Characterization of the pathway-specific positive transcriptional regulator for actinorhodin biosynthesis in Streptomyces coelicolor A3(2) as a DNA-binding protein

Abstract

The ActII-ORF4 protein has been characterized as a DNA-binding protein that positively regulates the transcription of the actinorhodin biosynthetic genes. The target regions for the ActII-ORF4 protein were located within the act cluster. These regions, at high copy number, generate a nonproducer strain by in vivo titration of the regulator. The mutant phenotype could be made to revert with extra copies of the wild-type actII-ORF4 gene but not with the actII-ORF4-177 mutant. His-tagged recombinant wild-type ActII-ORF4 and mutant ActII-ORF4-177 proteins were purified from Escherichia coli cultures; both showed specific DNA-binding activity for the actVI-ORF1-ORFA and actIII-actI intergenic regions. DNase I footprinting assays clearly located the DNA-binding sites within the -35 regions of the corresponding promoters, showing the consensus sequence 5'-TCGAG-3'. Although both gene products (wild-type and mutant ActII-ORF4) showed DNA-binding activity, only the wild-type gene was capable of activating transcription of the act genes; thus, two basic functions can be differentiated within the regulatory protein: a specific DNA-binding activity and a transcriptional activation of the act biosynthetic genes.

FIG. 1

High-resolution S1 mapping within the actVI-ORF1–ORFA intergenic region in S. coelicolor PM218. Transcriptional analysis of the actVI-ORF1 (A) and actVI-ORFA (B) genes is shown. The RNAs were extracted from the S. coelicolor strains: J1501 (lane 1), PM218 (lane 2), PM218 carrying pPAS3 as a control (lane 3), PM218 carrying the wild-type actII-ORF4 gene in pPAS4 (lane 4), PM218 carrying the actII-ORF4-177 gene in pPAS1 (lane 5), and E. coli tRNA as a control (lane 6). The probes, indicated by double-headed arrows, were the 846-bp KpnI-BssHII fragment (A) and the 474-bp CelII-SmaI fragment (B). The fragments were labeled at the positions indicated by the asterisks. Protected fragments of the expected size are indicated by arrows. As size marker, the HinfI-digested pBR329 was used.

FIG. 2

Transcriptional analysis of some act genes in the recombinant strains carrying the actVI-ORF1–ORFA region in high copy number. Transcription was determined for the actI-ORF1 (A), actVI-ORFA (B), and actVI-ORF1 (C) genes. The RNAs were extracted from the following S. coelicolor strains: J1501 carrying pIJ486 as a control (lane 1); J1501 containing pMF1123, the actVI-ORF1–ORFA intergenic region, in high copy number (lane 2); the same strain carrying, in addition to pMF1123, the wild-type actII-ORF4 gene (lane 3) or the actII-ORF4-177 gene (lane 4) respectively, in the compatible plasmids pPAS4 or pPAS1; J1501 (lane 5); and E. coli tRNA as a control (lane 6). The probes and their respective sizes are indicated accordingly. The labeled positions are indicated by asterisks. The respective S1-protected fragments are indicated by arrows. The size markers are as in Fig. 1.

FIG. 3

Transcriptional analysis of some act genes in the recombinant strains carrying the actIII-actI region in high copy number. Transcription was determined for the actI-ORF1 (A), actVI-ORFA (B), and actVI-ORF1 (C) genes. The RNAs were extracted from the following S. coelicolor strains: J1501 carrying pIJ486 as a control (lane 1); J1501 carrying pMF1135, the actIII-actI intergenic region, in high copy number (lane 2); the same strain carrying, in addition to pMF1135, the wild type actII-ORF4 gene (pPAS4) (lane 3) or the actII-ORF4-177 gene (pPAS1) (lane 4); J1501 (lane 5); and E. coli tRNA (lane 6) as a control. The probes were as in Fig. 2 and were labeled at the restriction site indicated by asterisks. The sizes of the protected fragments are indicated. The size markers are as in Fig. 1.

FIG. 4

Gel mobility shift analysis of the actVI-ORF1–ORFA intergenic region with crude extracts from Streptomyces. Lanes: 1, probe actVI-ORF1–ORFA intergenic region (329 bp); 2 and 4, cell extract and 60% ammonium sulfate precipitated from S. coelicolor PM218(pIJ702), respectively; 3 and 5, cell extract and 60% ammonium sulfate fraction from S. coelicolor PM218(pMF1125), respectively.

FIG. 5

Purification pattern of the wild-type His-tagged ActII-ORF4 protein. Lanes: 1 and 2, total-cell lysate of E. coli BL21(DE3)pLysS/pET-19b not induced and induced, respectively; 3 and 4, total-cell lysate of E. coli BL21(DE3)pLysS/pPAC10 not induced and induced, respectively; 5 and 6, soluble and insoluble fractions, respectively, from a cell extract of E. coli BL21(DE3)pLysS/pPAC10; 7, fraction from a Ni²⁺ column eluted with 1 M imidazole; 8, fraction eluted with 0.1 M imidazole plus 5 mM ATP; 9, purified His-tagged ActII-ORF4 protein. Molecular masses are indicated in kilodaltons.

FIG. 6

DNA-binding assays. (A) Gel mobility shift analysis of the actVI-ORF1–ORFA intergenic region and supershift assays. Lanes: 1, probe, F139 fragment (154 bp); 2, wild-type His-tagged ActII-ORF4 protein (0.7 μg); 3, wild-type His-tagged ActII-ORF4 protein (0.7 μg) plus anti-His tag antibody; 4, His-tagged ActII-ORF4-177 protein (0.7 μg); 5, His-tagged ActII-ORF4-177 protein (0.7 μg) plus anti-His tag antibody. (B) Cross-competitions in gel mobility shift analysis of the actVI-ORF1–ORFA intergenic region. Lanes: 1, probe, F139 fragment; 2, 3, and 4, His-tagged ActII-ORF4 protein (0.7, 0.8, and 0.9 μg, respectively); 5, 6, and 7, wild-type His-tagged ActII-ORF4 protein (0.9 μg) plus cold F139 fragment (50, 100, and 200 ng, respectively; 8, 9, and 10, wild-type His-tagged ActII-ORF4 protein (0.9 μg) plus cold F1031 fragment (50, 100, and 200 ng, respectively).

FIG. 7

DNase I footprinting analysis in the actVI region. (A) Upper strand: F139 fragment 5′-labeled (G1 primer) as described in Materials and Methods. (B) Lower strand: F139 fragment 5′-labeled (G39 primer). Lanes: 1, probes; 2, wild-type His-tagged ActII-ORF4 protein (1.5 μg); 3, His-tagged ActII-ORF4-177 protein (1.5 μg). Sequencing reactions (ACGT) generated with the corresponding primers (G1 or G39) were run in parallel. The hypersensitive regions are indicated by arrows. The −10 and −35 regions of the promoters are indicated.

FIG. 8

Summary of DNase I footprinting analysis for the actVI-ORF1–ORFA (A) and actIII-actI-ORF1 (B) intergenic regions. The transcriptional and translational start points from the act genes are indicated by thin and thick arrows, respectively. For the actVI-ORF1 and ORFA, transcriptional and translational start points are the same (20). Transcription start points for the actIII (23) and actI-ORF1 (50a) genes are also indicated, along with the −10 and −35 regions. The protected (boxes) and hypersensitive (vertical arrows) regions are marked in the sequence.