Genetics and Molecular Biology

A Cotransformation Method To Identify a Restriction-Modification Enzyme That Reduces Conjugation Efficiency in Campylobacter jejuni

Ximin Zeng, Zuowei Wu, Qijing Zhang, Jun Lin
Christopher A. Elkins, Editor
DOI: 10.1128/AEM.02004-18

ABSTRACT

Conjugation is an important mechanism for horizontal gene transfer in Campylobacter jejuni, the leading cause of human bacterial gastroenteritis in developed countries. However, to date, the factors that significantly influence conjugation efficiency in Campylobacter spp. are still largely unknown. Given that multiple recombinant loci could independently occur within one recipient cell during natural transformation, the genetic materials from a high-frequency conjugation (HFC) C. jejuni strain may be cotransformed with a selection marker into a low-frequency conjugation (LFC) recipient strain, creating new HFC transformants suitable for the identification of conjugation factors using a comparative genomics approach. To test this, an erythromycin resistance selection marker was created in an HFC C. jejuni strain; subsequently, the DNA of this strain was naturally transformed into NCTC 11168, an LFC C. jejuni strain, leading to the isolation of NCTC 11168-derived HFC transformants. Whole-genome sequencing analysis and subsequent site-directed mutagenesis identified Cj1051c, a putative restriction-modification enzyme (aka CjeI) that could drastically reduce the conjugation efficiency of NCTC 11168 (>5,000-fold). Chromosomal complementation of three diverse HFC C. jejuni strains with CjeI also led to a dramatic reduction in conjugation efficiency (∼1,000-fold). The purified recombinant CjeI could effectively digest the Escherichia coli-derived shuttle vector pRY107. The endonuclease activity of CjeI was abolished upon short heat shock treatment at 50°C, which is consistent with our previous observation that heat shock enhanced conjugation efficiency in C. jejuni. Together, in this study, we successfully developed and utilized a unique cotransformation strategy to identify a restriction-modification enzyme that significantly influences conjugation efficiency in C. jejuni.

IMPORTANCE Conjugation is an important horizontal gene transfer mechanism contributing to the evolution of bacterial pathogenesis and antimicrobial resistance. Campylobacter jejuni, the leading foodborne bacterial organism, displays significant strain diversity due to horizontal gene transfer; however, the molecular components influencing conjugation efficiency in C. jejuni are still largely unknown. In this study, we developed a cotransformation strategy for comparative genomics analysis and successfully identified a restriction-modification enzyme that significantly influences conjugation efficiency in C. jejuni. The new cotransformation strategy developed in this study is also expected to be broadly applied in other naturally competent bacteria for functional comparative genomics research.

INTRODUCTION

Campylobacter jejuni is a leading bacterial cause of human gastroenteritis in the United States and developed countries. C. jejuni infection may also result in long-lasting sequelae, such as irritable bowel syndrome (1), reactive arthritis (2), and even the life-threatening Guillain-Barré syndrome (3–6). Campylobacter is highly prevalent in food-producing animals; contaminated poultry meat is the major risk factor for human campylobacteriosis (7, 8). However, to date, there are still no effective intervention strategies to prevent and control Campylobacter infections in humans and animal reservoirs (8, 9), partly due to the significant genome plasticity and strain diversity observed in C. jejuni.

Horizontal gene transfer (HGT) plays a critical role in bacterial diversity, consequently contributing to bacterial evolution and adaptation in different environmental niches (10). Of the three general HGT mechanisms in bacteria (natural transformation, conjugation, and transduction) (11, 12), natural transformation has been well documented in C. jejuni (8, 13–18). However, information concerning conjugation, an important mechanism for the dissemination of antibiotic resistance and virulence factors, is still very limited in C. jejuni (19–22).

In our previous study, we observed that a simple heat shock treatment drastically enhanced conjugation efficiency in the standard C. jejuni NCTC 11168 and 81-176 strains, which display low-frequency conjugation (LFC) (23). This finding indicates the existence of a genetic determinant controlling conjugation efficiency in C. jejuni; however, the underlying molecular basis is still unknown after ruling out several factors, such as secreted substances, CRISPR, and selected restriction-modification components (23). In the same study, we also observed that some C. jejuni strains, such as CG8486, displayed extremely high-frequency conjugation (HFC) as a recipient strain compared to the LFC NCTC 11168 and 81-176 strains (23). Despite the availability of the whole-genome sequences of these C. jejuni strains, there exist great differences in their genetic background, which make it very challenging to identify the factor(s) controlling conjugation efficiency using a comparative genomics approach. For example, a preliminary genome comparison between NCTC 11168 and CG8486 revealed 106 different genes (24).

