A-factor and streptomycin biosynthesis inStreptomyces griseus

Accumulating data have shown that the metabolites with a -butyrolactone ring functions as an autoregulatory factor or a microbial hormone for the expression of various phenotypes not only in a variety ofStreptomyces spp. but also in the distantly related bacteria. A-factor, as a representative of this type of autoregulators, triggers streptomycin biosynthesis and cellular differentiation inStreptomyces griseus. A model for the A-factor regulatory cascade on the basis of recent work is as follows. At an early step in the A-factor regulatory relay, the positive A-factor signal is first received by an A-factor receptor protein that is comparable in every aspect to eukaryotic hormone receptors, and then, via one or more regulatory steps, transmitted to an A-factor-responsive protein that binds to the upstream activation sequence of thestrR gene, a regulatory gene in the streptomycin biosynthetic gene cluster. The StrR protein thus induced appears to activate the other streptomycin biosynthetic genes. This review summarizes the characteristics of A-factor as a microbial hormone and the A-factor regulatory relay leading to streptomycin production.     

Physiological factors affecting streptomycin production by Streptomyces griseus ATCC 12475 in batch and continuous culture

Abstract Conditions of growth are described for the production of streptomycin by Streptomyces griseus ATCC 12475 using chemically defined minimal medium and complex medium. It was found using batch cultures that early synthesis of the antibiotic occurred during growth in minimal medium but was delayed until the onset of stationary phase in complex medium. This effect was independent of whether spores or vegetative cells were used as inoculum. Stability of streptomycin biosynthesis in continuous culture was dependent on dilution rate and medium employed. Cultures were highly unstable when grown on complex medium but could be maintained in steady states in continuous culture using minimal medium when the dilution rate was increased in a stepwise manner, starting at a dilution rate of 0.02 h⁻¹ (15% of μ _max). The effect of changing dilution rate on growth, streptomycin production and the level of streptomycin phosphotransferase was examined using this technique.     

Transformation using in vivo and in vitro methylation in Streptomyces griseus

Streptomyces griseus does not readily take up foreign DNA isolated from other Streptomyces species or Escherichia coli, presumably due to its unique restriction-modification systems that function as a barrier for interspecific DNA transfer. To efficiently transform S. griseus by avoiding the restriction barriers, we methylated incoming DNA in vivo and in vitro and treated protoplasts with heat prior to transformation. Whereas heat treatment of protoplasts or methylation of the E. coli-Streptomyces shuttle vectors (pXE4 and pKK1443) did not prominently improve the transformation efficiency, HpaII methylation of the vectors from any E. coli strains tested in this study highly increased the transformation efficiency. The highest transformation efficiency was observed when the shuttle vectors were isolated from the dam, hsd strain of E. coli (GM161) and methylated by AluI and HpaII methyltransferases, and the efficiency was approximately the same as that of the vectors from S. griseus. We identified several restriction-modification systems that decrease the transformation efficiency. This research also led us to understand methylation profiles and restriction-modification systems in S. griseus.     

In vitro protein phosphorylation associated with cellular differentiation of Streptomyces griseus

In vitro phosphorylation reactions with crude cellular extracts revealed that phosphorylation of a 17-kDa protein is associated with the onset of aerial mycelium formation in solid culture (but not submerged spore formation in liquid culture) of Streptomyces griseus. The possible importance of the 17-kDa protein phosphorylation in cellular differentiation was further indicated by inducing aerial mycelium formation in the presence of decoyinine and in studies using certain developmental mutants (relC, afsA, and M-1). It is proposed that the 17-kDa protein may play a role in cellular differentiation of S. griseus via its phosphorylation. Received: 17 July 1997 / Accepted: 20 September 1997     

