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Efficiency of purine utilization by Helicobacter pylori: roles for adenosine deaminase and a NupC homolog.

Miller EF, Vaish S, Maier RJ - PLoS ONE (2012)

Bottom Line: Using mutant analysis we show that a strain lacking the gene encoding a NupC homolog (HP1180) was growth-retarded in a defined medium supplemented with certain purines.This strain was attenuated for uptake of radiolabeled adenosine, guanosine, and inosine, showing a role for this transporter in uptake of purine nucleosides.Deletion of the GMP biosynthesis gene guaA had no discernible effect on mouse stomach colonization, in contrast to findings in numerous bacterial pathogens.

View Article: PubMed Central - PubMed

Affiliation: Microbiology Department, The University of Georgia, Athens, Georgia, United States of America.

ABSTRACT
The ability to synthesize and salvage purines is crucial for colonization by a variety of human bacterial pathogens. Helicobacter pylori colonizes the gastric epithelium of humans, yet its specific purine requirements are poorly understood, and the transport mechanisms underlying purine uptake remain unknown. Using a fully defined synthetic growth medium, we determined that H. pylori 26695 possesses a complete salvage pathway that allows for growth on any biological purine nucleobase or nucleoside with the exception of xanthosine. Doubling times in this medium varied between 7 and 14 hours depending on the purine source, with hypoxanthine, inosine and adenosine representing the purines utilized most efficiently for growth. The ability to grow on adenine or adenosine was studied using enzyme assays, revealing deamination of adenosine but not adenine by H. pylori 26695 cell lysates. Using mutant analysis we show that a strain lacking the gene encoding a NupC homolog (HP1180) was growth-retarded in a defined medium supplemented with certain purines. This strain was attenuated for uptake of radiolabeled adenosine, guanosine, and inosine, showing a role for this transporter in uptake of purine nucleosides. Deletion of the GMP biosynthesis gene guaA had no discernible effect on mouse stomach colonization, in contrast to findings in numerous bacterial pathogens. In this study we define a more comprehensive model for purine acquisition and salvage in H. pylori that includes purine uptake by a NupC homolog and catabolism of adenosine via adenosine deaminase.

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Growth of H. pylori 26695 in the chemically defined medium EMF12.Liquid growth medium EMF12 was supplemented with 60 μM hypoxanthine (solid line) or contained no purines (dashed line). H. pylori cells were inoculated at an initial OD600 of 0.015 (approx. 1.3×107 cfu/ml). Growth was monitored over time by measuring the absorbance at 600 nm. Results are the mean ± SD of three independent cultures.
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pone-0038727-g002: Growth of H. pylori 26695 in the chemically defined medium EMF12.Liquid growth medium EMF12 was supplemented with 60 μM hypoxanthine (solid line) or contained no purines (dashed line). H. pylori cells were inoculated at an initial OD600 of 0.015 (approx. 1.3×107 cfu/ml). Growth was monitored over time by measuring the absorbance at 600 nm. Results are the mean ± SD of three independent cultures.

Mentions: When grown in EMF12 supplemented with hypoxanthine, H. pylori 26695 reached an average final OD600 of 0.13±0.02 (approx. 8.1×107 cfu/ml), which corresponded to three generations of growth (Figure 2). H. pylori grown in F12 generally achieves a maximum growth yield of between 107–108 cfu/ml [12]. In the absence of a purine source the optical density of the cell culture decreased, confirming reports that H. pylori requires a purine source for growth [5]. Serial dilutions were plated at 0 and 18 hours post-inoculation to verify that the number of cells in the presence of hypoxanthine increased during log phase of growth (data not shown). To further assess whether purine auxotrophy is strain-specific, we grew two other wild-type strains, 43504 and X47, under these same conditions and likewise observed growth that was dependent on the presence of hypoxanthine (data not shown).


Efficiency of purine utilization by Helicobacter pylori: roles for adenosine deaminase and a NupC homolog.

Miller EF, Vaish S, Maier RJ - PLoS ONE (2012)

Growth of H. pylori 26695 in the chemically defined medium EMF12.Liquid growth medium EMF12 was supplemented with 60 μM hypoxanthine (solid line) or contained no purines (dashed line). H. pylori cells were inoculated at an initial OD600 of 0.015 (approx. 1.3×107 cfu/ml). Growth was monitored over time by measuring the absorbance at 600 nm. Results are the mean ± SD of three independent cultures.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3368855&req=5

pone-0038727-g002: Growth of H. pylori 26695 in the chemically defined medium EMF12.Liquid growth medium EMF12 was supplemented with 60 μM hypoxanthine (solid line) or contained no purines (dashed line). H. pylori cells were inoculated at an initial OD600 of 0.015 (approx. 1.3×107 cfu/ml). Growth was monitored over time by measuring the absorbance at 600 nm. Results are the mean ± SD of three independent cultures.
Mentions: When grown in EMF12 supplemented with hypoxanthine, H. pylori 26695 reached an average final OD600 of 0.13±0.02 (approx. 8.1×107 cfu/ml), which corresponded to three generations of growth (Figure 2). H. pylori grown in F12 generally achieves a maximum growth yield of between 107–108 cfu/ml [12]. In the absence of a purine source the optical density of the cell culture decreased, confirming reports that H. pylori requires a purine source for growth [5]. Serial dilutions were plated at 0 and 18 hours post-inoculation to verify that the number of cells in the presence of hypoxanthine increased during log phase of growth (data not shown). To further assess whether purine auxotrophy is strain-specific, we grew two other wild-type strains, 43504 and X47, under these same conditions and likewise observed growth that was dependent on the presence of hypoxanthine (data not shown).

Bottom Line: Using mutant analysis we show that a strain lacking the gene encoding a NupC homolog (HP1180) was growth-retarded in a defined medium supplemented with certain purines.This strain was attenuated for uptake of radiolabeled adenosine, guanosine, and inosine, showing a role for this transporter in uptake of purine nucleosides.Deletion of the GMP biosynthesis gene guaA had no discernible effect on mouse stomach colonization, in contrast to findings in numerous bacterial pathogens.

View Article: PubMed Central - PubMed

Affiliation: Microbiology Department, The University of Georgia, Athens, Georgia, United States of America.

ABSTRACT
The ability to synthesize and salvage purines is crucial for colonization by a variety of human bacterial pathogens. Helicobacter pylori colonizes the gastric epithelium of humans, yet its specific purine requirements are poorly understood, and the transport mechanisms underlying purine uptake remain unknown. Using a fully defined synthetic growth medium, we determined that H. pylori 26695 possesses a complete salvage pathway that allows for growth on any biological purine nucleobase or nucleoside with the exception of xanthosine. Doubling times in this medium varied between 7 and 14 hours depending on the purine source, with hypoxanthine, inosine and adenosine representing the purines utilized most efficiently for growth. The ability to grow on adenine or adenosine was studied using enzyme assays, revealing deamination of adenosine but not adenine by H. pylori 26695 cell lysates. Using mutant analysis we show that a strain lacking the gene encoding a NupC homolog (HP1180) was growth-retarded in a defined medium supplemented with certain purines. This strain was attenuated for uptake of radiolabeled adenosine, guanosine, and inosine, showing a role for this transporter in uptake of purine nucleosides. Deletion of the GMP biosynthesis gene guaA had no discernible effect on mouse stomach colonization, in contrast to findings in numerous bacterial pathogens. In this study we define a more comprehensive model for purine acquisition and salvage in H. pylori that includes purine uptake by a NupC homolog and catabolism of adenosine via adenosine deaminase.

Show MeSH
Related in: MedlinePlus