A recA null mutation may be generated in Streptomyces coelicolor

Abstract

The recombinase RecA plays a crucial role in homologous recombination and the SOS response in bacteria. Although recA mutants usually are defective in homologous recombination and grow poorly, they nevertheless can be isolated in almost all bacteria. Previously, considerable difficulties were experienced by several laboratories in generating recA null mutations in Streptomyces, and the only recA null mutants isolated (from Streptomyces lividans) appeared to be accompanied by a suppressing mutation. Using gene replacement mediated by Escherichia coli-Streptomyces conjugation, we generated recA null mutations in a series of Streptomyces coelicolor A3(2) strains. These recA mutants were very sensitive to mitomycin C but only moderately sensitive to UV irradiation, and the UV survival curves showed wide shoulders, reflecting the presence of a recA-independent repair pathway. The mutants segregated minute colonies with low viability during growth and produced more anucleate spores than the wild type. Some crosses between pairs of recA null mutants generated no detectable recombinants, showing for the first time that conjugal recombination in S. coelicolor is recA mediated, but other mutants retained the ability to undergo recombination. The nature of this novel recombination activity is unknown.

FIG. 1.

Creation of recA null mutants of S. coelicolor. (A) Maps of the recA⁺ and ΔrecA::aac(3)IV alleles on the chromosome. The recA alleles are shown with their neighboring genes (arrows). The filled bar represents the aac(3)IV (white arrow) cassette used for replacement of the recA gene. The hybridization probe is indicated by the bar below. The PstI cutting sites (Ps) are marked, and the size of the probe-hybridizing PstI fragment is indicated. (B) Hybridization analysis of selected Apra^r Kan^s exconjugants. Genomic DNA was digested with PstI and subjected to Southern hybridization after electrophoretic separation in an agarose gel. Each panel shows the result of the recA⁺ parent and a representative mutant. The respective 1.3- and 0.85-kb hybridizing fragments expected in the parent and the mutant are indicated.

FIG. 2.

Linearity of the chromosome in the recA mutants. (A) Restriction map of the terminal region of the S. coelicolor chromosome. The terminal protein is indicated by the circle. Restriction sites for BamHI (Ba) and PstI (Ps) are marked, with their distances from the chromosomal terminus indicated in kb. The 1.3-kb BamHI fragment (on pLUS221) was used as a hybridization probe (filled bar). (B) Genomic DNA was isolated from strains 3454 and 3456 and their recA mutants, digested with PstI, and subjected to Southern hybridization. The hybridizing 6.2-kb PstI fragments are indicated.

FIG. 3.

UV and mitomycin C sensitivity of the recA mutants. The recA⁺ strains (filled symbols) and their recA mutants (open symbols) were subjected to UV irradiation (A) and mitomycin C cross-linking (B), and the surviving spores were scored.

FIG. 4.

Growth defects of the recA mutants. (A) Minute colonies (arrows) produced by the recA null mutants. Spores of the recA mutants were spread on MM (20) medium with appropriate supplements and incubated at 30°C for 6 days. (B) Anucleate spores produced by the recA mutants. Aerial mycelium and spore chains were collected on glass slides inserted into MM agar supplemented with mannitol (21), on which the recA mutants were grown at 30°C (21). The collected material was stained with DAPI and examined with a fluorescence microscope. The top photographs are phase-contrast images of the spore chains. The bottom photographs are fluorescence images of the spore chains. Examples of anucleated spores are indicated by the arrowheads. The numbers and percentages (in parentheses) of nucleate (+) and anucleate (−) spores from more than 15 fields for each strain are tabulated at the bottom.

FIG. 5.

Scheme of recombination during conjugation. Chromosomal recombination in the recA⁺ SCP1^NF × recA mutant SCP1⁻ crosses (cross IV) in the three series (Table 2) is illustrated. The linear chromosomes are depicted by the horizontal lines, and the long (1-Mb) and short (22-kb) TIR by the bars. The relative positions of the hisA1 (h), uraA1 (u), strA1 (s), and recA alleles are marked, and the selection markers are labeled by triangles. The position of SCP1^NF (NF) is marked by diamonds. The approximate RF[UxS] and RF[HxS] are listed to the right. The expected crossovers required to produce Ura⁺ Str^r and His⁺ Str^r recombinants are indicated by the solid and dashed lines, respectively.