Marine Mikrobiologie, Fachbereich Biologie/Chemie, Zentrum für Umweltforschung und Umwelttechnologie, Universität Bremen, D-28359 Bremen, Germany1
Genetik, Fachbereich Biologie, Universität Oldenburg, Postfach 2503, D-26111 Oldenburg, Germany2
Author for correspondence: Michael G. Lorenz. Tel: +49 421 218 7224. Fax: +49 421 218 7222. e-mail: mglorenz{at}biotec.uni-bremen.de
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ABSTRACT |
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Keywords: Pseudomonas stutzeri, genomovars,, heterogamic transformation, sexual isolation, rpoB phylogeny
The EMBL accession numbers for the sequences reported in this paper are given in Methods.
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INTRODUCTION |
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An increasing body of evidence points at natural genetic transformation as an important mechanism of the horizontal exchange of genes (Dubnau, 1999 ; Lorenz & Wackernagel, 1994
; Paget & Simonet, 1994
). Natural genetic transformation (termed transformation in the following) is a gene-transfer process which involves the binding of exogenous DNA to specific cell surface receptors, DNA processing (including degradation of one strand) and the subsequent active transport of single-stranded DNA into the cytoplasm where it is recombined with the recipient chromosome (for reviews see Dubnau, 1999
; Grossman, 1995
; Lorenz & Wackernagel, 1994
). Transformation is unique to the domains Bacteria and Archaea (Lorenz, 1998
). More than 40 species have been described as being transformable so far belonging to phylogenetic branches such as the Proteobacteria, Green Sulphur Bacteria, Cyanobacteria, low and high G+C bacteria (Firmicutes), the DeinococcusThermus group and the Euryarchaeota. This, together with the homology of competence genes among Gram-negative and Gram-positive bacteria (Dubnau, 1999
), suggests that transformation is a phylogenetically old property. Transformation appears to take place at all environmental sites where bacteria live, among them the marine, freshwater, soil and plant environments (Bertolla et al., 1999
; Lorenz & Wackernagel, 1994
; Paget & Simonet, 1994
). Studies performed so far with respect to sexual isolation in naturally transformable species and the mechanisms behind it have included mainly B. subtilis and Haemophilus influenzae (Albritton et al., 1984
; Roberts & Cohan, 1993
; for a review see Lorenz & Wackernagel, 1994
). The most intensive study performed so far included a desert soil population of B. subtilis where sexual isolation among subpopulations and between B. subtilis and other named Bacillus species was shown to rely on DNA restriction (albeit only marginally) and nucleotide sequence divergence of the donor DNA from the recipient chromosome (Majewski & Cohan, 1999
; Roberts & Cohan, 1993
; Zawadzki et al., 1995
).
The naturally transformable ubiquitous saprophyte, Pseudomonas stutzeri, is a promising organism to investigate gene transfer and sexual isolation mechanisms because the species has a well defined taxonomic substructure. DNADNA hybridizations have shown the existence of seven genomovars (Rosselló et al., 1991 ; Rosselló-Mora et al., 1996
). The genomovar structure of P. stutzeri is consistent with the finding of seven genotypic groups in a genomic fingerprint analysis (Sikorski et al., 1999
). Variable grouping of various strains in cluster analyses of genomic fingerprints and macrorestriction patterns indicated that chromosomal rearrangements, presumably also a result of horizontal gene transfer, have occurred and contributed to the extremely high genotypic diversity within this species (Bennasar et al., 1998
; Ginard et al., 1997
; Rainey et al., 1994
; Sikorski et al., 1999
).
Here quantitative data are presented on the transformability and heterogamic transformation of P. stutzeri strains affiliated to the seven genotypic groups. Possible mechanisms and evolutionary implications of the observed sexual isolation among and within groups are discussed.
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METHODS |
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Amplification and sequencing of part of the RNA polymerase ß gene.
