Characterisation of Campylobacter jejuni genes potentially involved in phosphonate degradation
© Hartley et al; licensee BioMed Central Ltd. 2009
Received: 07 May 2009
Accepted: 25 June 2009
Published: 25 June 2009
Potential biological roles of the Campylobacter jejuni genes cj0641, cj0774c and cj1663 were investigated. The proteins encoded by these genes showed sequence similarities to the phosphonate utilisation PhnH, K and L gene products of Escherichia coli. The genes cj0641, cj0774c and cj1663 were amplified from the pathogenic C. jejuni strain 81116, sequenced, and cloned into pGEM-T Easy vectors. Recombinant plasmids were used to disrupt each one of the genes by inserting a kanamycin resistance (KmR) cassette employing site-directed mutagenesis or inverse PCR. Campylobacter jejuni 81116 isogenic mutants were generated by integration of the mutated genes into the genome of the wild-type strain. The C. jejuni mutants grew on primary isolation plates, but they could not be purified by subsequent passages owing to cell death. The mutant C. jejuni strains survived and proliferated in co-cultures with wild-type bacteria or in media in which wild-type C. jejuni had been previously grown. PCR analyses of mixed wild-type/mutant cultures served to verify the presence of the mutated gene in the genome of a fraction of the total bacterial population. The data suggested that each mutation inactivated a gene essential for survival. Rates of phosphonate catabolism in lysates of E. coli strain DH5α were determined using proton nuclear magnetic resonance spectroscopy. Whole-cell lysates of the wild-type degraded phosphonoacetate, phenylphosphonate and aminomethylphosphonate. Significant differences in the rates of phosphonate degradation were observed between lysates of wild-type E. coli, and of bacteria transformed with each one of the vectors carrying one of the C. jejuni genes, suggesting that these genes were involved in phosphonate catabolism.
Phosphorus is an essential element for living organisms. In bacteria it has roles in biosynthesis and energy metabolism, as well as in the structure of biomolecules. Phosphonates (Phn) are a class of organophosphates structurally similar to phosphate esters, but characterised by a carbon-to-phosphorus (C-P) bond [1–3]. The C-P bond is highly stable compared to oxygen-to-phosphorus, nitrogen-to-phosphorus or sulphur-to-phosphorus bonds. The C-P bond is resistant to chemical hydrolysis, thermal decomposition, photolysis , and to the action of phosphatases [5, 6]. During phosphate limitation stress, some bacteria can utilise phosphonates as an alternative source of phosphorus by breaking the C-P bonds of these compounds [7–9].
Four routes of phosphonate catabolism are known in microorganisms, namely, the phosphonoacetaldehyde hydrolase (phosphonatase), phosphonoacetate hydrolase, phosphonopyruvate hydrolase and C-P lyase pathways [6, 10]. Cleavage mechanisms and substrate specificity are different for each pathway. C-P lyase pathways are able to cleave a variety of phosphonates including aminomethylphosphonate (AmePhn) and phenylphosphonate (PhePhn), the latter is not degraded by other enzymes of phosphonate catabolism [1, 11–13].
Genes encoding C-P lyase pathways have been found in bacteria of the genera Enterobacter, Escherichia, Klebsiella and Kluyvera [1, 11–14]; and phosphonatase pathways are found in the genera Enterobacter and Salmonella [11, 15]. Recently, it was demonstrated that Campylobacter spp. are capable of degrading some phosphonate-bearing compounds although in silico analyses indicated that no genes orthologous to those encoding C-P bond-cleaving enzymes in other bacteria are present in the genome of Campylobacter jejuni . Two different C-P bond cleavage activities were discovered in Campylobacter spp. The first one is associated with the cell wall and has the ability to catalyse phenylphosphonate and phenylphosphinate (PhePhi). The second cleavage activity is found in both the cell wall and cytosolic fractions and catalyses phosphonoalkyl carboxylates . Phosphonate metabolism in Campylobacter does not require phosphate starvation conditions, unlike other bacteria in which the expression of Phn-degrading enzymes are under control of the Pho regulon [7, 13, 16].
