Identification of Cj1051c as a Major Determinant for the Restriction Barrier of Campylobacter jejuni Strain NCTC11168

Abstract

Campylobacter jejuni is a leading cause of human diarrheal illness in the world, and research on it has benefitted greatly by the completion of several genome sequences and the development of molecular biology tools. However, many hurdles remain for a full understanding of this unique bacterial pathogen. One of the most commonly used strains for genetic work with C. jejuni is NCTC11168. While this strain is readily transformable with DNA for genomic recombination, transformation with plasmids is problematic. In this study, we have identified a determinant of this to be cj1051c, predicted to encode a restriction-modification type IIG enzyme. Knockout mutagenesis of this gene resulted in a strain with a 1,000-fold-enhanced transformation efficiency with a plasmid purified from a C. jejuni host. Additionally, this mutation conferred the ability to be transformed by plasmids isolated from an Escherichia coli host. Sequence analysis suggested a high level of variability of the specificity domain between strains and that this gene may be subject to phase variation. We provide evidence that cj1051c is active in NCTC11168 and behaves as expected for a type IIG enzyme. The identification of this determinant provides a greater understanding of the molecular biology of C. jejuni as well as a tool for plasmid work with strain NCTC11168.

INTRODUCTION

Campylobacter jejuni is a Gram-negative, microaerophilic pathogen and a major cause of human diarrheal disease worldwide. C. jejuni is the second leading cause of bacterial food-borne illness in the United States behind Salmonella (6) and is the leading cause of food-borne illness in the United Kingdom (29, 30). While an understanding of the genetics of C. jejuni trails behind our understanding of many other enteric pathogens, completed genomic sequences and molecular tools have been developed, which are allowing a greater understanding of this unique pathogen. Genome sequencing has revealed a number of homopolymeric tracts (49), which can lead to phase variation due to slipped-strand mispairing, affecting many cellular processes, including capsule (3), motility (23, 24), bacteriophage infection (7, 56), and restriction-modification (R-M) (5, 46).

The primary function of R-M systems is likely the prevention of extracellular DNA from establishing in the cell. Specifically, the need for this protection is most apparent for bacteriophages and transposons but may also play a role in preventing horizontal transfer facilitating speciation (32). R-M systems are diverse in specificity and strategy, but their general function is to degrade nonself DNA, which is determined by methylation patterns on the DNA. At specific recognition sites, fully methylated DNA is ignored, hemimethylated DNA is methylated, and nonmethylated DNA is digested (restricted) (59). These systems complicate molecular biology work by inhibiting the transfer of plasmids between species and even between strains of the same species of bacteria (31), as multiple systems within a single strain can all participate in the restriction barrier (16). The identification and deletion of these systems are useful for developing strains which allow plasmid transformation (10, 26, 34, 35). Other approaches used to work around a restriction barrier have been to appropriately methylate the plasmid before transformation into the target species (9, 11, 20, 37, 42, 48, 66), to eliminate the target sites in the plasmid to evade digestion (37), or to use plasmids with few or no naturally occurring target sites (41).

R-M systems are categorized into 4 types based on a variety of features. Type II R-M systems are the most common systems commercially available, as they recognize specific sequences and cleave within, or close to, those sequences and are therefore of greatest use for molecular biology techniques. These systems usually have independent methyltransferase (MTase) and restriction endonuclease (REase) enzymes recognizing the same specificity sequence, serving to methylate and restrict, respectively. Type II systems are further divided into subtypes, of which subtype G designates those which have both MTase and REase functions in the same enzyme (53).

C. jejuni strain NCTC11168 is one of the most commonly used laboratory strains in Campylobacter research. It was originally isolated from a patient with diarrhea (17, 55) and was the first Campylobacter strain to be sequenced (49). While C. jejuni NCTC11168 is naturally transformable and amenable to electroporation, for the chromosomal insertion of DNA via genomic recombination, plasmid transformation by either method is problematic. This is not the case for all strains of C. jejuni; several are capable of plasmid transformation from C. jejuni and Escherichia coli hosts (44, 61, 63). Additionally, several strains are not naturally transformable, and their barriers have begun to be elucidated (14, 15). Complementation assays with NCTC11168 are therefore commonly performed by the use of expression constructs inserted into the genome (into a pseudogene [25, 28, 43, 58] or near ribosomal genes [33]). Not only does this add complications for strain construction, there are also always risks of side effects when disrupting the genome. Additionally, a single genomic copy of an expression construct potentially gives lower expression levels than expression from a multicopy plasmid. Often, this copy number enhancement of expression levels is useful to obtain sufficient expression from synthetic constructs for comparison to the native form.

