Variation of the natural transformation frequency of Campylobacter jejuni in liquid shake culture

David L. Wilson1,2, Julia A. Bell1, Vincent B. Young1,3,4, Stacey R. Wilder1,3, Linda S. Mansfield1,3 and John E. Linz1,2,3

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


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Natural transformation, a mechanism that generates genetic diversity in Campylobacter jejuni, was studied in a novel liquid shake culturing system that allowed an approximately 10 000-fold increase in cell density. C. jejuni transformation frequency was analysed in this system under 10 %, 5·0 % and 0·7 % CO2 atmospheres. At 5·0 % and 10 % CO2 concentrations, when purified isogenic chromosomal DNA was used to assess competence, transformation frequency ranged from 10-3 to 10-4 at low cell concentrations and declined as cell density increased. Transformation frequency under a 0·7 % CO2 atmosphere was more stable, maintaining 10-3 levels at high cell densities, and was 10- to 100-fold higher than that under a 10 % CO2 atmosphere. Three of four C. jejuni strains tested under a 5·0 % CO2 atmosphere were naturally competent for isogenic DNA; competent strains demonstrated a lack of barriers to intraspecies genetic exchange by taking up and incorporating chromosomal DNA from multiple C. jejuni donors. C. jejuni showed a preference for its own DNA at the species level, and co-cultivation 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. Transformation frequency during co-cultivation of isogenic populations was also influenced by CO2 concentration. Under a 0·7 % CO2 atmosphere, co-cultivation transformation frequency increased approximately 500-fold in a linear fashion with regard to cell density, and was 1000- to 10 000-fold higher during late-exponential-phase growth when compared to cultures grown under a 10 % CO2 atmosphere.


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The bacterial pathogen Campylobacter jejuni is frequently isolated from poultry, dairy cows, beef cattle (Jacobs-Reitsma, 2000) and other livestock, and has been identified as a major cause of foodborne bacterial gastroenteritis in the United States and other developed countries (Friedman et al., 2000). Erythromycin and the fluoroquinolone ciprofloxacin are the antibiotics of choice for the treatment of severe illness caused by C. jejuni (Altkreuse et al., 1999; Smith et al., 1999). Ciprofloxacin resistance in C. jejuni is predominantly chromosomally mediated by mutations in the genes encoding subunits of DNA gyrase (gyrA) and DNA topoisomerase IV (parC) (Wang et al., 1993; Gibreel et al., 1998; Wilson et al., 2000; Engberg et al., 2001). The increasing frequency of ciprofloxacin resistance in C. jejuni populations has correlated with the increased use of fluoroquinolones in food animals (Smith et al., 1999; Engberg et al., 2001; McDermott et al., 2002). Fluoroquinolone use provides a selective pressure for a ciprofloxacin-resistant phenotype and its spread in C. jejuni may be hastened by the relatively high degree of intraspecies genetic recombination that characterizes this organism (Dingle et al., 2001, 2002; Suerbaum et al., 2001). Natural transformation has been demonstrated in C. jejuni, providing one mechanism by which the species can generate genetic diversity and accelerate the dissemination of genetic traits such as ciprofloxacin resistance. Studies of Neisseria, Streptococcus and Campylobacter have implicated natural transformation as a means for spreading genetic determinants that increase antibiotic resistance and virulence (Dowson et al., 1989; Laible et al., 1991; Spratt et al., 1992; Spratt, 1994; Nuijten et al., 2000). C. jejuni is also characterized by a high degree of antigenic diversity (Lee et al., 1999; Karlyshev et al., 2000; Dorrell et al., 2001; Guerry et al., 2002), and intraspecies genetic recombination via natural transformation is of concern as a mechanism by which C. jejuni might evade vaccine-induced host immunity or by which attenuated strains used as live vaccines might regain pathogenicity (Guerry et al., 1994a).

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.


   METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains, DNA and media.
Campylobacter jejuni strains 3124, 3130, 3130 CR2161, 33292, 33292 CR2162, 33560, 33560 CR2162, 43429 CR2161, 43470 CR2161, 81-176 and 81-176 CR2161, and Campylobacter coli 18493, were described previously (Wilson et al., 2000). CR216 indicates a ciprofloxacin-resistant isolate. Chromosomal DNA was isolated by using hexadecyltrimethylammonium bromide (Wilson et al., 2000), or by using Easy–DNA (Invitrogen). Plasmid DNA was propagated in E. coli DH5{alpha} F- {phi}80dlacZ{Delta} M15 {Delta}(lacZYAargF)U169 deoR recA1 endA1 hsdR17() phoA supE44 {lambda}- thi-1 gyrA96 relA1 (Life Technologies) and purified by CsCl density-gradient centrifugation (Ausubel et al., 1997) using a Sorvall Discovery 90SE ultracentrifuge (Kendro Laboratory Products) at 20 °C with vacuum for 3 h at 310 000 g followed by 3 h at 192 000 g. DNA was quantified using a DU 530 spectrophotometer (Beckman Instruments).

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 917–2845 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{alpha}; clones were identified by blue–white 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 (12–24 h, depending on the strain). A 100 µl inoculum of this mid-exponential-phase culture (1–5x108 c.f.u. ml-1) was transferred to 75 ml Bolton broth (equilibrated to incubator conditions for 10–14 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 15–30 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 3–5 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 {Omega} and 25·0 µF. Time constants were recorded in the 6·0–7·0 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, 5·0–10 % 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 (2–3 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 20–30 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 (1–5x108 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 10–12 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.


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Comparison of natural competence in multiple strains of C. jejuni
C. jejuni strains 3130, 33292, 33560 and 81-176 were evaluated for their ability to be transformed with purified ciprofloxacin-resistance-encoding isogenic chromosomal DNA in liquid shake culture under a 5·0 % CO2 atmosphere (Table 1). Competence was assayed at 2 h intervals as cell density increased 1000- to 10 000-fold. Transformation frequencies for C. jejuni 3130, 33560 and 81-176 were highest during early exponential growth and declined as population size increased. Data for 81-176 are shown in Fig. 1. Decline in transformation frequency during growth in liquid shake culture was not due to a reduced viability of ciprofloxacin-resistant transformants, because co-cultivation of wild-type 81-176 and 81-176 CR2161 at the end of exponential growth did not result in a greater decrease in culturability of 81-176 CR2161 cells (data not shown). The decrease in transformation frequency as population density increased was most dramatic in 33560, which demonstrated a more than 10 000-fold decline. Strain 33560 also exhibited the lowest overall growth rate, characterized by a multi-phasic growth curve with short periods of exponential growth (data not shown). Because of this fluctuation in growth rate it was unclear whether the end of exponential growth was reached with 33560 in our experiments. Transformation frequencies for 3130 and 81-176 decreased approximately 300-fold and 400-fold respectively, with most of this decline occurring at the end of exponential growth. Strain 33292 was not transformable under these culture conditions despite using isogenic DNA.


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Table 1. Natural competence of wild-type C. jejuni strains determined with isogenic chromosomal DNA

Chromosomal DNA from strains 3130 CR2161, 33292 CR2162, 33560 CR2162 and 81-176 CR2161, each containing a single base pair mutation in the gyrA gene that confers resistance to ciprofloxacin, was used to assess the competence of wild-type strains. Transformation frequency was determined at 2 h intervals during 16–22 h incubation in liquid shake culture under a 5·0 % CO2 atmosphere as described in Methods. A saturating amount of chromosomal DNA (either 1·0 µg or 10 µg) was used as the transforming agent at each timepoint. Transformation frequency is reported as transformants per c.f.u. A plot of the C. jejuni 81-176 transformation frequency vs c.f.u. ml-1 is shown in Fig. 1. Strains 3130 and 33560 produced plots with similar patterns, demonstrating a decline in natural transformation frequency as cell density increased (data not shown). ND, Not detected.

