链霉菌次级代谢调控相关的双组分系统研究进展

链霉菌具有强大的次级代谢能力, 能够产生众多具有生物活性的次级代谢产物, 如目前广泛应用的抗生素、抗肿瘤药物以及免疫抑制剂等。在链霉菌中, 次级代谢产物的生物合成受到包括途径特异性、多效性以及全局性调控基因在内的多层次严格调控。关键调控基因的缺失或过表达可以显著影响次级代谢产物的生物合成, 提示对于链霉菌次级代谢重要调控基因的功能及其作用机制的研究具有巨大的潜在应用价值。其中, 作为细菌信号传导系统的双组分系统(Two-component system, TCS)一直是大家研究的关注点。越来越多的研究表明TCS在链霉菌次级代谢过程中发挥着全局性的调控功能。本文重点介绍链霉菌模式菌株——天蓝色链霉菌中TCS(包括典型TCS)、孤立的组氨酸蛋白激酶(HK)以及应答调控蛋白(RR)参与次级代谢调控的研究进展。这些TCS的功能鉴定及机制解析为工业链霉菌的定向遗传改造以提高重要次级代谢产物的含量提供了理论依据。     

Disruption of sscR encoding a γ-butyrolactone autoregulator receptor in Streptomyces scabies NBRC 12914 affects production of secondary metabolites

We report the cloning and sequence analysis of a gamma-butyrolactone autoregulator regulatory island that includes an sscR gene encoding the gamma-butyrolactone autoregulator receptor from Streptomyces scabies NBRC 12914, a plant pathogenic strain. gamma-Butyrolactone autoregulators trigger secondary metabolism, and sometimes morphological differentiation in the Gram-positive genus Streptomyces through binding to a specific autoregulator receptor. This gene cluster showed close similarity to other regulatory islands of Streptomyces origin that are responsible for the control of secondary metabolism. The recombinant SscR protein expressed in Escherichia coli prefers a gamma-butyrolactone autoregulator containing a long C-2 side chain and beta-hydroxyl group at the C-6 position. An inactivation experiment confirmed that this gamma-butyrolactone autoregulator receptor was involved in secondary metabolism but had no effects on the morphological differentiation. In the sscR-deleted mutant, the binding activity of the gamma-butyrolactone autoregulator was completely abolished, suggesting that its primary role is to detect the presence of an autoregulator in the environment. HPLC analysis of the culture broth showed that some peaks disappeared and new peaks that were not present in the broth of the wild-type strain appeared.     

Global changes in gene expression related to antibiotic synthesis in Streptomyces hygroscopicus

Two-dimensional gel electrophoresis was used to follow changes in gene expression associated with antibiotic (bialaphos) biosynthesis in Streptomyces hygroscopicus. Cultures were pulse-labelled with [35S]-methionine before, during, and after the switch from primary to secondary metabolism in order to compare kinetic profiles of bialaphos (antibiotic) production (bap) genes during this metabolic transition. Separation of gene products on two-dimensional gels revealed that 27 were dependent on brpA for optimal expression and were activated as the culture approached stationary phase. Genes which encoded 10 brpA-dependent proteins were mapped to a 10 kb SstI fragment of the 35 kb bap gene cluster by expressing them in Streptomyces lividans using the thiostrepton-inducible tipA promoter. N-terminal amino acid sequences of two brpA-dependent proteins, obtained by direct microsequencing of protein spots excised from two-dimensional gels, identified them as gene products mapping to the same region and involved in secondary metabolic conversions of the bap pathway. The kinetics of synthesis of 16 brpA-dependent gene products were characterized using QUEST computer software. Cluster analysis performed on the kinetics of synthesis of 346 of the most highly expressed gene products of HP5-29, including 16 brpA-dependent ones, identified 75 families having distinct patterns of expression. Many brpA-dependent proteins were clustered together; 10 were found in one kinetic family. These kinetic families also included brpA-independent gene products perhaps subject to similar regulatory mechanisms and thus possibly involved in bialaphos biosynthesis. The activation/derepression of bap expression took place as cultures approached stationary phase and was temporally related to synthesis of ppGpp.     

Overlapping binding of PhoP and AfsR to the promoter region of glnR in Streptomyces coelicolor

Growth of soil bacteria is often limited by the availability of essential nutrients such as carbon, nitrogen and phosphate. The reaction to a specific nutrient starvation triggers interconnected responses to equilibrate the metabolism. It is known that PhoP (response regulator involved in phosphate control) specifically binds to several promoters of genes involved in nitrogen metabolism which are also regulated by GlnR (regulator involved in nitrogen control). In this article we report a novel cross-talk between GlnR and the SARP-like regulator, AfsR. AfsR binds to some PhoP-regulated promoters including those of afsS (a small regulatory protein of secondary metabolism), pstS (a component of the phosphate transport system) and phoRP (encoding the two component system itself). We have characterized the regulation exerted upon the nitrogen regulator glnR gene by AfsR, using EMSA and DNase I footprinting assays as well as in vivo expression studies with ΔphoP, ΔafsR and ΔafsR-ΔphoP mutants. Both PhoP and AfsR proteins are able to bind to overlapping regions within the glnR promoter producing different effects. This work demonstrates a cross-talk of three different regulators of both primary and secondary metabolism.     