A well-recognized feature of C. jejuni is its high efficiency in natural transformation, a process widely observed in different bacteria to directly take up foreign DNA in the environment (11). With the aid of next-generation sequencing and comparative genomics technologies, recent natural transformation studies in different bacterial organisms (25, 26) have demonstrated that multiple recombination loci occurred independently (or cotransformed) within one recipient cell during natural transformation. This finding prompted us to speculate that some unique genetic materials from an HFC donor strain could be cotransformed together with a selection marker into an LFC recipient strain, consequently creating HFC transformants either through a knock-in or knockout mechanism; the new HFC transformants and LFC strain with the same genetic background can be used for efficient identification of conjugation factors using a comparative genomics approach. In this study, we developed a two-step cotransformation method with incorporation of an enrichment step for successful identification and characterization of a restriction-modification enzyme (Cj1051c, or CjeI) contributing to conjugation efficiency in Campylobacter jejuni.

RESULTS

ChuB mutation did not affect the conjugation efficiency in C. jejuni CG8486.The isogenic chuB::erm mutant (JL1126, Table 1) was successfully generated. This strain contains an erythromycin resistance (Eryr) selection marker in the HFC strain CG8486. The wild-type CG8486 and the chuB::erm mutant displayed similar conjugation efficiency, at (1.5 ± 1.0) × 10−4 CFU/recipient and (1.0 ± 0.02) × 10−4 CFU/recipient, respectively, as determined using the biparental conjugation method reported previously (23). Therefore, the isogenic chuB mutant of CG8486, which not only displays an HFC phenotype as its parent strain but also carries an Eryr selection marker, was suitable to serve as a genomic DNA donor for the two-step cotransformation approach used in this study.

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TABLE 1

Bacterial plasmids and strains used in this study

Two-step screening of the transformants with high-frequency conjugation.In the first step of screening (natural transformation), the Eryr transformants, which resulted from transformation of the chuB::erm, should be cotransformed with other variable regions (likely including those related to HFC) from the donor CG8486 (see Fig. 1A). If the Eryr transformant library contains a population with an HFC phenotype, the pooled Eryr transformants were expected to generate more progenies when serving as a recipient for conjugation. Compared to the conjugation using the LFC NCTC 11168 strain, the pooled Eryr transformant population displayed approximately 10-fold higher conjugation efficiency (data not shown), leading to the selection of 22 Eryr kanamycin-resistant (Kanr) transconjugants with potential HFC phenotype acquired, which were observed on the plates with a conjugation mix at higher dilution.

FIG 1
FIG 1

Identification and characterization of HFC transformants from C. jejuni NCTC 11168 using a two-step cotransformation strategy. (A) Schematic diagram of the two-step screening based on natural transformation. The donor genomic DNA from CG8486 with Eryr marker (chuB::erm, solid triangle) is used to transform NCTC 11168 with LFC phenotype. Variable regions (open rectangles), including the desired HFC factor (HFCF, solid circle), in the genome of C. jejuni CG8486 can be cotransformed with the Eryr marker (solid triangle). All the Eryr transformants are pooled and used as recipient for conjugation, together with the donor JL1116 containing shuttle vector pRY107 (Table 1); the LFC strain NCTC 11168 is used as a control recipient. Based on the difference in transconjugant emergence pattern, potential HFC transconjugants containing pRY107 (open oval) are obtained; the transconjugants with cured plasmid are used as recipient for conjugation to validate the HFC phenotype. Strains A1 and A5 are confirmed to be real HFC strains which originated from the same parent strain. Whole-genome sequencing and comparative genomics analysis identify the putative HFCF from the HFC CG8486 (within the dashed box). (B) The representative screening result showing the dramatic difference in conjugation efficiency when using the NCTC 11168 (left) and its derivative HFC transformant strain 1 (right) as a recipient. The undiluted conjugation mixture was spread on a selective plate. Compared to very limited transconjugants resulting from NCTC 11168 (left), large quantities of transconjugants were generated by using the HFC transformant strain 1 (>3,000-fold) (right).

To validate acquisition of the HFC phenotype in these 22 transconjugants, the conjugatively transferred pRY107 plasmid was cured from each potential HFC transconjugant, and the corresponding parental Eryr transformant was tested again for conjugation efficiency. A total of 9 transformants (no. 1, 11, 12, 13, 14, 17, 18, 19, and 21) were confirmed to have an HFC phenotype (>5 × 10−5 transconjugant CFU/recipient cell). Other transformants, such as transformants 20 and 22, displayed an LFC phenotype similar to that of the parental strain NCTC 11168 at a conjugation level of <5 × 10−7 transconjugant CFU/recipient cell (Fig. 2A). Therefore, using this unique two-step screening method, the plasmid-free Eryr derivatives of NCTC 11168 that displayed an HFC phenotype were successfully obtained (Fig. 2A).

