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Identification of the genes that contribute to lactate utilization in Helicobacter pylori.

Iwatani S, Nagashima H, Reddy R, Shiota S, Graham DY, Yamaoka Y - PLoS ONE (2014)

Bottom Line: The hp1222 gene product functions as an NAD-independent D-LDH and also contributes to the oxidation of L-lactate; the isogenic mutant of this gene failed to grow on D-lactate-dependent medium.Interestingly an alternate route must also exist for lactate transport as the knockout of genes did not completely prevent growth on D- or L-lactate.This study identified the genes contributing to the lactate utilization and demonstrated the ability of H. pylori to utilize both D- and L-lactate.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine-Gastroenterology, Baylor College of Medicine and Michael E. DeBakey Veterans Affairs Medical Center, Houston, Texas, United States of America; Department of Environmental and Preventive Medicine, Oita University Faculty of Medicine, Oita, Japan.

ABSTRACT
Helicobacter pylori are Gram-negative, spiral-shaped microaerophilic bacteria etiologically related to gastric cancer. Lactate utilization has been implicated although no corresponding genes have been identified in the H. pylori genome. Here, we report that gene products of hp0137-0139 (lldEFG), hp0140-0141 (lctP), and hp1222 (dld) contribute to D- and L-lactate utilization in H. pylori. The three-gene unit hp0137-0139 in H. pylori 26695 encodes L-lactate dehydrogenase (LDH) that catalyzes the conversion of lactate to pyruvate in an NAD-dependent manner. Isogenic mutants of these genes were unable to grow on L-lactate-dependent medium. The hp1222 gene product functions as an NAD-independent D-LDH and also contributes to the oxidation of L-lactate; the isogenic mutant of this gene failed to grow on D-lactate-dependent medium. The parallel genes hp0140-0141 encode two nearly identical lactate permeases (LctP) that promote uptake of both D- and L-lactate. Interestingly an alternate route must also exist for lactate transport as the knockout of genes did not completely prevent growth on D- or L-lactate. Gene expression levels of hp0137-0139 and hp1222 were not enhanced by lactate as the carbon source. Expression of hp0140-0141 was slightly suppressed in the presence of L-lactate but not D-lactate. This study identified the genes contributing to the lactate utilization and demonstrated the ability of H. pylori to utilize both D- and L-lactate.

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Growths of H. pylori 26695 wild type (A), the Δ140 mutant (B), the Δ138 mutant (C), the Δ1222 mutant (D), and the Δ138/Δ1222 mutant (E) cultured in PP medium with/without a particular carbon source; no primary carbon source (blue circle), 5 mM D-glucose (red diamond), 10 mM L-lactate (green triangle), 10 mM D-lactate (purple square).The cell growth was monitored by measuring the optical density at 600± SD of three independent experiments.
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pone-0103506-g002: Growths of H. pylori 26695 wild type (A), the Δ140 mutant (B), the Δ138 mutant (C), the Δ1222 mutant (D), and the Δ138/Δ1222 mutant (E) cultured in PP medium with/without a particular carbon source; no primary carbon source (blue circle), 5 mM D-glucose (red diamond), 10 mM L-lactate (green triangle), 10 mM D-lactate (purple square).The cell growth was monitored by measuring the optical density at 600± SD of three independent experiments.

Mentions: To investigate the effect of primary carbon source on the growth of H. pylori, we cultured 26695 wild-type (WT) strain in conditioned medium with/without different carbon sources (BB, BBG, BBL, or BBD, see Materials and Methods). However, none of the carbon-supplemented media (BBG, BBL, and BBD) improved bacterial growth compared to basal medium (BB). We hypothesized that carbon supplied by the basal components such as peptone, yeast extract, and/or FBS was sufficient so that H. pylori 26695 growth was independent of the additional carbon source. Therefore, a minimal conditioned medium (PP, see Materials and Methods) was prepared in order to minimize the effects of the basal composition to allow the potential benefits of additional carbon sources to be identified. When cultured in PP, 26695 WT strain showed relatively weak growth rate, which was further improved in the addition of 5 mM glucose (PPG), 10 mM L-lactate (PPL), or 10 mM D-lactate (PPD), suggesting that H. pylori 26695 could utilize D- and L-lactate as well as glucose (Fig. 2A). Similarly, the Δ140 mutant showed improved growth with glucose and D- and L-lactate but retained relatively higher growth rates compared to the WT strain (Fig. 2B). The ability to grow with L-lactate was significantly decreased in the Δ138 mutant. Interestingly, the growth of this mutant was more favorable with D-lactate than with glucose (Fig. 2C). In contrast, the Δ1222 mutant grew relatively better with L-lactate, but failed to grow with D-lactate (Fig. 2D). When the two sets of genes (hp0137–0139 and hp1222) were disrupted, the Δ138/Δ1222 mutant failed to grow with either D- or L-lactate (Fig. 2E).


