1 National Food Safety and Toxicology Center, Michigan State University, East Lansing, Michigan, USA
2 Department of Food Science and Human Nutrition, Michigan State University, East Lansing, Michigan, USA
3 Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan, USA
4 Infectious Disease Division, Department of Medicine, Michigan State University, East Lansing, Michigan, USA
Correspondence
John E. Linz
jlinz{at}msu.edu
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ABSTRACT |
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INTRODUCTION |
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Natural transformation in bacteria is defined as a process by which exogenous DNA is taken up and incorporated into the genome of a competent cell without special treatment of recipient cells. C. jejuni was determined to be competent for natural transformation in studies directed toward developing systems for genetic manipulation (Wang & Taylor, 1990a; Wassenaar et al., 1993
). This work indicated that strain-to-strain variation in natural competence exists in C. jejuni and supported the conclusion of Miller et al. (1988)
that transformation appears to be highly specific for Campylobacter DNA. In vitro experiments with Campylobacter coli have demonstrated that natural transformation can mediate flagellin gene transfer in a non-motile mutant population of bacteria, resulting in motile progeny (Alm et al., 1993
), and a similar study with isogenic C. jejuni populations established that flagellin gene exchange can occur by natural transformation at a frequency of approximately 10-5 (Wassenaar et al., 1995
).
Genetic exchange between non-isogenic C. jejuni strains was recently demonstrated in vitro and in chickens using selectable markers inserted in the hipO and htrA genes (de Boer et al., 2002). This study supported the interpretation of earlier work (Nuijten et al., 2000)
that flagellin gene duplication in non-motile C. jejuni populations followed by gene transfer via natural transformation in the caecum of the chicken was one possible mechanism for the development of motile progeny. The genetic exchange of genes (hipO and htrA) which are presumably not subject to selective pressure during chicken colonization suggests that co-cultivation can result in genome-wide exchange.
Natural transformation has been best described in Neisseria gonorrhoeae, Haemophilus influenzae, Streptococcus pneumoniae and Bacillus subtilis, where preference for transforming DNA, in each case, is species specific. Specificity is sequence mediated in the two Gram-negative organisms and regulated by growth-rate-dependent cell-to-cell signalling in the two Gram-positive organisms (Solomon & Grossman, 1996; Dubnau, 1999
). Natural transformation is one possible mechanism for generating genetic diversity in C. jejuni but it remains poorly described with regard to specificity, efficiency and molecular mechanisms. Understanding the mechanisms that control genetic variation in C. jejuni populations should lead to a broader understanding of the ability of this pathogen to discern and adapt to its environment.
In this study we developed a liquid shake culturing system to examine C. jejuni competence. We investigated natural competence during the growth phase and demonstrated that ciprofloxacin resistance in C. jejuni can be acquired through natural transformation. Although not all C. jejuni strains were naturally transformable under the limited conditions examined, competent strains were able to take up and incorporate ciprofloxacin-resistance-encoding chromosomal DNA from multiple C. jejuni sources. C. jejuni also demonstrated a species-specific preference in chromosomal DNA during the transformation process. Co-cultivation of isogenic C. jejuni populations showed DNA transfer via natural transformation at relatively low cell densities. Both co-cultivation transformation frequency and tranformation frequency as determined by the addition of purified isogenic chromosomal DNA to culture were relatively high under a 0·7 % CO2 atmosphere but decreased dramatically at higher CO2 concentrations.
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METHODS |
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C. jejuni was routinely cultured in Bolton broth or agar (Oxoid) and supplemented with ciprofloxacin (2 µg ml-1), chloramphenicol (20 µg ml-1), and/or kanamycin (30 µg ml-1) for selection of transformants. E. coli was grown in LB medium (Ausubel et al., 1997) supplemented with ampicillin (50 µg ml-1) or kanamycin (30 µg ml-1) as needed.
