Cj0734c/HisJ *

Campylobacter is an asaccharolytic microorganism which uses amino acids as a source of carbon and energy. CjaC/HisJ is a ligand-binding protein, a component of the ABC transport system. Campylobacter CjaC/HisJ is post-translationally modified by glycosylation. The number of glycosylation motifs present in the CjaC protein is species-specific. C. coli CjaC has two and C. jejuni one motif (E/DXNYS/T) which serves as a glycan acceptor. Although the two C. coli CjaC motifs have identical amino-acid sequences they are not glycosylated with the same efficiency. The efficacy of CjaC glycosylation in Escherichia coli containing the Campylobacter pgl locus is also rather low compared to that observed in the native host. The CjaC localization is host-dependent. Despite being a lipoprotein, CjaC is recovered in E. coli from the periplasmic space whereas in Campylobacter it is anchored to the inner membrane.


INTroDuCTIoN
Campylobacter spp., Gram-negative microorganisms, members of the ε-proteobacteria, are considered to be one of the emerging pathogens (Woolhouse, 2002;Moore et al., 2005).They are presently recognized as a leading bacterial cause of food-borne illnesses in Europe and the USA, and a major agent of bacterial diarrhoea worldwide.Campylobacter infections have been increasing steadily over the past decade.In the USA, an estimated 1.2 to 2.4 million cases of human campylobacteriosis occur every year.The disease symptoms of Campylobacter-mediated enteritis range from mild, watery diarrhoea to severe inflammatory diarrhoea.Infection with C. jejuni has been also associated with GBS (Guillain-Barre syndrome), an autoimmune disorder of the peripheral nervous system that may lead to respiratory muscle compromise or even death.The majority of cases of human enteritis caused by Campylobacter species are due to C. jejuni (80-85% of all enteric Campylobacter infections) and C. coli infections (10-15% of all enteric Campylobacter infections) (Taylor, 1992;Nachamkin et al., 1998;Skirrow & Blaser, 2000;Swartz, 2002).
Experimental documentation and data confirmed by sequencing have determined that C. jejuni NCTC 11168 lacks the phosphofructokinase gene necessary for the activity of the glycolytic pathway.This finding confirmed that C. jejuni NCTC 11168 is an asaccharolytic organism (Parkhill et al., 2000;Kelly, 2001;Velayudhan & Kelly, 2002).No phosphofructokinase gene has been identified in the genomes of other Campylobacter species sequenced so far or in the genomes of two closely related microorganisms:

2007
A. Wyszyńska and others Helicobacter pylori and H. hepaticus.Among the members of the ε-proteobacteria only Wolinella succinogenes, which is a cattle commensal, possesses a complete set of the glycolytic pathway genes (Fouts et al., 2005).Thus, Campylobacter relies on amino acids as a source of carbon and energy and its genome contains many genes responsible for amino acid uptake, mainly components of the ABC transport system.Consistent with this notion, inactivation of some genes potentially involved in amino acid transport resulted in attenuation of growth in the bird gut as documented by STM mutagenesis (Hendrixson & DiRita, 2004).Additionally, removal of single amino acids from the defined growth medium decreased the microorganism growth rate in vitro (Velayudhan et al., 2004).
In this report we analyzed the localization and post-translational modification of C. coli CjaC in comparison to its ortholog from C. jejuni 81176.Both genes encode 28 kDa immunopositive proteins, homologues of the solute binding components of the ABC transport system.For C. coli 72Dz/92 the protein was designated as CjaC, its ortholog from C. coli M275 was named HisJ and the one derived from the first Campylobacter strain to be sequenced (C.jejuni NCTC 11168) was annotated as Cj0734c.As it has been previously shown, the orthologs from C. jejuni and C. coli have only 90% of nucleotide sequence identity, whereas those from different C. coli clinical isolates (CjaC from C. coli 72Dz/92 and M275) display 100% identity.It has also been documented that specific primers which were designed based on the nucleotide sequence of the C. coli cjaC gene do not amplify the gene from C. jejuni (Garvis et al., 1996;Pawelec et al., 1998;2000).

