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).     

The genetic control of cell growth and development in yeast Saccharomyces cerevisiae. Disturbed sporulation in diploids with decreased activity of the Ras/cAMP signal transduction pathway

Seven haploid strains (four with the MAT alpha mating type and three with the MATa mating type) were selected from the Peterhof genetic collection of yeast. Previous phenotypic analysis assigned six of these strains to a physiological group of strains with a lower activity of the Ras/cAMP signal transduction pathway. The haploids were crossed, and the resulting 12 diploids showed higher glycogen accumulation, tolerance to heat shock and nitrogen starvation, and sporulation in complete media. Ten of the diploids expressed the hypersporulation phenotype (higher sporulation efficiency). The phenotypic characters of these ten diploids suggested a reduced activity of the Ras/cAMP pathway. All 12 diploids were tested for sporulation and production of two groups of asci (those with one or two spores and those with three or four spores) as dependent on culture conditions (21, 30, or 34 degrees C; standard sporulation medium or a complete medium containing potassium acetate or glycerol in place of glucose). Sporulation proved to depend on temperature and medium composition. The results are collated with the data on yeast phenotypes associated with a lower activity of the Ras/cAMP signal transduction pathway.     

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.     

Construction of a novel lipolytic fusion biocatalyst GDEst-lip for industrial application

The gene encoding esterase (GDEst-95) from Geobacillus sp. 95 was cloned and sequenced. The resulting open reading frame of 1497 nucleotides encoded a protein with calculated molecular weight of 54.7 kDa, which was classified as a carboxylesterase with an identity of 93–97% to carboxylesterases from Geobacillus bacteria. This esterase can be grouped into family VII of bacterial lipolytic enzymes, was active at broad pH (7–12) and temperature (5–85 °C) range and displayed maximum activity toward short acyl chain p-nitrophenyl (p-NP) esters. Together with GD-95 lipase from Geobacillus sp. strain 95, GDEst-95 esterase was used for construction of fused chimeric biocatalyst GDEst-lip. GDEst-lip esterase/lipase possessed high lipolytic activity (600 U/mg), a broad pH range of 6–12, thermoactivity (5–85 °C), thermostability and resistance to various organic solvents or detergents. For these features GDEst-lip biocatalyst has high potential for applications in various industrial areas. In this work the effect of additional homodomains on monomeric GDEst-95 esterase and GD-95 lipase activity, thermostability, substrate specificity and catalytic properties was also investigated. Altogether, this article shows that domain fusing strategies can modulate the activity and physicochemical characteristics of target enzymes for industrial applications.     

Atypical non-lipid-dependent strains of <Emphasis Type="Italic">Malassezia furfur</Emphasis>

Members of the yeast genus Malassezia, including atypical strains, are lipophilic except for Malassezia pachydermatis. New physiological features that characterize atypical Malassezia strains are mainly associated with alteration in Tween assimilation pattern — such isolates still require lipids for growth. We isolated three non-lipid-dependent strains of Malassezia from patients with diagnosed atopic dermatitis (AD). These isolates could not be identified to the species level via their physiological properties. Phylogenetic trees, based on the D1/D2 regions of the 26S rDNA gene sequences and nucleotide sequences of the ITS1-5.8S-ITS2 rRNA region, showed the isolates to belong to Malassezia furfur. Three non-lipid dependent isolates from AD skin were conspecific, and sequences analysis proved them to be M. furfur.     

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.     

Genetic control of growth and development of yeast Saccharomyces cerevisiae cells. Phenotypic selection of mutants among strains of the Peterhof genetic collection

Results of identifying phenotypes intrinsic to mutations of genes that regulate the activity of the signal transduction pathway of RAS-cyclic adenosinemonophosphate in six strains belonging to the Peterhof genetic collection of the yeast Saccharomyces cerevisiae are presented: an increase or decrease in the amount of the accumulated glycogen, resistance or sensitivity to heat shock and nitrogen starvation, and the growth and viability in media containing unfermentable carbon sources (potassium acetate, ethanol, and glycerol) at temperatures 30 and 70 degrees C. Collectively these phenotypic characteristics in five examined yeast strains can be interpreted as indicating disturbances in the activity of the RAS/cAMP pathway. However, the discrepancies revealed between cellular phenotypes in these strains and in the strains with a decreased or increased activity of the RAS/cAMP pathway did not allow them to be assigned to a particular functional activity group. The inconsistencies between phenotypes detected in this study may be prerequisites for the identification of new genes responsible for this signal transduction pathway or new mutations in the known genes that determine other phenotypic combinations.