Plasmid pJB23SK was prepared by amplifying C. jejuni genomic DNA with primers 5'-ACCCTATCAAACTCCGAATACC-3' and 5'-CACACCCAGCCTATCAAAC-3'. This primer pair was designed from and is exactly matched to all three copies of the 23S rRNA gene in the C. jejuni 11168 genome (sequence data produced by the C. jejuni Sequencing Group at the Sanger Institute; ftp://ftp.sanger.ac.uk/pub/pathogens/cj/); amplification produced a 1928 bp fragment that spans bases 9172845 of each copy of the 2890 bp 23S rRNA gene. A single SphI restriction site occurs at bp 343 of the PCR product; a single BamHI restriction site occurs at bp 1100 of the PCR product. The 1928 bp PCR fragment was ligated into SmaI-digested pUC19 and transferred to E. coli DH5; clones were identified by bluewhite screening, and fragment orientation verified by restriction enzyme digestion. A single clone, pJB23S, contained the PCR fragment in the orientation that placed the SphI site in the cloned fragment approximately 375 bp from the HindIII site in the pUC19 MCS. Plasmid pJB23S was then digested with SphI and the approximately 4200 bp fragment was gel-isolated and recircularized, effectively removing the BamHI site in the pUC19 MCS. This pJB23S derivative was then digested with BamHI and ligated with the approximately 1·4 kb gel-purified BamHI fragment from pILL600, which carries the C. coli aphA-3 (Trieu-Cuot et al., 1985
) kanamycin-resistance cassette. A single kanamycin-resistant clone, carrying plasmid pJB23SK (approx. 5·7 kb), was passaged and cassette insertion confirmed by restriction enzyme analysis.
Natural transformation.
C. jejuni colonies were grown for 48 h on Bolton agar at 37 °C, 5·0 % CO2 in a water-jacketed, IR autoflow automatic CO2 incubator (Nuaire). Individual colonies were transferred by toothpick to a Costar six-well cell culture tray (Corning), with each well containing 4 ml Bolton broth. The tray was incubated at 37 °C, 250 r.p.m. agitation on a KS125 orbital shaker (IKALABORTECHNIK), under a 0·7 %, 5·0 % or 10 % CO2 atmosphere until at least one well produced growth that gave an optical density reading (562 nm) between 0·02 and 0·03 (1224 h, depending on the strain). A 100 µl inoculum of this mid-exponential-phase culture (15x108 c.f.u. ml-1) was transferred to 75 ml Bolton broth (equilibrated to incubator conditions for 1014 h prior to inoculation) in a 250 ml Erlenmeyer flask. The 75 ml culture was incubated at 37 °C, 250 r.p.m. agitation, under a 0·7 %, 5·0 % or 10 % CO2 atmosphere. The pH of Bolton broth equilibrated under a 0·7 % and 10 % CO2 atmosphere was 7·05 and 6·45 respectively. Bolton broth pH at room atmosphere was 7·35.
At 2 h intervals, the cell density of the 75 ml culture was determined by serial dilution and spreading onto Bolton agar, and 1·5 ml of culture was transferred in duplicate to 35x10 mm cell culture dishes (Corning). A saturating level of ciprofloxacin- or chloramphenicol-resistance-encoding chromosomal DNA (1·0 or 10 µg) was added at time zero to one dish, while the other dish received an equivalent volume of TE (10 mM Tris, 1 mM EDTA) pH 8·0, and served as a negative control for monitoring spontaneous mutation background. The culture dishes were incubated at 37 °C, 250 r.p.m. agitation, with 0·7 %, 5·0 % or 10 % CO2. At 30 min, DNase I (Roche) was added to a concentration of 1 U µl-1 to the culture containing DNA, and after an additional 2·5 h incubation, aliquots were plated onto selective medium and the final culturable cell density from the transformation mix was determined by serial dilution and spreading onto Bolton agar. All plates were incubated at 37 °C, 5·0 % CO2 for 3 days.
Transposition.
The transposon used in this work is a modified version of Tn5, supplied commercially in the transposon construction vector pMOD-2<MCS> (Epicentre Technologies). The transposon was modified by insertion of a C. jejuni compatible chloramphenicol-resistance marker (Wang & Taylor, 1990b). Briefly, plasmid pBHpC8 (Ge et al., 1995
) was digested with HincII, and the resulting 800 bp blunt-ended fragment containing the marker was ligated into the HincII site of pMOD-2<MCS>. The new construct was named pMOD-2Cm.
In vitro transposition of C. jejuni 81-176 genomic DNA was performed using purified hyperactive Tn5 transposase (Epicentre) according to the manufacturer's instructions with modification. Briefly, the transposon was amplified from pMOD-2Cm using primers FP-1 and RP-1 (Epicentre) and digested with PvuII. One hundred nanograms of purified transposon (Tn5CmR) was incubated with 1 unit of transposase and 1530 µg of C. jejuni 81-176 chromosomal DNA for 2 h at 37 °C. The DNA was ethanol-precipitated and treated with T4 DNA polymerase and ligase to mend damaged DNA. The DNA was again ethanol-precipitated, redissolved in 10 µl TE pH 8·0, and placed on ice prior to electroporation.
