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Department of Infectious Diseases and Immunology, Utrecht University, Yalelaan 1, 3584 CL Utrecht, The Netherlands
Correspondence
Marc M. S. M. Wösten
M.Wosten{at}uu.nl
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The natural habitat of C. jejuni is the intestine of warm-blooded animals and a wide variety of watery environmental sources (Diergaardt et al., 2004; Rosef et al., 2001). This diversity in ecological niche demands that the bacterium is able to adapt rapidly to changing environments, such as alterations in temperature or the availability of oxygen and nutrients. In recent years a number of signal transduction systems have been identified in C. jejuni that may contribute to bacterial adaptation (MacKichan et al., 2004). One two-component signal transduction system important for bacterial survival is the PhosS-PhosR system, which senses changes in phosphate concentration and responds by altering the expression of a number of genes (the pho regulon) involved in phosphate acquisition and diverse metabolic processes (Wösten et al., 2006).
For most bacterial species, inorganic phosphate (Pi) is the preferred source of phosphate. Most bacteria assimilate Pi via specific uptake systems after hydrolysis of phosphate-containing substrates in the periplasm. An important enzyme involved in the hydrolysis of a variety of exogenous phosphate sources is alkaline phosphatase (PhoA). This metalloenzyme catalyses the non-specific hydrolysis of phosphomonoesters to an alcohol and Pi and thus makes Pi available for utilization by the bacterium. The production of PhoA is regulated by the phosphate concentration in the environment and upregulated during phosphate limitation. The enzyme is transported into the periplasm via the general secretory (Sec) system and becomes active after dimerization and binding of two Zn2+ and one Mg2+ cations (Torriani, 1990; Wang et al., 2005).
Intriguingly, search of the whole genome of C. jejuni for phoA homologues suggested that the pathogen lacks the typical PhoA. Characterization of the pho regulon of C. jejuni, however, indicated that the bacterium possesses a phosphatase that is involved in phosphate assimilation (Wösten et al., 2006). The corresponding gene (Cj0145) was upregulated during phosphate limitation and under the control of the PhosS-PhosR two-component system (Wösten et al., 2006). To further decipher the nature of this seemingly atypical enzyme, which was provisionally designated PhoACj, we further investigated the regulation, transport, activation and function of the enzyme. Here we provide evidence that the C. jejuni phosphatase deviates from the classical Escherichia coli PhoA in that it is transported over the cytoplasmic membrane via a previously unidentified twin-arginine translocation (Tat) secretion system, requires Ca2+ for its activity, and exclusively utilizes phosphomonoesters as a substrate.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. C. jejuni strains were routinely cultured under microaerophilic conditions (5 % O2, 10 % CO2 and 85 % N2) at 37 °C on plates containing blood agar base II (Oxoid) and 5 % horse blood lysed with 0.5 % saponin (Sigma) or in Heart Infusion broth (HI) (Oxoid). E. coli strains were grown on Luria–Bertani (LB) agar plates or in LB medium at 37 °C. When appropriate, media were supplemented with ampicillin (100 µg ml–1), kanamycin (50 µg ml–1) or chloramphenicol (20 µg ml–1).
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) using C. jejuni 81116 chromosomal DNA as template. The resulting 2181 bp PCR product was cloned into the pGEM-T easy vector (Promega) to form plasmid pGEM578. Inverse PCR was performed to introduce a deletion of 424 bp in the tatC gene and to create an unique BamHI restriction site by using the primers TAT-3BamHI and TAT-4BamHI (Table 2). After self-ligation, the plasmid was digested with BamHI and ligated to the Kmr cassette gene of pILL550 (Labigne-Roussel et al., 1987). Natural transformation (Wassenaar et al., 1993) was used to introduce the resulting knockout plasmid pGEM578 : : Km into C. jejuni to obtain the 81116 tatC : : Km mutant.
