An additional regulatory gene for actinorhodin production in Streptomyces lividans involves a LysR-type transcriptional regulator

Abstract

The sequence of a 4.8-kbp DNA fragment adjacent to the right-hand end of the actinorhodin biosynthetic (act) cluster downstream of actVB-orf6 from Streptomyces coelicolor A3(2) reveals six complete open reading frames, named orf7 to orf12. The deduced amino acid sequences from orf7, orf10, and orf11 show significant similarities with the following products in the databases: a putative protein from the S. coelicolor SCP3 plasmid, LysR-type transcriptional regulators, and proteins belonging to the family of short-chain dehydrogenases/reductases, respectively. The deduced product of orf8 reveals low similarities with several methyltransferases from different sources, while orf9 and orf12 products show no similarities with other known proteins. Disruptions of orf10 and orf11 genes in S. coelicolor appear to have no significant effect on the production of actinorhodin. Nevertheless, disruption or deletion of orf10 in Streptomyces lividans causes actinorhodin overproduction. The introduction of extra copies of orf10 and orf11 genes in an S. coelicolor actIII mutant restores the ability to produce actinorhodin. Transcriptional analysis and DNA footprinting indicate that Orf10 represses its own transcription and regulates orf11 transcription, expression of which might require the presence of an unknown inducer. No DNA target for Orf10 protein was found within the act cluster.

FIG. 1

Restriction map of the S. coelicolor A3(2) chromosome next to the right-hand end of the act cluster. The organization of ORFs within this region as deduced by DNA sequencing is given. Only relevant restriction sites are indicated; numbers in parentheses correspond to those reported by Malpartida and Hopwood (32). The MboI restriction site at which DNA rearrangement has occurred is indicated by asterisks. The solid bar shows the extent of the sequenced DNA fragment. The direct repeat sequences at the 3′ ends of orf10 and orf11 are represented by shaded boxes. The DNA fragments used for gene disruptions with the φC31-derived PM1 vector are indicated.

FIG. 2

Multiple alignment of the N terminus of S. coelicolor Orf10 protein (Orf10_Strco) with those of other LysR-type transcriptional regulators. The HTH motif involved in DNA binding is indicated. Origins of the amino acid sequences and (in parentheses) their Swissprot database accession numbers are as follows: BudR_Klete, Klebsiella terrigena (P52666); AlsR_Bacsu, Bacillus subtilis (Q04778); CbbR_Xanfl, Xanthobacter flavus (P25545); XapR_Ecoli, E. coli (P23841); Ttua_Agrvi, Agrobacterium vitis (P52669); and CynR_Ecoli, E. coli (P27111). Black boxes indicate positions in the alignment where the same amino acid is found in at least four of the seven sequences; gray boxes indicate residues similar to those marked in black.

FIG. 3

Alignment of the putative NAD(H)-NADP(H) binding (A) and catalytic (B) sites of S. coelicolor Orf11 protein (Orf11_Strco) with those of other SDRs. Origins of the proteins and (in parentheses) their Swissprot database accession numbers are as follows: Dhb1_human, human (P14061); Fabg_Ecoli, E. coli (P25716); Enta_Ecoli, E. coli (P15047); 2bhd_Strex, Streptomyces exfoliatus (P19992); Ridh_Kleae, Klebsiella aerogenes (P00335); Phbb_Zoora, Zoogloea ramigera (P23238); Act3_Strco, S. coelicolor (P16544); and Dhkr_Strcm, Streptomyces cinnamonensis (P41177). Conserved and similar residues are in black and gray boxes, respectively (plurality, 5). The amino acids reported to play a role in either site are indicated.

FIG. 4

Subcloning of the orf10-orf11 DNA region to elucidate the genes required for complementation analysis of actIII mutation. Plasmid construction was as described in Materials and Methods and Table 1. Plasmids pSCNB01 and pSCNB010 contain a TAG stop codon (generated by the cloning procedure) downstream of the 3′ end of the orf10 deletion. Plasmid pIJ2314 contains the actIII gene. Symbols: −, no actinorhodin production; +, actinorhodin production.

FIG. 5

Transcription analysis of the actII-orf4 gene. Total RNA was isolated from 3-day-old cultures of S. lividans strains TK21, TK21::pSCNB06A, and CNB073 (lanes 1, 3, and 4, respectively) and of S. coelicolor J1501 (lane 5). E. coli tRNA was used as a control (lane 2). A protected fragment of the expected size (384 nucleotides) was observed. End-labeled HinfI-digested pBR329 was used as a size marker. p, promoter.

