DNA restriction is a barrier to natural transformation in Pseudomonas stutzeri JM300

Berndt, Cecilia; Meier, Petra; Wackernagel, Wilfried

doi:10.1099/mic.0.26033-0

Cited by 51 publications

(40 citation statements)

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“…Plasmid transfer from P. putida to the restrictionproficient strain P. aeruginosa PAO2002N was typically 3 to 4 orders of magnitude less frequent than that to the restrictiondeficient strain PAO1162N (approximately 10 Ϫ11 versus 10 Ϫ7 ). Similar quantitative effects of restriction proficiency on plasmid transfer efficiency by conjugation and transformation have been observed (3,11,24). Clearly, the restriction-modification system can represent a significant barrier to interspecific horizontal transfer for the PAO strains, contributing to their sexual isolation (15).…”

Section: Discussionsupporting

confidence: 55%

Conjugal TOL Transfer from Pseudomonas putida to Pseudomonas aeruginosa : Effects of Restriction Proficiency, Toxicant Exposure, Cell Density Ratios, and Conjugation Detection Method on Observed Transfer Efficiencies

Pinedo¹,

Smets

2005

Appl Environ Microbiol

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The effects of restriction proficiency and premating exposure to toxicants on conjugal transfer of the TOL plasmid between Pseudomonas spp. was investigated by examinations of filter matings. A Pseudomonas putida KT2442-derived strain carrying a gfp-tagged variant of the TOL plasmid was used as a donor, and both restriction-deficient (PAO1162N) and -proficient (PAO2002N) Pseudomonas aeruginosa strains were used as recipients. The in situ enumeration of conjugation events allowed us to obtain frequency estimates that were unbiased by transconjugant growth or plasmid retransfer. We observed a strong dependence of the plasmid transfer frequency on the initial donor-to-recipient ratio of surface matings, which invalidated the use of mass action-based plasmid transfer kinetic estimators. Careful control of the initial parental cell densities permitted evaluations of the true effects of restriction proficiency and toxicant exposure on TOL transfer. At standard donor-to-recipient ratios (10 ؊3 for PAO1162N and 2 ؋ 10 1 for PAO2002N) and total cell densities (10 5 cells/mm 2 for PAO1162N and 10 6 cells/mm 2 for PAO2002N), plasmid transfer frequencies without toxicant exposure were approximately 10 ؊7 (events/ mm 2 ) ؊1 for PAO1162N and 10 ؊11 (events/mm 2 ) ؊1 for PAO2002N based on in situ observations of conjugation events. The enumeration of transconjugants via selective plating yielded transfer frequencies that were up to 1 order of magnitude lower. Premating exposure to sodium dodecyl sulfate (1 to 10 mM) significantly increased the transfer frequency for the restriction-proficient strain PAO2002N (P < 0.05) but not for the restriction-deficient strain PAO1162N. On the other hand, premating exposure to ethanol, toluene, or phenol had no positive effect on the plasmid transfer frequency. Clearly, restriction proficiency provides a strong barrier to interspecific transfer of the TOL plasmid, and this barrier was only marginally attenuated by recipient exposure to toxicants within the ranges examined.Plasmid transfer is considered an important factor in the adaptation of microbial communities to environmental changes and in bacterial evolution (16). The transfer of plasmids has been demonstrated in situations in which it confers a selective advantage (e.g., the transfer of plasmids that carry catabolic genes in pollutant-laden environments [4,14,19] and of plasmids that carry metal resistance genes in metal-stressed environments [33]) but also when there is apparently no selective advantage (20, 23). The kinetics of conjugal plasmid transfer are influenced by many factors, including the type of organisms involved, the physiological state of the donor (18, 28), energy or nutrient availability (21, 27), and bacterial distribution and density (2, 20, 30), among others. If plasmid transfer is indeed part of an adaptive response to stress, then stress should increase the ability of donors to transfer or of recipients to acquire a plasmid. For identification of the direct effects of toxicant stress on plasmid transfer rates,...

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Section: Discussionsupporting

confidence: 55%

Conjugal TOL Transfer from Pseudomonas putida to Pseudomonas aeruginosa : Effects of Restriction Proficiency, Toxicant Exposure, Cell Density Ratios, and Conjugation Detection Method on Observed Transfer Efficiencies

Pinedo¹,

Smets

2005

Appl Environ Microbiol

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“…These two DNA species could be included in the experiments because the transformation frequencies were generally higher with high-molecular-weight chromosomal DNA than with PCR fragments which increased the values for Rif r transformation frequencies above the background values for spontaneous Rif r mutants (2 ϫ 10 Ϫ6 for mutS). (The higher transformation frequencies with chromosomal DNA were not due to DNA restriction of the nonmodified DNA produced by PCR, because P. stutzeri ATCC 17587 is naturally restriction negative [4].) The results (Table 5) show three things.…”

Section: ϫ4mentioning

confidence: 64%

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

Meier

Wackernagel

2005

J Bacteriol

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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...