 


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Fig. 1. Transformation frequency of C. jejuni 81-176 during growth in liquid shake culture under a 5·0 % CO2 atmosphere. c.f.u. ml-1 on the x-axis represents the colony-forming units at the time DNA was added to culture; c.f.u. on the y-axis was determined after allowing 3 h for DNA uptake and expression. {circ}, {bullet}, 81-176 transformation with chromosomal DNA from strain 81-176 CR2161 (contains gyrA point mutation conferring ciprofloxacin resistance) ({bullet}, exponential growth; {circ}, end of exponential growth or beginning of stationary phase). Data are the result of multiple experiments performed on separate days.

 
Natural transformation of C. jejuni in liquid shake culture using donor chromosomal DNA from several different strains
Natural transformation frequency in C. jejuni strains 3130 and 81-176 was analysed using chromosomal DNA from ciprofloxacin-resistant strains 3130 CR2161, 33292 CR2162, 33560 CR2162, 43429 CR2161, 43470 CR2161 and 81-176 CR2161. Each donor DNA carried a single basepair mutation in the gyrA gene conferring ciprofloxacin resistance (Wilson et al., 2000). C. jejuni 81-176 (Fig. 2a) and 3130 (Fig. 2b) were shown to be competent for transformation by all sources of C. jejuni donor chromosomal DNA tested. The pattern of variation in transformation frequencies when different donor DNAs were used was similar in strains 3130 and 81-176. C. jejuni 43470 CR2161 DNA produced approximately fivefold lower transformation frequencies than isogenic DNA, whereas C. jejuni 43429 CR2161 DNA consistently produced transformation frequencies slightly higher than isogenic DNA, with an approximately 1·8-fold increase in strain 81-176 and 1·1-fold increase in strain 3130. DNA from the non-competent strain 33292 CR2162 produced a high level of variation in transformation frequency in both 3130 and 81-176.



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Fig. 2. Transformation frequencies of C. jejuni strains 81-176 (a) and 3130 (b) using chromosomal DNA from several strains. Recipient cells were aliquoted from a 75 ml Bolton broth culture after incubation for 10 h under a 5·0 % CO2 atmosphere as described in Methods. Transformation frequencies were determined with 1·0 µg donor DNA from strains 81-176 CR2161, 33560 CR2162, 33292 CR2162, 43470 CR2161, 43429 CR2161 and 3130 CR2161, each of which contains a point mutation in the gyrA gene conferring resistance to ciprofloxacin. Self-DNA indicates transformation frequency with isogenic DNA. Transformations for both 3130 and 81-176 were performed twice on separate days. Error bars represent the standard error.

 
DNA competition experiments were performed to determine whether C. jejuni can distinguish between available DNAs during the natural transformation process. Donor chromosomal DNA of strain 81-176 CR2161 was combined with competitor chromosomal DNA of E. coli, C. coli or C. jejuni strains that did not encode ciprofloxacin resistance. These DNA cocktails were introduced into C. jejuni 81-176 cultures and transformation frequencies were determined (Fig. 3). Transformation frequency was progressively reduced by increasing the amount of competitor DNA of both C. jejuni 81-176 and 3124 in the cocktail. The decrease in transformation frequency ranged from approximately fourfold when 1 µg chromosomal DNA was added to the transformation mix, to approximately 10-fold when 5 µg competitive chromosomal DNA was added. C. coli and E. coli chromosomal DNA failed to compete.



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Fig. 3. C. jejuni 81-176 transformation frequencies using 81-176 CR2161 chromosomal DNA mixed with competing chromosomal DNA from C. jejuni 81-176, C. jejuni 3124, C. coli 18493, or E. coli DH5{alpha}. Bolton broth cultures of 81-176 were prepared under a 5·0 % CO2 atmosphere as described in Methods with modification. Briefly, a late-exponential-phase seed culture from a six-well Costar tissue culture tray was used to inoculate 50 ml pre-conditioned Bolton broth. After 5 h incubation, 300 µl aliquots were transferred to 14 wells of a 24-well Costar tissue culture tray. Each of the 14 wells contained 200 ng 81-176 CR2161 chromosomal DNA and 0, 1, 2 or 5 µg competing DNA. The tissue culture tray was incubated for 3 h, with DNase I added at 30 min. Transformant and total cell populations were determined by serial dilution. Transformations were performed in duplicate on the same day. Error bars represent the standard error.

