Enzyme production-based approach for determining the functions of microorganisms within a community.
(73/325)
The functions of specific microorganisms in a microbial community were investigated during the composting process. Cerasibacillus quisquiliarum strain BLx(T) and Bacillus thermoamylovorans strain BTa were isolated and characterized in our previous studies based on their dominance in the composting system. Strain BLx(T) degrades gelatin, while strain BTa degrades starch. We hypothesized that these strains play roles in gelatinase and amylase production, respectively. The relationship between changes in the abundance ratios of each strain and those of each enzyme activity during the composting process was examined to address this hypothesis. The increase in gelatinase activity in the compost followed a dramatic increase in the abundance ratio of strain BLx(T). Zymograph analysis demonstrated that the pattern of active gelatinase bands from strain BLx(T) was similar to that from the compost. Gelatinases from both BLx(T) and compost were partially purified and compared. Homologous N-terminal amino acid sequences were found in one of the gelatinases from strain BLx(T) and that of compost. These results indicate strain BLx(T) produces gelatinases during the composting process. Meanwhile, the increase in the abundance ratio of strain BTa was not concurrent with that of amylase activity in the compost. Moreover, the amylase activity pattern of strain BTa on the zymogram was different from that of the compost sample. These results imply that strain BTa may not produce amylases during the composting process. To our knowledge, this is the first report demonstrating that the function of a specific microorganism is directly linked to a function in the community, as determined by culture-independent and enzyme-level approaches. (+info)
Biochemical characterization of different genotypes of Paenibacillus larvae subsp. larvae, a honey bee bacterial pathogen.
(74/325)
Paenibacillus larvae subsp. larvae (P. l. larvae) is the aetiological agent of American foulbrood (AFB), the most virulent bacterial disease of honey bee brood worldwide. In many countries AFB is a notifiable disease since it is highly contagious, in most cases incurable and able to kill affected colonies. Genotyping of field isolates of P. l. larvae revealed at least four genotypes (AB, Ab, ab and alpha B) present in Germany which are genotypically different from the reference strain DSM 7030. Therefore, based on these data, five different genotypes of P. l. larvae are now identified with genotype AB standing out with a characteristic brown-orange and circled two-coloured colony morphology. Analysing the metabolic profiles of three German genotypes (AB, Ab and ab) as well as of the reference strain using the Biolog system, a characteristic biochemical fingerprint could be obtained for each strain. Cluster analysis showed that while genotypes Ab, ab and the reference strain DSM 7030 are rather similar, genotype AB is clearly different from the others. Analysis of all isolates for plasmid DNA revealed two different plasmids present only in isolates belonging to genotype AB. Therefore, genotype AB is remarkable in all aspects analysed so far. Future analysis will show whether or not these differences will expand to differences in virulence. (+info)
Cloning and overexpression of the Exiguobacterium sp. F42 gene encoding a new short chain dehydrogenase, which catalyzes the stereoselective reduction of ethyl 3-oxo-3-(2-thienyl)propanoate to ethyl (S)-3-hydroxy-3-(2-thienyl)propanoate.
(75/325)
Exiguobacterium sp. F42 was screened as a producer of an enzyme catalyzing the NADPH-dependent stereoselective reduction of ethyl 3-oxo-3-(2-thienyl)propanoate (KEES) to ethyl (S)-3-hydroxy-3-(2-thienyl)propanoate ((S)-HEES). (S)-HEES is a key intermediate for the synthesis of (S)-duloxetine, a potent inhibitor of the serotonin and norepinephrine uptake carriers. The responsible enzyme (KEES reductase) was partially purified, and the gene encoding KEES reductase was cloned and sequenced via an inverse PCR approach. Sequence analysis of the gene for KEES reductase revealed that the enzyme was a member of the short chain dehydrogenase/reductase family. The probable NADPH-interacting site and 3 catalytic residues (Ser-Tyr-Lys) were fully conserved. The gene was highly expressed in Escherichia coli, and the gene product was purified to homogeneity from the recombinant E. coli by simpler procedures than from the original host. The molecular mass of the purified enzyme was 27,500 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and 55,000 as determined by gel filtration chromatography. Our results show that this enzyme can be used for the practical production of (S)-HEES. (+info)
Cerasibacillus quisquiliarum gen. nov., sp. nov., isolated from a semi-continuous decomposing system of kitchen refuse.
