Helicobacter ISSN 1523-5378
Phenylphosphonate Transport by Helicobacter pylori Ford O Phenylphosphonate r2007 i g iet ncompilation aUK al. l Publishing AAuthors r t i c l eTransport s Ltd by H. pylori Blackwell Oxford, © 1083-4389 Helicobacter HEL Journal XXX The © 2007 Blackwell Publishing Ltd, Helicobacter xx: xx–xx
Justin L. Ford,* Paul A. Gugger,† S. Bruce Wild† and George L. Mendz* *School of Medical Sciences, The University of New South Wales, Sydney, NSW 2052, Australia, †School of Chemistry, The Australian National University, Canberra, ACT 2601, Australia
Keywords Helicobacter pylori, phosphonate metabolism, phosphonate transport, phenylphosphonate, centrifugation through oil. Reprint requests to: Assistant Professor George L. Mendz, School of Medical Sciences, The University of New South Wales, Sydney, NSW 2052, Australia. Tel.: +61293852042; Fax: +61293851389; E-mail:
[email protected]
Abstract Background: Helicobacter pylori can utilize phenylphosphonate as a sole source of phosphorus, and it is able to transport the phosphonate N-phosphonoacetylL-aspartate. However, H. pylori does not have any genes homologous to those of the known pathways for phosphonate degradation in bacteria, indicating that it must have novel pathways for the transport and metabolism of phosphonates. Methods: Phenylphosphonate transport by H. pylori was studied in strains LC20, J99 and N6 by the centrifugation through oil method using [14C]-labeled phenylphosphonate. Results: The Michaelis constants of transport K t and Vmax for phenylphosphonate showed similar kinetics in the three strains. The Arrhenius plot for phenylphosphonate transport rates at permeant concentrations of 50 µmol/L was linear over the temperature range 10–40 °C with an activation energy of 3.5 kJ/mol, and a breakpoint between 5 and 10 °C. Transport rates increased with monovalent cation size. The effects of various inhibitors were investigated: iodoacetamide, amiloride, valinomycin, and nigericin reduced the rate of phenylphosphonate transport; sodium azide and sodium cyanide increased the transport rate; and monensin had no effect. Conclusions: The kinetics and properties of H. pylori phenylphosphonate transport were characterized, and the data suggested a carrier-mediated transport mechanism.
The Gram-negative, microaerophilic bacterium Helicobacter pylori belongs to the order of Campylobacterales [1]. Since it was first identified in 1983 [2], H. pylori has intensively investigated. The bacterium is thought to infect around 50% of the world population, and is considered to have evolved to colonize human hosts persistently for tens of thousands of years [3]. H. pylori colonizes the mucosa of the human gastric epithelium [4], predominately in the antrum, with the corpus also being colonized [5]. The majority of cases of H. pylori infection are asymptomatic [6], but the bacterium can cause chronic gastritis, loss of the mucosal lining of the stomach (ulcer, atrophy) and adenocarcinoma [7]. Phosphonates are stable, main-group organometallics that contain a phosphorus atom directly bonded to a carbon atom. The carbon–phosphorus bond energy has been calculated at 62 kCal, which is similar to that of the C-C bond (64 kCal) [8]. Accordingly, phosphonates are stable to degradation under a wide range of conditions [9]. The phosphorus group polarizes the adjoining carbon
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chain by partially donating an electron to the nearest carbon [10]. Phosphonates are rarely toxic to humans [9], and the most common industrial phosphonates are directly toxic to the environment [9,11]. Their strong chelation of metal ions can remove nutrients vital for biologic growth [11], albeit not at their environmental levels. Phosphonates are not common in nature but play vital biologic roles as phosphorus sources, phosphorus storage compounds [6], and in antibiotics such as for example, those produced by Streptomyces fradiae [3]. Phosphonates may have been significantly more prevalent in the environment during the initial evolution of life [6], and these compounds are found in a wide variety of organisms including eubacteria, fungi, mollusks, and insects [9]. Phosphonates are structurally similar to phosphate esters (C-O-P) and often act as inhibitors of enzymes primarily owing to the high stability of the C-P bond [9]. Metabolism of phosphonates can provide a source of phosphorus for some microorganisms [12]. Four phosphonate metabolism pathways have been elucidated: the
