Journal: Journal of microbiology and biotechnology
Tricholoma matsutake, an ectomycorrhizae that has mutual relationships with the rootlet of Pinus denisflora, forms a fruiting body that serves as a valuable food in Asia. However, the artificial culture of this fungus has not been successful. Soil fungi, including T. matsutake, coexist with many other microorganisms and plants; therefore, complex microbial communities have an influence on the fruiting body formation of T. matsutake. Here, we report on the structures of fungal communities associated with the fairy ring of T. matsutake through the pyrosequencing method. Soil samples were collected inside the fairy ring zone, in the fairy ring zone, and outside the fairy ring zone. A total of 37,125 sequencing reads were obtained and 728 to 1,962 Operational Taxonomic Units (OTUs) were observed in the sampling zones. The fairy ring zone had the lowest OTUs and the lowest fungal diversity of all sampling zones. The number of OTUs and fungal taxa inside and outside the fairy ring zone was, respectively, about two times and 1.5 times higher than the fairy ring. Taxonomic analysis showed that each sampling zone has different fungal communities. In particular, out of 209 genera total, six genera, in the fairy ring zone, such as genus Hemimycena, were uniquely present and 31 genera, such as genus Mycena, Boletopsis, and Repetophragma, were specifically absent. The results of metagenomic analysis based on the pyrosequencing indicate a decrease of fungal communities in the fairy ring zone and changes of fungal communities depending on the fairy ring growth of T. matsutake.
Yeast surface-displayed antibody libraries provide an efficient and quantitative screening resource for given antigens, but suffer from typically modest library sizes owing to low yeast transformation efficiency. Yeast mating is an attractive method for overcoming the limit of yeast transformation to construct a large, combinatorial antibody library, but the optimal conditions have not been reported. Here we report a large synthetic human Fab (antigen binding fragment) yeast surface-displayed library generated by stepwise optimization of yeast mating conditions. We first constructed HC (heavy chain) and LC (light chain) libraries, where all of the six CDRs (complementarity-determining regions) of the variable domains were diversified mimicking the human germline antibody repertoires by degenerate codons, onto single frameworks of VH3-23 and Vkappa1-16 germline sequences, in two haploid cells of opposite mating types. Yeast mating conditions were optimized in the order of cell density, media pH, and cell growth phase yielding a mating efficiency of ~58% between the two haploid cells carrying HC and LC libraries. We constructed two combinatorial Fab libraries with CDR-H3 of 9 or 11 residues in length with colony diversities of more than 109 by one round of yeast mating between the two haploid HC and LC libraries with modest diversity sizes of ~107. The synthetic human Fab yeast-displayed libraries exhibited relative amino acid compositions in each position of the six CDRs that were very similar to those of the designed repertoires, suggesting that they are a promising source for human Fab antibody screening.
The effects of microbial iron reduction and oxidation on the immobilization and mobilization of copper was investigated in a high concentration of sulfate with synthesized Fe(III) minerals and red earth soils rich in amorphous Fe (hydr)oxides. Batch microcosm experiments showed that red earth soil inoculated with subsurface sediments had a faster Fe(III) bioreduction rate than pure amorphous Fe(III) minerals and resulted in quicker immobilization of Cu in the aqueous fraction. Coinciding with the decrease of aqueous Cu, SO4(2-) in the inoculated red earth soil decreased acutely after incubation. The shift in the microbial community composite in the inoculated soil was analyzed through denaturing gradient gel electrophoresis. Results revealed the potential cooperative effect of microbial Fe(III) reduction and sulfate reduction on copper immobilization. After exposure to air for 144 h, more than 50% of the immobilized Cu was remobilized from the anaerobic matrices; aqueous sulfate increased significantly. Sequential extraction analysis demonstrated that the organic matter/sulfide-bound Cu increased by 52% after anaerobic incubation relative to the abiotic treatment but decreased by 32% after oxidation, indicating the generation and oxidation of Cu-sulfide co-precipitates in the inoculated red earth soil. These findings suggest that the immobilization of copper could be enhanced by mediating microbial Fe(III) reduction with sulfate reduction under anaerobic conditions. The findings have an important implication for bioremediation in Cu-contaminated and Fe-rich soils, especially in acid mine drainage (AMD)-affected sites.