Amplification of part of the RNA polymerase ß gene of all strains except LO147, LO151, LO139 and LO145 was done in 50 µl reaction mixtures including 25 ng chromosomal DNA, 1 µM primer +43F (5'-CCGCAAGACCTGATCAACGCC-3'; P. putida position 15141534, accession no. X15849) and primer +688R (5'-TGCCGGTACCCACCAGCG-3'; position 21422159), 50 µM dNTPs (Pharmacia) and 1·0 U Taq DNA Polymerase (Promega) in supplied reaction buffer. The following cycling protocol was performed: one cycle at 94 °C for 5 min, 40 cycles of 94 °C for 1 min, 64 °C for 1 min and 72 °C for 3 min, and a final primer extension at 72 °C for 10 min in a Perkin Elmer 480 apparatus. For strains LO147, LO151, LO139 and LO145, the amplification protocol was as above, except that primer annealing was at 58 °C and internal primers rpoB-Fint (5'-CCAGCCAGCTNTCGCNGTTC-3'; position 15721591) and rpoB-Rint (5'-CGGTTGGCGTCGTCGTGCTC-3'; position 20632082) were used. The PCR products were resolved on agarose gels (1·1%) including ethidium bromide. The bands of expected fragment size (646 bp; 511 bp for LO147, LO139 and LO145) were extracted from the gel using the Qiagen Gel Extracting Kit. The purified PCR products were reamplified using the initial amplification protocol. The obtained PCR products were purified as above and then sequenced with primer +43F (primer rpoB-Fint for LO151, LO139 and LO145) employing the dye terminator technology. The nucleotide sequence data of partial rpoB genes can be retrieved from the EMBL nucleotide sequence database under the following accession numbers: AJ279953 (LO177), AJ279954 (LO179), AJ279955 (LO151), AJ279956 (LO147), AJ279958 (LO137), AJ279959 (LO199), AJ279960 (LO159), AJ279961 (LO145), AJ279962 (LO169T), AJ279963 (LO171), AJ279964 (LO167), AJ279965 (LO139), AJ279966 (LO165T), AJ279967 (LO191T).
Phylogenetic reconstruction and sequence divergence.
Alignment of rpoB sequences was done using CLUSTAL X version 1.64b (Thompson et al., 1997 ) with default parameters (gap opening 10·00, gap extension 0·05, delay divergent sequences 40%, DNA transition weight 0·50). The alignment was corrected manually. For phylogenetic analysis, a 422 nt stretch from the alignment (Pseudomonas putida position 15912012) was used to generate a neighbour-joining dendrogram from a Jukes & Cantor distance matrix (TREECON software; Van de Peer & De Wachter, 1994
). Bootstrap values were obtained with 100 replicates.
Statistical analysis.
Experiments were repeated at least once. Statistical significance of differences between means was calculated using the Students t-test.
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RESULTS |
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Using LO147 as a recipient, the transformation frequency with DNA from the other group A strain, LO151, was at a level (P=0·72, n=2) very similar to the frequency of homogamic transformation (Fig. 1a). This confirmed, on a genetic basis, the close relationship of LO147 with LO151. In contrast, heterogamic transformation of LO147 with donor DNA of P. mendocina LO191T and P. alcaligenes LO165T was nearly two orders of magnitude lower than in homogamic transformation (Fig. 1a
). This indicated that P. stutzeri LO147 was sexually isolated from strains of the other species employed. Also, transformation frequencies with donors from P. stutzeri groups other than group A were significantly lower than in homogamic transformation (P=0·05, n=2), except LO169T (group D; P=0·14) and LO137 (group G; P=0·17, n=2). The data indicated that LO147 is sexually isolated from at least eight of the ten P. stutzeri strains belonging to other groups. The levels of sexual isolation from other groups ranged from 0·5 to 1·2 orders of magnitude lower heterogamic than homogamic transformation frequencies and thus were lower than those from the other pseudomonad species.