Electroporation.
Competent cells for electroporation were prepared as follows. A 75 ml Bolton broth culture of C. jejuni 81-176 was incubated for approximately 14 h at 5·0 % CO2 as described for natural transformation. Then 1·5 ml of culture was transferred to a sterile, chilled microfuge tube and placed on ice. Cells were pelleted at 11 000 g, 4 °C, for 2 min. Supernatant was removed and cells gently resuspended in 1 ml of an ice-cold 15 % (v/v) glycerol, 7 % sucrose solution (V. B. Young, personal communication). The cells were pelleted twice more as above and finally resuspended in 100 µl ice-cold 15 % glycerol, 7 % sucrose, and placed on ice for 1·5 h.
A 50 µl aliquot of chilled competent cells was gently mixed with 10 µl of chilled transposed chromosomal DNA (or 35 µg of suicide vector DNA) in a microfuge tube and placed on ice for 30 min. The cell/DNA mixture was transferred to a pre-chilled 0·2 cm cuvette and electroporated using a Gene Pulser II (Bio-Rad) with settings of 2·5 kV, 300 and 25·0 µF. Time constants were recorded in the 6·07·0 ms range. Cells were immediately stabilized after electroporation by gently mixing with 100 µl SOC medium (Guerry et al., 1994b
) and holding for 10 min at room temperature. Cells were then transferred to 1·5 ml pre-warmed Bolton broth in a 35x10 mm tissue culture dish. The transformation mixture was incubated for approximately 8 h at 37 °C, 5·010 % CO2, with agitation of 250 r.p.m. to allow for cell recovery and marker gene expression. Cells were pelleted at 8200 g for 2 min and resuspended in 200 µl Bolton broth prior to spreading on selective medium. Plates were incubated at 37 °C, 5·0 % CO2 for 3 days before transferring transformant colonies to fresh selective medium.
Southern blotting and hybridization.
Chromosomal DNA (23 µg per sample) was digested with endonuclease (Invitrogen) and loaded into a 0·8 % agarose gel (0·5x TBE buffer, 0·5 µg ethidium bromide ml-1). Electrophoresis was performed for approximately 12 h at 50 V. DNA was partially depurinated by treating the gel with 5 vols 0·25 M HCl for 30 min. Denaturation, neutralization, and transfer to positively charged nylon membrane (Roche) were performed by standard methods (Maniatis et al., 1989). Ultraviolet irradiation was used to cross-link DNA to the membrane (FisherBiotech FB-UVXL-1000 UV Cross-linker).
Probe DNA for hybridization was produced with the PCR Digoxigenin (DIG) Probe Synthesis Kit (Roche). Probe DNA created to anneal to C. jejuni chromosomal DNA was prepared using primers designed from sequence data produced by the C. jejuni Sequencing Group at the Sanger Institute and available at ftp://ftp.sanger.ac.uk/pub/pathogens/cj/. Primers JL 625 (5'-GTGATCTATCCATGAGCAAG-3') and JL 626 (5'-CTAGACCAGTGAGCTATTAC-3') were designed to amplify probe DNA for the C. jejuni 23S rRNA loci. Probe hybridization and detection was performed using a CSPD chemiluminescent Detection Starter Kit (Roche). Luminescent blots were exposed to X-OMAT AR film (Eastman Kodak).
DNA sequencing.
A 2030 µg sample of purified transposed chromosomal DNA was ClaI (Invitrogen) digested and ethanol-precipitated. Dye terminator fluorescent cycle sequencing was performed on chromosomal DNA using primers SqFP and SqRP (Epicentre), and subsequent analysis performed with an ABI PRISM 3100 Genetic Analyser (Applied Biosystems) at the Michigan State University Genomics Technology Support Facility. Initial sequence data were confirmed by sequencing of PCR amplification products from transposon insertion site.