灰色链霉菌启动子活性片段在大肠杆菌中的亚克隆及其序列分析

通过对重组质粒No.8-1的亚克隆,灰色链霉菌插入到大肠杆菌载体质粒中的序列已缩小到410bp,仍具有启动子功能。序列分析表明,启动子活性片段的G C碱基组成为50.5%。内含链霉菌启动子区域常有的正向重复序列;有1个Alul位点,1个Clal位点,2个Mbol位点,3个Nla Ⅲ位点,1个Pvu Ⅱ位点;具有类似于E.coli启动子的保守序列-10区和-35区,两者间隔18bp;在相应位置上分别有一段序列与E.coli的SD序列和在苄铅青链霉菌的SEP(Streptomyces-E.coil-type promoter)序列中存在的保守序列具有一定的相似性。     

Streptomycin biosynthesis in Streptomyces griseus I. Characterization of streptomycin-idiotrophic mutants

Abstract A total of 16 idiotrophic mutants unable to produce the aminoglycoside antibiotic streptomycin ( smi ) were isolated from Streptomyces griseus N2-3-11. Cosynthesis of streptomycin, its formation from various precursors and analysis of accumulated intermediates allowed grouping of the mutants in 3 classes, blocked: (I) in the first transamination step of the streptidine pathway; (II) in later steps of the streptidine pathway; or (III) outside streptidine biosynthesis.     

灰色链霉菌中乙酰木聚糖酯酶基因的克隆、表达及酶学性质研究

根据Gen Bank数据库中已报道的灰色链霉菌(Streptomyces griseus)全基因组序列,分析得到假定的乙酰木聚糖酯酶基因序列并设计引物;利用分子克隆的方法得到该菌株基因组中乙酰木聚糖酯酶基因,并构建原核表达载体p ET28a-Sgraxe,经IPTG诱导表达重组Sgr Axe,Ni-NTA亲和层析法纯化该蛋白。结果显示,克隆得到乙酰木聚糖酯酶基因axe,其序列全长1 008 bp,编码336个氨基酸。SDS-PAGE检测带有p ET28a-Sgraxe转化菌株诱导表达产物相对分子量约为37 k D,与理论值相符。纯化的重组Sgr Axe酶学性质表明,该酶最适反应温度为50℃,最适p H8.0,热稳定性较强,p H作用范围广;金属离子对酶均表现为抑制作用,尤其是Zn2+严重抑制酶活力;重组酶特征的分析揭示了其在工业中潜在的应用价值。     

Production of streptomycin from chitin using Streptomyces griseus in bioreactors of different configuration

Streptomyces griseus was cultured in three different bioreactors in a medium containing chitin flakes. When a conventional bioreactor stirred by two sets of Rushton impellers and operated at high speed was used, the yield of streptomycin (3.1 mg/l) was the highest observed and occurred at approximately 500 h. Cultivation of S. griseus in a bioreactor stirred at low speed by a U-shaped paddle resulted in a lower yield of streptomycin (1.8 mg/l) but this was achieved in a shorter period of time (400 h). Increasing the concentration of chitin from 5% to 10% w/v had no significant effect on either of these two parameters. The use of a novel vertical basket bioreactor in which the chitin flakes were contained within a wire mesh basket and were gently fluidised by air, enabled comparatively high yields of streptomycin (2.8 mg/l) in the relatively short time of 300 h.     

Self-cloning in Streptomyces griseus of an str gene cluster for streptomycin biosynthesis and streptomycin resistance.

An str gene cluster containing at least four genes (strR, strA, strB, and strC) involved in streptomycin biosynthesis or streptomycin resistance or both was self-cloned in Streptomyces griseus by using plasmid pOA154. The strA gene was verified to encode streptomycin 6-phosphotransferase, a streptomycin resistance factor in S. griseus, by examining the gene product expressed in Escherichia coli. The other three genes were determined by complementation tests with streptomycin-nonproducing mutants whose biochemical lesions were clearly identified. strR complemented streptomycin-sensitive mutant SM196 which exhibited impaired activity of both streptomycin 6-phosphotransferase and amidinotransferase (one of the streptomycin biosynthetic enzymes) due to a regulatory mutation; strB complemented strain SD141, which was specifically deficient in amidinotransferase; and strC complemented strain SD245, which was deficient in linkage between streptidine 6-phosphate and dihydrostreptose. By deletion analysis of plasmids with appropriate restriction endonucleases, the order of the four genes was determined to be strR-strA-strB-strC. Transformation of S. griseus with plasmids carrying both strR and strB genes enhanced amidinotransferase activity in the transformed cells. Based on the gene dosage effect and the biological characteristics of the mutants complemented by strR and strB, it was concluded that strB encodes amidinotransferase and strR encodes a positive effector required for the full expression of strA and strB genes. Furthermore, it was found that amplification of a specific 0.7-kilobase region of the cloned DNA on a plasmid inhibited streptomycin biosynthesis of the transformants. This DNA region might contain a regulatory apparatus that participates in the control of streptomycin biosynthesis.     