Here we attempted to identify factors in NCTC11168 which may contribute to its restriction barrier by screening random transposon mutants for plasmid transformation. We successfully identified one factor, cj1051c, which, when knocked out, allows a 1,000-fold increase in plasmid transformational efficiency with plasmids derived from a C. jejuni host. Furthermore, this mutation allows transformation with plasmids derived from an E. coli host. This work provides increased knowledge of the molecular biology of C. jejuni and allows for the increased use of plasmid-based techniques with this important pathogen.

MATERIALS AND METHODS

Culture conditions.

C. jejuni strain NCTC11168 was obtained from the ATCC (Manassas, VA) and routinely grown on Mueller-Hinton (MH) agar with 5 μg/ml trimethoprim (TrM) supplemented with 20 μg/ml chloramphenicol (Cm), 2 μg/ml tetracycline (Tet), 50 μg/ml kanamycin (Kan), or 5% horse blood, where appropriate. All C. jejuni cultures were grown in a Binder (Bohemia, NY) CB150 incubator set to 37°C, 5% O₂, 10% CO₂, and 85% N₂. E. coli strains DH5α and BW25141 were obtained from The E. coli Genetic Stock Center (New Haven, CT) and grown on LB agar aerobically at 37°C with appropriate antibiotics, as described above. Plasmid pWM1007 (45) was maintained in either a C. jejuni host (RM1849) or an E. coli host (DH5α) and extracted with Omega Plasmid Minikit II (Omega Bio-Tek, Norcross, GA). Bacterial strains and plasmids used in this study are listed in Table 1.

Table 1.

Bacterial strains and plasmids used in this study

Strain or plasmid	Descriptiona	Reference(s)
Strains
E. coli
DH5α	Plasmid host
BW25141	Plasmid rescue host	9
C. jejuni
81-176	pTet source	37
RM1849	D781	43
NCTC11168	Wild-type C. jejuni	16, 52
JH1	Knockout mutant of cj1051c	This work
JH2	Complementation strain for cj1051c	This work
Plasmids
pUC19	E. coli cloning plasmid	59
pMALC9	Transposase expression plasmid	1
pAJG40-134	Transposon source plasmids	18
pRY112	Cmr source	60
pWM1007	GFP expression plasmid, KanPr source	43
pJH19	pUC19 + cj0046	This work
pJH35	pJH19 + cj1051c expression construct	This work

^aGFP, green fluorescent protein; KanPr, promoter region of the kanamycin resistance cassette.

Generation of random mutants.

Mutagenesis was performed similarly to methods described in previous work (1, 19), by in vitro transposition followed by transformation and insertion into the chromosome by homologous recombination. Transposase C9 (39), a hyperactive mutant of Himar1, was purified, as previously described, as a maltose binding fusion protein expressed from pMALC9 (1) but in BL21(DE3)/pLysS (Promega, Madison, WI), with freeze-thaw lysis. Tagged transposons were previously developed and maintained on plasmids pAJG40 to pAJG134, each containing a unique tag sequence (19). Briefly, 2 μg of NCTC11168 genomic DNA, 1 μg of one of the transposon source plasmids pAJG40 to pAJG134, and 150 ng transposase were combined in a buffer (10% [vol/vol] glycerol, 25 mM HEPES [pH 7.9], 250 μg/ml acetylated bovine serum albumin, 2 mM dithiothreitol [DTT], 100 mM NaC1, 5 mM MgC1₂) at 30°C for 6 h (1). Transposed DNA was then purified on a column (Qiagen, Valencia, CA) and repaired. To repair the DNA, 1.5 U of T4 DNA polymerase, NEB buffer 2, and 25 μM each deoxynucleoside triphosphate (dNTP) were added, and the mixture was incubated for 20 min at 37°C, followed by inactivation at 75°C for 10 min. The reaction mixture was then treated with 10 U ligase overnight in 1× ligase buffer and column purified (Qiagen, Valencia, CA). Repaired mutated DNA was used to naturally transform NCTC11168 (see below). Mutant colonies were picked after 3 days of incubation on plates with MH agar, TrM, Cm, and 5% blood. The location of the transposon insertion in mutant strains was determined by plasmid rescue. Mutant genomic DNA (1 μg) was digested overnight with SspI, purified by phenol-chloroform extraction and ethanol precipitation, and ligated overnight with 400 U T4 DNA ligase. Ligation reaction mixtures were ethanol precipitated and electroporated into E. coli BW25141 (8), which contains the λpir gene necessary for the oriR6kγ origin of replication, according to Bio-Rad protocols. Colonies were picked from plates of LB agar with Cm after 48 to 72 h of incubation at 37°C. The insertion site was determined by the sequencing of the transposon in the subsequent plasmid with either primer ajg227 or CC001 (19).

