Journal of General and Applied Microbiology最新文献

There are a number of reporter systems that are useful for gene expression analysis in bacteria. However, at least in Salmonella, a versatile and simple luciferase reporter system that can be integrated precisely behind a promoter or gene of interest on a chromosome is not currently available. The luciferase operon luxCDABE from Photorhabdus luminescens has several advantages, including brightness, wide linear range, absence in most bacteria, stability at high temperature, and no substrate addition required for the assay. Here, a conjugation-mediated site-specific single-copy luciferase fusion system is developed. A reporter plasmid containing the conditional replication origin R6Kgγ, FRT-luxCDABE, and Km^R marker was designed to be incorporated into the FRT site behind the promoter or gene of interest on the chromosome in cells expressing FLP. However, when this reporter plasmid was electroporated directly into such a S. enterica strain, no colonies appeared, likely due to the low transformation efficiency of this relatively large plasmid DNA. Meanwhile, the same reporter plasmid was successfully introduced and launched as an insert of an FRT-containing conjugative transfer plasmid from a mating E. coli strain to the same recipient S. enterica strain, as well as Citrobacter koseri. RcsB-dependent inducible luminescence from the constructed wzc-luxCDABE strains was confirmed. This system is feasible for detecting very low levels of transcription, even in Gram-negative bacterial species that are relatively difficult to genetically manipulate.

Proteolytic enzymes stand out as the most widely employed category utilized in manufacturing industry. A new protease was separated from Planococcus sp.11815 strain and named as nprS-15615 in this research. The gene of this protease has not been reported, and its enzymatic properties have been studied for the first time. To enhance enzyme production, the Planococcus sp. protease gene was expressed in Bacillus licheniformis 2709. The expression level of nprS-15615 was observed under the control of regulatory elements P_aprE. nprS-15615 protease activity reached 1186.24±32.87 U/mL after 48 hours of cultivation in shake flasks which was nearly four times the output of the original bacteria (291.38±25.73U/mL). The optimum temperature and pH of the recombinant protease were 30 ℃ and 8.0, respectively.The enzyme exhibited the highest capacity for hydrolyzing casein and demonstrated resilience towards a NaCl concentration of 10.0% (wt/v). Furthermore, in the presence of 0.5% surfactants, the recombinant protease activity can maintain above 75%, and with the existence of 0.5% liquid detergents, there was basically no loss of enzyme activity which indicated that nprS-15615 had good compatibility with surfactants and liquid detergents. In addition, npS-15615 performed well in the washing experiment, and the washing effect at 20 ℃ can be significantly improved by adding crude enzyme solution in the washing process.

The fission yeast Schizosaccharomyces pombe ecl family genes respond to various starvation signals and induce appropriate intracellular responses, including the extension of chronological lifespan and induction of sexual differentiation. Herein, we propose that the colonization of hemocoel 1 (COH1) protein of Metarhizium robertsii, an insect-pathogenic fungus, is a functional homolog of S. pombe Ecl1 family proteins.

Endophytic actinobacteria are known to produce various enzymes with potential industrial applications. Alpha-amylase is an important class of industrial enzyme with a multi-dimensional utility. The present experiment was designed to characterize a moderately thermostable α-amylase producing endophytic Streptomyces mobaraensis DB13 isolated from Costus speciosus (J. Koenig) Sm. The enzyme was purified using 60% ammonium sulphate precipitation, dialysis, and Sephadex G-100 column chromatography. Based on 12% SDS-PAGE, the molecular weight of the purified α-amylase was estimated to be 55 kDa. The maximum α-amylase activity was achieved at pH 7.0, 50°C and it retained 80% of its activity at both pH 7.0 and 8.0 after incubation for 2 h. The α-mylase activity is strongly enhanced by Ca²⁺, Mg²⁺, and inhibited by Ba²⁺. The activity remains stable in the presence of Tween-80, SDS, PMSF, and Triton X-100; however, β-mercaptoethanol, EDTA, and H₂O₂ reduced the activity. The kinetic parameters K_m and V_max values for this α-amylase were calculated as 2.53 mM and 29.42 U/mL respectively. The α-amylase had the ability to digest various raw starches at a concentration of 10 mg/mL at pH 7.0, 50°C, where maize and rice are the preferred substrates. The digestion starts after 4 h of incubation, which reaches maximum after 48 h of incubation. These results suggest that S. mobaraensis DB13 is a potential source of moderately thermostable α-amylase enzyme, that effciently hydrolyzes raw starch. It suggesting that this α-amylase is a promising candidate to be use for industrial purposes.