FIG 2
FIG 2

Identification of CjeI as a limiting factor for conjugation. (A) Conjugation efficiency of representative Eryr transformants with potential HFC efficiency. Each bar represents mean of the results from two independent experiments, with duplicate measurements for each experiment. (B) Comparison of CjeI locus between C. jejuni NCTC 11168 and CG8486. The CjeI gene in C. jejuni NCTC 11168 is replaced by a two-gene operon in strain CG8486. (C) PCR analysis of CjeI locus. Two LFC transformants (20 and 22) and validated HFC transformants (1, 11, 12, 13, 14, 17, 18, 29, and 21) were used for PCR analysis. The length of corresponding PCR product at CjeI locus is 5,539 bp in NCTC 11168 and 3,471 bp in CG8486. (D) The effect of CjeI mutation on conjugation efficiency. The open bar denotes the heat shock untreated control, while the solid black bar denotes the recipient cells that were heat shocked at 50°C for 30 min. Each bar represents mean of the results from two independent experiments, with duplicate measurements for each experiment. The asterisk denotes statistically significant difference (P < 0.05).

Comparative genomic analysis among HFC, LFC, and NCTC 11168 reference genome sequences.A total of 6 HFC transformants (no. 1, 12, 14, 17, 19, and 21) and 2 LFC transformants (no. 20 and 22) were used for genome sequencing and comparative genomics analysis. Paired-end sequencing of these transformants in the Illumina MiSeq version 2 platform resulted in >200-fold genome coverage for each strain. De novo-assembled draft genome sequences from HFC and LFC transformants are compared with C. jejuni NCTC 11168. An initial examination (Table 2) revealed that two single-nucleotide polymorphisms (SNPs) were present in all HFC strains but absent from the two LFC strains. Based on annotation, the two SNPs, C→T at CheA and poly(C)10→poly(C)11 at Cj1429c, probably did not cause HFC. For example, the (C)10→poly(C)11 SNP might be due to a sequencing or assembly error.

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TABLE 2

Comparative analysis between HFC and LFC strains

Further analysis identified one gene replacement in which a two-gene operon from CG8486 replaced Cj1051c (CjeI) in two HFC transformants (no. 14 and 17; Fig. 2B). It is interesting that Cj1051c is annotated as a unique restriction enzyme that recognizes asymmetrical interrupted sequences (27) and is reported to be a restriction barrier of NCTC 11168 for plasmid transformation (28). PCR analysis confirmed that the gene replacement at the CjeI locus occurs in most (5 out of 9) HFC transformants (Fig. 2C) but is absent from all LFC transformants (data not shown). Therefore, the gene replacement at the CjeI locus is highly associated with the HFC phenotype.

Inactivation of CjeI increased conjugation efficiency and diminished the heat shock-enhanced conjugation.The comparative genomic analysis indicated that the LFC phenotype might be attributed to the function of CjeI. To test this, CjeI was inactivated in the LFC strain NCTC 11168. As shown in Fig. 2D, compared with wild-type NCTC 11168, the isogenic CjeI mutant displayed drastically increased conjugation efficiency to a level that is comparable to that of the HFC strain CG8486 (10−4 CFU/recipient cell).

Since we have observed that heat shock treatment drastically enhanced conjugation efficiency in NCTC 11168 (23), we further investigated if this phenotype is mediated through CjeI. As observed in a previous study (23), heat shock treatment of NCTC 11168 recipient cells led to an approximately 100-fold increase in conjugation efficiency (Fig. 2D). However, the CjeI mutant displayed a dramatic reduction in heat shock-enhanced conjugation, only resulting in an approximately 4.4-fold increase in conjugation efficiency (Fig. 2D). Together, these findings indicated CjeI is a limiting factor for the efficiency of conjugation in Campylobacter spp. and is also a major player involved in the heat shock-enhanced conjugation in LFC strains observed in a recent study (23).

Chromosomal complementation of CjeI in HFC strains decreased conjugation efficiency.The above-described CjeI inactivation experiment could not exclude the possibility of the polar effect caused by the insertion of chloramphenicol resistance marker cat. To further validate the role of CjeI in conjugation, CjeI was introduced into three HFC strains via chromosomal complementation. To perform chromosomal complementation, a new chromosomal integration vector, pROC, was created (Fig. 3A), which facilitates the addition of essential components (such as the appropriate promoter and selection marker) for gene expression at a ribosomal site. As shown in Fig. 3B, the introduction of CjeI in three HFC strains (CG8486, transformants 11 and 12) drastically decreased the conjugation efficiency (>1,000-fold reduction) to a level that was close to that of the LFC strain NCTC 11168 (<2 × 10−7 CFU/recipient cell). Of note, transformant 12 possesses CjeI but displayed the HFC phenotype. The complementation of CjeI could abolish the HFC phenotype, indicating that CjeI in transformant 12 might not be appropriately expressed or functional. Further analysis of assembled contigs of HFC transformant 12 revealed a C→T mutation at bp 929 of the predicted CjeI open reading frame (ORF), resulting in a GCT (Ala) to GTT (Val) mutation. Interestingly, this mutation was also observed in two other HFC transformants (no. 19 and 21). The assembled contigs of HFC transformant 1 showed another type of mutation, a T missing at bp 3455 of the predicted CjeI ORF, indicating a frameshift mutation. Therefore, those CjeI gene-containing HFC transformants (no. 1, 12, 19, and 21) all have a point mutation in the ORF of CjeI which is not observed in LFC transformants (20 and 22).

