Genetik, Fachbereich Biologie, Geo- und Umweltwissenschaften, Carl von Ossietzky Universität Oldenburg, POB 2503, D-26111 Oldenburg, Germany
Correspondence
Wilfried Wackernagel
wilfried.wackernagel{at}uni-oldenburg.de
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ABSTRACT |
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INTRODUCTION |
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RM systems are particularly frequent in the genomes of naturally transformable species (Kobayashi, 2001). However, it is not immediately expected that DNA restriction plays a major role during natural transformation since only a single strand of the transforming DNA is transported into the cytoplasm of competent cells (Dubnau, 1999
) and restriction enzymes generally recognize and cleave double-stranded DNA (Redaschi & Bickle, 1996
). In fact, in the three naturally transformable bacteria studied in this respect (Bacillus subtilis, H. influenzae and Streptococcus pneumoniae), a strong effect of restriction was not seen (Bron et al., 1980
; Stuy, 1976
; Lacks & Springhorn, 1984
). Here we examined the influence of the source of DNA on the natural transformation of Pseudomonas stutzeri JM300. Members of the species P. stutzeri have been found worldwide in terrestrial and aquatic habitats, and a large fraction of the isolates obtained from a variety of environmental samples are naturally transformable (Sikorski et al., 1999
, 2002b
). Using cloned P. stutzeri DNA for transformation we found that DNA replicated in Escherichia coli was much less effective in natural transformation and electroporation in strain JM300, while transformation of restriction-deficient mutants derived from JM300 was independent of the source of DNA.
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METHODS |
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Natural transformation assay.
The plate transformation procedure (Meier et al., 2002) was used. The plates with cells and DNA were incubated for 20 h at 37 °C. Transformation frequencies are expressed as transformed cells per total viable count.
DNA manipulations.
Chromosomal DNA from P. stutzeri was isolated using the Qiagen Genomic-tip 100/G. For preparation of plasmid DNA from E. coli or P. stutzeri the Qiagen Plasmid Kit was employed. DNA restriction and cloning of DNA fragments followed standard procedures. The fd phage was isolated from R408 helper phage-infected E. coli KK2186 pPM1 cells and purified by ultracentrifugation and treatment for 15 min with DNase I as described (Meier et al., 2002). The DNA from fd was purified by extraction with phenol four times (Sambrook et al., 1989
). DNA fragments were recovered from agarose gels by a sedimentation procedure (Weichenhan, 1991
).
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RESULTS |
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Isolation and characterization of restriction-deficient mutants
Restriction-deficient (r-) mutants were isolated from a culture of strain JM300 after mutagenesis by nitrosoguanidine. We enriched the culture for r- mutants by three subsequent electroporation steps with non-modified shuttle vector plasmid DNA (prepared from E. coli SF8) able to replicate in P. stutzeri and carrying different antibiotic resistance markers. It was assumed that lack of restriction would increase establishment of non-modified plasmids. For the first electroporation we used pUCPKS (ApR). From about 5000 transformed clones selected on Ap plates an electrocompetent cell suspension was electroporated with pNS1 (GmR, KmR). About 5000 GmR transformed clones were selected and used for the third electroporation step with plasmid pKT210 (CmR, StrR; selection: CmR). Then individual transformant clones were screened for lack of restriction by testing for increased levels of natural transformation with a fourth non-modified plasmid conferring TcR (pUCPT7-4). The screening was performed on agar plates where cells were streaked on a small area of about 1 cm2, then DNA (5 µl of a 60 µg ml-1 solution) was spotted on the area and after 6 h at 37 °C, selection for TcR transformants was applied by spraying the plate with a tetracycline solution. Among 200 screened strains, 12 strains (6 %) with decreased restriction efficiency were identified. Six of these strains (with similar growth and competence as the parental strain) were further characterized after plasmid-free isolates were obtained (strains CB1 to CB6).
Electroporation of strain CB6 with pUCPT7-4 showed that the level of transformation was independent of the source of DNA (Table 2). Similar results were obtained with CB1 to CB5. To test whether the loss of restriction was accompanied by a loss of DNA modification, pUCPT7-4 was isolated from one transformant of each r- mutant and used for electroporation of strain JM300. The level of transformation by plasmid DNA (50 ng per assay) from CB6 (2·7±1·1x10-4; n=3) was as high as that obtained by the plasmid isolated from JM300 (1·8±1·1x10-4; n=5), whereas transformation by control DNA (plasmid from E. coli SF8) was about 100-fold lower (1·7±0·05x10-6; n=4). Results with plasmids from CB1 to CB5 were similar. These data suggest that the mutants CB1 to CB6 had lost their restriction capacity but retained their ability for DNA modification.