Phytophthora species are highly destructive soilborne oomycetes pathogens that spread through infested soil and water. Ochrobactrum pseudogrignonense NC1 has been shown to inhibit plant parasitic nematodes via volatile organic compounds (VOCs). In this study, we investigated the inhibitory effect of O. pseudogrignonense NC1 against four Phytophthora species on agar plates and in vivo bioassay. We found that NC1 significantly inhibited the mycelial growth and zoospore production of all four species of Phytophthora in a dose-dependent manner. The half maximal inhibitory concentration (IC₅₀) values for inhibition of mycelial growth (or zoospore production) were 26% (14.8%), 18.9% (14.2%), 20.3% (8.3%) and 46.9% (4%) for Phytophthora capsici Leonian, Phytophthora infestans, Phytophthora parasitica var. nicotiana and Phytophthora sojae, respectively. The biocontrol efficiency of NC1 was 46.3% in pepper seedlings against P. capsici, almost 100% in potato tubers against P. infestans, 60% in tomato leave against P. parasitica and 100% in soybean leave against P. sojae, respectively. Our findings suggest that O. pseudogrignonense NC1 has great potential as a biocontrol agent for managing Phytophthora diseases.

Plant-derived phenolic gallic acid (GA) is an important raw material for antioxidants and food additives. Efforts to ferment GA using microbial processes have aimed at minimizing production costs and environmental load using enzymes that hydroxylate p-hydroxybenzoate and protocatechuate (PCA). Here, we found a p-hydroxybenzoate hydroxylase (PobA) in the bacterium Hylemonella gracilis NS1 (HgPobA) with 1.5-fold more hydroxylation activity than that from Pseudomonas aeruginosa PAO1 and thus converted PCA to GA more efficiently. The PCA hydroxylation activity of HgPobA was improved by introducing the amino acid substitutions L207V/Y393F or T302A/Y393F. These mutants had 2.9- and 3.7-fold lower K_m^app for PCA than wild-type HgPobA. An Escherichia coli strain that reinforces shikimate pathway metabolism and produces HgPobA when cultured for 60 h generated 0.27 g L^-1 of GA. This is the first report of fermenting glucose to generate GA using a natural enzyme from the PobA family. The E. coli strain harboring the HgPobA L207V/Y393F mutant increased GA production to 0.56 g L^-1. During the early stages of culture, GA was fermented at a 10-fold higher rate by a strain producing either HgPobA L207V/Y393F or T302A/Y393F compared with wild-type HgPobA, which agreed with the high k_cat^app/K_m^app PCA values of this mutant. We enhanced a PobA isozyme and its PCA hydroxylating function to efficiently and cost-effectively ferment GA.

In tidal flats, which are located at the transition zone between terrestrial and marine ecosystems, environmental factors such as temperature, sediment particle size, and tidal range exhibit geographic variation. Accordingly, the composition and structure of the microbial communities in the tidal flats are likely to vary in geographically different habitats. To clarify these differences with environmental factors causing them, we analyzed microbial communities consisting of bacteria and ciliates in sediments collected from nine tidal flats in geographical diverse region from Hokkaido to Kagoshima, Japan. The results confirmed that the community structures of bacteria and ciliophora in tidal flat sediments differed at the geographical scale of the Japanese archipelago. However, the variation could not be explained by the physical distance between the tidal flats nor by the differences in the trophic conditions among the tidal flats. Instead, the OTU richness of both the bacterial and ciliophoran communities was significantly related to the tidal range. The results also showed that bacteria and ciliophora tended to form similar communities among the tidal flats with similar median particle sizes. Furthermore, ciliophoran communities were similar among the tidal flats with similar bacterial communities. The results suggest that bacteria and ciliophora interact each other through trophic relationships or physical and chemical processes in the sediment habitats.

