Journal of General and Applied Microbiology最新文献

Kusaya shows a high preservability due to the microorganism-derived antibiotics contained in kusaya gravy, which is important for kusaya manufacturing. However, the antimicrobial compounds and its producing bacteria, as well as the antimicrobial activity of the kusaya gravy itself, have remained unknown. In this study, we isolated antibiotic-producing bacteria of the genus Streptomyces from kusaya gravy from Hachijojima and found that they produced antibacterial substances against various fungi and bacteria. In addition, we demonstrated that kusaya gravy itself shows antimicrobial activity, which was consistent with that of the isolates. This is the first report to directly indicate that kusaya gravy contains microorganism-derived antibiotics, which are assumed to be produced by actinomycetes.

In cyanobacteria that perform oxygenic photosynthesis, alternative sigma factors can play critical roles in environmental acclimation at the transcriptional initiation step. Here, we found in Synechococcus elongatus PCC 7942 that transcription of the pilA1 gene, encoding the type IV pilin, is dependent on one of the group 3 sigma factors, SigF1. We analyzed the promoter sequence determinants and proposed herein that the -10 and -35 boxes upstream of the transcriptional start site are critical for transcription. Interestingly, while the pilA1 promoter is activated by illumination, RNA polymerase containing SigF1 is already located on the promoter region under dark conditions, prior to illumination. This strongly suggests that promoter activation by light follows the recruitment of RNA polymerase during transcriptional initiation.

Gene expression controllers are useful tools for microbial production of recombinant proteins and valued bio-based chemicals. Despite its usefulness, they have rarely been applied to the practical industrial bioprocess, due to the lack of systems that meets the three requirements: low cost, safety, and tight control, to the inducer molecules. Previously, we have developed the high-spec gene induction system controlled by safe and cheap inducer choline. However, the system requires relatively high concentration (~100 mM) of choline to fully induce the gene under control. In this work, we attempted to drastically improve the sensitivity of this induction system to further reduce the induction costs. To this end, we devised a simple circuit which couples gene induction system with positive-feedback loop (P-loop) of choline importer protein BetT. After the tuning of translation level of BetT (strength of the P-loop) and deletion of endogenous betI (noise sources), highly active yet stringent control of gene expression was achieved using about 100 times less amount of inducer molecules. The choline induction system developed in this study has the lowest basal expression, the lowest choline needed to be activated, and the highest amplitude of induction as the highest available promoter such as those known as P_T5 system. With this system, one can tightly control the expression level of genes of interest with negligible cost for inducer molecule, which has been the bottleneck for the application to the large-scale industrial processes.

Fusarium meridionale is one of the pathogens causing maize ear rot, it produce bioactive secondary metabolites may threaten humans food safty, however, the production mechanism of the secondary metabolites and their interaction with maize ear remains poorly understood. To facilitate related studies, we sequenced and assembled the genome of F. meridionale strain JX18-4. The size of F. meridionale JX18-4 genome is 37.11 Mbp, include four nuclear chromosome contigs that consists of 11920 coding genes and one mitochondrial contig. 95.64% gene synteny collinearity was found between the assembly and the reference genomes F. graminearum strain PH-1. Compared to the sequences of seconary matabolism gene clusters sequences reported previously, the stain JX18-4 was predicted potential producing 8 clusters, including nivalenol, zearalenone, aurofusarin, fusarielin, fusaristatin A, fusarin, fusarubin and butenolide. This study aims to reveal the molecular mechanism of secondary metabolites producing, and the genomic information of JX18-4 will provide resources for the study of biological control mechanisms and plant-microbe interactions.

