Identification andin silico characterisation of putative conjugative transfer genes onGeobacillus stearothermophilus plasmids

We repor the first data demonstrating the presence of putative conjugative transfer genes on plasmids of the speciesGeobacillus stearothermophilus. Partial sequence analysis of the plasmid pGS18 fromG. stearothermophilus 18 was determined. It contained eleven complete open reading frames. Five of them encoded proteins which are homologous toBacillus megaterium pBM300 Mob/TraA,Lactococcus lactis pMRC01 TrsD and TrsE,Staphylococcus aureus pGO1 TrsG andS. aureus subsp.aureus pUSA03 TraL, the proteins that are associated with conjugative plasmid transfer. Southern hybridizations were performed on two other plasmids isolated fromG. stearothermophilus 3 andG. stearothermophilus 19 strains using the most homologous parts of those five genes as probes. Data from different hybridization patterns show a close homology of putative conjugative transfer genes between pGS18 and pGS3 hypothesizing a similar molecular organization of putative conjugative plasmid transfer region of both plasmids.     

Characterization of Thermophilic Proteolytic Spore-Forming Bacteria from a Geothermal Site in Lithuania Based on 16S rDNA RFLP and ITS-PCR Analyses1

Forty-two strains of gram-positive, aerobic, heterotrophic, obligately thermophilic, spore-forming bacteria were isolated from a geothermal site near the Baltic Sea in Lithuania. All of the strains were able to hydrolyze collagen and/or casein. Since characteristics of proteolytic activity are correlated with taxonomic positions of bacteria, the strains were grouped on the basis of molecular biological analyses. On the basis of RFLP patterns of 16S rDNA and 16S–23S rDNA ITS-PCR analysis, the strains were subdivided into nine groups.     

Paenibacillus tylopili sp.nov., a chitinolytic bacterium isolated from the mycorhizosphere of Tylopilus felleus

Two chitinolytic bacterial strains (designated MK2^T and V7) were isolated from the mycorhizosphere of the fungus Tylopilus felleus. The strains were facultatively anaerobic G⁺ endospore formers. Physiological analysis and 16S rRNA gene PCR-RFLP assays revealed nearly identical profiles for both strains, demonstrating their relationship at the species level. Sequences specific for the genus Paenibacillus were found within the 16S rRNA gene sequence of the strain MK2^T. The 16S rRNA gene sequence showed the highest similarity to the sequences of Paenibacillus amylolyticus, P. pabuli and P. xylanilyticus. DNA-DNA relatedness of the strain with the type strain of P. amylolyticus was 4.95 %, of P. pabuli 38.0 %, and of P. xylanilyticus 46.3 %, indicating no relatedness between MK2^T and any of them at the species level. The most abundant fatty acids in strains MK2^T and V7 were anteiso-C_15:0, iso-C_16:0, iso-C_15:0 and n-C_16:0. DNA-DNA relatedness, morphological, physiological and chemotaxonomic analyses, and phylogenetic data based on 16S rRNA gene sequencing made it possible to describe both strains as the novel species of the genus Paenibacillus, for which the name Paenibacillus tylopili is proposed, the type strain being MK2^T (DSM 18927^T, LMG 23975^T).     

Identification of the genus Geobacillus using genus-specific primers, based on the 16S-23S rRNA gene internal transcribed spacer

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.     

Unusual intragenomic and interspecific variability of <Emphasis Type="Italic">Geobacillus</Emphasis> 16S-23S rRNA internal transcribed spacers

The aim of this study was to evaluate the inter-and intraspecific as well as intragenomic variability of Geobacillus 16S–23S rRNA internal transcribed spacers without tRNA genes and to compare these sequences with sequences bearing tRNA genes. In this study the structural analysis was performed in a unique way because the length and the sequence of the structural blocks were adjusted to fit the structure of 16S–23S rRNA internal transcribed spacers of five different Geobacillus species. Our study demonstrated the mosaic-like structure of 16S–23S rRNA internal transcribed spacers in Geobacillus. Some characteristics of these spacers of geobacilli were not previously reported for other bacteria: unusually short conserved sequence in the 5′ end region, some identical conserved blocks in both 5′ and 3′ regions of 16S–23S rRNA internal transcribed spacers, the same sequence blocks in both 16S–23S and 23S–5S rRNA intergenic spacers. Our study demonstrated quite uniform arrangement of the sequence blocks in Geobacillus thermodenitrificans. This species diverged early in the phylogenetic tree of the genus Geobacillus. For the phylogenetically recent species Geobacillus kaustophilus and Geobacillus lituanicus the low inter-and intraspecific, but high intragenomic variability, as a consequence of recent phylogenetic events, was established.     