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) and tatC : : Km mutants, two E. coli–C. jejuni shuttle conjugative expression plasmids, pMA1 and pMA2, were constructed. To this end, the C. jejuni metK (Cj1096) promoter was amplified with the primers metKFSphI and metKRNsiI (Table 2
). After digestion with SphI and NsiI, the resulting 115 bp PCR product was ligated into SphI- and NsiI-digested pWM1007 (Miller et al., 2000), yielding plasmid pWM1007-pr.metK. The metK promoter, polylinker and the gene coding for green fluorescent protein (GFP) were amplified from pWM1007-pr.metK with the primers pILL550-pWM1007F and pILL550-pWM1007R (Table 2) and the 1.9 kb product was digested with BglII and EcoRI. The shuttle conjugative expression plasmid pMA1 was obtained by ligation of this 1.9 kb fragment into the BglII/EcoRI-digested plasmid pILL550. Plasmid pMA2 contains a chloramphenicol cassette (Cmr) instead of the kanamycin resistance gene. For construction of this plasmid, the Cmr of pAV35 (van Vliet et al., 1998) was amplified with the primers T7 and 22CATDO (Table 2). The PCR product of 880 bp containing one EcoRI site was digested with EcoRI and ligated into an EcoRI- and SwaI-digested pMA1, resulting in plasmid pMA2.
To complement the tatC : : Km mutant, the tatC gene was amplified with the primers TATcplforw.xbaI and TATcplrev1.sacI (Table 2). The restriction enzymes SacI and XbaI were used to clone the 800 bp tatC PCR fragment into pMA2, generating the complementation plasmid pMA2-tatC.
Plasmid pMA1 was used to introduce the phoACj, the GGphoACj construct (a gene coding for a PhoACj protein where the twin arginine residues are substituted by glycine residues) and an E. coli phoA gene into the C. jejuni phoA : : Cm mutant. By using the primer combinations phoAF1/phoAR and phoAF2/phoAR (Table 2) and C. jejuni 81116 chromosomal DNA as template, phoACj and GGphoACj PCR products were generated. These PCR products of 1800 bp in size were digested with SacI and SacII and ligated into pMA1, resulting in the plasmids pMA1-phoACj and pMA1-GGphoACj, respectively. The phoA gene of E. coli was PCR amplified from pCB267 (Schneider & Beck, 1986) with the primers phoAECF/phoAECR (Table 2). The 1800 bp PCR product was digested with SacI and SacII and introduced into pMA1 to form plasmid pMA1-phoAEc. All PCR products in this study were obtained with the proofreading enzyme Pfu (Promega) according to the instructions of the manufacturer. Nucleotide sequences of the cloned PCR products were verified by sequencing both strands. Complementation plasmids were introduced into C. jejuni mutants via conjugation (Labigne-Roussel et al., 1987).
Alkaline phosphatase assay.
Alkaline phosphatase activity was determined as previously described (Wösten et al., 2006), except that EDTA was omitted from the lysis buffer. In short, alkaline phosphatase activity of a bacterial culture grown in defined medium (Leach et al., 1997) containing 1.6 mM Pi (high) or 0.08 mM Pi (low) was assayed by monitoring the release of p-nitrophenol from p-nitrophenyl phosphate (PNPP) (Sigma). The units of alkaline phosphatase were calculated using the formula 103x[A420–(1.75xA550)]/txOD600xV.
Nitrite assay.
Nitrate reductase activity was determined as previously described (Sellars et al., 2002). Briefly, C. jejuni strains were grown overnight in HI medium containing 50 mM potassium nitrate. To the culture supernatants 1 % (w/v) sulphanilamide dissolved in 1 M HCl and 0.02 % (w/v) naphthylethylenediamine were added and after 15 min absorbance was measured at 540 nm. Nitrite concentrations, adjusted to the cell density, were determined by reference to a standard curve.
Growth experiments.
Overnight cultures grown in HI medium or defined medium were diluted to a starting OD600 of 0.05 and grown at 37 °C under microaerophilic (5 % O2) or oxygen-limited (0.3 % O2) conditions using the anoxomat system (MART Microbiology BV). Bacterial growth and cell density were monitored by measuring OD600 and counting c.f.u. at intervals.
Localization of the alkaline phosphatase activity.
A 10 ml C. jejuni culture grown with 0.08 mM Pi was subjected to cellular fractionation as described by Myers & Kelly (2005).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) (Wösten et al., 2006). To ensure that this phenotype was caused by disruption of PhoACj, we complemented the phoACj : : Cm mutant in trans with the shuttle plasmid pMA1-phoACj. This plasmid constitutively expresses the C. jejuni phoA gene via the metK promoter. Plasmid pMA1-phoACj restored the phosphatase activity when bacteria were grown in low-phosphate medium (Fig. 1, filled bars), indicating that PhoACj is responsible for the phosphatase activity.
