Effect of Temperature on the Activities of Cytochrome c Oxidase and Respiration in Cassava Root Mitochondria

Cosmid pR4Cl is a derivative of multicopy plasmid pIJ365 which has an insertion of the cos (cohesive end site) region of actinophage R4 [T. Morino, H. Takahashi and H. Saito, Mol. Gen. Genet., 198, 228 (1985)]. When the donor R4 phage was propagated in S. lividans carrying the plasmid, the phage lysate contained transducing particles which encapsulated head-to-tail concatemers of the plasmid DNA. These particles could mediate transfer of the plasmid at a high frequency. We examined conditions that gave a maximum transduction frequency of cosmid pR4Cl. Conditions which depress R4 phage propagation, such as incubation of recipient S. parvulus at a high temperature, improved the frequency. Obviously such conditions minimized the lethal effect of viable phage propogation. The highest transduction frequency obtained so far was around 3 × 10^-3 transductants per infected phage when S. lividans was used as the recipient. This was about 30 per cent of the cosmid transducing particles estimated from the cosmid DNA content in the transducing lysate. The significance of cosmid transduction for gene manipulation in Streptomyces strains is also discussed.     

Synthesis of Several Analogs of Esterified Cerebroside Isolated from Human and Pig Epidermis

A cosmid, pR4C1, composed of the actinophage R4 cos sequence and Streptomyces plasmid pIJ365, was encapsidated in R4 phage particles in vivo. [T. Morino et al., Mol. Gen. Genet., 198, 228–233 (1985)] In this report a cosmid derivative, pR4C4, is shown to be also encapsidated by heterogeneous actinophages, SPA10 and SPA38, and transferred to Streptomyces lividans. Use of this transduction and conservation of the DNA packaging mechanism are discussed.     

A Non-Restricting and Non-Methylating <Emphasis Type="Italic">Escherichia coli</Emphasis> Strain for DNA Cloning and High-Throughput Conjugation to <Emphasis Type="Italic">Streptomyces coelicolor</Emphasis>

Escherichia coli strains are used in secondary metabolism research for DNA cloning and transferring plasmids by intergeneric conjugation. Non-restricting strains are desirable for DNA cloning and non-methylating strains are beneficial for transferring DNA to methyl-restricting hosts, like Streptomyces coelicolor. We have constructed a non-methylating E. coli strain, JTU007, by deleting the DNA methylation genes dcm and dam from the widely used non-restricting cloning host DH10B. JTU007 was tested as donor for the conjugative transfer of a plasmid containing the 39 kb actinorhodin biosynthesis gene cluster to S. lividans and S. coelicolor. The Dcm⁻ Dam⁻ strain JTU007 transferred DNA into S. coelicolor A(3)2 derivatives at high frequency. To demonstrate the usefulness of E. coli JTU007 for gene cloning, we constructed a comprehensive S. toxytricini genomic cosmid library, and transferred it using high-throughput conjugation to the methyl-restricting S. coelicolor. One of the cosmid clones produced a brown pigment, and the clone was revealed to carry a tyrosinase operon. JTU007 is more useful than ET12567 because it does not restrict methylated DNA in primary cloning, and gives higher transformation and cosmid infection frequencies.     

S-Adenosylmethionine (SAM) and antibiotic biosynthesis: effect of external addition of SAM and of overexpression of SAM biosynthesis genes on novobiocin production in Streptomyces

The production of antibiotics in different Streptomyces strains has been reported to be stimulated by the external addition of S-adenosylmethionine (SAM) and by overexpression of the SAM synthetase gene metK. We investigated the influence of SAM addition, and of the expression of SAM biosynthetic genes, on the production of the aminocoumarin antibiotic novobiocin in the heterologous producer strain Streptomyces coelicolor M512 (nov-BG1). External addition of SAM did not influence novobiocin accumulation. However, overexpression of a SAM synthase gene stimulated novobiocin formation, concomitant with an increase of the intracellular SAM concentration. Streptomyces genomes contain orthologs of all genes required for the SAM cycle known from mammals. In contrast, most other bacteria use a different cycle for SAM regeneration. Three secondary metabolic gene clusters, coding for the biosynthesis of structurally very different antibiotics in different Streptomyces strains, were found to contain an operon comprising all five putative genes of the SAM cycle. We cloned one of these operons into an expression plasmid, under control of a strong constitutive promoter. However, transformation of the heterologous novobiocin producer strain with this plasmid did not stimulate novobiocin production, but rather showed a detrimental effect on cell viability in the stationary phase and strongly reduced novobiocin accumulation.     

