Soil bacteria Streptomyces are the most important producers of secondary metabolites, including most known antibiotics. These bacteria and their close relatives are unique in possessing linear chromosomes, which typically harbor 20 to 30 biosynthetic gene clusters of tens to hundreds of kb in length. Many Streptomyces chromosomes are accompanied by linear plasmids with sizes ranging from several to several hundred kb. The large linear plasmids also often contain biosynthetic gene clusters. We have developed a targeted recombination procedure for arm exchanges between a linear plasmid and a linear chromosome. A chromosomal segment inserted in an artificially constructed plasmid allows homologous recombination between the two replicons at the homology. Depending on the design, the recombination may result in two recombinant replicons or a single recombinant chromosome with the loss of the recombinant plasmid that lacks a replication origin. The efficiency of such targeted recombination ranges from 9 to 83% depending on the locations of the homology (and thus the size of the chromosomal arm exchanged), essentially eliminating the necessity of selection. The targeted recombination is useful for the efficient engineering of the Streptomyces genome for large-scale deletion, addition, and shuffling.
Although Streptococcus anginosus constitutes a proportion of the normal flora of the gastrointestinal and genital tracts, and the oral cavity, it has been reported that S. anginosus infection could be closely associated with abscesses at various body sites, infective endocarditis, and upper gastrointestinal cancers. The colonization in an acidic environment due to the aciduricity of S. anginosus could be the etiology of the systemic infection of the bacteria. To elucidate the aciduricity and acid tolerance mechanisms of the microbe, we examined the viability and growth of S. anginosus under acidic conditions. The viabilities of S. anginosus NCTC 10713 and Streptococcus mutans ATCC 25175 at pH 4.0 showed as being markedly higher than those of Streptococcus sanguinis ATCC 10556, Streptococcus gordonii ATCC 10558, and Streptococcus mitis ATCC 49456; however, the viability was partially inhibited by dicyclohexylcarbodiimide, an H+-ATPase inhibitor, suggesting that H+-ATPase could play a role in the viability of S. anginosus under acidic conditions. In addition, S. anginosus NCTC 10713 could grow at pH 5.0 and showed a marked arginine deiminase (ADI) activity, unlike its ΔarcA mutant, deficient in the gene encoding ADI, and other streptococcal species, which indicated that ADI could also be associated with aciduricity. These results suggest that S. anginosus has significant aciduric properties, which can be attributed to these enzyme activities.
The rapa whelk Rapana venosa is a commercially important gastropod in China, but a major invader worldwide. There is little information on the bacterial community composition in the digestive system of this species, despite the microflora has important roles in digestion, nutrition, disease resistance, and immune response. The present study investigated the bacterial flora community in the esophagus and intestinal tract of wild R. venosa by 16S rRNA gene sequencing. The esophagus and intestine had 1175 and 526 unique operational taxonomic units, respectively, with 616 common to both tissues. The 10 most highly represented microbial genera in the esophagus were Mycoplasma, Bifidobacterium, Escherichia, Shewanella, Vibrio, Lactobacillus, Octadecabacter, Enterococcus, Streptococcus, and Sphingomonas, which accounted for 41.35% of microbes. In the intestine, the most abundant genera were Mycoplasma, Bifidobacterium, Escherichia, Shewanella, Vibrio, Psychrilyobacter, Peptoniphilus, Sporobacterium, Octadecabacter, and Mobiluncusin (representing 62.74% of total microbes). A linear discriminant analysis, coupled with effect size, revealed that 31 taxa were differentially represented in esophagus and intestine bacterial communities. These results demonstrate the diversity of microbiota in the esophagus and intestinal tract of R. venosa and provide a basis for investigations into the physiological and immunological roles of these microorganisms in whelk.
We compared the gene expression levels of the blue-light-responsive genes, appA (encoding photosynthesis promoting protein AppA), ppsR (encoding photosynthesis suppressing protein PpsR), and EL368 (encoding a blue-light-activated histidine kinase with a light, oxygen, or voltage domain) between aerobic and anaerobic conditions in spheroplasts of the aerobic photosynthetic bacterium Erythrobacter litoralis. The spheroplasts conducted photosynthesis under red light but not under blue light. All three blue-light-responsive genes showed higher expression under aerobic conditions than under anaerobic conditions under blue light. In contrast, under red light, although the expression level of appA was higher in the presence of oxygen than in the absence of oxygen, the expression levels of ppsR and EL368 were similar in the presence and absence of oxygen. Our findings demonstrate that the expression of blue-light-responsive genes is strongly affected by oxygen in E. litoralis spheroplasts.
Candida glycerinogenes is an osmotolerant yeast used for commercial glycerol production, as well as a glycerol utilization yeast which produces high biomass on glycerol medium. In the present study, two STL1 homologues CgSTL1 and CgSTL2 encoding the putative glycerol transporters were identified, and their products were found to be localized to plasma membranes by tagging GFP protein. The functions of CgSTL1 and CgSTL2 on glycerol transport were confirmed by their expression in S. cerevisiaeSTL1 null mutant and simultaneous deletion in C. glycerinogenes. The expression of CgSTL1 were osmotic-induced, whereas that of CgSTL2 was constitutive. Over-expression of CgSTL1 and CgSTL2 in C. glycerinogenes resulted in improved glycerol consumption rate and cell growth. Our study provided more details on the glycerol transporter of C. glycerinogenes, the potential cell factory for using glycerol as a carbon source.