C. jejuni co-cultivation.
Tissue culture tray cultures of C. jejuni 81-176-Tn5CmR19 and C. jejuni 81-176-23SK4 were prepared as described for natural transformation. A 100 µl inoculum of each mid-exponential-phase culture (15x108 c.f.u. ml-1) was transferred to independent 250 ml Erlenmeyer flasks containing 75 ml Bolton broth that had been equilibrated in the incubator for 1012 h prior to inoculation. The 75 ml cultures were incubated at 37 °C, 250 r.p.m. agitation, under a 0·7 %, 5·0 % or 10 % CO2 atmosphere. At 2 h intervals, 750 µl of each culture was transferred to a 35x10 mm tissue culture dish. These mixed cultures were incubated at 37 °C, 250 r.p.m. agitation, under a 0·7 %, 5·0 % or 10 % CO2 atmosphere for a total of 3 h. At 30 min, DNAse I was added to the mixed culture at a final concentration of 1 U µl-1, which would allow only 30 min for DNA transfer by transformation between populations. For early timepoints, when transformation frequencies were relatively low, the entire 1·5 ml volumes were plated in 200 µl aliquots (seven plates) onto Bolton agar supplemented with kanamycin and chloramphenicol, except for 10 µl that was used in serial dilutions to determine culturable cell densities. At later timepoints, as transformation frequencies increased under a 0·7 % CO2 atmosphere, dilutions of the cell mixtures were plated onto selective medium. Plates were incubated at 37 °C, 5·0 % CO2, for 3 days.
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RESULTS |
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DISCUSSION |
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Two characteristics of transformation in C. jejuni have been established in the literature: (1) certain strains of C. jejuni are highly transformable with C. jejuni chromosomal DNA, demonstrating transformation frequencies in the range of 10-410-3 (Wang & Taylor, 1990a; Wassenaar et al., 1993
; Guerry et al., 1994a
), while other strains appear to be non-competent; and (2) C. jejuni populations are inefficiently transformed by electroporation with cloned Campylobacter DNA harvested from E. coli when compared with electroporation of the same plasmid harvested from C. jejuni. This suggests that C. jejuni harvested DNA is specifically modified and that this modification allows for efficient C. jejuni transformation (Labigne-Roussel et al., 1987
; Miller et al., 1988
). Thus, some strains of C. jejuni are highly competent yet selective for Campylobacter DNA. Natural competence in C. jejuni has also been demonstrated to be recA-dependent (Guerry et al., 1994a
) and to be influenced by the expression of galE, a gene involved in lipopolysaccharide biosynthesis (Fry et al., 2000
), and virB10, a gene located on the virulence plasmid of C. jejuni strain 81-176 (Bacon et al., 2000
, 2002
).
The conclusion that not all strains of C. jejuni are naturally competent was based on data from studies in which only a single source of donor chromosomal DNA was used in transformation experiments. Strains that were negative for transformation were not tested by transforming with isogenic DNA (Wang & Taylor, 1990a; Wassenaar et al., 1993
). Intra-species differences in DNA methylation patterns have been demonstrated in C. jejuni (Edmonds et al., 1992
); the differences in restrictionmodification systems between strains (Dorrell et al., 2001
; Ahmed et al., 2002
) could result in digestion of transforming DNA and thereby the perceived lack of competence observed in some C. jejuni strains. However our data, gathered using isogenic DNA in transformation experiments, showed that only three of four strains were transformable under our culturing conditions (5·0 % CO2 atmosphere), supporting the conclusion that many C. jejuni strains are competent, while certain strains are not naturally transformable. All competent C. jejuni strains were highly transformable with ciprofloxacin-resistance-encoding donor DNA at low cell densities, but transformation frequency declined as cell density increased when C. jejuni was cultured under a 5·0 % CO2 atmosphere. The magnitude of this decline varied between strains and could be dramatic, as observed with the approximately 10 000-fold decrease in strain 33560. The identification of factors that cause variation in natural competence will help reveal the mechanisms and regulation of natural transformation in C. jejuni.
One factor identified in this study as influencing natural transformation of C. jejuni is CO2 concentration. Natural transformation frequency, as determined both by adding purified isogenic DNA to culture and when isogenic populations are co-cultivated, is relatively high when C. jejuni is cultured under a 0·7 % CO2 atmosphere. As CO2 concentration increases, creating a more favourable environment for growth, transformation frequency decreases. Increasing CO2 concentration from 0·7 % to 10 % decreases transformation frequency approximately 100-fold at relatively high cell densities when purified DNA is used as the transforming agent; and during co-cultivation at high cell densities the decline extends to 10 000-fold. Changes in CO2 concentration may indirectly influence natural competence by affecting other culture conditions such as pH or O2 concentration, especially if CO2 and O2 levels vary inversely. A decrease in O2 concentration as population density increases in our culturing system may explain the decrease in transformation frequency observed during late exponential growth under 5·0 % and 10 % CO2 atmospheres.