Cloning and identification of saprolmycin biosynthetic gene cluster from Streptomyces sp. TK08046

Saprolmycins A–E are anti-Saprolegnia parasitica antibiotics. To identify the gene cluster for saprolmycin biosynthesis in Streptomyces sp. TK08046, polymerase chain reaction using aromatase and cyclase gene-specific primers was performed; the spr gene cluster, which codes for angucycline biosynthesis, was obtained from the strain. The cluster consists of 36 open reading frames, including minimal polyketide synthase, ketoreductase, aromatase, cyclase, oxygenase, and deoxy sugar biosynthetic genes, as defined by homology to the corresponding genes of the urdamycin, Sch-47554, and grincamycin biosynthetic gene clusters in Streptomyces fradiae, Streptomyces sp. SCC-2136, and Streptomyces lusitanus, respectively. To establish the function of the gene cluster, an expression cosmid vector containing all 36 open reading frames was introduced into Streptomyces lividans TK23. The transformant was confirmed to express the biosynthetic genes and produce saprolmycins by liquid chromatography–mass spectrometry analysis of the extract.     

珠江口沉积物来源链霉菌SCSIO 40020中bafilomycins的鉴定及其生物合成基因簇的异源表达

【目的】从珠江口沉积物来源的菌株SCSIO40020中分离bafilomycins,并对其生物合成基因簇进行克隆和异源表达研究。【方法】通过分析菌株SCSIO 40020的16S rRNA基因序列并构建系统发育树以鉴定菌种,以柱层析法和制备色谱法对次级代谢产物进行分离纯化,借助波谱学手段完成单体化合物的结构鉴定,采用生物信息学分析定位bafilomycins的生物合成基因簇,通过筛选菌株SCSIO 40020基因组的细菌人工染色体文库和接合转移将bafilomycins生物合成基因簇导入3种链霉菌进行异源表达,利用高效液相色谱检测异源表达菌株的发酵产物。【结果】菌株SCSIO 40020被鉴定为链霉菌属菌株,从其发酵产物中分离鉴定了2个单体化合物bafilomycinsA1和D。克隆了链霉菌SCSIO40020中bafilomycins的生物合成基因簇并推导了其生物合成途径,在3种链霉菌中表达产生了bafilomycins。【结论】从珠江口环境中获得了一株产生bafilomycins的链霉菌SCSIO 40020,成功建立了该菌株次级代谢产物生物合成基因簇的异源表达体系,并首次在链霉菌...     

链霉菌次级代谢产物高产菌株的构建策略

随着基因测序技术的发展,人类获得了大量有关链霉菌次级代谢产物合成基因簇的信息,通过合理的构建策略可以激活其中的隐性基因簇或提高基因簇的表达水平,从而获得新的链霉菌次级代谢产物,或显著提高已知次级代谢产物的发酵水平。从基因表达调控、转座子突变、合成生物学方法、组学方法等四个方面,综述了提高链霉菌次级代谢产物产量的构建策略及其研究进展。     

Effect of the Bacterial Hemoglobin vhbGene on the Efficiency of Intergeneric Conjugation Escherichia coli–Streptomyces and Biosynthesis of Antibiotics in Streptomyces

The bacterial hemoglobin vhbgene was cloned from sliding bacterium Vitreoscillasp. as an element of the system ensuring survival of this microorganism in an environment that contains insufficient amount of oxygen. The vhbgene was transferred fromEscherichia colito some Streptomycesstrains, producers of antibiotics, by the method of intergeneric conjugation using conjugative–integrative plasmid vectors pIH1 and pCH2. The stability of plasmid DNA inheritance was analyzed in the genomes of exconjugants. A positive effect of the vhbgene on processes of conjugation and antibiotic production in a number of examined strains was shown.     

