圈卷产色链霉菌7100分化相关基因——sawE的结构与功能

以天蓝色链霉菌的whiB基因为探针,从圈卷产色链霉菌7100的总DNA部分文库中克隆了含有whiB同源序列的28kb DNA片段,并对其中的14kb片段进行了序列测定。序列分析表明,该片段含有一个完整的开放阅读框—sawE。预测的蛋白质结构及同源性分析显示,sawE与天蓝色链霉菌孢子形成早期的关键基因whiB高度同源,编码产物为一个调控蛋白。sawE的破坏使圈卷产色链霉菌7100的分化终止在气生菌丝阶段,在延长培养时间的情况下仍保持白色的表型,菌丝不能分隔,不能形成成熟的灰色孢子,结果表明sawE基因是一个与圈卷产色链霉菌分化有关的重要基因。     

The genome of <Emphasis Type="Italic">Streptomyces rimosus</Emphasis> subsp. <Emphasis Type="Italic">rimosus</Emphasis> shows a novel structure compared to other <Emphasis Type="Italic">Streptomyces</Emphasis> using DNA/DNA microarray analysis

DNA/DNA genome microarray analysis together with genome sequencing suggests that the genome of members of the genus Streptomyces would seem to have a common structure including a linear genomic structure, a core of common syntenous Actinomycete genes, the presence of species specific terminal regions and two intermediate group of syntenous genes that seem to be genus specific. We analyzed Streptomyces species using DNA/DNA microarray comparative genome analysis. Only Streptomyces rimosus failed to give a congruent genome pattern for the genes found in Streptomyces coelicolor. We expanded the analysis to include a number of strains related to the type strain of S. rimosus and obtained a similar divergence from the main body of Streptomyces species. These strains showed very close identity to the original strain with no gene deletion or duplication detected. The 16S rRNA sequences of these S. rimosus strains were confirmed as very similar to the S. rimosus sequences available from the Ribosomal Database Project. When the SSU ribosomal RNA phylogeny of S. rimosus is analyzed, the species is positioned at the edge of the Streptomyces clade. We conclude that S. rimosus represents a distinct evolutionary lineage making the species a worthy possibility for genome sequencing.     

Characteristics of the surface-located carbohydrate-binding protein CbpC from Streptomyces coelicolor A3(2)

Streptomyces coelicolor A32 produces a 35.6-kDa carbohydrate-binding protein (named CbpC) in the presence of cellobiose, cellulose or chitin as sole carbon source. The protein was found secreted (a typical signal sequence was present at the N-terminus) and linked to the peptidoglycan layer of the mycelia. At its C-terminal end a putative cell-wall sorting signal was identified, consisting of (1) Streptomyces specific recognition site for a transpeptidase (LAETG instead of generic LPXTP consensus), (2) a hydrophobic region and (3) a tail of positively charged residues. The deletion of this sorting signal abolished the cell-wall attachment because the resulting CbpC-form was found extracellular. After purification this protein was shown to interact strongly with crystalline cellulose; different crystalline chitin-forms were recognised moderately and chitosan not. As demonstrated by analysing further truncated CbpC-forms a glycine-aspartate/serine rich region, which separates the carbohydrate-binding module from the sorting signal, plays an important role in protein stability.     

Cloning and DNA sequence analysis of the mercury resistance genes of Streptomyces lividans

Summary A broad-spectrum mercury resistance locus (mer) from a spontaneous chloramphenicol-sensitive (Cm^s), arginine auxotrophic (Arg^–) mutant of Streptomyces lividan 1326 was isolated on a 6 kb DNA fragment by shotgun cloning into the mercury-sensitive derivative S. lividans TK64 using the vector pIJ702. The mer genes form part of a very large amplifiable DNA sequence present in S. lividans 1326. This element was amplified to about 20 copies per chromosome in the Cm^s Arg^– mutant and was missing from strains like S. lividans TK64, cured for the plasmid SLP3. DNA sequence analysis of a 5 kb region encompassing the whole region required for broad-spectrum mercury resistance revealed six open reading frames (ORFs) transcribed in opposite directions from a common intercistronic region. The protein sequences predicted from the two ORFs transcribed in one direction showed a high degree of similarity to mercuric reductase and organomercurial lyase from other gram-negative and gram-positive sources. Few, if any, similarities were found between the predicted polypeptide sequences of the other four ORFs and other known proteins.     

