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PubMed2005

Predicted highly expressed genes in the genomes of Streptomyces coelicolor and Streptomyces avermitilis and the implications for their metabolism.

Highly expressed genes in bacteria often have a stronger codon bias than genes expressed at lower levels, due to translational selection. In this study, a comparative analysis of predicted highly expressed (PHX) genes in the Streptomyces coelicolor and Streptomyces avermitilis genomes was performed using the codon adaptation index (CAI) as a numerical estimator of gene expression level. Although it has been suggested that there is little heterogeneity in codon usage in G+C-rich bacteria, considerable heterogeneity was found among genes in these two G+C-rich Streptomyces genomes. Using ribosomal protein genes as references, approximately 10% of the genes were predicted to be PHX genes using a CAI cutoff value of greater than 0.78 and 0.75 in S. coelicolor and S. avermitilis, respectively. The PHX genes showed good agreement with the experimental data on expression levels obtained from proteomic analysis by previous workers. Among 724 and 730 PHX genes identified from S. coelicolor and S. avermitilis, 368 are orthologue genes present in both genomes, which were mostly 'housekeeping' genes involved in cell growth. In addition, 61 orthologous gene pairs with unknown functions were identified as PHX. Only one polyketide synthase gene from each Streptomyces genome was predicted as PHX. Nevertheless, several key genes responsible for producing precursors for secondary metabolites, such as crotonyl-CoA reductase and propionyl-CoA carboxylase, and genes necessary for initiation of secondary metabolism, such as adenosylmethionine synthetase, were among the PHX genes in the two Streptomyces species. The PHX genes exclusive to each genome, and what they imply regarding cellular metabolism, are also discussed.

Bacterial Proteins↗
PubMed1995

Stress-activated expression of a Streptomyces pristinaespiralis multidrug resistance gene (ptr) in various Streptomyces spp. and Escherichia coli.

A promoter which controls expression of the pristinamycin multidrug resistance gene (ptr) in Streptomyces pristinaspiralis could be induced by physiological stresses in both Streptomyces spp. and Escherichia coli. In S. pristinaspiralis, the ptr promoter (Pptr) was induced by pristinamycin I (PI) or pristinamycin II (PII). Streptomyces lividans was adopted as a convenient heterologous host for studies of Pptr regulation since it has no known pristinamycin biosynthetic genes. Two key regulatory features were documented in these studies: many (19 of 70) antibiotics and chemicals with no common targets or structural features induced the Pptr; induction with PI was most efficient during a transition phase when antibiotic biosynthetic genes are switched on. In Streptomyces coelicolor, Pptr activity was similarly inducible by PI and not dependent on sigma factors HrdA, HrdC, or HrdD. In E. coli, Pptr cloned in the bifunctional promoter probe vector pIJ2839 was functional and activated upon entry into stationary phase in the absence of exogenous inducer. Finally, gel-retardation studies demonstrated a Pptr-binding protein in S. lividans (where its activity was PI-inducible), S. coelicolor and S. pristinaespiralis. The fact that this activity was not detected in E. coli suggested the existence of another regulatory system perhaps also present in Streptomyces.

Anti-Bacterial Agents↗
PubMed1992

Regulation of secondary metabolism in Streptomyces spp. and overproduction of daunorubicin in Streptomyces peucetius.

Two DNA segments, dnrR1 and dnrR2, from the Streptomyces peucetius ATCC 29050 genome were identified by their ability to stimulate secondary metabolite production and resistance. When introduced into the wild-type ATCC 29050 strain, the 2.0-kb dnrR1 segment caused a 10-fold overproduction of epsilon-rhodomycinone, a key intermediate of daunorubicin biosynthesis, whereas the 1.9-kb dnrR2 segment increased production of both epsilon-rhodomycinone and daunorubicin 10- and 2-fold, respectively. In addition, the dnrR2 segment restored high-level daunorubicin resistance to strain H6101, a daunorubicin-sensitive mutant of S. peucetius subsp. caesius ATCC 27952. Analysis of the sequence of the dnrR1 fragment revealed the presence of two closely situated open reading frames, dnrI and dnrJ, whose deduced products exhibit high similarity to the products of several other Streptomyces genes that have been implicated in the regulation of secondary metabolism. Insertional inactivation of dnrI in the ATCC 29050 strain with the Tn5 kanamycin resistance gene abolished epsilon-rhodomycinone and daunorubicin production and markedly decreased resistance to daunorubicin. Sequence comparison between the products of dnrIJ and the products of the Streptomyces coelicolor actII-orf4, afsR, and redD-orf1 genes and of the Streptomyces griseus strS, the Saccharopolyspora erythraea eryC1, and the Bacillus stearothermophilus degT genes reveals two families of putative regulatory genes. The members of the DegT, DnrJ, EryC1, and StrS family exhibit some of the features characteristic of the protein kinase (sensor) component of two-component regulatory systems from other bacteria (even though none of the sequences of these four proteins show a significant overall or regional similarity to such protein kinases) and have a consensus helix-turn-helix motif typical of DNA binding proteins. A helix-turn-helix motif is also present in two of the proteins of the other family, AfsR and RedD-Orf1. Both sets of Streptomyces proteins are likely to be trans-acting factors involved in regulating secondary metabolism.