FIG 3
FIG 3

Chromosomal complementation of CjeI. (A) Schematic diagram of the chromosomal integration vector. The ribosomal fragment (rrs) from C. jejuni NCTC 11168 was cloned into NdeI/SphI-digested pUC19 to generate the plasmid pCjR. The fragment containing multiple cloning sites (indicated in figure) was subsequently introduced into pCjR, generating pCjR-MCS (Table 1), which facilitates downstream gene cloning. The overexpression promoter of Cj0299 (50) and chloramphenicol resistance marker cat were then incorporated into pCjR-MCS, leading to a new chromosomal integration vector, pROC (Table 1). The cjeI gene was finally cloned into pROC, generating the suicide vector pROC-CjeI (Table 1) for chromosomal complementation of cjeI. CjeI_Seq_F and Cm_Seq_R were used to confirm the integration of the complementation construct in the chromosome. (B) Conjugation efficiency of HFC strains with or without chromosomal complementation of CjeI. Besides the CG8486 wild type (CjeI gene-lacking), two representative HFC transformants, transformant 11 (CjeI gene-lacking) and transformant 12 (CjeI gene-containing), were also complemented with the CjeI gene (rrs::cjeI-cat). Each bar represents mean of triplicate measurements.

Expression and purification of rCjeI.In this study, we generated two recombinant strains, JL1165 and JL1166 (Table 1), that produce C-terminal His-tagged recombinant CjeI (rCjeI) and double His-tagged rCjeI, respectively. Upon induction by isopropyl thio-β-d-galactopyranoside (IPTG) at a final concentration of 0.5 mM at 37°C for 3 h, the rCjeI with 6×His tag at both the N terminus and C terminus was induced to a significantly higher level in JL1166 than the production of the rCjeI with C-terminal 6×His tag alone in JL1165 (data not shown). Therefore, the JL1166 construct was chosen for rCjeI purification. However, the growth of JL1166 at 37°C led to almost all of rCjeI going to the inclusion body. Although Empigen BB (Sigma, St. Louis, MO), a mild detergent, has been successfully used for the solubilization of functional His-tagged recombinant proteins from inclusion bodies in our previous studies (29–31), in this study, we failed to solubilize rCjeI from the inclusion body using Empigen BB (data not shown). Subsequently, we examined the production of rCjeI under various conditions and successfully expressed soluble rCjeI by growing cells at low temperature (4°C) with a long induction time (72 h) using a low concentration of IPTG (0.1 mM). As shown in Fig. 4A, the rCjeI with an approximate molecular mass of 157 kDa was significantly induced by a low concentration of IPTG by 24 h, and the production level continued to increase by 72 h postinduction. Approximately 50% of rCjeI was distributed in the soluble fraction. Finally, soluble rCjeI was successfully purified using nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography (Fig. 4A). The yield of rCjeI is approximately 1.8 mg/liter culture.

FIG 4
FIG 4

Functional characterization of CjeI. (A) SDS-PAGE analysis of rCjeI production and purification. The E. coli JL1166 was grown at 4°C for 72 h upon induction by a low concentration of IPTG (0.1 mM). The samples used for SDS-PAGE analysis include a whole-cell lysate of noninduced E. coli (0 h), whole-cell lysate of E. coli induced by IPTG at different time points (24, 48, and 72 h), and the soluble, insoluble, and eluted fractions during purification using Ni-NTA affinity chromatography. (B) Endonuclease activity analysis. The pRY107 extracted from E. coli DH5α was digested by rCjeI. The reaction mixture was sampled at different time points and subjected to DNA agarose gel (0.8%) electrophoresis. (C) The effect of heat shock on the activity of rCjeI. The rCjeI was heat shocked for different times (5, 15, 30, 45, and 60 min), followed by reaction with substrate DNA (pRY107) for 30 min. The reaction mixture without rCjeI (no rCjeI) and the mixture containing heat shock untreated rCjeI (no HS) were used as control. Samples were analyzed using agarose gel (0.8%) electrophoresis. HS, heat shock.

