degrees C) and at pH 7.0-9.0 (the pH growth range was 6.0 to 9.5) with 5% (w/v) NaCl (the NaCl concentration growth range was 0.9%, w/v). The DNA base composition was 43 +/- 1 mol % G+C. Phylogenetic analysis indicated that it was a member of the family Bacillaceae, Bacillus infernus and Bacillus firmus being the closest phylogenetic neighbours (having a mean similarity value of 96%); hence, strain COOI3B(T) is designated as a novel species, Bacillus subterraneus sp. nov.

Anaerobiosis↗

Bacillus taeanensis sp. nov., a halophilic Gram-positive bacterium from a solar saltern in Korea.

A halophilic bacterium, strain BH030017(T), showing optimum growth at 2-5 % (w/v) NaCl was isolated from solar-saltern sediment from the Tae-An area of Korea and was characterized taxonomically. The cells of strain BH030017(T) were Gram-positive, motile, short rods containing cell-wall peptidoglycan based on meso-diaminopimelic acid. The major cellular fatty acids were anteiso-C(15 : 0) and iso-C(15 : 0). The DNA G+C content was 36 mol% and the predominant lipoquinone was MK-7. The major cellular phospholipids were phosphatidylglycerol, diphosphatidylglycerol and phosphatidylethanolamine. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain BH030017(T) formed a cluster with Bacillus clarkii DSM 8720(T) and Bacillus agaradhaerens DSM 8721(T) within the family Bacillaceae. 16S rRNA gene sequence similarities with respect to closely related type strains were less than 95.1 %. On the basis of its phylogenetic, phenotypic and chemotaxonomic properties, strain BH030017(T) represents a novel species within the genus Bacillus, for which the name Bacillus taeanensis sp. nov. is proposed. The type strain is BH030017(T) (=KCTC 3918(T)=DSM 16466(T)).

Bacillus↗

Caldalkalibacillus thermarum gen. nov., sp. nov., a novel alkalithermophilic bacterium from a hot spring in China.

A thermophilic, alkaliphilic and catalase-positive bacterium, designated strain HA6(T), was isolated from a hot spring in China. The strain was aerobic and chemo-organotrophic and grew optimally at 60 degrees C, pH 8.5 and 1.5 % (w/v) NaCl. The cells were Gram-positive rods, forming single terminal endospores. The predominant cellular fatty acids were iso-C(15 : 0) and iso-C(17 : 0). The cell-wall peptidoglycan contained meso-diaminopimelic acid. The genomic DNA G+C content was 45.2 mol%. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that strain HA6(T) formed a distinct lineage within the family Bacillaceae and was most closely related to Bacillus horti K13(T) and Bacillus smithii DSM 4216(T), with sequence similarities of 91.8 and 93.1 %, respectively. On the basis of its physiological and molecular properties, strain HA6(T) should be placed in a novel genus and species, for which the name Caldalkalibacillus thermarum gen. nov., sp. nov. is proposed. The type strain of Caldalkalibacillus thermarum is strain HA6(T) (=CGMCC 1.4242(T)=JCM 13486(T)).

China↗

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↗

Cloning and nucleotide sequence of the thrB gene from the cyanobacterium Calothrix PCC 7601.

The cyanobacterium Calothrix PCC 7601 thrB gene, encoding homoserine kinase (EC 2.7.1.39), was cloned via complementation of an Escherichia coli threonine auxotroph, and its nucleotide sequence was determined. The comparison of the homoserine kinase amino acid sequences from Calothrix PCC 7601, E. coli K12 and Bacillus subtilis 168 indicates a closer relationship between cyanobacteria and bacillaceae than between cyanobacteria and enterobacteriaceae. Sequence analysis of the 5' and 3' flanking regions of the Calothrix thrB gene revealed the existence of a 169-codon-long open reading frame downstream from thrB: this sequence may be the second gene of a Calothrix thr operon. Two types of tandemly repeated sequences, sharing similarities with other prokaryotic transcriptional regulatory elements, were detected in the region upstream from the thrB gene.

Amino Acid Sequence↗

A gzip-based algorithm to identify bacterial families by 16S rRNA.