FIG. 6

Transcriptional analysis of orf10 (A) and orf11 (B) genes. Lanes: 1, E. coli tRNA; 2 and 3, total RNA extracted from 3-day-old cultures of S. lividans TK21 and CNB073, respectively; A+G and T+C, Maxam-and-Gilbert sequence ladders of the corresponding labeled probes. Asterisks indicate the most probable transcription start sites. (C) DNA sequence within the orf10-orf11 intergenic region. orf10 and orf11 mRNAs are initiated at the indicated nucleotides. Arrows indicate direction of transcription. Amino acids are represented in single-letter code below the DNA sequence. The boxed sequence represents the TN₁₁A consensus motif for LysR-type regulators contained in the DNase I-protected region. The GG nucleotides mutated to AT within this region are shown in italics.

FIG. 7

SDS-PAGE analysis of orf10 expression in E. coli and at various stages of its purification. Lanes: 1 and 2, whole-cell extracts from cultures of E. coli K12ΔH1Δtrp carrying the vector plasmid pAZe3ss, grown at 30 and 42°C, respectively; 3 and 4, whole-cell extracts from cultures of the same strain harboring plasmid pCNB019, grown at 30 and 42°C, respectively; 5, supernatant of 25,000 × g centrifugation; 6, pellet of this centrifugation (inclusion bodies) after the refolding step; 7, heparin-agarose chromatography (1 μg of protein). The recombinant purified Orf10 protein is indicated by arrowheads. The positions and molecular masses of marker proteins are shown on the left.

FIG. 8

DNA binding assays. Gel mobility shift analysis with the orf10-orf11 intergenic region was performed as described in Materials and Methods, using the 316-bp EspI-AvaI fragment as the probe. Lanes: 1 and 12, without protein addition; 2 and 3, with crude extracts from cultures of E. coli K12ΔH1Δtrp carrying the control plasmid pAZe3ss, grown at 30 and 42°C, respectively; 4 and 5, with crude extracts from cultures of the same strain harboring plasmid pCNB019, grown at 30 and 42°C, respectively; 6 to 9, with 1.2, 2.5, 12.5 and 25 ng of the purified S. coelicolor Orf10 protein, respectively; 10, with 25 ng of Orf10 and fivefold molar excess of unlabeled orf10-orf11 intergenic region; 11, with 25 ng of Orf10 previously treated for 15 min at 100°C.

FIG. 9

Synthesis of ³⁵S-labeled Orf10 protein. Samples were analyzed by SDS-PAGE and autoradiography as described in Materials and Methods and include whole-cell extract from a culture of E. coli K38/pGP-1-2 carrying plasmid pT7.7 grown at 42°C with rifampin addition (lane 1), whole-cell extract from a culture of E. coli K38/pGP-1-2 carrying plasmid pCNB023 grown at 42°C without and with rifampin addition (lanes 2 and 3, respectively), and supernatant of 25,000 × g centrifugation (lane 4) and its pellet (inclusion bodies) after the refolding step (lane 5). Positions of size markers are indicated on the left. The 34-kDa labeled protein is labeled with an arrow.

FIG. 10

Analysis of DNA binding activity of ³⁵S-labeled Orf10 purified from inclusion bodies. The binding reaction was carried out as described in Materials and Methods, and products were analyzed by native PAGE and autoradiography. Lanes: 1, in the absence of DNA; 2, with EcoRI-digested pUC19; 3, with EcoRI-digested M13mp18; 4, with the 246-bp NruI-AvaI fragment within the orf10-orf11 intergenic region (pCNB033 insert); 5, with the 206-bp AhaII fragment within the orf10-orf11 intergenic region (pCNB034A insert); 6 and 7, with the 393-bp PstI-SacII and 227-bp SmaI-HincII fragments containing direct repeat sequences at the 3′ ends of orf10 and orf11, respectively; 8, with the 484-bp EspI-XhoI fragment within the orf10-orf11 intergenic region containing the NdeI-engineered restriction site; 9, with the 455-bp MboII fragment within the actIII-actI intergenic region. Arrows indicate mobilities of the complexes between Orf10 protein and its DNA target.

FIG. 11

DNase I footprinting analysis of the interactions between Orf10 protein and the orf10-orf11 intergenic region. Upper (A) and lower (B) strands correspond to pCNB033 and pCNB034A inserts. DNase I protection experiments were done as described in Materials and Methods. Lanes: 1, without DNase I; 2, with DNase I but no Orf10; 3 to 5, with 20, 200, and 600 ng of Orf10, respectively; 6, with 600 ng of Orf10 and 10-fold molar excess of unlabeled orf10-orf11 intergenic region. The corresponding sequence reactions (lanes A, C, G, and T) were run in parallel. The boxed regions correspond to the TN₁₁A consensus motif for LysR-type regulators.