Sequence analysis.

R-M systems of NCTC11168 were identified and classified with REBASE (54) and the annotated genome sequence (21, 49). The homology and genetic context of cj1051c in NCTC11168 were compared against those of other common laboratory strains, 81116 (50) and 81-176 (27, 38), using ClustalW2 (40) on EBI (18). A search for homologs of cj1051c was performed with protein BLAST (2); sequences were aligned with ClustalW2, and the consensus was scored in JalView (62) and visualized with Microsoft Excel and Pfam (52).

Natural transformation of Campylobacter.

The natural transformation of C. jejuni strains was performed by a plate biphasic method (19, 60). Bacteria grown overnight on plates of MH agar with TrM (MH+TrM plates) were harvested in 20 ml MH broth to an optical density at 600 nm (OD₆₀₀) of 1.0, pelleted at 3,220 × g for 20 min, resuspended in 1 ml MH broth, pipetted onto an MH+TrM plate, and incubated upright at 37°C microaerophilically for 3 h. Purified DNA was then mixed into the bacteria, and the plate was incubated for a further 4 h. Bacteria were harvested by scraping into 1 ml MH broth, pelleted at 3,220 × g for 20 min, resuspended in 1 ml MH broth to equalize volumes after harvest, diluted 1:10 or 1:100 when anticipated to be necessary, and plated onto selective medium.

Electroporation of Campylobacter.

The production of electrocompetent C. jejuni strains and electroporation were performed as described previously (60). Bacteria from a lawn grown overnight on MH+TrM plates were harvested into 10 ml MH broth, adjusted to an OD₆₀₀ of ∼5, pelleted at 3,220 × g for 20 min at 4°C, and resuspended in 5 ml ice-cold wash buffer (272 mM sucrose, 15% glycerol). This wash step was repeated three more times, and finally, the bacteria were resuspended in 1 ml ice-cold wash buffer. Cells were aliquoted in 100 μl and stored at −80°C until needed. DNA was added to each 100-μl electrocompetent-cell aliquot thawed on ice and gently mixed, and the mixture was added to a 0.2-cm cuvette on ice. Electroporation was performed with a Bio-Rad MicroPulser (1 pulse of 2.5 kV, 600 Ω, and 10 μF), and the electroporated cells were transferred into 200 μl SOC broth, mixed, and spread onto MH+TrM plates. Plates were incubated upright for 5 h, and bacteria were harvested into 1 ml MH broth, spun at 10,000 × g for 2 min, resuspended in 100 μl MH broth, and plated onto selective medium. Negative-control experiments (no DNA) were performed in parallel.

Mutagenesis of cj1051c.

Knockout mutagenesis of cj1051c was performed by overlapping-extension PCR (oePCR) (22), maintaining the original frame to avoid polar effects. The region upstream of cj1051c was PCR amplified with primers jph239 and jph240 (see Table S1 in the supplemental material). The region downstream of cj1051c was PCR amplified by using primers jph241 and jph242. The tetracycline resistance gene, tetO, was PCR amplified from pTet by using primers jph247 and jph249, containing the same overlapping regions as those of C1 and C2 (primers for Cm^r amplification in a previously developed oePCR system [22]), for the cross compatibility of the markers. After phenol-chloroform extraction and ethanol precipitation of the individual amplicons, 500 ng each of upstream and downstream amplicons was combined with 1.6 μg of the tetO amplicon, and thermal cycling was performed without primers to anneal overlapping fragments and to fill in gaps. The mutagenesis construct was created by the PCR amplification of 2 μl of the annealing reaction mixture described above with the outside primers jph239 and jph242. The resulting amplicon was purified by phenol-chloroform extraction and ethanol precipitation. Mutagenesis was performed by electroporating the purified mutagenesis construct into NCTC11168 cells as described above and selecting Tet^r colonies to produce strain JH1. Mutants were verified for correct mutagenesis by PCR with primers jph243 and jph244, which flank the mutated region. All PCRs were performed with Phusion polymerase (New England BioLabs), and gel extractions were performed with an Omega gel extraction kit (Omega Bio-Tek, Norcross, GA).

