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Keywords:

  • Natural transformation;
  • Single-stranded DNA;
  • Pseudomonas stutzeri;
  • pilA;
  • pilC;
  • comA

Abstract

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

Pseudomonas stutzeri, in addition to being transformed by duplex DNA, is also transformed by the sense or antisense strand of the genetic marker employed (hisX+) or by heat-denatured chromosomal DNA. Transformation was absent in non-competent cells and in mutants defective for pilus biogenesis (pilA, pilC) and function (pilT) or DNA translocation into the cytoplasm (comA). Uptake of 3H-thymidine-labeled single-stranded DNA was hardly detectable reflecting the 20- to 60-fold lower transformation compared to duplex DNA. The results suggest that the steps in natural transformation also accommodate single-stranded DNA and that DNA translocation from the periplasm into the cytoplasm is not necessarily coupled to the degradation of a complementary strand. Small DNA single-stranded fragments are thus not excluded from horizontal gene transfer by transformation.


1Introduction

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

Natural transformation in prokaryotes is the active uptake of DNA from the environment of the cell and its heritable integration and expression. For DNA uptake competence a considerable number of gene functions are required [1,2]. Using duplex chromosomal or plasmid DNA the transformation of members of more than 60 bacterial species has been demonstrated [3,4].

In the soil bacterium Pseudomonas stutzeri transformation requires the competence phase and the formation of functional type IV pili [5]. These are cell organelles widely observed among Gram-negative bacteria and are involved in the flagella-independent translocation on surfaces (twitching motility [6]). In P. stutzeri the pili mediate the uptake of duplex DNA into the periplasmic space by which DNA becomes resistant against added DNase I [5]. Transformation further requires the ComA protein which has the characteristics of a polytopic integral membrane protein, and is facilitated by the ExbB protein which has two membrane domains [7]. ComA and its orthologs found in other transformable bacteria are necessary for translocation of DNA into the cytoplasm [1].

Here we report that P. stutzeri is naturally transformable by purified single-stranded DNA and that the cellular components required for transformation by duplex DNA are the same for single strands.

2Materials and methods

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

2.1Bacterial strains and plasmids

The P. stutzeri strain LO15 (hisX1) and the transformation-deficient mutants Tf300 (pilA::GmR), Tf81 (pilC::Tn5B20), Tf233 (comA::Tn5B20), Tf800 (exbB::KmR), and Tf59 (pilT::Tn5B20) have been described [5,7,8]. P. stutzeri JM300 was the donor of his+ DNA [9]. Escherichia coli XL10 (Stratagene, Amsterdam, The Netherlands) was used for plasmid preparation and E. coli KK2186 for preparation of phage f1 [10]. Cells were grown in Luria–Bertani liquid media (LB) or on LB-agar plates [11]. The medium for selection of his+ clones was MS agar [12]. Incubations were at 37°C. Plasmid pPM1 is pBluescriptII SK+ (Stratagene) with the 2.18-kb PstI fragment covering the hisX+ gene [13] in antisense orientation in its PstI site relative to the f1 origin (pPM2: sense orientation). Plasmid and genomic DNA were prepared using Qiagen columns (Qiagen, Hilden, Germany).

2.2Binding and uptake of 3H-labeled single-stranded DNA

Purified chromosomal DNA of P. stutzeri was labeled with 3H-deoxythymidine triphosphate as described [5]. Shortly before the assay, DNA (in 10 mM Tris–HCl, pH 8.0, 1 mM EDTA) was heated at 100°C for 5 min and cooled on ice. Binding and uptake was assayed as described using cell suspensions from the competence peak stored at −80°C [5] and aerated for 2 h at 37°C before use.

2.3Transformation on agar plate and in liquid culture

Cells of an overnight culture of P. stutzeri LO15 were washed three times with equal volumes of 0.9% (w/v) NaCl solution. The suspension (20 μl) together with transforming his+ DNA (in 10 μl of 10 mM Tris–HCl, pH 8.0) was spotted on LB agar. After 20 h at 37°C the spot was cut out with the agar, the cells resuspended in 1 ml of 15 mM MgCl2, 1 mM CaCl2 and 100 μg DNase I. After 15 min at 37°C the viable count was determined on LB agar and the his+ transformants on MS agar. Liquid transformation was performed as described [14], except that 200 μl of competent cell was used per assay. The DNase I treatment was for 15 min (see above). Transformation frequencies are expressed as transformant CFU per total CFU.

