Identification of a gene negatively affecting antibiotic production and morphological differentiation in Streptomyces coelicolor A3(2)

Abstract

SC7A1 is a cosmid with an insert of chromosomal DNA from Streptomyces coelicolor A3(2). Its insertion into the chromosome of S. coelicolor strains caused a duplication of a segment of ca. 40 kb and delayed actinorhodin antibiotic production and sporulation, implying that SC7A1 carried a gene negatively affecting these processes. The subcloning of SC7A1 insert DNA resulted in the identification of the open reading frame SCO5582 as nsdA, a gene negatively affecting Streptomyces differentiation. The disruption of chromosomal nsdA caused the overproduction of spores and of three of four known S. coelicolor antibiotics of quite different chemical types. In at least one case (that of actinorhodin), this was correlated with premature expression of a pathway-specific regulatory gene (actII-orf4), implying that nsdA in the wild-type strain indirectly repressed the expression of the actinorhodin biosynthesis cluster. nsdA expression was up-regulated upon aerial mycelium initiation and was strongest in the aerial mycelium. NsdA has DUF921, a Streptomyces protein domain of unknown function and a conserved SXR site. A site-directed mutation (S458A) in this site in NsdA abolished its function. Blast searching showed that NsdA homologues are present in some Streptomyces genomes. Outside of streptomycetes, NsdA-like proteins have been found in several actinomycetes. The disruption of the nsdA-like gene SCO4114 had no obvious phenotypic effects on S. coelicolor. The nsdA orthologue SAV2652 in S. avermitilis could complement the S. coelicolor nsdA-null mutant phenotype.

FIG. 1.

Streptomyces cosmid SC7A1 and some subclones carrying nsdA on pHJL401, a medium-copy-number SCP2*-based vector, delay actinorhodin production of M145. The cultures were grown on R2YE for 5 days at 30°C. The back of the plate is shown.

FIG. 2.

Identification of nsdA, which negatively affects Streptomyces differentiation by subcloning in M145. Lines with double arrows represent insert DNAs in each subclone. Open arrows represent genes, with their names shown above. The gray arrow represents nsdA. Some restriction enzyme sites are also shown. +, Act production was delayed. −, Act production was not delayed.

FIG. 3.

In nsdA mutants, aerial mycelium formation was delayed but more spores were eventually produced. Strains were grown on MS medium at 30°C. The upper sides of the plates are shown. Time points of observation are shown at the top. Strain names are to the left. M145, wild-type strain. YX2, an nsdA mutant strain in which amino acids 77 to 367 of NsdA were replaced by a 1.7-kb aac(3)IV cassette. LW9 was an nsdA-null mutant strain in which the whole nsdA gene was replaced by an oriT+aac(3)IV cassette. LW9/pHL128, LW9 complemented by pHL128 (a pHL127-derived plasmid containing nsdA).

FIG. 4.

nsdA disruption results in the overproduction of actinorhodin on (A) SMMS (6-day culture) and (B) MS media (4-day culture). The backs of the plates are shown. YX2 and LW9, nsdA disruption mutants. M145, parent strain. pHZ2731 carried nsdA inserted into pIJ8600. pHL127 is a pSET152-derived plasmid with aac(3)IV replaced by aadA. pHL128 carried nsdA inserted into pHL127. pHL129 carried the nsdA with the S458A mutation inserted in pHL127. The nsdA-null mutation phenotype was complemented by pHL128 but not by pHL129.

FIG. 5.

nsdA gene disruption results in the overproduction of CDA (A) and methylenomycin (B). Compared to that of M145, YX2 culture plugs generated a larger inhibition zone with the CDA-sensitive Bacillus mycoides indicator strain. Similarly, compared to that of M145/SCP1, YX2/SCP1 gave a larger inhibition zone on the Mmy-sensitive indicator S. coelicolor J1501. The CDA and methylenomycin overproduction phenotype was complemented by pHZ2731 but not by pIJ8600.

FIG. 6.

Northern blot analysis indicating an increase of actII-orf4 mRNA in the nsdA mutant strain. Total RNA was isolated from solid SMMS cultures grown for 28, 40, 52, 64, and 76 h. The same blot was stripped and hybridized with probe for 16S rRNA.

FIG. 7.

Localization and time of nsdA expression. (A) EGFP fluorescence microscopy (A1 to A4). M145/pHL117 (A1 and A2), in which a 544-bp fragment from −18 to −561 upstream of the nsdA GTG start codon was fused to a promoterless egfp gene, and M145 (A3 and A4) were grown on MS for 37 h. Phase-contrast (A1 and A3) and fluorescence (enhanced green fluorescent protein) (A2 and A4) micrographs are shown. Fluorescence signals of M145/pHL117 were relatively high in aerial mycelium and spore chains. SM, substrate mycelium; AM, aerial mycelium; SC, spore chain. Bar, 10 μm. (B) XylE activity assay. Strains were grown on SMMS medium for 28 to 76 h. They began to form aerial mycelium and sporulate at about 40 and 64 h, respectively. C23O activities were measured quantitatively (25). C23O activities were obviously detected in only M145/pHL134-nsdAp at 40, 52, 64, and 76 h. pHL134, M145/pHL134; PnsdA, M145/pHL134-nsdAp. Error bars indicate standard deviations. (C) RT-PCR. M145 was grown on SMMS medium for 18 to 76 h. It began to form aerial mycelium (Am) and sporulate (Sp) at about 28 and 52 h, respectively. nsdA was expressed at about 28 h. hrdB, as a control, was expressed throughout growth.

FIG. 8.

Sequence alignment of the SXR site (shown in the dashed box) in several NsdA-like proteins.