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). This indicates that, at least when the gene is expressed from a multicopy plasmid, no additional components present under low-phosphate conditions are necessary for the PhoACj activity.
pH optimum of C. jejuni phosphatase activity
To assess whether PhoACj is an alkaline phosphatase, we measured the phosphatase activity at different pH values. Wild-type 81116 and the phoACj : : Cm mutant were grown in defined medium under low-phosphate conditions and lysed in Tris/HCl buffer containing SDS and lysozyme with different pH values. Maximum phosphatase activity was obtained at pH 10 (Fig. 2a). No phosphatase activity was observed for the phoACj : : Cm mutant over the entire pH range (data not shown), indicating that PhoACj is the sole phosphomonoesterase of C. jejuni.
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), although they all contained an intact phoACj gene (data not shown). One explanation for this diversity may be the variable presence of enzyme cofactors. As alkaline phosphatases of other species require divalent cations for their activity, we tested C. jejuni phosphatase activity in bacteria lysed in the absence of the metal chelator EDTA. All C. jejuni strains tested showed a strong increase in alkaline phosphatase activity in the absence of EDTA (Fig. 2b), indicating that PhoACj needs a cation for its activity.
The nature of the cation that activates the PhoACj was sought by selective addition of cations to bacteria grown overnight in the presence of 1 mM EDTA (Fig. 2c). Enzyme activity was measured 30 min after the addition of the cation(s) to the culture. Alkaline phosphatase activity in the EDTA-treated strains was almost fully restored by the addition of 10 mM Ca2+, while other ions including Mn2+, K+, Mg2+, Zn2+ (Fig. 2c) or Na+, Cu2+, Co2+, Ni2+, Mo6+, 
, Fe2+ or Fe3+ or combinations thereof (data not shown), did not restore enzyme activity.
Substrate specificity of C. jejuni PhoACj
The main function of bacterial alkaline phosphatases is to release Pi from various exogenous organophosphate compounds for use in bacterial growth. To assess which type of compounds may serve as a substrate for PhoACj, we tested various potential phosphate donors for their ability to support growth of C. jejuni 81116 and the phoACj : : Cm mutant strain. Measurement of the optical density and c.f.u. counting of overnight cultures showed that the wild-type strain but not the mutant grew with 1.6 mM of the organophosphate monoesters, glucose 6-phosphate (G6P) and glycerol 3-phosphate (G3P) as sole phosphate source, while no growth was observed with the phosphodiesters cAMP or DNA (Fig. 2d).
PhoACj requires a twin-arginine sequence for enzyme activity
A large number of bacterial alkaline phosphatases are secreted via the Sec-dependent pathway. Analysis of the PhoACj protein revealed that it contains a conserved twin-arginine domain near the N terminus, characteristic of proteins that are exported by the Tat secretion machinery (Stanley et al., 2000). To address whether PhoACj might be transported by the C. jejuni Tat system, we replaced the coding sequence for two arginine residues with two glycine residues in the phoACj located on plasmid pMA1-phoACj, containing the constitutively expressed metK promoter. The resulting plasmid pMA1-GGphoACj was introduced into phoACj : : Cm mutant strain and alkaline phosphatase activity was measured under high- and low-phosphate concentrations. This strain lacked alkaline phosphatase activity under conditions of phosphate limitation, while minimal activity (less than 5 % of wild-type) was observed under high-phosphate conditions (Fig. 3a). For the control strain carrying the plasmid with the original phoACj (pMA1-PhoACj), high levels of enzyme activity were present independent of the phosphate concentration in the medium (Fig. 3a). These results strongly suggest that PhoACj is secreted via a thus far unidentified C. jejuni Tat secretion machinery.
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; Parkhill et al., 2000) (Fig. 3b). To test the functionality of the Tat system in C. jejuni, we inactivated the putative tatC homologue by insertion of a kanamycin-resistance cassette. A severe growth defect as measured by optical density and c.f.u. counting was observed for the tatC : : Km mutant compared to the wild-type 81116 strain (Fig. 3c). This defect was rectified by introduction of plasmid pMA2-tatC harbouring a constitutively expressed C. jejuni tatC gene.
Further evidence of the functionality of the C. jejuni Tat system was obtained by measurement of nitrate reductase activity. A search of the C. jejuni proteome for putative Tat substrates based on the presence of the specific Tat recognition consensus sequence (RRxFLK) (Lee et al., 2006) indicated 11 potential Tat substrates (Table 3) (Dilks et al., 2003), including NapA. In other bacterial species this Tat substrate reduces nitrate to nitrite, which may be used by the bacterium as an alternative electron acceptor (Pittman & Kelly, 2005). Analysis of nitrate reductase activity indicated that C. jejuni 81116 exhibited this enzyme activity, while the tatC : : Km mutant did not (Fig. 3d). This defect was restored by complementation of the tatC : : Km mutant with pMA2-tatC.