The molecular basis of Phn catabolism by C. jejuni wasinvestigated in this study. In silico analyses were performed to identify C. jejuni genes encoding proteins with similarity to those involved in C-P bond hydrolysis in E. coli K12 . Isogenic mutants of wild-type C. jejuni strain 81116 were constructed by disrupting with a kanamycin cassette three genes encoding proteins potentially implicated in phosphonate degradation, and the viability of the mutants was determined. Mutants of E. coli DH5α were generated by transforming the wild-type strain with plasmids carrying each one of the C. jejuni genes putatively involved in Phn catabolism. The rates of degradation of three phosphonate compounds by the mutant E. coli were compared to those of the wild-type.
In silico analyses of genes putatively involved in phosphonate catabolism
Proteins found by sequence and domain searches of the genome C. jejuni strain NCTC 11168 using E. coli enzymes involved in phosphonate utilisation
E. coli Protein
C. jejuni Protein/Function
Sequence Similarity (%)
Cj0774c/Probable binding protein.
Investigation of the genomic organisation of the genes cj0641, cj0774c and cj1663 indicated that they were not co-located, and none of the genes appeared to be co-transcribed with other genes.
Isolation of Campylobacter jejuni genes
Primers used to clone C. jejuni strain 81116 genes
5'-CTT TTG CTT TGC TAA GAT TTG AT
5'-TAA TCA TCA ATT TCC CCA GTC
5'-CTT CAT TCA TGA TGC CAC CTC C
5'-TGA ACT TCA AAA TCT AAG AGG T
5'-GTG TGT GAA AAT TTG AAA GGT G
5'-TCA TTT TAA CAC CCC ATG TTG
cj1663 p2A INV
5'-GAA GAT CTC CCA TTG TCT AAG ATA TAC TCC C
cj1663 p2B INV
5'-GAA GAT CTC CAA TAC TAT CAC TCA TGG ACA T
In vitro mutagenesis of Campylobacter jejuni genes
Disruption of cj0641 in plasmid pGU0303 and of cj0774c in plasmid pGU0304 was performed by insertion of a non-polar KmR cassette  within unique restriction sites Cla I and Hind III, respectively. No unique restriction sites existed in cj1663 which would serve to insert an antibiotic cassette in this gene. Hence, inverse PCR of pGU0305 was employed to generate Bgl II restriction sites for insertional inactivation of cj1663. The primers cj1663 p2A INV and cj1663 p2B INV were designed based on the nucleotide sequence of cj1663 cloned into recombinant plasmid pGU0305 (Table 1). Inverse PCR of pGU0305 generated a linearised plasmid with Bgl II sites incorporated into the primer termini, which was used to insert the KmR cassette into cj1663. The insertion of the KmR cassette within each gene was verified by restriction enzyme digest and sequence analysis.
The recombinant plasmids with disrupted C. jejuni genes pGU0306 (pGEM-T EasyΩcj064 1ΩKmR), pGU0307 (pGEM-T EasyΩcj0774c ΩKmR) and pGU0308 (pGEM-T EasyΩcj1663 ΩKmR) were subsequently employed to generate isogenic mutants of C. jejuni 81116.
Generation and identification of C. jejuni 81116 isogenic mutants
The disrupted cj0641, cj0774c and cj1663 genes were introduced into the genome of wild-type C. jejuni by electroporating electro-competent C. jejuni 81116 cells with plasmid DNA containing the mutated genes. Transformed bacteria could only be recovered on primary isolation plates.
Growth rates of mixed cultures of C. jejuni strain 81116 wild-type and isogenic mutants in fresh and conditioned Brucella Broth with and without 50 μg/ml kanamycin.
