Gene encoding a dextransucrase-like protein in Leuconostoc mesenteroides NRRL B-512F

A gene, dsrT, encoding a dextransucrase-like protein was isolated from the genomic DNA libraries of Leuconostoc mesenteroides NRRL B-512F dextransucrase-like gene. The gene was similar to the intact open reading frames of the dextransucrase gene dsrS of L. mesenteroides NRRL B-512F, dextransucrase genes of strain NRRL B-1299 and streptococcal glucosyltransferase genes, but was truncated after the catalytic domain, apparently by the deletion of five nucleotides. dsrT mRNA was produced in this strain L. mesenteroides when cells were grown in a sucrose medum, but at a level of 20% of that of dsrS mRNA. The molecular weight of the dsrT gene product was 150,000 by SDS-PAGE. The product did not synthesize dextran, but had weak sucrose cleaving activity. The insertion of five nucleotides at the putative deletion point in dsrT resulted in an enzyme with a molecular weight of 210,000 and with dextransucrase activity.     

Antimicrobial activity of Paenibacillus peoriae strain NRRL BD-62 against a broad spectrum of phytopathogenic bacteria and fungi

AIMS: To investigate the potential antagonistic activity of Paenibacillus peoriae strain NRRL BD-62 against phytopathogenic micro-organisms and to determine the physiological and biochemical characteristics of the antimicrobial compound produced by this strain. METHODS AND RESULTS: Strain NRRL BD-62 showed a broad inhibition spectrum with activity against various phytopathogenic bacteria and fungi. Physico-chemical characterization of the antimicrobial activity showed that it was stable during heat treatment and was retained even after autoclave at 121 degrees C for 10 min. The compound was also stable after the treatment with organic solvents, hydrolytic enzymes and its activity was preserved at a wide range of pH. The partial purification carried out by Sephadex G25 gel filtration showed two profiles of inhibition against the indicator strains tested, suggesting at least two different substances with distinct molecular weight. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on the production of antimicrobial substances in P. peoriae. Besides the antimicrobial inhibition capability, the strain NRRL BD-62 is also able to effectively fix molecular nitrogen, and produce chitinases and proteases as well, suggesting that further studies should be addressed to use P. peoriae strain NRRL BD-62 as a plant growth promoter and/or as a biocontrol agent in field experiments.     

PCR screening reveals unexpected antibiotic biosynthetic potential in Amycolatopsis sp. strain UM16

AIMS: To assess the antibiotic biosynthetic potential of Amycolatopsis sp. strain UM16 and eight other Amycolatopsis species. METHODS AND RESULTS: Amycolatopsis genomic DNA was screened by PCR for the glycopeptide, Type-II (aromatic) polyketide and ansamycin biosynthetic gene clusters. Amycolatopsis sp. strain UM16, which exhibits weak antitubercular activity, was shown to have the glycopeptide oxyB gene and the Type-II (aromatic) polyketide-synthase KSalpha-KSbeta tandem gene pair, but not the AHBA synthase gene. The ristocetin (glycopeptide) producer, Amycolatopsis lurida NRRL 2430(T), was shown to have the oxyB gene and the Type-II polyketide-synthase KSalpha-KSbeta tandem gene pair. Amycolatopsis alba NRRL 18532(T) was shown to have the glycopeptide oxyB gene and the AHBA synthase gene. Phylogenetic analyses using Amycolatopsis oxyB and KSalpha-KSbeta gene sequences were conducted. CONCLUSIONS: Amycolatopsis sp. strain UM16 appears to have the biosynthetic potential to produce glycopeptide and Type-II polyketide antibiotics, but not ansamycins. The potential to synthesize aromatic polyketides may be more widely distributed in Amycolatopsis than is currently recognized. SIGNIFICANCE AND IMPACT OF THE STUDY: PCR screening is a very useful tool for rapidly identifying the biosynthetic potential of an antibiotic-producing actinomycete isolate. Advanced knowledge of the type of antibiotic(s) produced will allow appropriate methods to be selected for antibiotic purification.     

