An extracellular cutinase from Pseudomonascepacia NRRL B 2320 was purified to apparent homogeneity. Upon biochemical characterization, the purified cutinase was found to be tolerant to organic solvents and surfactants under assay conditions. The molecular mass of cutinase was found to be 26.25 kDa by MALDI-TOF-MS analysis. The enzyme was able to show activity towards synthetic esters of chain length C4‒C16. The activity of cutinase was enhanced by mono cations and various effectors, whereas it was moderately inhibited by various divalent cations and serine blocking reagent, phenyl methyl sulphonyl fluoride (PMSF). The optimal pH and temperature for highest activity were found to be 7.9 and 36.5°C, respectively. An overall 1.42-fold increase in activity was observed after optimization of both assay and process conditions. The exposure of hydrophobic amino acid to an aqueous environment and change in secondary structure of cutinase was observed from thermodynamic parameters (ΔH*, ΔS*), fluorescence and circular dichorism spectra during the deactivation process. Two cutinase encoding genes were identified in P.cepacia, cloned and expressed in E.coli BL21 (DE3).
The bacterial strain C1112T was isolated from seafood processing wastewater collected from a treatment pond of the seafood factory in Songkhla Province, Thailand. Phylogenetic analysis based on concatenated sequences from the 16S rRNA gene and five housekeeping genes, fusA, lepA, leuS, gyrB and ileS respectively showed that the strain C1112T belonged to the genus Providencia, and share 91.75% similarity with P. stuartii DSM 4539T. DNA-DNA hybridization between the strain C1112T and P. stuartii KCTC 2568T was 48.1% relatedness. Moreover, some results from biochemical properties indicated that the strain C1112T was distinguished from the phylogenetically closest relatives. The major fatty acids of the strain C1112T were C16:0, iso-C15:0, C14:0 and C17:0 cyclo and the DNA G+C content was 41 mol%. Based on the genotypic and phenotypic considerations, it should be classified as a novel species of the genus Providencia for which the name Providencia thailandensis sp. nov. is proposed. The type strain is C1112T (= KCTC 23281T =NBRC 106720T).
Three strains (K59T, K60 and K70 T) representing two novel yeast species were isolated from the external surface of leaves of different wine grape (Vitis vinifera) plants, which were collected from the Kanchanaburi Research Station (N14°07′15.1″ E099°19′05.6″), Wang Dong Sub-district, Mueang District, Kanchanaburi Province, Thailand, by an enrichment technique. The sequences of the D1/D2 domain of the large subunit (LSU) rRNA gene of two strains (K59T and K60) were identical and differed from that of strain K70T. In terms of pairwise sequence similarity of the D1/D2 domain, the closest species to the three strains was Candida asparagi but with 2.3% nucleotide substitutions for strains K59T and K60, and 2.1% nucleotide substitutions for strain K70T. On the basis of morphological, biochemical, physiological and chemotaxonomic characteristics and the sequence analysis of the D1/D2 domain of the large subunit (LSU) rRNA gene, the three strains were assigned to be two novel Candida species. Two strains (K59T and K60) were assigned as Candida phyllophila sp. nov. (type strain K59T=BCC 42662T=NBRC 107776T=CBS 12671T). Candida vitiphila sp. nov. is proposed for strain K70T (=BCC 42663T=NBRC 107777T=CBS 12672T).
A rubber-degrading bacterium, the strain NS21, that was isolated from a soil sample in a botanical garden in Japan (Imai et al., 2011) was examined by phenotypic, phylogenetic and chemotaxonomic approaches to determine its taxonomic position. The strain NS21 was motile, possessing a single polar flagellum and a facultatively anaerobic straight rod. Analysis of the 16S rRNA and gyrB gene sequences of NS21 revealed a close relationship to the genus Rhizobacter. The predominant quinone type was Q-8. The G+C content of the NS21 genomic DNA was 70.8 mol%. The major fatty acids were C16:0, C17:0 cyclo, C18:1ω7c and C16:1ω7c and/or iso-C15:0 2-OH. C12:0 2-OH was present. The DNA-DNA hybridization experiments indicated that the DNA relatedness values of the strain NS21 to R. dauci H6T and R. fulvus Gsoil 322T were lower than 24%. The phenotypic characteristics showed obvious dissimilarities when compared to closely related species. On the basis of these taxonomic properties, a novel species is proposed as Rhizobacter gummiphilus sp. nov., with the strain NS21T (NBRC 109400T, BCC 58006T) as the type strain. The emended description of the genus Rhizobacter was also presented.