In recent years, a convenient phosphatase-coupled sulfotransferase assay method has been proven to be applicable to most sulfotransferases. The central principle of the method is that phosphatase specifically degrades 3'-phosphoadenosine-5'-phosphate (PAP) and leaves 3'-phosphoadenosine-5'-phosphosulfate (PAPS). Our group previously acquired a yeast 3',5'-bisphosphate nucleotidase (YND), which showed a higher catalytic activity for PAP than PAPS and could be a potential phosphatase for the sulfotransferase assay. Here, we obtained a beneficial mutant of YND with markedly improved substrate specificity towards PAP via rational design. Of 9 chosen mutation sites in the active site pocket, the mutation G236D showed the best specificity for PAP. After optimization of the reaction conditions, the mutant YND^G236D displayed a 4.8-fold increase in the catalytic ratio PAP/PAPS compared to the wild-type. We subsequently applied YND^G236D to the assay of human SULT1A1 and SULT1A3 with their known substrate 1-naphthol, indicating that the mutant could be used to evaluate sulfotransferase activity by colorimetry. Analysis of the MD simulation results revealed that the improved substrate specificity of the mutant towards PAP may stem from a more stable protein conformation and the changed flexibility of key residues in the entrance of the substrate tunnel. This research will provide a valuable reference for the development of efficient sulfotransferase activity assays.

Cellulose is an abundant biomass on the planet. Various cellulases from environmental microbes have been explored for industrial use of cellulose. Marine fish intestine is of interest as one source of new enzymes. Here, we report the discovery of genes encoding two β-glucosidases (Bgl3A and Bgl3B) and four endo-1,4-β-glucanases (Cel5A, Cel8, Cel5B, and Cel9) as part of the genome sequence of a cellulolytic marine bacterium, Microbulbifer sp. Strain GL-2. Five of these six enzymes (excepting Cel5B) are presumed to localize to the periplasm or outer membrane. Transcriptional analysis demonstrated that all six genes were highly expressed in stationary phase. The transcription was induced by cello-oligosaccharides rather than by glucose, suggesting that the cellulases are produced primarily for nutrient acquisition following initial growth, facilitating the secondary growth phase. We cloned the genes encoding two of the endo-1,4-β-glucanases, Cel5A and Cel8, and purified the corresponding recombinant enzymes following expression in Escherichia coli. The activity of Cel5A was observed across a wide range of temperatures (10-40 ˚C) and pHs (6-8). This pattern differed from those of Cel8 and the commercial cellulase Enthiron, both of which exhibit decreased activities below 30 ˚C and at alkaline pHs. These characteristics suggest that Cel5A might find use in industrial applications. Overall, our results reinforce the hypothesis that marine bacteria remain a possible source of novel cellulolytic activities.

To enhance the value of surimi, efforts have been made to develop a fermentation method with lactic acid bacteria (LAB) to proteolyze fish protein. However, fermenting unheated surimi poses a spoilage risk due to its high bacterial content. Surimi heat treatment can prevent spoilage, but gel formation induced by heating introduces another technical issue: it hinders uniform fermentation. Thus, this study aims to observe the proteolysis and enhance the functionality of seafood product through lactic acid fermentation of kamaboko, a heated surimi. Upon analyzing the kamaboko fermented with Lactobacillus helveticus JCM1004, we observed that LAB produced protease, resulting in the degradation of myosin heavy chain and actin during fermentation. Lactic acid fermentation significantly augmented the peptide content of kamaboko, subsequently elevating the angiotensin Ⅰ-converting enzyme (ACE) inhibitory activity in 200-fold diluted extract of fermented kamaboko to approximately 70% and higher. Notably, our investigation revealed that proteolysis was confined to the surface of kamaboko, as evidenced by SDS-PAGE analysis. This observation implies that the surface area of kamaboko influences the ACE inhibitory activity. Through a comparative analysis of various bacterial strains, we demonstrated that the increase in ACE inhibitory activity is contingent on the protease generated by LAB. These results suggest that LAB-mediated proteolysis of fish proteins liberates bioactive peptides, thereby manifesting in the ACE inhibitory activity. In summary, this study underscores that the fermentation of kamaboko employing proteolytic LAB holds promise in the development of novel functional seafood products.

