Characterization of a plasmid from Streptomyces coelicolor A3(2).

Covalently closed circular deoxyribonucleic acid (DNA) with a molecular weight of 20 X 10(6) was identified in strains of Streptomyces coelicolor A3(2) of various fertility types. Hybridization studies and digestion by various restriction endonucleases indicated that the circular DNAs (pSH1) were identical regardless of the fertility type (UF, IF, or NF) of the strain from which it was isolated. The pSH1 DNA was cleaved to many fragments by the endonucleases HincII, SmaI, and SalI and to three or four fragments by BamHI and PstI. Plasmid pSH1 carries single sites for each of the two restriction enzymes, EcoRI and HindIII. These sites are 7.6 X 10(6) daltons apart. Attempts to isolate the fertility factor SCP1 as covalently closed circular DNA were unsuccessful. These data suggest that the biochemically isolated plasmid pSH1 is not identical to the genetically characterized fertility factor SCP1, which has been identified in an autonomous state in IF-type strains and in an integrated state in NF-type strains.     

Apical hyphal extension in Streptomyces coelicolor A3(2).

Hyphal extension in the filamentous actinomycete Streptomyces coelicolor A3(2) was shown to occur by addition of newly synthesized wall material in an apical extension zone. Incubation of mycelia with tritiated N-acetyl-D-glucosamine (GlcNAc), a precursor of peptidoglycan, resulted in localized incorporation of label at the apex, as indicated by light microscopic and electron microscopic autoradiography. Within the hyphal extension zone there was a sharp decrease in incorporation with increasing distance from the apex. Hyphal tip shape, examined by low-temperature scanning electron microscopy, approximated to a semi-ellipsoid of revolution and was not hemispherical. Tip shape could be represented accurately by polynomial equations of degree less than seven. The surface stress theory was successfully applied to hyphal tip growth, with tip shape related qualitatively to the inverse of surface tension within the wall of the extension zone. Surface tension was assumed to be inversely proportional to the rate of incorporation of tritiated GlcNAc. Treatment of surface-grown hyphae with beta-lactam antibiotics resulted in localized swelling of hyphal tips. Lysozyme caused swelling of tips and of other regions of hyphae, frequently giving a beaded morphology associated with septa.     

High-multiplicity of chitinase genes in Streptomyces coelicolor A3(2).

Six different genes for chitinase from ordered cosmids of the chromosome of Streptomyces coelicolor A3(2) were identified by hybridization, using the chitinase genes from other Streptomyces spp. as probes, and cloned. The genes were sequenced and analyzed. The genes, together with an additional chitinase gene obtained from the data bank, can be classified into either family 18 or family 19 of the glycosyl hydrolase classification. The five chitinases that fall into family 18 show diversity in their multiple domain structures as well as in the amino acid sequences of their catalytic domains. The remaining two chitinases are members of family 19 chitinases, since their C-terminus shares more than 70% identity with the catalytic domain of ChiC of Streptomyces griseus, the sole gene for family 19 chitinase so far found in an organism other than higher plants.     

Phosphorylation of the AfsR product, a global regulatory protein for secondary-metabolite formation in Streptomyces coelicolor A3(2).

The AfsR protein is essential for the biosynthesis at the wild-type level of A-factor, actinorhodin, and undecylprodigiosin in Streptomyces coelicolor A3(2) and Streptomyces lividans. Because overexpression of the afsR gene caused some deleterious effect on these strains, a multicopy plasmid carrying the whole afsR gene was introduced into Streptomyces griseus, from which a crude cell lysate was prepared as a protein source. The AfsR protein was purified to homogeneity from the cytoplasmic fraction through several steps of chromatography, including affinity column chromatography with ATP-agarose and use of anti-AfsR antibody for its detection. The molecular weight of AfsR was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by gel filtration to be 105,300, which is in good agreement with that deduced from the nucleotide sequence of afsR. The purified AfsR protein was found to be phosphorylated through the transfer of the gamma-phosphate group of ATP in the presence of the cell extracts of S. coelicolor A3(2) and S. lividans. This phosphorylation proceeded very rapidly, and no competition was observed with CTP, GTP, UTP, or cyclic AMP. In the cell extract of S. griseus, no activity phosphorylating the AfsR protein was detected, suggesting that this activity is not generally present in Streptomyces spp. but is specific to certain species. It is conceivable that the extent of phosphorylation of the AfsR protein modulates its regulatory activity which, in turn, regulates expression of some target gene(s) involved in the secondary-metabolite formation in S. coelicolor A3(2).     

