Involvement of two A-factor receptor homologues in Streptomyces coelicolor A3(2) in the regulation of secondary metabolism and morphogenesis

Nucleotide sequences homologous to arpA encoding the A-factor receptor protein (ArpA) of Streptomyces griseus are distributed in a wide variety of streptomycetes. Two genes, cprA and cprB , each encoding an ArpA-like protein were found and cloned from Streptomyces coelicolor A3(2). CprA and CprB shared 90.7% identity in amino acid sequence and both showed about 35% identity to ArpA. Disruption of cprA by use of an M13 phage-derived single-stranded vector resulted in severe reduction of actinorhodin and undecylprodigiosin production. In addition, the timing of sporulation in the cprA disruptants was delayed by 1 day. The cprA gene thus appeared to act as a positive regulator or an accelerator for secondary metabolite formation and sporulation. Consistent with this idea, introduction of cprA on a low-copy-number plasmid into the parental strain led to overproduction of these secondary metabolites and accelerated the timing of sporulation. On the other hand, cprB disruption resulted in precocious and overproduction of actinorhodin. However, almost no effect on undecylprodigiosin was detected in the cprB disruptants. Sporulation of the cprB disruptant began 1 day earlier than the parental strain. The cprB gene thus behaved as a negative regulator on actinorhodin production and sporulation. Consistent with this, extra copies of cprB in the parental strain caused reduced production of actinorhodin and delay in sporulation. It is thus concluded that both cprA and cprB play regulatory roles in both secondary metabolism and morphogenesis in S. coelicolor A3(2), just as the arpA /A-factor system in Streptomyces griseus .     

一株白芍内生放线菌的分离、活性及系统发育分析

目的:分离白芍中拮抗农作物致病菌和人类常见病原菌的内生放线菌,并进行系统发育分析。方法:采用3种分离培养基,从白芍根部分离内生放线菌;通过滤纸片法筛选具有拮抗活性的菌株,观察菌丝形态,并进行16S rDNA序列系统发育分析。结果:从白芍中分离得到16株内生放线菌,其中从FYSCA培养基中分离到9株;16株内生放线菌中有6株具有拮抗作用,菌株S-BS033004对5种病原菌有拮抗活性,尤其是对棉花黄萎病菌和小孢拟盘多毛孢菌和耐青霉素类金黄色葡萄球菌的拮抗作用显著,抑菌圈≥20mm。经16S rDNA系统发育分析表明该菌株与Streptomyces anulatus NBRC13369T等6株链霉菌模式菌株亲缘关系较近,相似性均为99.7%。结论:白芍内生放线菌S-BS033004是一株杀菌谱较广的链霉菌,具有很好的开发潜力。     

Cloning and characterization of a gene involved in aerial mycelium formation in Streptomyces griseus.

A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) is essentially required for aerial mycelium formation and streptomycin production in Streptomyces griseus. A DNA fragment which induced aerial mycelium formation and sporulation in an A-factor-deficient mutant strain, S. griseus HH1, was cloned from this strain on a high-copy-number plasmid. Subcloning and nucleotide sequencing revealed that one open reading frame with 218 amino acids, named AmfC, served as a multicopy suppressor of the aerial mycelium-defective phenotype of the A-factor-deficient strain. The amfC gene did not restore A-factor or streptomycin production, indicating that amfC is involved in aerial mycelium formation independently of secondary metabolic function. Disruption of the chromosomal amfC gene in the wild-type S. griseus strain caused a severe reduction in the abundance of spores but no effect on the shape or size of the spores. The infrequent sporulation of the amfC disruptant was reversed by introduction of amfC on a plasmid. The amfC-defective phenotype was also restored by the orf1590 gene but not by the amfR-amfA-amfB gene cluster. Nucleotide sequences homologous to the amfC gene were distributed in all of 12 Streptomyces species tested, including Streptomyces coelicolor A3(2). The amfC homolog of S. coelicolor A3(2) was cloned and its nucleotide sequence was determined. The AmfC products of S. griseus and S. coelicolor A3(2) showed a 60% identity in their amino acid sequences. Introduction of the amfC gene of S. coelicolor A3(2) into strain HH1 induced aerial mycelium formation and sporulation, which suggests that both play the same functional role in morphogenesis in the strains.     