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An unexpected finding was that the heterogamic transformation frequency of LO179 with DNA of the other group H strain, LO177, was more than two orders of magnitude lower than the transformation frequency with LO179 DNA. In fact, the level of transformation with the LO177 donor was at a level comparable with that of several strains from other groups (e.g. LO159, group F2; LO171, group D; Fig. 1c). Still other donors (e.g. LO151, group A; LO145, group F1) were as inefficient in transforming LO179 as were donors from the other pseudomonad species employed, the transformation frequencies lying in the range of the spontaneous mutation frequency (see Fig. 1
). These results showed that LO179 is sexually isolated not only from other species and other P. stutzeri groups as observed with LO147 and LO177, but even from its next relative in the same group.
DNA competition
The finding that LO179 was poorly transformed by DNA of the other group member, LO177 (Fig. 1c), prompted us to perform experiments on the specificity of DNA entry in LO179. Transformation of this strain is reported to be greatly insensitive to the presence of heterologous DNA [calf thymus, E. coli (Carlson et al., 1983
; Lorenz et al., 1998
; Lorenz & Wackernagel, 1990
)]. Therefore, DNA competition studies were done using homogamic transforming DNA (RifR) and competing non-transforming (RifS) DNA from various sources at increasing mass excess (Fig. 2
).
In the control, inhibition of transformation matched the theoretical curve (Fig. 2a), that is, the transformation frequency declined proportionally to the amounts of competing non-transforming homogamic DNA added. Transformation was inhibited also by calf thymus DNA (Fig. 2b
) albeit to a much lower extent than by competing homogamic DNA (Fig. 2a
). For instance, at a 10-fold excess inhibition by calf thymus DNA was similarly low (35%) as observed in previous experiments using competing E. coli DNA (22%; Lorenz & Wackernagel, 1990
). Somewhat more inhibition of transformation was obtained with phage P22 DNA at 10-fold mass excess (Fig. 2b
). However, the level of inhibition (68·5%) was lower than expected (91%). With increasing amounts of competing heterogamic RifS LO177 DNA transformation of LO179 was reduced proportionally (Fig. 2c
) similar to what was observed with competing non-transforming homogamic DNA (Fig. 2a
). Hence we conclude that heterogamic LO177 DNA entered competent LO179 cells as freely as homogamic LO179 DNA.
Phylogeny of P. stutzeri based on rpoB nucleotide sequence
PCR primers were designed for amplification of a part of the gene encoding the ß subunit of RNA polymerase (rpoB). For this purpose the rpoB sequence of P. aeruginosa covering the region of rifampicin resistance (EMBL accession no. M99386) was aligned with the homologous sequence of P. putida (EMBL X15849). From this, primers 43f (P. putida position 1514) and 688r (position 2142) were constructed which gave amplification products of the expected size (646 bp) with all strains except strains LO147, LO151, LO139 and LO145. With these strains, an internal primer pair, rpoB-Fint and rpoB-Rint (P. putida positions 1572 and 2063, respectively), of the 646 bp region resulted in an amplification product of the expected size (511 bp). Determination of the nucleotide sequence of the PCR products showed 82·286·7% nucleotide sequence identity to the rpoB region of P. aeruginosa (alignments with 422 bp).
A tree was constructed on the basis of rpoB nucleotide sequences to analyse the genealogical relationships of the strains used. The results (Fig. 3) showed that, in accord with a previous tree obtained on the basis of 16S rDNA sequences (Sikorski et al., 1999
), all P. stutzeri strains grouped in a monophyletic branching pattern. Strains clustered in groups which were in harmony with the grouping pattern according to DNA hybridization (genomovars; Rosselló et al., 1991
; Rosselló-Mora et al., 1996
), 16S rDNA and genomic fingerprinting analyses (RREBM; Sikorski et al., 1999
; see Table 1
). However, the branching order of rpoB clusters could not be resolved as indicated by the low values of the bootstrap analysis. This finding is equivalent to that of 16S rDNA analysis (Sikorski et al., 1999
).
