Novel genes that influence development in Streptomyces coelicolor

Abstract

Filamentous soil bacteria of the genus Streptomyces carry out complex developmental cycles that result in sporulation and production of numerous secondary metabolites with pharmaceutically important activities. To further characterize the molecular basis of these developmental events, we screened for mutants of Streptomyces coelicolor that exhibit aberrant morphological differentiation and/or secondary metabolite production. On the basis of this screening analysis and the subsequent complementation analysis of the mutants obtained we assigned developmental roles to a gene involved in methionine biosynthesis (metH) and two previously uncharacterized genes (SCO6938 and SCO2525) and we reidentified two previously described developmental genes (bldA and bldM). In contrast to most previously studied genes involved in development, the genes newly identified in the present study all appear to encode biosynthetic enzymes instead of regulatory proteins. The MetH methionine synthase appears to be required for conversion of aerial hyphae into chains of spores, SCO6938 is a probable acyl coenzyme A dehydrogenase that contributes to the proper timing of aerial mycelium formation and antibiotic production, and SCO2525 is a putative methyltransferase that influences various aspects of colony growth and development.

FIG. 1.

Comparison of wild-type S. coelicolor and five insertion mutants. The wild-type (WT) parent strain, M145, and the mutants were grown on solid R2YE medium plates at 30°C for 2 days (A) and 5 days (B). The mutant phenotypes were caused by transposon disruption of an unknown gene in the case of NY247 and NY1868, transposon disruption of metH in the case of AG1440, transposon disruption of bldM in the case of DB2571, and transposon disruption of bldA in the case of DB5931.

FIG. 2.

Comparison of bldM alleles in different genetic backgrounds. The strains were grown on R2YE solid medium at 30°C for 4 days. A bldM::hyg allele was previously introduced into the J1915 genetic background to obtain bald strain J2151, which is blocked for aerial mycelium formation (27). Transfer of the bldM::hyg allele to M145 by genomic transformation yielded strain DB2321, which produces an aerial mycelium but fails to sporulate. Strain DB2571, which bears a bldM::Tn5apr allele and was generated in the M145 genetic background, also produces a nonsporulating aerial mycelium.

FIG. 3.

Scanning electron micrographs showing the colony surfaces of wild-type S. coelicolor and a metH mutant. The aerial mycelium of wild-type strain M145 produces abundant spores (A), while the aerial hyphae of metH mutant AG1440 appear to be wrinkled and lack any visible signs of sporulation (B). Colonies were grown on solid R2YE medium for 4 days at 30°C prior to scanning electron microscopy.

FIG. 4.

Genomic location of the insertion in mutant NY1868 and complementation of the insertion. (A) The Tn5apr transposon was found to be inserted into the 3′ end of the SCO6939 gene in the chromosome of NY1868. This gene and the flanking genes are shown, and the scale at the top indicates the base pair positions of these genes in the S. coelicolor chromosome sequence determined by the Sanger Centre genome sequencing project (4). The Sanger Centre annotation is indicated below each gene. The DNA regions that were PCR amplified, cloned into pSET152S, and used in complementation experiments are indicated at the bottom. (B) The delay in development exhibited by NY1868 was not complemented by a pSET152S derivative containing SCO6939 but was complemented by a construct containing SCO6938. The strains were grown on R2YE solid medium at 30°C for 2 days. WT, wild type.

FIG. 5.

Genomic location of the insertion in mutant NY247. The Tn5apr transposon was found to be inserted into gene SCO2525 in the chromosome of NY247. (A) SCO2525 gene and flanking genes. The scale bar at the top indicates the base pair positions in the S. coelicolor genome sequence (4). SCO2525 and SCO2524 have been annotated as unknown hypothetical proteins, while SCO2526 has been annotated as a possible acetyltransferase by the Sanger Centre. The DNA regions that were PCR amplified, cloned into pSET152S, and shown to complement the defects in NY247 are indicated at the bottom. (B) Partial alignment of SCO2525 with human PNMT (hPNMT). Identical residues are shaded, and residues that participate in binding to the SAM cofactor in human PNMT are indicated by asterisks (24). These active site residues are well conserved in the two proteins.

FIG. 6.

Requirement of SCO2525 for proper aerial mycelium formation and colony morphology. The defects apparent in NY247 (upper right) were complemented by introduction of pSET152S derivatives bearing SCO2525 either alone or in combination with the downstream gene (lower half). The strains were grown on R2YE solid medium for 5 days at 30°C. WT, wild type.