The nusG gene of Streptomyces griseus: Cloning of the gene and analysis of the A-factor binding properties of the gene product

Abstract The nusG gene of Streptomyces griseus was cloned and the nucleotide sequence determined. It encodes a protein with an identify of 76% to the reported receptor (VbrA) for VB-C, an autoregulatory factor in Streptomyces virginae . NusG protein was expressed in Escherichia coli . However, no binding activity for A-factor, an butyrolactone autoregulator in S. griseus very similar to VB-C, could be detected. The nusG gene of S. griseus does not seem to encode the A-factor-binding protein.     

Cloning and analysis of a gene cluster from Streptomyces coelicolor that causes accelerated aerial mycelium formation in Streptomyces lividans.

We describe the cloning and analysis of two overlapping DNA fragments from Streptomyces coelicolor that cause aerial mycelium to appear more rapidly than usual when introduced into Streptomyces lividans on a low-copy-number plasmid vector. Colonies of S. lividans TK64 harboring either clone produce visible aerial mycelia after only 48 h of growth, rather than the usual 72 to 96 h. From deletion and sequence analysis, this rapid aerial mycelium (Ram) phenotype appears to be due to a cluster of three genes that we have designated ramA, ramB, and ramR. Both ramA and ramB potentially encode 65-kDa proteins with homology to ATP-dependent membrane-translocating proteins. A chromosomal ramB disruption mutant of S. lividans was found to be severely defective in aerial mycelium formation. ramR could encode a 21-kDa protein with significant homology to the UhpA subset of bacterial two-component response regulator proteins. The overall organization and potential proteins encoded by the cloned DNA suggest that this is the S. coelicolor homolog of the amf gene cluster that has been shown to be important for aerial mycelium formation in Streptomyces griseus. However, despite the fact that the two regions probably have identical functions, there is relatively poor homology between the two gene clusters at the DNA sequence level.     

EshA accentuates ppGpp accumulation and is conditionally required for antibiotic production in Streptomyces coelicolor A3(2)

Disruption of eshA, which encodes a 52-kDa protein that is produced late during the growth of Streptomyces coelicolor A3(2), resulted in elimination of actinorhodin production. In contrast, disruption of eshB, a close homologue of eshA, had no effect on antibiotic production. The eshA disruptant accumulated lower levels of ppGpp than the wild-type strain accumulated. The loss of actinorhodin production in the eshA disruptant was restored by expression of a truncated relA gene, which increased the ppGpp level to the level in the wild-type strain, indicating that the reduced ppGpp accumulation in the eshA mutant was solely responsible for the loss of antibiotic production. Antibiotic production was also restored in the eshA mutant by introducing mutations into rpoB (encoding the RNA polymerase β subunit) that bypassed the requirement for ppGpp, which is consistent with a role for EshA in modulating ppGpp levels. EshA contains a cyclic nucleotide-binding domain that is essential for its role in triggering actinorhodin production. EshA may provide new insights and opportunities to unravel the molecular signaling events that occur during physiological differentiation in streptomycetes.     

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).     

Cloning and characterization of a gene involved in aerial mycelium formation in Streptomyces griseus.

A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) is essentially required for aerial mycelium formation and streptomycin production in Streptomyces griseus. A DNA fragment which induced aerial mycelium formation and sporulation in an A-factor-deficient mutant strain, S. griseus HH1, was cloned from this strain on a high-copy-number plasmid. Subcloning and nucleotide sequencing revealed that one open reading frame with 218 amino acids, named AmfC, served as a multicopy suppressor of the aerial mycelium-defective phenotype of the A-factor-deficient strain. The amfC gene did not restore A-factor or streptomycin production, indicating that amfC is involved in aerial mycelium formation independently of secondary metabolic function. Disruption of the chromosomal amfC gene in the wild-type S. griseus strain caused a severe reduction in the abundance of spores but no effect on the shape or size of the spores. The infrequent sporulation of the amfC disruptant was reversed by introduction of amfC on a plasmid. The amfC-defective phenotype was also restored by the orf1590 gene but not by the amfR-amfA-amfB gene cluster. Nucleotide sequences homologous to the amfC gene were distributed in all of 12 Streptomyces species tested, including Streptomyces coelicolor A3(2). The amfC homolog of S. coelicolor A3(2) was cloned and its nucleotide sequence was determined. The AmfC products of S. griseus and S. coelicolor A3(2) showed a 60% identity in their amino acid sequences. Introduction of the amfC gene of S. coelicolor A3(2) into strain HH1 induced aerial mycelium formation and sporulation, which suggests that both play the same functional role in morphogenesis in the strains.     

Cloning of Streptomyces griseus and Streptomyces lividans genes for glycerol dissimilation

Streptomyces lividans gyl DNA (for glycerol utilisation) was cloned by complementation of a Streptomyces coelicolor gyl mutant. Restriction mapping showed that the cloned DNA was highly homologous (perhaps 99%) to S. coelicolor gyl DNA. Using phage-mediated mutational cloning, an internal fragment of the S. coelicolor gyl operon was used to generate a gyl mutant of S. lividans, which subsequently served as recipient in the cloning of gyl DNA from S. griseus. A 7.5-kb SstI-generated fragment of S. griseus DNA was obtained which, as judged by analysis of restriction sites, was only perhaps 87% homologous with the S. coelicolor gyl operon. The cloned S. griseus DNA appears to contain intact gylA and gylB genes and probably also an upstream gene related to the putative gyl regulatory '0.9-kb' gene of S. coelicolor. Cloning of the fragment on a high-copy-number vector in S. lividans did not lead to high levels of the enzymes encoded by gylA and gylB. The S. griseus gylA and gylB genes were not detectably expressed in Escherichia coli glp mutants.     

