Expression of the melC operon in several Streptomyces strains is positively regulated by AdpA, an AraC family transcriptional regulator involved in morphological development in Streptomyces coelicolor

Abstract

Dark brown haloes of melanin around colonies are an easily visualized phenotype displayed by many Streptomyces strains harboring plasmid pIJ702 carrying the melC operon of Streptomyces antibioticus IMRU3270. Spontaneous melanin-negative mutants of pIJ702 occur with a frequency of ca. 1%, and often mutation occurs in the melC operon, which removes the BglII site as part of an inverted repeat. Other melanin-negative mutations seem to occur spontaneously in Streptomyces lividans, resulting in white colonies from which intact, melanin-producing pIJ702 can be isolated by introduction into a new host. S. lividans ZX66 was found to be such a mutant and to have a secondary mutation influencing expression of the melC operon on the chromosome. A 3.3-kb DNA fragment was isolated from its progenitor strain, JT46, and a gene able to restore melC operon expression was found to encode a member of an AraC family of transcriptional regulators, which was equivalent to AdpA(c) in Streptomyces coelicolor and therefore was designated AdpA(l). Lack of melC operon expression was correlated with a single A-to-C transversion, which altered a single key amino acid residue from Thr to Pro. The transcription of the melC operon was found to be greatly reduced in the adpA mutant background. The counterpart gene (adpA(a)) in the S. antibioticus strain in which the melC operon carried on pIJ702 originated was also isolated and was found to have an identical regulatory role. Thus, we concluded that the melC operon is under general direct positive control by AdpA family proteins, perhaps at the transcriptional level and certainly at the translational level via bldA, in Streptomyces.

FIG. 1.

melC operon is expressed in JT46 (right) but not in ZX66 (left). Both strains were transformed with pIJ702 carrying melC1 and melC2 and were grown on MMT containing thiostrepton.

FIG. 2.

Localization of a gene activating melC expression. The DNA sequence and genetic organization of the original 3,336-bp insert isolated from S. lividans in pJTU1452 are almost identical to the DNA sequence and genetic organization of the corresponding region carrying three complete ORFs (SCO2790 to SCO2792) in S. coelicolor, which is shown at the top. pJTU1452 derivatives are shown below pJTU1452, and their abilities to activate melC operon expression (black colonies on plates) are shown on the left.

FIG. 3.

Overall characteristics of the adpA_l gene and alignment of adpA_c (from S. coelicolor M145), adpA_l (from S. lividans ZX64), and adpA_l* (from S. lividans ZX66) and the protein sequences encoded by these genes. Nucleotides are numbered from the ATG start codon. L is the rare leucine (TTA) codon. HTH_AraC is a region with a helix-turn-helix motif for the typical AraC family of bacterial regulatory proteins, and the shaded region (with two alternative sequences) has the potential to form a stem-loop structure. Only the regions with differences at the nucleotide (nt) and amino acid (aa) levels between alleles are shown, and mutant positions are indicated by triangles.

FIG. 4.

(A) Lack of mel operon expression in adpA mutants of S. coelicolor ZD2, S. lividans ZD3, and S. antibioticus ZD4 compared with their progenitors, M145 (left), JT46 (middle), and IMRU3720 (right), respectively. (B) Dosage effect of adpA. The top half of each plate contained wild-type S. antibioticus IMRU3720, S. lividans JT46 and ZX64 carrying pIJ702, or S. lividans ZX64 and ZX66 carrying pIJ702, while the bottom half of each plate contained strains corresponding to the strains in the top half but with an additionally integrated copy of adpA_l (carried on pJTU1457) except that the two patches for the same strain (ZX64/pIJ702::pJTU1457) are shown in the bottom half of the left plate. For the strains at the bottom there was both increased production of black pigment and enhanced sporulation compared with the strains at the top.

FIG. 5.

(A and B) Gel mobility shift assay (B) for detection of the binding of AdpA_l to a γ-³²P-labeled 295-bp DNA fragment (A) covering the promoter region of the melC operon. The solid arrowhead in panel B indicates the position when 0.01 μg (lane 2) or 0.02 μg (lane 3) of total proteins isolated from E. coli BL21(DE3) carrying pJTU1464 was added to the radioactively labeled probe fragment (open arrowhead). Addition of 0.02 μg of total proteins isolated from E. coli BL21(DE3) carrying pET15b to the same probe was used as a negative control (lane 1). (C) Alignment of the three regions upstream of the melC operon (A) with the consensus AdpA-binding sequence (51) shown at the top (5′-TGGCSNGWWY-3′, where S is G or C, W is A or T, Y is T or C, and N is any nucleotide).