Amino Acid Sequence↗
PubMed1990

Cloning and characterization of a gene of Streptomyces griseus that increases production of extracellular enzymes in several species of Streptomyces.

A 7.2 kb Bg/II restriction fragment, which increases the production of several extracellular enzymes, including alkaline phosphatase, amylase, protease, lipase and beta-galactosidase, was cloned in Streptomyces lividans from the DNA of S. griseus ATCC 10137. This gene (named saf) showed a positive gene dosage effect on production of extracellular enzymes. When the saf gene was introduced into cells in high copy numbers it delayed the formation of pigments and spores in S. lividans and also retarded actinorhodin production in Streptomyces coelicolor. The saf gene hybridized with specific bands in the DNA of several Streptomyces strains tested. A 1 kb fragment containing the saf gene was sequenced and contains an open reading frame (ORF) of 306 nucleotides which encodes a polypeptide of Mr 10,500. This ORF is contained within a fragment of 432 bp which retained activity in Streptomyces. A fragment with promoter activity is present upstream of the saf reading frame. The predicted Saf polypeptide has a strong positive charge, and does not show a typical amino acid composition for a membrane protein, and contains a DNA-binding domain similar to those found in several regulatory proteins.

Alkaline Phosphatase↗
PubMed1991

Interactions of Streptomyces serine-protease inhibitors with Streptomyces griseus metalloendopeptidase II.

Streptomyces griseus metalloendopeptidase II (SGMPII) was shown to form tight complexes with several Streptomyces protein inhibitors which had been believed to be specific to serine proteases, such as Streptomyces subtilisin inhibitor (SSI), plasminostreptin (PS), and alkaline protease inhibitor-2c' (API-2c'), as well as with Streptomyces metalloprotease inhibitor (SMPI). The dissociation constants of complexes between SGMPII and these inhibitors were successfully determined by using a novel fluorogenic bimane-peptide substrate. The values ranged from nM to pM. The results of studies by gel chromatographic and enzymatic analyses indicated that SGMPII is liberated from the complex with SSI by the addition of subtilisin BPN'. SGMPII and subtilisin BPN' proved, therefore, to interact with SSI in a competitive manner, despite the difference in the chemical nature of their active sites.

Base Sequence↗
PubMed1983

Molecular cloning and expression in streptomyces lividans of a hygromycin B phosphotransferase gene from Streptomyces hygroscopicus.

The gene encoding the phosphotransferase enzyme that modifies hygromycin B in its producing organism Streptomyces hygroscopicus, has been cloned in the Streptomyces vector pIJ41. Two plasmids, pFM4 and pFM6, containing 2.1 and 19.6 kb inserts of Streptomyces hygroscopicus DNA, respectively, which express the modifying enzyme, have been isolated. A 3.1 kb PstI restriction fragment from pFM4 was inserted in the Streptomyces vector pIJ350 and the resulting plasmids, pMZ11.1 and pMZ11.2, express the hygromycin B-resistance phenotype. The utility of this dominant marker for cloning experiments is discussed in the text.

Bacillus subtilis↗
PubMed1984

Cloning and expression in Streptomyces lividans of a paromomycin phosphotransferase from Streptomyces rimosus Forma paromomycinus.

The paromomycin producing organism Streptomyces rimosus forma paromomycinus is resistant to this antibiotic and contains a phosphotransferase which inactivates paromomycin. The gene encoding this enzyme has been inserted in the Streptomyces vector pIJ702 and then cloned in Streptomyces lividans, selecting for paromomycin-resistance. Three plasmids have been isolated and one of them, pMJ1, contains a 2.2 kb insert with a single HindIII restriction site. Insertion of foreign DNA in this site blocks the expression of the phosphotransferase enzyme indicating that it is within the cloned gene. These findings provide a new dominant selective marker for Streptomyces cloning vectors with the versatility of insertional inactivation.