Diversity of the culturable lichen-derived actinobacteria and the taxonomy of Streptomyces parmotrematis sp. nov.

A total of 37 actinobacteria were isolated from eighteen lichen samples collected in Thailand. Based on the 16S rRNA gene sequences, they were identified into five genera including Actinoplanes (1 strain), Actinomadura (1 strain), Pseudosporangium (1 strain), Wangella (1 strain) and Streptomyces (33 strains). Among these isolates, strain Ptm05^T, Ptm01 and Ptm12 showed low 16S rRNA gene similarity and was selected for the further taxonomic study using the polyphasic approach. These strains showed the highest 16S rRNA gene sequence similarity with Streptomyces sparsogenes ATCC 25498^T (97.44–97.72%). Strain Ptm05^T was selected for the type strain. The chemical cell composition of the strain was similar to the members of Streptomyces genus. LL-diaminopimelic acids were detected in the peptidoglycan. Menaquinones were MK-9(H₈) and MK-9(H₆). Phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannoside, one unidentified phospholipid, one unidentified glycolipid and one unidentified lipid were detected as the polar lipids. The predominant cellular fatty acids are anteiso-C_15:0, iso-C_15:0, iso-C_16:0, iso-C_17:0 and C_16:0. The dDNA-DNA hybridization values among strain Ptm05^T and its closely related Streptomyces type strains were 17.2–18.0%. In addition, the ANIb and ANIm between strain Ptm05^T and related Streptomyces type strains were ranged from 75.69 to 76.13% and 85.21 to 85.35%, respectively. Based on phenotypic and genomic evidence, strain Ptm05^T (=?TBRC 14546^T?=?NBRC 115203^T) represents the novel species of the genus Streptomyces for which the name Streptomyces parmotrematis sp. nov. is proposed. This study showed that the lichens are the promising source of the novel actinobacterial taxa.

     

Chromium accumulation by two Streptomyces spp. isolated from riverine sediments

Strains designated R22 and R25, isolated from Salí River sediments, Argentina, were highly resistant to chromium. These strains were shown by 16S rRNA sequencing studies to be Streptomyces spp.; this affiliation was consistent with morphological and chemical characteristics. Growth of strains R22 and R25 in medium containing 100 mg l⁻¹ chromate was reduced by only 23% and 34%, respectively, compared with growth in medium without added chromium. Streptomyces sp. strains R22 and R25 both accumulated chromium with yields of 10.0 and 5.6 mg Cr g⁻¹ of dry weight, respectively, and a chromate concentration of 50 mg ml⁻¹. Cell fractionation studies with strain R22 showed that the great majority of the chromium were associated with the cell wall fraction. Streptomyces strains R22 and R25 may have applications in bioremediation of chromium contamination. Journal of Industrial Microbiology & Biotechnology (2001) 26, 210–215. Received 23 June 2000/ Accepted in revised form 24 January 2000     

地黄内生放线菌的分离及地黄轮纹病拮抗菌Streptomyces folium leaf-16的新种鉴定

药用植物内生放线菌具有合成天然活性化合物的潜力,放线菌新种是寻找新型抗生素先导化合物的一个重要来源。【目的】挖掘药用植物地黄内生放线菌资源,并对地黄轮纹病拮抗菌株leaf-16进行新种鉴定。【方法】本研究采用五步消毒法分离河南道地药材地黄的内生放线菌,以地黄轮纹病原真菌草茎点霉(Phoma herbarum)为指示菌,采用平板对峙法筛选对该病菌有抑制作用的菌株,16S rRNA基因测序发现一株抗地黄轮纹病的放线菌新种leaf-16。通过形态、生理生化、细胞壁化学组分和分子生物学等特征对菌株leaf-16进行多相分类学鉴定。【结果】经平板对峙实验得到8株抗地黄轮纹病的放线菌,其中菌株leaf-16经16S rRNA基因测序、形态比较、生理生化、化学组分和分子生物学以及DNA-DNA杂交分析,确定菌株leaf-16为1株链霉菌新种,并命名为Streptomyces folium。【结论】菌株leaf-16为1株链霉菌新种,具有抑制地黄轮纹病原真菌的活性,为进一步分离新型抗地黄轮纹病的生物制剂奠定物质基础。     