Metabolic switches and adaptations deduced from the proteomes of Streptomyces coelicolor wild type and phoP mutant grown in batch culture

Bacteria in the genus Streptomyces are soil-dwelling oligotrophs and important producers of secondary metabolites. Previously, we showed that global messenger RNA expression was subject to a series of metabolic and regulatory switches during the lifetime of a fermentor batch culture of Streptomyces coelicolor M145. Here we analyze the proteome from eight time points from the same fermentor culture and, because phosphate availability is an important regulator of secondary metabolite production, compare this to the proteome of a similar time course from an S. coelicolor mutant, INB201 (ΔphoP), defective in the control of phosphate utilization. The proteomes provide a detailed view of enzymes involved in central carbon and nitrogen metabolism. Trends in protein expression over the time courses were deduced from a protein abundance index, which also revealed the importance of stress pathway proteins in both cultures. As expected, the ΔphoP mutant was deficient in expression of PhoP-dependent genes, and several putatively compensatory metabolic and regulatory pathways for phosphate scavenging were detected. Notably there is a succession of switches that coordinately induce the production of enzymes for five different secondary metabolite biosynthesis pathways over the course of the batch cultures.     

Virus antibody dynamics in primary and secondary dengue infections

Dengue viral infections show unique infection patterns arising from its four serotypes, (DENV-1,2,3,4). Its effects range from simple fever in primary infections to potentially fatal secondary infections. We analytically and numerically analyse virus dynamics and humoral response in a host during primary and secondary dengue infection for long periods using micro-epidemic models. The models presented here incorporate time delays, antibody dependent enhancement, a dynamic switch and a correlation factor between different DENV serotypes. We find that the viral load goes down to undetectable levels within 7–14 days as is observed for dengue infection, in both cases. For primary infection, the stability analysis of steady states shows interesting dependence on the time delay involved in the production of antibodies from plasma cells. We demonstrate the existence of a critical value for the immune response parameter, beyond which the infection gets completely cured. For secondary infections with a different serotype, the homologous antibody production is enhanced due to the influence of heterologous antibodies. The antibody production is also controlled by the correlation factor, which is a measure of similarities between the different DENV serotypes involved. Our results agree with clinically observed humoral responses for primary and secondary infections.     

Investigation of proteomic responses of Streptomyces lydicus to pitching ratios for improving streptolydigin production

Streptomyces lydicus has been reported to produce antibiotic streptolydigin. Pitching ratios play crucial roles in primary and secondary metabolism of Streptomyces bacteria. The higher pitching ratio (30%, v/v) significantly enhanced the levels of streptolydigin products in S. lydicus. Proteome analysis revealed that betaglucosidase and UTP-glucose-1-phosphate uridylyltransferase were up-regulated to accelerate the starch hydrolyzation at the high pitching ratios. Enhancement in the levels of UDPN-acetylmuramoylalanyl-D-glutamate-2, 6-diaminopimelate ligase and glycine cleavage system aminomethyltransferase were involved in the conversion of amino acids into secondary metabolites. Additionally, the expression levels of PfkA2, PfkA3, Zwf2, SucD, GalE1, GatB, TktA1 and ThcA, associated with glycolysis, pentose phosphate pathway, TCA cycle and amino acid metabolism, were dramatically elevated at high pitching ratios, which play important roles in the enhanced streptolydigin production in S. lydicus E9. Interestingly, the levels of proteins (glutamine synthetase I, glutamate synthase subunit beta and glutamine synthetase) were down-regulated with the increases of pitching ratios and fermentation progress, revealing that pitching ratio altered the glutamine synthetase levels and consequently regulated the streptolydigin production of S. lydicus E9. The up-regulation of proteins (eg, aldehyde dehydrogenase and alkyl hydroperoxide reductase) was involved in the redox-based regulation network triggered by an imbalance of the intracellular cell redox homeostasis and by crosstalk with secondary metabolism at the higher pitching ratio. These results settle new insights into physiological facts of S. lydicus E9 in responses to pitching ratios and will eventually improve the antibiotic production schemes in industry.     

