Structure of Carbohydrate Moiety in Asterosaponin A. Isolation of Three New Disaccharides

ABSTRACT

This study aimed to investigate the role of serine/threonine kinase PkaE in Streptomyces coelicolor A3(2). Liquid chromatography tandem mass spectrometry was performed for comparative phosphoproteome and proteome analyses of S. coelicolor A3(2), followed by an in vitro phosphorylation assay. Actinorhodin production in the pkaE deletion mutant was lower than that in wild-type S. coelicolor A3(2), and the spores of the pkaE deletion mutant were damaged. Furthermore, phosphoproteome analysis revealed that 6 proteins were significantly differentially hypophosphorylated in pkaE deletion mutant (p < 0.05, fold-change ≤ 0.66), including BldG and FtsZ. In addition, the in vitro phosphorylation assay revealed that PkaE phosphorylated FtsZ. Comparative proteome analysis revealed 362 differentially expressed proteins (p < 0.05) and six downregulated proteins in the pkaE deletion mutant involved in actinorhodin biosynthesis. Gene ontology enrichment analysis revealed that PkaE participates in various biological and cellular processes. Hence, S. coelicolor PkaE participates in actinorhodin biosynthesis and morphogenesis.     

Isolation and Physical Properties of New Piericidins Produced by Streptomyces pactum

During the screening of metabolites of microorganisms for new insecticides, many piericidin-like substances were found in the mycelial extract of Streptomyces pactum. The isolation and physical properties of these piericidins are described.     

天蓝色链霉菌代谢物组测定方法优化及其代谢特征

链霉菌能够产生多种抗生素,具有重要的研究与应用价值。代谢物组学能够定性和定量测定胞内外主要低分子量代谢产物。相对于其他组学,代谢物组学在监控胞内代谢状态、指导物种理性改造方面具有独特优势。本文旨在建立一种快速、准确的链霉菌胞内代谢物分析方法。以模式菌株天蓝色链霉菌为研究对象,基于GC-MS分析平台优化了代谢物组学样品制备流程中的细胞淬灭时间、菌体分离方法、代谢物提取及代谢物衍生化条件,并利用该方法对天蓝色链霉菌不同生长时期各代谢途径的相对活性进行了初步分析。采用"低温淬灭(–40℃,4 min)-快速过滤分离-反复冻融(45 s/3 min)-衍生化(40℃,90 min)"的流程能够鉴定出中心代谢途径(糖酵解、戊糖磷酸途径和TCA循环)、氨基酸代谢途径、脂肪酸代谢途径、核酸代谢途径及部分次级代谢途径中的103种主要代谢物。利用该流程测定发现天蓝色链霉菌细胞生长周期中存在显著的代谢时序差异,并且发现氨基酸与脂肪酸代谢在衔接初级代谢与次级代谢生物合成中具有重要作用。本研究建立的测定方法能够有效地用于天蓝色链霉胞内代谢物分析,该方法将有助于深入刻画链霉菌细胞代谢过程,为菌株代谢工程改造增加次级代谢产物产量提供理性指导。     

Expression and Characterization of the Streptomyces coelicolor Serine/Threonine Protein Kinase PkaD

We identified and characterized the gene encoding a new eukaryotic-type protein kinase from Streptomyces coelicolor A3(2) M145. PkaD, consisting of 598 amino acid residues, contained the catalytic domain of eukaryotic protein kinases in the N-terminal region. A hydrophobicity plot indicated the presence of a putative transmembrane spanning sequence downstream of the catalytic domain, suggesting that PkaD is a transmembrane protein kinase. The recombinant PkaD was found to be phosphorylated at the threonine and tyrosine residues. In S. coelicolor A3(2), pkaD was transcribed as a monocistronic mRNA, and it was expressed constitutively throughout the life cycle. Disruption of chromosomal pkaD resulted in a significant loss of actinorhodin production. This result implies the involvement of pkaD in the regulation of secondary metabolism.     

Streptomyces genes involved in aerial mycelium formation

Abstract Cloning of genes as suppressors of the aerial mycelium-defective phenotype of Streptomyces griseus HH1 resulting from A-factor-deficiency has led to the identification of several genes, including amfR, amfAlamfB, amfC , and orf1590 . These genes are involved in aerial mycelium formation independent of secondary metabolic function. Among these, AmfR which belongs to the family of response regulators of two-component regulatory systems and AmfA/AmfB similar to ATP-dependent membrane translocators are analogous to the multicomponent phosphorelay and the Spo0K system, respectively, both of which are required for the initiation of sporulation in Bacillus subtilis . Involvement of a protein serine/threonine kinase in aerial mycelium formation is also suggested, because the Streptomyces coelicolor A3(2) afsK gene encoding a 'eukaryotic'-type protein kinase reverses the aerial mycelium-defective phenotype of strain HH1, independent of secondary metabolic function.     

