A mutation at proline-115 in the A-factor receptor protein of Streptomyces griseus abolishes DNA-binding ability but not ligand-binding ability.

A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) and its specific receptor protein (ArpA) are required for streptomycin production and aerial mycelium formation in Streptomyces griseus. A mutant strain HO1 that produced streptomycin and formed aerial mycelium and spores was derived from an A-factor-deficient mutant, S. griseus HH1. The phenotypes of mutant HO1 were found to result from a single amino acid replacement of ArpA; the proline residue at position 115 in the wild-type ArpA was replaced by serine, yielding mutant ArpA (P115S). The mutant ArpA (P115S) was still able to form a homodimer and possessed A-factor-binding ability but lost the ability to bind DNA. The properties of P115S suggest that ArpA consists of two independently functional domains, one for A-factor binding and one for DNA binding, and that proline-115 plays an important role in DNA binding. This is in agreement with the idea that A-factor binding to the COOH-terminal domain of ArpA causes a subtle conformational change of the distal NH2-terminal DNA-binding domain, resulting in dissociation of ArpA from DNA.     

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.     

The A-factor regulatory cascade and cAMP in the regulation of physiological and morphological development in Streptomyces griseus

In the A-factor regulatory cascade leading to the onset of streptomycin biosynthesis and aerial mycelium formation in Streptomyces griseus, the A-factor receptor protein (ArpA) serves as a DNA-binding repressor and A-factor releases the repression by binding to ArpA and dissociating it from the DNA. Mutants defective in arpA therefore produce streptomycin and aerial hyphae in the absence of A-factor. A gene that inhibits streptomycin production and aerial hyphae formation in an arpA mutant was cloned on a high-copy-number plasmid and found to encode a eukaryotic-type adenylate cyclase (CyaA). Consistent with this, an exogenous supply of cAMP at high concentration almost abolished streptomycin production and aerial hyphae formation. On the other hand, cAMP at lower concentrations stimulated or accelerated these developmental processes. The effects of cAMP were detectable only in arpA mutants, and not in the wild-type strain; an exogenous supply of cAMP or cyaA disruption in the wild-type strain caused almost no effect on these phenotypes. Thus the effects of cAMP became apparent only in the arpA-defective background. cAMP at high concentrations inhibited stringent response factor ppGpp production, which is important for the onset of antibiotic biosynthesis. cAMP also influenced the timing of tyrosine phosphorylation of more than nine proteins. These findings show that a cAMP regulatory relay for physiological and morphological development functions in a concerted and interdependent way with other signal transduction pathways. Journal of Industrial Microbiology & Biotechnology (2001) 27, 177–182. Received 21 September 1999/ Accepted in revised form 14 September 2000     

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 .     

The aerial mycelium-defective phenotype of Streptomyces griseus resulting from A-factor deficiency is suppressed by a Ser/Thr kinase of S. coelicolor A3(2)

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     

DNA-binding activity of the A-factor receptor protein and its recognition DNA sequences

The A-factor receptor protein (ArpA) containing an α-helix-turn-α-helix DNA-binding consensus sequence at its N-terminal portion plays a key role in the regulation of secondary metabolism and cell differentiation in Streptomyces griseus . A binding site forming a palindrome 24 bp in length was initially recovered from a pool of random-sequence oligonucleotides by rounds of a binding/immunoprecipitation/amplification procedure with histidine-tagged ArpA and anti-ArpA antibody. By means of further binding/gel retardation/amplification experiments on the basis of the recovered sequence, a 22 bp palindromic binding site with the sequence 5'-GG(T/C)CGGT(A/T)(T/C)-G(T/G)-3' as one half of the palindrome was deduced as a consensus sequence recognized and bound by ArpA. ArpA did not bind to the binding site in the presence of its ligand, A-factor. In addition, exogenous addition of A-factor to the ArpA–DNA complex induced immediate release of ArpA from the DNA. All of these data are consistent with the idea, obtained from previous genetic studies, that ArpA acts as a repressor-type regulator for secondary metabolism and cellular differentiation by preventing the expression of a certain key gene(s) during the early growth phase. A-factor, produced in a growth-dependent manner, releases ArpA from the DNA, thus switching on the expression of the key gene(s), leading to the onset of secondary metabolism and aerial mycelium formation.     

