RESUMO
In this work, the variability of spo0A gene in the genus Geobacillus and applicability of this gene for the taxonomy within this genus were evaluated. The protein Spo0A is the master regulator of the endospore-forming process in the all endospore-forming bacteria. Geobacillus genus-specific primers GEOSPO were designed based on the sequences of Geobacillus spo0A gene available through the public databases. Inter and intraspecific variability of Geobacillus spo0A gene was determined after sequencing of the GEOSPO-PCR products. Geobacillus spo0A sequence analysis showed that three species--Geobacillus thermodenitrificans, G. stearothermophilus, and G. jurassicus--could be easily identified. Similarity between the sequences of these species and the other species were in the range of 83.3%-92.0%. In contrast, intraspecific similarity of G. thermodenitrificans and G. stearothermophilus was high--above 99.0%. Similarity of spo0A sequences of G. subterraneus-G. uzenensis species cluster also matched this interval. Intercluster similarity between G. lituanicus-G. thermoleovorans-G. kaustophilus-G. vulcani and G. thermocatenulatus-G. gargensis-G. caldoxylosilyticus-G. toebii-G. thermoglucosidasius species clusters, as well as interspecific similarity within these two clusters was in the range of the intraspecific similarity determined for G. thermodenitrificans and G. stearothermophilus. It was also determined that spo0A cannot be used as the phylogenetic marker for the genus Geobacillus.
Assuntos
Bacillaceae/classificação , Proteínas de Bactérias/genética , DNA Intergênico/genética , Filogenia , Bacillaceae/genética , Técnicas de Tipagem Bacteriana , DNA Bacteriano/genética , DNA Ribossômico/genética , Dados de Sequência Molecular , RNA Ribossômico 16S/genética , Análise de Sequência de DNARESUMO
Four bacterial strains, designated K9(T), K105, K1012A and K101, were isolated from soil in Lithuania. All these strains could use n-butanol as a sole carbon source. The strains grew in a medium containing 12-120 mM n-butanol. The strains were strictly aerobic, Gram-positive endospore-formers. The best growth was achieved at 25 degrees C and pH 7.0 in medium containing 1 % (w/v) NaCl. The strains showed identical profiles of 16S-23S rRNA internal transcribed spacer PCR and nearly identical 16S rRNA gene PCR-RFLP electrophoretic patterns and physiological characteristics, demonstrating their relationship at the species level. The cellular fatty acid profile of K9(T) consisted of significant amounts of the C(15) branched-chain fatty acids iso-C(15 : 0) (16.78 %) and anteiso-C(15 : 0) (45.80 %). The diagnostic cell-wall diamino acid was meso-diaminopimelic acid. The 16S rRNA gene sequence of K9(T) showed the highest similarity to the sequences of Bacillus simplex DSM 1321(T) and Bacillus muralis LMG 20238(T) (98.3 and 97.7 %, respectively). The DNA G+C content was 37.4 mol%. Studies of DNA-DNA relatedness, morphological, physiological and chemotaxonomic analyses and phylogenetic data based on 16S rRNA gene sequencing allowed strains K9(T), K105, K1012A and K101 to be described as members of a novel species of the genus Bacillus, for which the name Bacillus butanolivorans sp. nov. is proposed. The type strain is K9(T) (=DSM 18926(T) =LMG 23974(T)).
Assuntos
Bacillus/classificação , Bacillus/isolamento & purificação , Butanóis/metabolismo , Microbiologia do Solo , Bacillus/genética , Bacillus/fisiologia , Técnicas de Tipagem Bacteriana , Composição de Bases , DNA Bacteriano/análise , DNA Espaçador Ribossômico/análise , Ácidos Graxos/análise , Genes de RNAr , Microbiologia Industrial/métodos , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , RNA Ribossômico 16S/genética , RNA Ribossômico 23S/genética , Análise de Sequência de DNA , Especificidade da EspécieRESUMO
The aim of this study was to develop an easy and accurate technique for the identification of the genus Geobacillus. For this purpose, Geobacillus genus-specific primers GEOBAC (GEOBAC-F and GEOBAC-R) based on the 16S-23S rRNA gene internal transcribed spacer (ITS) region sequences have been designed. In total, 52 sequences from three species of the genus Geobacillus (Geobacillus stearothermophilus, Geobacillus kaustophilus and Geobacillus lituanicus) were examined for the design of these primers. Analysis of the sequences revealed three highly conservative regions common to these species: 5' and 3' end regions of 16S-23S rRNA gene ITSs and box A. Some sequences possessed two additional conservative regions - genes of tRNA(Ile) and tRNA(Ala). These particular sequences were chosen for the construction of the primers. The designed primers targeted the gene of tRNA(Ile) and the 3' end region of ITSs. This technique was validated with both the reference strains of the genus Geobacillus and the thermophilic aerobic endospore-forming environmental isolates. Different Geobacillus species could be grouped according to the number and size of GEOBAC-PCR products and identified on the basis of the AluI and TaqI restriction analysis of these products.
Assuntos
Bacillaceae/isolamento & purificação , Técnicas Bacteriológicas/métodos , DNA Bacteriano/genética , DNA Espaçador Ribossômico/genética , Reação em Cadeia da Polimerase/métodos , Análise do Polimorfismo de Comprimento de Fragmentos Amplificados/métodos , Bacillaceae/classificação , Bacillaceae/genética , Sequência Conservada , Primers do DNA/genética , DNA Bacteriano/química , DNA Espaçador Ribossômico/química , Dados de Sequência Molecular , Polimorfismo de Fragmento de Restrição , RNA de Transferência de Alanina/genética , RNA de Transferência de Isoleucina/genética , Análise de Sequência de DNARESUMO
Obligately thermophilic, aerobic, proteolytic, endospore-forming strain N-3(T) was isolated from a high-temperature oilfield in Lithuania. 16S rRNA gene sequence analysis placed this strain in genetic group 5 of the endospore formers. Geobacillus thermoleovorans appeared to be the closest phylogenetic neighbour (99.4 % sequence similarity). The G+C content of strain N-3(T) was 52.5 mol% and matched the range established for the genus Geobacillus. Studies of DNA-DNA relatedness and morphological and physiological analyses enabled strain N-3(T) to be described as a member of the genus Geobacillus, but could not assign this strain to any other known species of this genus. Results of this polyphasic study allowed characterization of strain N-3(T) as a novel species in the genus Geobacillus - Geobacillus lituanicus sp. nov. This species can be distinguished from G. thermoleovorans and Geobacillus stearothermophilus on the basis of 16S rRNA gene PCR-RFLP assays with the restriction endonucleases AluI, HaeIII and TaqI. The type strain of the novel species is N-3(T) (=DSM 15325(T)=VKM B-2294(T)).