格尔德霉素基因工程高产菌株的构建和培养

在格尔德霉素产生菌吸水链霉菌17997(Streptomyces hygroscopicus 17997)中存在两种3-氨基-5-羟基苯甲酸(3-amino-5-hydroxybenzoic acid, AHBA)的生物合成基因簇, 根据同源性可分为苯醌类和萘醌类。已证明其中苯醌类的AHBA生物合成基因簇负责格尔德霉素(geldanamycin, Gdm)起始单位的合成, 而萘醌类的AHBA基因簇可能参与未知安莎化合物的生物合成。为提高吸水链霉菌17997菌种的Gdm发酵产量, 并研究高产菌种在固体培养基上孢子的生长周期。采用基因阻断技术, 将吸水链霉菌17997中的萘醌类AHBA生物合成基因簇(shnSOP)进行破坏, 以获得DSOP菌株, 从而减少对合成所需共同底物AHBA的争夺。HPLC分析结果表明DSOP菌株Gdm的发酵产量比原株提高185%。同时, 通过孢子计数发现该菌株在固体培养基上的孢子生长经历2个周期, 第2代孢子菌种的Gdm产量较高。     

Detection and properties of A-factor-binding protein from Streptomyces griseus.

The optically active form of tritium-labeled A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone), a pleiotropic autoregulator responsible for streptomycin production, streptomycin resistance, and sporulation in Streptomyces griseus, was chemically synthesized. By using the radioactive A-factor, a binding protein for A-factor was detected in the cytoplasmic fraction of this organism. The binding protein had an apparent molecular weight of approximately 26,000, as determined by gel filtration. Scatchard analysis suggested that A-factor bound the protein in the molar ratio of 1:1 with a binding constant, Kd, of 0.7 nM. The number of the binding protein was roughly estimated to be 37 per genome. The "inducing material" virginiae butanolide C (VB-C), which has a structure very similar to that of A-factor and is essential for virginiamycin production in Streptomyces virginiae, did not inhibit binding. In addition, no protein capable of specifically binding 3H-labeled VB-C was found in S. griseus. Together with the observation that VB-C had almost no biological activity on the restoration of streptomycin production or sporulation in an A-factor-deficient mutant of S. griseus, these results indicated that the binding protein had a strict ligand specificity. Examination for an A-factor-binding protein in Streptomyces coelicolor A3(2) and Streptomyces lividans showed the absence of any specifically binding protein.     

The A-factor-binding protein of Streptomyces griseus negatively controls streptomycin production and sporulation.

A-factor, 2-(6'-methylheptanoyl)-3R-hydroxymethyl-4-butanolide, is an autoregulator essential for streptomycin production and sporulation in Streptomyces griseus. S. griseus 2247 that requires no A-factor for streptomycin production or sporulation was found to have a defect in the A-factor-binding protein. This observation implied that the A-factor-binding protein in the absence of A-factor repressed the expression of both phenotypes in the wild-type strain. Screening among mutagenized S. griseus colonies for strains producing streptomycin and sporulating in the absence of A-factor yielded three mutants that were also deficient in the A-factor-binding protein. Reversal of the defect in the A-factor-binding protein of these mutants led to the simultaneous loss of streptomycin production and sporulation. These data suggested that the A-factor-binding protein played a role in repressing both streptomycin production and sporulation and that the binding of A-factor to the protein released its repression. Mutants deficient in the A-factor-binding protein began to produce streptomycin and sporulate at an earlier stage of growth than did the wild-type strain. These mutants produced approximately 10 times more streptomycin than did the parental strain. These findings are consistent with the idea that the intracellular concentration of A-factor determines the timing of derepression of the gene(s) whose expression is repressed by the A-factor-binding protein.     

An allatostatic factor and juvenile hormone synthesis by corpora allata in Locusta migratoria

The hemolymph juvenile hormone (JH) titer in sexually immature female adults of Locusta migratoria (Ibaraki strain, Japan) was lower than in sexually mature females; nevertheless, JH synthetic activity by the corpora allata (CA) in vitro was considerably higher in immature females than in sexually mature females ([Okuda et al., 1996]). We carried out experiments to explain this contradiction. The CA activity of sexually immature female adults was very low when the CA were incubated as a complex together with the corpora cardiaca (CC) and brain. When the same complex was assayed after cutting the nerve cord connecting the CC and CA (NCA1), JH synthesis by the CA was enhanced tenfold. When this pair of CA was incubated in fresh medium without the CC and brain, JH synthesis was further increased. Therefore, the higher in-vitro JH production by CA from immature female adults was the result of isolation of the CA from the brain and CC. A methanolic extract of brain-CC complexes contained a factor that inhibited JH synthetic activity by CA in vitro in both immature and mature insects, and this inhibition was reversible. The factor was heat-resistant but lost allatostatic activity after pronase digestion. These results indicate that the allatostatic factor is probably a heat-stable peptide.     

Sporulation of Streptomyces griseus in submerged culture.

