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The conservon (cvn) of Streptomyces species encodes a putative membrane-associated signaling complex resembling the eukaryotic G-protein-coupled receptor (GPCR) system. The cvn is widely distributed in the genomes of Actinobacteria, indicating that it plays an important role in this group of bacterial species; however, the exact role of this regulatory system is hitherto poorly understood. In the present study, we generated null mutants for all 13 copies of the cvn operon distributed in the genome of Streptomyces coelicolor A3(2) and observed that the aerial mycelium formation and antibiotic production in a cvn1 mutant were markedly impaired. The cvn1 mutant formed aerial mycelium and produced actinorhodin and undecylprodigiosin at remarkably low levels on solid medium containing 1-2% glucose and at high levels on medium containing 6-10% glucose. The same phenotype as this was observed with a cvnA1 mutant. Transcriptional analyses revealed that the expression of sigU encoding a vegetative sigma factor was upregulated in the cvn1 mutant. Overexpression of rsuA encoding the σ(SigU) antagonist restored aerial mycelium formation and pigment production in the cvn1 mutant, suggesting that the developmental defect in the cvn1 mutant is based on the high expression level of sigU.

A-factor (2-isocapryloyl-3R-hydroxymethyl-γ-butyrolactone) is essential for aerial mycelium formation and streptomycin (Sm) production in Streptomyces griseus . A protein Ser/Thr kinase (AfsK), the product of the Streptomyces coelicolor A3(2) afsK gene, controlling secondary metabolism in this strain, reversed the aerial mycelium-negative phenotype of an A-factor-deficient mutant strain, S. griseus HH1, and induced sporulation without affecting A-factor productivity or Sm production. A mutant AfsK protein lacking kinase activity failed to induce aerial mycelium formation which indicates the importance of the kinase activity for suppression in S. griseus . These data suggest that a Ser/Thr kinase functionally similar to S. coelicolor A3(2) AfsK plays a regulatory role in aerial mycelium formation in S. griseus , either as a member in the A-factor regulatory network or independently of this network

A gene named sksl+was cloned as a suppressor of the K-252a-sensitivity phenotype of Schizosaccharomyces pombe (Sp) from a gene library of the parental Sp chromosomal DNA constructed with a multicopy vector pDB248'. The gene encoded a 308-amino-acid (aa) protein similar to the Caenorhabditis elegans F37A4.5 gene product and to the mouse and Drosophila Mov34 gene products. The sksl+ null mutants obtained by gene disruption were non-viable, indicating that sksl+ is essential for vegetative growth. The parental Sp strain carrying multiple copies of sksl+ showed distinct cross-resistance to staurosporine, thiabendazole and vanadate in addition to K-252a, although Sksl has no similarity in aa sequence to those of ATP-binding cassette (ABC)-type transporters. The multicopy plasmid containing sksl+conferred multidrug resistance (MDR), even in a mutant cell defective in pmdl+ encoding an ABC-type transporter. It is therefore unlikely that the function of pmdl+is involved in MDR conferred by sksl+. These results suggest that sksl+is a functionally novel MDR gene.

Two macrolide-resistance determinants were cloned from a mycinamicin (Mm)-producing Micromonospora griseorubida strain in Streptomyces lividans and Streptomyces parvulus. One of the cloned genes, designated myrA, was cloned as a gene which conferred strong resistance to Mm and tyiosin (Ty), but not to erythromycin (Er) or josamycin (Jm) on S. lividans. Another gene, named myrB, was cloned as an ErRencoding gene which conferred MLS resistance (to macrolide, lincosamide and streptogramine B antibiotics) on S. parvulus. Both myrA and myrB were sequenced and the corresponding ORFs were determined. The deduced amino acid (aa) sequence of myrA showed no similarity to proteins in the available databases, suggesting that an unknown mechanism of macrolide resistance is exerted by the MyrA protein. The deduced aa sequence of myrB exhibited high similarity to 23S rRNA methyltransferases (MTases), such as ErmE and CarB, from a variety of microorganisms.