Whereas transformation frequency determined with purified chromosomal DNA indicates uptake, integration and expression of DNA, co-cultivation transformation frequency measures those same steps involved in natural competence plus DNA release during culture growth. Our co-cultivation experiments provide evidence for the presence of significant levels of natural DNA in culture. These experiments demonstrate that DNA transfer via natural transformation can begin to occur in our growth system when cell densities approach 5x106 c.f.u. ml-1. Under a 0·7 % CO2 atmosphere, the increase in transformation frequency (approx. 500 fold) above this threshold level can be attributed either to an increase of DNA in culture (beyond that due to natural cell death), or to an increase in population competence once DNA has accumulated in the culture. Since transformation frequency is relatively constant under a 0·7 % CO2 atmosphere when saturating levels of purified chromosomal DNA are used as the transforming agent, the increase in co-cultivation transformation frequency is more likely to be the result of an amplification and release of DNA during culturing. The naturally competent bacteria N. gonorrhoeae and S. pneumoniae exhibit lysis mechanisms which release DNA into culture (Gibbs et al., 1989; Steinmoen et al., 2002
), and the nature of the C. jejuni 33560 growth curve, which demonstrates several apparent phases of exponential growth spaced at regular intervals, may indicate that a similar mechanism exists in C. jejuni. Experiments are being designed to characterize the quantity and nature of endogenous DNA in C. jejuni culture.
This study has shown that E. coli and C. coli chromosomal DNAs are ineffective competitiors in transformation experiments with marked C. jejuni chromosomal DNA, indicating a preference of C. jejuni for DNA of its own species during natural transformation. However, other laboratories have demonstrated natural transformation of C. jejuni with C. coli chromosomal DNA (Wang & Taylor, 1990a; Alm et al., 1991
; Guerry et al., 1994a
), and in addition Wang & Taylor (1990a)
showed that C. jejuni and C. coli chromosomal DNA will compete for DNA uptake with equal efficiency in C. coli while E. coli chromosomal DNA lacks the specificity to be competitive. The signature that confers DNA specificity during natural transformation has not been identified in C. jejuni, nor is it clear whether the DNA signature would allow passage through multiple barriers (which include DNA uptake, host restriction nucleases, and recombination into the chromosome), or only through a single barrier in the process. Electroporation data with plasmid DNA have indicated that C. jejuni discriminates against non-Campylobacter DNA, recognizing and eliminating foreign DNA presumably through restrictionmodification systems (Miller et al., 1988
). If the specificity of DNA uptake shown in C. coli is combined with C. jejuni DNA restriction systems that eliminate foreign DNA during transformation, this would provide a dual barrier to protect the fidelity of the Campylobacter chromosome. Such redundant features would argue that DNA uptake and recombination are commonplace in the life cycle of this genus.
Co-cultivation experiments demonstrated that DNA transfer via natural transformation occurred between isogenic populations during short periods of exposure in liquid medium when cell density and presumably DNA concentrations were low. Our data support the hypothesis that this type of genetic transfer is genome-wide (Alm et al., 1993; Wassenaar et al., 1995
; de Boer et al., 2002
). It has been suggested that the presence of multiple strains is required to generate genetic diversity in C. jejuni through horizontal gene transfer. The ability of C. jejuni strains 3130 and 81-176 to take up, integrate and express chromosomal DNA from multiple C. jejuni strains demonstrates a lack of barriers to genetic exchange within the species. However, genetic exchange within a population derived from a single cell may also be significant. Sequence analysis of one C. jejuni genome has identified several genes with hypervariable sequences (Parkhill et al., 2000
; Wassenaar et al., 2002
). These so-called contingency genes, which are susceptible to slip-strand mispairing during DNA replication (van Belkum et al., 1998
), have recently been linked to phase variation in motility (Karlyshev et al., 2002
) and lipooligosaccharide structure (Linton et al., 2000
; Guerry et al., 2002
), both proposed virulence factors. Natural transformation with these hypervariable sequences may significantly increase genotypic and phenotypic diversity in C. jejuni populations. A mechanism utilizing such chromosomal mutation followed by genetic transfer via natural transformation could accelerate the ability of C. jejuni to generate genetic diversity and thereby more rapidly acquire and pass on DNA conferring competitive advantages in a dynamic environment. In effect such a mechanism would allow C. jejuni to evolve similarly to sexually reproducing organisms (Zhang et al., 2002
), with beneficial and detrimental genetic determinants being shuffled within a population of cells and natural selection providing the filter that would allow for the survival of competitive complex progeny.
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ACKNOWLEDGEMENTS |
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Received 2 June 2003;
revised 6 August 2003;
accepted 3 September 2003.
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