Methods of assessment of transfer of a gentamycin-resistance gene (aacC1) from genetically engineered microorganisms into agriculturally important soil bacteria

In order to assess the risk associated with the deliberate release of genetically engineered microorganisms (GEMs) into the agricultural environment, the transfer of plasmids between bacterial strains was investigated under laboratory conditions. Genetically modified Rhizobium leguminosarum and Agrobacterium tumefaciens strains carrying the gentamycin acetyltransferase resistance gene (aacC1) on various plasmids were investigated for their ability to transfer the aacC1 gene to their wild-type (w.t.) counterparts, as well as to Pseudomonas syringae. Conjugation experiments between the various strains, were carried out after the relevant characteristics and conditions for selective growth of each bacterial strain had been ascertained. After conjugations on filters had been completed, the putative transconjugants were grown in media containing antibiotics and assessed for the presence of aacC1 gene by: (a) DNA plasmid profile; (b) expression of AAC(3)-I enzyme activity; (c) colony hybridization using a ³²P-labelled DNA probe complementary to the aacC1 gene. The results obtained indicate that transfer of the aacC1 gene from genetically modified strains of R. leguminosarum into a plasmid-free strain of A. tumefaciens occurred via self-transmissible plasmids. Alternatively, genetically modified A. tumefaciens bearing the aacC1 gene on plasmids acquired from R. leguminosarum strains, transferred it ineffectively to a hardly detectable frequency. No transfer of the aacC1 gene from genetically modified R. leguminosarum or A. tumefaciens strains into P. syringae has been observed. These data indicate that in the absence of the RP4 element, genetically modified A. tumefaciens is not able to efficiently transfer aacC1 into w.t. R. leguminosarum and P. syringae. Correspondence to: A. S. Tsiftsoglou     

Transfer of genes for utilization of starch (sta2) and melibiose (mel) to industrial strains of Saccharomyces cerevisiae by single-chromosome transfer,using a kar1 mutant as vector

Summary A method has been developed for the transfer of genes from other yeast strains and species to industrial yeast strains, using a haploid, kar1-1 mutant strain of Saccharomyces cerevisiae as a vector. The sta2 gene, conferring the ability to metabolize starch was transferred from an autotrophic haploid strain of S. cerevisiae (S. diastaticus) and the melibiose-metabolism (mel) gene(s), from S. kluyveri, to the kar1-1 mutant [K5-5A; ( ade2 his4 can1 gal) by normal mating and protoplast fusion. From this strain, the genes were transferred to baker's yeast and brewing yeast strains, which did not utilize starch, and to baker's yeast strains, which did not utilize melibiose, by protoplast fusion, spore-cell pairing, or rare-mating. Strains that utilized starch or melibiose were obtained by all three methods. Pulsed-field gel electrophoresis preparations showed little change in the mobility of the chromosomes of the hybrids. The most probable explanation for the results obtained is that single chromosomes were transferred, first, from the donor strains to the kar1-1 haploid mutant strain, and then from the kar1-1 vector to the recipient industrial strain of S. cerevisiae. The transfer of the genes is probably accomplished through formation of disomic strains and the, in the case of the hybrids that metabolize starch, by integration of the sta2 gene into the genome of the industrial yeast strains.     

Rapid detection and high-resolution discrimination of the genus Streptomyces based on 16S–23S rDNA spacer region and denaturing gradient gel electrophoresis