AIMS: Microbial family identification of 16S rDNA sequences by applying a strategy based on algorithms for data compression. METHODS AND RESULTS: Perl scripts were developed to analyse similarities in microbial sequences, based on a gzip data compression technique. For each bacterial family (n=196) a 16S rRNA reference file was constructed to compare new queries looking at compression performance. An online user-friendly bioinformatics tool was built up to attribute a bacterial family to a 16S rRNA sequence. It was successfully applied to recognize different bacterial families, including Legionellaceae, Bacillaceae, Enterobacteriaceae, Acetobacteriaceae and Rhizobiaceae. The percentage of positive identifications is higher than 95% for fragments over 450 bp. CONCLUSIONS: A new bioinformatics approach has been developed to assign a taxonomic classification to a 16SrDNA sequence. An online tool provides quick and easy sequence attribution. The general principle can be applied to other genes of taxonomic interest. SIGNIFICANCE AND IMPACT OF THE STUDY: Availability of simple bioinformatics tools can support the development of molecular-based analysis and classification of bacteria, especially for environmental or uncultured strains.

Algorithms↗

Phylogenetic diversity of thermophilic actinomycetes and Thermoactinomyces spp. isolated from mushroom composts in Korea based on 16S rRNA gene sequence analysis.

Forty one strains isolated from 21 samples of various mushroom composts in Korea were analyzed by 16S rRNA gene sequencing to investigate the phylogenetic diversity of thermophilic actinomycetes. The 25 strains of thermophilic actinomycete isolates were related to the five genera, Pseudonocardia, Saccharomonospora, Saccharopolyspora, Streptomyces, and Thermobifida, within the order Actinomycetales, and 16 strains were classified into the genus Thermoactinomyces within the family Bacillaceae. Most of 41 isolates were encompassed by two genera, Streptomyces and Thermoactinomyces, that were isolated mainly in composts prepared from waste cotton and hay, respectively. Among them, M104 and M109 were placed in distinct taxonomic positions although these strains formed phylogenetic lineages related to the genus Streptomyces and to the family Streptosporangiaceae, respectively. Therefore, a phenetic and genetic characterization of these strains will be needed to pinpoint their taxonomic position.

Agaricales↗

Effect of sulfur-containing compounds on Bacillus cellulosome-associated 'CMCase' and 'Avicelase' activities.

The isolation of cellulosomes from clostridial sources has been extensively studied; however, the isolation of cellulosomes from facultative soil anaerobes of the family Bacillaceae is not as well characterized. The Bacillus cellulosome (celluloxylanosome) essentially consists of two complex components: C-I and C-II. This multi-component complex enables Bacillus to degrade a variety of carbonaceous compounds as it is composed of several enzymes, such as cellulases, xylanases and other degradative enzymes. The cellulosomal cellulases from Bacillus megaterium were purified using cellulose affinity chromatography, followed by Sepharose 4B gel filtration chromatography. The objective of this investigation was to establish the effect of sulfate and sulfide on cellulosomal 'cellulase' activity. An increase in sulfide concentration led to a general enhancement of cellulosomal-associated cellulolytic activity, whereas an increase in sulfate concentration resulted in an inhibition of the cellulosome-associated cellulolytic activity.

Bacillus↗

Comparative phylogenetic assignment of environmental sequences of genes encoding 16S rRNA and numerically abundant culturable bacteria from an anoxic rice paddy soil.

We used both cultivation and direct recovery of bacterial 16S rRNA gene (rDNA) sequences to investigate the structure of the bacterial community in anoxic rice paddy soil. Isolation and phenotypic characterization of 19 saccharolytic and cellulolytic strains are described in the accompanying paper (K.-J. Chin, D. Hahn, U. Hengstmann, W. Liesack, and P. H. Janssen, Appl. Environ. Microbiol. 65:5042-5049, 1999). Here we describe the phylogenetic positions of these strains in relation to 57 environmental 16S rDNA clone sequences. Close matches between the two data sets were obtained for isolates from the culturable populations determined by the most-probable-number counting method to be large (3 x 10(7) to 2.5 x 10(8) cells per g [dry weight] of soil). This included matches with 16S rDNA similarity values greater than 98% within distinct lineages of the division Verrucomicrobia (strain PB90-1) and the Cytophaga-Flavobacterium-Bacteroides group (strains XB45 and PB90-2), as well as matches with similarity values greater than 95% within distinct lines of descent of clostridial cluster XIVa (strain XB90) and the family Bacillaceae (strain SB45). In addition, close matches with similarity values greater than 95% were obtained for cloned 16S rDNA sequences and bacteria (strains DR1/8 and RPec1) isolated from the same type of rice paddy soil during previous investigations. The correspondence between culture methods and direct recovery of environmental 16S rDNA suggests that the isolates obtained are representative geno- and phenotypes of predominant bacterial groups which account for 5 to 52% of the total cells in the anoxic rice paddy soil. Furthermore, our findings clearly indicate that a dual approach results in a more objective view of the structural and functional composition of a soil bacterial community than either cultivation or direct recovery of 16S rDNA sequences alone.