The nusG gene of Streptomyces griseus: Cloning of the gene and analysis of the A-factor binding properties of the gene product

Abstract The nusG gene of Streptomyces griseus was cloned and the nucleotide sequence determined. It encodes a protein with an identify of 76% to the reported receptor (VbrA) for VB-C, an autoregulatory factor in Streptomyces virginae . NusG protein was expressed in Escherichia coli . However, no binding activity for A-factor, an butyrolactone autoregulator in S. griseus very similar to VB-C, could be detected. The nusG gene of S. griseus does not seem to encode the A-factor-binding protein.     

Urea denaturation of the immobilized proteases of Streptomyces griseus (pronase)

The protease preparation (pronase, EC 3.4. group) from Streptomyces griseus has been covalently immobilized on porous succinamidopropyl glass using a carbodiimide carboxyl activation procedure. The separate activities of the individual proteases in this preparation were assayed using specific synthetic substrates. Stabilities of both soluble and immobilized preparations were determined and compared by assaying for each activity in urea solutions of various concentration. The loss of activity by the immobilized enzymes was shown to be reversible under most conditions. Analysis of the data in terms of a two-state transition showed that the urea concentration resulting in 50% loss of activity was increased for each enzyme as a result of immobilization. Also the m-value in the relation ΔG_D = ΔG^H₂O_D - m[urea] decreased for each enzyme upon immobilization. Thus, all of the enzymes were stabilized by immobilization and the apparent broadening of the transitions, as measured by the decreased m-value, was interpreted as the formation of a population of molecules with different stabilities. The degree of apparent stabilization upon immobilization varied with the magnitude decreasing as: aminopeptidases > carboxypeptidase ? trypsin > proteases A and B. Furthermore, it is suggested that stabilization may result from multipoint attachment since the magnitude was correlated with the number of potential enzyme reaction sites as reflected by their lysine contents.     

Expression of the Streptomyces griseusα-amylase gene in Escherichia coli

Abstract The amy gene of Streptomyces griseus was not expressed in Escherichia coli cells due to the lack of recognition of the amy promoter by the E. coli RNA polymerase, as confirmed by using promoter-probe vectors. The expression of the amy gene in E. coli was detected only when the promoter-less gene was placed under the control of the lacZ promoter and was dependent on the level of IPTG added to the medium. The extracellular α-amylase detected in the culture broth seems to be released by cellular lysis. When the amy gene lacking both leader peptide and promoter was transcribed from the lacZ promoter, no α-amylase activity was detected but larger E. coli cells and inclusion bodies were observed.     

Biocontrol of Fusarium oxysporum f.sp. cubense tropical race 4 by formulated cells and cell-free extracts of Streptomyces griseus in sterile soil environment