Complementation of cj1051c.

Complementation was performed by the expression of cj1051c driven by the Kan^r promoter, as it was known to have activity in this strain and the native promoter of cj1051c was not obvious. This expression construct, along with the Cm^r marker, was cloned within a presumed pseudogene (cj0046) of JH1. The cj0046 open reading frame (ORF) was cloned by PCR amplification with primers jph38 and jph39 (see Table S1 in the supplemental material), containing BamHI and EcoRI sites, respectively, followed by ligation into pUC19 (64), to create plasmid pJH19. The expression construct for cj1051c complementation was created by overlapping-extension PCR similarly to a method described previously by Hansen et al. (22), with SpeI sites included at either side for subsequent cloning. The Cm^r cassette was PCR amplified from pRY112 (65) with primers jph250 (C1 [22] with an SpeI site added) and C2 (22). The Kan^r promoter was PCR amplified from pWM1007 with primers jph251 and jph252, and the cj1051c ORF was PCR amplified from NCTC11168 genomic DNA with primers jph253 and jph254, containing an SpeI site. After phenol-chloroform extraction and ethanol precipitation of the three amplicons described above individually, 100 ng of the Cm^r cassette, 100 ng of the Kan promoter, and 500 ng of the cj1051c ORF were thermally cycled without primers to anneal overlapping fragments and to fill in gaps. The expression construct was created by PCR with 2 μl of the above-described annealing reaction mixture as the template, using outside primers jph250 and jph254. The complementation construct for cj1051c was created by cloning the above-described expression construct into the native unique SpeI site of cj0046 previously cloned into pJH19, to create pJH35. Sequences from pUC19 in pJH35 were removed by linearization with BamHI followed by gel extraction, NspI digestion, and gel extraction to yield the linear cj1051c complementation construct. This construct was then ethanol precipitated and electroporated into JH1 cells, resulting in a double crossover at the cj0046 locus, to create complementation strain JH2. Insertion at the target site was verified by PCR with primers jph43 and jph44, which flank the mutated region. All PCRs were performed with Phusion polymerase (New England BioLabs), and gel extractions were performed with an Omega gel extraction kit (Omega Bio-Tek, Norcross, GA).

Growth curves.

Growth curves were determined by using rocking T75 filter-top flasks at 37°C under microaerophilic conditions. Bacteria grown on MH+TrM plates overnight were harvested into 20 ml MH broth to an OD₆₀₀ of 0.7. Two milliliters of this culture was inoculated into 20 ml of MH broth preincubated overnight and placed into rocking flasks. Samples (200 μl) were taken at various time points, and the OD₆₀₀ of each was measured.

Motility.

Motility was determined by soft-agar stabs. Bacterial cultures were resuspended in MH broth to an OD₆₀₀ of ∼0.5, and a metal wire was dipped into each culture and stabbed into a plate of MH broth with 0.4% agar. Each plate contained a positive control (NCTC11168) and a nonmotile negative control (NCTC11168 ΔflgK) (12), to account for plate-to-plate variation. After a 48-h incubation, the diameters of growth for each sample were measured and compared to that for NCTC11168 with paired t tests.

Autoagglutination.

Measurements of autoagglutination were performed similarly to methods described previously (47). Bacteria from cultures grown overnight on MH+TrM plates were resuspended in phosphate-buffered saline (PBS) to an OD₆₀₀ of 1.0, and 1.5 ml was aliquoted into 2.0-ml centrifuge tubes in triplicate. The C. jejuni NCTC11168 ΔflgK strain has almost entirely absent flagella (12) and was therefore used as a likely negative control (47). Bacterial suspensions were incubated for 24 h at room temperature, followed by the removal of 200 μl from the top of the sample for OD₆₀₀ measurements.

RESULTS

Mutant screen for transformation.

