Exogenous AdoMet and its analogue sinefungin differentially influence DNA cleavage by R.EcoP15I--usefulness in SAGE

While it has been demonstrated that AdoMet is required for DNA cleavage by Type III restriction enzymes, here we show that in the presence of exogenous AdoMet, the head-to-head oriented recognition sites are cleaved only on a supercoiled DNA. On a linear DNA, exogenous AdoMet strongly drives methylation while inhibiting cleavage reaction. Strikingly, AdoMet analogue sinefungin results in cleavage at all recognition sites irrespective of the topology of DNA. The cleavage reaction in the presence of sinefungin is ATP dependent. The site of cleavage is comparable with that in the presence of AdoMet. The use of EcoP15I restriction in presence of sinefungin as an improved tool for serial analysis of gene expression is discussed.     

Cloning of repeated DNA sequences from Streptomyces cattleya

Abstract A number of DNA sequences were cloned from Streptomyces cattleya which hybridized to more than one chromosomal DNA sequence. These sequences were unrelated and have a minimum copy number of between 4 and 10. One of these sequences showed hybridization to multiple DNA fragments from a wide range of other Streptomyces .     

Plasmodium falciparum: Antimalarial activity in culture of sinefungin and other methylation inhibitors

A culture line of Plasmodium falciparum (FCR-3/Gambia) was exposed in vitro for a 2-day period to several analogs of adenosylhomocysteine. Minimal concentrations giving complete inhibition of growth were 0.2 mM for 3-deazaadenosine, 0.2 mM for 5′-deoxy-5′-(isobutylthio)-3-deazaadenosine, and 0.3 μM for sinefungin. The effects of the first two of these compounds were potentiated by homocysteine-thiolactone, suggesting that they were inhibiting methylation reaction(s) indirectly via adenosylhomocysteine hydrolase (EC 3.3.1.1).     

Context-dependent conservation of DNA methyltransferases in bacteria

DNA methytransferases (MTs) in bacteria are best understood in the context of restriction-modification (R-M) systems, which act as bacterial immune systems against incoming DNA including phages, but have also been described as selfish elements. But several orphan MTs, which are not associated with any restriction enzyme, have also been characterized and may protect against parasitism by R-M systems. The occurrence of MTs in these two contexts, namely as part of R-M systems or as orphans, is poorly understood. Here we report the results of a comparative genomic survey of DNA MTs across ～1000 bacterial genomes. We show that orphan MTs overwhelm R-M systems in their occurrence. In general, R-M MTs are poorly conserved, whereas orphans are nearly as conserved within a genus as any average gene. However, oligonucleotide usage and conservation patterns across genera suggest that both forms of MTs might have been horizontally acquired. We suggest that many orphan MTs might be 'degradation' products of R-M systems, based on the properties of orphan MTs encoded adjacent to highly diverged REs. In addition, several fully degraded R-M systems exist in which both the MT and the RE are highly divergent from their corresponding reference R-M pair. Despite their sporadic occurrence, conserved R-M systems are present in strength in two highly transformable genera, in which they may contribute to selection against integration of foreign DNA.     

Cloning and characterization of an amplified DNA sequence in chromosomal DNA of Streptomyces aureofaciens 2201

In strain 2201 of Streptomyces aureofaciens, a high copy number amplified DNA sequence (ADS-Sa2201) was found and characterized. The amplified sequence in the chromosomal DNA of this strain forms a stretch of about 10 kb tandemly repeated 100-500 times. In this strain also, extrachromosomal copies of the repeated unit of the ADS-Sa2201 were found. In cloning experiments any autonomous replicon was found on ADS-Sa2201 and it thus can be presumed that the presence of the extrachromosomal copies of the repeated unit of ADS-Sa2201 is only a result of its excision from the chromosome. A part of the repeated unit of ADS-Sa2201 was also found in chromosomal DNA of strain 13 of S. aureofaciens. In this strain no part of ADS-Sa2201 is present extrachromosomally.     

S-adenosylhomocysteine inhibition of three purified tRNA methyltransferases from rat liver.

Three tRNA methyltransferases from rat liver have been fractionated and purified greater than 100-fold. These enzymes have been examined for their sensitivity to inhibition by S-adenosylhomocysteine (SAH). The methyltransferase which forms m2-guanine in the region between the dihydrouridine loop and the acceptor stem of tRNA (m2-guanine methyltransferase I) is least sensitive to SAH inhibition, with a Ki of 8 muM. The enzyme responsible for forming m2-guanine between the dihydrouridine and anticodon loops (m2-guanine methyltransferase II) has a Ki of 0.3 muM, while m1-adenine methyltransferase shows intermediate sensitivity to SAH (Ki = 2.4 muM). All three methyltransferases have similar Km's for the S-adenosylmethionine substrate (1.5-2.0 muM). These results are consistent with the hypothesis that activity of individual tRNA methyltransferases may be controlled by enzyme systems which alter cellular SAH levels.     