Cloning, Molecular↗
PubMed1990

Comparison of secretory expression in Escherichia coli and Streptomyces of Streptomyces subtilisin inhibitor (SSI) gene.

To elucidate differences in the mechanism of gene expression between Streptomyces and Escherichia coli, the regulatory region for expression of the gene for a proteinaceous proteinase inhibitor, Streptomyces subtilisin inhibitor (SSI), was altered to express efficiently in E. coli. This was carried out by inserting a pre-SSI-encoding region downstream of the tac promoter and ribosome-binding site in a multi-copy plasmid. When the resultant plasmid pMKSI161-9 was introduced into E. coli JM105, SSI protein was found to be expressed and secreted into the periplasmic space by Western blot analysis. When introduced into 'leaky' E. coli strains, this protein was detected in the medium as well as in the periplasmic space in bacteria. NH2-terminal sequencing analysis of the SSI purified from E. coli JM105 indicated two processing sites, Ala(-4)/Ala(-3)-Pro(-2)-Gly and Ala(-4)-Ala-3/Pro(-2)-Gly-1, of pre-SII. These sites were different from those in Streptomyces albogriseolus S-3253 and Streptomyces lividans 66. The inhibitor activity of the processed protein toward subtilisin BPN' was almost the same as that of authentic SSI.

Amino Acid Sequence↗
PubMed1992

Cosmid pJAR4, a novel Streptomyces-Escherichia coli shuttle vector for the cloning of Streptomyces operons.

A novel shuttle cosmid vector (pJAR4), based on pK505, was constructed for the cloning of Streptomyces DNA. It is a low-copy-number vector which determines hygromycin B-resistance as a selective marker and was used to clone the puromycin biosynthesis pathway from Streptomyces alboniger. Cosmids pJAR4 and pKC505 (which determines apramycin-resistance) stably co-transform both Streptomyces lividans and Streptomyces griseofuscus.

Cloning, Molecular↗
PubMed1987

The active sites of the beta-lactamases of Streptomyces cacaoi and Streptomyces albus G.

The active-site serine of the extracellular beta-lactamases of Streptomyces cacaoi and Streptomyces albus G has been labelled with beta-iodopenicillanate. The determination of the sequence of the labelled peptides obtained after trypsin digestion of the denatured proteins indicate both enzymes to be class A beta-lactamases. Surprisingly the two Streptomyces enzymes do not appear to be especially homologous, and none of them exhibited a high degree of homology with the Streptomyces R61 DD-peptidase. Our data confirm that, as a family of homologous enzymes, class A is rather heterogeneous, with only a small number of conserved residues in all members of the class.

Amino Acid Sequence↗
PubMed1998

The conjugative plasmid pSG5 from Streptomyces ghanaensis DSM 2932 differs in its transfer functions from other Streptomyces rolling-circle-type plasmids.

The Streptomyces ghanaensis plasmid pSG5 is self-transmissible but does not form the growth-retardation zones (pocks) normally characteristic of the Streptomyces plasmid-transfer process. The complete nucleotide sequence of pSG5 was determined on both strands. pSG5 is 12,208 bp in length and has a GC content of 68 mol%. Characterization of the open reading frames by insertion and deletion analysis revealed that only a single gene, traB, is involved in the transfer of pSG5. The deduced amino acid sequence of TraB is similar to the SpoIIIE protein that is responsible for chromosome translocation during prespore formation of Bacillus subtilis. In contrast to the tra genes of the other Streptomyces plasmids, the pSG5 traB does not represent a kill function. Although pSG5 transfer is not associated with pock formation, pSG5 was shown to possess putative spd genes that are responsible for the pock phenotype of other Streptomyces plasmids. However, promoter-probe experiments revealed that the spd genes of pSG5 are not transcribed, thus explaining the deficiency in pock formation.

Amino Acid Sequence↗
PubMed1995

Sequence and functional analysis of the Streptomyces phaeochromogenes plasmid pJV1 reveals a modular organization of Streptomyces plasmids that replicate by rolling circle.

pJV1 is an 11 kb, high-copy-number conjugative Streptomyces phaeochromogenes plasmid that replicates by the rolling circle mechanism (RCR). Sequencing combined with functional analysis of deletion, insertion and frameshift mutations was used to characterize the genes involved in plasmid transfer and chromosome mobilization (Cma), the single-strand origin for RCR and an associated strong incompatibility (Sti) determinant. pJV1 contains two essential transfer genes whose expression is regulated by an adjacent repressor gene with similarity to the GntR family of regulators. A consensus sequence specific for the helix-turn-helix motifs of repressor proteins of Streptomyces plasmids is proposed. Unregulated expression of the transfer genes by inactivation of the repressor is lethal. Three additional genes increase intramycelial plasmid spread resulting in pock formation but, unlike the essential transfer genes, are not required for Cma. The pJV1 transfer genes and their regulatory region, but not the minimal replication region encoding the double-strand replication origin and replication protein, are similar in their sequence and arrangement to those of the Streptomyces nigrifaciens plasmid pSN22, revealing a modular organization of Streptomyces RCR plasmids.