A random screen was performed to try to identify factors of NCTC11168 which inhibited plasmid transformation. Approximately 40,000 random transposon mutant isolates of NCTC11168 were produced, harvested as a pool, grown overnight, and made electrocompetent. One microgram of pWM1007 purified from either an E. coli (DH5α) host or a C. jejuni (RM1849) host was electroporated into this pool, followed by selection for the plasmid antibiotic marker. No transformants were obtained with the plasmid from the E. coli host; however, 164 transformants were obtained with the plasmid from the C. jejuni host. The transposon insertion sites for 2 of the transformants were found to be in the same location in the cj1051c gene, a probable restriction-modification enzyme. The transposon insertion of the other transformants was not determined but may be part of a future study.

Sequence analysis of cj1051c.

Analysis of the predicted R-M and methylation systems of NCTC11168 (49) revealed seven potential systems (Fig. 1): one type I system, one type IV system, three type IIG systems with restriction and modification components fused (including cj1051c), one type II system containing only a methylation component, and a dam-like methylation enzyme. A comparison of the genetic region of cj1051c across other common laboratory strains, 81116 and 81-176, revealed differences between each strain (Fig. 2A). The genome sequence of 81116 at the equivalent locus contains a poly(A)₇ tract generating a stop codon and two ORFs, C8J_0993 and C8J_0992. The restoration of a full-length ORF by deletion to a poly(A)₆ tract resulted in a gene with a high level of nucleotide identity across the majority of the cj1051c ORF, except for regions in the predicted specificity domains. In strain 81-176, it appears that the cj1051c locus is entirely absent, and a BLAST search did not find it elsewhere in the 81-176 genome. A BLASTp search for cj1051c found 66 C. jejuni and Campylobacter coli strains with a high level of similarity across the protein sequence but variation in the predicted specificity regions (Fig. 2B). Only two strains, C. jejuni 110-21 and C. jejuni DFVF1099 (57), had complete sequence identity to the NCTC11168 sequence. Additionally, 33 strains (not including NCTC11168) contained a poly(G)₉ tract between the two predicted specificity regions. We found evidence of possible phase variation in 3 strains due to insertions or deletions in this tract leading to premature termination (Fig. 3).

Fig 1.

Predicted methylation and R-M systems of C. jejuni strain NCTC11168 based on REBASE and the genomic sequence. White arrows indicate methylation or R-M system genes, and gray arrows indicate the genomic context of the adjacent genes.

Fig 2.

(A) Comparison of common laboratory strains NCTC11168, 81116, and 81-176 in the region of cj1051c. The insertional mutation indicated by an asterisk in 81116 results in a frameshift and premature ORF termination. Gray regions indicate nucleotide identity. (B) Percent consensus of the Cj1051c protein sequence across 67 C. jejuni and C. coli strains below the following Pfam-predicted regions: N, HSDR_N_2 (Pfam accession number PF13588), type I restriction enzyme R protein N terminus; M, N6_Mtase (accession number PF02384), N-6 DNA methylase; S, Methylase_S (accession number PF01420), type I restriction-modification DNA specificity domain. aa, amino acids.

Fig 3.

Portion of the ClustalW2 comparison of the cj1051c loci in C. coli 1417, C. coli 7-1, and C. jejuni 1336, with apparent phase variation in the G₉ tract prematurely terminating the reading frame compared to a representative full-length gene from C. coli strain H6. Both C. coli 1417 and C. jejuni 1336 appear to have single-nucleotide deletions, whereas C. coli 7-1 appears to have a 2-bp insert. This region is between the two specificity domains predicted in the protein; therefore, these mutations may not inactivate the enzyme. The boxed region highlights the poly(G) tract, and boldface type indicates the now-in-frame stop codons. “Cj” represents Campylobacter jejuni, and “Cc” represents Campylobacter coli, followed by the strain designation.

Effect of cj1051c on transformation efficiency.

In order to test the transformational efficiency of the transposon mutant of cj1051c, several attempts were made to cure it of pWM1007 with marker-free subculturing, heat and oxidative stress, and rifampin treatment (4). As this method failed, a Tet^r knockout strain of cj1051c (JH1) as well as a complementation strain (JH2) were produced. The transformation efficiency was tested by electroporation with 1 μg of pWM1007 purified from a C. jejuni host (RM1849) on strains NCTC11168, JH1, and JH2. Wild-type strain NCTC11168 did take up pWM1007 at a low efficiency of 12 (±3.3) CFU per microgram of plasmid, while knockout strain JH1 had an approximately 1,000-fold increase (1.9 × 10⁴ ± 9.9 × 10² CFU/μg) in transformation efficiency over this value, which was highly significant (P < 0.001). Complementation strain JH2 had an approximately 2-fold-lower (1.1 × 10⁴ ± 1.1 × 10³ CFU/μg; P < 0.05) transformational efficiency than did JH1. The above-described data are representative of replicates across different electrocompetent stocks and plasmid purifications and were compared with Student's t test.

