Cloning and characterization of a gene (msdA) encoding methylmalonic acid semialdehyde dehydrogenase from Streptomyces coelicolor.

A homolog of the mmsA gene of Pseudomonas aeruginosa, which encodes methylmalonic acid semialdehyde dehydrogenase (MSDH) and is involved in valine catabolism in pseudomonads and mammals, was cloned and sequenced from Streptomyces coelicolor. Of the two open reading frames (ORFs) found, which are convergently transcribed and separated by a 62-nucleotide noncoding region, the deduced amino acid sequence of the msdA ORF (homologous to mmsA) is similar to a variety of prokaryotic and eukaryotic aldehyde dehydrogenases that utilize NAD+, particularly to the MmsA protein from P. aeruginosa. No significant similarity was found between the deduced product of ORF1 and known proteins in the databases. An S. coelicolor msdA mutant, constructed by insertion of a hygromycin resistance gene (hyg) into the msdA coding region, lost the MSDH activity and the ability to grow in a minimal medium with valine or isobutyrate as the sole carbon source but grew on propionate. The msdA::hyg mutation was complemented by introduction of the msdA gene on a plasmid. When the S. coelicolor msdA gene was overexpressed in Escherichia coli under the control of the T7 promoter, a protein of 51-kDa, corresponding to the approximate mass of the predicted S. coelicolor msdA product (52.6 kDa), and specific MSDH activity were detected. These results strongly suggest that msdA indeed encodes the MSDH that is involved in valine catabolism in S. coelicolor.     

Gene controlled by promoter--P_(TH4) depending on whiG of Streptomyces coelicolor

The downstream gene controlled by promoter--PTH4 which is related to Streptomycesdifferentiation was cloned, and its sequence was determined by the dideoxy chain termination method. The results indicated that the 1597 bp of DNA fragment conferred a complete open reading frame (ORF). In searches of databases, the deduced product of the ORF was not homologous with any known proteins; it may be a new protein. The function of the gene was studied using the strategy of gene disruption; the actinorhodin could not be produced when this gene was disrupted. Therefore, this gene may be related to actinorhodin biosynthesis in Streptomyces coelicolor, and the result also shows that this gene may play a role in multiple level regulation of differentiation genes in Streptomyces.     

Gene controlled by promoter--PTH4 depending on whiG of Streptomyces coelicolor

The downstream gene controlled by promoter--P_TH4 which is related to Streptomyces differentiation was cloned, and its sequence was determined by the dideoxy chain termination method. The results indicated that the 1597 bp of DNA fragment conferred a complete open reading frame (ORF). In searches of databases, the deduced product of the ORF was not homologous with any known proteins; it may be a new protein. The function of the gene was studied using the strategy of gene disruption; the actinorhodin could not be produced when this gene was disrupted. Therefore, this gene may be related to actinorhodin biosynthesis in Streptomyces coelicolor, and the result also shows that this gene may play a role in multiple level regulation of differentiation genes in Streptomyces.     

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.     

Cloning and nucleotide sequence of the Streptomyces coelicolor gene encoding glutamine synthetase

The Streptomyces coelicolor glutamine synthetase (GS) structural gene (glnA) was cloned by complementing the glutamine growth requirement of an Escherichia coli strain containing a deletion of its glnALG operon. Expression of the cloned S. coelicolor glnA gene in E. coli cells was found to require an E. coli plasmid promoter. The nucleotide sequence of an S. coelicolor 2280-bp DNA segment containing the glnA gene was determined and the complete glnA amino acid sequence deduced. Comparison of the derived S. coelicolor GS protein sequence with the amino acid sequences of GS from other bacteria suggests that the S. coelicolor GS protein is more similar to the GS proteins from Gram-negative bacteria than it is with the GS proteins from two Gram-positive bacteria, Bacillus subtilis and Clostridium acetobutylicum.     

Nucleotide sequence and deduced functions of a set of cotranscribed genes of Streptomyces coelicolor A3(2) including the polyketide synthase for the antibiotic actinorhodin.

A 5.3-kb region of the Streptomyces coelicolor actinorhodin gene cluster, including the genes for polyketide biosynthesis, was sequenced. Six identified open reading frames (ORF1-6) were related to genetically characterized mutations of classes actI, VII, IV, and VB by complementation analysis. ORF1-6 run divergently from the adjacent actIII gene, which encodes the polyketide synthase (PKS) ketoreductase, and appear to form an operon. The deduced gene products of ORF1-3 are similar to fatty acid synthases (FAS) of different organisms and PKS genes from other polyketide producers. The predicted ORF5 gene product is similar to type II beta-lactamases of Bacillus cereus and Bacteroides fragilis. The ORF6 product does not resemble other known proteins. Combining the genetical, biochemical, and similarity data, the potential activities of the products of the six genes can be postulated as: 1) condensing enzyme/acyl transferase (ORF1 + ORF2); 2) acyl carrier protein (ORF3); 3) putative cyclase/dehydrase (ORF4); 4) dehydrase (ORF5); and 5) "dimerase" (ORF6). The data show that the actinorhodin PKS consists of discrete monofunctional components, like that of the Escherichia coli (Type II) FAS, rather than the multifunctional polypeptides for the macrolide PKSs and vertebrate FASs (Type I).     

