Construction and characterization of a Streptomyces rimosus recA mutant: the RecA-deficient strain remains viable
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ORIGINAL PAPER
A. Mikoc á I. Ahel á V. Gamulin
Construction and characterization of a Streptomyces rimosus recA mutant: the RecA-de®cient strain remains viable Received: 26 January 2000 / Accepted: 5 May 2000 / Published online: 26 July 2000 Ó Springer-Verlag 2000
Abstract Although previously reported attempts to construct recA null mutants in Streptomyces spp. have been unsuccessful, we have used the suicide plasmid pErmDRecA to inactivate the recA gene in Streptomyces rimosus by gene disruption. pErmDRecA carries the erythromycin resistance gene ermE and a 451-bp fragment of the S. rimosus recA gene (encoding amino acids 2±151). An erythromycin-resistant clone with single plasmid integration into the recA gene on the chromosome was analyzed in detail. This clone possesses one inactive copy of recA which lacks the entire promoter region and the ATG start codon, and a second, truncated gene that encodes only ®rst 151 amino acids of the RecA protein. This S. rimosus recA mutant can therefore be considered a completely RecA-de®cient strain. The mutant strain is highly sensitive to UV light. Introduction of a plasmid carrying the wild type S. rimosus recA gene completely restored the UV resistance of the recA mutant to wild-type levels. recA genes encoding RecA proteins with short deletions at the C-terminus (21 and 51 amino acids) could not fully rescue the UV sensitivity of the S. rimosus recA strain, when introduced in the same way. Key words recA mutant á Streptomyces á Gene disruption á UV sensitivity
Introduction Genetic instability is one of the most striking features of streptomycetes. These mycelium-forming and antibioticCommunicated by W. Goebel A. Mikoc á I. Ahel á V. Gamulin (&) Department of Molecular Genetics, Ruder Boskovic Institute, P.O. Box 1016, 10000 Zagreb, Croatia E-mail: [email protected] Tel.: +385-1-4561177; Fax: +385-1-4561115
producing bacteria can spontaneously lose more than 10% of their linear chromosome. Large deletions are often associated with ampli®cations of speci®c sequences to high copy number (Chen 1995; Vol and Altenbuchner 1998). It has been proposed that the RecA protein could be involved in generating deletions and ampli®cations in these bacteria (Chen 1995; Young and Cullum 1987), and might therefore play a role in genetic instability. Early attempts to isolate recA mutants of Streptomyces spp. (Harold and Hopwood 1970a, b; Stonesifer and Baltz 1985; Tsai and Chen 1987; Kieser et al. 1989) were not entirely successful. The recA genes of four streptomycetes: S. lividans (Nussbaumer and Wohlleben 1994), S. venezuelae (Yao and Vining 1994), S. ambofaciens (Aigle et al. 1997) and S. rimosus (Mikoc et al. 1997) have been cloned and sequenced only recently. RecA proteins in bacteria are phylogenetically very conserved, and the same is true for the four known RecAs of Streptomyces. They all display high homology with the Escherichia coli RecA protein and contain all functional domains and modules identi®ed in E. coli RecA (Kowalczykowski et al. 1994). A unique characteristic
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