Journal of General and Applied Microbiology最新文献

S-adenosylmethionine (SAM) is an important biomolecule that mainly acts as a methyl donor and plays many roles in a variety of biological functions. SAM is also required for the biosynthesis of valuable methylated compounds, but its supply is a bottleneck for these biosynthetic pathways. To overcome this bottleneck and to reconfigure SAM homeostasis, a high-throughput sensing system for changes in intracellular SAM availability is required. We constructed a plasmid that can detect the factors that can alter SAM availability using minimal components. It does so by placing a fluorescent protein under a promoter controlled by endogenous MetJ, a transcription factor that represses its own regulons upon binding with SAM. Next, to validate SAM-responsive behavior, we systematically reconstructed 10 synthetic promoters with different positions and with different number of metbox sites. We found that a position between the -35 box and the -10 box was the most effective for repression and that this setup was suitable for detecting the genetic or environmental factors that can deplete and recover the intracellular SAM availability. Overall, the response patterns of the synthetic MetJ-regulated promoters characterized in this study may be useful for the development of better SAM biosensing systems.

We investigated the presence and functionality of the carbon storage regulator (Csr) system in Aeromonas salmonicida SWSY-1.411. CsrA, an RNA-binding protein, shared 89% amino acid sequence identity with Escherichia coli CsrA. CsrB/C sRNAs exhibited a typical stem-loop structure, with more GGA motifs, which bind CsrA, than E. coli. CsrD had limited sequence identity with E. coli CsrD; however, it contained the conserved GGDEF and EAL domains. Functional analysis in E. coli demonstrated that the Csr system of A. salmonicida influences glycogen biosynthesis, biofilm formation, motility, and stability of both CsrB and CsrC sRNAs. These findings suggest that in A. salmonicida, the Csr system affects phenotypes like its E. coli counterpart. In A. salmonicida, defects in csr homologs affected biofilm formation, motility, and chitinase production. However, glycogen accumulation and protease production were unaffected. The expression of flagellar-related genes and chitinase genes was suppressed in the csrA-deficient A. salmonicida. Northern blot analysis indicated the stabilization of CsrB and CsrC in the csrD-deficient A. salmonicida. Similar to that in E. coli, the Csr system in A. salmonicida comprises the RNA-binding protein CsrA, the sRNAs CsrB and CsrC, and the sRNA decay factor CsrD. This study underscores the conservation and functionality of the Csr system and raises questions about its regulatory targets and mechanisms in A. salmonicida.

Phthalates esters (PAEs) are a kind of polymeric material additives widely been added into plastics to improve products' flexibility. It can easily cause environmental pollution which are hazards to public health. In this study, we isolated an efficient PAEs degrading strain, Janthinobacterium sp. E1, and determined its degradation effect of di-2-ethylhexyl phthalate (DEHP) under stress conditions. Strain E1 showed an obvious advantage in pollutants degradation under various environmental stress conditions. Degradation halo clearly occurred around the colony of strain E1 on agar plate supplemented with triglyceride. Strain E1's esterase is a constitutively expressed intracellular enzyme. The esterase purified from strain E1 showed a higher catalytic effect on short-chain PAEs than long-chain PAEs. The input of DEHP, DBP (dibutyl phthalate) and DMP (dimethyl phthalate) into the tested soil did not change the species composition of soil prokaryotic community, but altered the dominant species in specific environmental conditions. And the community diversity and richness decreased to a certain extent. However, the diversity and richness of the microbial community were improved after the contaminated soil was treated with the strain E1. Our results also suggested that strain E1 exhibited a tremendous potential in environmental bioremediation in the real environment, which provides a new insight into the elimination of the pollutants contamination in the urban environment.

Zn-deficiency, a global health challenge affects one-third of the world population. Zn-biofertilizer offer an efficient and cost-effective remedy. As Zn-biofertilizer can improve plant growth and grain's Zn-content ensuring improved dietary Zn-supply. This study sought to understand how silver and TiO₂ nanoparticles in the rhizosphere affect the activity of Zn-solubilization bacteria (ZSB) and plant growth. Two ZSB strains Bacillus sp. D-7 and Pseudomonas sp. D-117 with excellent Zn-solubilization efficiency of 254 and 260%, respectively were isolated and characterized using polyphasic characterization including 16S rRNA gene sequencing to formulate an effective Zn-biofertilizer. The plant growth promoting activity of this biofertilizer in Mung bean was checked in the presence and absence of various doses of TiO₂ and Ag-NPs and was compared with plant grown without biofertilizer. The change in rate of seed germination, vegetative growth (shoot and root length, fresh and dry weight), photosynthetic pigment and Zn-content was checked. Lower doses of nanomaterials (50 and 100 mg kg⁻¹ soil) slightly promoted the plant growth compared to control. While, higher doses (200 and 400 mg kg⁻¹ soil) inhibited the growth. A maximum decrease of shoot length, root length, fresh-weight, and dry-weight of 57.1, 53.9, 53.1, and 10.4% respectively was observed with 400 mg kg⁻¹ of Ag-NPs. However, in the presence of ZSB, the decrease at the same Ag-NP concentration was 41.6, 31.5, 27.4, and 6.6, respectively. These results strongly suggest that Zn-solubilizing bacteria improve resilience to nanoparticles toxicity and helps in Zn fortification in Mung bean even under nanomaterial stress.

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.

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.

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.