NdgR,an IclR-like regulator involved in amino-acid-dependent growth,quorum sensing,and antibiotic production in <Emphasis Type="Italic">Streptomyces coelicolor</Emphasis>

NdgR (regulator for nitrogen source-dependent growth and antibiotic production), an IclR-like regulator, has been initially identified as a binding protein to the promoters of doxorubicin biosynthetic genes in Streptomcyes peucetius by DNA affinity capture assay method. NdgR is well conserved throughout the Streptomcyes species and many other bacteria such as Mycobacteria and Corynebacteria. In Streptomcyes coelicolor, ndgR deletion mutant showed slow cell growth and defects in differentiation and enhances the production of actinorhodin (ACT) in minimal media containing certain amino acids where wild-type strain could not produce ACT. Although deletion mutant of ndgR showed different antibiotic production in minimal media containing Leu or Gln, it only showed reduced mRNA expression levels of the genes involved in leucine metabolism. Neither NdgR-dependent expression of glnA nor direct binding of NdgR protein to glnA, glnII, and glnR promoters was observed. However, ScbR, which is governed by NdgR shown by gel mobility shift assay, binds to promoter of glnR, suggesting indirect regulation of glutamine metabolism by NdgR. NdgR protein binds to intergenic region of ndgR–leuC, and scbR–scbA involved in γ-butyrolactone. Two-dimensional gel analysis has shown a global effect of ndgR deletion in protein expression, including up-regulated proteins involved in ACT synthesis and down-regulation of chaperones such as GroEL, GroES, and DnaK. These results suggest a global regulatory role for NdgR in amino acid metabolisms, quorum sensing, morphological changes, antibiotic production, and expression of chaperonines in S. coelicolor. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The Histidinol Phosphate Phosphatase Involved in Histidine Biosynthetic Pathway Is Encoded by <Emphasis Type="Italic">SCO5208</Emphasis> (<Emphasis Type="Italic">hisN</Emphasis>) in <Emphasis Type="Italic">Streptomyces coelicolor</Emphasis> A3(2)

Through the screening of a Streptomyces coelicolor genomic library, carried out in a histidinol phosphate phosphatase (HolPase) deficient strain, SCO5208 was identified as the last unknown gene involved in histidine biosynthesis. SCO5208 is a phosphatase, and it can restore the growth in minimal medium in this HolPase deficient strain when cloned in a high or low copy number vector. Moreover, it shares sequence homology with other HolPases recently identified in Actinobacteria. During this work a second phosphatase, SCO2771, sharing no homologies with SCO5208 and all so far described phosphatases was identified. It can complement HolPase activity mutation only at high copy number. Sequence analysis of SCO5208 and SCO2771, amplified from the HolPase mutant strain, revealed that SCO5208 shows a mutation in a conserved amino acid, whereas SCO2771 does not show any mutation. All these results show that S. coelicolor SCO5208, recently renamed hisN, is the HolPase involved in histidine biosynthesis.     

Exploring the conformational roles of signal sequences: synthesis and conformational analysis of lambda receptor protein wild-type and mutant signal peptides

Secretion of the Escherichia coli lambda receptor protein (LamB protein) appears from genetic evidence to be correlated with the predicted tendency of its signal sequence to adopt an alpha-helical conformation [Emr, S. D., & Silhavy, T. J. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 4599]. We have tested this hypothesis by synthesizing major portions of signal sequences from the wild-type and mutant LamB proteins and analyzing their conformations by circular dichroism. The wild-type signal sequence contains a seven-residue hydrophobic region flanked by a proline and a glycine. Chou-Fasman rules predict that this segment will adopt an alpha-helical conformation. An export-deficient mutant is missing four residues from this region; the helix-breaking glycine and proline are thus separated by only three residues, and an alpha helix is not predicted to form. In each of the export-restored revertants, either the glycine or the proline is replaced with a residue which promotes helix formation. The helix content of the synthetic signal sequence fragments on the basis of CD measurements supports the secondary structure hypothesis described above. The relative helicity in aqueous sodium dodecyl sulfate, lysolecithin, or trifluoroethanol is as follows: wild type greater than R2 (Pro----Leu) greater than R1 (Gly----Cys) much greater than deletion mutant.     