Amino Acid Sequence↗
PubMed1997

Electroporation of intact cells of Streptomyces parvulus and Streptomyces vinaceus.

Various assays of classical PEG-assisted transformation as well as electrotransformation of Streptomyces parvulus IMET41380 and Streptomyces vinaceus NCIB8852 are described. Contrary to the so far reported assays of electrotransforming Streptomyces strains, electroporation in S. parvulus and S. vinaceus was carried out on intact cells, without any lysozyme treatment. In these two strains, the classical PEG-assisted transformation of protoplasts does not work efficiently (10(3) to 10(4) transformants per micrograms of pIJ702 DNA) and electrotransformation gives 10 to 100 times higher yields (10(5) transformants per micrograms of pIJ702 DNA). The electroporation method described here is not applicable to other Streptomyces strains (S. lividans or S. coelicolor).

Electroporation↗
PubMed1995

Studies of Streptomyces reticuli cel-1 (cellulase) gene expression in Streptomyces strains, Escherichia coli, and Bacillus subtilis.

Various streptomyces strains [Streptomyces lividans 66, Streptomyces vinaceus, and Strepotmyces coelicolor A3 (2)] acquired the ability to utilize crystalline cellulose (Avicel) after transformation with a multicopy vector containing the cel-1 gene from Streptomyces reticuli. The expression level in these hosts was two to three times lower than in S. reticuli, indicating the absence of positive regulatory elements. Like S. reticuli, they processed the Avicelase to its catalytic domain and to an enzymatically inactive part. The cel-1 gene with its original upstream region was not expressed within Escherichia coli. When cel-1 had been fused in phase with the lacZ gene, large quantities of the fusion protein were produced in E. coli. However, this protein was enzymatically inactive and proteolytically degraded to a series of truncated forms. As the cellulase (Avicelase) synthesized by S. reticuli is not cleaved by the E. coli proteases, its posttranslational modification is proposed. With Bacillus subtilis as host, the cel-1 gene was expressed neither under its own promoter nor under the control of a strong Bacillus promoter.

Amino Acid Sequence↗
PubMed1986

Nucleotide sequence of afsB, a pleiotropic gene involved in secondary metabolism in Streptomyces coelicolor A3(2) and "Streptomyces lividans".

The nucleotide sequence of afsB from Streptomyces coelicolor A3(2), a pleiotropic gene which positively controls the biosynthesis of A-factor and the pigmented antibiotics actinorhodin and undecylprodigiosin in S. coelicolor A3(2) and "Streptomyces lividans," was determined. The determinant of the afsB gene, which includes the putative AfsB protein consisting of 243 amino acids, was mapped functionally by tests for A-factor and pigment production in "S. lividans" and S. coelicolor A3(2) after introduction of recombinant plasmids containing various restriction endonuclease fragments on the vector plasmids pIJ41 and pIJ702. The putative AfsB protein contains two regions separated by 167 residues which resemble conserved domains of known DNA-binding proteins. High-resolution nuclease S1 protection mapping revealed that the afsB mRNA, approximately 1,300 base pairs (bp) long, which was determined by Northern blot hybridization, had its start point 340 bp upstream of the putative methionine start codon. The Northern hybridization experiment also suggested that the afsB gene was constitutively transcribed throughout growth. Also shown by the Northern hybridization was the presence of an unidentified gene with an extraordinary amount of 880-bp mRNA located downstream from afsB. Dot hybridization with the brown pigment production genes, possibly involved in polyketide biosynthesis, as the probe suggested that the afsB gene did not stimulate transcription of the pigment production genes. In Southern blot DNA-DNA hybridization analysis with the afsB sequence as the probe, sequences exhibiting various degrees of homology were found in several Streptomyces spp. A DNA sequence showing strong homology to the afsB in Streptomyces griseus FT-1, a high streptomycin producer, behaved like an extrachromosomal element, homologous to the afsA gene, a structural gene for A-factor biosynthesis.

4-Butyrolactone↗
PubMed1996

Induction of actinorhodin production by rpsL (encoding ribosomal protein S12) mutations that confer streptomycin resistance in Streptomyces lividans and Streptomyces coelicolor A3(2).