Bacterial growth (cfu/ml)
7.0 × 109
6.0 × 109
6.5 × 109
7.0 × 109
Brucella Broth + Kanamycin
Conditioned Brucella Broth
7.0 × 109
8.5 × 109
7.2 × 109
7.8 × 109
Conditioned Brucella Broth + Kanamycin
2.0 × 108
2.0 × 108
4.4 × 108
Measurement of phosphonate degradation by E. coli lysates
Degradation of PhePhn, phosphonoacetate (PhnAce), AmePhn and PhePhi, was measured in whole-cell lysates of wild-type E. coli DH5α grown on LBA plates. The catabolism of phosphonate was confirmed as resulting from enzyme activities by heating the lysates for 4 h at 80°C, or by suspending them in 1% SDS and observing the abolition of catabolic activity.
Previous studies demonstrated that Campylobacter spp. catabolise various phoshonates , but sequence analyses of the C. jejuni strains 11168 and RM1221 genomes did not identify in this species orthologues of genes involved in phosphonate degradation in E. coli or Salmonella typhimurium. Nonetheless, the coding sequences cj0641, cj0774c and cj1663 showed some sequence or domain homology to genes encoding proteins of the C-P lyase of E. coli. These genes were disrupted in C. jejuni strain 81116 to determine whether the resultant mutants were altered in their ability to degrade phosphonates. The low sequence similarity between E. coli PhnH, PhnK and PhnL and C. jejuni proteins was in contrast with the high sequence similarity found with Enterobacter sp. 638 and K. pneumoniae proteins of the C-P lyase pathway, suggesting that the C. jejuni enzymes belong to different pathways.
The three genes from C. jejuni strain 81116 were cloned, and analyses of their sequences showed nucleotide identities of 96–99% with the cj0641, cj0774c and cj1663 genes of C. jejuni strain 11168. The data indicated that the genes were highly conserved, even more than the highly conserved flaA genes of Campylobacter spp. which have similarities between 73.6 and 92.3% . The three genes were not co-located or co-transcribed in the C. jejuni 11168 or 81116 genomes, and did not form part of operons.
The genes cj0641, cj0774c or cj1663 were inactivated by inserting a KmR cassette, and C. jejuni 81116 isogenic mutants were constructed by incorporation of each the disrupted genes into the genome of the wild-type strain through double allelic exchanges. The mutant bacteria were not viable, but were able to grow in mixed cultures with the wild-type strain. Growth experiments suggested that the mutants required for viability some factor(s) expressed by the wild-type strain. It is highly unlikely that a mutation in each of the three genes would result in identical phenotypes by chance. Inactivation of these genes by insertion of a KmR cassette may have resulted in polar mutations. However, none of three genes belongs to a recognisable cluster or to an operon, and analyses of the genes downstream of cj0641, cj0774c and cj1663 indicated that they were not co-transcribed. In addition, the insertion of the KmR cassette has been performed in numerous studies without having polar effects on downstream genes [18, 20]. Thus, polar mutations probably were not the cause of the decreased survival. Alternatively, it is possible that these genes encode multifunctional proteins given that the C. jejuni genome is one of the most densely transcribed genomes known to date , and some of the functions of those proteins could be essential to the survival of C. jejuni.
The results of the bacterial cell culture experiments indicated the dependence of mutated C. jejuni on the wild-type strain for survival, suggesting that the mutants lacked the ability to produce, regulate, or utilise some compound(s) produced by the wild-type C. jejuni strain 81116.
Indirect evidence for the involvement of the proteins CJ0641, CJ0774c and CJ1663 in phosphonate degradation was obtained by expressing them in E. coli DH5α transformed with plasmids carrying each one of the genes. The C-P lyase enzyme of E. coli is expressed under conditions of phosphate starvation . To ensure that this endogenous E. coli lyase was not expressed, bacteria were grown in the presence of inorganic phosphate. Hence, the catabolism of phosphonates observed in the transformed E. coli was the result of other enzyme activities (Figure 2).
