Journal of General and Applied Microbiology最新文献

Mycosporine-like amino acids (MAAs) are low-molecular-weight UV-protective compounds, and porphyra-334 and shinorine are common MAAs. Porphyra-334 is synthesized via the conjugation of mycosporine-glycine with threonine, whereas substitution with serine yields shinorine. The terrestrial cyanobacterium Nostoc commune KU002 (NIES-2538) produces 7-O-(β-arabinopyranosyl)-porphyra-334, and the mysABCD gene cluster responsible for MAA biosynthesis has been isolated. The heterologous expression of the mysABC genes from N. commune KU002 in Escherichia coli cells led to mycosporine-glycine production regardless of the culture medium supplemented with serine, threonine, or xylose. When the mysABCD genes from N. commune KU002 were expressed in E. coli cells, porphyra-334 production occurred, and shinorine production was observed upon serine supplementation in the culture medium. Notably, threonine and xylose supplementation in the culture medium increased the amounts of porphyra-334 in both cellular extracts and culture medium extracts. When the mysD gene was replaced with that from the shinorine producer Actinosynnema mirum JCM 3225, shinorine was primarily synthesized instead of porphyra-334. Interestingly, the transformant expressing the chimeric KU002-mysABC-JCM3225-mysD produced a novel MAA derivative with an absorption maximum at 334 nm and a molecular mass of 346 when cultured in the medium supplemented with threonine and xylose. These results suggest that the substrate specificity of MysD, which catalyzes the conjugation of mycosporine-glycine and serine or threonine, alters the production of porphyra-334 or shinorine and that the supplements added to the culture medium affect the amount and composition of MAAs produced in the E. coli transformant.

Amplicon sequencing is a widely used method for surveying biological diversity. However, the technique is disturbed by PCR bias leading to errors in community composition analyses. In this study, microbial community composition was evaluated in twenty-eight locations of hot spring water with temperatures between 87-48°C at Nakabusa Hot Springs, Japan, using amplicon sequencing analysis with the V4 region of the 16S rRNA gene. In discrepancy with the greenish color and the absorption spectra of the microbial samples, the relative abundance of amplicon sequence variants (ASVs) in the major photosynthetic organisms, Chloroflexus spp., were scarce in any sample when using the annealing temperature of 50°C in amplicon PCR. Changing the annealing temperature to 68ºC significantly improved the detection efficiency of Chloroflexus ASVs, and the obtained numbers were consistent with the presence of the photosynthetic pigments. The abundance of many other microbial ASVs was also dependent on the annealing temperature. The log ratio in the abundance of major ASVs between two annealing temperatures was correlated with the GC content of the 16S rRNA gene, suggesting that even some other major ASVs in the community are seriously affected by PCR bias due to the GC content. Combined usage of results from two different annealing temperatures, rather than a result using a single annealing temperature, seems to be a better way to obtain community structure information with less PCR bias in thermophilic organisms of high 16S rRNA GC content.

Methyl methacrylate (MMA), the primary raw material of acrylic resin, is an important polymeric material due to its increasing demand and ease of recycling. The most promising biosynthetic route for MMA involves the condensation of methanol with methacrylyl-CoA (MAA-CoA), an intermediate in the valine degradation pathway. The toxicity of MAA-CoA, poor stability and low activity of the heterologous pathway enzymes make this biosynthetic pathway less feasible. For enabling the evolutionary engineering of this pathway and its components (enzymes), we constructed a biosensor system in which the cellular level of key intermediate MAA-CoA can be evaluated in a high-throughput manner. With the aid of this MAA-CoA sensory system, we could establish the functional pathway from isobutyric acid to MAA-CoA. The sensor described in this paper should be valuable tool in the design-build-test-learn cycle for optimizing and breeding this MMA pathway.

In the biosynthesis of peptidoglycan (PG), murE protein (MurE) adds a diamino acid at position 3 of the peptide chain of peptidoglycan. The diamino acid that is added by MurE and makes cross-linkage with adjacent peptide chain differs depending on the bacterial species: Gram-negative bacteria add meso-diaminopimelic acid (DAP), while most Gram-positive bacteria add L-lysine (Lys). In this study, the murE gene of Levilactobacillus brevis that transfers Lys in PG synthesis was cloned into Escherichia coli that has DAP-type PG. The transformant cells harboring L. brevis murE showed reduction of colony forming units during cultivation, and were elongated or burst when murE was expressed. Amino acid analysis of solubilized PG revealed that the Lys/DAP ratio increased in the PG of the transformants. Interestingly, aspartic acid that is responsible for the formation of cross-linkages between Lys and other peptide chain in the PG of L. brevis also increased, suggesting that Lys-type PG with Asp cross-linkage was partially formed by the cloning of murE gene.

The extracellular export of target chemicals is essential for achieving the target productivity of microbial cell factories (MCFs). We demonstrated that MscCG, a mechanosensitive channel responsible for glutamate export in glutamate-producing MCF of Corynebaterium glutamicum, can export various intracellular low-molecular-weight chemicals outside the cell. The mechanosensitive channels exporter improved L-Lys productivity and conferred substantial 5'-IMP fermentative production ability to the Escherichia coli MCF, which lacks inherent 5'-IMP exporters, indicating that mechanosensitive channels, which are low selective, functioned effectively as MCF exporters. We also demonstrated the effectiveness of a gain-of-function (GOF) mutation of mechanosensitive channels as MCF exporters; however, the essential mechanism underlying this GOF mutation remains unknown. Therefore, we performed molecular dynamics simulations to identify this mechanism at the atomic level. Consequently, we partially elucidated the underlying mechanism of G46D-induced GOF in MscL, which was effective as a 5'-IMP exporter. Specifically, the kink at A38 in the inner transmembrane helix of MscL forming its pore can affect GOF behavior. Based on these results, we conclude that mechanosensitive channels have potential as innovative and versatile exporters of MCFs, capable of enhancing the production efficiency of target chemicals and enabling their production in the absence of natural exporters.