Identification and cloning of a umu locus in Streptomyces coelicolor A3(2)

The umuDC operon of Escherichia coli is required for efficient mutagenesis by UV and many other DNA-damaging agents. E. coli umu mutants are defective in mutagenesis and slightly more sensitive to DNA-damaging agents. The existence of a umuDC analogue in Streptomyces coelicolor was suggested by data of our previous works. We cloned from Streptomyces coelicolor a fragment of DNA homologous to the E. coli umuDC region that is able to complement the E coli umuC122::Tn5 mutation. Therefore our data suggest that S. coelicolor contains a functional umu-like operon.     

Expression of a functional fungal polyketide synthase in the bacterium Streptomyces coelicolor A3(2).

The multifunctional 6-methylsalicylic acid synthase gene from Penicillium patulum was engineered for regulated expression in Streptomyces coelicolor. Production of significant amounts of 6-methylsalicylic acid by the recombinant strain was proven by nuclear magnetic resonance spectroscopy. These results suggest that it is possible to harness the molecular diversity of eukaryotic polyketide pathways by heterologous expression of biosynthetic genes in an easily manipulated model bacterial host in which prokaryotic aromatic and modular polyketide synthase genes are already expressed and recombined.     

A breakdown in macromolecular synthesis preceding differentiation in Streptomyces coelicolor A3(2).

A transitory cessation of growth was recorded in Streptomyces coelicolor A3(2) at the end of vegetative mycelium formation on solid medium. In the same phase a striking reduction in protein and nucleic acid synthesis was detected. Growth and macromolecular synthesis resumed, nearly reaching the original values, when morphological differentiation occurred. It is concluded that a physiological stress occurs within the bacterial population just before the onset of the morphological differentiation.     

The obligate aerobic actinomycete Streptomyces coelicolor A3(2) survives extended periods of anaerobic stress

The actinomycete Streptomyces coelicolor is an obligate aerobe that is found in soil and aqueous habitats. The levels of oxygen in these environments can vary considerably, which raises the question of how these bacteria survive during periods of anaerobiosis. Although S. coelicolor cannot grow in the complete absence of oxygen, we demonstrate here that it is capable of microaerobic growth and maintaining viability through several weeks of strict anaerobiosis. Both resting and germinated spores are able to survive abrupt exposure to anaerobiosis, which contrasts the situation with Mycobacterium species where gradual oxygen depletion is required to establish a latent state in which the bacterium is able to survive extended periods of anaerobiosis. Growth of S. coelicolor resumes immediately upon re-introduction of oxygen. Taken together these findings indicate that survival is not restricted to spores and suggest that the bacterium has evolved a mechanism to maintain viability and a membrane potential in the hyphal state. Furthermore, although we demonstrate that several members of the genus also survive long periods of anaerobic stress, one species, Streptomyces avermitilis, does not have this capacity and might represent a naturally occurring variant that is unable to adopt this survival strategy.     

天蓝色链霉菌调控基因tcrA功能的初步研究

天蓝色链霉菌的开放阅读框SCO5433编码一个含有TPR(Tetratricopeptide repeat)结构域的调控蛋白。该基因的阻断突变株表现出孢子颜色加深和色素产量增加的表型变化。孢子颜色的加深在以葡萄糖或甘露醇为碳源的MM培养基上表现明显;色素产量的增加在以甘露醇为碳源的MM培养基和MS培养基上表现最为明显;插片培养结合光学显微镜观察并没有发现突变株在形态分化上有显著变化;这些发现预示着可能存在一个SCO5433参与的调控途径,在一定条件下,这一途径对天蓝色链霉菌次级代谢可能起着负调控作用,而与形态分化无关。     

Identification of genes for mycothiol biosynthesis in Streptomyces coelicolor A3(2)

Mycothiol is a low molecular weight thiol compound produced by a number of actinomycetes, and has been suggested to serve both anti-oxidative and detoxifying roles. To investigate the metabolism and the role of mycothiol in Streptomyces coelicolor, the biosynthetic genes (mshA, B, C, and D) were predicted based on sequence homology with the mycobacterial genes and confirmed experimentally. Disruption of the mshA, C, and D genes by PCR targeting mutagenesis resulted in no synthesis of mycothiol, whereas the mshB mutation reduced its level to about 10% of the wild type. The results indicate that the mshA, C, and D genes encode non-redundant biosynthetic enzymes, whereas the enzymatic activity of MshB (acetylase) is shared by at least one other gene product, most likely the mca gene product (amidase).     