Defective restriction renders natural transformation independent of the source of transforming DNA
Natural transformation of JM300 with pCB25 (selection for GmR) was about 43-fold lower when the plasmid came from SF8 and not from JM300 (Table 2). In the restriction-defective mutant CB6 (and similarly in CB1 to CB5) the ratio of transformation frequencies with the two DNA preparations was close to one and at the level seen in JM300 with properly modified DNA. This suggests that (i) the restriction was eliminated in the mutants and (ii) the uptake of P. stutzeri DNA replicated in E. coli SF8 was as efficient as that of DNA replicated in P. stutzeri JM300.
Since during natural transformation only a single strand enters the cytoplasm (Dubnau, 1999), which is normally not a target of restriction enzymes, we considered the possibility that due to the rather high plasmid DNA concentration in these assays, frequently strands of the transforming DNA with opposite polarity would enter a cell and after annealing, might be attacked by restriction enzymes. Such a situation has been described previously in natural transformation of B. subtilis with phage DNA (transfection) where strong restriction of the phage DNA was observed (Bron et al., 1980
). At low DNA concentrations, when cells take up at best only one strand, restriction should be minimal if annealed DNA was the major substrate for restriction. This was not the case. When derivatives of JM300 and CB6 having a chromosomal hisX : : GmR gene (strains JB12 and CB61, respectively) were transformed (selection: His+) with pPM1 (carrying hisX+) isolated from SF8, the approximately 50-fold lower transformation of JB12 compared to CB61 seen at high DNA concentrations (33 µg ml-1) was also seen at concentrations down to 0·03 µg ml-1 (Fig. 2
), which corresponds to about 0·1 plasmid per recipient cell (Fig. 2
). This result indicates that restriction during natural transformation with cloned chromosomal DNA cannot be explained by restriction of annealed donor DNA strands.
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DISCUSSION |
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It has been stated that restriction does not act during natural transformation (Majewski, 2001) because a single strand (which is not a normal substrate of restriction endonucleases) is delivered into the cell and subsequently recombines with the chromosome, giving a hemimethylated duplex, which is normally not restricted either. Transformation studies in species (B. subtilis, H. influenzae and S. pneumoniae) in which restricting and non-restricting isogenic strains were available supported this view (Bron et al., 1980
; Stuy, 1976
; Lacks & Springhorn, 1984
), while transfection was strongly decreased by restriction in H. influenzae and B. subtilis. The latter presumably resulted from cytoplasmic annealing of phage DNA strands leading to restriction-sensitive non-modified duplexes. On the other hand, it was found later that the vast majority of restriction endonucleases also cleave non-modified single-stranded DNA, although at a lower rate than corresponding duplexes (Nishigaki et al., 1985
; Horiuchi & Zinder, 1975
; Blakesley & Wells, 1975
). This opens the general possibility of restriction during natural transformation. Interestingly, in B. subtilis the BsuI restriction enzyme cleaves purified single-stranded phage DNA in vitro quite effectively but in vivo it does not reduce the transfecting activity of purified phage single strands in marker rescue assays employing coinfection with modified phage (Bron et al., 1980
). It was concluded that the complexing of the single strand with single-strand binding protein upon entering the cytoplasm (see Lacks, 1999
) protects the DNA from restriction. This protection gets lost upon annealing with a non-modified complementary strand. A different situation is observed in S. pneumoniae harbouring the DpnII RM system. These cells express the single-strand-specific modification methyltransferase during competence leading to effective protection of transforming DNA (Lacks et al., 2000
).
