Streptomyces coelicolorA3（2）M145中丝／苏氨酸蛋白激酶基因prkC的功能初探

目的对Streptomyces coelicolorA3（2）M145中编码丝／苏氨酸蛋白激酶PrkC的基因SC03848进行功能初探。方法对PrkC蛋白序列进行生物信息学分析,在S．coelicolorA3（2）M145中敲除prkC基因,并进行互补、和过表达实验,对比突变菌株生长、次生代谢物产量、孢子萌发效率等。结果prkC基因在S．coelicolorA3（2）M145孢子萌发、生长、次生代谢等方面均起重要作用。结论prkC是一个多效调节基因,其具体生理功能和作用机制有待深入研究。     

Accumulation of S-adenosylmethionine induced oligopeptide transporters including BldK to regulate differentiation events in Streptomyces coelicolor M145

S-Adenosylmethionine (SAM), the major methyl donor in diverse biological processes, was recently found to be involved in the regulation of differentiation in streptomycetes. Exogenous SAM, in a quantity as low as 2muM, enhanced antibiotic production and inhibited morphological development of Streptomyces coelicolor M145. Total protein profiling of S. coelicolor M145 revealed that SAM enhanced the expression of oligopeptide-binding components related to ABC transporters that included BldK, a well-known regulatory factor in S. coelicolor differentiation. A radiolabeled SAM feeding experiment verified that exogenous SAM can be imported into the cell, which is under the control of the bld cascade. This study substantiated that BldK serves as a transducer of the SAM signal and uncovered the possible role of oligopeptide import in the regulation of Streptomyces differentiation, particularly in relation to SAM.     

A new gene, sigG, encoding a putative alternative sigma factor of Streptomyces coelicolor A3(2)

An oligonucleotide probe encoding a peptide motif conserved in all sigma factors was used to isolate a new gene, sigG, from a Streptomyces coelicolor A3(2) genomic library. The deduced protein of 263 amino acids with an M(r) of 29,422 showed the greatest similarity to the previously identified sporulation sigma factor (sigma F) of Streptomyces coelicolor, and general stress response sigma factor (sigma B) of Bacillus subtilis, mostly in domains suggested to be involved in recognition of -10 and -35 promoter regions. Southern-blot hybridization with DNA from several Streptomyces spp. revealed the presence of a similar gene in all strains tested. Disruption of the S. coelicolor sigG gene appeared to have no obvious effect on growth, morphology, differentiation, and production of pigmented antibiotic actinorhodin and undecylprodigiosin.     

Disruption of SCO5461 gene coding for a mono-ADP-ribosyltransferase enzyme produces a conditional pleiotropic phenotype affecting morphological differentiation and antibiotic production in Streptomyces coelicolor

The SCO5461 gene of Streptomyces coelicolor A3(2) codes for an ADP-ribosyltransferase enzyme that is predicted to be a transmembrane protein with an extracellular catalytic domain. PCR-targeted disruption of the gene resulted in a mutant that differentiated normally on complex SFM medium; however, morphological differentiation in minimal medium was significantly delayed and this phenotype was even more pronounced on osmotically enhanced minimal medium. The mutant did not sporulate when it was grown on R5 medium, however the normal morphological differentiation was restored when the strain was cultivated beside the wild-type S. coelicolor M145 strain. Comparison of the pattern of ADP-ribosylated proteins showed a difference between the mutant and the wild type, fewer modified proteins were present in the cellular crude extract of the mutant strain. These results support our previous suggestions that protein ADP-ribosylation is involved in the regulation of differentiation and antibiotic production and secretion in Streptomyces.     

Conversion of Kepone by Methanosarcina thermophila

Abstract Amino acid sequences of protease inhibitors ( Streptomyces subtilisin inhibitor-like proteins) widely distributed in Streptomyces were compared to clarify the taxonomic status of three strains of Streptomyces spp., S. coelicolor A3(2), S. lividans 66 and S. coelicolor Müller, which are closely related by conventional taxonomical procedures. The sequence comparison indicated that S. coelicolor A3(2) is distinct from the type strain S. coelicolor Müller, but belongs to the same taxon as S. lividans 66.     

Genome plasticity in Streptomyces: identification of 1 Mb TIRs in the S. coelicolor A3(2) chromosome

The chromosomes of several widely used laboratory derivatives of Streptomyces coelicolor A3(2) were found to have 1.06 Mb inverted repeat sequences at their termini (i.e. long-terminal inverted repeats; L-TIRs), which are 50 times the length of the 22 kb TIRs of the sequenced S. coelicolor strain M145. The L-TIRs include 1005 annotated genes and increase the overall chromosome size to 9.7 Mb. The 1.06 Mb L-TIRs are the longest reported thus far for an actinomycete, and are proposed to represent the chromosomal state of the original soil isolate of S. coelicolor A3(2). S. coelicolor A3(2), M600 and J1501 possess L-TIRs, whereas approximately half the examined early mutants of A3(2) generated by ultraviolet (UV) or X-ray mutagenesis have truncated their TIRs to the 22 kb length. UV radiation was found to stimulate L-TIR truncation. Two copies of a transposase gene (SCO0020) flank 1.04 Mb of DNA in the right L-TIR, and recombination between them appears to generate strains containing short TIRs. This TIR reduction mechanism may represent a general strategy by which transposable elements can modulate the structure of chromosome ends. The presence of L-TIRs in certain S. coelicolor strains represents a major chromosomal alteration in strains previously thought to be genetically similar.     

