SCO6992, a Protein with β-Glucuronidase Activity, Complements a Mutation at the absR Locus and Promotes Antibiotic Biosynthesis in Streptomyces coelicolor

Abstract

IntroductionStreptomycetes is a group of gram-positive and filamentous soil bacteria with a unique and complex differentiation process that includes simultaneous morphological (sporulation) and physiological (secondary metabolism) differentiation. Many scientific findings have elucidated that the two differentiation processes are genetically closely related and thus controlled concurrently by many regulatory factors. Generally, genes for secondary metabolite formation and morphological differentiation are clustered in the chromosome, and their expression is precisely controlled by a pathway-specific regulatory gene(s) within the cluster as well as by global regulatory genes [1][2][3].Several classes of mutants that simultaneously block the synthesis of more than one antibiotic constitute a good genetic evidence of a global regulatory system. Streptomyces coelicolor has been known to produce four genetically and structurally distinct antibiotics: the pigmented antibiotics actinorhodin and undecylprodigiosin and the unpigmented antibiotics methylenomycin and CDA (Ca 2+ -dependent antibiotic) [4]. According to an S. coelicolor genome sequence analysis, 18 gene clusters are expected to code exclusively for enzymes characteristic of its secondary metabolism [5]. abs (antibiotic synthesis deficient) mutants, for example, affect global regulatory genes involved only in physiological differentiation. Among them, absmutations absA -, absB -, and absCwere found to lack all four antibiotics, but retained normal sporulation. The molecular nature of the absA1/A2 gene products that can restore the bacterial ability for biosynthesizing all four antibiotics was identified as a two-component regulatory system, composed by the sensor histidine kinase AbsA1 and the aspartate kinase response regulator AbsA2 [4]. The phosphorylated AbsA2 is known to act as a global negative regulator of antibiotic biosynthesis in S.Streptomyces coelicolor is a filamentous soil bacterium producing several kinds of antibiotics. S. coelicolor abs8752 is an abs (antibiotic synthesis deficient)-type mutation at the absR locus; it is characterized by an incapacity to produce any of the four antibiotics synthesized by its parental strain J1501. A chromosomal DNA fragment from S. coelicolor J1501, capable of complementing the absphenotype of the abs8752 mutant, was cloned and analyzed. DNA sequencing revealed that two complete ORFs (SCO6992 and SCO6993) were present in opposite directions in the clone. Introduction of SCO6992 in the mutant strain resulted in a remarkable increase in the production of two pigmented antibiotics, actinorhodin and undecylprodigiosin, in S. coelicolor J1501 and abs8752. However, introduction of SCO6993 did not show any significant difference compared to the control, suggesting that SCO6992 is primarily involved in stimulating the biosynthesis of antibiotics in S. coelicolor. In silico analysis of SCO6992 (359 aa, 39.5 kDa) revealed that sequences homologous to SCO6992 were all annotated as hypothetical proteins. Although a metall...

Comparative Proteomic Analysis of Transcriptional and Regulatory Proteins Abundances in S. lividans and S. coelicolor Suggests a Link between Various Stresses and Antibiotic Production

“…Interestingly, the sub-units of the complex I of the respiratory chain were far more abundant in SC than in SL , in both Pi conditions (Figure 5 of [ 9 ]). SCO6992/AbsR1, a member of the AbrC3 regulon [ 76 , 78 ] that has a positive impact on RED and ACT biosynthesis [ 104 ]. The RR CutR/SCO5862 of the TCS CutS/CutR, thought to be involved in the regulation of copper homeostasis [ 105 ], has a negative impact on RED and ACT biosynthesis [ 106 ].…”

“…SCO6992/AbsR1, a member of the AbrC3 regulon [ 76 , 78 ] that has a positive impact on RED and ACT biosynthesis [ 104 ].…”

LuxR-Type SCO6993 Negatively Regulates Antibiotic Production at the Transcriptional Stage by Binding to Promoters of Pathway-Specific Regulatory Genes in Streptomyces coelicolor

“…Streptomyces strains were routinely maintained on minimal medium or R2YE medium [ 17 ] at 30°C. For cultivation of S. coelicolor J1501, the medium was supplemented with histidine and uracil at final concentrations of 50 μg/ml and 7.5 μg/ml, respectively [ 12 ]. Thiostrepton (25 μg/ml), apramycin (50 μg/ml), and kanamycin (50 μg/ml) were added to the media, when necessary.…”

“…For actinorhodin and undecylprodigiosin quantitation in broth culture, an exponential culture (5 ml) of S. coelicolor was transferred to 100 ml of R2YE broth in a 500 ml baffled flask and incubated at 30°C on a reciprocal shaker. Portions (5.0 ml) of the culture broth were taken out at intervals and the concentrations of actinorhodin and undecylprodigiosin were measured at 633 nm (A 633 ) and 530 nm (A 530 ), respectively, as previously described [ 12 ].…”

“…Recently, we reported that the introduction of SCO6992 gene encoding a novel β-glucuronidase dramatically increased the biosynthesis of undecylprodigiosin and actinorhodin in abs R - mutant of S. coelicolor J1501. This antibiotic biosynthesis-promoting effect of SCO6992 was abolished by the presence of SCO6993 , which acts in opposition to SCO6992 [ 12 ]. Based on this, SCO6993 is expected to be a regulatory gene that suppresses antibiotic biosynthesis.…”