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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: 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.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.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 gua and pur mutants in EMF12 supplemented with individual purines.H. pylori strains were grown in EMF12 supplemented with one of seven purine sources. After 20 hours, the optical density was measured. Positive growth was defined as a statistically significant increase in OD600 relative to the baseline OD600 of 0.025 (student's t-test, P<0.05). Results are the mean ± SD of three independent growth cultures.
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pone-0038727-g003: Growth of gua and pur mutants in EMF12 supplemented with individual purines.H. pylori strains were grown in EMF12 supplemented with one of seven purine sources. After 20 hours, the optical density was measured. Positive growth was defined as a statistically significant increase in OD600 relative to the baseline OD600 of 0.025 (student's t-test, P<0.05). Results are the mean ± SD of three independent growth cultures.

Mentions: In most organisms, GMP is synthesized from IMP by the enzymes IMP dehydrogenase (GuaB) and GMP synthetase (GuaA), while adenylosuccinate synthetase (PurA) and adenylosuccinate lyase (PurB) catalyze the formation of AMP from IMP (Figure 1). The conversion of GMP back to IMP is carried out in one step by GMP dehydrogenase (GuaC). H. pylori possesses homologs for purA, purB, guaA, guaB, and guaC[17], [18]. Recent reports using H. pylori G27 showed that the gua and pur genes perform similar roles in purine salvage as do their homologs in other bacteria [5]. In order to confirm these results in H. pylori 26695, we constructed gene deletions in guaA, guaB, guaC, purA and purB (Table S2, Materials and Methods). To study the phenotypes of each mutant, strains were grown for 20 h in the chemically defined medium EMF12 supplemented with one of seven different purine sources (Figure 3). As predicted based on the canonical purine conversion pathway outlined in Figure 1, strain EM202k (guaA) required guanine or guanosine for significant growth (see Figure 3 legend). EM203k (guaB) was able to utilize xanthine in addition to guanine and guanosine as a sole purine source. EM204 (guaC) lacked the ability to degrade GMP back into IMP and therefore grew in the presence of all purines except guanine, guanosine, and xanthine. Similarly, strains EM205 (purA) and EM206 (purB) grew only in the presence of exogenous adenine or adenosine. These results support the conclusion that the gua and pur genes are responsible for inter-conversion between GMP and AMP in H. pylori.


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 gua and pur mutants in EMF12 supplemented with individual purines.H. pylori strains were grown in EMF12 supplemented with one of seven purine sources. After 20 hours, the optical density was measured. Positive growth was defined as a statistically significant increase in OD600 relative to the baseline OD600 of 0.025 (student's t-test, P<0.05). Results are the mean ± SD of three independent growth cultures.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038727-g003: Growth of gua and pur mutants in EMF12 supplemented with individual purines.H. pylori strains were grown in EMF12 supplemented with one of seven purine sources. After 20 hours, the optical density was measured. Positive growth was defined as a statistically significant increase in OD600 relative to the baseline OD600 of 0.025 (student's t-test, P<0.05). Results are the mean ± SD of three independent growth cultures.
Mentions: In most organisms, GMP is synthesized from IMP by the enzymes IMP dehydrogenase (GuaB) and GMP synthetase (GuaA), while adenylosuccinate synthetase (PurA) and adenylosuccinate lyase (PurB) catalyze the formation of AMP from IMP (Figure 1). The conversion of GMP back to IMP is carried out in one step by GMP dehydrogenase (GuaC). H. pylori possesses homologs for purA, purB, guaA, guaB, and guaC[17], [18]. Recent reports using H. pylori G27 showed that the gua and pur genes perform similar roles in purine salvage as do their homologs in other bacteria [5]. In order to confirm these results in H. pylori 26695, we constructed gene deletions in guaA, guaB, guaC, purA and purB (Table S2, Materials and Methods). To study the phenotypes of each mutant, strains were grown for 20 h in the chemically defined medium EMF12 supplemented with one of seven different purine sources (Figure 3). As predicted based on the canonical purine conversion pathway outlined in Figure 1, strain EM202k (guaA) required guanine or guanosine for significant growth (see Figure 3 legend). EM203k (guaB) was able to utilize xanthine in addition to guanine and guanosine as a sole purine source. EM204 (guaC) lacked the ability to degrade GMP back into IMP and therefore grew in the presence of all purines except guanine, guanosine, and xanthine. Similarly, strains EM205 (purA) and EM206 (purB) grew only in the presence of exogenous adenine or adenosine. These results support the conclusion that the gua and pur genes are responsible for inter-conversion between GMP and AMP in H. pylori.

Bottom Line: 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.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.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