S-Adenosyl-L-methionine Activates Actinorhodin Biosynthesis by Increasing Autophosphorylation of the Ser/Thr Protein Kinase AfsK in Streptomyces coelicolor A3(2)

S-Adenosyl-L-methionine (SAM) is one of the major methyl donors in all living organisms. The exogenous treatment with SAM leads to increased actinorhodin production in Streptomyces coelicolor A3(2). In this study, mutants from different stages of the AfsK-AfsR signal transduction cascade were used to test the possible target of SAM. SAM had no significant effect on actinorhodin production in afsK, afsR, afsS, or actII-open reading frame 4 (ORF4) mutant. This confirms that afsK plays a critical role in delivering the signal generated by exogenous SAM. The afsK-pHJL-KN mutant did not respond to SAM, suggesting the involvement of the C-terminal of AfsK in binding with SAM. SAM increased the in vitro autophosphorylation of kinase AfsK in a dose-dependent manner, and also abolished the effect of decreased actinorhodin production by a Ser/Thr kinase inhibitor, K252a. In sum, our results suggest that SAM activates actinorhodin biosynthesis in S. coelicolor M130 by increasing the phosphorylation of protein kinase AfsK.     

天蓝色链霉菌胞内蓝色素提取方法研究

对天蓝色链霉菌— 10 0胞内蓝色素提取方法进行了研究 ,结果表明碱提取法、SDS法、研磨法的色素提取得率分别为 90 2 %、95 2 %和 54 6 % ;酶水解法的色素提取得率 <30 % ;细胞在pH9缓冲液中自溶 ,浓度为 1/4原发酵浓度 ,4 0℃保温搅拌 4 8h ,色素提取得率为 33 8%。     

天蓝色链霉菌中长链3-酮脂酰ACP合成酶的功能

【背景】链霉菌属于放线菌科,在土壤环境中广泛分布。链霉菌具有复杂的形态分化和多样性的次生代谢网络,能产生大量具有生物活性的次级代谢产物,被广泛深入研究。【目的】天蓝色链霉菌是链霉菌的模式菌株,其脂肪酸合成代谢与次级代谢联系紧密,但目前脂肪酸合成代谢途径还不清楚,其长链3-酮脂酰ACP合成酶还未见报道。【方法】利用大肠杆菌FabF序列进行同源比对,发现天蓝色链霉菌A3(2)的基因组中,SCO2390(ScoFabF1)、SCO1266(ScoFabF2)、SCO0548(ScoFabF3)和SCO5886 (ScoRedR)具有较高的相似性,并具有保守的Cys-His-His催化活性中心,可能具有长链3-酮脂酰ACP合成酶活性。采用PCR扩增方法分别获得以上基因,连入表达载体pBAD24M后分别互补大肠杆菌fabB(ts)突变株和fabB(ts)fabF双突变株,并检测转化子的生长情况。以上基因与pET-28b连接后,在大肠杆菌BL21(DE3)中表达,并利用Ni-NTA纯化获得蛋白,体外测定其催化活性。将以上基因分别互补大肠杆菌fabF突变株后,GC-MS测定互补株的脂肪酸组成。【结果】4个同源基因中,只有ScofabF1能恢复fabB(ts)fabF双突变株42°C时在添加油酸条件下的生长,其他3个基因均不能恢复生长。而这4个基因都不能恢复fabB(ts)突变株42°C时生长。体外活性测定ScoFabF1具有长链3-酮脂酰ACP合成酶活性,其他3个蛋白都不具有该活性。仅ScofabF1能显著提高大肠杆菌fabF突变株的顺-11-十八碳烯酸(C18:1)比例,其他3个基因都不具有该功能。【结论】天蓝色链霉菌中ScofabF1编码长链3-酮脂酰ACP合成酶II,在脂肪酸利用过程中发挥重要作用。天蓝色链霉菌中没有发现编码长链3-酮脂酰ACP合成酶I的基因,其可能通过其他途径合成少量的不饱和脂肪酸。以上研究结果为进一步研究天蓝色链霉菌中脂肪酸合成机制奠定了基础。     

