A method for the identification of promoters recognized by RNA polymerase containing a particular sigma factor: cloning of a developmentally regulated promoter and corresponding gene directed by the Streptomyces aureofaciens sigma factor RpoZ

We have developed a method for the identification of promoters recognized by a particular sigma factor of RNA polymerase, based on a two-compatible plasmid system in Escherichia coli (Ec). Using the method, a DNA fragment containing the promoter, P_REN40, recognized by sporulation-specific Streptomyces aureofaciens (Sa) sigma factor RpoZ, was cloned. High-resolution S1 nuclease mapping using RNA prepared from Ec, and Sa from various developmental stages has shown a high degree of similarity of P_REN40 to consensus sequence of flagellar and chemotaxis promoters. The promoter was induced at the time of aerial mycelium formation, and was off in the Sa strain with the rpoZ-disrupted gene. A promoter-bearing DNA fragment was inserted into the promoter-probe plasmid pARC1 to give expression patterns consistent with the results of direct RNA analysis. The region downstream of the promoter was cloned in Sa. Sequence analysis revealed an open reading frame (ORF) of 283 amino acids (M_r 30 006), encoding a highly basic (pI 12.35) protein with high percentage of serine, threonine and alanine (41.8%).     

Formation of the ternary complex EF-Tu.GTP.Phe-tRNA in the translation system ofStreptomyces aureofaciens

The efficiency of formation of the ternary complex consisting of the elongation factor Tu and Phe-tRNA’s fromEscherichia coli andStreptomyces aureofaciens was tested to explain the lower activity of thein vitro poly(U) translation system fromS. aureofaciens. Both factors were shown to be functionally interchangeable in the ternary complex formation with Phe-tRNA from eitherE. coli orS. aureofaciens. However, the efficiency of binding ofS. aureofaciens Phe-tRNA to EF-Tu was much lower with both factors.     

A novel gene——samfR involved in early stage of Streptomyces ansochromogenes differentiation

A 4.6 kb DNA fragment was cloned from the DNA library ofStreptomyces ansochromogenes using a partial DNA fragment located in the downstream of promoter-P_TH4 as probe. The experiments revealed that this DNA fragment consists ofsaw D gene and a 1.4 kbPvu II fragment which can accelerate mycelium formation ofS. ansochromogenes. The nucleotide sequence of 1.4 kb DNA fragment was determined and analysed; the result indicated that the fragment contains one complete open reading frame (ORF) which encodes a protein with 213 amino acids, and this gene was designated assamfR. The deduced protein has 36% amino acid identities and 52% amino acid similarities in comparison with that encoded byhppR gene, which is involved in the regulation of catabolism for 3-(3-hydroxyphenyl) propionate (3HPP) inRhodococcus globerulus. The function ofsamfR gene was studied using strategy of gene disruption, and the resultingsamfR mutant failed to form aerial hyphae and spores, its development and differentiation stopped at the stage of substrate mycelium in contrast with wild type strain. The results showed that thesamfR gene is closely related toS. ansochromogenes differentiation. Project supported by the National Natural Science Foundation of China (Grant No. 39830010).     

Cloning of a two-component regulatory system probably involved in the regulation of chitinase inStreptomyces cœlicolor A3(2)

Using the method for the identification of promoters recognized by the sporulation specific σ factor (σ^F), we identified a positive 950 pbSau3Al DNA fragment inStreptomyces cœlicolor A3(2). High-resolution S1-nuclease mapping identified a potential promoter, P_F35, in theE. coli two-plasmid system similar to the consensus sequence ofBacillus subtilis promoters recognized by the general stress-response σ factor (σ^B). However, the putativesigF-dependent promoter, P_F35, was inactive inS. cœlicolor in the course of diffenentiation and it was located divergently in the promoter region directing expression of thechiC gene encoding chitinase. Sequence analysis of the region potentially governed by P_F35 revealed two translationally coupled genes encoding proteins similar to bacterial two-component regulatory systems, and with the highest similarity to the two-component systemchiS, chiR, regulating chitinase activity inStreptomyces thermoviolaceus. However, the genes had a divergent orientation with respect to the P_F35 promoter. Disruption of theS. cœlicolor chiR gene appeared to have no obvious effect on growth, morphology, differentiation, and production of pigmented antibiotic actinorhodin and undecylprodigiosin. Moreover, thechiR disruption did not affect the overall chitinase activity.     