2.4Isolation of single-stranded DNA

The preparation of single-stranded DNA from the phagemids pPM1 and pPM2 was performed as published [15]. E. coli KK2186 with one of the two phagemids was grown in LB with 1000 μg ampicillin ml−1 and the f1 helper phage R408 used at a multiplicity of infection of 5. The lysate was treated with DNase I (100 μg ml−1) in 15 mM MgCl2 and 1 mM CaCl2 for 15 min at 37°C. Under these conditions plasmid DNA added to the lysate at 10 μg ml−1 was degraded to completion within 1 min as determined by gel electrophoresis (reaction terminated by excess of EDTA). Phage f1 containing the single strands of pPM1 or pPM2 was purified from the lysate by CsCl density gradient centrifugation in a swinging bucket rotor of a Beckman ultracentrifuge as described [16] to remove any double-stranded DNA possibly present as free DNA or in residual cells. The DNA of the purified phage was extracted four times as described [11]. DNA concentrations were determined by UV photometry.

3Results

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

3.1Transformation efficiency of duplex and single-stranded DNA

In preliminary experiments transformation of the histidine-auxotrophic hisX1 mutant LO15 of P. stutzeri with heat-denatured chromosomal wild-type DNA occurred but the level was about 25-fold lower than that with native DNA. We repeated the experiment with a preparation of the antisense strand of the genetic marker employed. For this, the single strand was isolated from purified f1 phage with the cloned 2.18-kb chromosomal PstI fragment covering hisX+[13]. The transforming activity was compared to that of duplex plasmid pPM1 DNA (its colE1 origin of replication is inactive in P. stutzeri). We used plate transformation [12] in which cells during incubation with DNA on the surface of a non-selective agar plate grow for several generations during which they pass through the competence phase before reaching the stationary phase. As shown in Fig. 1 a linear dependence of the transformation frequency on the concentration of the antisense strand DNA between 0.03 and 50 μg ml−1 was obtained. The inclination angle in the double log plot was about 45° indicating a one-hit mechanism as is canonically found in transformation studies with chromosomal markers on duplex DNA (Fig. 1). Compared on a mass basis the transformation frequency by single-stranded DNA was about 1.6% of that by duplex DNA at 2.5 μg ml−1 and about 2% at 10 μg ml−1.

image

Figure 1. His+ transformation frequency of P. stutzeri LO15 hisX1 at various concentrations of single-stranded (?) and duplex DNA (•) with the hisX+ allele. The duplex DNA was pPM1 and the single-stranded DNA the plus strand of pPM1 (antisense strand of hisX1). The data are means of three independent determinations. The lines show the linear regression of the data.

Download figure to PowerPoint

As we used circular single-stranded DNA for transformation it was considered that a molecule entering the cytoplasm as an intact molecule could be converted to a duplex circular molecule by complementary strand synthesis. Although autonomous replication would not occur because the colE1 origin of replication present in pPM1 and pPM2 is inactive in P. stutzeri, the molecule might form a cointegrate with the chromosome through homologous recombination at the hisX gene (phenotype: His+ AmpR). However, we did not find His+ AmpR clones among transformants obtained with either single- or double-stranded DNA (more than 100 tested for each) indicating that cointegrate formation and hence formation of duplex circular molecules was rare or did not occur.

3.2Comparison of transformation by sense and antisense strands

During transformation with duplex DNA either of two strands is transported into the cytoplasm. Therefore, the transformation frequency reflects the mean of the efficiencies of the two strands which form different mismatches upon recombinational integration. The hisX1 mutation in strain LO15 consists of a C to T transition in the first position of codon Q87 in the hisX ORF [13] resulting in an amber triplet (Table 1). In Section 3.1 the antisense strand of hisX+ was used forming a TG mismatch after strand transfer. With the sense strand (prepared from pPM2) the transformation activity was not significantly different from that of the antisense strand (Table 1). Assuming equally efficient uptake of the two single strands, the similar transformation frequencies suggest that TG formed during integration of the antisense strand is eliminated (or maintained) with similar efficiency by the mismatch repair system as CA formed by the sense strand. In E. coli TG and CA mismatches are repaired with about equally high efficiency [18].