Detection of the single-stranded DNA of Streptomyces plasmid pSA1.1 and a binding histone-like protein

Abstract Streptomyces plasmid pSA1.1 accumulated single-stranded DNA as replication intermediates in S. lividans ; therefore, this plasmid was considered to replicate by a rolling-circle mechanism. A DNA-binding protein (pI > 9.7 and about 10 kDa) was purified on a denatured DNA-Cellulose column, then on a native DNA-Cellulose column. The N-terminal amino acid sequence of this protein has a high homology with bacterial histone-like proteins. In the gel retardation assay, this protein bound with the single-stranded DNA of pSA1.1. We propose that this protein may participate in the replication of pSA1.1.     

Introduction of some new endophytic bacteria from Bacillus and Streptomyces genera as successful biocontrol agents against Sclerotium rolfsii

Endophytic bacteria live inside plant tissues without causing disease and not only promote plant growth but can also protect plants against plant pathogens. During 2010–2011 crop years, some endophytic bacteria were collected and are then biochemically and molecularly identified (16srRNA) from bean farms of East Azarbaijan, Iran. Among these bacteria isolates, four isolates from Bacillus genera and four isolates from Streptomyces genera were selected for evaluation of their ability for biocontrol of Sclerotium rolfsii in laboratory and glasshouse conditions. Except one isolate named Streptomyces parvus, the rest of isolates could significantly inhibit mycelial growth in dual culture on PDA medium. All seven selected isolates showed significant inhibition in disease treatments in glasshouse experiments. Biological traits, such as length, wet and dry weight of roots and stems in endophytic bacterial treatment showed no differences with healthy control.     

黑暗链霉菌DNA同源重组系统的构建

以黑暗链霉菌Tt-49基因组为模板,利用PCR方法,扩增安普霉素生物合成关键基因aprF-G的上、下游序列,作为同源交换臂,并将红霉素抗性基因筛选标记及其启动子插入两交换臂之间,以温敏型质粒pKC1139为基础,构建用于阻断黑暗链霉菌Tt-49安普霉素生物合成的重组质粒pFD8.该质粒通过E.coil ET12567/pUZ8002去甲基化修饰后,经接舍转移进入黑暗链霉菌Tt-49,利用红霉素抗性筛选得到3株阳性转化子,分别命名为Tt-49 AG1、Tt-49 AG2和Tt-49 AG3.通过PCR鉴定,证明pFD8已插入黑暗链霉菌Tt-49基因组的目标位点.以亲株作对照,对3株工程菌进行红霉素抗性能力考察,发现3株工程菌的抗红霉素能力均高迭1 000 μg/mL以上.     

Distribution of oxoacyl synthase homology sequences within Streptomyces DNA

Abstract A genomic DNA sequence of Streptomyces strain ISP 5485 was cloned, sequenced and compared with corresponding information from nucleic acid data banks. The DNA sequence was unique, but showed homology to DNA coding for the condensing enzyme, 2-oxoacyl synthase, of the deoxyerythronolide B synthase complex (DEBS) from Saccharopolyspora erythraea NRRL 2338. A subfragment of the sequenced DNA was used to construct a gene-specific probe that formed part of the putative 2-oxoacyl synthase gene. The PCR-amplified and labelled probe was used in hybridization experiments involving 33 streptomycete strains that produced different classes of antibiotics. The probe showed widespread homology with DNA considered to be part of analogous genes within genomes of different polyketide producers. The implications of the probe homology to bacterial chromosomal DNA are discussed.     

Purification and characterization of S-adenosylhomocysteine deaminase from streptonigrin-producing Streptomyces flocculus.

An S-adenosylhomocysteine deaminase has been isolated and purified from streptonigrin-producing Streptomyces flocculus ATCC 13257. Deamination represents the major metabolic route of S-adenosylhomocysteine in this organism. The protein was found to be monomeric with a molecular weight of 56,100 +/- 1,600. The activity was optimal at pH 7.0 and 37 degrees C, and the deaminase was inactivated by p-chloromercuribenzoate but not by metal chelators. The Km for S-adenosylhomocysteine is 2.5 mM, and the Ki for inhibition by deoxycoformycin is 1.6 nM.     