A DNA fragment of 42 kb encompassing one of the mycinamicin II (Mm)-resistance-encoding genes, myrB, from a Mm-producing strain, Micromonospora griseorubida, was cloned in Escherichia coli using the cosmid vector pJB8. Nucleotide sequencing of the neighboring region of myrB and a computer-aided analysis of the sequence predicted the presence of an open reading frame (ORF) with 254 amino acids which showed great similarity to the macrocin O-methyltransferase (tylF gene product) in tylosin (Ty)-producing Streptomyces fradiae. When a 1.0-kb AluI fragment containing the complete ORF was fused to the lacZ promoter in the correct orientation and expressed in E. coli, a mycinamicin III (MIII) O-methyltransferase (MOMT) activity was detected only upon induction with isopropyl-beta-D-thiogalactopyranoside (IPTG). All these data indicate that this ORF codes for the structural gene of MOMT, and it is designated mycF.

Streptomyces griseus possesses multiple stress-response sigma factors including sigma(H). Previously, we have suggested that sigma(H) and related sigma factors are involved in the developmental control of S. griseus. Herein, we studied the role of two sigma(H) paralogs--sigma(F) and sigma(N)--which are encoded in tandem coding sequences of sigF-sigN in S. griseus [sigma(N) has been described as sigma(L) previously (Gene 320:127, 2003)]. A sigF mutant produced decreased levels of intracellular melanin and formed irregular spores. A triple mutant for sigHNF exhibited defective melanin production. While sigN was transcribed by three tandem promoters during the early to late growth phases, sigF was transcribed in the late developmental phase by a single promoter. The activity of the promoter preceding the rpp operon (Prpp), which is responsible for the intracellular melanin biosynthesis, was decreased in the sigF mutant and abolished in the sigHNF, adpA and A-factor biosynthesis mutants. The in vitro transcription assay demonstrated that Esigma(F) transcribed the rpp promoter. Both Esigma(F) and Esigma(N) transcribed a sigma(H)-dependent promoter that preceded the sigH operon, and their activities were repressed by the addition of RshA, an anti-sigma(H) protein. Overall, the results suggest that the three sigma factors have similar functions and that they are required for spore development and pigmentation. The transcription of the rpp operon is regulated both by the stress-response sigma factors and the A-factor regulatory cascade.

We cloned and characterized sigH encoding a stress-response sigma factor, sigma(H), of Streptomyces griseus. Nucleotide sequencing of the sigH gene cluster revealed an identical gene organization as in the orthologous region of Streptomyces coelicolor A3(2). Transcriptional analysis by S1 nuclease mapping showed the presence of three tandem promoters (P1-P3) that direct transcription of sigH. The activity of P1 was markedly reduced in a sigH-depleted mutant, suggesting its dependence on sigma(H). P1 was induced by addition of 0.7 M NaCl, and P2 was induced by heat shock at 45 degrees C or addition of 4% ethanol. The sigH mutant of S. griseus showed conditional defect in aerial mycelium formation and streptomycin production depending on high concentration of glucose (>2%). Meanwhile, the wild-type strain of S. griseus introduced with rshA encoding a probable anti-sigma factor for sigma(H) on a high-copy-number plasmid was unable to perform development on media containing 1% glucose while it showed wild-type phenotype on media containing 1% maltose. RshA inhibited the sigma(H)-dependent in vitro run-off transcription at P1, which confirmed its role as a negative regulator for sigma(H). Analysis of RshA-sigma interaction by an Escherichia coli two-hybrid system showed specific interaction of RshA with sigma(H) and two related sigma factors, sigma(L) and sigma(F). We speculate that the high copy number of rshA represses morphological and physiological development of S. griseus through simultaneous inactivation of sigma(H) and related stress-response sigma factors, which play an essential role in the onset of cellular differentiation and antibiotic production on glucose media.

We report cloning and characterization of a 2.8 kb DNA fragment that suppressed the aerial mycelium-deficient phenotype of an amfR mutant of Streptomyces griseus when it was introduced on a high-copy-number plasmid. Nucleotide sequencing revealed that the cloned DNA fragment contained a part of a regulatory operon homologous to one of the conserved operons identified in the genome of Streptomyces coelicolor A3(2). The operon appeared to consist of 5 CDSs (rarA–E; restoration of aerial mycelium formation in an amfR mutant): rarA encoded a membrane protein with weak similarity to the histidine kinase of the two-component regulatory system; rarB and rarC products did not show marked similarity to other proteins with known function; rarD encoded a G-protein carrying two GTP-binding consensus sequences conserved in the eukaryotic Ras-like proteins; rarE product showed end-to-end homology to cytochrome P450. The 2.8 kb fragment contained a 5′-end incomplete rarA and complete rarB-D in the downstream from the promoter region of mel operon of the vector plasmid. Subcloning showed that the region containing rarA only is sufficient for the aerial mycelium-inducing activity. The truncation of rarA at its 5′ terminus was essential for the restoration activity, which implied that the mutated rarA product causes unusual signaling that directs the onset of morphogenesis without amfR function. Inactivation of both rarA in Streptomyces griseus and cvnD9, a rarD ortholog in S. coelicolor resulted in precocious and glucose-resistant formation of aerial mycelium and secondary metabolites, which suggested that the operon negatively regulates the onset of differentiation. S1 nuclease protection analysis showed that the transcriptional activity of the promoter preceding rarA is developmentally regulated in an amfR- and glucose-dependent manner.