Effect of the plasmid host on transformation.

Plasmid pWM1007, purified from several C. jejuni hosts (NCTC11168, JH1, and RM1849) and E. coli (DH5α), was used to transform wild-type strain NCTC11168 and strain JH1 naturally and by electroporation (Table 2). Transformation by the electroporation of wild-type strain NCTC11168 was most efficient with the plasmid prepared from NCTC11168, reduced with the plasmid from JH1, and least efficient with the plasmid from RM1849. The electroporation of NCTC11168 with the plasmid purified from an E. coli host failed. The electroporation of JH1 with a plasmid purified from any C. jejuni strain was highly efficient and was successful with a plasmid purified from an E. coli host.

Table 2.

Effect of plasmid host on transformational efficiency into the wild-type (NCTC11168) and cj1051c knockout (JH1) strains

Method	Plasmid host	Mean transformation efficiency into target strain (CFU/μg) ± SD
		NCTC11168	JH1
Electroporation	NCTC11168	1.1 × 103 ± 1.9 × 102	1.2 × 104 ± 1.5 × 103
	JH1	1.1 × 102 ± 8.1	2.9 × 104 ± 5.1 × 103
	RM1849	1.2 × 101 ± 3.3	1.9 × 104 ± 9.9 × 102
	DH5α	0	2.6 × 101 ± 2.3
Natural	JH1	0	6.6 × 102 ± 3.2 × 102
transformation	RM1849	0	4.9 × 101 ± 2.1 × 101
	DH5α	0	0

Effect of cj1051c on natural transformation.

Natural transformation of the wild-type strain and JH1 by pWM1007 purified from C. jejuni strains RM1849 and JH1 and E. coli strain DH5α was attempted (Table 2). Natural transformation with the plasmid purified from C. jejuni failed for the wild-type strain but was successful for JH1, having a higher efficiency with the plasmid derived from JH1 than with the plasmid derived from RM1849. Transformation by the plasmid purified from E. coli failed for both the C. jejuni wild-type strain and JH1.

Characterization of strain JH1.

Several assays were performed to characterize other phenotypes of JH1 which may be important for its use in further studies. Growth curves showed that JH1 was very similar to NCTC11168, although at early points OD₆₀₀ measurements for JH1 were higher (P < 0.05) (Fig. 4). There was no difference found in motility between NCTC11168 (10.7 ± 1.0 mm) and JH1 (10.0 ± 1.0 mm), and as expected, the motility of the negative control was lower (2.7 ± 0.3 mm) (P < 0.05). Both NCTC11168 (OD₆₀₀ = 0.079 ± 0.006) and JH1 (OD₆₀₀ = 0.012 ± 0.002) had increased autoagglutination over the NCTC11168 ΔflgK negative control (OD₆₀₀ = 0.651 ± 0.054) (P < 0.05); however, JH1 also had increased autoagglutination over NCTC11168 (P < 0.05).

Fig 4.

Growth curves for C. jejuni strain NCTC11168 and cj1051c knockout strain JH1. Both curves are highly similar, although JH1 does have higher levels at several time points. ∗, P < 0.05.

DISCUSSION

C. jejuni strain NCTC11168 is one of the most common laboratory strains used for Campylobacter research; however, the restriction barrier inhibiting transformation with plasmids from E. coli hosts and other C. jejuni hosts is a major limiting factor in its use. While it can readily take up DNA for homologous recombination by natural transformation or electroporation, plasmid transformation is problematic. This often necessitates the generation of chromosomal integrants for complementation studies with this strain. Analysis of the methylation and R-M systems of C. jejuni strain NCTC11168 revealed several systems present. Some or all of these systems potentially play a role in the restriction barrier of NCTC11168, and we therefore screened random mutants to identify which factors may be involved. To date, cj0031 has been shown to be subject to phase variation (5), cj1461 has been shown to have dam-like activity (36), and the type I system was analyzed and shown to have a grouping similar to those of RM1849 and 81-176 but not 81116 as well as to be subject to phase variation (46). To our knowledge, this is the first study to examine the R-M systems involved in plasmid transformation in C. jejuni.

Mutagenesis and complementation of cj1051c.