rCjeI displayed endonuclease activity.The CjeI is annotated as a type I restriction-modification enzyme. Given that the inactivation of CjeI drastically enhanced conjugation efficiency (Fig. 2D), we speculated that CjeI has endonuclease activity by digesting the pRY107 from E. coli DH5α effectively. As shown in Fig. 4B, in the absence of rCjeI (0 min), most pRY107 are in the supercoil form (a sharp bright band in the front). However, after being incubated with rCjeI for as short as 5 min, the pRY107 plasmid DNA consistently showed a low migration rate, indicating that plasmids became nicked and/or linearized. This finding demonstrated that CjeI possesses endonuclease activity on the pRY107 from E. coli DH5α.

rCjeI is heat labile.Based on above-mentioned finding that the heat shock-enhanced conjugation efficiency is primarily dependent on CjeI, we speculated that the endonuclease activity of CjeI can be greatly reduced upon temporary heat shock treatment. To test this, the thermostability of CjeI on the endonuclease activity was further examined in this study. As shown in Fig. 4C, temporary heat treatment of rCjeI at 50°C for as short as 5 min completely abolished its endonuclease activity, reflected by the same plasmid profile as that for the original pRY107. Only the rCjeI without heat treatment hydrolyzed pRY107 (Fig. 4C, third lane from left).

DISCUSSION

It is well known that the delivery of a heterogeneous shuttle vector into C. jejuni host is frustrating, whether using natural transformation, electroporation, or conjugation approaches, and it has become a hurdle for scientists to perform molecular manipulation for C. jejuni. Although C. jejuni strains are naturally competent, the shuttle vector derived from different species (e.g., E. coli) is still very difficult to be directly transformed into a C. jejuni host strain (13, 26), likely due to the presence of powerful restriction-modification systems in C. jejuni. To address this challenge, some natural transformation-based gene delivery approaches, such as chromosomal integration at ribosomal (32) or pseudogene (33) sites, have been developed; however, these approaches require complicated molecular manipulation. Thus, triparental or biparental conjugation has been widely used to deliver desired plasmids into the C. jejuni host. However, as observed in many laboratories and as reported in our recent study (23), different C. jejuni strains displayed drastic differences in the efficiency to acquire plasmid via conjugation. In particular, some commonly used standard C. jejuni strains, such as NCTC 11168, displayed extremely low conjugation efficiency. Recently, we have established a convenient heat shock protocol to enhance the conjugation efficiency of C. jejuni; however, several attempts to explore underlying molecular mechanisms were not successful (23).

In this study, a delicate two-step cotransformation approach was designed to identify the factors involved in conjugation in C. jejuni using a comparative genomics approach. The idea of cotransformation of potential conjugation-associated components with a selective marker was inspired by recent in-depth genomics studies on the mechanism of natural transformation (25, 26). These studies revealed that each transformant clone contains 1,000 donor polymorphisms in 3 to 6 contiguous runs (with the length of 8.16 ± 4.5 kb), which means the cotransfer of multiple genetic fragments from donor DNA can occur within every single transformant cell. On the basis of this evidence, therefore, the screening based on an Eryr selection marker (chuB::erm in this case) could generate a pool of transformants in which a variety of other donor DNA fragments (including the genetic determinant of interest) were expected to be introduced as well. In addition, each Eryr transformant colony obtained from nature transformation represents an enriched population of specific transformant cells; the pooled transformants with an enriched population are suitable for a comparative conjugation assay, which has been demonstrated in this study.

A restrictive component, CjeI, contributing to C. jejuni conjugation was successfully identified and characterized in this study. Based on annotation, CjeI belongs to the unusual restriction endonuclease, which requires S-adenosyl-l-methionine for endonuclease activity and can recognize interrupted sequence and asymmetrically cut the DNA (27). In a random transposon mutagenesis study (28), CjeI was identified as the restriction barrier to the transformation of a C. jejuni-derived plasmid into C. jejuni NCTC 11168. However, in the same study, the E. coli-derived plasmid was still very difficult to be transformed into the isogenic CjeI mutant of NCTC 11168, indicating that other restriction component(s) may be actively involved in the transformation process. In our study, a CjeI mutation could result in thousands-fold increase in conjugation efficiency when using E. coli DH5α as the conjugative donor, indicating that CjeI is a major limiting factor for conjugation in NCTC 11168. In addition, using purified rCjeI in conjunction with an in vitro endonuclease activity assay, we also demonstrated that CjeI is very sensitive to temporary heat shock treatment (at 50°C for 5 min); this finding is consistent with the fact that the heat shock-enhanced conjugation is primarily mediated through CjeI (Fig. 2D). Previous studies also showed that a heat-labile restriction-modification enzyme system was responsible for the heat-enhanced conjugation in Corynebacterium glutamicum (34, 35).