Table 1.  Transformation of P. stutzeri LO15 (hisX1) with purified sense and antisense strands of the hisX+ allele
DNAAllelehis+ transformation frequencya
  1. aObtained with 23.3 μg ml−1 DNA in plate transformation; the data are given with standard deviation (n=3).

5′-.…CAG….his+ (sense strand)1.1 (±0.4)×10−7
5′-.…TAG….hisX1 (amber; recipient duplex) 
3′-.…ATC….  
3′-.…GTC….his+ (antisense strand)2.0 (±0.5)×10−7

3.3Requirement of competence

In P. stutzeri transformation competence with duplex DNA occurs as a sharp peak in late exponential phase broth cultures [14]. Competence is rapidly lost in early stationary phase and absent in overnight culture [5,14]. In liquid culture transformation with highly competent cells the transforming activity of heat-denatured DNA was about 1/20 of that of duplex DNA (Table 2) and corresponded to the data of Fig. 1 obtained by plate transformation with a purified single strand. Similarly, alkali denaturation of chromosomal DNA decreased its transforming activity at 6.6 μg ml−1 to 3.7±1.0% (n=3) compared to native DNA. With cells taken shortly after the competence maximum, transformation with single-stranded DNA declined and was below detection level with overnight culture cells, a result similar to what was found with duplex DNA (Table 2). With post-maximum competent cells also the transformation with purified sense strands (5 μg ml−1) decreased to 4% (n=2).

Table 2.  Transformation of P. stutzeri LO15 (hisX1) of different competence states by heat-denatured and duplex chromosomal his+ DNA in liquid culture
P. stutzeri LO15aRelative transformation frequenciesn
 single-stranded DNAduplex DNA 
  1. aThe competence maximum of a culture was determined and the post-maximum competent cells were taken from the culture 1 h after the maximum [9,13].

  2. bThe transformation frequencies were 2.9 (±0.7)×10−6 at 2.5 μg duplex DNA ml−1 and 1.5 (±0.7)×10−7 at 2.5 μg single-stranded DNA ml−1.

Maximum competent cells1b1b3
Post-maximum competent cells0.050.12
Cells from overnight culture≤0.02≤0.0012

3.4Requirement of type IV pili

Although type IV pili do not bind DNA, they are essential for the uptake of duplex DNA in Neisseria gonorrhoeae[19] and P. stutzeri[5]. A mutant (Tf300) in which the pilA coding for the structural protein of type IV pili was inactivated by an inserted gentamicin resistance cassette [5] was no longer transformable by single-stranded and duplex DNA (Table 3). Similarly, a strain in which the pilC gene was inactivated coding for an essential accessory protein for pilus biosynthesis and which therefore does not form pili [5] was transformation-deficient (Table 3). Thus, pili are required for transformation with single-stranded DNA, probably for the DNA uptake. The pilT mutant Tf59 which has excessive pili which are, however, non-functional in twitching motility and pilus-specific phage PO4 infection [8] was not transformable with pPM1 (≤4.3×10−11) and the purified antisense single strand (≤5.4×10−11).

Table 3.  Natural transformation of P. stutzeri LO15 and various transformation-deficient mutants by single-stranded (ss DNA; antisense strand) and duplex DNA (pPM1) and binding and uptake of 3H-thymidine-labeled ss DNA
P. stutzeriRelevant genotypeRelative transformation frequencyBinding of ss DNAc (pg DNA per 5×108 cells)Uptake of ss DNAc (pg DNA per 5×108 cells)n
  ss DNAduplex DNA   
  1. ahis+ transformation frequency (plate transformation) with 33.3 μg ml−1 single-stranded DNA per spot was 1.9 (±0.5)×10−7 (data are means of 2–3 determinations).

  2. bhis+ transformation frequency (plate transformation) with 33.3 μg ml−1 duplex DNA per spot was 7.6 (±1.9)×10−6 (data are means of 2–3 determinations).