Uricase is an important microbial enzyme which can be used in clinical treatment of gout, hyperuricemia, and tumor lysis syndrome. A total of 127 clinical isolates of Pseudomonas aeruginosa were tested for uricase production. Pseudomonas strain named Ps43 showed the highest level of native uricase enzyme expression. The open reading frame of uricase enzyme was amplified from Ps43 and cloned into the expression vector pRSET-B. Uricase was expressed using E. coli BL21 (DE3). The ORF was sequenced and assigned gene bank accession number KJ718888. The nucleotide sequence analysis was identical to the coding sequence of uricase gene puuD of P. aeruginosa PAO1. We report successful expression of P. aeruginosa uricase in Escherichia coli. E. coli showed an induced protein with a molecular weight of ~58 KDa that confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot. We also established efficient protein purification using Ni-NTA column with activity of the purified enzyme 2.16 IU and 2 fold increase in the specific activity of the pure enzyme compared to the crude enzyme.
Recently, the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR/Cas9) system, a genome editing technology, was shown to be versatile in treating several antibiotic-resistant bacteria. In the present study, we applied the CRISPR/Cas9 technology to kill extended spectrum beta-lactamase (ESBL)-producing Escherichia coli. ESBL bacteria are mostly multi-drug resistant (MDR), and have plasmid-mediated antibiotic resistance genes that can be easily transferred to other members of the bacterial community by horizontal gene transfer. To restore sensitivity to antibiotics in these bacteria, we searched for a CRISPR/Cas9 target sequence that was conserved among > 1,000 ESBL mutants. There was only one target sequence for each TEM- and SHV-type ESBL, with each of these sequences found in ~200 ESBL strains of each type. Furthermore, we showed that these target sequences can be exploited to re-sensitize MDR cells in which resistance is mediated by genes that are not the target of CRISPR/Cas9 system, but by genes that are present on the same plasmid as target genes. We believe our Re-Sensitization to Antibiotics from Resistance (ReSAFR) technology, which enhances the practical value of the CRISPR/Cas9 system, will be an effective method of treatment against plasmid-carrying MDR bacteria.
The combination of trimethoprim (TMP) and sulfamethoxazole (SMX) has been shown to be active against Mycobacterium tuberculosis (Mtb) in clinical tuberculosis (TB) treatment. However, the mechanism of action of TMP-SMX against Mtb is still unknown. To unravel this, we have studied the effect of TMP and SMX by deleting folP2 gene in Mycobacterium smegmatis (Msm), and overexpressing the Mtb and Msm folP1/2 genes in Msm. Knocking out of the folP2 gene in Msm reduced the minimum inhibitory concentration (MIC) of SMX 8-fold compared with wild type. Overexpression of the folP1 genes from Mtb and Msm increased the MICs by 4 and 2-fold in Msm for SMX and TMP respectively. We show a strong correlation between the expression of folP1 and folP2 genes and TMP-SMX resistance in mycobacteria. This suggests that a combination of FolP2 inhibitor and SMX could be used for TB treatment with a better outcome.
Rabbit hemorrhagic disease virus (RHDV) is high contagious and causes often fatal disease that affects both wild and domestic rabbits of the species Oryctolagus cuniculus. A highly pathogenic RHDV variant (RHDVa) has been circulated in the Korean rabbit population since 2007 and has a devastating effect on rabbit industry in Korea. A highly pathogenic RHDVa was isolated from naturally infected rabbits, and the gene encoding the VP60 protein was cloned into a baculovirus transfer vector and expressed in insect cells. The hemagglutination titer of the sf-9 cell lysate infected with recombinant VP60 baculovirus was 131,072 units/50 microliter and of the supernatant 4,096 units/50 microliter. Guinea pigs immunized twice intramuscularly with a trial inactivated RHDVa vaccine containing recombinant VP60 contained 2,152 hemagglutination inhibition (HI) geometric mean titers. The 8-week-old white rabbits inoculated with one vaccine dose were challenged with a lethal RHDVa 21 days later and showed 100% survival rates. Recombinant VP60 protein expressed in a baculovirus system induced high HI titers in guinea pigs and rendered complete protection, which led to the development of a novel inactivated RHDVa vaccine.