疱疹病毒蛋白激酶功能的研究进展

丝氨酸／苏氨酸激酶存在于所有已知的疱疹病毒中,它们具有多种功能,参与病毒感染的整个过程,尤其是病毒与机体的相互作用。主要阐述了两类保守的疱疹病毒丝氨酸／苏氨酸激酶（单纯疱疹病毒HSV的ULl3激酶和US3激酶）在病毒感染过程的重要作用。两者都参与调控细胞和病毒基因的表达,介导病毒衣壳出核以及免疫逃避。虽然这些激酶对病毒在体外培养细胞中复制的影响各不相同,但是对于病毒的毒力非常重要,因此,可用作抗病毒药物设计的靶位。     

Several enzymes of the central metabolism are phosphorylated in Staphylococcus aureus

Staphylococcus aureus is an important human and animal pathogen that harbors protein kinases. The proteins phosphorylated in this bacterium grown on glucose minimal medium have been in vivo labeled with[(32)P]-orthophosphate and analyzed by two-dimensional gel electrophoresis followed by MS. A total of 11 glycolytic phosphoproteins have been identified and verified. In vitro analyses have revealed that phosphorylation of these glycolytic enzymes is catalysed primarily through the activity of an endogenous serine/threonine kinase and to a lesser extent by a tyrosine kinase. The identification of these phosphoproteins should prove helpful in understanding and unravelling of the role of phosphorylation with respect to pathogenesis and virulence in this organism.     

Multiple phosphorylation of membrane-associated calcium-dependent protein serine/threonine kinase in Streptomyces fradiae

In Streptomyces fradiae, calcium ions induce alterations in intensity and specificity of the secondary metabolism and stimulate aerial mycelium formation and sporulation. Using in vitro labeling, we demonstrate that in S. fradiae in the late exponential growth phosphorylation of 65-kDa membrane-associated protein is also influenced by Ca(2+) added exogenously. Calcium ions at physiological concentration stimulate intensive Ca(2+)-dependent phosphorylation of 65-kDa protein at multiple sites on serine, threonine, and tyrosine residues. Assay of protein kinases in situ demonstrated in the fraction of membrane-associated proteins the presence of two autophosphorylating protein serine/threonine kinases with molecular masses of 127 kDa and 65 kDa. Autophosphorylation of both proteins is also Ca(2+)-dependent.     

A calmodulin-like protein in the bacterial genus Streptomyces

The gene, named cabB, encoding a calmodulin-like protein of 70 amino acids containing two helix-loop-helix EF-hand motifs was cloned from Streptomyces coelicolor A3(2). cabB was transcribed from a single promoter throughout growth. The CabB protein produced in Escherichia coli was a monomer in solution, although it corresponded to one half of a dumbbell shape of the eukaryotic calmodulins. CabB bound calcium and upon binding of calcium its alpha-helix content was increased, as determined by circular dichroism spectroscopy. The growth of cabB-disruptants (mutant DeltacabB) on minimal agar medium containing calcium higher than 20 mM was delayed, suggesting that CabB has a role in calcium homeostasis by serving as a calcium buffer or transporter, as suggested for calerythrin in actinomycetes and the invertebrate sarcoplasmic calcium-binding proteins. Wide distribution of cabB almost exclusively in actinomycetes suggests a common role of EF-hand CabB-type proteins in these filamentous, soil-dwelling Gram-positive bacterial genera.     

Two New Butenolides Produced by an Actinomycete Streptomyces sp.

Two new butenolides, (4S)‐4,10‐dihydroxydodec‐2‐en‐1,4‐olide ( 1 ) and (4S)‐4,8,10‐trihydroxy‐10‐methyldodec‐2‐en‐1,4‐olide ( 2 ), together with three known compounds, MKN‐003B ( 3 ), MKN‐003C ( 4 ), and cyclo(Ala‐Leu) ( 5 ), were isolated from the culture broth of a bacterium of the genus Streptomyces derived from soil environment. The structures of these compounds were elucidated on the basis of spectroscopic analysis. The inhibitory activities of the butenolides against eight pathogenic fungi were evaluated. All of the butenolides showed moderate‐or‐weak antifungal activities in a broth microdilution assay.     