A Penicillium expansum glucose oxidase-encoding gene, GOX2, is essential for gluconic acid production and acidification during colonization of deciduous fruit

Penicillium expansum, the causal agent of blue mold rot, causes severe postharvest maceration of fruit through secretion of total, d-gluconic acid (GLA). Two P. expansum glucose oxidase (GOX)-encoding genes, GOX1 and GOX2, were analyzed. GOX activity and GLA accumulation were strongly related to GOX2 expression, which increased with pH to a maximum at pH 7.0, whereas GOX1 was expressed at pH 4.0, where no GOX activity or extracellular GLA were detected. This differential expression was also observed at the leading edge of the decaying tissue, where GOX2 expression was dominant. The roles of the GOX genes in pathogenicity were further studied through i) development of P. expansum goxRNAi mutants exhibiting differential downregulation of GOX2, ii) heterologous expression of the P. expansum GOX2 gene in the nondeciduous fruit-pathogen P. chrysogenum, and iii) modulation of GLA production by FeSO(4) chelation. Interestingly, in P. expansum, pH and GLA production elicited opposite effects on germination and biomass accumulation: 26% of spores germinated at pH 7.0 when GOX activity and GLA were highest whereas, in P. chrysogenum at the same pH, when GLA did not accumulate, 72% of spores germinated. Moreover, heterologous expression of P. expansum GOX2 in P. chrysogenum resulted in enhanced GLA production and reduced germination, suggesting negative regulation of spore germination and GLA production. These results demonstrate that pH modulation, mediated by GLA accumulation, is an important factor in generating the initial signal or signals for fungal development leading to host-tissue colonization by P. expansum.     

Expression of the Aspergillus niger glucose oxidase gene in Saccharomyces cerevisiae and its potential applications in wine production

There is a growing consumer demand for wines containing lower levels of alcohol and chemical preservatives. The objectives of this study were to express the Aspergillus niger gene encoding a glucose oxidase (GOX; beta- d-glucose:oxygen oxidoreductase, EC 1.1.3.4) in Saccharomyces cerevisiae and to evaluate the transformants for lower alcohol production and inhibition of wine spoilage organisms, such as acetic acid bacteria and lactic acid bacteria, during fermentation. The A. niger structural glucose oxidase (gox) gene was cloned into an integration vector (YIp5) containing the yeast mating pheromone alpha-factor secretion signal (MFalpha1(S)) and the phosphoglycerate-kinase-1 gene promoter (PGK1(P)) and terminator (PGK1(T)). The PGK1(P)- MFalpha1(S)- gox- PGK1(T) cassette (designated GOX1) was introduced into a laboratory strain (Sigma1278) of S. cerevisiae. Yeast transformants were analysed for the production of biologically active glucose oxidase on selective agar plates and in liquid assays. The results indicated that the recombinant glucose oxidase was active and was produced beginning early in the exponential growth phase, leading to a stable level in the stationary phase. The yeast transformants also displayed antimicrobial activity in a plate assay against lactic acid bacteria and acetic acid bacteria. This might be explained by the fact that a final product of the GOX enzymatic reaction is hydrogen peroxide, a known antimicrobial agent. Microvinification with the laboratory yeast transformants resulted in wines containing 1.8-2.0% less alcohol. This was probably due to the production of d-glucono-delta-lactone and gluconic acid from glucose by GOX. These results pave the way for the development of wine yeast starter culture strains for the production of wine with reduced levels of chemical preservatives and alcohol.     