Clearly, CjeI is not the sole genetic determinant significantly affecting conjugation efficiency in C. jejuni, because conjugation is a complex process in which many factors are involved. In this study, we observed that heat shock treatment of the CjeI mutant still led to an approximately 4-fold increase in conjugation efficiency (Fig. 2D), suggesting the existence of another heat-labile restriction system to limit conjugation in C. jejuni. In addition, with respect to our screening, a total of nine transformants were confirmed to have an HFC phenotype (Fig. 2A). However, only five transformants (11, 13, 14, 17, and 18) have a gene replacement in the CjeI locus resulting from the knockout mechanism of our approach, leading to the characterization of CjeI in this study. Notably, as discussed above, the cotransformation strategy used in this study also has a knock-in feature. Therefore, the other four HFC transformants (no. 1, 11, 19, and 21), which are CjeI positive, may have acquired unique determinant(s) from HFC donor DNA; However, we also observe single nucleotide mutations in these four CjeI gene-containing but HFC strains. Furthermore, the introduction of CjeI in transformant 12 by chromosomal complementation drastically decreased conjugation efficiency (Fig. 3), indicating low CjeI activity in these four transformants. Therefore, we could not completely rule out the possibility that CjeI could not express or function appropriately in these four HFC strains. The identification of new determinants in these transformants needs further in-depth sequence analysis and molecular characterization. Finally, we speculate that the unique cotransformation approach used in this study could be used for the functional genomics study of many other bacterial organisms with high efficiency in natural transformation, such as Helicobacter pylori, Haemophilus influenzae, and Vibrio cholerae (15, 36, 37).

MATERIALS AND METHODS

Bacterial strains, plasmids, and culture conditions.The major bacterial strains and plasmids used are summarized in Table 1. The human isolates NCTC 11168 (38) and CG8486 (24) were characterized in a previous study (23) and used in this study. The C. jejuni strains were grown routinely in Mueller-Hinton (MH) broth or on agar at 42°C in Heracell 150i tri-gas incubator (85% N2, 10% CO2, 5% O2; Thermo Scientific). An E. coli donor strain containing both transmissible plasmid pRY107 (39) and helper plasmid RK212.1 (40) for biparental conjugation was constructed in a previous study (23). E. coli strains were grown routinely in Luria-Bertani (LB) broth with shaking (250 rpm) or on agar at 37°C (for cloning purpose) or 4°C (for expression of recombinant protein). When needed, culture media were supplemented with ampicillin (100 μg/ml), kanamycin (50 μg/ml), tetracycline (12.5 μg/ml), or/and chloramphenicol (20 and 6 μg/ml for E. coli and C. jejuni, respectively).

Construction of isogenic chuB::erm mutant.ChuB is prevalent in different C. jejuni strains (41) and was inactivated by insertion of the erythromycin resistance cassette erm (42). In brief, a 2.1-kb PCR fragment was amplified from C. jejuni NCTC 11168 using the ChuB_F/ChuB_R primer pair (Table 3) with Taq enzyme (Promega), which was further purified and ligated to pGEM-T Easy vector (Promega). The recombinant plasmid was subsequently digested with SwaI (NEB). Then, the vector backbone was ligated with the blunt-end erm PCR product that was used in our previous study (42). Approximately 200 ng of the resulting suicide vector was then introduced into high-frequency conjugation strain C. jejuni CG8486 through electroporation (43). Transformants were selected on an MH plate containing 5 μg/ml erythromycin (Ery), and the insertional mutation was confirmed by PCR using the chuB_F/Erm_seq_R primer pair (Table 3).

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TABLE 3

Major primers used in this studya

Two-step screening of transformants with HFC phenotype.The schematic diagram of the two-step screening of the transformants with HFC is partly shown in Fig. 1A.

Step 1, natural transformation.The genomic DNA of the isogenic CG8486 chuB::erm mutant was extracted using the Wizard genomic DNA purification kit (Promega). Approximately 4 μg of genomic DNA was used for natural transformation using the LFC strain NCTC 11168 as the recipient strain, as previously described (14, 23). The Ery-resistant (Eryr) transformants were grown and selected on MH plates containing 5 μg/ml Ery. Two independent natural transformation experiments were performed, leading to two libraries composed of approximately 1,000 and 2,500 Eryr colonies, respectively, in each transformation experiment.