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.     

Hybridization in Actinomycetes]

Ways of transferring genetic information in Actinomycetes and their use for the transmission of genetic material in intervarietal crosses are discussed. The producing of hybrids is shown to be an efficient method to obtain Actinomycetes strains with new properties. Properties of recombinants obtained in intervarietal crosses Streptomyces coelicolor A3(2)XS. lividans 66 are shown to possess new antibiotic properties differing from those of parental strains: they suppress a number of Actinomycetes and bacterial strains which are not affected by parental strains. It is found that at least two groups of genes located on chromosomes of S. coelicolor A3(2) and S. lividans 66 participate in the synthesis of the antibiotic. The obtaining of recombinants between S. coelicolor A3(2) and S. griseus 15 makes possible to select variants which are capable to produce the antibiotic grisine and are resistant to actinophages, specifically attacking S. griseus. Properties of recombinants between S. coelicolor A3(2) and S. griseus 15 make possible to localize on parental chromosomes regions containing genes which control the synthesis of the antibiotic, the formation of a receptor for the adsorption of actinophages, and genes controlling the restriction and t he modification of actinophage development in S. coelicolor and S. griseus. A sex plasmid (SGP1) is found in S. griseus 15.     

Deletion of scbA enhances antibiotic production in Streptomyces lividans

Antibiotic production in many streptomycetes is influenced by extracellular gamma-butyrolactone signalling molecules. In this study, the gene scbA, which had been shown previously to be involved in the synthesis of the gamma-butyrolactone SCB1 in Streptomyces coelicolor A3(2), was deleted from the chromosome of Streptomyces lividans 66. Deletion of scbA eliminated the production of the antibiotic stimulatory activity previously associated with SCB1 in S. coelicolor. When the S. lividans scbA mutant was transformed with a multi-copy plasmid carrying the gene encoding the pathway-specific activator for either actinorhodin or undecylprodigiosin biosynthesis, production of the corresponding antibiotic was elevated significantly compared to the corresponding scbA(+) strain carrying the same plasmid. Consequently, deletion of scbA may be useful in combination with other strategies to construct host strains capable of improved bioactive metabolite production.     

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。     

Heterologous expression of novobiocin and clorobiocin biosynthetic gene clusters

A method was developed for the heterologous expression of biosynthetic gene clusters in different Streptomyces strains and for the modification of these clusters by single or multiple gene replacements or gene deletions with unprecedented speed and versatility. Lambda-Red-mediated homologous recombination was used for genetic modification of the gene clusters, and the attachment site and integrase of phage phiC31 were employed for the integration of these clusters into the heterologous hosts. This method was used to express the gene clusters of the aminocoumarin antibiotics novobiocin and clorobiocin in the well-studied strains Streptomyces coelicolor and Streptomyces lividans, which, in contrast to the natural producers, can be easily genetically manipulated. S. coelicolor M512 derivatives produced the respective antibiotic in yields comparable to those of natural producer strains, whereas S. lividans TK24 derivatives were at least five times less productive. This method could also be used to carry out functional investigations. Shortening of the cosmids' inserts showed which genes are essential for antibiotic production.     

Construction and properties of hybrids obtained in interspecific crosses between Streptomyces coelicolor A3(2) and Streptomyces griseus Kr-15.

Recombinants between Streptomyces coelicolor A3(2) and Streptomyces griseus Kr-15 were obtained using methods of hybrid construction. Recombinant Rcg1, obtained from a cross between S. griseus and a S. coelicolor UF (SCPI-) strain, phenotypically resembled S. coelicolor UF strains and in crosses with a S. coelicolor NF donor strin produced recombinatn progeny at a frequency of 100%. Recominant Rcg3, like SCP1-carrying S. coelicolor strains, inhibited SCP1-strains of S. coelicolor and in crosses with a UF recipient strain of S. coelicolor generated recombinants at high frequency. In crosses between S. griseus and Rcgi the frequency of recombinant formation was increased about 100-fold relative to crosses between S. griseus and S. coelicolor. Effective transfer of S. grieseus and Rcg3 chromosomal markers into Rcg1 and S. coelicolor, respectively, indicated that S. griseus had donor properties. Studies of the ability of recombinants to support phage growth indicated that parental chromosomal fragments containing genes involved in control of phage-receptor formation and intracellular growth were present in the hybrids. Grisin-producing recombinants, capable of restricting phages attacking S. coelicolor and S. griseus, were obtained.