 
Southern hybridization and sequence analysis of C. jejuni 81-176 transformants
C. jejuni strains marked with chloramphenicol acetyltransferase (confers chloramphenicol resistance) and 3'-aminoglycoside phosphotransferase (confers kanamycin resistance) were developed in order to monitor chromosomal DNA transfer between distinct populations in co-cultivation experiments. The use of chloramphenicol and kanamycin resistance markers in these DNA transfer experiments eliminated any concern about background mutant populations because spontaneous mutation conferring chloramphenicol or kanamycin resistance was not observed under our selective conditions. The suicide vector pJB23SK was constructed for targeted insertion of a kanamycin marker into one copy of the C. jejuni 23S rRNA gene (Nuijten et al., 1990; Kim et al., 1993). Southern hybridization schemes that identified homologous integration sites for pJB23SK were developed with the aid of sequence data produced by the C. jejuni Sequencing Group at the Sanger Institute and available at ftp://ftp.sanger.ac.uk/pub/pathogens/cj/. HincII chromosomal digests probed with rRNA operon DNA produced three distinct hybridization signals in the 81-176 wild-type strain (Fig. 4a), indicative of the presence of three rRNA operons. Homologous recombination of pJB23SK into separate 81-176 chromosomes clearly resulted in disruption of two of these operons (81-176-23SK2, 23SK3, 23SK4, 23SK5, 23SK6, 23SK8, 23SK9, 23SK10). Hybridization analysis with the aphA-3 probe DNA (Fig. 4b) confirmed that each transformant represented a single recombination event. A single-crossover recombination would increase HincII fragment size by approximately 5·7 kb since a HincII restriction site is absent in pJB23SK, while a double-crossover integration of the kanamycin-resistance marker would result in a fragment size increase of approximately 1·4 kb. The majority if not all of the recombination events were double-crossover integrations. The nature of the recombination events into the largest HincII fragment (23SK1 and 23SK7) was difficult to confirm from the given data because resolving 20 kb fragments by standard gel electrophoresis is inefficient. Transformant 81-176-23SK4, which contains the 1·4 kb kanamycin-resistance cassette in one of the smaller 23S rRNA HincII chromosomal fragments, was selected for co-cultivation experiments.



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Fig. 4. Southern hybridization analysis of C. jejuni 81-176 and randomly selected pJB23SK transformants. Chromosomal DNA was digested with HincII and hybridized with DIG-labelled (a) 23S rRNA–DNA, and (b) aphA-3 DNA (kanamycin-resistance marker). Hybridization with 23S rRNA–DNA produced three unique fragments in each DNA sample (one for each rRNA operon). Homologous recombination with pJB23SK results in integration at one of these three loci and an increase in the size of the corresponding wild-type fragment. Hybridization with aphA-3 DNA confirms that each transformant resulted from a single recombination event. The aphA-3 hybridization signal is absent in the wild-type sample. The positions of lambda DNA markers are shown on the left. The arrow indicates the isolate used in co-cultivation experiments.

 
We developed an in vitro transposition and transformation method for C. jejuni. A single transposition/electroporation experiment using C. jejuni 81-176 as the recipient strain repeatedly generated approximately 1500–3000 transformants, of which 14 were analysed by Southern hybridization (Fig. 5). The transposon used in this work carries a cat gene that was used as a probe to evaluate transposon insertion in transformants. The random nature of transposon insertion is supported by the multiple locations of hybridization signal in Fig. 5. Each transformant carries a single copy of the cat gene. Transformant 81-176-Tn5CmR19 was selected for co-cultivation experiments because its growth rate was similar to 81-176-23SK4 (data not shown). Sequence analysis of 81-176-Tn5CmR19 using the C. jejuni sequence database (ftp://ftp.sanger.ac.uk/pub/pathogens/cj/) indicated an insertion site (CATCAGCCTTAACTTTCGTG-Tn5CmR-ACTTTCGTGTTTTTACCTTG) within a putative sensory transduction histidine kinase gene (Cj 0889c). Transposon integration produced an 8 bp direct repeat at the insertion site. Insertion of Tn5CmR into the Cj 0889c locus did not affect transformation frequency (data not shown).