Characteristics of a Streptomyces coelicolor A3(2) extracellular protein targeting chitin and chitosan

Upstream of the Streptomyces coelicolor A3(2) chitinase G gene, a small gene (named chb3) is located whose deduced product shares 37% identical amino acids with the previously described CHB1 protein from Streptomyces olivaceoviridis. The chb3 gene and its upstream region were cloned in a multicopy vector and transformed into the plasmid-free Streptomyces lividans TK21 strain. The CHB3 protein (14.9 kDa) was secreted by the S. lividans TK21 transformant during growth in the presence of glucose, N-acetylglucosamine, yeast extract, and chitin. The protein was purified to homogeneity using anionic exchange, hydrophobic interaction chromatographies, and gel filtration. In contrast to CHB1, CHB3 targets alpha-chitin, beta-chitin, and chitosan at pH 6.0 but does so relatively loosely. The ecological implications of the divergence of substrate specificity of various types of chitin-binding proteins are described.     

Structural and functional characterization of the recR gene of Streptomyces

The recR gene product is necessary for homologous recombination and recombinational DNA repair in eubacteria. We report the isolation and sequencing of the recR gene from Streptomyces coelicolor. It encodes a protein of 198 amino acids, with a predicted molecular mass of 22 kDa. The deduced amino acid sequence shows significant similarity to that of RecR proteins from other bacteria, including Escherichia coli and Bacillus subtilis. Like these, Streptomyces RecR contains potential helix-hairpin-helix, zinc finger and ATP-binding motifs, as well as the Toprim domain which is present also in topoisomerases of Types IA and II, primases and nucleases of the OLD family. The recR genes of Escherichia coli and Bacillus subtilis are immediately preceded by a small ORF (orf12 and orf107, respectively). An equivalent ORF (orf1) is also found in Streptomyces. S. lividans recR mutants, obtained either by insertional inactivation of recR or by deletion of the gene together with the preceding ORF, displayed increased sensitivity to DNA-damaging agents (such as UV light and methylmethanesulfonate), when compared with the wild-type strain. Both mutants could be complemented by the wild-type orflrecR genes and also by the recR gene alone. Based on these results, orf1 appears to be dispensable for the repair function of Streptomyces RecR. In studies of heterologous complementation, the B. subtilis recR region (orf107recR) was found to complement the S. lividans deltaorflrecR mutant, but the equivalent region from E. coli (orf12recR) could not. However, in the absence of orf107, B. subtilis recR was unable to restore the wild-type phenotype to the Streptomyces deletion mutant.     

A glgC gene essential only for the first of two spatially distinct phases of glycogen synthesis in Streptomyces coelicolor A3(2).

By using a PCR approach based on conserved regions of ADP-glucose pyrophosphorylases, a glgC gene was cloned from Streptomyces coelicolor A3(2). The deduced glgC gene product showed end-to-end relatedness to other bacterial ADP-glucose pyrophosphorylases. The glgC gene is about 1,000 kb from the leftmost chromosome end and is not closely linked to either of the two glgB genes of S. coelicolor, which encode glycogen branching enzymes active in different locations in differentiated colonies. Disruption of glgC eliminated only the first of two temporal peaks of ADP-glucose pyrophosphorylase activity and glycogen accumulation and prevented cytologically observable glycogen accumulation in the substrate mycelium of colonies (phase I), while glycogen deposition in young spore chains (phase II) remained readily detectable. The cloned glgC gene therefore encodes an ADP-glucose pyrophosphorylase essential only for phase I (and it is therefore named glgCI). A second, phase II-specific, glgC gene should also exist in S. coelicolor, though it was not detected by hybridization analysis.     

Nucleotide sequence of the tcmII-tcmIV region of the tetracenomycin C biosynthetic gene cluster of Streptomyces glaucescens and evidence that the tcmN gene encodes a multifunctional cyclase-dehydratase-O-methyl transferase.

Mutations in the tcmII-tcmIV region of the Streptomyces glaucescens chromosome block the C-3 and C-8 O-methylations of the polyketide antibiotic tetracenomycin C (Tcm C). The nucleotide sequence of this region reveals the presence of two genes, tcmN and tcmO, whose deduced protein products display similarity to the hydroxyindole O-methyl transferase of the bovine pineal gland, an enzyme that catalyzes a phenolic O-methylation analogous to those required for the biosynthesis of Tcm C. The deduced product of the tcmN gene also has an N-terminal domain that shows similarity to the putative ActVII and WhiE ORFVI proteins of Streptomyces coelicolor. The tcmN N-terminal domain can be separated from the remainder of the tcmN gene product, and when coupled on a plasmid with the Tcm C polyketide synthase genes (tcmKLM), this domain enables high-level production of an early, partially cyclized intermediate of Tcm C in a Tcm C- null mutant or in a heterologous host (Streptomyces lividans). By analogy to fatty acid biosynthesis, the tcmKLM polyketide synthase gene products are probably sufficient to produce the linear decaketide precursor of Tcm C; thus, the tcmN N-terminal domain is most likely responsible for one or more of the early cyclizations and, perhaps, the attendant dehydrations that lead to the partially cyclized intermediate. The tcmN gene therefore appears to encode a multifunctional cyclase-dehydratase-3-O-methyl transferase. The tcmO gene encodes the 8-O-methyl transferase.     