The mouse L-histidine decarboxylase gene: structure and transcriptional regulation by CpG methylation in the promoter region

To investigate the regulation of mouse L-histidine decarboxylase (HDC) gene expression, we isolated genomic DNA clones encoding HDC. Structural analysis revealed that the mouse HDC gene was composed of 12 exons, spanning approximately 24 kb. Northern blotting analysis indicated that, among the cell lines examined, a high level of HDC gene expression was restricted to mature mast cell lines and an erythroblastic cell line. The gene was induced strongly in the mouse immature mast cell line P815 after incubation in the peritoneal cavity of BDF1 mice. We observed that the promoter region was demethylated in the HDC-expressing cell lines and in induced P815 cells. Interestingly, forced demethylation by 5-azacytidine (5-azaC) treatment induced high expression of HDC mRNA in P815 cells. The activity of a mouse HDC promoter-reporter construct stably transfected in P815 cells was repressed by in vitro patch-methylation. This low promoter activity of the patch-methylated reporter construct was restored after 5-azaC treatment, which demethylated the patch-methylated promoter. These results indicate that DNA methylation state of the promoter region controls HDC gene expression.     

Two New Native β-Glucosidases from Clavispora NRRL Y-50464 Confer Its Dual Function as Cellobiose Fermenting Ethanologenic Yeast

Yeast strain Clavispora NRRL Y-50464 is able to produce cellulosic ethanol from lignocellulosic materials without addition of external β-glucosidase by simultaneous saccharification and fermentation. A β-glucosidase BGL1 protein from this strain was recently reported supporting its cellobiose utilization capability. Here, we report two additional new β-glucosidase genes encoding enzymes designated as BGL2 and BGL3 from strain NRRL Y-50464. Quantitative gene expression was analyzed and the gene function of BGL2 and BGL3 was confirmed by heterologous expression using cellobiose as a sole carbon source. Each gene was cloned and partially purified protein obtained separately for direct enzyme assay using varied substrates. Both proteins showed the highest specific activity at pH 5 and relatively strong affinity with a K_m of 0.08 and 0.18 mM for BGL2 and BGL3, respectively. The optimum temperature was found to be 50°C for BGL2 and 55°C for BGL3. Both proteins were able to hydrolyze 1,4 oligosaccharides evaluated in this study. They also showed a strong resistance to glucose product inhibition with a K_i of 61.97 and 38.33 mM for BGL2 and BGL3, respectively. While BGL3 was sensitive showing a significantly reduced activity to 4% ethanol, BGL2 demonstrated tolerance to ethanol. Its activity was enhanced in the presence of ethanol but reduced at concentrations greater than 16%. The presence of the fermentation inhibitors furfural and HMF did not affect the enzyme activity. Our results suggest that a β-glucosidase gene family exists in Clavispora NRRL Y-50464 with at least three members in this group that validate its cellobiose hydrolysis functions for lower-cost cellulosic ethanol production. Results of this study confirmed the cellobiose hydrolysis function of strain NRRL Y-50464, and further supported this dual functional yeast as a candidate for lower-cost cellulosic ethanol production and next-generation biocatalyst development in potential industrial applications.     

Glucose oxidase production by Penicillium variabile P16: effect of medium composition

The effect of medium composition on glucose oxidase (GOD) production by the strain Penicillium variabile P16 was studied in shake culture. All the experiments were performed in a medium containing glucose and sodium nitrate as carbon and nitrogen sources, respectively. Addition of Fe²⁺ to the medium was important to stimulate GOD production. Calcium carbonate was extremely important in maintaining the medium at a pH high enough so that the enzyme was not inactivated. The time course of growth and enzyme production by P. variabile P16 in the optimized medium is reported.     