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Fig. 5. Southern hybridization analysis of C. jejuni 81-176 and randomly selected Tn5CmR transposon transformants. Chromosomal DNA was digested with ClaI and hybridized with DIG-labelled chloramphenicol acetyltransferase (cat) DNA. A single ClaI site is present in the transposon. The hybridization pattern indicates a unique transposon insertion site in each transformant. A second Southern scheme (data not shown) performed using BseRI chromosomal digests of the above isolates supported the uniqueness of each transformant and thereby the randomness of transposon insertion. The cat hybridization signal is absent in the wild-type sample. The positions of lambda DNA markers are shown on the left. The arrow indicates the isolate used in co-cultivation experiments.

 
Comparison of C. jejuni 81-176 natural transformation frequencies during incubation under different CO2 atmospheres
The transformation frequency of C. jejuni was evaluated when cultured under 0·7 %, 5·0 % and 10 % CO2 atmospheres. Cultures were grown and transformation frequency determined at 2 h intervals for 12–14 h during the growth phase as described in Methods. A saturating amount (10 µg) of 81-176-Tn5CmR19 chromosomal DNA was used at each timepoint to determine transformation frequency. Growth curves for each culture condition are shown in Fig. 6. An atmosphere of 10 % CO2 produced the highest growth rate, and an atmosphere of 0·7 % CO2 the lowest. Transformation frequency plots (Fig. 7) demonstrated an opposite trend from the growth rate data, with an increase in a dose–response fashion as CO2 level declined. At relatively low cell densities, an approximately 20-fold difference in transformation frequency was observed between 0·7 % and 10 % CO2 conditions. As cell density increased, the difference in transformation frequency between 0·7 % and 10 % CO2 also increased, resulting in an approximately 100-fold difference at relatively high cell densities (109 c.f.u. ml-1 range) (Fig. 7). This increase in the difference of transformation frequencies at higher cell densities was due to the decrease in transformation frequency at 10 % CO2, with transformation frequencies remaining fairly stable (10-3 range) under a 0·7 % CO2 atmosphere. A similar decrease in transformation frequency at high cell densities was observed under a 5·0 % CO2 atmosphere when either 81-176 CR2161 (Fig. 1) or 81-176-Tn5CmR19 chromosomal DNA (Fig. 7) was used as the transforming agent.



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Fig. 6. Cell concentrations of C. jejuni 81-176 during exponential growth in liquid shake culture under different CO2 atmospheres. {bullet}, 0·7 % CO2 atmosphere; {circ}, 5·0 % CO2 atmosphere; {blacktriangleup}, 10 % CO2 atmosphere. The data for each curve are the result of multiple experiments from different days; error bars represent the standard error.

 


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Fig. 7. Transformation frequencies of C. jejuni 81-176 during growth in liquid shake culture under different CO2 atmospheres. Ten-microgram aliquots of purified isogenic chromosomal DNA from strain 81-176-Tn5CmR19 (confers chloramphenicol resistance) were used to assess competence at 2 h intervals during exponential growth. {bullet}, 0·7 % CO2 atmosphere; {circ}, 5·0 % CO2 atmosphere; {blacktriangleup}, 10 % CO2 atmosphere. The data for each plot are the result of multiple experiments from different days.