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phosphonatase pathway, the phosphonoacetate hydrolase pathway, the phosphonopyruvate hydrolase pathway, and the C-P lyase pathway [12]. 2-Aminoethylphosphonate (2-AEP) is a substrate of the Bacillus cereus phosphonatase pathway. Transport of 2-AEP by this bacterium shows Michaelis–Menten kinetics and is enhanced by aminomethylphosphonate [13]. Studies on phosphonate utilization by Escherichia coli indicated that its C-P lyase pathway was induced under phosphate starvation conditions. Genetic analyses of this pathway established that the 14 genes phnC to phnP are involved in phosphonate metabolism [14]. The phnC, phnD, and phnE genes of the C-P lyase pathway were proposed to form a binding-proteindependent phosphonate transporter [15], but its function and physiologic characteristics remain to be determined. H. pylori transports and degrades the phosphonate N-phosphonoacetyl-L-aspartate (PALA) [16]. PALA transport is saturable, with a Kt of 14.8 mmol/L and a Vmax of 19.11 nmol/minutes per microliter of cell water. The addition of phosphonoacetate significantly reduced the rate of PALA transport [16], suggesting that both compounds entered the cell via the same route. The bacterium H. pylori can grow on phenylphosphonate (Phephn) as the sole source of phosphorus, as can Campylobacter jejuni [17], which indicates that both bacteria take up the phosphonate. Phephn is a substrate of the C-P lyase pathway but H. pylori does not have genes orthologous to those encoding the C-P lyase pathway or to any of the known phosphonate metabolism pathways [12]. Phenylphosphonate transport was studied in three H. pylori strains using radioactive tracer analysis and the centrifugation through oil method. The transport kinetics was characterized, and the effects on transport rates of temperature, potential cofactors, and inhibitors were determined.
Methods Materials Blood agar base No. 2, defibrinated horse blood, and horse serum were from Oxoid (Heidelberg West, Victoria, Australia). Amphotericin (Fungizone®) was from BristolMyers Squibb (Noblepark, Victoria, Australia). Yeast Nitrogen Base without phosphate (YNB) and Synthetic Complete Supplement Mixture (without uracil) (SC-URA) were from Bio101 (Carlsbad, CA, USA). Phephn, αaminomethylphosphonate (Amephn), phosphonoacetate (Phnace), 2,5-diphenyloxazole (PPO), dibutylphthalate, dioctylphthalate, monensin, iodoacetamide, amiloride, nigericin, valinomycin, sodium azide (NaN3), sodium cyanide (NaCN), and dithiothreitol (DTT) were from Sigma-Aldrich (Castle Hill, NSW, Australia). Phosphate
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(Na2HPO4 and NaH2PO4) was from Merck (Kilsyth, Victoria, Australia). Bromo[U-14C]benzene (50 mCi/mmol) and [U-14C]taurine were purchased from GE Healthcare Biosciences (Sydney, NSW, Australia). [3H]H2O was from New England Nuclear (Du Pont; North Ryde, NSW, Australia). Toluene was from BDH Chemicals (Kilsyth, Victoria, Australia). Triton X-100 was from Ajax Fine Chemicals (Seven Hills, NSW, Australia). All other reagents were of analytical grade.
Synthesis of [U-14C] Phenylphosphonic Acid The palladium catalyst Pd(PPh3)4 was prepared by the method of Coulson [18]. Diethyl [U-14C]phenylphosphonate was synthesized by the palladium-catalyzed reaction of bromo[U-14C]benzene (0.13 g) with 1.2 equivalents each of diethylphosphite (0.138 g) and triethylamine (0.101 g), and 0.22 equivalent (0.21 g) of catalyst, according to the method of Hirao et al. [19]. The phosphonate ester was purified by column chromatography on silica gel by elution with diethyl ether and was isolated from the eluate as a pale yellow oil after evaporation of the ether, yielding 0.059 g (33%). Hydrolysis of this material by heating for 8 hours in 10 mol/L hydrochloric acid afforded the desired product as a grayish solid, which was purified by dissolving in water and passing the solution through a plug of cotton wool to remove a trace of palladium. Evaporation of the aqueous filtrate provided the pure product with c. 98% yield as a colorless solid (0.043 g, 12.6 mCi/mmol). Radioactivity measurements were performed on a Beckmann LC-6000 IC scintillation counter using the ‘Ready Safe’ liquid scintillation cocktail (Beckmann Coulter, Gladesville, NSW, Australia).