Biological pretreatment using microbial enzymes appears to be the most promising pre-treatment technology for the breakdown of recalcitrant lignin structure. This research focuses on the identification and characterization of lignin-depolymerizing enzymes in Bacillus subtilis strain S11Y, previously isolated from palm oil wastes in Malaysia. The draft genome sequences of this highly lignin-depolymerizing strain revealed that the genome lacked any of the well-known dye-decolorizing peroxidase or catalase-peroxidase that are commonly reported to be involved in lignin depolymerization by bacteria, indicating that strain S11Y has distinct sets of potential lignin depolymerization genes. The oxidative stress-related enzymes Cu/Zn type-superoxide dismutase (Sod2) and a heme-containing monofunctional catalase (Kat2) were identified in the genome sequences that are of interest. Their lignin-depolymerizing ability were evaluated by treating Alkali lignin (AL) with each enzyme and their degradation ability were evaluated using gel-permeation chromatography (GPC), ultrahigh-pressure liquid chromatography-mass spectrometry (UHPLC/MS), and gas chromatography-mass spectrometry (GC/MS), which successfully proved lignin depolymerizing ability. Successful evaluation of lignin depolymerizing enzymes can be applicable for lignin pretreatment process in green energy production and generation of valuable chemicals in bio-refinery.

5-Aminolevulinic acid (ALA) is a precursor of heme and a natural amino acid synthesized in the cells of most living organisms. Currently, ALA is used as an ingredient in pharmaceuticals, supplements, cosmetics, feed, fertilizers, and other products. ALA is mainly produced by industrial fermentation by the photosynthetic bacterium Rhodobacter sphaeroides. In this study, we tried to improve the ALA productivity by R. sphaeroides using a genetic strategy to highly express ALA synthase (ALAS) genes. We inserted a constitutive promoter (P_rrnB or P_{rsp_7571}) upstream of genes encoding ALAS (hemA and/or hemT) to construct strains that constitutively express ALAS. The highest transcript levels of hemA were observed in the strain where P_rrnB was inserted into the hemA promoter region and were 3.5-fold higher than those in the wild-type. The highest transcript levels of hemT were observed in the strain where P_rrnB was inserted into the hemT promoter region and were 46-fold higher than those in the wild-type. The maximum ALAS activity was observed in crude cell extracts of the strain where P_rrnB was inserted into the hemT promoter region under optimized growth conditions that was 2.7-fold higher than that in the wild type. This strain showed 12-fold accumulation of ALA compared to the wild-type. Thus, we improved ALA productivity without using exogenous DNA sequences. In the future, further improvement in ALA productivity may be expected by applying this approach to current industrial ALA-producing bacteria.

　Microalgae are promising cell factories for producing value-added products. Large-scale microalgal cultivation suffers from invasion by contaminating microorganisms. Since most contaminating organisms cannot utilize phosphite as a unique phosphorus source, phosphite-utilizing ability may provide a growth advantage against contaminating organisms and solve this problem. Studies showed that microorganisms, typically unable to metabolize phosphite, can utilize phosphite by expressing exogenous phosphite dehydrogenase. Here, we constructed Cyanidioschyzon merolae strains introduced with the phosphite dehydrogenase gene, ptxD, from Ralstonia sp. 4506. The ptxD-introduced strains grew in a phosphite-dependent manner, with the phosphite-related growth rate almost matching that with phosphate as sole phosphorus source.