Molecular characterization of a novel chitinase from Bacillus thuringiensis subsp. kurstaki

AIMS: The present work aims to study a new chitinase from Bacillus thuringiensis subsp. kurstaki. METHODS AND RESULTS: BUPM255 is a chitinase-producing strain of B. thuringiensis, characterized by its high chitinolytic and antifungal activities. The cloning and sequencing of the corresponding gene named chi255 showed an open reading frame of 2031 bp, encoding a 676 amino acid residue protein. Both nucleotide and amino acid sequences similarity analyses revealed that the chi255 is a new chitinase gene, presenting several differences from the published chi genes of B. thuringiensis. The identification of chitin hydrolysis products resulting from the activity, exhibited by Chi255 through heterologous expression in Escherichia coli revealed that this enzyme is a chitobiosidase. CONCLUSIONS: Another chitinase named Chi255 belonging to chitobiosidase class was evidenced in B. thuringiensis subsp. kurstaki and was shown to present several differences in its amino acid sequence with those of published ones. The functionality of Chi255 was proved by the heterologous expression of chi255 in E. coli. SIGNIFICANCE AND IMPACT OF THE STUDY: The addition of the sequence of chi255 to the few sequenced B. thuringiensis chi genes might contribute to a better investigation of the chitinase 'structure-function' relation.     

Disruption of the Zymomonas mobilis extracellular sucrase gene (sacC) improves levan production

AIMS: Disruption of the extracellular Zymomonas mobilis sucrase gene (sacC) to improve levan production. METHODS AND RESULTS: A PCR-amplified tetracycline resistance cassette was inserted within the cloned sacC gene in pZS2811. The recombinant construct was transferred to Z. mobilis by electroporation. The Z. mobilis sacC gene, encoding an efficient extracellular sucrase, was inactivated. A sacC defective mutant of Z. mobilis, which resulted from homologous recombination, was selected and the sacC gene disruption was confirmed by PCR. Fermentation trials with this mutant were conducted, and levansucrase activity and levan production were measured. In sucrose medium, the sacC mutant strain produced threefold higher levansucrase (SacB) than the parent strain. This resulted in higher levels of levan production, whilst ethanol production was considerably decreased. CONCLUSIONS: Zymomonas mobilis sacC gene encoding an extracellular sucrase was inactivated by gene disruption. This sacC mutant strain produced higher level of levan in sucrose medium because of the improved levansucrase (SacB) than the parent strain. SIGNIFICANCE AND IMPACT OF THE STUDY: The Z. mobilis CT2, sacC mutant produces high level of levansucrase (SacB) and can be used for the production of levan.     

植酸酶产生菌黑曲霉N14的诱变选育及其基因分析

以植酸酶产生菌黑曲霉03214为出发菌株,经紫外线和亚硝基胍诱变,获得了产酶活性较出发菌株提高了22．3％,达422IU／ml发酵液的突变菌株黑曲霉N14,其最适pH值为2．5,最适温度为50℃。通过对黑曲霉N14植酸酶phyA基因进行PCR扩增,获得了一条长约1．5kb的特异性产物。以pMD18-T为载体,构建了含有目的基因片段的重组质粒。DNA序列测定表明,目的基因片段含有植酸酶phyA基因的完整序列(GenBank Accession：AY426977),phyA基因全长1506bp,其中包含一段长102bp的内含子,编码467个氨基酸,有10个潜在的糖基化位点,5’端有一编码19个氨基酸的信号肽序列。实验结果为植酸酶基因工程菌的构建奠定了基础。     