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). To exclude that the lack of enzyme activity in the TatC mutant was caused by a more general protein secretion defect, we introduced into the tatC : : Km mutant strain a plasmid carrying the E. coli PhoA, which utilizes the Sec secretion pathway (Palmer & Berks, 2003). This enzyme was active in this background, indicating that the Sec system in the C. jejuni tatC mutant was still functional (data not shown). These results, in conjunction with the requirement of the twin-arginine motif, indicate that PhoACj is transported across the cytoplasmic membrane by the Tat secretion pathway. Like the E. coli PhoA, the C. jejuni PhoACj remains cell associated, as measurement of enzyme activity in the culture supernatant indicated that virtually all the activity remained associated with the cell fractions (Fig. 4b). The enzyme activity for the E. coli PhoA in C. jejuni was primarily found in the periplasmic fraction, while active C. jejuni PhoA was mostly found in the membrane fraction.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Here we provide evidence that the C. jejuni alkaline phosphatase (PhoACj) is a key enzyme in the acquisition of Pi. The enzyme differed from the typical PhoA present in most bacterial species as it required calcium as a cofactor and is transported into the periplasm via the Tat secretion pathway. The transport of PhoACj via the Tat system is the first functional evidence of the existence of this pathway in C. jejuni.
Key evidence that C. jejuni Cj0145 encodes the only functional alkaline phosphatase in C. jejuni was the successful complementation in trans of the mutant strain with an intact copy of this gene. We placed the intact phoACj gene on a shuttle plasmid under a constitutive promoter rather than its own PhosS-PhosR-regulated promoter to investigate whether additional phosphate-regulated factors were required for the enzyme activity. The strong enzyme activity in the complemented strain measured under high-phosphate conditions (Fig. 1) indicates that the C. jejuni phosphatase can act independently of other phosphate-regulated molecules. The enzyme utilizes calcium as a cofactor. This became evident when multiple C. jejuni strains were tested for phosphatase activity. In some strains, enzyme activity was only observed when EDTA, which was used to lyse the bacteria, was omitted from the lysis buffer or when an excess of calcium was added to the EDTA-containing buffer. The need for a divalent cation as a cofactor is not unusual, as alkaline phosphatases from other bacterial species also require divalent cation(s) to hydrolyse different types of phosphate compounds (Monds et al., 2006; Wang et al., 2005; Wu et al., 2007). E. coli PhoA requires Mg2+ and Zn2+ as cofactors. The requirement of Ca2+ as cofactor has thus far only been reported for PhoAVc of Vibrio cholerae (Roy et al., 1982) and PhoX of Pasteurella multocida (Wu et al., 2007). In P. multocida an aspartic acid together with a stretch of hydrophobic amino acids near the C terminus of the protein are essential for Ca2+ binding and phosphatase activity (Wu et al., 2007). Analysis of the PhoACj protein sequence indicates the presence of a similar motif.
Bacterial alkaline phosphatase provides bacteria with Pi by reducing different phosphoester compounds available in the environment. The alkaline phosphatase of P. multocida (Wu et al., 2007) can cleave both phosphomonoester and phosphodiester bonds. Our results indicate that the C. jejuni enzyme is able to utilize phosphomonoesters such as glucose 6-phosphate and glycerol 3-phosphate, but not phosphodiesters (Fig. 2d). This may limit the ecological niches of the bacterium.
Alkaline phosphatases generally exert the hydrolysis of phosphomonoester-containing substrates in the bacterial periplasm. E. coli PhoA and most of its homologues in other bacterial species are transported across the cytoplasmic membrane by the main protein secretion system, the Sec system (Palmer & Berks, 2003). Analysis of the protein sequence of PhoACj indicated the presence of a typical twin-arginine (Tat) consensus motif in the N terminus. This RRxFLK motif, in which the twin-arginine is highly conserved (Stanley et al., 2000), led to the discovery that the C. jejuni phosphatase exploits the Tat secretion machinery to gain access to the periplasm. Evidence that the Tat system serves as transport machinery for PhoACj includes the lack of enzyme activity after substitution of the twin-arginine residues (Fig. 3a) and after inactivation of the tatC gene, which results in dysfunction of the Tat system (Fig. 4a). The absence of enzyme activity in these strains even in the presence of calcium (data not shown) implies that the enzyme is not active in the cytosol, consistent with its assumed function. Although phoACj was transcribed, PhoACj is probably not transported through the E. coli Tat system, because no difference was observed in alkaline phosphatase activity between E. coli wild-type and a tatC mutant (data not shown). Furthermore, the E. coli tatC mutant could not be complemented with an intact C. jejuni tatC (data not shown). This may indicate that, in contrast to the Sec systems, at least some components of the Tat systems of C. jejuni and E. coli are not compatible.