(76/325)
A moderately thermophilic and alkaliphilic bacillus, which had been reported and designated BLx (Haruta et al., 2002), was isolated from a semi-continuous decomposing system of kitchen refuse. Cells of strain BLxT were strictly aerobic, rod-shaped, motile and spore forming. The optimum temperature and pH for growth were approximately 50 degrees C and pH 8-9. Strain BLxT was able to grow at NaCl concentrations from 0.5 to 7.5%, with optimum growth at 0.5% NaCl. The predominant menaquinone was MK-7, and the major fatty acid was iso-C(15 : 0). Phylogenetic analysis showed that strain BLxT was positioned in an independent lineage within the cluster that includes the genera Virgibacillus and Lentibacillus in Bacillus rRNA group 1. Strain BLxT exhibited 16S rDNA similarity of 92.8-94.8% to Virgibacillus species and 92.3% to Lentibacillus salicampi. Phenotypic, chemotaxonomic and phylogenetic analyses supported the classification of strain BLxT in a novel genus and species. Cerasibacillus quisquiliarum gen. nov., sp. nov. is proposed on the basis of phenotypic, chemotaxonomic and phylogenetic data. The type strain is BLxT (DSM 15825T=IAM15044T=KCTC 3815T). (+info)
Anoxybacillus voinovskiensis sp. nov., a moderately thermophilic bacterium from a hot spring in Kamchatka.
(77/325)
A novel moderately thermophilic bacterium, strain TH13T, was isolated from a hot spring in Kamchatka. It was found to be a Gram-positive, facultative aerobe; the straight, non-motile rods grew at 30-64 degrees C (optimum 54 degrees C). The isolate was positive for catalase and oxidase tests and reduced nitrate to nitrite, but was negative for H2S production and growth in more than 3% NaCl (w/v). The isolate grew at pH 7-8, but not at pH values higher than 9. The DNA G+C content was 43.9 mol%. Phylogenetic analysis based on 16S rRNA gene sequencing indicated that strain TH13T was a member of the genus Anoxybacillus. DNA-DNA hybridization revealed a low relatedness (less than 30.2%) between the isolate and its close phylogenetic neighbours Anoxybacillus pushchinoensis and Anoxybacillus flavithermus. On the basis of phenotypic characteristics, phylogenetic data and DNA-DNA hybridization data, it was concluded that the isolate merited classification as a novel species, for which the name Anoxybacillus voinovskiensis sp. nov. is proposed. The type strain of this species is TH13T (=NCIMB 13956T=JCM 12111T). (+info)
Marinibacillus campisalis sp. nov., a moderate halophile isolated from a marine solar saltern in Korea, with emended description of the genus Marinibacillus.
(78/325)
A Gram-positive, motile, round to ellipsoidal, endospore-forming, rod-shaped bacterial strain, SF-57T, was isolated from a marine solar saltern in Korea. This organism grew between 4 and 39 degrees C, with optimum growth at 30 degrees C. Strain SF-57T grew in the presence of 0.5-15.0% NaCl, with optimum growth at 2-3% NaCl. The peptidoglycan type of strain SF-57T was A1alpha linked directly through l-Lys. In strain SF-57TT, menaquinone-7 (MK-7) was the predominant isoprenoid quinone and anteiso-C(15 : 0) was the major fatty acid. The DNA G+C content was 41.8 mol%. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain SF-57T formed a coherent cluster with Marinibacillus marinus, with a bootstrap resampling value of 100%. The level of 16S rRNA gene sequence similarity between strain SF-57T and M. marinus DSM 1297T was 98.9%. The mean DNA-DNA relatedness level between strain SF-57T and the type strain of M. marinus was 20.6%. Based on phenotypic properties, phylogenetic analyses and genomic data, strain SF-57T merits placement in the genus Marinibacillus as a representative of a novel species, for which the name Marinibacillus campisalis sp. nov. is proposed. The type strain is SF-57T (=KCCM 41644T=JCM 11810T). (+info)
Anoxybacillus ayderensis sp. nov. and Anoxybacillus kestanbolensis sp. nov.