Bacterial Cultures and Preparation The H. pylori strains used in this study were J99 (with annotated genome) [20], N6 from the Institut Pasteur (Paris, France), and LC20, an isolate obtained from a patient with gastritis. H. pylori was grown on Campylobacter selective agar supplemented with 8% (v/v) defibrinated horse blood, 2.0 µg/mL amphotericin, 5.0 µg/ mL vancomycin, 1250 U/mL polymixin B, and 2.5 µg/mL trimethoprim (CSA). For studies of H. pylori growth with different phosphonates, bacteria were grown on phosphatefree liquid media (PFDM) containing SC-URA (1.9 g/L) and YNB (5.78 g/L). All H. pylori cultures were incubated at 37 °C under the microaerobic conditions 5% CO2, 5% O2, and 90% N2. The purity of the cultures was verified by positive urease and catalase tests, and motility and morphology were observed under phase contrast microscopy [21]. Cells were harvested in 150 mmol/L NaCl and centrifuged at 16,000 ×g for 10 minutes. The pellet was collected and
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the supernatant discarded. The pellet was resuspended in 150 mmol/L NaCl solution [21]. Growth of H. pylori in PFDM supplemented with 10 mol/L of either phosphate, Phephn, Amephn, or Phnace was measured at 24 and 48 hours by plating out serial dilutions on CSA plates and counting the number of colony-forming units per milliliter (cfu/mL). NaCl (150 mmol/L) was used as negative control. All samples were measured in triplicate. Errors are quoted as ± standard deviations.
Transport Assays by Centrifugation Through Oil Method Measurement of the entry rates of Phephn into bacterial cells was carried out by the centrifugation through oil method [22]. Aliquots of 100 µL of cell suspensions were combined with 100 µL of a Phephn solution containing trace amounts of [14C]Phephn at time zero. The combined cell and Phephn suspensions were then layered over 500 µL of oil (4 : 1 (v/v) di-n-butylphthalate:di-isooctyl phthalate) and centrifuged using a Hettich Mikro 22R centrifuge (Andreas Hettich GmbH, Tuttlingen, Germany) at 36,220 ×g for 1 minute to separate the cells from the Phephn and stop the transport of permeant. To remove thoroughly any Phephn not in the cell pellet, the remaining permeant aqueous solution layer was removed, and the oil layer was washed three times with Milli-Q water. Next, the oil layer was removed, and 400 µL of 1% (v/v) Triton X-100 was added to the cell pellet and the mixture was left to stand for 12–16 hours. Perchloric acid (750 µL, 6%, v/v) was then added to the detergent suspension and incubated on ice for 30 minutes. The cold suspension was centrifuged at 36,220 ×g for 5 minutes at 6 °C. One milliliter of supernatant from this suspension was added to 10 mL of scintillation fluid for radioactivity counting.
Measurement of Radioactivity in Transport The scintillant liquid contained 2,5-diphenyloxazole (0.5% w/v) in 5% Triton:toluene (1 : 2, v/v). Disintegrations per minute (dpm) were measured on a Packard Tri-Carb 2100TR Scintillation System (Packard Instrument Co.; Meriden, CT, USA). Samples were analyzed for 5 minutes to determine the average dpm. A 50 µL standard was used in place of the cell pellet to calculate moles of Phephn from dpm.
Calculation of Cell Pellet Size In each experiment, the volume of the pellet was determined by the same method as the total water space
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using tritium (3H2O) instead of the Phephn. 3H2O was left for 20 minutes to permeate the cells before pelleting. The extracellular space of the pellet was measured using the impermeant [14C]taurine [22], and the cell suspension was centrifuged within 20 seconds of adding the taurine. To determine the quantity of Phephn taken up by the cells, the dpm due to Phephn in the extracellular water space were subtracted from the Phephn dpm of the total pellet.
Temperature Dependence of Transport Rates The effect of temperature on transport rates was investigated by incubating cell suspensions, substrate solutions, and oil mixtures at the required temperature for 30 minutes before beginning transport assays as described above. The centrifuge was chilled to the required temperature prior and during the assay at temperatures under 25 °C. Temperatures were independently measured using a thermometer.