The cj1051c gene is predicted to encode a type II (subtype GSγ) R-M system enzyme with both restriction and methyltransferase activities (54). REBASE (54) predicts its recognition sequence to be GAG(N)₅GT, which occurs 11 times on the plasmid used, pWM1007. We initially identified this gene from two transposon mutants which apparently facilitated the electroporation of pWM1007. Both of these mutants had the same transposon insertion site, suggesting that they were sibling strains, either from the same pretransformation parent or due to replication during posttransformation recovery. The other 162 transformed mutant isolates did not have their mutation sites mapped in this study but may contain unique mutations relevant to plasmid transformation. This may be the topic of future work. Subsequent knockout mutagenesis of cj1051c resulted in a strain (JH1) which permitted the natural transformation of a plasmid from a C. jejuni host, permitted the electroporation of a plasmid from an E. coli host, and had an approximately 1,000-fold increase in electroporation efficiency with a plasmid from a heterologous C. jejuni host. This implies that this single factor, cj1051c, could be a major contributor to the restriction barrier in this strain. The rate of transformation of JH1 with the plasmid from an E. coli host was lower than that with the plasmid from a C. jejuni host, and this relationship is similar to that of C. jejuni strains 129108 (63), 81116 (61), and C31 (44). However, this is not the case for all C. jejuni strains, as strain 480 had a high transformational efficiency with a plasmid derived from an E. coli host (61). The complementation of JH1 by the genomic expression of cj1051c resulted in a strain (JH2) with an approximately 2-fold-lower (P < 0.05) transformational efficiency than that of JH1. Small effects were expected from the complementation strain based on previous work in the laboratory with genomic complementation. Nonnative promoter and ribosome binding sites as well as the cj1051c ORF being so large (4.0 kbp; the fourth largest in the genome [49]) coupled with only a single copy (genome, not plasmid, based) resulted in a low expected activity level. Indeed, the knockout mutant was not fully complemented to wild-type levels, but its levels were significantly lower than those of the knockout mutant. We believe that this verifies that cj1051c is a major factor in the restriction barrier of NCTC11168.

Phase variation of cj1051c.

A comparison of cj1051c across other common C. jejuni laboratory strains, 81116 and 81-176, showed that it has not been maintained. An insertional mutation in a poly(A) tract of the cj1051c locus in 81116 has caused a frameshift leading to the premature termination of the open reading frame, likely eliminating any activity. Strain 81116 is one of the most commonly used C. jejuni strains for plasmid work, and this mutation may explain its enhanced transformation efficiency. In 81-176, the cj1051c locus appears to be completely absent, which was previously noted for another clinical strain (51). Further support for phase variation at this locus is provided by the finding of a poly(G)₉ tract in most of the BLASTp hits for Cj1051c, which would be expected to be highly susceptible to phase variation. We found evidence that this has occurred in 3 strains with truncated ORFs due to mutations in this tract (Fig. 3). However, as this tract occurs between the two predicted specificity regions, and not toward the beginning of the ORF, it is unclear whether the truncation would result in an inactive enzyme or one with modified activity. This being the third R-M system to be identified in C. jejuni that may be susceptible to phase variation could explain why all of the type II REases present are fused to their MTases (Fig. 1). Otherwise, if nonfused enzymes were common, any inactivation of an MTase, but not the paired REase, by phase variation would be fatal.

Specificity domain variation.

In R-M systems, the specificity domain determines the recognition site of the targeted DNA to which the enzyme binds. The cj1051c loci in NCTC11168 and 81116 differ in the predicted specificity region of the gene, which, based on the sequences deposited in the NCBI database, appears to be common (Fig. 2B). A BLASTp search for Cj1051c found only two strains with complete identity, whereas 66 strains of C. jejuni and C. coli appear to have maintained the cj1051c locus with high sequence similarity throughout the protein, except with variation in the predicted specificity region (Fig. 2B). This finding is in agreement with a previous sequence analysis of this locus, describing it as the first case of a type IIG enzyme with divergence in the specificity domains between closely related strains (13). Likely, type II enzymes with both restriction and methylation components fused would be most available to this variation, as they would share the same specificity domain, and therefore, their targets would change together. However, any change in the target sequence would likely target a nonmethylated region and would therefore require the methylation of the new site to occur before restriction to prevent a lethal effect. The high level of variation in the specificity domain may serve to protect the organism against quickly evolving bacteriophages or to limit horizontal transfer between strains. C. jejuni is noted for its high degree of phase variation, which can affect bacteriophage infection (7, 56), possibly driving up the rate of bacteriophage variation, which may then be countered by a more flexible restriction system. Flexibility in the specificity domain would also minimize the horizontal transfer of genetic material between strains, as evidenced by the plasmid work here, which would tend to maintain more diverse populations (32).