MATErIAlS AND METHoDS
Bacterial strains and plasmids.The bacterial strains and plasmids used in this study are listed in Table 1.The C. coli 72Dz/92 strain (formerly classified by biochemical tests as C. jejuni) was obtained from Child Health Centre (Warszawa, Poland), from a patient with diarrhoea.The strain belongs to the most commonly isolated serotype in Poland, Lior 71.The C. jejuni 81-176 strain, isolated from an outbreak of Campylobacter diarrhoea associated with raw milk consumption, and widely used in pathogenesis studies, was a gift of M. J. Blaser (Korlath et al., 1985).Campylobacter and E. coli strains were cultured as previously described (Pawelec et al., 2000).Antibiotics (ampicillin (100 µg mL -1 ), kanamycin (40 µg mL -1 ) or chloramphenicol (20 µg mL -1 )) were added to the media when appropriate.
recombinant DNA techniques.Procedures for plasmid DNA isolation and DNA analysis (di-gestion with restriction enzymes, T4 ligation), agarose gel electrophoresis and transformation of E. coli competent cells were carried out as described by Sambrook and Russel (2001).Preparations of plasmid DNA for electroporation as well as isolation of DNA from agarose gels were performed according to manufacturer's instructions (A&A Biotechnology).Polymerase chain reactions (PCR) were performed with Taq polymerase (Qiagen) on a Mastercycler Personal (Eppendorf) under standard conditions.Oligonucleotide primers used in this work were synthesized by Sigma-Ark GmbH.Sequences of the primers are given in Table 2.
recombinant plasmid construction.Inverse PCR was employed to disrupt the cjaC gene from C. coli 72Dz/92.PUWM410, a derivative of pBluescript II KS carrying a 0.84 kb DNA fragment containing the cjaC gene, was used as a template.Deletion of 300 bp and a unique restriction EcoRI site were introduced into the cjaC gene by inverse PCR with primers CL and CR.The resulting plasmid was designated pUWM411.A 1.4-kb EcoRI restriction DNA fragment of the pBF14 plasmid containing a gene encoding resistance to kanamycin was cloned into the unique EcoRI site of pUWM411 generating pUWM416.
Transformation of Campylobacter.Inactivated genes were introduced into the Campylobacter genome by allelic exchange as described earlier (Wassenaar et al., 1993).C. coli 72Dz/92, in which the cjaC gene was disrupted, was designated AW6.The expected disruption of the chromosomal locus as a result of double cross-over recombination event was verified by PCR amplification.The loss of CjaC in C. coli AW6 was also demonstrated by Western blotting of whole-cell proteins with specific rCjaC antibodies.The mutated strain had normal colony morphology and exhibited normal growth rate when cultured on blood agar plates or MH medium.
Site-directed mutagenesis.Point mutations were generated using the Quick-Change site-directed mutagenesis kit by following the procedures recommended by the supplier (Stratagene).Plasmid pUWM77 (derivative of pBluescript II SK containing cjaC gene from C. coli 72Dz/92 (Pawelec et al., 1998)) was used as a template for PCR-mediated mutagenesis.Point mutations: N28A, N34A, N132A were introduced with primers: CNAS1/CNAS2, CNTT1/ CNTT2, CNDS1/CNDS2, respectively.The mutagenic oligonucleotide primers are summarized in Table 2. Plasmids containing the cjaC gene with various point mutations were transformed into E. coli DH5α and the presence of the desired mutations was verified by DNA sequencing.Fragments containing the cjaC gene with various point mutations were cloned into the pRY111 shuttle vector.Resulting plasmids were named pUWM758 (cjaC with N28A Post-translational modification of Campylobacter coli CjaC mutation), pUWM764 (cjaC with N34A mutation), pUWM763 (cjaC with N132A mutation).The shuttle plasmid containing a wild-type copy of the C. jejuni cjaC gene was named pUWM771.All derivatives of pRY111 were introduced into C. coli AW6 by electroporation.
Preparation of cellular fractions.Proteins from the periplasmic space were released by chloroform as described by Ames and coworkers (1984).The cell envelope was fractionated into inner and outer membranes by selective solubilization of the inner membrane (IM) with sarcosyl detergent (N-lauroyl sarcosine sodium salt).Preparation of Campylo-bacter membrane fractions was performed according to the method of Blaser and coworkers (1983) and E. coli membrane fractions were prepared using the procedure described by Filip and coworkers (1973).CjaCx6His protein was purified by affinity chromatography (Qiagen) under non-denaturing conditions, following cloning of the mature protein-encoding nucleotide sequence amplified by PCR into the pQE31 plasmid.
labeling of CjaC with [ 3 H]palmitate.E. coli XL1Blue cells harboring pUWM77 (pBluescript II SK containing the cjaC gene expressed from its own promoter) were grown in LB medium at 37°C to an  DNA, and proteins from cellular fractions (periplasm, cell envelope, outer-membrane) were separated by electrophoresis in 12% polyacrylamide gels containing SDS or by TSDS/PAGE and electrotransferred onto nitrocellulose membrane.Blots were developed using rabbit anti-Campylobacter or anti-rCjaC antibodies.To eliminate nonspecific reactivity, rabbit sera were absorbed first with heat-killed E. coli cells, and then with an E. coli cell lysate obtained by sonication.Afterwards, the sera were sterilized by filtration and kept frozen at -20°C.The serum against rCjaC was raised in rabbits immunized with rCjaCx6His as described earlier (Pawelec et al., 2000).Omp50 serum, obtained from J. M. Bolla, was raised in rabbit by three successive subcutaneous injections of the purified protein (Bolla et al., 2000).