海洋放线菌Streptomyces sp.大片段DNA基因组文库的构建

目的:构建稀有海洋放线菌Streptomyces sp.基因组文库.方法:以稀有海洋放线菌Streptomyces sp.为实验材料,随机剪切提取的总DNA,5'-磷酸末端补平回收40kb左右的DNA片段,与pWEBTM载体连接,经包装蛋白包装成噬菌体后侵染宿主细胞E.coli EPI100,构建该菌株的基因组文库,并对该文库进行质量鉴定.结果:成功构建了稀有海洋放线菌Streptomyces sp.的基因组文库,效价达9.0×104CFU/mL,得到4000个阳性克隆子,远远大于按覆盖率为99%计算至少所需的837个阳性克降子数,且平均插入片段长度为36kb,重组率100%.阳性克隆子保存于96孔板中,-80℃保存.结论:所构建文库的各项指标均达到要求,为了进一步评估Streptomyces sp.所能合成的所有潜在天然产物,还需要进一步检测该文库中包含有生物合成基因簇的大肠杆菌的表达情况.     

链霉菌来源生物碱及药理活性研究进展

不同来源的链霉菌所产生的次级代谢产物具有结构新颖、复杂多样且生物活性良好,是具有研究潜力的药物资源;生物碱类化合物是链霉菌代谢产物中重要活性成分之一。近十年从链霉菌中已经报道了许多生物碱的成分,本文按菌株来源综述了2007~2017年间报道的链霉菌来源的生物碱及其生物活性,为其他研究生物碱及其活性研究提供指导。     

Cloning and analysis of DNA sequences from Streptomyces hygroscopicus encoding geldanamycin biosynthesis

A gene library constructed from large (20 kb) fragments of total DNA from the geldananmycin-producing strain Streptomyces hygroscopicus 3602 cloned in the plasmid vector pIJ61 were used to transform S. lividans TK24. Three transformants of about 800 tested were found to have acquired the ability to produce an antibiotic lethal to a geldanamycin-sensitive strain of Bacillus subtilis. The plasmids isolated from these transformants, pIA101, pIA102 and pIA103, each contained an insert of 15 kb. A 4.5 kb DNA fragment from the insert in pIA102 hybridised to DNA from S. hygroscopicus 3602 and to DNA encoding part of the erythromycin polyketide synthase but not to S. lividans TK24 DNA. The integration-defective phage vector C31 KC515 containing this 4.5 kb fragment was able to lysogenise S. hygroscopicus 3602 to produce lysogens defective in geldanamycin production. Loss of the prophage restored the ability to produce geldanamycin. Extracts of fermentation broth cultures of S. lividans containing pIA101, pIA102 and pIA102 and pIA103 analysed by thin-layer chromatography (TLC) contained compounds identical or very similar to purified geldanamycin, which were not present in S. lividans. These compounds showed a mass spectrum indistinguishable from geldanamycin. The evidence suggests that the clones contain DNA sequences encoding functions required for geldanamycin biosynthesis including components of the polyketide synthase.     

Bacterial colony screening with a Streptomyces DNA probe

Restriction fragments of 1.5 kb-3.5 kb length were selected from a SalI digest of Streptomyces coriofaciens ISP5485 DNA. After radiolabelling, these fragments were used as a molecular probe. A number of actinomycetes was screened in colony hybridization. Streptomyces and Streptoverticillium strains were recognized by the probe and the hybridization sensitivity was high with isolated DNA.     

Streptomyces cloning vector derived from Streptomyces lavendulae-grasserius mini-plasmid pSLG33

Abstract The cryptic multicopy plasmid designated pSLG33 (2.65 kb) was isolated from the vegetative mycelium of Streptomyces lavendulae-grasserius RIA 746 and physically characterized. pRS410 vector (5.4 kb) was constructed by insertion of aph and tsr genes coding for neomycin and thiostrepton resistance, respectively, in a non-essential part of the plasmid molecule. The pRS410 is compatible with multicopy Streptomyces plasmid vectors derived from pIJ101 plasmid.     

CbiX-homologous protein (CbiXhp), a metal-binding protein, from Streptomyces seoulensis is involved in expression of nickel-containing superoxide dismutase

To find the accessory proteins participating in expression and maturation of nickel-containing superoxide dismutase (NiSOD), a metal-binding protein (CbiXhp) homologous to cobaltochelatase (CbiX) of Bacillus megaterium was isolated by nickel-nitrilotriacetic acid resin from Streptomyces seoulensis. The deduced amino acid sequence of cbiXhp showed 87% and 39% identity to CbiX of Streptomyces coelicolor and that of B. megaterium, respectively. Overexpression of CbiXhp increased the activity and the expression of NiSOD in the presence and absence of nickel, but to a lesser extent in its absence. This result indicates that CbiXhp is involved in the expression of NiSOD.