Streptomycin-induced expression in Bacillus subtilis of YtnP, a lactonase-homologous protein that inhibits development and streptomycin production in Streptomyces griseus

Bacillus subtilis induces expression of the gene ytnP in the presence of the antimicrobial streptomycin, produced by the Gram-positive bacterium Streptomyces griseus. ytnP encodes a lactonase-homologous protein that is able to inhibit the signaling pathway required for the streptomycin production and development of aerial mycelium in S. griseus.     

The regulator of streptomycin gene expression, StrR, of Streptomyces griseus is a DNA binding activator protein with multiple recognition sites

In Streptomyces griseus the expression of at least one streptomycin biosynthetic gene, strB1 , is dependent on the pathway-specific activator protein StrR. We show here that StrR is a DNA-binding protein which specifically interacts with the strB1 promoter fragment. Footprinting experiments demonstrate that the StrR protein binds to an inverted repeat located upstream of the strB1 promoter. Further StrR-binding sites having the consensus sequence GTTCGActG(N)₁₁CagTcGAAc were identified in the str—sts gene clusters of S. griseus and Streptomyces glaucescens by sequence comparison, gel retardation, and footprinting studies. The genetic and biochemical evidence strongly supports the model of the StrR protein activating the expression of streptomycin biosynthetic genes by interacting with multiple binding sites within the str—sts gene clusters of S. griseus and S. glaucescens .     

Involvement of two A-factor receptor homologues in Streptomyces coelicolor A3(2) in the regulation of secondary metabolism and morphogenesis

Nucleotide sequences homologous to arpA encoding the A-factor receptor protein (ArpA) of Streptomyces griseus are distributed in a wide variety of streptomycetes. Two genes, cprA and cprB , each encoding an ArpA-like protein were found and cloned from Streptomyces coelicolor A3(2). CprA and CprB shared 90.7% identity in amino acid sequence and both showed about 35% identity to ArpA. Disruption of cprA by use of an M13 phage-derived single-stranded vector resulted in severe reduction of actinorhodin and undecylprodigiosin production. In addition, the timing of sporulation in the cprA disruptants was delayed by 1 day. The cprA gene thus appeared to act as a positive regulator or an accelerator for secondary metabolite formation and sporulation. Consistent with this idea, introduction of cprA on a low-copy-number plasmid into the parental strain led to overproduction of these secondary metabolites and accelerated the timing of sporulation. On the other hand, cprB disruption resulted in precocious and overproduction of actinorhodin. However, almost no effect on undecylprodigiosin was detected in the cprB disruptants. Sporulation of the cprB disruptant began 1 day earlier than the parental strain. The cprB gene thus behaved as a negative regulator on actinorhodin production and sporulation. Consistent with this, extra copies of cprB in the parental strain caused reduced production of actinorhodin and delay in sporulation. It is thus concluded that both cprA and cprB play regulatory roles in both secondary metabolism and morphogenesis in S. coelicolor A3(2), just as the arpA /A-factor system in Streptomyces griseus .     

Cloning of a pleiotropic gene that positively controls biosynthesis of A-factor, actinorhodin, and prodigiosin in Streptomyces coelicolor A3(2) and Streptomyces lividans.

A-factor (2S-isocapryloyl-3S-hydroxymethyl-gamma-butyrolactone), an autoregulating factor originally found in Streptomyces griseus, is involved in streptomycin biosynthesis and cell differentiation in this organism. A-factor production is widely distributed among actinomycetes, including Streptomyces coelicolor A3(2) and Streptomyces lividans. A chromosomal pleiotropic regulatory gene of S. coelicolor A3(2) controlling biosynthesis of A-factor and red pigments was cloned with a spontaneous A-factor-deficient strain of S. lividans HH21 and plasmid pIJ41 as a host-vector system. The restriction endonuclease KpnI-digested chromosomal fragments were ligated into the plasmid vector and introduced by transformation into the protoplasts of strain HH21. Three red transformants thus selected were found to produce A-factor and to carry a plasmid with the same molecular weight, and a 6.4-megadalton fragment was inserted in the KpnI site of pIJ41. By restriction endonuclease mapping and subcloning, a restriction fragment (1.2 megadaltons, approximately 2,000 base pairs) bearing the gene which causes concomitant production of A-factor and red pigments was determined. The red pigments were identified by thin-layer chromatography and spectroscopy to be actinorhodin and prodigiosin, both of which are the antibiotics produced by S. coelicolor A3(2). The cloned fragment was introduced into the A-factor-negative mutants (afs) of S. coelicolor A3(2) by using pIJ702 as the vector, where it complemented one of these mutations, afsB, characterized by simultaneous loss of A-factor and red pigment production. We conclude that the cloned gene pleiotropically and positively controls the biosynthesis of A-factor, actinorhodin, and prodigiosin.     

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.