Transformation of the wild-type E. coli strain with the plasmid pGEM-T Easy did not affect the rates of degradation of PhePhn, PhnAce and AmePhn. Transformation with a plasmid bearing cj0641, cj0774c or cj1663 altered the rate of PhePhn and PhnAce degradation relative to the wild-type (Figure 2). Transformation with a plasmid bearing one of the three genes inactivated with a kanamycin cassette had no effect on phosphonate degradation (Figure 2). The largest inhibition of PhePhn degradation was observed in E. coli transformed with cj0641, while the largest inhibition of PhnAce was due to transformation with cj1663. The relative decrease of phosphonate degradation between the three strains suggested that CJ0641 was more specific to PhePhn and CJ1663 was more specific to PhnAce. Comparison of the relative decrease of phosphonate degradation between substrates for each strain showed that E. coli transformed with pGU0303 and pGU0304 decreased PhnAce and PhePhn catabolism in equal amounts, while pGU0305 inhibited PhnAce degradation significantly more than that of PhePhn.
A reasonable expectation for the expression of an exogenous enzyme in a bacterium which also has a similar endogenous activity is that the overall activity measured in the transformed bacterium will be higher than in the parent organism. However, the measured activity is modulated by a number of factors including the relative abundance of both enzymes, their affinity for the substrate, their velocities and the substrate concentration. Regarding the latter, the overall measured activity will be higher when the concentration of the substrate is sufficient for both the endogenous and exogenous enzymes to operate as if the other were not present. Otherwise, both enzymes will compete for the substrate and depending on their relative affinities for the substrate and velocities, the overall measured activity may be higher, not significantly changed or lower. The latter case could occur when the exogenous enzyme has a much greater affinity and significantly lower velocity. In the case of PhePhn and PhnAce the substrate concentrations are well below saturation of the C. jejuni enzymes , hence the enzymes expressed in E. coli will compete with the endogenous activities, and the observed decrease of overall rates in the transformed bacteria could be explained by a competition of enzymes for the substrate
Several factors could be responsible for the lack of change in the rates of AmePhn degradation. C. jejuni has an enzyme system that degrades PhePhn and PhnAce and an independent system that degrades AmePhn, with the catabolism of AmePhn approximately half of that of the other two phosphonates . This suggested that the former activities were due to different enzyme(s) than that of the latter. Also, the absence of changes in the rates of AmePhn degradation may be due to homologues of the broad specificity C-P lyase of E. coli  having different specificities in C. jejuni. Finally, the rate of AmePhn degradation may have undergone changes which were below the levels of detection owing to its significantly smaller value than the rate of PhePhn and PhnAce.
No change was observed in the degradation of PhePhi between all strains showing that the changes in activity are specific to phosphonate degradation, thus, supporting the interpretation that these genes are involved in phosphonate metabolism.
The data showed that the gene products of cj0641, cj0774c and cj1663 were essential for the survival of the bacterium. In addition, evidence supported that CJ0641, CJ0774c and CJ1663 would be involved in phosphonate metabolism in the bacterium. The specific molecular events leading to changes in phosphonate degradation in transformed E. coli remain to be fully elucidated, but the study demonstrated that the C. jejuni proteins expressed in E. coli modulated the rates of phosphonate catabolism in the latter.
Brain Heart Infusion (BHI) Broth, Brucella Broth, Columbia Agar, Luria Broth, Tryptone Soya Agar (TSA) and defibrinated horse blood were from Oxoid (Heilderberg, VIC, Australia). Ampicillin and kanamycin, were purchased from Sigma (St Louis, MO, USA); and isopropyl-β-D-1-thioglactopyranoside (IPTG) and 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-Gal) were from Progen Biosciences (Archefield, QLD, Australia). Phosphonoacetic acid (PhnAce), α-aminomethylphosphonic acid (AmePhn), phenylphosphonic acid (PhePhn), and phenylphosphinic acid (PhePhi) were from Aldrich (Milwaukee, WI, USA). Sodium dodecyl sulfate (SDS) was from Amresco (Solon, OH, USA). All other reagents were of analytical grade.