As the leading source of antibiotics, Streptomyces species are the subject of widespread investigation. Many approaches have been tried to aid in the classification of Streptomyces isolates to the genus, species, and strain levels. Genetic methods are more rapid and convenient than classification methods based on phenotypic characteristics, but a method that is universal in detecting all Streptomyces yet selective in detecting only Streptomyces is needed. The highly conserved nature of the 16S rRNA gene (16S rDNA) combined with the need to discriminate between closely related strains results in analyses of ribosomal intergenic spacer (RIS) regions being more productive than analyses of 16S rRNA genes. PCR primers were designed to amplify the RIS region as well as a sufficient length of the 16S rRNA gene to enable phylogenetic analyses of Streptomyces. Improved selectivity and specificity for the amplification of RIS sequences from Streptomyces with environmental samples was demonstrated. The use of RIS–PCR and denaturing gradient gel electrophoresis (DGGE) was shown to be a convenient means to obtain unique genetic “fingerprints” of Streptomyces cultures allowing them to be accurately identified at species, and even strain classification levels. These RIS–PCR and DGGE approaches show potential for the rapid characterization of environmental Streptomyces populations.     

Suppression of tumorigenicity by the IncW R plasmid pSa in Agrobacterium tumefaciens

Summary The effect of the IncW R plasmid, pSa, on tumorigenicity and on the expression and maintenance of the Ti plasmid in tumorigenic strains of A. tumefaciens was determined. Plasmid pSa could be transferred into and stably maintained by both octopine-and nopaline-utilizing A. tumefaciens strains. The R plasmid had no effect on Ti plasmid maintenance or on Ti plasmid functions, such as octopine utilization or conjugal bacterial transfer. However, A. tumefaciens strains harboring both the R plasmid and the Ti plasmid in most instances failed to induce tumors on a number of plant species. This effect on tumorigenicity is specific to pSa. When pSa is cured from the A. tumefaciens transconjugants or when their Ti plasmids are genetically transferred to an appropriate recipient, the resultant strains lacking the R plasmid regain tumorigenicity. Restriction endonuclease analysis of plasmid DNA isolated from transconjugants harboring pSa showed no difference in Ti plasmid cleavage patterns when compared to plasmid DNA isolated from the tumorigenic parent strain. These results indicate that pSa does not induce detectable permanent genetic alteration of the Ti plasmid. Rather, it appears that the R plasmid suppresses some Ti plasmid function(s) necessary for tumorigenicity.     

Studies on Xylanase from Microorganisms

As xylanase-producing microorganisms, 64 strains belonging to the genus Streptomyces were isolated from the barn-yard manures, silages and litters collected in Hokkaido district. Among these isolates the strain 102–1–4, which was found to be a new species under taxonomical studies and named Streptomyces xylophagus nov. sp., had the most outstanding ability for the enzyme production. In addition to the isolates, 38 strains of Streptomyces and 480 strains of filamentous fungi which have been preserved in our culture collection were also examined on their ability to produce the enzyme. 1) Among the strains of Streptomyces tested, only two strains, St. albogriseolus IAM 0031 and St. olivaceus IAM 0025 were found to have the ability, but their abilities were less than that of St. xylophagus nov. sp. 2) Out of 480 strains of fungi tested, Chaetomium, Schyzophyllum, Trametes, Echinodontium, Alternaria, Cepharosporium, Cercospora, Gibberella, Glomerella and Macrosporium produced the enzyme. Especially, Ch. trilateral 2264 was the most excellent.     

Identification of a novel <Emphasis Type="Italic">Streptomyces chattanoogensis</Emphasis> L10 and enhancing its natamycin production by overexpressing positive regulator ScnRII

A novel Streptomyces strain, L10, which is capable of producing natamycin, was isolated from a soil sample collected from Zhejiang province, China. On the basis of phylogenetic analysis of rpoB gene and 16S rDNA sequences, as well as phenotypic comparison, strain L10 (CGMCC 2644) is proposed to be a previously uncharacterized strain of S. chattanoogensis. By screening a cosmid library of strain L10 and primer walking, a partial sequence of scnRI and the entire sequence of scnRII were obtained, which are orthologues to the pathway-specific positive regulator genes of natamycin biosynthesis in S. natalensis. The engineered S. chattanoogensis Dl, generated by inserting an additional copy of scnRII into the chromosome of strain L10, increased its natamycin production by 3.3 fold in YSG medium and 4.6 fold in YEME medium without sucrose.     