Step 2, comparative conjugation.We modified our previous biparental conjugation protocol (23) to screen the NCTC 11168-derived Eryr (chuB::erm) transformants that potentially display the HFC phenotype. Each conjugation experiment was performed in two independent experiments with duplicate measurements in each experiment. In brief, the Eryr C. jejuni transformants from each library were harvested from plates using MH broth and mixed well. Approximately 500 μl of the Eryr C. jejuni transformant suspension (optical density at 600 nm, around 10) was used as the recipient and mixed with 500 μl of the logarithmic-phase E. coli donor strain JL1116 (optical density at 600 nm, around 1.2) containing shuttle vector pRY107 and the RK212.1 helper plasmid (23). The mixture was then pelleted down, resuspended in 100 μl of MH broth, and then spotted onto MH agar plate and incubated for 7 h under microaerophilic conditions. Following incubation, the mixed C. jejuni and E. coli cells were harvested from MH agar plates using 700 μl MH broth. The cell suspension was serially diluted (10-fold) in MH broth and differentially plated onto two types of MH plates, with one containing Campylobacter-specific selective antibiotics (SR117E; Oxoid) to recover the total C. jejuni recipient cells, and the other containing selective antibiotics (SR117E; Oxoid) plus kanamycin (50 μg/ml) to recover the C. jejuni transconjugants containing the pRY107 plasmid. The conjugation efficiency was determined on the basis of transconjugant CFU per recipient. As a control, the wild-type NCTC 11168 that displayed LFC phenotype was used and subjected to the same conjugation procedure detailed above. The NCTC 11168-derived Eryr transformants, due to containing a population of potential HFC transformants, were expected to display significantly higher conjugation efficiency than the control NCTC 11168. Specifically, the kanamycin-resistant transconjugants observed at the dilution at which no single transconjugant was observed in control strain potentially acquire an HFC phenotype. The transconjugants of interest were picked and subjected to validation as described below.

Validation of conjugation efficiency.To determine if the transconjugants resulted from HFC transformants, the shuttle vector pRY107 that confers kanamycin resistance was cured from each transconjugant. Briefly, each transconjugant was purified, inoculated into antibiotic-free MH broth, and passaged every other day for 2 weeks. The culture was then serially diluted in MH broth and plated on MH plates to retrieve isolated colonies. A panel of colonies were individually picked and replicated onto plain MH agar plates and MH agar plates containing kanamycin (50 μg/ml); the colonies that did not grow in the presence of kanamycin were desired plasmid-cured strains. These plasmid-cured strains were subsequently used as recipient strains for conjugation efficiency evaluation by performing the biparental conjugation experiment as described before (23). Each conjugation efficiency measurement was performed in two independent experiments, with duplicate measurements in each experiment.

Genome sequencing and comparative genomic analysis.Genomic DNA from eight selected plasmid-cured strains was extracted using the Wizard genomic DNA purification kit (Promega) and purified using the Genomic DNA Clean & Concentrator kit (Zymo Research). Genomic DNA was sequenced using the MiSeq platform at the Iowa State University DNA Facility, where genomic DNA was end repaired, ligated to specific adaptors, and subject to paired-end sequencing. After filtering raw reads, the clean reads were de novo assembled into contigs using the CLC Genomics Workbench. The contigs were aligned against reference genome C. jejuni NCTC 11168 (38, 44) for comparative analysis by using Mauve (version 2.3.0) (45, 46).

Inactivation of Cj1051c.The Cj1051c (CjeI) gene was inactivated via allelic exchange using a suicide vector, as described previously (47–49). Briefly, approximately 4.6 kb of Cj1051c fragment was amplified from C. jejuni NCTC 11168 using PfuUltra II fusion HS DNA polymerase (Stratagene) and the CjeI_F/CjeI_R primer pair (Table 3), and cloned into SmaI-digested pUC19. The pUC19-CjeI recombinant plasmid was digested with SwaI and then ligated with a blunt-end chloramphenicol resistance cassette, cat (42). The resulting suicide vector, in which the orientation of cat is the same as that of CjeI (detected with CjeI_F1/Cm_Seq_R [Table 3]), was introduced into C. jejuni NCTC 11168 via natural transformation (14). Transformants were selected on an MH plate containing 6 μg/ml chloramphenicol, and the insertional mutation was confirmed by PCR using the primer pairs CjeI_F1/CjeI_R1 and CjeI_F1/Cm_Seq_R (Table 3).

