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Journal of Bacteriology, January 2005, p. 143-154, Vol. 187, No. 1
0021-9193/05/$08.00+0     doi:10.1128/JB.187.1.143-154.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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

Petra Meier and Wilfried Wackernagel*

Genetics, Department of Biology and Environmental Sciences, Carl von Ossietzky University Oldenburg, Oldenburg, Germany

Received 23 July 2004/ Accepted 17 September 2004

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 mismatch-binding 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.

* Corresponding author. Mailing address: Genetik, Institut für Biologie und Umweltwissenschaften, Fakultät für Mathematik und Naturwissenschaften, C.v.O. Universität Oldenburg, POB 2503, D-26111 Oldenburg, Germany. Phone: 49-441-798 3298. Fax: 49-441-798 5606. E-mail: wilfried.wackernagel{at}uni-oldenburg.de.

Journal of Bacteriology, January 2005, p. 143-154, Vol. 187, No. 1
0021-9193/05/$08.00+0     doi:10.1128/JB.187.1.143-154.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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