Phylogenetic analysis of the rplA genes encoding ribosomal protein L1

Previously we have identified therplA gene encoding ribosomal protein L1 inStreptomyces aureofaciens. Sequence comparison of ribosomal protein L1 among several bacterial genera revealed a high level of conservation. Based on this conservation, these proteins were used as a phylogenetic tool to compare evolutionary relationships among eubacteria and archaebacteria. This phylogenetic analysis of L1 ribosomal proteins including theS. aureofaciens rplA gene product revealed, except similar bacterial groupings, some new evolutionary relationships.     

A novel gene—samfR involved in early stage ofStreptomyces ansochromogenes differentiation

A 4.6 kb DNA fragment was cloned from the DNA library ofStreptomyces ansochromogenes using a partial DNA fragment located in the downstream of promoter-P_TH4 as probe. The experiments revealed that this DNA fragment consists ofsaw D gene and a 1.4 kbPvu II fragment which can accelerate mycelium formation ofS. ansochromogenes. The nucleotide sequence of 1.4 kb DNA fragment was determined and analysed; the result indicated that the fragment contains one complete open reading frame (ORF) which encodes a protein with 213 amino acids, and this gene was designated assamfR. The deduced protein has 36% amino acid identities and 52% amino acid similarities in comparison with that encoded byhppR gene, which is involved in the regulation of catabolism for 3-(3-hydroxyphenyl) propionate (3HPP) inRhodococcus globerulus. The function ofsamfR gene was studied using strategy of gene disruption, and the resultingsamfR mutant failed to form aerial hyphae and spores, its development and differentiation stopped at the stage of substrate mycelium in contrast with wild type strain. The results showed that thesamfR gene is closely related toS. ansochromogenes differentiation.     

Variations in levels of cAMP, DNA and RNA in Streptomyces alboniger under conditions of aerial mycelium formation and repression

Abstract The relationship between endogenous levels of cyclic adenosine 3',5'-monophosphate (cAMP) and the formation of aerial mycelia was investigated in Streptomyces alboniger under conditions of aerial mycelium formation and repression. The relationship between cellular levels of DNA and RNA and aerial mycelium formation was also investigated. In contrast to cellular differentiation in other Streptomyces , neither variations in cAMP, DNA or RNA levels were found to be associated with the development of aerial mycelia in S. alboniger . The regulation of adenylate cyclase in S. alboniger , however, was found to differ from that of Escherichia coli and related organisms in that glucose raised, rather than lowered, endogenous cAMP levels.     

In vitro protein phosphorylation associated with cellular differentiation of Streptomyces griseus

In vitro phosphorylation reactions with crude cellular extracts revealed that phosphorylation of a 17-kDa protein is associated with the onset of aerial mycelium formation in solid culture (but not submerged spore formation in liquid culture) of Streptomyces griseus. The possible importance of the 17-kDa protein phosphorylation in cellular differentiation was further indicated by inducing aerial mycelium formation in the presence of decoyinine and in studies using certain developmental mutants (relC, afsA, and M-1). It is proposed that the 17-kDa protein may play a role in cellular differentiation of S. griseus via its phosphorylation. Received: 17 July 1997 / Accepted: 20 September 1997     

Sporulation-inducing factor inStreptomyces avermitilis

The excretion of a diffusible factor was detected in sporulating, avermectin-producing strain ofStreptomyces avermitilis. The factor induced the formation of aerial mycelium and spores by a nonsporulating mutant.     

Identification and characterization of an indigoidine-like gene for a blue pigment biosynthesis in <Emphasis Type="Italic">Streptomyces aureofaciens</Emphasis> CCM 3239

An incomplete oligoketide (PK; ‘polyketide’) gene cluster, aur1, responsible for the production of an angucycline-like antibiotic auricin was identified in Streptomyces aureofaciens CCM 3239. A region downstream of the aur1 was cloned and sequenced, revealing 28 new genes encoding putative protein products involved in deoxysugar biosynthesis and other putative PK-related biosynthetic functions. In addition, a gene, bpsA, encoding a protein similar to non-ribosomal peptide synthetases (NRPSs) was identified in this region. A deduced protein product of the gene showed the highest similarity to NRPSs IndC from Erwinia chrysanthemi and BpsA from Streptomyces lavendulae, both involved in the biosynthesis of a blue pigment indigoidine. S. aureofaciens CCM 3239 was found to produce an extracellular blue pigment with identical properties as indigoidine. A deletion mutant of bpsA in S. aureofaciens CCM 3239 failed to produce the blue pigment. In addition, the deletion of bpsA had a positive effect on auricin production. The results indicate the involvement of the bpsA gene in biosynthesis of the indigoidine blue pigment in S. aureofaciens CCM 3239.