Ca2+-dependent modulation of antibiotic resistance in Streptomyces lividans 66 and Streptomyces coelicolor A3(2)

The level of resistance to antibiotics of various chemical structure in actinobacteria of the genus Streptomyces is shown to be regulated by Ca²⁺ ions. The inhibitors of Ca²⁺/calmodulin and Ca²⁺/phospholipid-dependent serine/threonine protein kinases (STPK) are found to reduce antibiotic resistance of actinobacteria. The effect of Ca²⁺-dependent phosphorylation on the activity of the enzymatic aminoglycoside phosphotransferase system protecting actinobacteria from aminoglycoside antibiotics was studied. It is shown that inhibitors of Ca²⁺/calmodulin and Ca²⁺/phospholipid-dependent STPK reduced the Ca²⁺-induced kanamycin resistance in Streptomyces lividans cells transformed by a hybrid plasmid which contained the aminoglycoside phosphotransferase VIII (APHVIII) gene. In S. coelicolor A3(2) cells, the protein kinase PK25 responsible for APHVIII phosphorylation in vitro was identified. It is suggested that STPK play a major role in the regulation of antibiotic resistance in actinobacteria.     

Mycobacterium tuberculosis serine/threonine kinases PknB, PknD, PknE, and PknF phosphorylate multiple FHA domains

The physiologic roles and the substrates of the Mycobacterium tuberculosis (Mtb) serine/threonine kinases are largely unknown. Here, we report six novel interactions of PknB, PknD, PknE, and PknF with the Forkhead-Associated (FHA) domains of Rv0020c and the putative ABC transporter Rv1747. Purified PknB and PknF kinase domains phosphorylated multiple FHA-domain proteins in vitro. Although they remain to be verified in vivo, these reactions suggest a web of interactions between STPKs and FHA domains.     

Dependence of aminoglycoside 3′-phosphotransferase VIII activity on serine/threonine protein kinases in <Emphasis Type="Italic">Streptomyces rimosus</Emphasis>

In Streptomyces rimosus, selection for resistance to the aminoglycoside antibiotic kanamycin triggers the normally silent aminoglycoside 3-phosphotransferase VIII gene (aphVIII). The expression of APHVIII is accompanied by amplification of the chromosomal DNA fragment containing the aphVIII gene. Earlier, S. rimosus aphVIII gene was isolated and sequenced. Using in vitro labeling and immunoprecipitation with anti-APHVIII antibodies, we have demonstrated that endogenous protein kinases (PKs) in extracts of S. rimosus strain S683 actively phosphorylate two serine residues in the APHVIII molecule. The amount of phosphate incorporated into APHVIII in the presence of Ca²⁺ is 1.84-fold greater than that without Ca²⁺. Analysis of ingel autophosphorylation and phosphorylation of the substrate incorporated into the gel matrix has shown that modification of APHVIII is catalyzed by two serine/threonine PKs (74 kDa and 55 kDa). The activity of 55-kDa PK is dependent on Ca²⁺ and calmodulin. The specific kanamycin phosphotransferase activity of exhaustively phosphorylated APHVIII is 3.72 times higher than that of the unmodified enzyme. It is proposed that the above PKs may be involved in the regulation of kanamycin resistance in S. rimosus.__________Translated from Molekulyarnaya Biologiya, Vol. 39, No. 2, 2005, pp. 255–263.Original Russian Text Copyright © 2005 by Elizarov, Sergienko, Sizova, Danilenko.     

Inflorescence-specific expression of AtK-1, a novel Arabidopsis thaliana homologue of shaggy/glycogen synthase kinase-3

We report here the isolation of the Arabidopsis thaliana gene AtK-1. The predicted protein sequence of AtK-1 show 70% identity to the Arabidopsis ASK and alfalfa MsK kinases that are homologs of the Drosophila shaggy and rat GSK-3 serine/threonine protein kinases playing an important role in signal transduction processes in animals. Northern analysis of different organs revealed exclusive expression in inflorescences suggesting an involvement of the AtK-1 kinase in reproduction-specific processes.     

Structural basis for prokaryotic calcium- mediated regulation by a Streptomyces coelicolor calcium binding protein

The important and diverse regulatory roles of Ca²⁺ in eukaryotes are conveyed by the EF-hand containing calmodulin superfamily. However, the calcium-regulatory proteins in prokaryotes are still poorly understood. In this study, we report the three-dimensional structure of the calcium-binding protein from Streptomyces coelicolor, named CabD, which shares low sequence homology with other known helix-loop-helix EF-hand proteins. The CabD structure should provide insights into the biological role of the prokaryotic calcium-binding proteins. The unusual structural features of CabD compared with prokaryotic EF-hand proteins and eukaryotic sarcoplasmic calcium-binding proteins, including the bending conformation of the first C-terminal α-helix, unpaired ligand-binding EF-hands and the lack of the extreme C-terminal loop region, suggest it may have a distinct and significant function in calcium-mediated bacterial physiological processes, and provide a structural basis for potential calcium-mediated regulatory roles in prokaryotes.