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Heterogamic transformation experiments were conducted that employed PCR-amplified rpoB donor DNA with transforming activity, and LO179 and LO177 as recipients, respectively. Transformation of LO179 was very inefficient and the heterogamic transformation frequency with the LO177 PCR product already reached the detection limit. In contrast, LO177 was transformed at much higher frequencies by heterogamic PCR products (see below). Therefore, LO177 was used as a recipient in the following experiments. Transformation of LO177 showed a linear response (1·8x10-64·9x10-5 transformation frequency) to the concentration of LO177 PCR amplified DNA (1·515 µg DNA ml-1) and saturation was approximated at 18 µg DNA ml-1 (4·1x10-5 transformation frequency). The correlation of sexual isolation, defined as the log-transformed data of the ratio of homogamic and heterogamic transformation frequencies, and rpoB sequence divergence (% mismatches) was investigated. The results of this analysis (Fig. 4) indicated that sexual isolation increased with rpoB sequence divergence. Sexual isolation increased over 1·85 log units up to 7·2% mismatches (LO159). Values of sexual isolation hardly increased (1·72·15 log units; limit of detection, 2·46 log units) at higher degrees of sequence divergence of amplified donor rpoB DNA (7·89·5% mismatches). The data of heterogamic transformations with chromosomal DNA from Fig. 1
were also included in the analysis. The results (Fig. 4
) indicated that sexual isolation increased by 2·5 log units up to 7·8% mismatches (LO139). At higher sequence divergence, sexual isolation increased only by another 0·5 log units (limit of detection, 4·26 log units). A marked difference between sexual isolation values of PCR-amplified and chromosomal DNA transformations was noticed, in particular at 1·7% (LO179) and 7·2% (LO159) mismatches.
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DISCUSSION |
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The potential of P. stutzeri for intraspecific horizontal gene transfer by transformation was investigated. For six strains falling into five of the seven genomic groups, transformability to rifampicin resistance was demonstrated (Table 2). For the other six strains, the RifR frequencies in experiments with and without DNA could not be distinguished. These strains may have developed only extremely low, if any, DNA uptake competence under the conditions employed here. The data are in overall accord with previous qualitative transformability assays (Carlson et al., 1983
), except that for one strain (LO151) reported transformability could not be supported here. A qualitative effect of the DNA preparation or a marker effect is probably not an explanation for the observed lack of transformability because LO151 DNA transformed LO147 at high frequency (Fig. 1
). The fact that transformability of P. stutzeri is confined to distinct strains and not a general phenomenon of the species matches observations made with other transformable species including a B. subtilis natural population (85% transformable strains; Cohan et al., 1991
) and H. influenzae clinical isolates (42% transformable strains; Rowji et al., 1989
). Among the transformable P. stutzeri strains, variation of the level of competence was high (three orders of magnitude, Table 2
) and thus comparable to ranges in competence found with B. subtilis and H. influenzae (Cohan et al., 1991
; Rowji et al., 1989
). It appears that some strains have a higher potential for gene acquisition than others developing low or no competence. However, whether this is true in the natural environment is not clear inasmuch as competence in P. stutzeri (LO179) can increase as a response to nutrient starvation (Lorenz & Wackernagel, 1991
). It can not be excluded that strains, including those with low competence under standardized high-nutrient conditions, may have an increased capability of DNA uptake in the environment.
The evolutionary impact leading to the observed polymorphism in transformability and competence levels in P. stutzeri is not known. In another transformable species, S. pneumoniae, lack of transformability was associated with a deficiency in the production of competence factor (Yother et al., 1986 ). Perhaps allelic variation of competence genes also accounts for the different phenotypes observed among P. stutzeri strains. Variation in competence levels could be a reflection of niche-specific selection as Rainey et al. (1994)
proposed with regard to the considerable heterogeneity in other phenotypic properties including fatty acid and substrate utilization profiles among strains. On the other hand, Cohan et al. (1991)
interpreted their results of high variability of competence levels within a natural population of B. subtilis as an indication of low, if any, selection on rates of recombination. Clearly, more information is necessary with regard to competence levels in natural populations of P. stutzeri from different geographical locations and habitats in order to address this problem.
