Table 1.
Bacterial strains and plasmids used in this study.
Figure 1.
Gene organization of H. pylori 26695 wild type and isogenic mutants (A), and RT-PCR products of relevant genes (B).
Colored arrows indicate intact genes, and void arrows indicate inactivated genes. cat; chloramphenicol resistance gene, aph3; kanamycin resistance gene. RT-PCR products were obtained from 30 cycles of amplification.
Figure 2.
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.
Figure 3.
Concentrations of L-lactate (A) and D-lactate (B) in culture supernatants of H. pylori 26695 wild type and isogenic mutants.
Exponentially growing 26695 wild type (blue), the Δ140 mutant (red), the Δ138 mutant (green), the Δ1222 mutant (purple), the Δ138/Δ1222 mutant (aqua), as well as blank medium (orange) were incubated with 5 mM L- or D-lactate, and lactate concentrations were determined by biochemical assay kits as in Materials and Methods. The data points and error bars represent the means ± SD of three independent experiments.
Figure 4.
NAD-independent D- and L-lactate dehydrogenase (D- and L-iLDH) activities in cell extracts of H. pylori strains 26695, J99, NCTC 11637, G27, TN2GF4, SS1, and C. jejuni ATCC 33292.
The activity was measured in cell extracts by a coupled colorimetric assay using 5- or L-lactate as an electron donor and a mixture of artificial acceptors, MTT and PMS. The activity in the first 30 min was spectrophotometrically monitored at 570 nm, and was normalized by the total protein concentration in cell extract. All the data represent the means ± SD of three independent experiments.
Figure 5.
NAD-independent D-lactate dehydrogenase (D-iLDH) and NAD-dependent L-lactate dehydrogenase (L-nLDH) activities in cell extracts of H. pylori 26695 wild type and isogenic mutants.
(A) D-iLDH activity was measured as described above. (B) L-nLDH activity was measured by monitoring the reduction of NAD to NADH, arising from the conversion of L-lactate to pyruvate, which was spectrophotometrically detected at 450 nm, and was calibrated based on enclosed NADH standards. All the data represent the means ± SD of three independent experiments, and one unit of the activity is defined as the amount of enzyme that catalyzes the conversion of lactate into pyruvate to generate 1.0 µmole of NADH per minute at 37°C.
Figure 6.
The relative gene expressions of hp0138, hp0140, and hp1222 in H. pylori 26695 wild type grown in a variety of carbon sources (5 mM glucose, 10 mM L-lactate, 10 mM D-lactate, 2.5 mM glucose+5 mM L-lactate, 2.5 mM glucose+5 mM D-lactate, or 5 mM L-lactate+5 mM D-lactate).
Real-time quantitative PCRs were performed in 7300 Real-Time PCR System (Applied Biosystems) using Taqman-designed primers and probes (Life technologies), and the data were analyzed by comparative Ct (ΔΔCt) method with 16S rRNA as the reference gene. All the quantifications were performed in triplicate and were shown as the means ± SD.