Effects of Inhibitors, Phosphonates, Phosphate, and Cations on Transport Rates The effects of potential transport inhibitors were investigated by incubating 100 µL of cells with 50 µL of each of the inhibitors for 30 minutes at 37 °C [23]. [14C]labeled Phephn (50 µL) was added to the suspensions, and after 20 seconds, the transport was measured at 25 °C. The effects of Amephn, Phnace, and phosphate were investigated by first mixing these compounds with [14C]-labeled Phephn, and then performing the centrifugation through oil assay. The effect of partially changing the primary extracellular cation was investigated. To ensure that cell viability and integrity was preserved, the suspensions contained 75 mmol/L NaCl, the ionic strength was kept constant, and cells were exposed to the various cations for only 20 seconds. Bacteria suspended in 400 µL of NaCl (150 mmol/L) were centrifuged at 4 °C, 10,000 ×g, and 200 µL of the supernatant were removed. At time zero, 200 µL of an aqueous solution of Phephn (100 µmol/L) in Li+, Na+, K+, Rb+, or Cs+ chloride salts (300 mmol/L) was added to this cell suspension. Aliquots of 200 µL of the mixed cation cell suspensions with permeant were layered on the oil, and the transport assay was performed as described.
Calculation of Kinetic Parameters Michaelis constants for transport K t and maximal velocities Vmax were calculated by nonlinear regression using the SIGMA PLOT 2002 (version 8.02) program (SPSS Inc., San Jose, CA, USA.). The errors in these calculations are determined by the program as ± standard deviation.
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Figure 1 Transport rates into H. pylori of Phephn as a function of permeant concentration. Phephn transport rates were measured at 25 °C in H. pylori strains J99 (), LC20 (), and N6 () suspended in 150 mmol/L NaCl, at a time-point of 20 seconds and Phephn concentrations between 1 and 200 µmol/L using the centrifugation through oil method. The rates represent an average of three replications. Errors were calculated as the standard deviation between the replications.
Results An optimal time-point to investigate the transport of Phephn was determined by measuring, as a function of time, the amount of permeant entering the cells at constant extracellular concentrations of Phephn between 2 and 200 µm. In this range of concentrations, the uptake of Phephn over time was linear for 60 seconds. Longer transport times allowed higher levels of substrate to be taken up by the cells, thus reducing the effects of noise occurring in these measurements. On the other hand, transport rates measured at times approaching saturation lose sensitivity. To optimize the accuracy and sensitivity of measuring Phephn transport, a time-point for transport assays of 20 seconds was chosen. Rates of Phephn transport at various concentrations were determined using the transport through oil technique in H. pylori strains J99, LC20, and N6 (Fig. 1). Kt and Vmax were calculated by nonlinear regression fit to the Michaelis–Menten equation [24]. All three strains showed similar transport kinetics (Table 1, Fig. 1). Cell pellets contain an amount of nontransported substrate in the extracellular aqueous compartment. The amount of substrate transported was calculated by subtracting the amount of extracellular substrate from the total substrate in the pellet. An increase in the extracellular volume will increase the amount of permeant not
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Figure 2 Arrhenius plot of [14C]-labeled Phephn transport rates into H. pylori strain N6 cells at a permeant concentration of 50 µmol/L. Initial rates were determined at a fixed time-point of 20 seconds using the centrifugation through oil method. The values represent the average of five measurements.
transported, yielding artifacts in the measurements. To determine if increasing Phephn concentrations affected the size of the cell pellet, the total pellet volume and extracellular aqueous spaces were measured at Phephn concentrations ranging from 10 µmol/L to 150 mmol/L; the extracellular permeant concentrations had no effect on the size of the cell pellet. The temperature dependence of Phephn transport rates at a fixed time-point of 20 seconds was measured over the temperature range 5–40 °C. The Arrhenius plot for Phephn transport at permeant concentrations of 50 µmol/L was linear between 10 and 40 °C (Fig. 2). An activation energy of 3.5 kJ/mol was calculated from the temperature data suggesting that Phephn transport is carrier mediated. At 5 °C, the values of the rates measured were much lower
Table 1 Kinetic parameters of Phephn transport in H. pylori. [14C]-labeled Phephn transport rates were measured in H. pylori strains J99, LC20, and N6 over a Phephn range of 1–200 µmol/L using the centrifugation through oil method. Kt and Vmax were calculated using nonlinear regression analysis. Errors are given as the standard deviation of measurements
Strain
Kt (µmol/L)
Vmax (nmole per microliter cellular water per second)
N6 J99 LC20
42 ± 9 46 ± 4 55 ± 9.5
571 ± 50 601 ± 23 658 ± 48
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Figure 3 Effects of monovalent cations on Phephn transport rates in H. pylori. Transport rates of [14C]-labeled Phephn were determined under the same conditions as per Fig. 1. H. pylori cells were suspended in solutions of different monovalent cations at a final concentration of 150 mmol/L, [14C]-labeled Phephn (final concentration 50 µmol/L) was added at time zero, and the effect on Phephn transport rates measured. Errors were calculated as the standard deviation between replications.