Chromosomal complementation of CjeI.The chromosomal complementation in the ribosomal sites was performed using strategy similar to that in a previous report (32), except a multiple-cloning site (MCS) was introduced in the chromosomal integration vector. The cloning process was illustrated in Fig. 3. First, approximately 1.9 kb of rrs fragment was amplified from C. jejuni NCTC 11168 using PfuUltra II fusion HS DNA polymerase (Stratagene) and primer pair rrsF (NdeI)/rrsR (SphI) (Table 3), and cloned into NdeI/SphI-digested pUC19, in which the MCS was removed by NdeI/SphI digestion. The resulting recombinant plasmid, pCjR, was digested with XbaI and MfeI and then ligated with annealed oligonucleotides pCjR_MCS_F1 and pCjR_MCS_R1, which introduce MCS (EcoRI-SacI-KpnI-BamHI-SalI-PstI-AfeI) into pCjR, resulting in the plasmid pCjR-MCS. Because CjeI is located within an operon and probably does not have an immediate adjacent promoter, a Cj0299 overexpression promoter from a beta-lactamase-resistant NCTC 11168 transformant JL974 (50) was amplified with the Cj0299_pMW10_F (XbaI)/Cj0299_pMW10_R1 (KpnI) primer pair, digested with XbaI/KpnI, and ligated with XbaI/KpnI-digested pCjR-MCS, resulting in plasmid pRO. The chloramphenicol resistance cassette cat (42) was amplified with the Cm_F (MfeI)/Cm_R (MfeI) primer pair, digested with MfeI, and ligated into MfeI-digested pRO, resulting in plasmid pROC. The cloning of the Cj0299 overexpression promoter and chloramphenicol resistance cassette cat into pCjR was confirmed by PCR (using the rrs_Seq_F/rrs_Seq_R and rrs_Seq_F/Cm-Seq_R primer pairs) and Sanger sequencing. The 4.1-kb fragment containing the whole ORF of CjeI was amplified with the CjeI_F4 (KpnI)/CjeI_R4 (BamHI) primer pair, digested with KpnI/BamHI, and ligated into KpnI/BamHI-digested pROC. The cloning of CjeI into pROC was confirmed by PCR (using the CjeI_F_Seq/Cm-Seq_R primer pair) and Sanger sequencing. The resulting suicide vector, pROC-CjeI, was introduced into C. jejuni Cj8486 and NCTC 11168 HFC transformants 11 and 12 via electroporation. Transformants were selected on an MH plate containing 6 μg/ml chloramphenicol, and the insertion of CjeI was confirmed by PCR using the CjeI_F_Seq/Cm-Seq_R primer pair (Table 3).

Production of recombinant CjeI.Histidine-tagged recombinant CjeI (rCjeI) was produced in E. coli using pET serial vectors. Briefly, a 4,017-bp fragment covering the full length of CjeI (amino acids [aa] 1 to 1339) was amplified from C. jejuni NCTC 11168 using the CjeI_F5 (NdeI)/CjeI_R5 (SalI) primer pair (Table 3). The NdeI/SalI doubly digested CjeI PCR product and expression vectors (pET-21b or pET-28b) were ligated and transformed into E. coli BL21(DE3). The pET-21b derivative clone (JL1165), which produces C-terminal-6×His (C-6×His)-tagged rCjeI, or the pET-28b derivative clone (JL1166), which produces 6×His-tagged rCjeI at both ends, was sequenced to confirm no errors. At first, the expression of both constructs was evaluated under normal conditions (3 h of incubation after adding 0.5 mM IPTG at 37°C). To increase the solubility of rCjeI, the expression of rCjeI from JL1166 was performed at 4°C for 3 days. Cell pellets were lyzed by incubation with lysozyme, followed by sonication. The soluble fraction containing rCjeI was purified using the procedures described in our previous publication (29). The eluted fractions containing pure rCjeI were pooled and dialyzed against buffer of 1× phosphate-buffered saline (PBS) with 10% glycerol. The concentration of the purified rCjeI was determined using a bicinchoninic acid (BCA) assay (51) and was aliquoted for storage at −80°C.

Endonuclease activity of rCjeI.The assay for the endonuclease activity of rCjeI was performed in the volume of 50 μl composed of 2.25 μg of purified rCjeI, 3 μg of shuttle vector pRY107, 50 mM potassium acetate, 20 mM Tris-acetate (pH 7.9), 10 mM magnesium acetate, 1 mM dithiothreitol (DTT), 100 μg/ml bovine serum albumin (BSA; NEB), and 80 μM S-adenosyl-methionine (catalog no. B9003S; NEB). The reaction mixture was incubated at 37°C; samples were taken at different time points, followed by immediate heat inactivation of rCjeI at 65°C for 15 min. The samples were subjected to agarose gel electrophoresis for examination of hydrolysis of pRY107 by rCjeI. When required, purified rCjeI was heat treated (at 50°C) before being added to the reaction mixture.

Data availability.The sequences of the eight Eryr NCTC 11168 transformants (Table 1) have been deposited into GenBank under BioProject PRJNA428364.

ACKNOWLEDGMENTS

Special thanks to Patricia Guerry for providing plasmids pRY107 and RK212.1 and C. jejuni isolate CG8486. We are grateful to Devarshi Ardeshna, Barbara Gillespie, and Samantha Brown for providing technical support. We also thank Barbara Gillespie for editing and proofreading the manuscript.

This project was supported by The University of Tennessee AgResearch. Ximin Zeng was supported by NIH grant R21AI119462.

FOOTNOTES

    • Received 16 August 2018.
    • Accepted 14 September 2018.
    • Accepted manuscript posted online 21 September 2018.

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KEYWORDS

Campylobacter jejuni
comparative genomics
conjugation
restriction-modification enzyme

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