At what stage in natural transformation of P. stutzeri could restriction act? Presynaptic and postsynaptic attacks of restriction endonucleases may be considered. Perhaps different from B. subtilis, in P. stutzeri the DNA binding protein associating with the single-stranded DNA does not protect DNA against restriction. This could largely eliminate its transforming potential if these sites are frequently present in the DNA. A postsynaptic attack could occur if the non-modified strand was integrated into hemimethylated genomic regions in a way producing duplex regions lacking methylation. The resulting double-strand break would be lethal in the absence of repair and would thus eliminate the transformant. Hemimethylated DNA is expected to be transient and close behind the replication fork. A double-strand break in this region can be repaired by recBCD-dependent recombination using the other replicated duplex as homologous DNA (Kuzminov, 1999). Since the repair involves degradation at DNA ends the genetic marker may often be lost. The 40-fold reduction in natural transformation would require that DNA is almost always incorporated at hemimethylated sites and in a way leading to replacement of the methylated strand. This is difficult to understand.
Any barrier that will limit intra- and interspecific genetic exchange in prokaryotes will contribute to sexual isolation and thereby will foster speciation (Majewski, 2001). In contrast to observations in B. subtilis, H. influenzae and S. pneumoniae, our data with P. stutzeri JM300 and ATCC 17587 suggest that restriction can contribute to sexual isolation among transformable bacteria. In a recent study on intra- and interspecific transformation in Pseudomonas, it was observed that strain ATCC 17587 described here as having no RM system was the strain least isolated from other species (including Pseudomonas alcaligenes and Pseudomonas mendocina) and also from members of other genomic groups of P. stutzeri (including members of six genomovars), whereas strain JM300 was most strongly isolated from all strains, even from the strain most closely related to JM300 in that study (Lorenz & Sikorski, 2000
). Moreover, the sexual isolation of JM300, when acting as a recipient for DNA from the other strains, was much stronger than expected on the basis of the nucleotide sequence divergence to the different donor DNAs. The authors suspected that besides sequence divergence and a different level of DNA uptake competence, other not yet identified genetic factors could contribute to the sexual isolation of P. stutzeri JM300. The restriction system of JM300 described here could be such a factor. Presently, only five RM systems have been detected in P. stutzeri (see http://rebase.neb.com), and it is not yet known how frequent and diverse RM systems are among the over 500 members of that species identified by molecular methods (Sikorski et al., 2002a
, b
). Several authors have pointed out that RM systems can contribute to an increase of genetic diversity in prokaryotes by allowing natural genetic engineering involving homologous and illegitimate recombination processes and by exerting certain selection pressure on the DNA sequence (Arber, 1991
; Kusano et al., 1997
; McKane & Milkman, 1995
; Rocha et al., 2001
). The data of this study add the notion that RM systems can contribute to speciation also by producing sexual isolation and thereby allowing the free divergence of the isolated lineage.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Arber, W. (2000). Genetic variation: molecular mechanisms and impact on microbial evolution. FEMS Microbiol Rev 24, 17.[CrossRef][Medline]
Bagdasarian, M., Lurz, R., Rückert, B., Franklin, F. C. H., Bagdasarian, M. M., Frey, J. & Timmis, K. N. (1981). Broad host range, high copy number, RSF1010-derived vectors, and a host-vector system for gene cloning in Pseudomonas. Gene 16, 237247.[CrossRef][Medline]
Bickle, T. A. & Krüger, D. H. (1993). Biology of DNA restriction. Microbiol Rev 57, 434450.[Medline]
Blakesley, R. W. & Wells, R. D. (1975). Single-stranded DNA from phiX174 and M13 is cleaved by certain restriction endonucleases. Nature 257, 421422.[Medline]
Bron, S., Luxen, E. & Venema, G. (1980). Restriction and modification in B. subtilis. Mol Gen Genet 179, 103110.[Medline]
Brunschwig, E. & Darzins, A. (1992). A two-component T7 system for the overexpression of genes in Pseudomonas aeruginosa. Gene 111, 3541.[CrossRef][Medline]
Canosi, U., Iglesias, A. & Trautner, T. A. (1981). Plasmid transformation in Bacillus subtilis: effects of insertion of Bacillus subtilis DNA into plasmid pC194. Mol Gen Genet 181, 434440.[Medline]
Carlson, C. A., Pierson, L. S., Rosen, J. J. & Ingraham, J. L. (1983). Pseudomonas stutzeri and related species undergo natural transformation. J Bacteriol 153, 9399.[Medline]
Dubnau, D. (1999). DNA uptake in bacteria. Annu Rev Microbiol 53, 217244.[CrossRef][Medline]
Horiuchi, K. & Zinder, N. D. (1975). Site-specific cleavage of single-stranded DNA by a Haemophilus restriction endonuclease. Proc Natl Acad Sci U S A 72, 25552558.[Abstract]
Kobayashi, I. (2001). Behavior of restriction-modification systems as selfish mobile elements and their impact on genome evolution. Nucleic Acids Res 29, 37423756.
