Impact of mutS Inactivation on Foreign DNA Acquisition by Natural Transformation in Pseudomonas stutzeri

Abstract

In prokaryotic mismatch repair the MutS protein and its homologs recognize the mismatches. The mutS gene of naturally transformable Pseudomonas stutzeri ATCC 17587 (genomovar 2) was identified and characterized. The deduced amino acid sequence (859 amino acids; 95.6 kDa) displayed protein domains I to IV and a mismatchbinding motif similar to those in MutS of Escherichia coli. A mutS::aac mutant showed 20-to 163-fold-greater spontaneous mutability. Transformation experiments with DNA fragments of rpoB containing single nucleotide changes (providing rifampin resistance) indicated that mismatches resulting from both transitions and transversions were eliminated with about 90% efficiency in mutS ؉ . The mutS ؉ gene of strain ATCC 17587 did not complement an E. coli mutant but partially complemented a P. stutzeri JM300 mutant (genomovar 4). The declining heterogamic transformation by DNA with 0.1 to 14.6% sequence divergence was partially alleviated by mutS::aac, indicating that there was a 14 to 16% contribution of mismatch repair to sexual isolation. Expression of mutS ؉ from a multicopy plasmid eliminated autogamic transformation and greatly decreased heterogamic transformation, suggesting that there is strong limitation of MutS in the wild type for marker rejection. Remarkably, mutS::aac altered foreign DNA acquisition by homology-facilitated illegitimate recombination (HFIR) during transformation, as follows: (i) the mean length of acquired DNA was increased in transformants having a net gain of DNA, (ii) the HFIR events became clustered (hot spots) and less dependent on microhomologies, which may have been due to topoisomerase action, and (iii) a novel type of transformants (14%) had integrated foreign DNA with no loss of resident DNA. We concluded that in P. stutzeri upregulation of MutS could enforce sexual isolation and downregulation could increase foreign DNA acquisition and that MutS affects mechanisms of HFIR.In all organisms repair of DNA is an essential process for maintaining DNA integrity and for avoiding mutations. Misincorporated bases remaining after DNA replication or spontaneous base modification in DNA produce mismatches which are repaired by an excision-type mechanism provided by the mismatch repair (MMR) system (11, 56). MMR proteins were first identified in Streptococcus pneumoniae, and the best-characterized MMR proteins are those in Escherichia coli (11,55). In prokaryotes MutS initiates the repair by recognition and binding to the mismatch; this is followed by a search together with MutL on both sides of the mismatch for a site from where excision can be initiated (30). The ubiquity of MutS homologs in all three biological kingdoms (19) underscores the importance of MutS in maintaining the genetic stability of cellular genomes (26). In bacteria loss of the mismatch repair capacity results in mutator strains with increased spontaneous mutation frequencies (23). Such mutator strains have been found among environmental, commensal, and pathogenic isolates of Pseudomonas aeruginosa (59), E. c...

Three nth homologs are all required for efficient repair of spontaneous DNA damage in Deinococcus radiodurans

“…The b subunit of RNA polymerase, which is involved in rifampicin binding, is highly conserved among prokaryotes, and Rif r mutants detected in many bacteria are the result of amino acid exchanges (Garibyan et al 2003). This mutation analysis system had been widely used (Garibyan et al 2003;Wolff et al 2004;Miller et al 2002;Davidsen et al 2005;Meier and Wackernagel 2005;Morlock et al 2000;Anthony et al 2005;Wang et al 2001;Nicholson and Maughan 2002;Maughan et al 2004;Fig. 2 Histogram showing the ratio of GC:AT per mutated rpoB sequences determined from wild-type and mutant strains of D. radiodurans.…”

Deletions of recBCD or recD influence genetic transformation differently and are lethal together with a recJ deletion in Acinetobacter baylyi

“…In this context, it might be recalled that MMR in transformable bacteria is directed by gaps or strand interruption towards elimination of the donor strand (Claverys & Lacks, 1986). In Pseudomonas stutzeri, the transformation with genomic DNA of strains with single nucleotide exchange mutations was only 1.9-fold increased by mutS deficiency, but on average was more than 10-fold increased when a 1.5 kbp fragment was used (Meier & Wackernagel, 2005), indicating that mismatches were formed relatively rarely during transformation with large DNA molecules and more frequently with short fragments. Similarly, in B. subtilis, the transformation with large DNA molecules having a single nucleotide exchange was also only about 1.3-fold increased in an MMR mutant (Majewski & Cohan, 1998).…”

Role of Hypermutability in the Evolution of the GenusOenococcus

“…Mutations in MMR-related genes have been implicated in the hypermutability of numerous clinical isolates, including E. coli (6), Salmonella enterica (27), Haemophilus influenzae (54), Staphylococcus aureus (41), Streptococcus pneumoniae (37), and Pseudomonas aeruginosa (39). Direct inactivation of mutS or mutL in E. coli (36), Bacillus anthracis (60), Staphylococcus aureus (40,41), or Pseudomonas stutzeri (34) has been shown to increase spontaneous mutation frequencies from 10-to 1,000-fold.…”