rESulTS AND DISCuSSIoN
The Campylobacter cjaC gene (cj0734c) was identified in a C. coli 72Dz/92 cosmid genomic li-brary by immunoscreening with rabbit anti-Campylobacter antibodies.Its product, of a predicted molecular mass of 28.6 kDa, exhibits an overall homology to solute-binding proteins (family 3) of the ABC transport system (Pawelec et al., 1998).Its homolog from C. jejuni/coli M275 complements a hisJ deletion in Salmonella.Originally designated as C. jejuni, the Campylobacter M275 strain is now classified as C. coli (Konkel et al., 1999).
The Campylobacter cjaC gene was mutagenized by gene replacement as described in the methods section.Proteins isolated from the cjaC mutant and wild-type strain were separated by SDS/PAGE, transferred onto nitrocellulose and reacted with a specific rabbit anti-rCjaC serum.Three immunoreactive bands of approx.30, 33 and 35 kDa were observed when proteins derived from the wild-type strain were examined, whereas knockout of cjaC resulted in a loss of all immunoreactive bands (Fig. 1, lanes 4  and 5).The CjaC molecular mass (30, 33 and 35 kDa) calculated from the migration rate are all higher than the predicted one.The discrepancy probably reflects protein post-translational modification (see below).These results encouraged us to study CjaC derived from different Campylobacter clinical isolates in regard to its post-translational modification.Western immunoblot analysis with anti-rCjaC antibodies was carried out using whole-cell extracts of fourteen human clinical isolates belonging to two species: C. jejuni and C. coli.The Campylobacter isolates were classified into species based on PCR amplification with primers complementary to 23S rRNA (Pawelec et al., 2000).Two or three forms of CjaC were recognized by anti-rCjaC antibodies.The number of CjaC forms detected was strain-dependent (Fig. 2A and B).In Post-translational modification of Campylobacter coli CjaC the majority of cases, two forms of the protein were recognized with anti-CjaC antibodies in lysates obtained from C. jejuni cells whereas three CjaC forms were detected when C. coli lysates were analyzed.
In silico analysis of the CjaC orthologs from C. jejuni (251 amino acids), C. coli ( 256 amino acids), C. upsaliensis (252 amino acids) and C. lari (252 amino acids) revealed differences in the amino-acid sequence of the N-terminal fragment of the protein (Fig. 2C).
In trans complementation of the cjaC:Km disruption with pUWM771 carrying the cjaC gene resulted in three forms of CjaC whose molecular masses corresponded to those present in wild-type cells (Fig. 3B, lane 4).These data suggested that C. coli CjaC is present in three forms of different molecular masses.The two protein bands of lower mobility were much more intense than the one of the highest migration rate.The product of the cjaC gene expressed in E. coli from pUWM77 is visible mainly as a band that corresponds in mass to the lowest band seen for C. coli (Fig. 1, lane 1; Fig. 3A, lane 2).Recombinant CjaC (rCjaCx6His) was obtained as a cytoplasmic protein of a molecular mass lower than that of mature CjaC (Fig. 1, lane 2).To examine the potential post-translational modifications of C. coli CjaC, the pACYC184/ pgl plasmid (which carries the Campylobacter pgl (protein glycosylation) gene cluster functioning in E. coli) was introduced into E. coli expressing cjaC from its own promoter.Next, whole-cell extracts were examined for reactivity with rabbit anti-Campylobacter serum.The activity of pgl gene product resulted in two extra forms of CjaC of a decreased mobility, proving that CjaC is its target (Fig. 3A).The intensities of the two glycosylated protein bands were much weaker than of the band corresponding to the unglycosylated CjaC, indicating that the glycosylation process is not as efficient as in the native host.It should be pointed out that in E. coli most of CjaC is periplasmic, whereas in Campylobacter the protein is anchored in the inner membrane (see below).A silmilar difference between the efficiency of glycosylation mediated by the pgl locus in Campylobacter and E. coli cells was also observed for the C. jejuni VirB10 (Cjp3) protein, which is a pVirencoded component of TFSS.VirB10 was isolated as a component of a glycine-acid extract fraction but its localization in either C. jejuni or E. coli has not been determined by fractionation (Bacon et al., 2002;Larsen et al., 2004).