Sexual isolation among and within P. stutzeri taxonomic groups
Isolates from different habitats were found in common genotypic groups (H and G; Tables 1 and 2
). Preliminary results obtained with wild isolates from a marine sediment suggested that P. stutzeri strains from different 16S rRNA gene sequence clusters can inhabit common ecological niches (J. Sikorski, N. Teschner & W. Wackernagel, unpublished results). It has been reported previously that DNA can be taken up in marine sediment and soil microcosms by added competent bacteria including P. stutzeri and that transformation among genetically marked isogenic strains can occur in non-sterile soil (Gallori et al., 1994
; Lorenz et al., 1988
; Nielsen et al., 1997
; Paul et al., 1991
; Romanowski et al., 1993
; Sikorski et al., 1998
; Stewart et al., 1991
).
A major finding of this study was that heterogamic transformation frequencies of three recipients from two groups with DNA of representatives of other groups and species were significantly lower than homogamic transformation frequencies (Fig. 1). This indicated that mechanisms exist in these two taxonomic groups that lead to sexual isolation from other groups and species. This may hold true also for transformable members of the other groups of P. stutzeri. Apparently, sexual isolation is a quantitative character in P. stutzeri transformation. This adds to findings obtained with a B. subtilis desert soil population (Cohan et al., 1991
). The levels of sexual isolation determined in P. stutzeri were considerably different among the three recipients tested (Fig. 1
). Group A strain LO147 (Fig. 1a
) was least isolated from the other P. stutzeri groups (maximally by a factor of 17 from group H), followed by group H strains with considerably higher sexual isolation (LO177) up to a factor of 300 from group E and LO179 up to a factor of 4000 from group F1. A striking observation was that within group H, LO179 was sexually isolated from LO177 (Fig. 1c
). This was not reciprocal: LO177 was transformed at similar frequencies by its own and by LO179 DNA (Fig. 1b
). Collectively these data add to the great genotypic diversity found in P. stutzeri. Some strains like LO147 (group A) may be transformed by heterogamic DNA from other P. stutzeri groups more readily than others like LO177 and LO179 (group H).
Free entry of heterogamic DNA into competent cells
DNA competition studies indicated that transformation of LO179 was only partially inhibited by an excess of heterologous calf thymus DNA (Fig. 2b). This suggested discrimination of unrelated DNA during uptake. However, it is improbable that a mechanism exists in LO179 that is like other transformation systems including those operative in N. gonorrhoeae and H. influenzae where discrimination against heterologous DNA during transport into the cell is strict when the donor DNA lacks specific recognition sequences required for uptake (for a review see Lorenz & Wackernagel, 1994
). The finding that unrelated phage P22 DNA showed considerable inhibition of LO179 transformation (although not as high as expected when DNA of any source enters the cell at equal probability; see Fig. 2b
) supports this conclusion. The observation of non-proportional inhibition of LO179 transformation by unrelated DNA requires further experimentation which, however, is out of the scope of the present study. With regard to the objective of this study it is relevant to note that similar inhibition patterns of transformation were observed when competing non-transforming homogamic and heterogamic LO177 DNA were employed in competition studies (compare Fig. 2a
and c
). This allows us to conclude that LO177 DNA was taken up as efficiently as homogamic DNA by competent LO179 cells. Similar results were obtained with LO177 as recipient and competing RifS homogamic and LO179 DNA (data not shown). Hence inefficient uptake of heterogamic DNA is not an explanation for sexual isolation of LO179 from LO177. This probably also holds true for transformations of LO179 employing DNAs from other groups and the other Pseudomonas species: DNA from a P. stutzeri strain that, according to genomic fingerprint and phylogenetic analysis (16S rDNA), was shown to be most closely related to P. mendocina (J. Sikorski & M. G. Lorenz, unpublished results) inhibited transformation of LO179 in a similar pattern as did homogamic non-transforming DNA (data not shown).