Bacterial strains, plasmids and growth conditions
Bacterial strains and plasmids used in the study
Bacterial Strain or Plasmid
Origin and phenotype/Plasmid description
C. jejuni 11168
Human origin. Serotype O2. 
C. jejuni 81116
Human origin, motile isolate. 
E. coli DH5α
Sup E44Ωlac U169 (80lacZ ΩM15) hsdR 17 rec A1 end A1 gyr A96 thi-1 rel-A1 
pGEM-T EasyΩcj0641 (This study)
pGEM-T EasyΩcj0774c (This study)
pGEM-T EasyΩcj1663 (This study)
pGEM-T EasyΩcj0641 ΩKmR (This study)
pGEM-T EasyΩcj0774c ΩKmR (This study)
pGEM-T EasyΩcj1663 ΩKmR (This study)
"Conditioned" Brucella Broth to grow mutant C. jejuni bacteria was prepared by growing wild-type C. jejuni strain 81116 in Brucella Broth for 24 hours, removing the cells by centrifugation, and collecting and filtering the supernatant. The absence of viable cells in the supernatant was verified by plating it and observing no growth on the plates.
Escherichia coli strain DH5α was grown on Luria Broth Agar (LBA) plates supplemented with ampicillin, kanamycin, IPTG or X-Gal when required, and incubated at 37°C for 24 hours. Liquid cultures of E. coli were grown in Luria Broth with antibiotic supplements, ampicillin or kanamycin, where necessary, and under the same conditions as for plates.
Sequences of E. coli K12 proteins  were employed to search in the fully annotated genomes of C. jejuni strains NCTC 11168 and RM1221, as well as the genomes of Klebsiella pneumoniae and Enterobacter sp. 638 for genes orthologous to those involved in C-P bond hydrolysis. The E. coli sequences were those encoded by the genes phnG, phnH, phnI, phnJ, phnK, phnL, and phnM (accession numbers P16685, P16686, P16687, P16688, P16678, P16679, and P16689) that are required for alkylphosphonate catalysis and likely constituting a membrane-associated carbon-phosphorus (C-P) lyase [2, 7].
Sequence homology searches were performed using the BLASTP program for microbial genomes http://www.ncbi.nlm.nih.gov/sutils/genom_table.cgi? of the National Center for Biotechnology Information (NCBI; Bethesda, MD, USA). Domain homology searches were carried out using the Conserved Domain Architecture Retrieval Tool (CDART) of NCBI http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi?.
Primers for the genes cj0641, cj0774c and cj1663 in C. jejuni strain 81116, were designed using the published nucleotide sequence of C. jejuni strain NCTC 11168 . Primer sequences are shown in Table 2. Protein similarity analyses comparing the sequenced genes of C. jejuni 81116 to the published sequences of C. jejuni 11168 were performed using BLASTP program for microbial genomes of the NCBI.
Standard PCR in 50 μl reaction mixtures were performed with the following parameters: 95°C for 4 minutes, 30 cycles of 94°C for 30 s, 52°C for 30 s, and 72°C for 2 min. Template DNA was prepared by the crude lysis boiling method, in which a single C. jejuni colony was boiled for 5 min in 150 μl of water and cellular debris were removed by centrifugation.
Cloning and screening of recombinant plasmids pGU0303, pGU0304 and pGU0305
Amplified PCR fragments were cloned into the bacterial cloning vector system pGEM-T Easy (Promega; Madison, WI, USA) in E. coli by standard cloning techniques . The plasmids pGU0303, pGU0304 and pGU0305 each containing one of the three C. jejuni genes are described in Table 4. Transformant colonies putatively carrying recombinant plasmids were screened by blue/white colony selection, and further verified by PCR, using plasmid specific primers for the T7 and SP6 polymerase promoters, which flank the multiple cloning region of pGEM-T Easy. Prior to mutagenesis, constructs were sequenced by di-dioxynucleotide sequencing, to verify the correct base pair order.