A wild-type strain of Streptomyces griseus forms spores both on solid media (aerial spores) and in liquid culture (submerged spores). Both spore types are highly resistant to sonication, but only aerial spores are resistant to lysozyme digestion. Electron micrographs suggest that lysozyme sensitivity may result from the thinner walls of the submerged spores. Studies of the life cycle indicate that neither streptomycin excretion nor extracellular protease activity is required for sporulation: the analysis of mutants, however, suggests that antibiotic production may be correlated with the ability to sporulate. A method was devised to induce the rapid sporulation of S. griseus in a submerged culture. This method, which depends on nutrient deprivation, was used to determine that either ammonia or phosphate starvation can trigger sporulation and that the enzyme glutamine synthetase may be useful as a sporulation marker after phosphate deprivation.     

Changes in juvenile hormone biosynthetic rate and whole body content in maturing virgin queens of Solenopsis invicta

Studies were conducted on the physiological and hormonal changes following the release of alates from developmentally suppressive pheromones produced by mature queens of the fire ant Solenopsis invicta Buren. Winged virgin queens were removed from the pheromonal signal and placed in colony fragments. The time for dealation, degree of ovarian development, and biosynthesis rate and whole body content of juvenile hormone (JH) were measured. The production rate and content of JH were highly correlated. Dealation and the initiation of oviposition corresponded to peak production of JH. JH production rose sharply following separation from the natal nest, peaking after 3 days. After 8 days of isolation, JH production gradually subsided to levels similar to that found in pre-release queens, but began to increase again after 12 days. Mature queens had highly elevated levels of JH relative to recently dealate females, probably reflecting the increased reproductive capability of these older females. The results support the hypothesis that the pheromone released by functional queens inhibits reproduction in virgin alates by suppressing corpora allata activity and the production of JH.     

Antineoplastic agents. Part 409: Isolation and structure of montanastatin from a terrestrial actinomycete.

A Montana soil actinomycete, Streptomyces anulatus, produced (1 x 10(-2)% yield) a new cancer cell growth inhibitory cyclooctadepsipeptide named montanastatin (1) accompanied by the potent anticancer antibiotic valinomycin (2) in very high (5.1%) yields. Valinomycin but not montanastatin inhibited growth of a number of pathogenic bacteria and fungi. Interpretation of high-field (500 MHz) NMR and high-resolution FAB mass spectral data allowed assignment of the structure cyclo-(D-Val-L-Lac-L-Val-D-Hiv) to montanastatin. Valinomycin (2) was also isolated from actinomycetes cultured from a tree branch and animal feces collected in Malaysia. Streptomyces exfoliatus, isolated from the tree branch, was found to contain valinomycin in 1.6% yield, while the fecal isolate, S. anulatus, gave valinomycin in 0.9% yield.     

Rapid detection and isolataion of DNA-binding compounds: from Streptomyces xanthochromogenes

J. HOLMALAHTI, S. JUNTTILA AND A. VON WRIGHT. 1996. The culture medium of Streptomyces xanthochromogenes JH903 was found to show selective activity against DNArepair-deficient Escherichia coli CM871 strain. In this report we describe a simple method to locate and isolate DNA binding compounds from the fermentation broth. The method is based on the retention of DNA-reacting compounds in cellulose complexed with DNA, and purification of these compounds with thin-layer chromatography. Screening of microbial metabolites from chloroform extracts of fermentation media resulted in detection of five genotoxic fractions.     

抗生素AGPM发酵过程中蛋白质表达差异的研究

由土壤中筛选到一能产生新型抗肿瘤抗生素AGPM的藤黄灰链霉菌株,应用蛋白质双向电泳方法,比较了藤黄灰链霉菌在发酵24h与72h蛋白质表达的差异,发现在发酵72h有17个蛋白质差异点出现,此外还发现一些蛋白质的含量明显高于24h的同种蛋白含量,这表明这些蛋白可能与抗生素AGPM的合成以及和菌体的生长等有关。     

圈卷产色链霉菌分化关键基因——saw1的克隆及酶切分析

链霉菌是现代生物学研究中一种重要的微生物,它有两个突出的特征:其一,有无与伦比的合成次生代谢产物的能力,世界上所知数千种抗生素的70％由其产生。其二,有一个复杂的发育分化的生命周期,是微生物分化研究的一个最好的模式材料。链霉菌分化主要为形态分化和生理分化,两者彼此独立又相互关联,构成复杂的分化调控网络,研究分化基因的调控不但有重要的理论意义,而且可用于控制抗生素的生物合成,因此弄清合成途径的分子机制,也有潜在的应用价值。圈卷产色链霉菌是从我国东北土     

Studies on the production of daunomycinonederived glycosides and related metabolites inStreptomyces coeruleorubidus andStreptomyces peucetius