A novel recombinant ethyl ferulate esterase from Burkholderia multivorans

AIMS: Isolation and identification of bacterial isolates with specific ferulic acid (FA) esterase activity and cloning of a gene encoding activity. METHODS AND RESULTS: A micro-organism with ethyl ferulate hydrolysing (EFH) activity was isolated by culture enrichment techniques. Detailed molecular identification based on species-specific primers and two conserved genes (16S rRNA and recA) led to the identification of the isolate as Burkholderia multivorans UWC10. A gene (designated estEFH5) encoding an EFH enzyme was cloned and its nucleotide sequence determined. Translational analysis revealed that estEFH5 encoded a polypeptide of 326 amino acids with an estimated molecular weight of 34.83 kDa. The EstEFH5 primary structure showed a typical serine hydrolase motif (G-H-S-L-G). The estEFH5 gene was over-expressed in Escherichia coli in an insoluble form. Following urea denaturation and in vitro refolding, the enzyme was purified using one-step His Select Nickel chromatographic column. CONCLUSION: Purified EstEFH5 showed a preference for short-chain rho-nitrophenyl esters (C2 and C3) a typical feature for carboxylesterase. Furthermore, the recombinant enzyme also retained the activity against ethyl ferulate (EF). SIGNIFICANCE AND IMPACT OF THE STUDY: A biocatalytic process for the production of FA from EF as a model substrate was demonstrated. This is the first report that describes the cloning and expression of a gene encoding FA esterase activity from the genus Burkholderia.     

木质素过氧化物酶基因（LipH8）的克隆及在甲醇毕赤酵母中的表达

以黄孢原毛平革菌 (Phanerochaetechrysosporium)RNA为模板 ,克隆LipH8基因片段 ,研究LipH8基因在甲醇毕赤酵母中的表达。构建了甲醇酵母表达质粒pMETA_LipH8载体 ,并将其线性化后用电穿孔法导入PichiamethabolicaPMAD16 ,部分阳性克隆的PCR结果表明LipH8基因已经整合到甲醇毕赤酵母染色体上 ,经摇瓶培养筛选出表达水平较高的酵母工程菌株。胞外木质素过氧化物酶活力达 932U L。     

编码1,3-丙二醇氧化还原酶基因的克隆和表达

采用PCR法克隆了巴氏梭菌（Clostridium pasteurianum）CpN86菌株编码1,3丙二醇氧化还原酶基因（dhaT基因）;完成了dhaT基因测序、表达载体构建和在大肠杆菌中表达;分离和纯化了dhaT基因表达的重组蛋白。实验结果：（1）PCR法克隆的dhaT基因和肺炎克雷伯氏菌Klebsiella pneumoniae菌株dhaT基因的序列同源性为829%;（2）dhaT基因表达蛋白的酶活为108U/mg;（3）dhaT基因表达的蛋白分子量为43kD;（4）Western blot确定了dhaT基因表达的蛋白和 CpN86菌株天然蛋白有相同的抗原反应。     

Enterococcus faecium F58, a bacteriocinogenic strain naturally occurring in Jben, a soft, farmhouse goat's cheese made in Morocco

AIMS: Characterization of Ent F-58 produced by Enterococcus faecium strain F58 isolated from Jben, a soft, farmhouse goat's cheese manufactured without starter cultures. METHODS AND RESULTS: E. faecium strain F58 was isolated because of its broad inhibitory spectrum, including activity against food-borne pathogenic and spoilage bacteria. The antimicrobial substance was produced during the growth phase, with maximum production after 16-20 h of incubation at 30 degrees C, and was stable over a wide pH range (4-8) and at high temperatures (5 min at 100 degrees C). The enterocin was purified to homogeneity using cation exchange and hydrophobic interaction on C-18 and reverse-phase high-performance liquid chromatography. The activity was eluted as two individual active fractions (F-58A and F-58B) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis showed masses of 5210.5 and 5234.3 Da respectively. Both peptides were partially sequenced by Edman degradation, and amino-acid sequencing revealed high similarity with enterocin L50 (I). PCR-amplified fragments containing the structural genes for F-58 A and B were located in a 22-kb plasmid harboured by this strain. We verified that it also holds the structural gene for P-like enterocin. CONCLUSION: E. faecium strain F58 from Jben cheese, a producer of enterocin L50, exerts an inhibitory effect against strains of genera such as Listeria, Staphylococcus, Clostridium, Brochothrix and Bacillus. Enterocin was characterized according to its functional and biological properties, purification to homogeneity and an analysis of its amino acid and genetic sequences. SIGNIFICANCE AND IMPACT OF THE STUDY: E. faecium strain F58 is a newly discovered producer of enterocin L50, the biotechnological characteristics of which indicate its potential for application as a protective agent against pathogens and spoilage bacteria in foods.     