Analysis of functions in plasmid pHZ1358 influencing its genetic and structural stability in <Emphasis Type="Italic">Streptomyces lividans</Emphasis> 1326

The complete DNA sequence of plasmid pHZ1358, a widely used vector for targeted gene disruption and replacement experiments in many Streptomyces hosts, has been determined. This has allowed a detailed analysis of the basis of its structural and segregational instability, compared to the high copy number plasmid pIJ101 of Streptomyces lividans 1326 from which it was derived. A 574-bp DNA region containing sti (strong incompatibility locus) was found to be a determinant for segregational instability in its original S. lividans 1326 host, while the structural instability was found to be related to the facile deletion of the entire Escherichia coli-derived part of pHZ1358, mediated by recombination between 36-bp direct repeats. A point mutation removing the BamHI site inside the rep gene encoding a replication protein (rep*) and/or a spontaneous deletion of the 694-bp region located between rep and sti including the uncharacterized ORF85 (orf85 ⁻) produced little or no effect on stability. A pHZ1358 derivative (pJTU412, sti ⁻ , rep*, orf85 ⁻) was then constructed which additionally lacked one of the 36-bp direct repeats. pJTU412 was demonstrated to be structurally stable but segregationally unstable and, in contrast to sti ⁺ pHZ1358, allowed efficient targeted gene replacement in S. lividans 1326.     

The rpoZ gene,encoding the RNA polymerase omega subunit,is required for antibiotic production and morphological differentiation in Streptomyces kasugaensis

The occurrence of pleiotropic mutants that are defective in both antibiotic production and aerial mycelium formation is peculiar to streptomycetes. Pleiotropic mutant KSB was isolated from wild-type Streptomyces kasugaensis A1R6, which produces kasugamycin, an antifungal aminoglycoside antibiotic. A 9.3-kb DNA fragment was cloned from the chromosomal DNA of strain A1R6 by complementary restoration of kasugamycin production and aerial hypha formation to mutant KSB. Complementation experiments with deletion plasmids and subsequent DNA analysis indicated that orf5, encoding 90 amino acids, was responsible for the restoration. A protein homology search revealed that orf5 was a homolog of rpoZ, the gene that is known to encode RNA polymerase subunit omega (omega), thus leading to the conclusion that orf5 was rpoZ in S. kasugaensis. The pleiotropy of mutant KSB was attributed to a 2-bp frameshift deletion in the rpoZ region of mutant KSB, which probably resulted in a truncated, incomplete omega of 47 amino acids. Furthermore, rpoZ-disrupted mutant R6D4 obtained from strain A1R6 by insertion of Tn5 aphII into the middle of the rpoZ-coding region produced neither kasugamycin nor aerial mycelia, similar to mutant KSB. When rpoZ of S. kasugaensis and Streptomyces coelicolor, whose deduced products differed in the sixth amino acid residue, were introduced into mutant R6D4 via a plasmid, both transformants produced kasugamycin and aerial hyphae without significant differences. This study established that rpoZ is required for kasugamycin production and aerial mycelium formation in S. kasugaensis and responsible for pleiotropy.     

Functional and genetic analysis of the Saccharomyces cerevisiae RNC1/TRM2: evidences for its involvement in DNA double-strand break repair