Spore colour in Streptomyces coelicolor A3(2) involves the developmentally regulated synthesis of a compound biosynthetically related to polyketide antibiotics

Streptomyces coelicolor produces spores whose development of a grey colour requires the activity of the whiE locus. The cloned whiE locus was identified after mobilization into a whiE mutant of a library of S. coelicolor DNA inserted into a transmissible plasmid vector. The whiE region of the cloned DNA was localized both by subcloning and by mutagenesis of the cloned DNA with the Streptomyces transposon Tn4560. Nucleotide sequencing of this region revealed seven open reading frames, of which six show homology at the level of deduced gene products with genes involved in the synthesis of polyketide antibiotics. A previously described S. coelicolor DNA segment encoding biosynthesis of a brown pigment (Horinouchi and Beppu, 1985) corresponds to the cloned whiE DNA. It is proposed that whiE is normally expressed only in the aerial hyphae, where the biosynthetic product is responsible for spore colour.     

Sequence analysis of the agrA gene encoding beta-agarase from Pseudomonas atlantica.

The nucleotide sequence of the agrA gene encoding an extracellular beta-agarase of Pseudomonas atlantica was determined. An open reading frame of 1,515 nucleotides which corresponded to agrA was found. The nucleotide sequence predicts a primary translation product of 504 amino acids and Mr 57,486. Comparison of the deduced amino acid sequences of beta-agarase from P. atlantica and the extracellular beta-agarase from Streptomyces coelicolor A3(2) suggests that these proteins share several domains in common.     

Molecular cloning and characterization of two lincomycin-resistance genes, ImrA and ImrB, from Streptomyces lincolnensls 78–11

Two different lincomycin-resistance determinants (lmrA and lmrB) from Streptomyces lincolnensis 78-11 were cloned in Streptomyces lividans 66 TK23. The gene lmrA was localized on a 2.16 kb fragment, the determined nucleotide sequence of which encoded a single open reading frame 1446 bp long. Analysis of the deduced amino acid sequence suggested the presence of 12 membrane-spanning domains and showed significant similarities to the methylenomycin-resistance protein (Mmr) from Streptomyces coelicolor, the QacA protein from Staphylococcus aureus, and several tetracycline-resistance proteins from both Gram-positive and Gram-negative bacteria, as well as to some sugar-transport proteins from Escherichia coli. The lmrB gene was actively expressed from a 2.7 kb fragment. An open reading frame of 837 bp could be localized which encoded a protein that was significantly similar to 23S rRNA adenine(2058)-N-methyltransferases conferring macrolide-lincosamide-streptogramin resistance. LmrB also had putative rRNA methyltransferase activity since lincomycin resistance of ribosomes was induced in lmrB-containing strains. Surprisingly, both enzymes, LmrA and LmrB, had a substrate specificity restricted to lincomycin and did not cause resistance to other lincosamides such as celesticetin and clindamycin, or to macrolides.     

Molecular cloning, heterologous expression, and functional characterisation of a malate synthase gene from Streptomyces coelicolor A3(2)

With the rapid generation of genetic information from the Streptomyces coelicolor genome project, deciphering the relevant gene products is critical for understanding the genetics of this model streptomycete. A putative malate synthase gene (aceB) from S. coelicolor A3(2) was identified by homology-based analysis, cloned by polymerase chain reaction, and fully sequenced on both strands. The putative malate synthase from S. coelicolor has an amino acid identity of 77% with the malate synthase of S. clavuligerus, and possesses an open reading frame which codes for a protein of 540 amino acids. In order to establish the identity of this gene, the putative aceB clones were subcloned into the expression vector pET24a, and heterologously expressed in Escherichia coli BL21(DE3). Soluble cell-free extracts containing the recombinant putative malate synthase exhibited a specific activity of 1623 (nmol.mg-1.min-1), which is an increment of 92-fold compared to the non-recombinant control. Thus, the gene product was confirmed to be a malate synthase. Interestingly, the specific activity of S. coelicolor malate synthase was found to be almost 8-fold higher than the specific activity of S. clavuligerus malate synthase under similar expression conditions. Furthermore, the genomic organisation of the three Streptomyces aceB genes cloned thus far is different from that of other bacterial malate synthases, and warrants further investigation.