Phosphorylated AbsA2 negatively regulates antibiotic production in Streptomyces coelicolor through interactions with pathway-specific regulatory gene promoters

Abstract

The AbsA two-component signal transduction system, comprised of the sensor kinase AbsA1 and the response regulator AbsA2, acts as a negative regulator of antibiotic production in Streptomyces coelicolor, for which the phosphorylated form of AbsA2 (AbsA2 approximately P) is the agent of repression. In this study, we used chromatin immunoprecipitation to show that AbsA2 binds the promoter regions of actII-ORF4, cdaR, and redZ, which encode pathway-specific activators for actinorhodin, calcium-dependent antibiotic, and undecylprodigiosin, respectively. We confirm that these interactions also occur in vitro and that the binding of AbsA2 to each gene is enhanced by phosphorylation. Induced expression of actII-ORF4 and redZ in the hyperrepressive absA1 mutant (C542) brought about pathway-specific restoration of actinorhodin and undecylprodigiosin production, respectively. Our results suggest that AbsA2 approximately P interacts with as many as four sites in the region that includes the actII-ORF4 promoter. These data suggest that AbsA2 approximately P inhibits antibiotic production by directly interfering with the expression of pathway-specific regulators of antibiotic biosynthetic gene clusters.

FIG. 1.

CDA bioassay (A) and Western analysis (B) indicate when the AbsA system may be negatively regulating antibiotic production. (A) CDA bioassay to determine when CDA is produced in S. coelicolor J1501 grown in liquid TSB medium. Seventy-five microliters of culture supernatant from cells grown for 12, 24, 36, 48, 60, and 72 h was spotted on discs placed on a lawn of Staphylococcus aureus on ONA with or without calcium nitrate and incubated first for 3 h at 4°C and then overnight at 37°C. Zones of inhibition were first observed at 36 h and indicate the presence of CDA. (B) Western analysis of membrane (M) and cytoplasmic (C) fractions of S. coelicolor grown at 30°C for 12, 24, 36, and 48 h on cellophane discs overlaid on solid R2YE medium. Anti-AbsA1 antibodies were used to detect the presence of AbsA1 in the membrane fraction of cells grown for 24 h.

FIG. 2.

In vivo identification of AbsA2 target promoters by ChIP. Anti-AbsA2 antibodies were used to immunoprecipitate AbsA2/DNA complexes from cells treated with formaldehyde. PCR was performed with primers flanking putative target promoters (absA primers 97/98, actII-ORF4 primers 85/86, cdaR primers 109/110, and redZ primers 121/122). DNA used for PCR was total DNA prior to immunoprecipitation (lanes 1) and immunoprecipitated DNA (lanes 2). PCR using primers that flank the absA promoter is representative of all primer sets that did not result in amplification of the immunoprecipitated DNA. Amplification of the actII-ORF4, cdaR, and redZ promoters from the immunoprecipitated DNA indicated that these three promoters are targets of AbsA2.

FIG. 3.

EMSAs with purified MBP-AbsA2 recombinant protein with each of the three target promoters identified by ChIP (cdaR primers 109/110, redZ primers 121/122, and actII-ORF4 primers 85/86). ³²P-labeled probe (0.89 ng) was incubated at 30°C for 15 min with various concentrations of protein that had been preincubated at 30°C for 30 min with or without 100 mM PA. Reaction mixtures were resolved on a native 1.5% glycerol-containing, 8% polyacrylamide gel with 1× TBE plus 1.5% glycerol as the running buffer. Lane 1 contains probe alone. Lanes 2 and 8 contain 0.527 μM of MBP-AbsA2. Lanes 3 and 9 contain 0.675 μM of MBP-AbsA2. Lanes 4 and 10 contain 0.810 μM of MBP-AbsA2. Lanes 5 and 11 contain 0.972 μM of MBP-AbsA2. Lanes 6 and 12 contain 1.166 μM of MBP-AbsA2. Lanes 7 and 13 contain 1.4 μM of MBP-AbsA2.

FIG. 4.

Identification of the region within the actII-ORF4 promoter that is required for AbsA2 binding. (A) Schematic representation of the actII-ORF4 promoter region. (B) EMSAs with various amounts of MBP-AbsA2 protein with small overlapping fragments shown in panel A. ³²P-labeled probe (0.89 ng) was incubated at 30°C for 15 min with various concentrations of protein that had been preincubated at 30°C for 30 min with or without 100 mM PA. Lanes 1 contain probe alone (0.89 ng of radiolabeled DNA). Lanes 2 and 4 contain 0.675 μM of MBP-AbsA2. Lanes 3 and 5 contain 1.4 μM of MBP-AbsA2.

FIG. 5.

Bypass of the absA1 mutant by actII-ORF4 and redZ. Genes were cloned into pIJ6902 under the control of the strong thiostrepton-inducible promoter. Induction of actII-ORF4 in the antibiotic-deficient strain C542 restored actinorhodin production, whereas induction of redZ restored undecylprodigiosin production. Strains were grown on R2YE containing 50 μg/ml thiostrepton for 7 days at 30°C.

FIG. 6.

Schematic representation of the AbsA two-component signal transduction system in S. coelicolor. AbsA2∼P represses transcription of the cdaR, actII-ORF4, and redZ promoters, thereby preventing the production of CDA, actinorhodin, and undecylprodigiosin, respectively.