Determining the functionality of putative Tat-dependent signal peptides in Streptomyces coelicolor A3(2) by using two different reporter proteins. (41/441)

The availability of the complete genome sequence of Streptomyces coelicolor A3(2) has allowed the prediction of the Tat-exported proteins of this Gram-positive bacterium. To predict secreted proteins that potentially use the Tat pathway for their secretion, the TATscan program was developed. This program identified 129 putative Tat substrates. To test the validity of these predictions, nine signal sequences, including three which were not identified by existing prediction programs, were selected and fused to the structural xlnC gene in place of its native signal sequence. Xylanase C (XlnC) is a cofactorless enzyme which is secreted in an active form exclusively through the Tat-dependent pathway by Streptomyces lividans. Among the nine chosen signal sequences, seven were shown to be Tat-dependent, one was Sec-dependent and one was probably not a signal sequence. The seven Tat-dependent signal sequences comprised two lipoprotein signal sequences and three sequences not predicted by previous programs. Pulse-chase experiments showed that the precursor-processing rate in the seven transformants was generally slower than wild-type XlnC, indicating that these signal peptides were not equivalent in secretion. This suggested that there might be some incompatibility between the signal peptide and the reporter protein fused to it. To test this possibility, the signal peptides were fused to a cofactorless chitosanase (SCO0677), a Tat-dependent protein validated in this work but structurally different from XlnC. With some fluctuations, similar results were obtained with this enzyme, indicating that the type of folding of the reporter protein had little effect on the Tat secretion process.  (+info)

Characterization of O-methyltransferase ScOMT1 cloned from Streptomyces coelicolor A3(2). (42/441)

The roles of O-methyltransferases (OMTs) in microorganisms are not well understood, and are suggested to increase antimicrobial activity. Studies on OMTs cloned from microorganisms may help elucidate their roles. Streptomyces coelicolor A3(2) produces many useful natural antibiotics such as actinorhodin. Based on sequence information from S. coelicolor A3(2) genome, it was possible to clone several methyltransferases. An OMT cloned from Streptomyces coelicolor A3(2), ScOMT1 was characterized by in vivo and in vitro assays. Of 23 compounds tested, 13 were found to serve as its substrates. Of the 13 substrates, the methylated positions of 7 compounds were determined by HPLC, NMR, and MS analyses. This OMT favored ortho-dihydroxyflavones. Among the compounds tested here, the best substrate is 6,7-dihydroxyflavone.  (+info)

The absB gene encodes a double strand-specific endoribonuclease that cleaves the read-through transcript of the rpsO-pnp operon in Streptomyces coelicolor. (43/441)

The absB locus of Streptomyces coelicolor encodes a homolog of bacterial RNase III. We cloned and overexpressed the absB gene product and purified a decahistidine-tagged version of the protein. We show here that AbsB is active against double-stranded RNA transcripts derived from synthetic DNAs but is inactive with single-stranded homopolymers. We thus designate the absB product RNase IIIS. Using T7 RNA polymerase and a cloned template containing the rpsO-pnp intergenic region, we synthesized an RNA substrate representing a portion of the read-through transcript normally produced in S. coelicolor. This transcript contains the sequences that form the putative rpsO terminator and those that form an intergenic stem-loop structure thought to be the site for RNase IIIS processing of the read-through transcript. We show that RNase IIIS does cleave that model transcript, with primary and secondary cleavage sites in an internal loop in the stem-loop structure. We have identified the primary and secondary cleavage sites by primer extension and demonstrate the further processing of the initial cleavage products. Thus, as is the case in Escherichia coli, the read-through transcript from rpsO-pnp is cleaved by RNase IIIS in S. coelicolor. However, the cleavage sites are different in the two systems. The positions of the cleavage sites in the stem-loop of the S. coelicolor transcript are more akin to those identified in the processing of bacteriophage T7 mRNAs. A kinetic assay for RNase IIIS was developed, and kinetic parameters for the reaction utilizing the model transcript from rpsO-pnp were determined.  (+info)

Effects of growth phase and the developmentally significant bldA-specified tRNA on the membrane-associated proteome of Streptomyces coelicolor. (44/441)

Previous proteomic analyses of Streptomyces coelicolor by two-dimensional electrophoresis and protein mass fingerprinting focused on extracts from total cellular material. Here, the membrane-associated proteome of cultures grown in a liquid minimal medium was partially characterized. The products of some 120 genes were characterized from the membrane fraction, with 70 predicted to possess at least one transmembrane helix. A notably high proportion of ABC transporter systems was represented; the specific types detected provided a snapshot of the nutritional requirements of the mycelium. The membrane-associated proteins did not change very much in abundance in different phases of growth in liquid minimal medium. Identification of gene products not expected to be present in membrane protein extracts led to a reconsideration of the genome annotation in two cases, and supplemented scarce information on 11 hypothetical/conserved hypothetical proteins of unknown function. The wild-type membrane proteome was compared with that of a bldA mutant lacking the only tRNA capable of efficient translation of the rare UUA (leucine) codon. Such mutants are unaffected in vegetative growth but are defective in many aspects of secondary metabolism and morphological differentiation. There were a few clear changes in the membrane proteome of the mutant. In particular, two hypothetical proteins (SCO4244 and SCO4252) were completely absent from the bldA mutant, and this was associated with the TTA-containing regulatory gene SCO4263. Evidence for the control of a cluster of function-unknown genes by the SCO4263 regulator revealed a new aspect of the pleiotropic bldA phenotype.  (+info)