 
Natural transformation during co-cultivation of isogenic C. jejuni populations under different CO2 atmospheres
Roughly equal cell concentrations of C. jejuni 81-176-Tn5CmR19 (chloramphenicol resistant) and C. jejuni 81-176-23SK4 (kanamycin resistant) from independent 75 ml Bolton broth cultures were mixed in 1·5 ml volumes over a 12–14 h period under 0·7 %, 5·0 % and 10 % CO2 atmospheres. DNase I was added after 30 min to the mixed cultures, which were then allowed to incubate for another 2·5 h to allow for expression of acquired DNA. Transformants were identified by plating on Bolton agar supplemented with chloramphenicol and kanamycin (Table 2). When DNase I was added at time zero to the mixed cultures, chloramphenicol- and kanamycin-resistant bacteria were never recovered. Co-cultivation experiments were performed at the same time and in the same incubator as the experiments that determined natural transformation frequency with 81-176-Tn5CmR19 chromosomal DNA (Fig. 7).


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Table 2. Genetic transfer between isogenic C. jejuni populations in liquid shake culture during the growth phase at different CO2 concentrations

81-176I shows the initial cell concentrations from 1·5 ml mixed cultures of C. jejuni 81-176-Tn5CmR19 (chloramphenicol resistant) and C. jejuni 81-176-23SK4 (kanamycin-resistant). The cell concentrations of the chloramphenicol- and kanamycin-resistant populations were roughly equal upon mixing. 81-176F indicates the final population size 3 h after mixing. Transformants or progeny were identified by their dual resistance to chloramphenicol and kanamycin. Transformation frequency is reported as transformants per c.f.u.F. The data shown were collected from independent experiments performed at least three times on separate days for each CO2 concentration.

 
Under 0·7 % CO2 and 5·0 % CO2 atmospheres, co-cultivation transformation of C. jejuni could be detected when cell density approached 5x106 c.f.u. ml-1 (Table 2). Co-cultivation transformation frequency increased in a linear fashion with regard to cell density, approximately 500-fold (3·0x10-8–1·4x10-5 transformants per c.f.u.) under a 0·7 % CO2 atmosphere. In contrast, co-cultivation transformation frequency under a 5·0 % CO2 atmosphere was relatively stable, fluctuating within a range of 4·1x10-9–6·1x10-8 transformants per c.f.u. At 10 % CO2, co-cultivation transformation was rare and somewhat erratic, occurring at relatively low frequency (9·0x10-10–1·4x10-8 transformants per c.f.u.) in 4 of 16 data points, with the four positive samples varying in cell density between 2·7x107 and 1·1x109 c.f.u. ml-1. Co-cultivation transformation frequency under a 0·7 % CO2 atmosphere was 1000–10 000-fold higher during late exponential growth when compared to culture grown at 10 % CO2. The determination of transformation frequency in these experiments was based on the acquisition of a mutant allele. Since there are both mutant and wild-type alleles in the 81-176 mixed cultures described above, and we were unable to monitor for the transformation of mutant phenotype to wild-type phenotype, the transformation frequencies as reported in Table 2 are probably lower (presumably by half) than what actually occurred in mixed culture.


   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Natural competence in Campylobacter species was first recognized with the work of Wang & Taylor (1990), and has since been exploited to develop genetic methodology in these organisms. However, the role that natural competence plays in the survival of Campylobacter has yet to be determined. We propose that natural transformation is predominantly a mechanism through which Campylobacter can acquire genetic determinants that offer competitive advantages in a dynamic environment. Experiments presented in this study demonstrating that chromosomally mediated ciprofloxacin resistance can be acquired in C. jejuni by means of natural competence and that natural transformation frequency varies in response to its environment support this proposition. The selective pressure of antibiotic use in food animals and the rapid spread of ciprofloxacin resistance in C. jejuni populations (Smith et al., 1999; McDermott et al., 2002) may provide one example of how mutation and DNA transfer via natural transformation may be working to effect C. jejuni adaptation and survival in a changing environment.

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 10-4–10-3 (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 restriction–modification 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 restriction–modification 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.


   ACKNOWLEDGEMENTS
 
This work was supported by funds from the National Food Safety and Toxicology Center at Michigan State University, the Michigan Agricultural Experiment Station, a USDA Regional Research Project (S-295), USDA Animal Health Formula Funds 71-9212, the Rackham Board of Govenors, and the National Institutes of Health (61-0954).


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Received 2 June 2003; revised 6 August 2003; accepted 3 September 2003.



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