A new gene encoding a heat shock protein 70 family protein of Schizosaccharomyces pombe (Sp), named sks2+, was cloned as a weak suppressor for the K-252a-sensitive mutation, ucm1. The nucleotide sequence of sks2+ revealed an open reading frame of a 613-amino-acid (aa) protein. The deduced aa sequence of sks2+ showed significant homology with Saccharomyces cerevisiae (Sc) Ssb1p and Ssb2p responsible for protein synthesis by non-organelle-localized ribosomes, as well as with other proteins of the HSP70 family. The cells lacking the functional sks2+ gene were viable and showed no increased sensitivity to K-252a but grew slowly with an elongated morphology. These results suggest that the sks2+ gene product plays a role in the cell cycle progression and is able to confer drug resistance in a multicopy state.

A-factor is a microbial hormone that functions as a key switch for secondary metabolite formation and morphogenesis in Streptomyces griseus. Genetic and biochemical studies on the A-factor-binding protein have implied that the binding protein present in the cytoplasm plays a role in repressing streptomycin (Sm) production and sporulation while the binding of A-factor to the binding protein releases this repression. The A-factor signal is transferred, probably via some additional regulatory proteins in the A-factor-regulatory cascade, to the strR gene, a regulator for Sm biosynthesis. A positive regulatory protein binds about 430-330 bp upstream from the transcription start point of the strR promoter and activates its transcription. The StrR product, in turn, activates the other Sm-biosynthesis genes. A global regulatory gene, afsR, of Streptomyces coelicolor A3(2) encodes a 993-amino acid protein that is phosphorylated by a specific phosphokinase, AfsK, encoded by the region just upstream from the afsR gene. Site-directed mutagenesis of afsR has revealed that phosphorylated AfsR globally stimulates transcription of antibiotic-production genes. It is most likely that AfsR and AfsK compose a two-component regulatory system. Although AfsR shows no significant homology with typical regulators of the two-component systems in other prokaryotes, such as OmpR and PhoB of Escherichia coli, it shows considerable homology with regulatory proteins in antibiotic biosynthetic gene clusters of Streptomyces spp., such as actII ORF4, dnrR1 ORF1 and redD ORF1.(ABSTRACT TRUNCATED AT 250 WORDS)

Several microbial secondary metabolites function as essential chemical signals for induction of cellular differentiation in the producing organisms. The role of A-factor and its analogues such as essential autoregulators in actinomycetes is discussed and a review is given of fungal metabolites with hormonal activities. Divergent secondary metabolites with the capability to induce cellular differentiation in other organisms are also discussed as to their possible involvement in a symbiotic relationship in the ecosystem.

Human pro-urokinase (pro-UK) and two pro-UK deletion mutants, one lacking the epidermal growth factor(EGF)-like domain, and the other lacking both the EGF-like domain and the kringle domain, were produced in Saccharomyces cerevisiae. This was done using the yeast GAL7 promoter and the prepeptide sequence of a fungal aspartic proteinase, Mucor pusillus rennin (MPR). Although biologically active and heavily glycosylated pro-UKs were secreted into the culture medium, the amounts were extremely small. On the other hand, large amounts of pro-UKs of a single-chain form were accumulated inside the cells, exceeding 3–4% of total cellular proteins. The intracellular pro-UKs were N-glycosylated, probably with a single core carbohydrate unit, and amino acid sequencing of their N termini revealed that the secretion signal of MPR was correctly processed. Biologically active pro-UKs were recovered in high yields by means of solubilization with 4.5 M guanidine · HC1 and subsequent dialysis for refolding. The refolded yeast pro-UK was indistinguishable from human kidney-derived pro-UK in terms of specific enzymatic activity and its secondary structure, as determined by circular dichroism spectroscopy.