Identification of proteins involved in starch and polygalacturonic acid degradation using LC/MS

Plant biomass in the form of cheap wastes, such as straw, corn stalks, wood chips, sawdust, bagasse, pomace, etc., is abundant throughout the world. To convert these wastes into the useful value-added compounds microbial enzymes are the preferred choice. In this paper, we identify enzymes involved in the degradation of starch and polygalacturonic acid using liquid chromatography/mass spectrometry based analysis. We analysed total protein from soil and compost samples. Extracellular proteins from enrichment cultures were analysed in parallel and used as controls in the sample preparation and identification of proteins. In general, both protein sequence coverage and the number of identified peptides were higher in the samples obtained from the enrichment cultures than from the total protein from soil and compost. The influence of the nature of gel (zymography vs. SDS/polyacrylamide) was negligible. Thus, starch and polygalacturonic acid degradation associated proteins can be directly excised from the zymograms without the need to align zymograms with the SDS/polyacrylamide gels. A range of starch and polygalacturonic acid degradation associated enzymes were identified in both total protein samples and extracellular proteins from the enrichment cultures. Our results show that proteins involved in starch and polygalacturonic acid degradation can be identified by liquid chromatography/mass spectrometry from the complex protein mixtures both with and without cultivation of microorganism     

Gene expression and activity analysis of the first thermophilic U32 peptidase

Peptidase family U32 is one of the few whose catalytic type and structure has not yet been described. It is generally accepted that U32 peptidases represent putative collagenases and contribute to the pathogenicity of some bacteria. Meanwhile, U32 peptidases are also found in nonpathogenic bacteria including thermophiles and hyperthermophiles. Here we report cloning of the U32.002 peptidase gene from thermophilic Geobacillus thermoleovorans DSM 15325 and demonstrate expression and characterization of the recombinant protein. It has been determined that U32.002 peptidase is constitutively expressed in the cells of thermophilic G. thermoleovorans DSM 15325. The recombinant oligomeric enzyme showed its activity only against heat-treated collagen. It was unable to degrade albumin, casein, elastin, gelatine and keratin. In contrast to this, the monomeric recombinant protein showed no activity at all. This paper is the first report about the thermophilic U32 peptidase. As the thermophilic bacteria are non-pathogenic, the role of constitutively expressed extracellular collagenolytic U32 peptidase in these bacteria is unclear.     

Characterization of the novel xylanase from the thermophilic Geobacillus thermodenitrificans JK1

Thermophilic strain JK1 was isolated from compost using xylan as a single carbon source. On the basis of 16S rRNA gene phylogenetic analysis and spo0A gene sequence similarity analysis, strain JK1 was identified as Geobacillus thermodenitrificans strain. During the exponential culture growth, the strain JK1 was found to produce the single xylan degrading enzyme ??45 kDa in size. Xylose was not an inducer of this xylanase. Cloning, expression and characterization of the recombinant xylanase were performed. Xylanase of G. thermodenitrificans JK1 was cellulase-free; pH and temperature optimums were found to be 6.0 and 70°C, respectively. The metal ions Na⁺, K⁺, Ca²⁺, and Co²⁺ showed partial inhibition of the activity, while Mn²⁺ had slight stimulating effect on the enzymatic activity. Recombinant xylanase was thermostable over the temperature range of 55?C70°C. It presented the highest stability after incubation at 55°C for 60 min showing 84% residual activity. 50% residual activity was revealed after incubation at 60°C for 60 min as well as at 65 and 70°C for 30 min. Results of the thermostability experiments showed xylanase of JK1 having quite low thermostability when compared with the respective enzymes of the other geobacilli.     

Phylogenetic,Inter, and Intraspecific Sequence Analysis of <Emphasis Type="Italic">spo0A</Emphasis> Gene of the Genus <Emphasis Type="Italic">Geobacillus</Emphasis>

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.     

Complete nucleotide sequence of pGS18, a 62.8-kb plasmid from <Emphasis Type="Italic">Geobacillus stearothermophilus</Emphasis> strain 18

The complete nucleotide sequence (62.8 kb) of pGS18, the largest sequenced plasmid to date from the species Geobacillus stearothermophilus, was determined. Computational analysis of sequence data revealed 65 putative open reading frames (ORFs); 38 were carried on one strand and 27 were carried on the other. These ORFs comprised 84.1% of the pGS18 sequence. Twenty-five ORFs (38.4%) were assigned to putative functions; four ORFs (6.2%) were annotated as pseudogenes. The amino acid sequences obtained from 29 ORFs (44.6%) had the highest similarity to hypothetical proteins of the other microorganisms, and seven (10.8%) had no significant similarity to any genes present in the current open databases. Plasmid replication region, strongly resembling that of the theta-type replicon, and genes encoding three different plasmid maintenance systems were identified, and a putative discontinuous transfer region was localized. In addition, we also found several mobile genetic elements and genes, responsible for DNA repair, distributed along the whole sequence of pGS18. The alignment of pGS18 with two other large indigenous plasmids of the genus Geobacillus highlighted the presence of well-conserved segments and has provided a framework that can be exploited to formulate hypotheses concerning the molecular evolution of these three plasmids.