A strain of Streptomyces lividans, TK24, was found to produce a pigmented antibiotic, actinorhodin, although S. lividans normally does not produce this antibiotic. Genetic analyses revealed that a streptomycin-resistant mutation str-6 in strain TK24 is responsible for induction of antibiotic synthesis. DNA sequencing showed that str-6 is a point mutation in the rpsL gene encoding ribosomal protein S12, changing Lys-88 to Glu. Gene replacement experiments with the Lys88-->Glu str allele demonstrated unambiguously that the str mutation is alone responsible for the activation of actinorhodin production observed. In contrast, the strA1 mutation, a genetic marker frequently used for crosses, did not restore actinorhodin production and was found to result in an amino acid alteration of Lys-43 to Asn. Induction of actinorhodin production was also detected in strain TK21, which does not harbor the str-6 mutation, when cells were incubated with sufficient streptomycin or tetracycline to reduce the cell's growth rate, and 40 and 3% of streptomycin- or tetracycline-resistant mutants, respectively, derived from strain TK21 produced actinorhodin. Streptomycin-resistant mutations also blocked the inhibitory effects of relA and brgA mutations on antibiotic production, aerial mycelium formation or both. These str mutations changed Lys-88 to Glu or Arg and Arg-86 to His in ribosomal protein S12. The decrease in streptomycin production in relC mutants in Streptomyces griseus could also be abolished completely by introducing streptomycin-resistant mutations, although the impairment in antibiotic production due to bldA (in Streptomyces coelicolor) or afs mutations (in S. griseus) was not eliminated. These results indicate that the onset and extent of secondary metabolism in Streptomyces spp. is significantly controlled by the translational machinery.

Alleles↗
PubMed1996

Influence of disease-suppressive strains of Streptomyces on the native Streptomyces community in soil as determined by the analysis of cellular fatty acids.

Analysis of cellular fatty acid profiles was used to distinguish among introduced pathogen- suppressive strains and indigenous strains of Streptomyces spp. isolated from soil of field plots established to test the efficacy of Streptomyces strains PonSSII and PonR in the biological control of potato scab. Reference libraries of fatty acid profiles were developed for a collection of known pathogenic strains and the introduced suppressive strains. Population densities of pathogen-related, suppressive, and saprophytic Streptomyces strains were determined from the relationship of field isolates to mean library profiles using cluster analysis and the unweighted pair-group method using arithmetic averages. Community diversity was similarly determined. Streptomyces strains PonSSII and PonR were distinguished from each other and from the pathogen group (which clustered together) based on fatty acid profiles. The introduced, suppressive strains successfully colonized the soil and represented 2-19% of the isolates sampled over 2 years. The introduction of the suppressive strains inhibited the population of strains related to the pathogen library at each sample date; the pathogen population was substantially lower in soil from treatments where the suppressive strains were introduced compared with the nonamended control. At harvest, the pathogen-related population was suppressed 85-93 and 36-44% in 1991 and 1992, respectively, in treatments with the suppressive strains compared with the nonamended control. Diversity of the community was not affected by the introduced strains, and diversity and equitability indices were similar among treatments at any sample time. The inhibition of the pathogen-related population was correlated with a reduction of scab symptoms observed in the field plots into which the suppressive strains were introduced. Implications of a fundamental shift in the pathogen-related population in response to the introduction of the suppressive strains for long-term biological control of potato scab are encouraging.

Fatty Acids↗
PubMed2006

[Activation of silent antibiotic synthesis in Streptomyces lividans by disruption of a negative regulator nsdA, a gene conserved in Streptomyces].

The global regulatory gene, nsdA, negatively regulates antibiotics production in Streptomyces coelicolor. Southern blot experiment, using an nsdA fragment of S. coelicolor as probe, indicated that nsdA gene existed in many Streptomyces. Primers were designed based on the published sequences of S. coelicolor and S. avermitilis. PCR amplification and sequencing showed that nsdA in Streptomyces was conservative and that of S. lividans ZX64 has a 100% identity in the nucleotide sequence comparing with that of S. coelicolor A3 (2). The nsdA disrupted mutant of S. lividans was constructed named as WQ2. WQ2 was able to produce actinorhodin but the wild-type strain ZX64 did not, which has a silent gene cluster contributing to the biosynthesis of actinorhodin. However, the ability was lost when another copy of the wild nsdA gene was introduced into WQ2. All the results above indicate that nsdA homologous gene is wildly existent and conserved in Streptomyces. And it plays a role in negatively regulating the actinorhodin synthesis in S. lividans and disruption of it can activate the silent gene cluster.

Anti-Bacterial Agents↗