Cellular lipids of Lactiplantibacillus species were extracted and neutral glyceroglycolipids (GGLs) were purified, and analyzed by thin-layer chromatography (TLC). Four GGLs with known structures were detected in GGL preparation of L. plantarum, and the same GGL profiles of TLC were observed for all other strains of Lactiplantibacillus species examined, suggesting that the GGL profile could be one of the chemotaxonomic characters of the genus Lactiplantibacillus. On the other hand, the quantity of each GGL showed some variation among species, and L. pentosus was found to have higher proportion of disaccharide-type GGL, designated GGL-3 in this study, compared with other species including L. plantarum. The quantitative difference of GGL-3 found in this study could be regarded as the characteristics of L. pentosus. The carbohydrate structure of L. pentosus GGL-3 was precisely analyzed by 1H NMR and methylation analysis, and the structure was confirmed to be αGal-(1→2)-αGlc-diacylglycerol, with the carbohydrate structure identical to that of L. plantarum, although fatty acid composition of the two GGL-3 showed some difference.
In the fermentative production of compounds by using microorganisms, control of the transporter activity responsible for substrate uptake and product efflux, in addition to intracellular metabolic modification, is important from a productivity perspective. However, there has been little progress in analyses of the functions of microbial membrane transporters, and because of the difficulty in finding transporters that transport target compounds, only a few transporters have been put to practical use. Here, we constructed a Corynebacterium glutamicum-derived transporter expression library (CgTP-Express library) with the fusion partner gene mstX and used a peptide-feeding method with the dipeptide L-Ala-L-Ala to search for alanine exporters in the library. Among 39 genes in the library, five candidate alanine exporters (NCgl2533, NCgl2683, NCgl0986, NCgl0453, and NCgl0929) were found; expression of NCgl2533 increased the alanine concentration in cell culture. The CgTP-Express library was thus effective for finding a new transporter candidate.
The Pseudomonas aeruginosa strain, PAO1, has three putative γ-glutamyltranspeptidase (GGT) genes: ggtI, ggtII, and ggtIII. In this study, the expression of each of these genes in P. aeruginosa PAO1 was analyzed, and the properties of the corresponding GGT proteins were investigated. This is the first report on biochemical characterization of GGT paralogs from Pseudomonas species. The crude extracts prepared from P. aeruginosa PAO1 exhibited hydrolysis and transpeptidation activities of 17.3 and 65.0 mU/mg, respectively, and the transcription of each gene to mRNA was confirmed by RT-PCR. All genes were cloned, and the expression plasmids constructed were introduced into an Escherichia coli expression system. Enzyme activity of the expressed protein of ggtI (PaGGTI) was not detected in the system, while the enzyme activities of the expressed proteins derived from ggtII and ggtIII (PaGGTII and PaGGTIII, respectively) were detected. However, the enzyme activity of PaGGTII was very low and easily decreased. PaGGTII with C-terminal his-tag (PaGGTII25aa) showed increased activity and stability, and the purified enzyme consisted of a large subunit of 40 kDa and a small subunit of 28 kDa. PaGGTIII consisted of a large subunit of 37 kDa and a small subunit of 24 kDa. The maximum hydrolysis and transpeptidation activities of PaGGTII25aa were obtained at 40ºC–50ºC, and the maximum hydrolysis and transpeptidation activities of PaGGTIII were obtained at 50ºC–60ºC. These enzymes retained approximately 80% of their hydrolysis and transpeptidation activities after incubation at 50ºC for 10 min, reflecting good stability. Both PaGGTII25aa and PaGGTIII showed higher activities of hydrolysis and transpeptidation in the alkali range than in the acidic range. However, they were highly stable at a wide pH range (5–10.5).
Fungi uniquely synthesize lysine through the α-aminoadipate pathway. The saccharopine reductase ScLys9 catalyzes the formation of saccharopine from ɑ-aminoadipate 6-semialdehyde, the seventh step in the lysine biosynthesis pathway in Saccharomyces cerevisiae. Here, we characterized the functions of TrLys9, an ortholog of S. cerevisiae ScLys9 in the industrial filamentous fungus Trichoderma reesei. Transcriptional level analysis indicated that TrLYS9 expression was higher in the conidial stage than in other stages. Disruption of TrLYS9 led to lysine auxotrophy. Phenotype analysis of the ΔTrlys9 mutant showed that TrLYS9 was involved in fungal development including vegetative growth, conidiation, and conidial germination and lysine biosynthesis. Cellulase production was also impaired in the ΔTrlys9 mutant due to the failure of conidial germination in liquid cellulase-inducing medium. Defects in radial growth and asexual development of the ΔTrlys9 mutant were fully recovered when exogenous lysine was added to the medium. These results imply that TrLys9 is involved in fungal development and lysine biosynthesis in T. reesei.
Certain mutations of the model cyanobacterium Synechococcus elongatus PCC 7942 during laboratory storage have resulted in some divergent phenotypes. One laboratory-stored strain (H1) shows a temperature-sensitive (ts) growth phenotype at 40 °C. Here, we investigated the reason for this temperature sensitivity. Whole genome sequencing of H1 identified a single nucleotide mutation in synpcc7942_R0040 encoding tRNA-Leu(CAA). The mutation decreases the length of the tRNA-Leu t-arm from 5 to 4 base pairs, and this explains the ts phenotype. Secondary mutations suppressing the ts phenotype were identified in synpcc7942_1640, which putatively encodes a NYN domain-containing protein (nynA). The NYN domain is thought to be involved in tRNA/rRNA degradation. Thus, the structural stability of tRNA-Leu is critical for growth at 40 °C in Synechococcus elongatus PCC 7942.
Bacillus velezensis S141, a plant growth-promoting rhizobacteria (PGPR), was isolated from a soybean field in Thailand. Previous studies demonstrated that S141 enhanced soybean growth, stimulating nodulation for symbiotic nitrogen fixation with soybean root nodule bacteria, including Bradyrhizobium diazoefficience USDA110. Isoflavone glycosides are produced in soybean roots and hydrolyzed into their aglycones, triggering nodulation. This study revealed that S141 efficiently hydrolyzed two isoflavone glycosides in soybean roots (daidzin and genistin) to their aglycones (daidzein and genistein, respectively). However, S141, Bacillus subtilis 168, NCIB3610, and B. velezensis FZB42 hydrolyzed isoflavone glucosides into aglycones. A BLASTp search suggested that S141 and the other three strains shared four genes encoding β-glucosidases corresponding to bglA, bglC, bglH, and gmuD in B. subtilis 168. The gene inactivation analysis of B. subtilis 168 revealed that bglC encoded the major β-glucosidase, contributing about half of the total activity to hydrolyze isoflavone glycosides and that bglA, bglH, and gmuD, all barely committed to the hydrolysis of isoflavone glycosides. Thus, an unknown β-glucosidase exists, and our genetic knowledge of β-glucosidases was insufficient to evaluate the ability to hydrolyze isoflavone glycosides. Nevertheless, S141 could predominate in the soybean rhizosphere, releasing isoflavone aglycones to enhance soybean nodulation.