The mithramycin gene cluster of Streptomyces argillaceus contains a positive regulatory gene and two repeated DNA sequences that are located at both ends of the cluster

Abstract

Sequencing of a 4.3-kb DNA region from the chromosome of Streptomyces argillaceus, a mithramycin producer, revealed the presence of two open reading frames (ORFs). The first one (orfA) codes for a protein that resembles several transport proteins. The second one (mtmR) codes for a protein similar to positive regulators involved in antibiotic biosynthesis (DnrI, SnoA, ActII-orf4, CcaR, and RedD) belonging to the Streptomyces antibiotic regulatory protein (SARP) family. Both ORFs are separated by a 1.9-kb, apparently noncoding region. Replacement of the mtmR region by an antibiotic resistance cassette completely abolished mithramycin biosynthesis. Expression of mtmR in a high-copy-number vector in S. argillaceus caused a 16-fold increase in mithramycin production. The mtmR gene restored actinorhodin production in Streptomyces coelicolor JF1 mutant, in which the actinorhodin-specific activator ActII-orf4 is inactive, and also stimulated actinorhodin production by Streptomyces lividans TK21. A 241-bp region located 1.9 kb upstream of mtmR was found to be repeated approximately 50 kb downstream of mtmR at the other end of the mithramycin gene cluster. A model to explain a possible route for the acquisition of the mithramycin gene cluster by S. argillaceus is proposed.

FIG. 1

(A) Structure of mithramycin. (B) Schematic representation of the location of mtmR in the chromosome of S. argillaceus with respect to mithramycin sugar biosynthetic genes and the mithramycin polyketide synthase (PKS) genes. The black bar indicates the region sequenced in this paper. pFL3R, pFL3R1, and pFL3R2 represent the inserts in the plasmid constructions used for activation of mithramycin production. The insert in pFL3R1 was generated by PCR amplification (see Materials and Methods). A, Asp700I; B, BamHI; H, HindIII; P, SphI; S, SmaI; T, StuI. Restriction sites A, P, S, and T are not unique sites in the chromosomal region shown.

FIG. 2

Alignment of the amino acid sequence of MtmR and different SARPs. MtmR, mithramycin positive regulator from S. argillaceus (this work); DnrI, daunorubicin positive regulator from S. peucetius (33); SnoA, nogalamycin positive regulator from S. nogalater (41); ActII-orf4, actinorhodin positive regulator from S. coelicolor (15); RedD, undecylprodigiosin positive regulator from S. coelicolor (24); and CcaR, cephamycin and clavulanic acid positive regulator from S. clavuligerus (25).

FIG. 3

HPLC analysis of mithramycin production by the wild-type S. argillaceus strains containing pIAGO (A) and pFL3R (B) and S. argillaceus M13R1 (C). The arrows indicate the mobility of mithramycin.

FIG. 4

Analysis of the gene replacement experiment to generate mutant M13R1. (A) Scheme representing the replacement event in the chromosome of wild-type S. argillaceus strain produced by a double crossover to construct mutant M13R1. The asterisks indicate the ends of the probe used for Southern hybridization (a 3.7-kb HindIII-SphI fragment). The thin lines represent the BamHI hybridizing bands both in the wild type and in the mutant. B, BamHI; G, BglII; H, HindIII; P, SphI; T, StuI. Am, apramycin resistance cassette; tsr, thiostrepton resistance cassette. (B) Southern hybridization by using a 3.7-kb HindIII-SphI fragment as a probe. Lane 1, BamHI-digested chromosomal DNA of the wild-type S. argillaceus strain; lane 2, BamHI-digested chromosomal DNA of M13R1 strain. Sizes of bands in kilobases are indicated on the left and right of the gel.

FIG. 5

(A) Southern hybridization of overlapping cosmid clones containing the mithramycin gene cluster by using the 3.7-kb HindIII-SphI fragment as a probe. Plasmid DNAs from cosAR3, cosAR7, and cosAR13 were digested with BamHI. Sizes of bands in kilobases are indicated on the left of the gel. (B) Schematic representation of the mithramycin gene cluster showing the locations of the repeated sequences (RS) at both ends of the cluster. B, BamHI; G, BglII; L, SalI. The black bars indicate the hybridizing bands formed against the probe, the 3.7-kb HindIII-SphI fragment. The shaded rectangles indicate the locations of the direct repeated sequences. cosAR13, cosAR7, and cosAR3 correspond to three cosmid clones comprising the mithramycin gene cluster.

FIG. 6

Alignment of the DNA sequences of the 0.6-kb BglII-SalI fragment from cosAR3 and the homologous region from the 4.3-kb sequence located in cosAR13 around orfA and mtmR. The sequences were compared by using the GAP program (9). The coding sequence of orfA is indicated in bold letters. The sequence that is identical in both DNA regions is enclosed within a frame.