Strains ofStreptomyces coeruleorubiduě ISP 5145,JA 10092 and 39–146, differing mutually in antibiotic activity, were found to produce identical spectrum of metabolites (at least nine antibiotically active glycosides, 13-dihydrodaunomycinone, ε-rhodomycinone and a larger number of unidentified compounds); only trace quantities of daunomycin and daunomycinone could be detected. A fraction of glycosides with a higher R_f (0.4–0.7), isolated from strain 39–146, could be transformed to daunomycin by mild hydrolysis and to daunomycinone by total hydrolysis.Streptomyces peucetius IMI 101 335 differed fromStreptomyces coeruleorubidus in an increased production of ε-rhodomycinone and a lower content of glycosides; the zone of daunomycin was most pronounced among the glycoside spots.Streptomyces coeruleorubidus 39-146 exhibited the highest activity in a medium containing 3.5% soluble starch, 3.0% soybean meal, 0.3% NaCl and 0.3% CaCo₃; glucose was a more useful carbon source for the remaining strains The activity ofStreptomyces coeruleorubidus was inhibited by 1-propanol, Na-propionate,5,5-diethylbarbiturate and bacitracin. Ferrous sulphate stimulated the production of glycosides only in strain JA 10092, decreasing simultaneously the production of aglycones.     

Role of adenosine kinase in the control of Streptomyces differentiations: Loss of adenosine kinase suppresses sporulation and actinorhodin biosynthesis while inducing hyperproduction of undecylprodigiosin in Streptomyces lividans

Adenosine kinase (ADK) catalyses phosphorylation of adenosine (Ado) and generates adenosine monophosphate (AMP). ADK gene (adk(Sli), an ortholog of SCO2158) was disrupted in Streptomyces lividans by single crossover-mediated vector integration. The adk(Sli) disruption mutant (Deltaadk(Sli)) was devoid of sporulation and a plasmid copy of adk(Sli) restored sporulation ability in Deltaadk(Sli), thus indicating that loss of adk(Sli) abolishes sporulation in S. lividans. Ado supplementation strongly suppressed sporulation ability in S. lividans wild-type (wt), supporting that disruption of adk(Sli) resulted in Ado accumulation, which in turn suppressed sporulation. Cell-free experiments demonstrated that Deltaadk(Sli) lacked ADK activity and in vitro characterization confirms that adk(Sli) encodes ADK. The intracellular level of Ado was highly elevated while the AMP level was significantly reduced after loss of adk(Sli) while Deltaadk(Sli) displayed no significant derivation from wt in the levels of S-adenosylhomocysteine (SAH) and S-adenosylmethionine (SAM). Notably, Ado supplementation to wt lowered AMP content, albeit not to the level of Deltaadk(Sli), implying that the reduction of AMP level is partially forced by Ado accumulation in Deltaadk(Sli). In Deltaadk(Sli), actinorhodin (ACT) production was suppressed and undecylprodigiosin (RED) production was dramatically enhanced; however, Ado supplementation failed to exert this differential control. A promoter-probe assay verified repression of actII-orf4 and induction of redD in Deltaadk(Sli), substantiating that unknown metabolic shift(s) of ADK-deficiency evokes differential genetic control on secondary metabolism in S. lividans. The present study is the first report revealing the suppressive role of Ado in Streptomyces development and the differential regulatory function of ADK activity in Streptomyces secondary metabolism, although the underlying mechanism has yet to be elucidated.     

链霉菌ZG0429的分类鉴定与链霉亲和素的分离纯化研究

从土壤中筛选得到1株高产链霉亲和素的放线菌ZG0429,根据形态观察、培养特征、生理生化鉴定以及16S rRNA序列分析,初步判定该菌株为链霉菌属中的淡紫灰链霉菌(Streptomyces lavendulae)。经发酵,ZG0429的链霉亲和素产量可达201.0mg/L。进一步采用硫铵沉淀和凝胶过滤层析纯化,链霉亲和素的回收率为76.87%,纯度可以达到97.03%。该方法简单易行,成本低廉,可得到高产量、高纯度、高活性的目的蛋白,为链霉亲和素发酵产品的大规模纯化提供了依据。     

Comparison of beta-Glucosidase Activities in Different Streptomyces Strains

Cellobiase (beta-glucosidase) production was compared for two streptomycetes: Streptomyces flavogriseus, a known producer of cellulase complex, and Streptomyces sp. strain CB-12, a strain isolated for its rapid growth on cellobiose. The optimal conditions for enzyme activity were established in relation to pH, temperature, enzyme stability, and substrate affinity. The production of beta-glucosidase by the two strains depended on the carbon substrate in the medium. Cellobiose was found to repress the biosynthesis of the enzyme in S. flavogriseus and to stimulate its production in strain CB-12. The biosynthesis of the enzyme correlated well with the accumulation of glucose in the culture filtrates. The combined action of the beta-glucosidases produced by the two Streptomyces strains might allow a better utilization of the reaction products which arise during the biodegradation of cellulose.