Expression of doxorubicin-daunorubicin resistance genes in different anthracycline-producing mutants of Streptomyces peucetius

Abstract

Two DNA fragments, ric1 and ric2, were isolated from the Streptomyces peucetius 7600 mutant, which produces daunorubicin and doxorubicin, on the basis of their abilities to confer doxorubicin and daunorubicin resistance to Streptomyces lividans. These two fragments are unrelated by restriction mapping and do not show any homology by Southern analysis, yet both of them increase the level of resistance 10-fold in transformed S. lividans. Functional analysis revealed that ric1 also contains two genes of daunorubicin biosynthesis: one coding for the aklavinone C-11 hydroxylase and the other corresponding to the putative dnrR2 regulatory gene of wild-type S. peucetius ATCC 29050 (K. J. Stutzman-Engwall, S. L. Otten, and C. R. Hutchinson, J. Bacteriol. 174:144-154, 1992). Northern (RNA) blot experiments, performed with a ric1 fragment containing daunorubicin-doxorubicin resistance gene(s), revealed a transcript of about 2,100 nucleotides that is present only during the phase of anthracycline metabolite production. The amount of this transcript is higher in strain 7600 than in strain 7900, a mutant which produces 5-fold more daunorubicin and 10-fold less doxorubicin than 7600. Furthermore, two 7900-derived blocked mutants, 8600 and 9700, do not express the 2,100-nucleotide transcript in spite of the absence of gross rearrangements in the ric1 region such as occur with the 7900 parental strain.

Regulation of daunorubicin production in Streptomyces peucetius by the dnrR2 locus

“…However, in the case of the dnrI::aphII strain, the nature of the mutation and the observed properties of the strain seem to exclude the possibility that any functional DnrI protein could be present (30,43). This implies that S. peucetius possesses some mechanism of DNR resistance that can be expressed independently from dnrI, such as the DNR resistance genes identified by Colombo et al (10), which are different from the drrAB (17) and drrC (23) resistance genes and lie outside the gene cluster containing dnrN and dnrI. The fact that the introduction of extra copies of dnrN carried on a high-copy-number plasmid results in a twofold increase in the DNR resistance of the dnrI::aphII mutant (34) implies that dnrN may be able to influence the expression of DNR resistance by some mechanism which does not require DnrI.…”

The Streptomyces peucetius drrC gene encodes a UvrA-like protein involved in daunorubicin resistance and production

“…Streptomyces peucetius, which produces the important antitumor drugs daunorubicin (DNR) (4,10) and doxorubicin (DXR) (2), contains the drrAB (17) and ric2 (8) resistance genes, which are assumed to provide self-resistance to these two antibiotics because they confer DNR and DXR resistance when introduced into Streptomyces lividans. The DrrA protein strongly resembles bacterial proteins that transport compounds by an ATP-dependent process (20), as well as the Mdr1 P glycoprotein responsible for DNR-DXR resistance of human cancer cells and known to act as an ATP-dependent transporter (19).…”

“…DrrA thus is a candidate for a DNR-DXR binding and transport protein, whereas the hydrophobic DrrB protein could be responsible for binding DrrA to the bacterial membrane. The ric2 locus, which unlike drrAB is not part of the cluster of DXR production genes (8), may also be important for self-resistance. A third gene, drrC, in the same cluster of genes as is drrAB, encodes the DrrC protein described here.…”

Streptomyces peucetius daunorubicin biosynthesis gene, dnrF: sequence and heterologous expression

“…The gene belongs to the Dxr-Dnr biosynthetic gene cluster and, as previously suggested (Colombo et al, 1992), it is closely associated with the Dxr-Dnr resistance gene drrAB. The dnrF gene encodes an enzyme of 489 amino acids that catalyses the conversion of aklavinone to E-rhodomycinone via 1 1 -hydroxylation.…”

“…This finding is consistent with the observation, made by Guilfoile & Hutchinson (1991), that, in S. petleetius ATCC 29050, a spacer of 233 nt separates the drrAB gene from an open reading frame (ORF) transcribed in the Downloaded from www.microbiologyresearch.org by IP: 54.191.190.102 On: Sat, 12 May 2018 10:12:59 S. F I L I P P I N I a n d O T H E R S opposite direction. T h e ORF was only partially sequenced but comparison of the deduced first 79 amino acids revealed a significant similarity with FAD-and NADPHdependent bacterial hydroxylases (Blanco e t a/., 1993 (Grein et al, 1963), 7600, 7800 and 9700 (Colombo et al, 1992) and the plasmids pWHM3 (Vara et al, 1989), pWHM603 (Guilfoile & Hutchinson, 1991) and ricl (Colombo et al, 1992) are described elsewhere. The ermE* promoter (Gramajo et al, 1991) was isolated from pIJ4070 kindly provided by M. J. Bibb (unpublished results).…”