  3. cHeat-denatured chromosomal 3H-thymidine-labeled DNA of P. stutzeri JM300; specific activity 5–8×106 cpm μg−1.

LO15 1a1b1064±43821±63
Tf300pilA::GmR≤3.0×10−4≤9.0×10−61491±45621±22
Tf81pilC::Tn5B20≤3.0×10−4≤8.0×10−63162±341730±83
Tf233comA::Tn5B20≤2.0×10−4≤7.0×10−62525±98528±132
Tf800exbB::KmR0.50.25957±32224±42

3.5Necessity of the translocation machinery

In P. stutzeri the translocation of DNA from the periplasm into the cytoplasm depends on ComA and is supported by a protein coded by exbB[7]. The data in Table 3 obtained with plasmid pPM1 DNA confirm the previous findings obtained with chromosomal DNA [7] that a comA mutation abolishes transformation by duplex DNA and an exbB mutation markedly decreases it. The transformation by single-stranded DNA was prevented by the comA mutation and reduced by the exbB mutation (Table 3). Thus, single-stranded DNA depends on the DNA translocation machinery as does duplex DNA.

3.6Binding and uptake of single-stranded DNA

As shown in Table 3, the amounts of 3H-DNA which bound to competent LO15 cells, the pil mutants, and the comA and exbB strains were between about 1000 and 3000 pg per 5×108 cells. The binding of single-stranded DNA to cells was more labile than that of duplex DNA since the DNA amounts recovered in the supernatants of the final third washing step of cells exceeded the amount of bound DNA by about three-fold. With duplex DNA the DNA in the final supernatant was always much less than the amount of DNA bound to the cells [5]. The fraction of single-stranded DNA taken up into the cells (measured as DNase I-resistant DNA) during the 90 min of incubation was only about 1–2.5% of the bound DNA in all strains (Table 3). Under the same conditions about 30% of the bound duplex DNA was taken up into cells of LO15 (roughly 50 pg DNA per 5×108 cells [5]), Tf233 and Tf800 [7]. Thus, the uptake of single-stranded DNA relative to its binding is much lower than for duplex DNA and perhaps corresponds to the relatively low transforming activity of single-stranded DNA. The data in Table 3 do not allow the conclusion that in the pilus-defective pilA and pilC mutants the uptake of single-stranded DNA is lower than in the wild-type and the other mutants.

4Discussion

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

Our results indicate that P. stutzeri is naturally transformable by single-stranded DNA. In the experiments employing purified single-stranded DNA the selected genetic marker (his+) was delivered with similar efficiencies by the sense and the antisense strand. Compared to duplex DNA the transformation with purified single strands was about 2.0% and with heat-denatured chromosomal DNA about 5%. Transformation of another Gram-negative strain, Haemophilus influenzae, with heat-denatured chromosomal DNA was previously shown to be at best 0.2% compared to duplex DNA and occurred only when DNA was associated with the competent cells at pH 4.8 followed by a shift to pH 7.4 [20]. Maintaining cells at the low pH prevented transformation [20]. In P. stutzeri transformation by single-stranded DNA occurred under standard conditions, i.e. at neutral pH in broth medium, either in liquid culture or on agar plates. In the Gram-positive strain Streptococcus pneumoniae natural transformation by single-stranded DNA obtained via purified f1 phage has been observed in a marker rescue system [21]. This result was questioned because purification of the f1 phage may not have been rigorous enough to remove any double-stranded DNA including residual cells [22]. In Bacillus subtilis natural transformation by single-stranded plasmid in the absence of marker rescue was not detected [22]. An experiment as we did with rigorously purified single-stranded DNA and employing a chromosomal marker is so far lacking in Gram-positives. Heat-denatured DNA may contain non-denatured or renatured portions.

The single strands of the hisX+ gene used in our experiments were isolated from E. coli so that DNA restriction might act on them in P. stutzeri. Since in natural transformation only single-stranded DNA reaches the cytoplasm and restriction endonucleases act on duplex DNA [17], restriction would, if it occurred, not change the ratio between the transforming activity of single-stranded and duplex DNA. This was supported by the finding that the transforming activity of native chromosomal P. stutzeri DNA relative to that of heat-denatured DNA was roughly the same as the activity of pPM1 relative to that of the purified single strands.