(79/325)
Two thermophilic bacilli were isolated from mud and water samples of the Ayder and Kestanbol hot springs in the provinces of Rize and Canakkale, respectively, in Turkey. Strains AB04T and K4T were sporulating, Gram-positive, rod-shaped bacteria. These isolates were moderately thermophilic (with an optimum temperature for growth of 50-55 degrees C), facultative anaerobes able to grow on a wide range of carbon sources including d-glucose, d-raffinose, d-sucrose, D-xylose, D-fructose, L-arabinose, maltose, D-mannose and D-mannitol. Analysis of the 16S rRNA gene sequences showed that these isolates resembled Anoxybacillus flavithermus DSM 2641T and Anoxybacillus gonensis NCIMB 13933T. DNA-DNA hybridization data revealed that thermophilic isolate AB04T has only 51.2 % relatedness to A. flavithermus, 45.1 % relatedness to Anoxybacillus pushchinoensis and 68.6 % relatedness to A. gonensis. Thermophilic isolate K4T showed only 60.4 % relatedness to A. flavithermus, 42.9 % relatedness to A. pushchinoensis and 38.5 % relatedness to A. gonensis. On the basis of the DNA-DNA hybridization data, isolates AB04T and K4T are not related to A. flavithermus DSM 2641T, A. pushchinoensis DSM 12423T or A. gonensis NCIMB 13933T at the species level, but show relatedness to one another of 40.5 %. On the basis of the data presented, it is proposed that strains AB04T (= NCIMB 13972T = NCCB 100050T) and K4T (= NCIMB 13971T = NCCB 100051T) be designated as the type strains of Anoxybacillus ayderensis sp. nov. and Anoxybacillus kestanbolensis sp. nov., respectively. (+info)
Effect of dimer dissociation on activity and thermostability of the alpha-glucuronidase from Geobacillus stearothermophilus: dissecting the different oligomeric forms of family 67 glycoside hydrolases.
(80/325)
The oligomeric organization of enzymes plays an important role in many biological processes, such as allosteric regulation, conformational stability and thermal stability. alpha-Glucuronidases are family 67 glycosidases that cleave the alpha-1,2-glycosidic bond between 4-O-methyl-D-glucuronic acid and xylose units as part of an array of hemicellulose-hydrolyzing enzymes. Currently, two crystal structures of alpha-glucuronidases are available, those from Geobacillus stearothermophilus (AguA) and from Cellvibrio japonicus (GlcA67A). Both enzymes are homodimeric, but surprisingly their dimeric organization is different, raising questions regarding the significance of dimerization for the enzymes' activity and stability. Structural comparison of the two enzymes suggests several elements that are responsible for the different dimerization organization. Phylogenetic analysis shows that the alpha-glucuronidases AguA and GlcA67A can be classified into two distinct subfamilies of bacterial alpha-glucuronidases, where the dimer-forming residues of each enzyme are conserved only within its own subfamily. It seems that the different dimeric forms of AguA and GlcA67A represent the two alternative dimeric organizations of these subfamilies. To study the biological significance of the dimerization in alpha-glucuronidases, we have constructed a monomeric form of AguA by mutating three of its interface residues (W328E, R329T, and R665N). The activity of the monomer was significantly lower than the activity of the wild-type dimeric AguA, and the optimal temperature for activity of the monomer was around 35 degrees C, compared to 65 degrees C of the wild-type enzyme. Nevertheless, the melting temperature of the monomeric protein, 72.9 degrees C, was almost identical to that of the wild-type, 73.4 degrees C. It appears that the dimerization of AguA is essential for efficient catalysis and that the dissociation into monomers results in subtle conformational changes in the structure which indirectly influence the active site region and reduce the activity. Structural and mechanistic explanations for these effects are discussed. (+info)