Post-translational modification of C. coli
Further analysis using site-directed mutagenesis was employed to study which of the three NXS/T motifs of CjaC is really modified by glycan binding.All N's from the three NXS/T motifs of CjaC were replaced by the neutral A. In effect, three derivative proteins with single amino acid substitutions were constructed as described in the methods section.The mutated versions of the proteins produced in C. coli AW6 were examined for their mobility by SDS/PAGE.Among the derivative proteins, two were impaired in glycosylation.Replacement of N by A at positions 28 or 34 resulted in proteins visible on the gel as two bands.The first band contained a protein of an apparent mass that corresponded to the nonglycosylated protein present in E. coli.The migration rate of the second band was like that of the middle band observed for wild-type CjaC from C. coli.Presumably, it contained a monoglycosylated form of the protein.Recently, it has been documented that the Campylobacter N-glycosylation system, unlike the eukaryotic one, requires a negatively charged amino acid at position -2 (motif D/EXNXS/T) (Kowarik et al., 2006).Although the two analyzed motifs (28-NAS-30 and 34-NTT-36) are preceded by an E residue in position -2 they are not equivalent substrates for glycosylation.Alanine substitution of N28 resulted in obtaining almost equal amounts of nonglycosylated and monoglycosylated forms of CjaC, whereas the mutated derivative with alanine substitution of N34 appeared on the gel as a band corresponding to the monoglycosylated form with a molecular mass equivalent to the middle band of the wild-type CjaC (Fig. 3 B, lanes 6 and 7).The same observation was made for the VirB10 protein (Larsen et al., 2004).Interestingly, the effectively glycosylated NAS motif is missing in the C. jejuni CjaC protein.In contrast to the NAS and NTT motifs, substitution of the NDS (position 132-134) amino-acid sequence by an ADS motif did not influence the number of CjaC forms.The latter motif, which is not preceded by E/D (position -2), is apparently not glycosylated in the C. jejuni CjaC protein.