Chemobiosynthesis of novel 6-deoxyerythronolide B analogues by mutation of the loading module of 6-deoxyerythronolide B synthase 1. (45/441)

Chemobiosynthesis (J. R. Jacobsen, C. R. Hutchinson, D. E. Cane, and C. Khosla, Science 277:367-369, 1997) is an important route for the production of polyketide analogues and has been used extensively for the production of analogues of 6-deoxyerythronolide B (6-dEB). Here we describe a new route for chemobiosynthesis using a version of 6-deoxyerythronolide B synthase (DEBS) that lacks the loading module. When the engineered DEBS was expressed in both Escherichia coli and Streptomyces coelicolor and fed a variety of acyl-thioesters, several novel 15-R-6-dEB analogues were produced. The simpler "monoketide" acyl-thioester substrates required for this route of 15-R-6-dEB chemobiosynthesis allow greater flexibility and provide a cost-effective alternative to diketide-thioester feeding to DEBS KS1(o) for the production of 15-R-6-dEB analogues. Moreover, the facile synthesis of the monoketide acyl-thioesters allowed investigation of alternative thioester carriers. Several alternatives to N-acetyl cysteamine were found to work efficiently, and one of these, methyl thioglycolate, was verified as a productive thioester carrier for mono- and diketide feeding in both E. coli and S. coelicolor.  (+info)

Ancestral antibiotic resistance in Mycobacterium tuberculosis. (46/441)

Chemotherapeutic options to treat tuberculosis are severely restricted by the intrinsic resistance of Mycobacterium tuberculosis to the majority of clinically applied antibiotics. Such resistance is partially provided by the low permeability of their unique cell envelope. Here we describe a complementary system that coordinates resistance to drugs that have penetrated the envelope, allowing mycobacteria to tolerate diverse classes of antibiotics that inhibit cytoplasmic targets. This system depends on whiB7, a gene that pathogenic Mycobacterium shares with Streptomyces, a phylogenetically related genus known as the source of diverse antibiotics. In M. tuberculosis, whiB7 is induced by subinhibitory concentrations of antibiotics (erythromycin, tetracycline, and streptomycin) and whiB7 null mutants (Streptomyces and Mycobacterium) are hypersusceptible to antibiotics in vitro. M. tuberculosis is also antibiotic sensitive within a monocyte model system. In addition to antibiotics, whiB7 is induced by exposure to fatty acids that pathogenic Mycobacterium species may accumulate internally or encounter within eukaryotic hosts during infection. Gene expression profiling analyses demonstrate that whiB7 transcription determines drug resistance by activating expression of a regulon including genes involved in ribosomal protection and antibiotic efflux. Components of the whiB7 system may serve as attractive targets for the identification of inhibitors that render M. tuberculosis or multidrug-resistant derivatives more antibiotic-sensitive.  (+info)

The Streptomyces subtilisin inhibitor (SSI) gene in Streptomyces coelicolor A3(2). (47/441)

Streptomyces subtilisin inhibitors (SSIs) are produced by a wide variety of Streptomyces species. Streptomyces coelicolor A3(2) contains two genes, SCO0762 and SCO4010, encoding an SSI-like protein. Of these two genes, SCO0762 was transcribed actively throughout growth. Gene disruption of SCO0762 (mutant DeltaSCO0762) resulted in overproduction of extracellular protease activity, showing that SCO0762 serves as a modulator of extracellular protease activities. Mutant DeltaSCO0762 showed no apparent phenotypic changes in morphological differentiation, forming aerial hyphae and spores in the same time course as the parental strain. SCO4010 appeared to be a pseudogene, because mutant DeltaSCO4010 showed the same protease activity as the parental strain, probably due to amino acid replacement of one (Arg-60) of the important residues for SSI activity, and because the transcription of this gene was extremely low.  (+info)

Novel method for detection of butanolides in Streptomyces coelicolor culture broth, using a His-tagged receptor (ScbR) and mass spectrometry. (48/441)

Gamma-butyrolactone derivative molecules in Streptomyces play a crucial role in cell density control, secondary metabolism, and cell differentiation. As their synthesis level in the cell is very low compared to those of similar N-acyl homoserine lactone molecules from gram-negative bacteria, it is very hard to analyze them even with several hundredfold concentration of the culture broth. We have developed a very quick and easy detection method using an affinity capture technique with His-tagged receptor proteins and electrospray tandem mass spectrometry. Using Streptomyces coelicolor as a model system, SCB1 was detected from only 100 ml of the culture broth after solvent extraction. This method can be further applied to detection and quantitative analysis of butanolides and inhibitor screening of the receptor molecules.  (+info)