Kusano, K., Sakagami, K., Yokochi, T., Naito, T., Tokinaga, Y., Ueda, E. & Kobayashi, I. (1997). A new type of illegitimate recombination is dependent on restriction and homologous interaction. J Bacteriol 179, 53805390.[Abstract]
Kuzminov, A. (1999). Recombinational repair of DNA damage in Escherichia coli and bacteriophage . Microbiol Mol Biol Rev 63, 751813.
Lacks, S. A. (1999). DNA uptake by transformable bacteria. In Transport of Molecules Across Microbial Membranes (Society for General Microbiology Symposium no. 58), pp. 138168. Edited by J. K. Broome-Smith, S. Baumberg, C. J. Stirling & F. B. Ward. Cambridge: Cambidge University Press.
Lacks, S. A. & Springhorn, S. S. (1984). Transfer of recombinant plasmids containing the gene for DpnII DNA methylase into strains of Streptococcus pneumoniae that produce DpnI or DpnII restriction endonucleases. J Bacteriol 158, 905909.[Medline]
Lacks, S. A., Ayalew, S., de la Campa, A. G. & Greenberg, B. (2000). Regulation of competence for genetic transformation in Streptococcus pneumoniae: expression of dpnA, a late competence gene encoding a DNA methyltransferase of the DpnII restriction system. Mol Microbiol 35, 10891098.[CrossRef][Medline]
Levin, B. R. & Bergstrom, C. T. (2000). Bacteria are different: observations, interpretations, speculations, and opinions about the mechanisms of adaptive evolution in prokaryotes. Proc Natl Acad Sci U S A 97, 69816985.
Lorenz, M. G. & Sikorski, J. (2000). The potential for intraspecific horizontal gene exchange by natural genetic transformation: sexual isolation among genomovars of Pseudomonas stutzeri. Microbiology 146, 30813090.
Lorenz, M. G. & Wackernagel, W. (1991). High frequency of natural genetic transformation of Pseudomonas stutzeri in soil extract supplemented with carbon/energy and phosphorus source. Appl Environ Microbiol 57, 12461251.
Lorenz, M. G. & Wackernagel, W. (1994). Bacterial gene transfer by natural genetic transformation in the environment. Microbiol Rev 58, 563602.[Medline]
Lorenz, M. G., Meyer, B., Wittstock, M., Graupner, S. & Wackernagel, W. (1998). Selective DNA uptake and DNA restriction as barriers to horizontal gene exchange by natural genetic transformation in Pseudomonas stutzeri JM300. In Horizontal Gene Transfer, pp. 131143. Edited by M. Syvanen & C. Kado. London: Chapman & Hall.
Majewski, J. (2001). Sexual isolation in bacteria. FEMS Microbiol Lett 199, 161169.[CrossRef][Medline]
McKane, M. & Milkman, R. (1995). Transduction, restriction and recombination patterns in Escherichia coli. Genetics 139, 3543.
Meier, P., Berndt, C., Weger, N. & Wackernagel, W. (2002). Natural transformation of Pseudomonas stutzeri by single-stranded DNA requires type IV pili, competence state and comA. FEMS Microbiol Lett 207, 7580.[CrossRef][Medline]
Meselson, M., Yuan, R. & Heywood, J. (1972). Restriction and modification of DNA. Annu Rev Biochem 41, 447466.[CrossRef][Medline]
Murray, N. E. (2000). Type I restriction systems: sophisticated molecular machines (a legacy of Bertani and Weigle). Microbiol Mol Biol Rev 64, 412434.
Nishigaki, K., Kaneko, Y., Wakuda, H., Husimi, Y. & Tanaka, T. (1985). Type II restriction endonucleases cleave single-stranded DNAs in general. Nucleic Acids Res 13, 57475760.[Abstract]
Ochman, H., Lawrence, J. G. & Groisman, E. A. (2000). Lateral gene transfer and the nature of bacterial innovation. Nature 405, 299304.[CrossRef][Medline]
Ostendorf, T., Cherepanov, P., de Vries, J. & Wackernagel, W. (1999). Characterization of a dam mutant of Serratia marcescens and nucleotide sequence of the dam region. J Bacteriol 181, 38803885.