CjaC localization
Analysis of the amino-acid sequence of CjaC showed that it is equipped with a putative signal sequence containing the LVAC motif that can be processed by signal peptidase II specific for lipoprotein precursors.The lipoprotein nature of CjaC was examined by growing E. coli XL-Blue harboring pUWM77 (pBluescript II SK containing the cjaC gene expressed from its own promoter) in the presence of [ 3 H]palmitic acid.The experiment suggested that CjaC is modified with palmitic acid, at least when expressed in E. coli.The data obtained was not completely clear, mainly because E. coli carrying the empty vector expressed a lipoprotein of a molecular mass comparable to that of CjaC (not shown).
The subcellular localization of CjaC in C. jejuni and C. coli was further examined by cell fractionation.Proteins obtained from different cell compartments were separated by SDS/PAGE, electrotransferred onto nitrocellulose membrane and detected with anti-rCjaC antibodies.Localization of the outer membrane protein Omp50 was also determined (Bolla et al., 2000).The CjaC localization was host-dependent.In both Campylobacter species the protein was recovered in the inner-membrane fraction (Fig. 4A, lane 5).Omp50 was mainly recovered from outer membrane, as expected (Fig. 4B, lane 6).In contrast most of CjaC expressed in E. coli cells was recovered as periplasmic (not shown).Also experiments carried out by Garvis et al. (1996) indicated that the CjaC/HisJ derived from the C. coli M275 strain in E. coli localizes in the periplasm.
Lipoproteins of Gram-negative bacteria are anchored in the inner or outer membrane via fatty acids attached to the N-terminal cysteine.Destination of E. coli lipoproteins to either the inner or outer membrane is dependent on the lipoprotein sorting signal, the amino-acid residue localized next to the lipid-modified cysteine, and on the activity of the LolA-LolB system (Tokuda & Matsuyama, 2004).According to the +2 rule, the presence of a G residue next to the lipid-modified cysteine in C. coli 72Dz/92 CjaC and an S residue in the same position in the sequence of the CjaC homologue from C. jejuni 81176, suggests that these proteins should be located in the outer membrane.The amino-acid sequences of E. coli Lol system proteins were used to search the non-redundant database at the National Center for Biotechnology Information (NCBI) with the PSI-BLAST algorithm to look for Lol proteins in C. jejuni NCTC 11168.Homologs of all of them but one (LolB) were found: LolA-Cj0943c, LolC-Cj0941c, LolD-Cj1277c, LolE-Cj1662c.The defectiveness of the C. jejuni Lol transport system may explain why some lipoproteins such as CjaC, CjaA or JlpA are partially released from the inner membrane and transported to the cell surface instead of being incorporated into the outer membrane (Jin et al., 2001).

CoNCluSIoNS
In summary, we have shown that localization of CjaC is host-dependent, as was also observed for Campylobacter CjaA lipoprotein (unpublished).The CjaC protein is probably not recognized by the E. coli Lol system and is released into the periplasm.Although the lipid-modified cysteine of CjaC is followed by the G/S residue, the protein was recovered in the inner-membrane fraction of the native host.The inner-membrane localization of CjaC is consistent with its physiological function.The post-translational modification of CjaC is species-dependent.C. jejuni CjaC exists in two forms, unglycosylated and monoglycosylated, whereas the protein from C. coli is present as three forms.Based on the predicted amino-acid sequences of CjaC from C. upsaliensis RM3195 and C. lari RM2100, which do not contain a potential glycosylation motif (E/DXNXS/T), one can suspect that these proteins are not glycosylated.Further experiments will be necessary to determine the physiological role of the post-translational modification of C. jejuni and C. coli CjaC.A simple PCR assay or Western blot analysis with anti-CjaC antibodies can be used to distinguish between two Campylobacter species: C. jejuni and C. coli.
CjaC Nita-Lazar et al. (2005) demonstrated that C. jejuni 81176 HisJ/CjaC, which contains two potential motifs of glycosylation, exists in two forms, an unglycosylated and a monoglycosylated one.That

Figure 4 .
Figure 4. Subcellular localization of CjaC (A) and omp 50 (B) in C. coli.Proteins derived from different cell fractions were separated electrophoretically on a 12% polyacrylamide gel, blotted onto a nitrocellulose membrane and probed with polyclonal anti-rCjaC (A) or anti-Omp50 (B) antibodies.The positions of molecular mass markers are indicated on the left.Lanes: 1, whole cell lysate; 2, osmotically shocked cells; 3, osmotic shock fluid-fraction of periplasmic proteins; 4, molecular size standard; 5, sarcosyl-soluble fraction (proteins of the inner membrane); 6, sarcosyl-insoluble fraction (proteins of the outer membrane); 7, proteins of both membranes.

Table 1 . Bacterial strains and plasmids used in this study
Preparation of bacterial protein extracts, SDS/PAGE and blotting were done by standard methods.Total proteins expressed by Campylobacter or E. coli containing C. coli -1 culture; DuPont-NEN) was added to the culture which was further incubated for 3 h.E. coli cells were then harvested, washed twice with 100% ethanol and the pellet air-dried.Bacteria were resuspended in PBS buffer and lysed with sample buffer.The released proteins were separated on a Tricine/ SDS/PAGE gel (TSDS/PAGE, 16.5% T, 3% C) and the radiolabeled lipoproteins detected by fluorography.Protein immunoblot analysis.

Table 2 . oligonucleotides used in this study
*Bold letters indicate C. coli sequences; restriction recognition sequences introduced for cloning purposes are underlined; mismatches are double underlined