than those obtained by extrapolation of the linear Arrhenius plot, indicating the existence of a breakpoint in the transport process between 5 and 10 °C. To determine potential effectors involved in Phephn transport, the effects of five monovalent cations and of sodium phosphate at various concentrations were investigated. Transport rates increased with increasing cation size (Fig. 3), but varying the type of extracellular cation did not affect the size of the cell pellet. The presence of 0– 125 mmol/L phosphate had no effect on Phephn transport rates. Several characteristics of Phephn transport were elucidated by measuring the effects of various transport inhibitors [17,22,23] on entry rates. Sodium azide and sodium cyanide increased the rate of transport; iodoacetamide, amiloride, valinomycin, and nigericin reduced the rate of transport; and monensin had no effect (Fig. 4). Competition of Amephn or Phnace with Phephn transport was investigated to determine the specificity of the transport mechanism. The presence of phosphonate at concentrations of 250 µmol/L or 50 mmol/L induced no change on the rate of Phephn (50 µmol/L) transport at 25 °C relative to controls without the other phosphonates. To determine whether Phephn, Amephn, or Phnace could be employed by H. pylori as a sole phosphorus source to support growth in vitro, the bacterium was grown in PFDM supplemented with either phosphate, one of the
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Figure 4 Effects of inhibitors on Phephn transport rates in H. pylori. Transport rates of [14C]-labeled Phephn were measured in H. pylori cells suspended in various inhibitors for 30 minutes and under the same conditions as per Fig. 1. NaCl (150 mmol/L) was used as a control in place of an inhibitor. Independent experiments were performed twice. Rates were determined as the average of three replications. Errors were calculated as the standard deviation between replications.
Figure 5 Growth of H. pylori after 24 and 48 hours in defined liquid media with YNB, SC-URA, 10 mmol/L KCl, without phosphate (shaded squares), with added 10 mmol/L of either sodium phosphate (shaded ovals), Phephn (inverted filled triangles), Amephn (upright filled triangles), or Phnace (filled circles).
three phosphonates or no phosphorus source. After 24 and 48 hours, bacteria grew in suspensions with phosphate or Phephn, but growth was inhibited in cultures without a phosphorus source, or with Amephn or Phnace (Fig. 5).
Discussion Transport of Phephn by H. pylori was saturable, showed Michaelis–Menten kinetics, and had similar parameters in the three strains tested (Table 1). Transport was specific to
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Phephn, and unaffected by the presence of Amephn and Phnace, both of which can be used as phosphorus sources by some microorganisms [12]. This result was in contrast to the transport of PALA into H. pylori which was inhibited by Phnace [16]. The linear temperature dependence of transport rates between 10 and 40 °C (Fig. 2) suggests that Phephn transport is carrier-mediated. The breakpoint in the plot may be caused by temperature-induced conformational changes of the proteins involved in Phephn transport and/or changes in membrane fluidity. This observation is consistent with previous studies on carrier-mediated transport in H. pylori that have shown similar breakpoints [25], suggesting changes in membrane fluidity in this temperature range are the likely cause of the temperature dependence. Transport rates of Phephn increased with monovalent cation size (Fig. 3). A similar effect was observed for fumarate import rates [22], whereas rates of glucose influx were markedly reduced in the presence of monovalent cations relative to those determined with Na+ ions [25]. The effects on Phephn transport rates may arise from interactions of the cations with this phosphonate, or with the transport mechanism. The phosphonate group of Phephn is polarized, making it soluble in water and resistant to diffusion across cell membranes [26]. Cations may bind to the charged phosphonate moiety and reduce the charge effect. On the other hand, cationic ligands can complex with proteins and change their conformations. The significant ion size differences between the five monovalent metal cations may induce subtle changes in the structure of the carrier that result in changes in Phephn rates of transport. Finally, some transporters such as symports and antiports commonly use the movement of cations down a concentration gradient to drive the translocation of solutes across a membrane. These types of mechanism may be involved in the transport of Phephn across the cell membrane. Increasing the extracellular phosphate concentration had no effect on the rate of Phephn transported, suggesting that both types of phosphorus compounds were taken up by different systems. Sodium cyanide and sodium azide increased the rates of Phephn transport (Fig. 4); both compounds inhibit the electron transport chain by disrupting the function of cytochrome c, an enzyme that is involved also in the transport of phosphate [27]. Both compounds decreased the rate of fumarate or glucose transport in H. pylori [22,25]. These results supported the conclusion that Phephn entered the cell via different routes to phosphate, fumarate, or glucose. The enhancement of Phephn transport rates by NaCN and NaN3 may reflect a strategy used by H. pylori to cope with phosphate limitation. The bacterium may compensate for disruption of phosphate transport by increasing the level of phospho-