Screening and purification for novel cytochrome b5 from uncultured environmental micro-organisms

AIMS: We describe a sequence-based PCR method suitable for the isolation of a novel soluble heme-binding domain of cytochrome b(5) (cyt b(5)) gene directly from metagenomic DNA is described. METHODS AND RESULTS: Using the degenerate primer set, a cyt b(5) gene was isolated directly from metagenomic DNA. Based on the sequence-based PCR method, the similar conserved motif of cyt b(5) from Rhodopseudomonas palustris strain makes the novel target gene. The gene encoding cyt b(5) was cloned and expressed in Escherichia coli BL21 (DE3) using pET expression system. The expressed recombinant enzyme was purified by Ni-nitrilotriacetic acid affinity chromatography and characterized. CONCLUSIONS: Sequence-based strategy is an effective method for application of the novel gene from metagenomic DNA. SIGNIFICANCE AND IMPACT OF THE STUDY: Investigation of novel genes from metagenome, most of the micro-organism species are largely untapped, could represent an interesting and useful reservoir for biological processes.     

<Emphasis Type="Italic">Paenibacillus seodonensis</Emphasis> sp. nov., isolated from a plant of the genus <Emphasis Type="Italic">Campanula</Emphasis>

Strain DCT-19^T, representing a Gram-stain-positive, rodshaped, aerobic bacterium, was isolated from a native plant belonging to the genus Campanula on Dokdo, the Republic of Korea. Comparative analysis of the 16S rRNA gene sequence showed that this strain was closely related to Paenibacillus amylolyticus NRRL NRS-290^T (98.6%, 16S rRNA gene sequence similarity), Paenibacillus tundrae A10b^T (98.1%), and Paenibacillus xylanexedens NRRL B-51090^T (97.6%). DNADNA hybridization indicated that this strain had relatively low levels of DNA-DNA relatedness with P. amylolyticus NRRL NRS-290^T (30.0%), P. xylanexedens NRRL B-51090^T (29.0%), and P. tundrae A10b^T (24.5%). Additionally, the genomic DNA G + C content of DCT-19^T was 44.8%. The isolated strain grew at pH 6.0–8.0 (optimum, pH 7.0), 0–4% (w/v) NaCl (optimum, 0%), and a temperature of 15–45°C (optimum 25–30°C). The sole respiratory quinone in the strain was menaquinone-7, and the predominant fatty acids were C_15:0 anteiso, C_16:0 iso, and C_16:0. In addition, the major polar lipids were diphosphatidylglycerol and phosphatidylethanolamine. Based on its phenotypic properties, genotypic distinctiveness, and chemotaxonomic features, strain DCT-19^T is proposed as a novel species in the genus Paenibacillus, for which the name Paenibacillus seodonensis sp. nov. is proposed (=KCTC 43009^T =LMG 30888^T). The type strain of Paenibacillus seodonensis is DCT-19^T.     

Cloning of the Thermostable Cellulase Gene from Newly Isolated Bacillus subtilis and its Expression in Escherichia coli

A bacterial strain with high cellulase activity (0.26 U/ml culture medium) was isolated from hot spring, and classified and named as B. subtilis DR by morphological and 16SrDNA gene sequence analysis. A thermostable endocellulase, CelDR, was purified from the isolated strain. The optimum temperature of the enzyme reaction was 50°C, and CelDR retained 70% of its maximum activity at 75°C after incubation for 30 min. The putative gene celDR, consisting an open reading frame (ORF) of 1,524 nucleotides and encoding a protein of 508 amino acids with a molecular weight of 55 kDa, was purified from B. subtilis DR and cloned into pET-28a for expression. The cellulase production in E. coli BL21 (DE3) was enhanced to approximately three times that of the wild-type strain.