Variations of chromosomal structures and nucleotide sequences around mating-type-like (MTL) loci among Zygosaccharomyces species have been reported. We have analyzed these differences in more detail and, on the basis of PCR- and next-generation sequencing data, we describe the MTL loci on chromosomes C and F for Z. rouxii type-strain NBRC1130, Z. rouxii NBRC0740 and Zygosaccharomyces sp. NBRC1876. We developed a mating strategy for Zygosaccharomyces sp. NBRC1876 and Z. rouxii NBRC0740, and found that the mated stains could be identified from parental strains on the basis of nucleotide sequence variations of the MTL loci. We further obtained evidence that Zygosaccharomyces sp. NBRC1876 is a natural interspecies hybrid between Z. rouxii and a related species.
We analyzed the stress response in a spore clone from Shirakami kodama yeast, Saccharomyces cerevisiae, with an exceptional high tolerance to oxidative stress. The levels of reactive oxygen species (ROS) in this clone were very low, whereas the genes for superoxide dismutase (SOD2) and catalase (CTT1) were highly expressed and those enzymes also had high activities even under non-stress conditions. Both genes are regulated by general stress-responsive transcription factors Msn2 and Msn4, and Yap1, a transcription factor required for oxidative stress tolerance, and the removal of Msn2 or Yap1 caused a significant decrease in CTT1-expression. Under non-stress conditions, Msn2 was ~3.6-fold more abundant in the nucleus of the spore clone compared with a laboratory strain, whereas the nuclear abundance of Yap1 remained unchanged. Thus, a high tolerance to oxidative stress in this spore clone results from a high expression of ROS-degrading enzymes by the abundant accumulation of Msn2 in the nucleus. We found that oxidative stress caused by the presence of furfural did not impair fermentation by this strain, which could make it attractive for ethanol production from lignocellulosic biomass.
Beauvericin is a mycotoxin which has insecticidal, anti-microbial, anti-viral and anti-cancer activities. Beauvericin biosynthesis is rapidly catalyzed by the beauvericin synthetase (BEAS) in Beauveria bassiana. Ca2+ plays crucial roles in multiple signaling pathways in eukaryotic cells. These Ca2+ signals are partially decoded by Ca2+ sensor calmodulin (CaM). In this report, we describe that B. bassiana BEAS (BbBEAS) can interact with CaM in a Ca2+-dependent manner. A synthetic BbBEAS peptide, corresponding to the putative CaM-binding motif, formed a stable complex with CaM in the presence of Ca2+. In addition, in vitro CaM-binding assay revealed that the His-tagged BbBEAS (amino acids 2421–2538) binds to CaM in a Ca2+-dependent manner. Therefore, this work suggests that BbBEAS is a novel CaM-binding protein in B. bassiana.
γ-aminobutyric acid (GABA) is an important non-protein amino acid involved in the response to various environmental stresses in plant cells. The objectives of this study was to test the hypothesis that intracellular accumulation of GABA improves osmotic tolerance in the unconventional yeast Candida glycerinogenes. In C. glycerinogenes, the expression of UGA4 encoding GABA-specific permease is highly induced by hyperosmotic stress. Exogenous GABA application enhanced intracellular GABA accumulation and promoted cell growth under hyperosmotic conditions. Overexpression of the glutamate decarboxylase gene GAD1 resulted in an increased intracellular GABA and improvement in cell growth under hyperosmotic conditions. These results indicated that improving intracellular GABA accumulation of C. glycerinogenes, either through exogenous application or cellular synthesis, is available for improving the tolerance to hyperosmotic stress. We demonstrate that GABA accumulation plays an important role in osmotic stress resistance of the unconventional yeast C. glycerinogenes.
The marine bacterium Rhodovulum sulfidophilum is a nonsulfur phototrophic bacterium, which is known to produce extracellular nucleic acids in soluble form in culture medium. In the present paper, constructing the response regulator ctrA-deficient mutant of R. sulfidophilum, we found that this mutation causes a significant decrease in the extracellular DNA production. However, by the introduction of a plasmid containing the wild type ctrA gene into the mutant, the amount of extracellular DNA produced was recovered. This is the first and clear evidence that the extracellular DNA production is actively controlled by the CtrA in R. sulfidophilum.