Identification of the genes that contribute to lactate utilization in Helicobacter pylori.

Iwatani S, Nagashima H, Reddy R, Shiota S, Graham DY, Yamaoka Y - PLoS ONE (2014)

Growths of H. pylori 26695 wild type (A), the Δ140 mutant (B), the Δ138 mutant (C), the Δ1222 mutant (D), and the Δ138/Δ1222 mutant (E) cultured in PP medium with/without a particular carbon source; no primary carbon source (blue circle), 5 mM D-glucose (red diamond), 10 mM L-lactate (green triangle), 10 mM D-lactate (purple square).The cell growth was monitored by measuring the optical density at 600± SD of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4117512&req=5

pone-0103506-g002: Growths of H. pylori 26695 wild type (A), the Δ140 mutant (B), the Δ138 mutant (C), the Δ1222 mutant (D), and the Δ138/Δ1222 mutant (E) cultured in PP medium with/without a particular carbon source; no primary carbon source (blue circle), 5 mM D-glucose (red diamond), 10 mM L-lactate (green triangle), 10 mM D-lactate (purple square).The cell growth was monitored by measuring the optical density at 600± SD of three independent experiments.
Mentions: To investigate the effect of primary carbon source on the growth of H. pylori, we cultured 26695 wild-type (WT) strain in conditioned medium with/without different carbon sources (BB, BBG, BBL, or BBD, see Materials and Methods). However, none of the carbon-supplemented media (BBG, BBL, and BBD) improved bacterial growth compared to basal medium (BB). We hypothesized that carbon supplied by the basal components such as peptone, yeast extract, and/or FBS was sufficient so that H. pylori 26695 growth was independent of the additional carbon source. Therefore, a minimal conditioned medium (PP, see Materials and Methods) was prepared in order to minimize the effects of the basal composition to allow the potential benefits of additional carbon sources to be identified. When cultured in PP, 26695 WT strain showed relatively weak growth rate, which was further improved in the addition of 5 mM glucose (PPG), 10 mM L-lactate (PPL), or 10 mM D-lactate (PPD), suggesting that H. pylori 26695 could utilize D- and L-lactate as well as glucose (Fig. 2A). Similarly, the Δ140 mutant showed improved growth with glucose and D- and L-lactate but retained relatively higher growth rates compared to the WT strain (Fig. 2B). The ability to grow with L-lactate was significantly decreased in the Δ138 mutant. Interestingly, the growth of this mutant was more favorable with D-lactate than with glucose (Fig. 2C). In contrast, the Δ1222 mutant grew relatively better with L-lactate, but failed to grow with D-lactate (Fig. 2D). When the two sets of genes (hp0137–0139 and hp1222) were disrupted, the Δ138/Δ1222 mutant failed to grow with either D- or L-lactate (Fig. 2E).

Bottom Line: The hp1222 gene product functions as an NAD-independent D-LDH and also contributes to the oxidation of L-lactate; the isogenic mutant of this gene failed to grow on D-lactate-dependent medium.Interestingly an alternate route must also exist for lactate transport as the knockout of genes did not completely prevent growth on D- or L-lactate.This study identified the genes contributing to the lactate utilization and demonstrated the ability of H. pylori to utilize both D- and L-lactate.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine-Gastroenterology, Baylor College of Medicine and Michael E. DeBakey Veterans Affairs Medical Center, Houston, Texas, United States of America; Department of Environmental and Preventive Medicine, Oita University Faculty of Medicine, Oita, Japan.

ABSTRACT
Helicobacter pylori are Gram-negative, spiral-shaped microaerophilic bacteria etiologically related to gastric cancer. Lactate utilization has been implicated although no corresponding genes have been identified in the H. pylori genome. Here, we report that gene products of hp0137-0139 (lldEFG), hp0140-0141 (lctP), and hp1222 (dld) contribute to D- and L-lactate utilization in H. pylori. The three-gene unit hp0137-0139 in H. pylori 26695 encodes L-lactate dehydrogenase (LDH) that catalyzes the conversion of lactate to pyruvate in an NAD-dependent manner. Isogenic mutants of these genes were unable to grow on L-lactate-dependent medium. The hp1222 gene product functions as an NAD-independent D-LDH and also contributes to the oxidation of L-lactate; the isogenic mutant of this gene failed to grow on D-lactate-dependent medium. The parallel genes hp0140-0141 encode two nearly identical lactate permeases (LctP) that promote uptake of both D- and L-lactate. Interestingly an alternate route must also exist for lactate transport as the knockout of genes did not completely prevent growth on D- or L-lactate. Gene expression levels of hp0137-0139 and hp1222 were not enhanced by lactate as the carbon source. Expression of hp0140-0141 was slightly suppressed in the presence of L-lactate but not D-lactate. This study identified the genes contributing to the lactate utilization and demonstrated the ability of H. pylori to utilize both D- and L-lactate.

Show MeSH
Related in: MedlinePlus