Anaerobiosis↗

Inhibition of the plaquing efficiency of T4r+ bacteriophage by subtilisin.

The mechanism by which the replicative cycle of T4r(+) phage is inhibited by certain nonhost bacterial systems was investigated. Some Bacillaceae, especially Bacillus subtilis, decreased the plaquing efficiency of this virus more than 95% within 24 hr of exposure. Sarcina lutea and Micrococcus sp. both failed to cause any significant change in the infectivity of T4r(+) phage. Preliminary investigations into the nature of the inhibitory substance(s) suggested that an extracellularly elicited protein was at least partially responsible for this effect. Further analysis has implicated subtilisin, an exoprotease from B. subtilis, as the cause of some, if not all, of the observed decrease in plaquing efficiency. Gel-filtration chromatography of control and treated (14)C-labeled T4r(+) phage showed a wide dispersal of phage-specific material of these particles after 24 hr of exposure to pure subtilisin or to expended medium exoprotease from B. subtilis. It was concluded that B. subtilis exoprotease is capable of chemically altering the structure of the phage capsid, thus causing a decrease in its plaquing efficiency.

Amylases↗

Tetrahydrofolate-dependent biosynthesis of ribothymidine in transfer ribonucleic acids of Gram-positive bacteria.

Trimethoprim, an inhibitor that prevents tetrahydrofolate-dependent transmethylation reactions inbacteria, was used in a comparative study to discriminate between two possible biosynthetic pathways, either the S-adenosylmethionine or the tetrahydrofolate-dependent formation of ribothymidine (rT) in transfer ribonucleic acids (tRNA's) of several strains of gram-positive and gram-negative microorganisms. rT-deficient tRNA's accumulate in trimethoprim-treated gram-positive Streptococcus faecium, Staphylococcus aureus, Corynebacterium bovis, Arthrobacter albidus, and all examined Bacillaceae, except Bacillus stearothermophilus. The rT-deficient rT-deficient tRNA's accept the methyl moiety from S-adenosylmethionine in vitro, with extracts from Escherichia coli (wild type) as a source of methylating enzymes; 90% of the incorporated methyl groups are present in rT. Trimethoprim does not inhibit the biosynthesis of rT in tRNA of gram-negative Enterobacteriaceae, Rhizobium lupini, and Pseudomonadaceae, suggesting that the rT-specific tRNA methyltransferases of these gram-negative strains use S-adenosylmethionine as coenzyme.

Arthrobacter↗

Bacterial parasite of a plant nematode: morphology and ultrastructure.

The life cycle of a bacterial endoparasite of the plant-parasitic nematode Meloidogyne incognita was examined by scanning and transmission electron microscopy. The infective stage begins with the attachment of an endospore to the surface of the nematode. A germ tube then penetrates the cuticle, and mycelil colonies form in the pseudocoelom. Sporulation is initiated when terminal cells of the mycelium enlarge to form sporangia. A septum within each sporangium divides the forespore from the basal or parasporal portion of the cell. The forespore becomes enclosed by several laminar coats. The parasporal cell remains attached to the forespore and forms the parasporal microfibers. After the newly formed spores are released into the soil, these microfibers apparently enable a mature spore to attach to the nematode. These results indicate that the endoparasite is a procaryotic organism having structural features that are more common to members of Actinomycetales and to the bacterium Pasteuria ramosa than to the sporozoans or to the family Bacillaceae, as previous investigatios have concluded.

Actinomycetales↗