A stationary-phase acyl-coenzyme A synthetase of Streptomyces coelicolor A3(2) is necessary for the normal onset of antibiotic production

Abstract

The fadD1 and macs1 genes of Streptomyces coelicolor are part of a two-gene operon. Both genes encode putative acyl coenzyme A synthetases (ACSs). The amino acid sequence of FadD1 has high homology with those of several ACSs, while MACS1 has the closest homology with medium-chain ACSs, broadly known as SA proteins. Like FadD of Escherichia coli, FadD1 also has a broad substrate specificity, although saturated long-chain fatty acids appears to be the preferred substrate. fadD1 is a growth-phase-regulated gene, and its mRNA is detected only during the stationary phase of growth. Interestingly, a mutation in fadD1 alters the levels of another ACS or ACSs, both at the stationary phase and at the exponential phase of growth, at least when glucose is used as a main carbon source. The mutant also shows a severe deficiency in antibiotic production, and at least for Act biosynthesis, this deficiency seems to be related to delayed expression of the Act biosynthetic genes. Antibiotic production is restored by the introduction of a wt fadD1 allele into the cell, demonstrating a strict link between ACS activity and the biosynthesis of secondary metabolites. The results of this study indicate that the ACSs may be useful targets for the design of rational approaches to improving antibiotic production.

FIG. 1.

Genetic organization of the macs1-fadD1 operon and the transcriptional regulator acsR. Arrows show oligonucleotides used for RT-PCR assays. The bar represents the fadD1 DNA fragment used as a probe in Northern and Southern analyses. S, SmaI sites within fadD1 that were used for the construction of the deletion mutant.

FIG. 2.

ACS levels throughout growth. ACS activities in crude protein extracts prepared from cells of S. coelicolor M145 (filled symbols) and MCB40 (open symbols) grown in SMM-glucose (circles) or SMM-oleate (diamonds) were determined. Palmitate was used as a substrate in the enzyme assays. The values are the averages of three independent determinations. EP, TP, and SP, exponential, transition, and stationary phases of growth, respectively.

FIG. 3.

Expression of fadD1 throughout growth. Northern blot analyses of total RNAs isolated during the exponential phase (EP), the transition phase (TP), and the stationary phase (SP) from cultures of S. coelicolor grown in SMM-glucose or SMM-oleate. The probes used were specific for fadD1 (A) or accB (B). Size markers in kilobases are indicated to the left of the panels.

FIG. 4.

RT-PCR analysis for determination of the transcriptional linkage of macs1 and fadD1. The pair of primers used is indicated in Fig. 1. RNAs were isolated from stationary-phase cultures grown in SMM-oleate (lane 1) or SMM-glucose (lane 2) or from exponential-phase SMM-oleate-grown cultures (lane 3). No specific amplification occurred when reverse transcriptase was omitted from the reaction mixture of RNA from stationary-phase cultures (lane 4). DNA was used as a positive control for PCR (lane 5). Lane 6, size markers (SM).

FIG. 5.

Effect of the growth medium on redD expression. RNAs (5 μg) were isolated at the times indicated from cultures grown in SMM-oleate (lane O) or SMM-glucose (lane G). The samples were blotted on a nitrocellulose filter and hybridized with ³²P-labeled redD.

FIG. 6.

Effect of the fadD1 mutation on Act production and on the expression of act biosynthetic genes. (A) Open and filled circles represent the growth curves for M145 and MCB40, respectively. Open, filled, and grey squares represent the levels of Act production in MCB40, M145, and the complemented mutant MCB40/pCB32, respectively. (B) RNAs were isolated at the times indicated, and the samples were dot blotted on nitrocellulose membranes. The probe used was ³²P-labeled actIII.