The transport of PhoACj via the Tat system is the first reported evidence that this secretion pathway is functional in C. jejuni. Genome analysis predicts at least 11 putative Tat substrates in C. jejuni strain NCTC 11168 (Table 3) (Dilks et al., 2003). Besides PhoACj, we provide evidence that nitrate reductase NapA, a well-documented Tat substrate in other bacterial species (Ding & Christie, 2003; Lavander et al., 2006; Weiner et al., 1998), requires the Tat system of C. jejuni (Fig. 3d). The lack of nitrate reductase activity in the TatC mutant but gain-of-function after introduction of an intact copy of the gene clearly demonstrates that TatC, which is an essential component of the Tat system (Bogsch et al., 1998; Jongbloed et al., 2000), is required for NapA function. Considering the nature of most predicted Tat substrates, which are mostly cofactor requiring enzymes involved in electron transport (Table 3), it seems plausible to assume that the main function of the Tat secretion system in C. jejuni is to translocate redox cofactor-containing proteins that contribute to the assembly of the electron transport chain and energy conservation under oxygen-limited conditions (Palmer & Berks, 2003). Why the alkaline phosphatase of C. jejuni also exploits this secretion pathway, rather than the classical Sec system often used by PhoA of other species, awaits further investigation.
The atypical use of the Tat system to transport alkaline phosphatase across the cytoplasmic membrane has also been demonstrated for Pseudomonas fluorescens and Thermus thermophilus (Monds et al., 2006; Angelini et al., 2001) and is assumed for V. cholerae and P. multocida (Wu et al., 2007). P. fluorescens, however, also possesses Tat-independent alkaline phosphatases (Monds et al., 2006). Interestingly, the sole alkaline phosphatase PhoACj of C. jejuni is 52 % identical to the V. cholerae PhoAVc protein, and 49 % and 41 % identical to the PhoX proteins of P. fluorescens and P. multocida, respectively. Furthermore, PhoAVc of V. cholerae and PhoX of P. multocida are activated by Ca2+, as we found for the C. jejuni enzyme. Similarly, the pH optimum of PhoACj (
pH 10, Fig. 2a) is comparable to that of the P. multocida homologue (Wu et al., 2007), which is higher than that for E. coli PhoA (pH 8.5) (Nesmeyanova et al., 1981). Because of their shared dependence on the Tat secretion system, the alkaline phosphatases of V. cholerae, P. fluorescens and P. multocida have been proposed to form a separate group of enzymes, designated the PhoX family (Wu et al., 2007). The sequence similarity and functional characteristics of the C. jejuni PhoACj indicate that PhoACj also belongs to this group and therefore we propose to rename PhoACj as PhoX. This further supports the designation of a novel PhoX family of Tat-dependent alkaline phosphatases.
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ACKNOWLEDGEMENTS |
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tatC). This work was supported by NWO-VIDI grant 917.66.330 to M. M. S. M. W. Edited by: J. M. Becker
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 29 July 2007;
revised 2 November 2007;
accepted 6 November 2007.
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), the tatC mutant (tatC : : Km) (
) and complemented tatC mutant carrying plasmid pMA2-tatC (
) in HI medium under microaerophilic conditions. Growth curves are from one representative of three independent experiments. The optical densities as well as the corresponding c.f.u. ml–1 are presented. Bacterial cultures were diluted to OD600 between 0.1 and 0.5 to enable accurate measurement of OD600. The actual c.f.u. counts fully reflected optical densities for each strain for the entire duration of the incubation, indicating that bacterial growth was measured. (d) Nitrite reductase assay demonstrating that the enzyme required a functional TatC. Nitrite accumulation was measured in culture supernatants of 1x109 c.f.u. of C. jejuni strain 81116, the tatC : : Km mutant and the mutant containing complementation plasmid pMA2-tatC grown in HI with 50 mM potassium nitrate. Standard errors based on four independent experiments are indicated.