Influence of n-propanol on growth and antibiotic production by an industrial strain of Streptomyces erythreus under different nutritional conditions

Summary The effect of propanol on primary and secondary metabolism of a glucose and phosphorus non-repressive strain of Streptomyces erythreus was studied. Propanol increased erythromycin final titer by 100% as well as biomass (20%), both occuring later in the course of the production phase. A growth-dissociated production metabolism is emphasized by propanol (higher value). A partly growth-associated production pathway tends to switch its production pattern (higher and lower values) with propanol.     

A butenolide signaling system synergized with biosynthetic gene modules led to effective activation and enhancement of silent oviedomycin production in Streptomyces

Secondary metabolic gene clusters widely exist in the genomes of Streptomyces but mostly remain silent. To awaken this hidden reservoir of natural products, various strategies concerning secondary metabolic pathways are applied. Here, we describe that butenolide signaling molecule deficiency and glucose addition can interdependently activate the expression of silent oviedomycin biosynthetic gene clusters in Streptomyces ansochromogenes and Streptomyces antibioticus. Since oviedomycin is a promising anti-tumor lead compound, in order to improve its yield, we use the cluster-situated genes (ovmF, ovmG, ovmI and ovmH) encoding the enzymes for acyl carrier protein modification and precursor biosynthesis, and the discrete precursor biosynthetic genes (pyk2, gap1 and accA2) involved in glycolysis to assemble two gene modules (pFGIH and pPGA). Their co-overexpression in ΔsabA (a disruption mutant of sabA encoding SAB synthase) has superimposed effect on the yield of oviedomycin, which can be further increased to 59-fold in the presence of galactose as optimal carbon source. This is the most unambiguous evidence that butenolide signaling system can synergize with the optimization of primary metabolism to regulate the expression of secondary metabolic gene clusters, providing efficient strategies for mining natural products of Streptomyces.     

Dual regulation between the two-component system PhoRP and AdpA regulates antibiotic production in Streptomyces

Journal of Industrial Microbiology & Biotechnology - Antibiotic production during secondary metabolism in Streptomyces spp. is elaborately controlled by multiple environmental signals and...     

“Pseudo” γ-Butyrolactone Receptors Respond to Antibiotic Signals to Coordinate Antibiotic Biosynthesis

In actinomycetes, the onset of secondary metabolite biosynthesis is often triggered by the quorum-sensing signal γ-butyrolactones (GBLs) via specific binding to their cognate receptors. However, the presence of multiple putative GBL receptor homologues in the genome suggests the existence of an alternative regulatory mechanism. Here, in the model streptomycete Streptomyces coelicolor, ScbR2 (SCO6286, a homologue of GBL receptor) is shown not to bind the endogenous GBL molecule SCB1, hence designated “pseudo” GBL receptor. Intriguingly, it could bind the endogenous antibiotics actinorhodin and undecylprodigiosin as ligands, leading to the derepression of KasO, an activator of a cryptic type I polyketide synthase gene cluster. Likewise, JadR2 is also a putative GBL receptor homologue in Streptomyces venezuelae, the producer of chloramphenicol and cryptic antibiotic jadomycin. It is shown to coordinate their biosynthesis via direct repression of JadR1, which activates jadomycin biosynthesis while repressing chloramphenicol biosynthesis directly. Like ScbR2, JadR2 could also bind these two disparate antibiotics, and the interactions lead to the derepression of jadR1. The antibiotic responding activities of these pseudo GBL receptors were further demonstrated in vivo using the lux reporter system. Overall, these results suggest that pseudo GBL receptors play a novel role to coordinate antibiotic biosynthesis by binding and responding to antibiotics signals. Such an antibiotic-mediated regulatory mechanism could be a general strategy to coordinate antibiotic biosynthesis in the producing bacteria.     

Cyclic AMP regulation of tylosin biosynthesis and secondary metabolism in Streptomyces fradiae

Adenosine 3':5' cyclic monophosphate seems to regulate antibiotic biosynthesis and secondary metabolism in tylosin-producing cultures of Streptomyces fradiae C373.1. A dose-dependent response is observed by exogenous additions of dibutyryl cyclic AMP (cAMP), and is related to the nutritional status of the culture. Addition of cAMP to cultures growing in nutritionally lean media caused higher cumulative antibiotic tigers and some cellular differentiation compared with the control. In nutritionally rich media, a qualitatively different behavior resulted: an almost instantaneous shift toward secondary metabolism occurred. The response is characterized by extensive cellular differentiation with little growth and only a trace of antibiotic production. The possible role of cyclic AMP n the regulation of tylosin biosynthesis and secondary metabolism and its relation to specific nutrient limitations in synthetic, defined media in Streptomyces fradiae is discussed. (c) 1994 John Wiley & Sons, Inc.