Transformation with single-stranded DNA was dependent on type IV pili (Table 3), which have been shown to be necessary also for the uptake of duplex DNA into the periplasm of P. stutzeri[5]. However, although relatively large amounts of single-stranded DNA bound to the cells, uptake measurements with the wild-type and non-piliated mutants were all at a low background level and therefore did not allow us to provide evidence for an involvement of pili in uptake of single-stranded DNA. When the pili were present but functionally inactive due to a pilT mutation transformation by single-stranded DNA was abolished as was previously observed for duplex DNA [8]. The pilT mutants are defective for twitching motility [8]. According to the hypothesis introduced by Bradley [23] and recently discussed by Wall and Kaiser [24] PilT is involved in the depolymerization or retraction of pili in several Gram-negative bacteria allowing twitching motility. Since pilT mutants of naturally transformable Synechocystis, N. gonorrhoeae and P. stutzeri are transformation-deficient [8,25,26] and the N. gonorrhoeae and P. stutzeri mutants no longer take up DNA [8,25] it was considered that during pilus retraction DNA bound to the cell is transported into the periplasm [8,25]. This transport may also extend to the small amounts of single-stranded DNA. Transformation by single strands further required the competence state of cells. In P. stutzeri competence occurs during a relatively short period in the late exponential phase [14] and probably involves other genes than those for pilus biogenesis. Presumably a gene for a DNA-binding protein on the cell surface is such a competence gene that is expressed during late exponential phase (N. Weger and W. Wackernagel, unpublished results). Our data further show that transformation by single strands was dependent on the machinery that translocates DNA through the cytoplasmic membrane. In this machinery a key component is ComA, a polytopic membrane protein which is assumed to form a pore in the inner membrane through which a single strand is transported formed from duplex DNA by an unknown DNase (perhaps being part of the translocation machinery) [1]. ComA and its orthologs are essential for natural transformation of N. gonorrhoeae, H. influenzae, P. stutzeri, S. pneumoniae and B. subtilis[1,7]. The requirement of comA for transformation with single-stranded DNA indicates that the generation of a single strand from duplex DNA is a process occurring separate from the translocation step. This finding does not exclude the possibility that the generation of single strands from duplex DNA is abolished in comA mutants which would be expected if the formation and translocation of single-stranded DNA were mechanistically coupled. From our experiments we can conclude that preformed single strands are guided to the ComA pore and can enter the cytoplasm only through that pore.

The requirement of the competence phase of cells, of functional type IV pili and of the translocation apparatus suggests that in P. stutzeri the manner in which single-stranded DNA enters the cell and the cytoplasm involves the same steps as those of duplex DNA. Since the transformation frequencies were about 20- to 60-fold lower than with equivalent amounts of duplex DNA one of the steps is apparently less effective with single-stranded DNA. Perhaps the putative DNA receptor protein in the cell wall of P. stutzeri interacts only relatively weakly with single-stranded DNA so that its transport into the periplasm is rather inefficient. This would explain why only a very small fraction of the radiolabeled single-stranded DNA associated with the cells became DNase I-resistant (Table 3). In B. subtilis the DNA receptor protein in the cell wall has been identified [27]. In vitro it has greatly reduced affinity for single-stranded DNA compared to duplex DNA [27]. Whether a defective DNA receptor of B. subtilis would prevent transformation by single-stranded DNA as it does with duplex DNA has not yet been determined.

Our studies show that the uptake and translocation mechanisms for duplex DNA also accommodate single-stranded DNA. We found that relatively short single strands of about 2 kb or less were active in transformation. It is possible that such single-stranded fragments are generated in the bacterial habitat by exonucleases degrading only one strand of duplex DNA or by denaturing conditions or are produced by transducing phages containing single-stranded DNA [28]. Such DNA would not be excluded from participating in horizontal gene transfer by natural transformation of P. stutzeri.

Acknowledgements

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

This work was supported by the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie.

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  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References
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