Possible involvement of the sco2127 gene product in glucose repression of actinorhodin production in Streptomyces coelicolor.

Streptomyces coelicolor mutants resistant to 2-deoxyglucose are insensitive to carbon catabolite repression (CCR). Total reversion to CCR sensitivity is observed by mutant complementation with a DNA region harboring both glucose kinase glkA gene and the sco2127 gene. The sco2127 is located upstream of glkA and encodes a putative protein of 20.1 kDa. In S. coelicolor, actinorhodin production is subject to glucose repression. To explore the possible involvement of both SCO2127 and glucose kinase (Glk) in the glucose sensitivity of actinorhodin production, this effect was evaluated in a wild-type S. coelicolor A3(2) M145 strain and a sco2127 null mutant (Δsco2127) derived from this wild-type strain. In comparison with strain M145, actinorhodin production by the mutant was insensitive to glucose repression. Under repressive conditions, only minor differences were observed in glucose utilization and Glk production between these strains. SCO2127 was detected mainly during the first 36 h of fermentation, just before the onset of antibiotic production, and its synthesis was not related to a particular carbon source. The glucose sensitivity of antibiotic production was restored to wild-type phenotype by transformation with an integrative plasmid containing sco2127. Our results support the hypothesis that SCO2127 is a negative regulator of actinorhodin production and suggest that the effect is independent of Glk.

Interaction of SCO2127 with BldKB and its possible connection to carbon catabolite regulation of morphological differentiation in Streptomyces coelicolor.

In Streptomyces coelicolor, the sco2127 gene is located upstream of the gene encoding for glucose kinase. This region restores sensitivity to carbon catabolite repression (CCR) of Streptomyces peucetius var. caesius mutants, resistant to 2-deoxyglucose (Dog(R)). In order to search for the possible mechanisms behind this effect, sco2127 was overexpressed and purified for protein-protein interaction studies. SCO2127 was detected during the late growth phase of S. coelicolor grown in a complex media supplemented with 100 mM glucose. Pull-down assays using crude extracts from S. coelicolor grown in the same media, followed by far-western blotting, allowed detection of two proteins bound to SCO2127. The proteins were identified by MALDI-TOF mass spectrometry as SCO5113 and SCO2582. SCO5113 (BldKB) is a lipoprotein ABC-type permease (∼66 kDa) involved in mycelium differentiation by allowing the transport of the morphogenic oligopeptide Bld261. SCO2582, is a putative membrane metalloendopeptidase (∼44 kDa) of unknown function. In agreement with the possible role of SCO2127 in mycelium differentiation, delayed aerial mycelium septation and sporulation was observed when S. coelicolor A3(2) was grown in the presence of elevated glucose concentrations (100 mM), an effect not seen in a Δ-sco2127 mutant derived from it. We speculate that SCO2127 might represent a key factor in CCR of mycelium differentiation by interacting with BldKB.

Carbon source regulation of antibiotic production.

Antibiotics are low-molecular-mass products of secondary metabolism, nonessential for the growth of producing organisms, but very important for human health. They have unusual structures and are most often formed during the late growth phase of the producing microorganisms. Their production arises from intracellular intermediates, which are condensed into more complex structures through defined biochemical pathways. Their synthesis can be influenced by manipulating the type and concentration of nutrients formulating the culture media. Among them, the effect of the carbon source has been the subject of continuous studies for both industry and research groups. Glucose and other carbohydrates have been reported to interfere with antibiotic synthesis and this effect depends on the rapid utilization of the preferred carbon source. Different mechanisms have been described in bacteria and fungi to explain the negative effects of carbon catabolites on antibiotic production. They show important differences depending on the microbe being considered. Their understanding and manipulation have been useful for both perfecting fermentation conditions to produce anti-infectives and for strain improvement. To improve the production of antibiotics, carbon source repression can be decreased or abolished by mutations resulting in antimetabolite resistance. Enzymes reported as regulated by the carbon source have been used as targets for strain improvement. During the last few years, important advances have been reported elucidating the essential aspects of carbon source regulation on antibiotic production at biochemical and molecular levels. The aim of this review is to describe these advances, giving special emphasis to those reported for the genus Streptomyces.

Production of microbial secondary metabolites: regulation by the carbon source.

Microbial secondary metabolites are low molecular mass products, not essential for growth of the producing cultures, but very important for human health. They include antibiotics, antitumor agents, cholesterol-lowering drugs, and others. They have unusual structures and are usually formed during the late growth phase of the producing microorganisms. Its synthesis can be influenced greatly by manipulating the type and concentration of the nutrients formulating the culture media. Among these nutrients, the effect of the carbon sources has been the subject of continuous studies for both, industry and research groups. Different mechanisms have been described in bacteria and fungi to explain the negative carbon catabolite effects on secondary metabolite production. Their knowledge and manipulation have been useful either for setting fermentation conditions or for strain improvement. During the last years, important advances have been reported on these mechanisms at the biochemical and molecular levels. The aim of the present review is to describe these advances, giving special emphasis to those reported for the genus Streptomyces.

Cloning and expression of the sco2127 gene from Streptomyces coelicolor M145.

It is known that Streptomyces peucetius var. caesius mutants resistant to 2-deoxyglucose (Dog(R)) exhibit glucose transport deficiency, low glucose kinase (Glk) activity and insensitivity to carbon catabolite repression (CCR). This phenotype can be pleiotropically complemented by a 576-bp gene encoding SCO2127 from Streptomyces coelicolor, suggesting the participation of this protein in the CCR process. In the present work, the sco2127 region was subcloned into pQE30 and its transcription product (SCO2127-His(6)) overexpressed. This procedure allowed purification of SCO2127 (with a Ni-sepharose resin) and production of polyclonal antibodies. In western blot assays, the antibodies gave a positive reaction against protein extracts from both S. coelicolor and S. peucetius var. caesius, appearing as a single band of 34 kDa. No protein was detected using extracts from a S. coelicolor mutant lacking the sco2127 gene (Deltasco2127). In agreement with its possible involvement in the CCR process, SCO2127 was detected during the logarithmic growth phase of S. coelicolor grown in minimal medium supplemented with 50 and 100 mM glucose. In addition, when 50 mM glucose was utilized, SCO2127 and residual glucose concentration simultaneously decreased at later stages of the microbial growth.