RNase III is required for actinomycin production in Streptomyces antibioticus

Abstract

Using insertional mutagenesis, we have disrupted the RNase III gene, rnc, of the actinomycin-producing streptomycete, Streptomyces antibioticus. Disruption was verified by Southern blotting. The resulting strain grows more vigorously than its parent on actinomycin production medium but produces significantly lower levels of actinomycin. Complementation of the rnc disruption with the wild-type rnc gene from S. antibioticus restored actinomycin production to nearly wild-type levels. Western blotting experiments demonstrated that the disruptant did not produce full-length or truncated forms of RNase III. Thus, as is the case in Streptomyces coelicolor, RNase III is required for antibiotic production in S. antibioticus. No differences in the chemical half-lives of bulk mRNA were observed in a comparison of the S. antibioticus rnc mutant and its parental strain.

Fig 1

Schematic representation of the rnc gene of S. antibioticus, showing several restriction sites in the gene. The line shown below the schematic of the gene represents the internal fragment used for insertional mutagenesis. The diagram is drawn to scale.

Fig 2

Southern blot of digests of chromosomal DNA from S. antibioticus strain IMRU3720 and the rnc mutant, JSE1980. Approximately 2 μg of DNA was digested with BclI. The probe used for hybridization was the complete S. antibioticus rnc gene synthesized by PCR using pJSE1900 as the template and the primers described in Materials and Methods. Lane 1, size standards (STDS) prepared using the rnc gene (0.8 kb), pJSE1980 digested with BamHI and SmaI (1.2 kb), and pJSE1980 linearized with EcoRI (4.2 kb); lane 2, DNA from IMRU3720; lanes 3 to 7, DNA from JSE1980. All cultures for DNA preparation were grown for 48 h on NZ-amine (growth) medium. The DNA sample used for the digest shown in lane 3 was isolated from the NZ-amine culture grown on 50 μg/ml of apramycin. The DNA samples used for lanes 4 and 5 were isolated from a culture grown on galactose-glutamic acid (GGA [actinomycin production]) medium for 19 and 60 h (HR), respectively. The GGA culture was inoculated with mycelium from an NZ-amine culture that had been grown on 50 μg/ml of apramycin. The DNA samples for lanes 6 and 7 were isolated from a culture grown on GGA medium for 19 and 60 h, respectively, but the GGA culture was inoculated from an NZ-amine culture that was not grown on apramycin. GGA cultures contained 1 mM phosphate and no apramycin.

Fig 3

Growth and actinomycin production of S. antibioticus cultures. (A) Mycelial dry weights of 1-ml portions of cultures of S. antibioticus IMRU3720, JSE1980, and their derivatives were determined as described previously (16). (B) Actinomycin production was measured by ethyl acetate extraction of culture medium as described previously (16). Strains were grown from NZ-amine inocula on GGA medium. The NZ-amine cultures were grown with apramycin at 50 μg/ml, and the GGA cultures were grown without apramycin. Strains JSE1980/pJSE1995 and JSE1980/pIJ8600 were grown in the presence of 15 μg/ml thiostrepton to induce the tipA promoter.

Fig 4

Western blotting of mycelial extracts of S. antibioticus IMRU3720 and JSE1980. Mycelial extracts were prepared and Western blotting was performed as described previously (28). Lane 1, authentic S. coelicolor RNase III (arrow), 75 ng; lanes 2 to 5, 50 μg of mycelial extracts from IMRU3720 grown for 12, 24, 51, and 72 h (HR) on GGA medium; lanes 6 to 8, 50 μg of mycelial extracts from JSE1980 grown for 12, 51, and 72 h on GGA medium. In these experiments, the NZ-amine medium used for the JSE1980 cultures contained 50 μg/ml of apramycin. The GGA cultures were all grown without apramycin. To avoid the possibility of contamination of the mycelial extracts by leakage from the standard well, the RNase III standard was run on a separate gel and the picture of the standard shown in lane 1 was pasted onto the figure from a separate Western blot.

Fig 5

Determination of the chemical half-lives of bulk mRNA from various S. antibioticus strains. Cultures were grown as described in Materials and Methods, and rifampin was used to inhibit RNA synthesis following pulse-labeling with [³H]uridine. The data in the figure are derived from duplicate assays and are expressed as means ± standard errors of the means.