The biocontrol activities of cells and cell-free extracts of Streptomyces griseus was tested against Fusarium oxysporum f.sp. cubense tropical race 4 (FOC race 4) in a sterile soil environment. They were first formulated in sodium alginate, kaolin clay and in alginate–kaolin combination, prior to introducing into sterile soil inoculated with 6 log₁₀ cfu FOC race 4 g^?1 soil. Results revealed that bioformulated cells of S. griseus, irrespective of the materials used, were generally more effective in inhibiting growth of FOC race 4 when compared to non-formulated cells of S. griseus. Kaolin was the most suitable inert material as formulation of S. griseus with kaolin effectively suppressed FOC race 4, with only 5.40 log₁₀ cfu g^?1 of FOC race 4 recovered after 20 days. Kaolin formulations also allowed good cell recovery post-formulation. Alginate was less desirable as poorer control was demonstrated, with 6.12 and 6.16 log₁₀ cfu g^?1 of FOC race 4 recovered from soils treated with alginate only and alginate–kaolin formulated S. griseus, respectively. Bioformulations did not benefit cell-free extracts at all. Our study suggests formulation of cells of S. griseus is more beneficial than cell-free extracts and kaolin is the preferred material for formulation.     

Characterization of a novel sesquiterpene cyclase involved in (+)-caryolan-1-ol biosynthesis in Streptomyces griseus

Most terpenoids have been isolated from plants and fungi and only a few from bacteria. However, an increasing number of genome sequences indicate that bacteria possess a variety of terpenoid cyclase genes. We characterized a sesquiterpene cyclase gene (SGR2079, named gcoA) found in Streptomyces griseus. When expressed in Streptomyces lividans, gcoA directed production of a sesquiterpene, isolated and determined to be (+)-caryolan-1-ol using spectroscopic analyses. (+)-Caryolan-1-ol was also detected in the crude cell lysate of wild-type S. griseus but not in a gcoA knockout mutant, indicating that GcoA is a genuine (+)-caryolan-1-ol synthase. Enzymatic properties were characterized using N-terminally histidine-tagged GcoA, produced in Escherichia coli. As expected, incubation of the recombinant GcoA protein with farnesyl diphosphate yielded (+)-caryolan-1-ol. However, a small amount of another sesquiterpene was also detected. This was identified as the bicyclic sesquiterpene hydrocarbon (+)-β-caryophyllene by comparison with an authentic sample using GC-MS. Incorporation of a deuterium atom into the C-9 methylene of (+)-caryolan-1-ol in an in vitro GcoA reaction in deuterium oxide indicated that (+)-caryolan-1-ol was synthesized by a proton attack on the C-8/C-9 double bond of (+)-β-caryophyllene. Several β-caryophyllene synthases have been identified from plants, but these cannot synthesize caryolan-1-ol. Although caryolan-1-ol has been isolated previously from several plants, the enzyme responsible for its biosynthesis has not been identified previously. GcoA is thus the first known caryolan-1-ol synthase. Isolation of caryolan-1-ol from microorganisms is unprecedented.     

Rsistance to streptomycin in a producing strain of Streptomyces griseus.

Streptomyces griseus S 104 was sensitive to streptomycin during exponential growth in a medium which, in the subsequent stationary phase, supported production of the antibiotic in yields above 200 mug/ml. When antibiotic production began cultures developed a tolerance toward their lethal metabolite. This was not due to an increase in pH associated with antibiotic production, since pH effects on streptomycin sensitivity in S. griseus were in the reverse direction. However, the degree of tolerance was directly related to the amount of cell material present. Streptomycin production caused no change in the proportion of resistant variants in the population, nor did it cause the severe inhibition of protein synthesis observed in non-producing cultures exposed to the antibiotic. The lack of an effect on protein synthesis is attributed to the absence of streptomycin with in the cytoplasm since soluble extracts from mycelium harvested in the production phase were inactive when bioassayed immediately after cell disruption. However, they developed antibacterial activity rapidly when heated, and more slowly when incubated at 25 degrees C. The addition of phosphatase inhibitors during incubation prevented the appearance of antibiotic activity, and it was concluded that a small amount of streptomycin phosphate is present in the mycelium during antibiotic production. Differences in (14C) streptomycin uptake suggested that the mycelium was appreciably less permeable to the antibiotic in the production phase than during exponential growth. However, a small amount was taken up and much of it was in the soluble fraction of disrupted cells. Bioassays showed that this 14C-labeled antibiotic within the cells had been partially inactivated, suggesting that conversion of streptomycin to an inactive derivative is involved in the mechanism which protects the organism from its metabolite.