Sensitivity of sexual isolation to nucleotide sequence divergence
The level of sexual isolation of LO177 increased with rpoB sequence divergence both with PCR-amplified and chromosomal DNA (Fig. 4). However, the quantitative effects on sexual isolation were very different. With PCR-amplified RifR DNA, the next closest relative, LO179, was clearly isolated from LO177, whereas an effect of sequence divergence was not apparent with chromosomal LO179 DNA. Further, there was a markedly (0·95 log units) higher extent of sexual isolation with PCR-amplified DNA than with chromosomal DNA at 7·2% mismatched sequences (LO159). In their studies employing a B. subtilis population, Roberts & Cohan (1993)
discussed the lower sensitivity of sexual isolation on sequence divergence in transformation with large chromosomal DNA fragments compared to small DNA molecules (PCR-amplified 3366 bp rpoB fragment) as an effect of the increased stability of long-stretched flanking sequences in the heteroduplex. This hypothesis would also explain the observations made with LO177. With B. subtilis 168 Marburg donor strain and natural isolates as recipients, Zawadzki et al. (1995)
found a log-linear relationship between sexual isolation and sequence divergence. The data of this study do not show such a relationship regardless of whether PCR-amplified or chromosomal DNA was used (Fig. 4
). Sexual isolation increased up to 7·2% mismatches and hardly responded to sequence divergence at more than 7·8% mismatches (LO139). This finding is reminiscent of other results obtained with B. subtilis YB886, a derivative of the Marburg strain (Zawadzki et al., 1995
), which showed a pattern of sensitivity of sexual isolation to sequence divergence that is considerably different to other B. subtilis recipients. The authors concluded that evolution in this organism can proceed in the direction of nonlog-linear patterns of sexual isolation. The genetic basis leading to modulation in the sensitivity of sexual isolation towards sequence divergence is obscure, but allelic variation of recombination pathways could play a role (Zawadzki et al., 1995
). Also, differences in proofreading enzyme activities may cause large differences in the sensitivity of sexual isolation to sequence divergence (discussed by Roberts & Cohan, 1993
). Whether this holds true for heterogamic transformation in P. stutzeri has yet to be shown. The enormous genomic and allelic diversity in housekeeping functions found in P. stutzeri (Sikorski et al., 1999
) leads us to expect differences in the response of sexual isolation to sequence divergence. It may be of interest to study this topic in more detail with other recipients including new wild isolates.
Conclusions
Sexual isolation with regard to transformation has been demonstrated here to exist in P. stutzeri. The potential to receive genes can vary greatly among strains as an effect of heterogeneity in transformability, quantitative differences in competence development, sequence divergence that could affect heteroduplex formation between donor and recipient systems and other genetic factors not identified so far (leading to sexual isolation even between closely related strains as observed with LO179). Further, differences in ecological constraints among microhabitats can influence recombination probabilities such as modulation of competence by nutrient availability and quality, temperature, and secretion of DNases (Lorenz & Wackernagel, 1991 , 1992
; Sikorski et al., 1998
). The results obtained here add to the view on the basis of work on B. subtilis (Zawadzki et al., 1995
) that distinct lineages (e.g. genomovars, pathovars) within transformable species, for many of which population structures have been found that infer panmixis (for a review see Lorenz, 1998
), are free to diverge in neutral sequence characters as a result of sexual isolation mechanisms which prevent allelic randomization. Nevertheless, this border is not absolute, and foreign sequences may be acquired and fixed in a way similar to what has driven the recruitment of nitrogen fixation genes in particular P. stutzeri strains (Vermeiren et al., 1999
).
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ACKNOWLEDGEMENTS |
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Received 25 April 2000;
revised 14 August 2000;
accepted 4 September 2000.