Actinoplanes missouriensis grows by forming branched substrate mycelia and produces terminal sporangia. Each sporangium contains a few hundred spores, which swim as zoospores after being released from sporangia. Previously, we disrupted 22 putative acyltransferase genes and examined their involvement in morphological differentiation. Here, we described the characterization of one of them, a putative 1-acyl-2-hydroxy-sn-glycerol-3-phosphate acyltransferase (AGPAT) encoded by plsC (AMIS_11360). The plsC null (∆plsC) mutant exhibited a conditional growth defect in a nutrient-poor medium. No differences were observed between the wild-type and ∆plsC strains in sporangium formation, spore release, or zoospore motility. We confirmed the AGPAT activity of PlsC; the recombinant polyhistidine-tagged PlsC protein transferred the acyl group from palmitoyl-coenzyme A to 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphate, resulting in the production of 1,2-dipalmitoyl-sn-glycero-3-phosphate.

We investigated the effect of a starter culture on the fungal communities of commercial karebushi. Aspergillus pseudoglaucus was initially identified as the starter fungus. In karebushi samples from two manufacturers relying on naturally occurring molds, Aspergillus chevalieri was the dominant species, accompanied by Aspergillus montevidensis and Aspergillus sydowii, while A. pseudoglaucus was not detected. Among samples from six manufacturers that used the starter culture, A. pseudoglaucus was dominant in only three; in the remaining three, A. chevalieri predominated despite the starter being used. These results suggest that indigenous fungi, particularly A. chevalieri, present in the processing environment can outcompete the starter culture, influence the fungal community, and potentially contribute to the qualitative diversity of karebushi.

We previously constructed an Escherichia coli strain expressing 16 nitrogen fixation (nif) and 2 nif-related genes from Azotobacter vinelandii and improved nitrogenase activity to some extent by enhancing NifH-related functions. In the present study, we analyzed the formation of dinitrogenase, a heterotetrameric NifD₂K₂, produced in E. coli, using gel-filtration chromatography and blue native PAGE to gain insight into further increases in nitrogenase activity. A certain proportion of NifD and NifK proteins produced in E. coli were present as the complete NifD₂K₂ component, but some remained in the intermediate stages of maturation. Overexpression of nafY, which is involved in holo-NifD₂K₂ formation, effectively increased nitrogenase activity.

Genome modification would be useful for developing breeding techniques for haploid Zygosaccharomyces rouxii and natural hybrid allodiploid Zygosaccharomyces sp. yeast strains used in miso and soy sauce production. In this study, genome editing using CRISPR-Cas9 was attempted in Zygosaccharomyces sp. strains. Based on techniques in Saccharomyces cerevisiae, the Cas9 gene and guide RNA (gRNA) were expressed from the same plasmid. Targeting of the ZygoLEU2 gene of haploid Z. rouxii strain DA2 led to of a single-nucleotide insertion in the ORF, resulting in termination of translation at 10 amino acids. This single-base insertion was 3-bp upstream of the protospacer-associated motif (PAM) sequence, suggesting that it occurred during the repair process following the Cas9-induced double-strand break. The transformant was auxotrophic for leucine, verifying that genome editing using CRISPR-Cas9 had occurred. Application of the CRISPR-Cas9 system to allodiploid Zygosaccharomyces sp. strains, which have T- and P-subgenomes, resulted in transformants with base insertions or deletions upstream of the PAM sequence, or insertions of different subgenome sequences. Leucine-auxotrophic transformants were obtained in which the ORF of the ZygoLEU2 gene in both subgenomes were mutated. In some genome-edited strains, a significant region of one subgenome chromosome was missing. Lastly, we applied CRISPR-Cas9 to the gene encoding Hog1, a protein kinase involved in adaptation to high-salt and high-osmolarity conditions. Mutation of the HOG1 genes of both the T- and P-subgenomes by CRISPR-Cas9 significantly reduced growth in high salt and high osmolarity conditions.

Ralstonia pseudosolanacearum is a plant-pathogenic bacterium that causes bacterial wilt in economically important crops. Chemotaxis is required for full virulence in R. pseudosolanacearum. R. pseudosolanacearum Ps29 possesses 20 methyl-accepting chemotaxis proteins (MCPs) and 2 MCP-like chemoreceptors. To understand the role of chemotaxis in plant infection, we are characterizing the functions of these 20 MCPs. Out of 20 MCPs, 8 MCPs have been characterized. To characterize the remaining MCPs, we deleted the 8 genes encoding characterized MCPs in R. pseudosolanacearum Ps29 to construct R. pseudosolanacearum D8. R. pseudosolanacearum D8 was examined for chemotactic responses to several chemically undefined materials including vegetable juices and tryptic soy broth (TSB) to find attractants. R. pseudosolanacearum D8 showed strong responses to green pepper and carrot juices and TSB. We constructed a mutant library of R. pseudosolanacearum D8 by deleting each of the MCP genes. Chemotaxis assays to TSB revealed that an MCP which we named McpD was responsible for sensing an attractant(s) in TSB. Because amino acids are the major constituents of TSB, we measured chemotactic responses of R. pseudosolanacearum D8 to 20 proteinogenic amino acids and found Asp and Glu as the major attractants of McpD and Cys as the minor attractant. R. pseudosolanacearum Ps29 can utilize Asp and Glu as sole carbon and nitrogen sources, suggesting that the role of McpD-mediated chemotaxis is finding growth substrates.