We previously isolated the RNC1/TRM2 gene and provided evidence that it encodes a protein with a possible role in DNA double strand break repair. RNC1 was independently re-isolated as the TRM2 gene encoding a methyl transferase involved in tRNA maturation. Here we show that Trm2p purified as a fusion protein displayed 5' --> 3' exonuclease activity on double-strand (ds) DNA, and endonuclease activity on single-strand (ss) DNA, properties characteristic of previously isolated endo-exonucleases. A variant of Trm2p, Trm2p(ctDelta76aa) lacking 76 amino acids at the C-terminus retained nuclease activities but not the methyl transferase activity. Both the native and the variant exhibited sensitivity to the endo-exonuclease inhibitor pentamidine. The Saccharomyces cerevisiae trm2(Delta232-1920nt) mutant (containing only the first 231 nucleotides of the TRM2 gene) displayed low sensitivity to methyl methane sulfonate (MMS) and suppressed the MMS sensitivity of rad52 mutants in trm2(Delta232-1920nt)rad52 double mutants. The deletion of KU80, in trm2(Delta232-1920nt) mutant background displayed higher MMS sensitivity supporting the view of the possible role of Trm2p in a competing repair pathway separate from NHEJ. In addition, trm2 exo1 double mutants were synergistically more sensitive to MMS and ionizing radiation than either of the single mutant suggesting that TRM2 and EXO1 can functionally complement each other. However, the C-terminal portion, required for its methyl transferase activity was found not important for DNA repair. These results propose an important role for TRM2 in DNA repair with a potential involvement of its nuclease function in homologous recombination based repair of DNA DSBs.     

Characterization of a Transcriptional Activator Controlling Trichothecene Toxin Biosynthesis

Trichothecene biosynthetic pathway genes are localized within a gene cluster in Fusarium sporotrichioides and require the zinc-finger containing protein, TRI6, for expression. We show here that TRI6 is able to bind within the promoter regions of nine different pathway genes and that TRI6 binding is involved in pathway gene activation. TRI6 binding occurs at three distinct sites in the TRI5 promoter, all of which contain the sequence TNAGGCCT. DNA fragments from the promoter regions of six other pathway genes containing this sequence are also substrates for TRI6 binding. Specific nucleotide changes in the TNAGGCCT sequence dramatically reduced TRI6 binding. Analysis of TRI6 binding within the TRI3 and TRI11 promoters and the TRI4-TRI6 intergenic region which do not contain the TNAGGCCT motif suggests that the minimum sequence required for TRI6 binding is YNAGGCC. Two potential TRI6 binding sites, T4A and T4B, were identified within the intergenic region for the divergently transcribed TRI4 and TRI6 genes. Alteration or deletion of the T4A site resulted in the loss of nearly all in vitro TRI6 binding and was correlated with the loss of promoter activity in vivo as measured by the expression of mutant TRI4(p)/GUS fusions. This establishes a physiological role for TRI6 binding and demonstrates that TRI6 is directly involved in the regulation of pathway gene expression. To determine if a predicted Cys2His2 zinc-finger motif at the C-terminus of TRI6 is involved in DNA binding, a C187A mutant was constructed in TRI6 using site-directed mutagenesis. The C187A mutant did not bind promoter DNA fragments, supporting the role of C187 in DNA binding. In addition, a TRI6 homologue in the distantly related macrocyclic trichothecene pathway of Myrothecium roridum (MRTRI6) was also shown to bind to the same TRI5 and TRI4 promoter fragments bound by TRI6. Together, these data confirm our previous proposal that TRI6 is an activator of trichothecene pathway gene expression and that DNA binding employs the C-terminal region of TRI6 containing three predicted Cys2His2 zinc fingers.     

Expression of the Streptomyces coelicolor A3(2) ptpA gene encoding a phosphotyrosine protein phosphatase leads to overproduction of secondary metabolites in S. lividans

Abstract A DNA fragment that caused pigment production in Streptomyces lividans was isolated from a gene library of Pst I-digested chromosomal fragments of S. coelicolor A3(2). Subcloning and nucleotide sequencing proved the identity of the cloned gene to ptpA encoding a low-molecular-mass phosphotyrosine protein phosphatase. The S. lividans transformant containing ptpA on pIJ41 with a copy number of 3–4 per genome produced large amounts of undecylprodigiosin and A-factor, in addition to the pigmented antibiotic actinorhodin, whereas the transformant containing ptpA on an SCP2* derivative with a copy number of 1–2 did not. The PtpA protein produced as a fusion to the maltose binding protein in Escherichia coli showed phosphatase activity toward o -phosphotyrosine, but not toward o -phosphoserine or o -threonine. Introduction of a mutant ptpA gene encoding an inactive protein with serine instead of the 9th cysteine caused no pigmentation. Disruption of the chromosomal ptpA gene of S. coelicolor A3(2), however, appeared to cause no detectable effect on the production of the pigmented antibiotics or A-factor and the ptpA disruptants developed aerial mycelium and spores normally.