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“…Previous studies of the influence of RM systems in conjugation have generated conflicting conclusions in relation to the effect of RM systems on plasmid transfer (8)(9)(10)(11)(12)(13)(14)(15). This has led to some controversy on how restriction-modification systems act on the uptake of single-stranded DNA, such as plasmids transferred by conjugation.…”

Section: Discussionmentioning

confidence: 96%

“…This has led to the view that the conjugational transfer of plasmids through a singlestranded DNA intermediate is immune to restriction by RM systems, as the great majority of these recognize only nonmethylated double-stranded DNA (12)(13)(14)(15)(16)(17). Other studies have, however, contradicted this.…”

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confidence: 99%

The EcoKI Type I Restriction-Modification System in Escherichia coli Affects but Is Not an Absolute Barrier for Conjugation

Roer¹,

Aarestrup²,

Hasman³

2014

J. Bacteriol.

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The rapid evolution of bacteria is crucial to their survival and is caused by exchange, transfer, and uptake of DNA, among other things. Conjugation is one of the main mechanisms by which bacteria share their DNA, and it is thought to be controlled by varied bacterial immune systems. Contradictory results about restriction-modification systems based on phenotypic studies have been presented as reasons for a barrier to conjugation with and other means of uptake of exogenous DNA. In this study, we show that inactivation of the R.EcoKI restriction enzyme in strain Escherichia coli K-12 strain MG1655 increases the conjugational transfer of plasmid pOLA52, which carriers two EcoKI recognition sites. Interestingly, the results were not absolute, and uptake of unmethylated pOLA52 was still observed in the wild-type strain (with an intact hsdR gene) but at a reduction of 85% compared to the uptake of the mutant recipient with a disrupted hsdR gene. This leads to the conclusion that EcoKI restriction-modification affects the uptake of DNA by conjugation but is not a major barrier to plasmid transfer. The exchange of chromosomal and/or extrachromosomal DNA, such as plasmids, viruses, and transposons, is crucial for the evolution of bacteria and their ability to adapt to new environments. Exchange of genetic material occurs among both related and unrelated species of bacteria and is driven by the three horizontal gene transfer (HGT) mechanisms: conjugation, transformation, and transduction (1-3). Restriction-modification (RM) systems are described as major barriers to HGT (4-6) and comprise restriction endonucleases with a cognate methyltransferase. These recognize and cleave DNA not modified by the methyltransferase, thereby making the bacterium able to distinguish between its own (methylated) DNA and incoming nonmethylated DNA.Based on their protein-complex subunit composition and functionality, RM systems can be divided into four types; this study focuses on type I. Type I systems require products of the three genes hsdR (restriction), hsdM (methylation), and hsdS (sequence specificity) and cleave randomly at a remote distance from the recognition sequence. Restriction occurs only when a protein complex of all three gene products (R 2 M 2 S) is formed, whereas methylation of the DNA requires formation of a complex of only the HsdM and HsdS proteins (M 2 S) (7).Some studies have indicated that transfer by conjugation is unaffected by RM systems but that unmodified phage or free DNA in transformation is readily degraded (8-11). This has led to the view that the conjugational transfer of plasmids through a singlestranded DNA intermediate is immune to restriction by RM systems, as the great majority of these recognize only nonmethylated double-stranded DNA (12-17). Other studies have, however, contradicted this. In 1964, Arber and Morse (18) proposed that host specificity (RM systems) might play a role in the acceptance or rejection of DNA transferred by conjugation in Escherichia coli. In many studies from the 1960s, the t...

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DNA restriction is a barrier to natural transformation in Pseudomonas stutzeri JM300

Cited by 51 publications

References 50 publications

Conjugal TOL Transfer from Pseudomonas putida to Pseudomonas aeruginosa : Effects of Restriction Proficiency, Toxicant Exposure, Cell Density Ratios, and Conjugation Detection Method on Observed Transfer Efficiencies

Conjugal TOL Transfer from Pseudomonas putida to Pseudomonas aeruginosa : Effects of Restriction Proficiency, Toxicant Exposure, Cell Density Ratios, and Conjugation Detection Method on Observed Transfer Efficiencies

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

The EcoKI Type I Restriction-Modification System in Escherichia coli Affects but Is Not an Absolute Barrier for Conjugation

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