Rayssiguier, C., Thaler, D. S. & Radman, M. (1989). The barrier to recombination between Escherichia coli and Salmonella typhimurium is disrupted in mismatch-repair mutants. Nature 342, 396401.[CrossRef][Medline]
Redaschi, N. & Bickle, T. A. (1996). DNA restriction and modification systems. In Escherichia coli and Salmonella, 2nd edn, pp. 773781. Edited by F. C. Neidhardt and others. Washington, DC: American Society for Microbiology.
Roberts, M. S. & Cohan, F. M. (1993). The effect of DNA sequence divergence on sexual isolation in Bacillus. Genetics 134, 401408.
Rocha, E. P. C., Danchin, A. & Viari, A. (2001). Evolutionary role of restriction/modification systems as revealed by comparative genome analysis. Genome Res 11, 946958.
Romanowski, G., Lorenz, M. G., Sayler, G. & Wackernagel, W. (1992). Persistence of free plasmid DNA in soil monitored by various methods, including a transformation assay. Appl Environ Microbiol 58, 30123019.[Abstract]
Rossello, R. A., Garcia-Valdes, E., Lalucat, J. & Ursing, J. (1991). Genotypic and phenotypic diversity of Pseudomonas stutzeri. Syst Appl Microbiol 14, 150157.
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Schweizer, H. P. (1991). Escherichia-Pseudomonas shuttle vectors derived from pUC18/19. Gene 97, 109112.[CrossRef][Medline]
Schweizer, H. P. (1993). Small broad-host-range gentamicin resistance gene cassettes for site-specific insertion and deletion mutagenesis. BioTechniques 15, 831833.[Medline]
Sikorski, J., Graupner, S., Lorenz, M. G. & Wackernagel, W. (1998). Natural genetic transformation of Pseudomonas stutzeri in a nonsterile soil. Microbiology 144, 569576.[Abstract]
Sikorski, J., Rossello-Mora, R. & Lorenz, M. G. (1999). Analysis of genotypic diversity and relationships among Pseudomonas stutzeri strains by PCR-based genomic fingerprinting and multilocus enzyme electrophoresis. Syst Appl Microbiol 22, 393402.[Medline]
Sikorski, J., Möhle, M. & Wackernagel, W. (2002a). Identification of complex composition, strong strain diversity and directional selection in local Pseudomonas stutzeri populations from marine sediment and soils. Environ Microbiol 4, 465476.[CrossRef][Medline]
Sikorski, J., Teschner, N. & Wackernagel, W. (2002b). Highly different levels of natural transformation are associated with genomic subgroups within a local population of Pseudomonas stutzeri from soil. Appl Environ Microbiol 68, 865873.
Smith, H. O., Gwinn, M. L. & Salzberg, S. L. (1999). DNA uptake signal sequences in naturally transformable bacteria. Res Microbiol 150, 603616.[CrossRef][Medline]
Stanier, R. Y., Palleroni, N. J. & Doudoroff, M. (1966). The aerobic pseudomonads: a taxonomic study. J Gen Microbiol 43, 159271.[Medline]
Stuy, J. H. (1976). Restriction enzymes do not play a significant role in Haemophilus homospecific or heterospecific transformation. J Bacteriol 128, 212220.[Medline]
Watson, A. A., Alm, R. A. & Mattick, J. S. (1996). Construction of improved vectors for protein production in Pseudomonas aeruginosa. Gene 172, 163164.[CrossRef][Medline]
Weichenhan, D. (1991). Fast recovery of DNA from agarose gels by centrifugation through blotting paper. Trends Genet 7, 109.
Wilson, G. G. & Murray, N. E. (1991). Restriction and modification systems. Annu Rev Genet 25, 585627.[CrossRef][Medline]
Zahrt, T. C. & Maloy, S. (1997). Barriers to recombination between closely related bacteria: MutS and RecBCD inhibit recombination between Salmonella typhimurium and Salmonella typhi. Proc Natl Acad Sci U S A 94, 97869791.
Received 2 October 2002;
revised 14 January 2003;
accepted 16 January 2003.
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