Corynebacterium glutamicum is a Gram-positive, rod-shaped, aerobic bacterium used for the fermentative production of L-glutamate. LldR (NCgl2814) is known as a repressor for ldhA and lldD encoding lactate dehydrogenases. LdhA is responsible for production of L-lactate, while LldD is for its assimilation. Since L-lactate production was observed as a by-product of glutamate production under biotin-limited conditions, LldR might play a regulatory role in the glutamate metabolism. Here for the first time, we investigated effects of overproduction or deletion of LldR on the glutamate metabolism under biotin-limited conditions in C. glutamicum. It was found that glutamate production under biotin-limited conditions was decreased by overproduction of LldR. In the wild-type cells, L-lactate was produced in the first 24 h and it was re-consumed thereafter. On the other hand, in the overproduced cells, L-lactate was produced like the wild type, but it was not re-consumed. This means that L-lactate assimilation, which is catalyzed by LldD, was suppressed by the overproduction of LldR, but L-lactate production, which is catalyzed by LdhA, was not affected, indicating that LldR mainly controls the expression of lldD but not of ldhA under biotin-limited conditions. This was confirmed by quantitative real-time RT-PCR. From these results, it is suggested that L-lactate metabolism, which is controlled by LldR, has a buffering function of the pyruvate pool for glutamate production.
Bacterial strain GB-01 was isolated from abamectin-contaminated soils by continuous enrichment culture. The preliminary identification of strain GB-01 as a Burkholderia species was based mainly on simple biochemical and substrate utilization tests; however, these tests alone cannot accurately differentiate all the species within the genus Burkholderia. The strain GB-01 was subjected to taxonomic analysis through a polyphasic approach, in which phenotypic, genotypic, and phylogenetic information was gathered to conclude the classification of this microbe. Phenotypic information comes from basic bacteriological tests and substrate utilization patterns using the Biolog GN2 MicroPlating system and automated miniature biochemical test kits, i.e. API 20 NE, ID 32 GN and API 50 CH, as well as analyzing the whole cell fatty acid profile. Genotypic information was gathered from whole genome DNA base composition (G+C mol%), and DNA-DNA hybridization with its closest species, while phylogenetic information was collected from the comparative analysis of 16S rRNA and recA gene sequences. The results of polyphasic analysis concluded that strain GB-01 is an atypical strain of the Burkholderia diffusa species.
Because of the growing market for sports drinks, prevention of yeast contamination of these beverages is of significant concern. This research was performed to achieve insight into the physiology of yeast growing in sports drinks through a genome-wide approach to prevent microbial spoilage of sports drinks. The genome-wide gene expression profile of Saccharomyces cerevisiae growing in the representative sports drink was investigated. Genes that were relevant to sulphate ion starvation response were upregulated in the yeast cells growing in the drink. These results suggest that yeast cells are suffering from deficiency of extracellular sulphate ions during growth in the sports drink. Indeed, the concentration of sulphate ions was far lower in the sports drink than in a medium that allows the optimal growth of yeast. To prove the starvation of sulphate ions of yeast, several ions were added to the beverage and its effects were investigated. The addition of sulphate ions, but not chloride ions or sodium ions, to the beverage stimulated yeast growth in the beverage in a dose-dependent manner. Moreover, the addition of sulphate ions to the sports drink increased the biosynthesis of sulphur-containing amino acids in yeast cells and hydrogen sulphide in the beverage. These results indicate that sulphate ion concentration should be regulated to prevent microbial spoilage of sports drinks.