The scarcity of enzymes having an optimal activity in lignocellulose deconstruction is an obstacle for industrial-scale conversion of cellulosic biomass into biofuels. With the aim of mining novel lignocellulolytic enzymes, a ~9 Gb metagenome of bacteria in Vietnamese native goats’ rumen was sequenced by Illumina platform. From the data, 821 ORFs encoding carbohydrate esterases (CEs) and polysaccharide lyases (PLs) serving for lignocellulose pre-treatment, 816 ORFs encoding 11 glycoside hydrolase families (GHs) of cellulases, and 2252 ORFs encoding 22 GHs of hemicellulases, were mined. The carbohydrate binding module (CBM) was also abundant with 763 ORFs, of which 480 ORFs are located with lignocellulolytic enzymes. The enzyme modularity analysis showed that CBMs are usually present in endoglucanase, endo 1,3-beta-D-glucosidase, and endoxylanase, whereas fibronectin 3-like module (FN3) mainly represents in GH3 and immunoglobulin-like domain (Ig) was located in GH9 only. Every domain located in each ORF was analyzed in detail to contribute enzymes’ modularity which is valuable for modelling, to study the structure, and for recombinant production. With the aim of confirming the annotated results, a mined ORF encoding CBM63 was highly expressed in E. coli in soluble form. The purified recombinant CBM63 exhibited no cellulase activity, but enhanced a commercial cellulase activity in the destruction of a paper filter.
Bilirubin oxidase has applications in the health and environmental sectors. Hence, several attempts have been made to increase enzyme yields. However, improvements were not very high. We report here the development of a mutant strain of Myrothecium verrucaria by using UV-rays, which produced 28.8 times more enzyme compared with the parent and was higher than the yields reported in earlier submerged cultures. The mutant strain produced 35.6 times more enzyme than the parent in solid-state fermentation, which is better than that previously reported for a solid-state fermentation process. The specific activity of the enzyme produced by the mutant was higher than that of the parental enzyme. Bilirubin oxidase from both strains showed an optimum activity at pH 7 and 40°C. However, the time required to inactivate half of the initial enzyme activity at 60°C was much higher in the case of the enzyme obtained from the mutant compared with the parental enzyme. The improved thermostability of the enzyme from the mutant strain could be due to the point mutations induced during the UV irradiation, since there was no change in the mass of the enzyme compared with the parental enzyme. The bilirubin oxidase of the mutant strain degraded the bilirubin faster than the enzyme obtained from the parent under similar conditions. Faster activity of the enzyme obtained from the mutant strain could be due to its lower Km (79.4 μM) compared with that of the parental enzyme (184 μM). Hence, the mutant enzyme showed a better functionality and thermostability, which will be beneficial for industrial applications.
Saccharomyces cerevisiae Ypr147cp was found localized to lipid droplets but the physiological role of Ypr147cp remains unknown. Sequence analysis of Ypr147cp revealed an α/β hydrolase domain along with the conserved GXSXG lipase motif. Recombinant Ypr147cp showed both triacylglycerol lipase and ester hydrolase activities. Knock out of YPR147C led to accumulation of TAG in ypr147cΔ when compared to wild type (WT). In addition, transmission electron microscopic analysis of ypr147cΔ cells revealed a greater number of lipid bodies, justifying the increase in TAG content, and the phenotype was rescued upon overexpression of YPR147C in ypr147cΔ. Moreover, the lipid profiling confirmed the accumulation of fatty acids derived from neutral and phospholipids in ypr147cΔ cells. Based on these results, Ypr147cp is identified as a lipid droplet associated triacylglycerol lipase along with an ester hydrolyzing capacity.
Lactococcus lactis is a probiotic bacterium that produces various bacteriocins. Periodontopathogens induce inflammation and halitosis through the actions of lipopolysaccharide (LPS) and trypsin-like enzymes. The purpose of this study was to investigate the inhibitory effects of L. lactis on the bioactivity of periodontopathogens. To investigate the antimicrobial peptide of L. lactis, the spent culture medium (SCM) of L. lactis was treated with or without proteinase K after collection by centrifugation, and the antibacterial activity of SCM against periodontopathogens was assessed. To evaluate the neutralizing effect of L. lactis on halitosis, SCM of periodontopathogens was mixed with an L. lactis suspension, and the levels of volatile sulfur compounds (VSCs) were measured by gas chromatography. LPS from the periodontopathogens was extracted by an LPS extraction kit with little modification, and THP-1 cells as a monocytic cell line were treated with the extracted LPS in the presence or absence of UV-killed L. lactis. The production of inflammatory cytokines was analyzed by ELISA. The SCM of L. lactis exhibited antimicrobial activity against the periodontopathogens, whereas the proteinase K-treated SCM showed little antimicrobial activity. In addition, the L. lactis suspension had a neutralizing effect on the VSCs produced by periodontopathogens, and UV-killed L. lactis inhibited the production of IL-6 and TNF-α induced by the LPS. These results suggest that L. lactis may be a useful probiotic to prevent and treat periodontitis and halitosis.
A new antibiotic, designated mumiamicin, was isolated from the cultured broth of the rare actinomycete strain, Mumia sp. YSP-2-79, by Diaion HP-20, silica gel and ODS column chromatography, followed by HPLC purification. The chemical structure of mumiamicin was elucidated as a new furan fatty acid by nuclear magnetic resonance and mass spectrometry. Mumiamicin showed antimicrobial activity and antioxidative activity.