The glycoside hydrolase (GH) 71 α-1,3-glucanase (Agn1p) from Schizosaccharomyces pombe consists of an N-terminal signal sequence and a catalytic domain. Meanwhile, the GH87 α-1,3-glucanase (Agl-KA) from Bacillus circulans KA-304 consists of an N-terminal signal sequence, a first discoidin domain (DS1), a carbohydrate-binding module family 6 (CBM6), a threonine and proline repeat linker (TP), a second discoidin domain (DS2), an uncharacterized domain, and a catalytic domain. DS1, CBM6, and DS2 exhibit α-1,3-glucan binding activity. This study involved genetically fusing TP, DS1, CBM6, TP, and DS2 to the C-terminus of Agn1p, generating the fusion enzyme Agn1p-DCD. The fusion enzyme was then expressed in Escherichia coli and purified from the cell-free extract. Agn1p-DCD and Agn1p exhibited similar characteristics, such as optimal pH, optimal temperature, pH stability, and thermostability. Insoluble α-1,3-glucan (1%) hydrolyzing assay showed that Agn1p-DCD and Agn1p released approximately 7.6 and 5.0 mM of reducing sugars, respectively, after 48 h of reaction. Kinetic analysis and an α-1,3-glucan binding assay indicated that the addition of DS1, CBM6, and DS2 enhanced the affinity of Agn1p for α-1,3-glucan. Moreover, Agn1p-DCD contributed to enhancing the fungal growth inhibition activity when combined with a mixture of GH19 chitinase and GH16 β-1,3-glucanase.

Fumarase is an enzyme catalyzing reversible reaction between fumarate and L-malate in the citric acid cycle. Fumarase is used in the industrial production of L-malate, and its immobilization is required for reuse of the fumarases to reduce the cost. Accordingly, understanding the properties of immobilized fumarase is crucial, and several groups report on the storage stability and kinetic parameters of immobilized fumarase. Here we have immobilized fumarase from the thermophilic red alga Cyanidioschyzon merolae (CmFUM) on ceramic beads and investigated its biochemical and physical properties. CmFUM demonstrated sufficient stability and reusability for industry use after immobilization. Notably, the thermostability was dramatically enhanced through immobilization. The K_m value and k_cat of immobilized CmFUM for fumarate were 1.7 mM and 22.7 s^-1 respectively. The K_m value for fumarate was lower than that of other reported immobilized fumarases, indicating a high substrate affinity of immobilized CmFUM. Furthermore, the enhanced stability resulting from immobilization partially compensated for the decrease in activity. The high affinity towards fumarate and good thermostability of immobilized CmFUM revealed in this study are advantageous traits for improving enzyme-mediated isomer-specific L-malate production.

The culture filtrates of the predominant bacterial strains isolated from soil samples have been shown to increase the microbial colony counts on agar plates used for the isolation of uncultured bacteria. One of the factors in the culture filtrates responsible for this increase was identified to be superoxide dismutase (SOD). The generation of reactive oxygen species (O₂^-, H₂O₂, and ・OH) was detected from conventional laboratory agar media. The use of agar media supplemented with radical scavengers (SOD, catalase, ascorbic acid, or rutin) effectively increased the colony counts and kinds of microbial strains that grew from soil samples. Taxonomical studies on these isolates revealed new taxa for phylum Actinomycetota; one family, three genera, and nine species were newly described. One of the strains, Patulibacter minatonensis KV-614^T belonging to the new family Patulibacteraceae, was isolated on agar medium supplemented with SOD. P. minatonensis KV-614^T represents a novel lineage within the phylum Actinomycetota. A polymerase chain reaction (PCR) study using specific primers for the detection of strains related to the genus Patulibacter, order Solirubrobacterales, showed a high distribution frequency, with detection in over 70% of the soil samples tested. These data suggest that the use of radical scavengers may facilitate the isolation of some hitherto-uncultivated microorganisms widely distributed in soil.