In vitro mutagenesis of cloned Campylobacter jejuni genes cj0641, cj0774c and cj1663 Mutagenesis was performed by insertional inactivation of the genes cj0641, cj0774c and cj1663 using a non-polar kanamycin antibiotic resistance cassette KmR [26, 27]. The KmR cassette was amplified from the plasmid pMW2 using primers with Bgl II sites incorporated into the termini (Table 2). The KmR cassette was inserted into unique restriction sites within the cloned C. jejuni inserts. Linearised plasmids for pGU0303 (pGEM-T EasyΩcj0641), and pGU0304 (pGEM-T EasyΩcj0774c) were generated by restriction enzyme digestion with Cla I and Hind III respectively, according to the manufacturer's instructions.
The plasmid pGU0305 (pGEM-T EasyΩcj1663) was linearised by inverse PCR, as no unique restriction sites existed within cj1663. The primers cj1663 p2A INV and cj1663 p2B INV were designed based on the nucleotide sequence of pGU0305 and created a small deletion within cj1663. Inverse PCR in 50 μL reaction mixtures was conducted as follows: 95°C for 5 min, 40 cycles 94°C for 30 s, 52°C for 1 min, and 72°C for 5 min. The Eppendorf TripleMaster Taq was the DNA polymerase used for inverse PCR (Eppendorf; North Ryde, NSW, Australia).
Campylobacter jejuni isogenic mutants
Electrocompetent C. jejuni strain 81116 cells were prepared  and transformed with mutated recombinant plasmids using a BioRad MicroPulser (BioRad; Regents Park, NSW, Australia) at a voltage of 2.48 V, and time constant of 5 min. The contents of the 2 mm cuvettes were flushed with BHI broth and allowed to recover at 37°C for 5 h under microaerobic conditions. Reactions were plated onto TSA/HBA plates.
Measurement of phosphonate catabolism using nuclear magnetic resonance spectroscopy
Wild-type and transformed E. coli strain DH5α cells were grown on LBA plates supplemented with 50 μg/ml ampicillin and incubated at 37°C for 6 h. The bacteria were harvested in 150 mM sodium chloride (NaCl) and centrifuged 16,000 × g at 4°C for 10 min. The pellet was collected and the supernatant discarded. The pellet was resuspended in 14 ml 150 mM NaCl solution and washed twice more. Cells were lysed by twice freezing in liquid nitrogen and thawing.
Catabolism of AmePhn, PhePhn, PhePhi and PhnAce was measured employing proton nuclear magnetic resonance (1H-NMR) spectroscopy. Lysates (150 μl) were suspended in 150 mM NaCl and mixed with 2H2O (50 μl), 150 mM KCl (50 μl), and 150 mM NaCl (150 μl) before phosphonate was added at time zero to give a final volume of 600 μl. Initial concentrations of phosphonate substrates were 120 mM. Suspensions of bacterial lysates were placed in 5 mm NMR tubes (Wilmad; Buena, NJ, USA) and measurements of enzyme activities were carried out at 37°C. 1H-NMR free induction decays were collected using a Bruker DMX-600 spectrometer, operating in the pulsed Fourier transform mode with quadrature detection. The instrumental parameters for the spectrometer were: operating frequency 600.13 MHz, spectral width 6009.61 Hz, memory size 16 K, acquisition time 3.61 s, number of transients 64, pulse angle 50° (ca. 3 μs) and relaxation delay with solvent presaturation 1.7 s. Spectral resolution was enhanced by Gaussian multiplication with line broadening of 0.7 Hz and Gaussian broadening factor of 0.19. Proton spectra were acquired with presaturation of the water resonance. The time-evolution of substrates and products were followed by acquiring sequential spectra of the reactions. Enzyme rates were obtained by measuring the decrease over time of the intensity of resonances arising from the phosphonate compounds. Calibrations of substrate peaks were performed by extrapolating the resonance intensity data to zero time and assigning to this intensity the original concentration value.
The work was supported by a grant from the Australian Research Council.
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