Cloning of whiG, a gene critical for sporulation of Streptomyces coelicolor A3(2).

In whiG mutants of Streptomyces coelicolor A3(2), aerial hyphae do not show any sign of sporulation. A library of S. coelicolor DNA was prepared in a phi C31 temperate phage vector (KC516), and one recombinant phage (KC750) that could restore the wild-type phenotype to a collection of whiG mutants when integrated into their genomes was found. Subcloning experiments with low- and high-copy-number Streptomyces plasmid vectors allowed partial localization of whiG in the cloned DNA and revealed that hypersporulation was associated with the presence of extra copies of whiG.     

Identification of genes involved in siderophore transport in Streptomyces coelicolor A3(2)

The potential iron siderophore transporter genes have been determined from the genome sequence of Streptomyces coelicolor A3(2). One of these gene clusters, cdtABC, was disrupted and characterized to determine its role in the uptake of the siderophores produced by S. coelicolor. Resistance to the siderophore-like antibiotics, salmycin and albomycin, was tested in the parent and cdtABC mutant, showing that the parent, but not the mutant, was sensitive to salmycin, while both were resistant to albomycin. Ferrioxamine competition assays against salmycin suggest that the uptake of salmycin is via a ferrioxamine transport system. However, Fe-55 ferrioxamine B uptake experiments did not reveal any difference between the parent and mutant. This suggests that CdtABC specifically transports salmycin, while ferrioxamine uptake maybe substituted by another transport system.     

Glucose repression in Streptomyces coelicolor A3(2): a likely regulatory role for glucose kinase

The glucose kinase gene (glkA-ORF3) of Streptomyces coelicolor A3(2) plays an essential role in glucose utilisation and in glucose repression of a variety of genes involved in the utilisation of alternative carbon sources. These genes include dagA, which encodes an extracellular agarase that permits agar utilisation. Suppressor mutants of glkA-ORF3 deletion strains capable of utilising glucose (Glc⁺) arise at a frequency of about 10^?5 on prolonged incubation. The Glc⁺ phenotype of the mutants is reversible (at a frequency of about 10^?3) and reflects either the activation of a normally silent glucose kinase gene or the modification of an existing sugar kinase. Although the level of glucose kinase activity in the Glc⁺ supressor mutants is similar to that in the glkA ⁺ parental strain, glucose repression of dagA remains defective. Expression of the glucose kinase gene of Zymomonas mobilis in glkA-ORF3 mutants restored glucose utilisation, but not glucose repression of dagA. Over-expression of glkA-ORF3 on a high-copy-number plasmid failed to restore glucose repression of dagA in glkA-ORF3 mutants and led to loss of glucose repression of dagA in a glkA ⁺ strain. These results suggest that glucose phosphorylation itself is not sufficient for glucose repression and that glkA-ORF3 plays a specific regulatory role in triggering glucose repression in S. coelicolor A3(2).     

Identification of a DNA sequence associated with plasmid integration in Streptomyces coelicolor A3(2)

Summary We identified a DNA element of length about 1 kb that is present in two copies in the chromosome of Streptomyces coelicolor A3(2) and is also present on the plasmid SCP1 which has been carefully defined genetically, but never isolated as extrachromosomal DNA.A copy of the element is close (within 5 kb) of a gene coding for an extracellular agarase in the chromosome of S. coelicolor A3(2) and in an NF strain, in which SCP1 has integrated into the chromosome, the agarase gene has been deleted. The element has properties reminiscent of Insertion Sequences in Escherichia coli, but it is not yet know if it can transpose.     

Isolation of covalently closed circular deoxyribonucleic acid from Streptomyces coelicolor A3(2).

Covalently closed circular deoxyribonucleic acid (DNA) was isolated from two strains of Streptomyces coelicolor A3(2), representing two of the known fertility types. In each of the two strains circular DNA of about 20 times 10-6 daltons could be detected, amounting to about 1.5% of the total cellular DNA. The possible function of this DNA is discussed.