Cj1051c behaves as expected for a type IIG enzyme.

Table 2 shows a range of rates of transformation for either the wild-type strain or JH1 based on the host from which the plasmid was extracted. When the plasmid is purified from another C. jejuni host (RM1849), it is able to transform JH1 with a much higher rate than for the wild-type strain. This is what would be expected if Cj1051c has REase activity, as predicted. The cleavage of the plasmid in the wild-type strain would be expected, assuming a lack of methylation at the recognition site, resulting in a lower transformation rate than that for a strain without Cj1051c activity. Additionally, the wild-type strain transforms better with a plasmid purified from the wild type than with a plasmid purified from JH1. This is what would be expected if Cj1051c has MTase activity, as predicted. The lack of this activity in JH1 would be expected to produce plasmids sensitive to restriction upon entry into the wild-type strain, as they would lack the appropriate methylation. This provides evidence that Cj1051c is active in NCTC11168 and behaves as expected for a type IIG enzyme predicted to have both restriction and methylation activities. The fact that the wild type is transformed better with a plasmid purified from the knockout strain than RM1849 also suggests that there may be another active R-M system present in the wild-type and knockout strains which is not present in RM1849. However, the finding that the rate of transformation into JH1 is high regardless of the plasmid's C. jejuni host strain suggests that cj1051c is a major determinant of the restriction barrier.

Application.

This study presents possibilities for future work to develop methods and tools for plasmid work with NCTC11168, as we anticipate that the effects observed here should be replicated with any plasmid, provided that it contains the recognition sequence. Due to its ability to be transformed by plasmids from an E. coli host, researchers could use JH1 as the parental strain for their own targeted mutagenesis studies followed by plasmid-based complementation. The ability of this strain to naturally transform plasmids may also be useful for laboratories without electroporation equipment. We therefore tested strain JH1 for any defects using common assays which may invalidate this application. No defects were found for the growth rate, motility, or autoagglutination, suggesting that, at least for this scope of phenotypes, JH1 may be a valid base strain for future work. Of note, however, is that JH1 did have increased autoagglutination over its parental strain, NCTC11168.

Since NCTC11168 can be transformed more efficiently by plasmids extracted from it, JH1 could also be used as an intermediate host to bridge the restriction barrier. Plasmid constructs developed in E. coli could be transformed into JH1, followed by transformation into wild-type strain NCTC11168. This would facilitate mutagenesis and plasmid-based complementation studies with wild-type strain NCTC11168 by using plasmid constructs developed in E. coli. Not only would this eliminate complications and potential side effects from genomic disruptions for complementation, it should additionally permit higher expression rates, as multiple copies of the expression construct would be present. Finally, since this factor plays a role in the restriction barrier of plasmids from an E. coli source, it may be possible to follow a strategy used for other species for artificial modification (66). The expression of cj1051c in an E. coli host may allow any plasmid construct developed in that host to be directly transformed into NCTC11168, as it would be appropriately methylated.

Our findings that C. jejuni strain NCTC11168 did not successfully transform a plasmid from an E. coli host and only very weakly transformed a plasmid from another C. jejuni host most likely explain the lack of plasmid work with this strain and the lack of success mentioned in the literature (33). We have shown that a major determinant of this appears be a restriction barrier mediated by cj1051c, which, when knocked out, allows an approximately 1,000-fold-enhanced transformation efficiency of a plasmid purified from another C. jejuni host. This mutation also allows the natural transformation of a plasmid from another C. jejuni host and the transformation of a plasmid from an E. coli host. Additionally, the strain commonly used for C. jejuni plasmid work, 81116, appears to have a homolog of cj1051c inactivated by an insertional event in a homopolymeric tract, which may explain the higher transformation rate for this strain. This finding, along with the other examples of homopolymeric tract inactivations in other strains at this locus, suggests that it is subject to phase variation. This increases the understanding of the active underlying molecular biology functions at work in C. jejuni and provides a tool which may increase the accessibility of strain NCTC11168 to common plasmid-based molecular biology techniques.