Rapid discrimination of potato scab-causing <Emphasis Type="Italic">Streptomyces</Emphasis> species based on the RNase P RNA gene sequences

Scab disease significantly damages potatoes and other root crops. Some Streptomyces species have been reported as potato-scab pathogens. Identification of the phytopathogenic Streptomyces is mainly done on the basis of the 16S rRNA gene, but use of this gene has some limitations for discriminating these strains because they share high similarities of 16S rRNA gene sequences. We tested the RNase P RNA (rnpB) gene as a taxonomic marker to clarify the relationship among closely related scab-causing Streptomyces strains. The rnpB genes were analyzed for 41 strains including 9 isolates from Jeju, Korea. There were 4 highly variable regions including nucleotide gaps in the rnpB genes. Interspecies similarity of the rnpB gene in tested Streptomyces strains was lower than about 97%, while the intraspecies similarity was higher than about 98%. Phylogenetic analysis demonstrated that the rnpB tree has similar topology to the 16S rRNA gene tree, but produces a more divergent phyletic lineage. These results revealed that the rnpB gene could be used as a powerful taxonomic tool for rapid differentiation of closely related Streptomyces species. In addition, it was also suggested that the variable regions marked as α, β, γ, and δ in the rnpB gene could be useful markers for the detection of specific Streptomyces species.     

Expression by Soil Bacteria of Nodulation Genes from Rhizobium leguminosarum biovar trifolii

Gram-negative, rod-shaped bacteria from the soil of white clover-ryegrass pastures were screened for their ability to nodulate white clover (Trifolium repens) cultivar Grasslands Huia and for DNA homology with genomic DNA from Rhizobium leguminosarum biovar trifolii ICMP2668 (NZP582). Of these strains, 3.2% were able to hybridize with strain ICMP2668 and nodulate white clover and approximately 19% hybridized but were unable to nodulate. Strains which nodulated but did not hybridize with strain ICMP2668 were not detected. DNA from R. leguminosarum biovar trifolii (strain PN165) cured of its symbiotic (Sym) plasmid and a specific nod probe were used to show that the relationship observed was usually due to chromosomal homology. Plasmid pPN1, a cointegrate of the broad-host-range plasmid R68.45 and a symbiotic plasmid pRtr514a, was transferred by conjugation to representative strains of nonnodulating, gram-negative, rod-shaped soil bacteria. Transconjugants which formed nodules were obtained from 6 of 18 (33%) strains whose DNA hybridized with that of PN165 and 1 of 9 (11%) strains containing DNA which did not hybridize with that of PN165. The presence and location of R68.45 and nod genes was confirmed in transconjugants from three of the strains which formed nodules. Similarly, a pLAFR1 cosmid containing nod genes from a derivative of R. leguminosarum biovar trifolii NZP514 formed nodules when transferred to soil bacteria.     

Cloning and characterization of the chlorophyll biosynthesis gene chlM from Synechocystis PCC 6803 by complementation of a bacteriochlorophyll biosynthesis mutant of Rhodobacter capsulatus

A bacteriochlorophyll a biosynthesis mutant of the purple photosynthetic bacterium Rhodobacter capsulatus was functionally complemented with a cosmid genomic library from Synechocystis sp. PCC 6803. The complemented R. capsulatus strain contains a defined mutation in the bchM gene that codes for Mg-protoporphyrin IX methyltransferase, the enzyme which converts Mg-protoporphyrin IX to Mg-protoporphyrin IX methylester using S-adenosyl-l-methionine as a cofactor. Since chlorophyll biosynthesis also requires the same methylation reaction, the Synechocystis genome should similarly code for a Mg-protoporphyrin IX methyltransferase. Sequence analysis of the complementing Synechocystis cosmid indicates that it contains an open reading frame exhibiting 29% sequence identity to BchM. In addition, expression of the Synechocystis gene in the R. capsulatus bchM mutant via the strong R. capsulatus puc promoter was shown to support nearly wild-type levels of bacteriochlorophyll a synthesis. To our knowledge, the Synechocystis sequence thus represents the first chlorophyll biosynthesis gene homolog of bchM. The complementing Synechocystis cosmid was also shown to code for a gene product that is a member of a highly conserved family of RNA binding proteins, the function of which in cyanobacteria remains undetermined.