Xylanase sourcing of different bacterial have significantly different enzymatic properties. Therefore, studying xylanase sourcing of different bacterial is important to their applications in different fields. Potential xylanase degradation genes in Massilia were recently discovered through genomic sequencing. However, its xylanase activity remains unexplored. The paper is the first to report xylanase (XynRBM26) belonging to the glycosyl hydrolase family (GH10) from the genus Massilia. The gene encodes a 383-residue polypeptide (XynRBM26) with the highest identity of 62% with the endoxylanase from uncultured bacterium BLR13. The XynRBM26 expressed in Escherichia coli BL21 is a monomer with a molecular mass of 45.0 kDa. According to enzymatic characteristic analysis, pH 5.5 is the most appropriate for XynRBM26, which could maintain more than 90% activity between pH 5.0-8.0. Moreover, XynRBM26 is stable at 37°C and could maintain at least 96% activity after being placed at 37°C for 1 h. This paper is the first to report that GH10 xylanase in animal gastrointestinal tract (GIT) has salt tolerance, which could maintain 86% activity in 5 M NaCl. Under the optimum conditions, Km, Vmax, and kcat of XynRBM26 to beechwood xylan are 9.49 mg/mL, 65.79 mol/min/mg, and 47.34 s-1, respectively. Considering that XynRBM26 comes from animal GIT, this xylanase has potential application in feedstuff. Moreover, XynRBM26 is applicable to high-salt food and seafood processing, as well as other high-salt environmental biotechnological fields, because of its high catalytic activity in high-concentration NaCl.
This study is the first report of the entire nucleotide sequence of an inositol phosphoceramide synthase gene from the stress-tolerant yeast Pichia kudriavzevii (PkAUR1). Sequence analysis revealed an open reading frame that spans 1,443 bp and encodes a 480-amino-acid-residue protein with the highest sequence similarity (41.7%) to Aur1 from Spathaspora passalidarum. A phenotypic assay with transformed S. cerevisiae and P. kudriavzevii indicated that two amino acid residues, Phe166 and Gly249, play crucial roles in the resistance to aureobasidin A, which is consistent with previous reports for other fungal Aur1s. The GenBank Accession No. for PkAUR1 is KP729614.
Pyrosequencing analysis of intestinal microflora from healthy Thai vegetarians and non-vegetarians exhibited 893 OTUs covering 189 species. The strong species indicator of vegetarians and non-vegetarians were Prevotella copri and Bacteroides vulgatus as well as bacterium closed to Escherichia hermanii with % relative abundance of 16.9 and 4.5-4.7, respectively. Core gut microbiota of vegetarian and non-vegetarian group consisted of 11 and 20 different bacterial species, respectively, belonging to Actinobacteria, Firmicutes and Proteobacteria commonly found in both groups. Two species of Faecalibacterium prausnitzii and Gemmiger formicilis had prevalence of 100% in both groups. Three species of Clostridium nexile, Eubacterium eligens and P. copri showed up in most vegetarians while more diversity of Collinsella aerofaciens, Ruminococcus torques, various species of Bacteroides, Parabacteroides, Escherichia, different species of Clostridium and Eubacterium were found in most non-vegetarians. Considering the correlation of personal characters, consumption behavior and microbial groups, the age of non-vegetarians showed strong positive correlation coefficient of 0.54 (p=0.001) to Bacteroides uniformis while exhibited a moderate ones to Alistipes finegoldii and B. vulgatus. Only positive moderate correlation of body mass index (BMI) and Parabacteroides distasonis appeared. Based on significant abundance of potential pathogens, the microbiota of non-vegetarian group showed the abundance of potential pathogen varieties of Bilophila wadsworthia, Escherichia coli and E. hermannii while the one of vegetarian served for only Klebsiella pneumonia. These results implied that the microbiota of vegetarian with high abundance of P. copri and low potential pathogen variety would be a way to maintain healthy in Thai.