The afsR gene of Streptomyces coelicolor A3(2) complements afsB mutations affecting production of pigmented antibiotics. It also directs pigment production in Streptomyces lividans when carried on a plasmid vector. Nucleotide sequencing of the afsR gene revealed that it codes for a 993-amino acid protein (Mr 105,600) with A- and B-type ATP-binding consensus sequences at its N-terminal portion and two DNA-binding consensus sequences with a helix-turn-helix motif at its C-terminal portion. Each of the N- and C-terminal halves was capable of conferring pigment production, to some extent, in S. lividans, when carried separately on a multicopy plasmid. In addition, expression in trans of the two regions on the same plasmid conferred pigment production to almost the same extent as did the intact afsR gene. Mutations at the two ATP-binding consensus sequences, that were generated by in vitro site-directed mutagenesis, revealed their functional importance. Disruption of the S. coelicolor A3(2) chromosomal afsR gene in either the N- or C-terminal half using phage phi C31 KC515 resulted in significant, but not complete, loss of pigment production. These data suggest that the AfsR protein comprises two domains, viz., an ATP-binding and a DNA-binding domain, each of which could function as a positive regulator for pigment production. These afsR mutants sporulate normally. In addition to an internal promoter, which we previously detected in the middle of the AfsR coding region, S1 nuclease mapping revealed two tandem transcriptional start points, separated by 64 bp, upstream from a putative ATG start codon of the AfsR product.

A gene encoding an endo-type semi-alkaline cellulase was cloned from an alkalophilic Streptomyces strain in Streptomyces lividans, and its nucleotide sequence was determined. Downstream from the transcriptional start point, which was determined by high-resolution S1 mapping, an open reading frame of 388 amino acids (aa) was present. The N-terminal amino acid sequence of the mature enzyme determined by an Edman degradation procedure suggested that the cellulase had an extraordinarily long leader sequence of about 70 aa. Comparison with the leader sequences of endoglucosidase H from Streptomyces plicatus and the cellulase from Cellulomonas fimi suggested that the semi-alkaline cellulase was processed in two steps during maturation.

A Streptomyces bikiniensis DNA fragment complementing a deficiency in streptomycin (Sm) production was cloned on the plasmid vector pIJ385. Host strain S. bikiniensis SD1 used as the recipient in cloning displayed deficiency in biosynthesis of N -methyl- l -glucosamine, one of the moieties of Sm. The cloned fragment on the multicopy plasmid pIJ385 conferred sevenfold increase in Sm production in comparison with the wild-type parental strain. By subcloning, the region complementing the Sm deficiency of SD1 was narrowed to a 3.0-kb fragment.

To increase yields of calf prochymosin (PC) produced in Escherichia coli , PC cDNA was cloned in a plasmid vector under control of the trp promoter. The hybrid plasmid pCRSOl constructed for this purpose contains cDNA coding for PC (from the 5th Arg to the C-terminal Ile) fused to the N-terminal fragment of the trpE gene preceded by the trp promoter and attenuator region. E. coli C600 harboring this plasmid produces approx. 300000 molecules of PC per cell. This is about a tenfold increase above the amount obtained using lac UV5 promoter [Nishimori et al., Gene 19 (1982) 337-344]. A similar plasmid, pCR601, which contains the same coding sequence fused to the trp promoter and N-terminal fragment of the trpL gene, directs the production of PC at the same rate as pCR501. In pCR601 the trp attenuator is deleted. Another plasmid, pCR701, in which construction of a sequence coding for f Met-PC cDNA that was aided by chemical synthesis, was placed under direct control of the trp promoter, produced PC at a much lower rate. Extracts prepared from all these bacterial transformants in the presence of urea showed distinct milk-clotting activity after renaturation and processing.

Calf prochymosin (cPC) gene was cloned from calf abomasum DNA by using a cloned cPC cDNA as a probe. The cPC gene spans approx. 10.5 kb and consists of 9 exons and 8 introns. The positions of exon-intron junctions coincide completely with those in the human pepsinogen gene. Analysis of 5′ -flanking region sequence and S1 nuclease mapping revealed that the transcriptional start point was located 25 bp upstream from the start codon and that the putative transcriptional promoter sequences (TATA box and CCAAT box) were located in − 30 and − 90 regions, respectively. Some distinctive sequences possibly functioning as regulatory signals for gene expression are present at the 5′-flanking region.