The A-factor-binding protein of Streptomyces griseus negatively controls streptomycin production and sporulation.

A-factor, 2-(6'-methylheptanoyl)-3R-hydroxymethyl-4-butanolide, is an autoregulator essential for streptomycin production and sporulation in Streptomyces griseus. S. griseus 2247 that requires no A-factor for streptomycin production or sporulation was found to have a defect in the A-factor-binding protein. This observation implied that the A-factor-binding protein in the absence of A-factor repressed the expression of both phenotypes in the wild-type strain. Screening among mutagenized S. griseus colonies for strains producing streptomycin and sporulating in the absence of A-factor yielded three mutants that were also deficient in the A-factor-binding protein. Reversal of the defect in the A-factor-binding protein of these mutants led to the simultaneous loss of streptomycin production and sporulation. These data suggested that the A-factor-binding protein played a role in repressing both streptomycin production and sporulation and that the binding of A-factor to the protein released its repression. Mutants deficient in the A-factor-binding protein began to produce streptomycin and sporulate at an earlier stage of growth than did the wild-type strain. These mutants produced approximately 10 times more streptomycin than did the parental strain. These findings are consistent with the idea that the intracellular concentration of A-factor determines the timing of derepression of the gene(s) whose expression is repressed by the A-factor-binding protein.     

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.     

Detection and properties of A-factor-binding protein from Streptomyces griseus.

The optically active form of tritium-labeled A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone), a pleiotropic autoregulator responsible for streptomycin production, streptomycin resistance, and sporulation in Streptomyces griseus, was chemically synthesized. By using the radioactive A-factor, a binding protein for A-factor was detected in the cytoplasmic fraction of this organism. The binding protein had an apparent molecular weight of approximately 26,000, as determined by gel filtration. Scatchard analysis suggested that A-factor bound the protein in the molar ratio of 1:1 with a binding constant, Kd, of 0.7 nM. The number of the binding protein was roughly estimated to be 37 per genome. The "inducing material" virginiae butanolide C (VB-C), which has a structure very similar to that of A-factor and is essential for virginiamycin production in Streptomyces virginiae, did not inhibit binding. In addition, no protein capable of specifically binding 3H-labeled VB-C was found in S. griseus. Together with the observation that VB-C had almost no biological activity on the restoration of streptomycin production or sporulation in an A-factor-deficient mutant of S. griseus, these results indicated that the binding protein had a strict ligand specificity. Examination for an A-factor-binding protein in Streptomyces coelicolor A3(2) and Streptomyces lividans showed the absence of any specifically binding protein.     

Site-directed mutagenesis of the A-factor receptor protein:: Val-41 important for DNA-binding and Trp-119 important for ligand-binding

The A-factor receptor protein (ArpA) plays a key role in the regulation of secondary metabolism and cellular differentiation in Streptomyces griseus. ArpA binds the target DNA site forming a 22 bp palindrome in the absence of A-factor, and exogenous addition of A-factor to the ArpA–DNA complex immediately releases ArpA from the DNA. An amino acid (aa) replacement at Val-41 to Ala in an α-helix–turn–α-helix (HTH) motif at the N-terminal portion of ArpA abolished DNA-binding activity but not A-factor-binding activity, suggesting the involvement of this HTH in DNA-binding. On the other hand, an aa replacement at Trp-119 to Ala generated a mutant ArpA that was unable to bind A-factor, thus resulting in an A-factor-insensitive mutant that bound normally to its target DNA in both the presence and absence of A-factor. These data suggest that ArpA consisting of two functional domains, one for HTH-type DNA-binding at the N-terminal portion and one for A-factor-binding at the C-terminal portion, is a member of the LacI family. Consistent with this, two ArpA homologues, CprA and CprB, from Streptomyces coelicolor A3(2), each of which contains a very similar aa sequence of the HTH to that of ArpA, also recognized and bound the same DNA target. However, neither CprA nor CprB recognized A-factor, probably due to much less similarity in the C-terminal domains.