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nate transported. This would be similar to the regulation of the Pho R protein in E. coli that is autophosphorylated in phosphate starvation conditions to initiate an upregulation of pho regulon genes involved in phosphonate metabolism [8]. In contrast, sodium cyanide and sodium azide both decrease the rate of fumarate and glucose transport rates in H. pylori [22,25]. Nigericin, which makes cell membranes permeable to H+ and K+ by dissipating their gradients, reduced the rate of Phephn transport in H. pylori. Monensin, which dissipates Na+ and H+ gradients, did not have an effect on the rate of Phephn transport (Fig. 4). Amiloride, a sodium transport inhibitor, and iodoacetamide an alkylating agent reduced Phephn transport rates. This is a similar to the effects on fumarate and glucose transport rates; however, the magnitude of inhibition in Phephn transport rates was much less than that found in fumarate and glucose [22,25]. The data suggested that H+ and Na+ gradients were not involved directly in the transport of Phephn, but that K+ gradients played a role in its uptake. The dissipation of the K+ gradient can have harmful effects on cells and the observed decrease in Phephn transport rates may be caused by the decline in cell viability rather than by a direct effect on the transport mechanism. The ionophore valinomycin, which makes membranes permeable to K+, also reduced Phephn transport rates. The reduction of transport rates by nigericin and valinomycin suggests that K+ ions are directly involved in the transport of Phephn. The transport mechanisms of Phephn and fumarate share some similarities with that of H. pylori. Fumarate and Phephn are acids and for both substrates the rate of transport was saturable and temperature dependent, and increased in the presence of larger cations. However, increasing K+ ion concentrations reduced the rate of fumarate transport, the opposite of the effect it had on Phephn rate of transport [22]. The transport of Phephn into H. pylori, measured by the centrifugation through oil method using radioactive tracer analysis, had similar properties in strains J99, LC20, and N6, suggesting that the uptake was characteristic of the species, and may allow the bacterium to survive in environments with low phosphate concentrations. Similarly to Campylobacter spp. [17] and E. coli [28], it was demonstrated that in vitro under conditions of phosphorus stress H. pylori can survive with Phephn as its sole phosphorus source (Fig. 5). Phosphonates can be used as alternative sources of phosphate in phosphate starvation conditions [12], but there is abundant phosphorus from organic and inorganic sources in the mucosa of the human stomach. Trace amounts of phosphonates are found in soils [9] from where they can be ingested by husbandry birds and animals and passed on to humans. In addition, many pesticides contain phosphonates and they appear as
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residues in raw or processed food, from where they can be assimilated by humans. Thus, it is difficult that in its natural habitat H. pylori would require phosphonates as phosphorus sources. On the other hand, it is not known whether the bacterium encounters phosphate limitation conditions in its route of transmission [30], and H. pylori may have developed phosphonate transport mechanisms to survive transient stages of phosphate starvation. Phosphonates were far more common in early evolutionary history [31], and this may have led to several phosphonate transport pathways being retained in modern bacteria. H. pylori can take up Phephn even when grown in the presence of abundant phosphate, but it is unlikely that this capability is simply an evolutionary vestige because it has a small genome [20], and more than 10,000 years of close evolution with its human host [32]. A relatively small genome may promote that some catabolic enzymes have secondary substrates, and Phephn transport and catabolism in H. pylori would then be due to proteins with either broad phosphonate specificity or an entirely different primary substrate. This possibility is supported by the ability of H. pylori to catabolize the synthetic phosphonate PALA, which is not a specific substrate of any enzyme [16]. The inability of H. pylori to catabolize the antibiotic glyphosate [16] and Amephn, the phosphonate compound formed from the catabolism of glyphosate before C-P bond cleavage [8], suggested that primarily, phosphonate degradation pathways are not directed primarily toward obtaining phosphorus. This work was made possible by the support of the Australian Research Council.
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