Hydrogenase-3 Contributes to Anaerobic Acid Resistance of Escherichia coli

BACKGROUND: Hydrogen production by fermenting bacteria such as Escherichia coli offers a potential source of hydrogen biofuel. Because H(2) production involves consumption of 2H(+), hydrogenase expression is likely to involve pH response and regulation. Hydrogenase consumption of protons in E. coli has been implicated in acid resistance, the ability to survive exposure to acid levels (pH 2-2.5) that are three pH units lower than the pH limit of growth (pH 5-6). Enhanced survival in acid enables a larger infective inoculum to pass through the stomach and colonize the intestine. Most acid resistance mechanisms have been defined using aerobic cultures, but the use of anaerobic cultures will reveal novel acid resistance mechanisms. METHODS AND PRINCIPAL FINDINGS: We analyzed the pH regulation of bacterial hydrogenases in live cultures of E. coli K-12 W3110. During anaerobic growth in the range of pH 5 to 6.5, E. coli expresses three hydrogenase isoenzymes that reversibly oxidize H(2) to 2H(+). Anoxic conditions were used to determine which of the hydrogenase complexes contribute to acid resistance, measured as the survival of cultures grown at pH 5.5 without aeration and exposed for 2 hours at pH 2 or at pH 2.5. Survival of all strains in extreme acid was significantly lower in low oxygen than for aerated cultures. Deletion of hyc (Hyd-3) decreased anoxic acid survival 3-fold at pH 2.5, and 20-fold at pH 2, but had no effect on acid survival with aeration. Deletion of hyb (Hyd-2) did not significantly affect acid survival. The pH-dependence of H(2) production and consumption was tested using a H(2)-specific Clark-type electrode. Hyd-3-dependent H(2) production was increased 70-fold from pH 6.5 to 5.5, whereas Hyd-2-dependent H(2) consumption was maximal at alkaline pH. H(2) production, was unaffected by a shift in external or internal pH. H(2) production was associated with hycE expression levels as a function of external pH. CONCLUSIONS: Anaerobic growing cultures of E. coli generate H(2) via Hyd-3 at low external pH, and consume H(2) via Hyd-2 at high external pH. Hyd-3 proton conversion to H(2) is required for acid resistance in anaerobic cultures of E. coli

Biomarkers of Nutrition for Development (BOND)—Iron Review

This is the fifth in the series of reviews developed as part of the Biomarkers of Nutrition for Development (BOND) program. The BOND Iron Expert Panel (I-EP) reviewed the extant knowledge regarding iron biology, public health implications, and the relative usefulness of currently available biomarkers of iron status from deficiency to overload. Approaches to assessing intake, including bioavailability, are also covered. The report also covers technical and laboratory considerations for the use of available biomarkers of iron status, and concludes with a description of research priorities along with a brief discussion of new biomarkers with potential for use across the spectrum of activities related to the study of iron in human health. The I-EP concluded that current iron biomarkers are reliable for accurately assessing many aspects of iron nutrition. However, a clear distinction is made between the relative strengths of biomarkers to assess hematological consequences of iron deficiency versus other putative functional outcomes, particularly the relationship between maternal and fetal iron status during pregnancy, birth outcomes, and infant cognitive, motor and emotional development. The I-EP also highlighted the importance of considering the confounding effects of inflammation and infection on the interpretation of iron biomarker results, as well as the impact of life stage. Finally, alternative approaches to the evaluation of the risk for nutritional iron overload at the population level are presented, because the currently designated upper limits for the biomarker generally employed (serum ferritin) may not differentiate between true iron overload and the effects of subclinical inflammation

Escherichia coli K-12 survives anaerobic exposure at pH 2 without RpoS, Gad, or hydrogenases, but shows sensitivity to autoclaved broth products.

Escherichia coli and other enteric bacteria survive exposure to extreme acid (pH 2 or lower) in gastric fluid. Aerated cultures survive via regulons expressing glutamate decarboxylase (Gad, activated by RpoS), cyclopropane fatty acid synthase (Cfa) and others. But extreme-acid survival is rarely tested under low oxygen, a condition found in the stomach and the intestinal tract. We observed survival of E. coli K-12 W3110 at pH 1.2-pH 2.0, conducting all manipulations (overnight culture at pH 5.5, extreme-acid exposure, dilution and plating) in a glove box excluding oxygen (10% H2, 5% CO2, balance N2). With dissolved O2 concentrations maintained below 6 µM, survival at pH 2 required Cfa but did not require GadC, RpoS, or hydrogenases. Extreme-acid survival in broth (containing tryptone and yeast extract) was diminished in media that had been autoclaved compared to media that had been filtered. The effect of autoclaved media on extreme-acid survival was most pronounced when oxygen was excluded. Exposure to H2O2 during extreme-acid treatment increased the death rate slightly for W3110 and to a greater extent for the rpoS deletion strain. Survival at pH 2 was increased in strains lacking the anaerobic regulator fnr. During anaerobic growth at pH 5.5, strains deleted for fnr showed enhanced transcription of acid-survival genes gadB, cfa, and hdeA, as well as catalase (katE). We show that E. coli cultured under oxygen exclusion (<6 µM O2) requires mechanisms different from those of aerated cultures. Extreme acid survival is more sensitive to autoclave products under oxygen exclusion

Effect of aeration on the extreme acid survival of W3110, Δ<i>hybC</i>, Δ<i>hycE</i>, and Δ<i>hypF</i>.

<p>The white bars represent cultures grown with aeration to stationary phase in LBK buffered at pH 5 that were diluted 200-fold into LBK pH 2 and exposed for 2 h with aeration at 37°C. The hatched and black bars represent anaerobic cultures grown to stationary phase in LBK buffered at pH 5.5 that were diluted 200-fold into LBK pH 2.5 (hatched) or pH 2 (black) and exposed for 2 h without aeration at 37°C. Aerobic and anaerobic cultures were maintained as stated in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010132#s4" target="_blank">Materials and Methods</a>. Error bars represent SEM, n = 5 or 6.</p

Effect of pH on the H₂ production by W3110, Δ<i>hybC</i>, and Δ<i>hycE</i>.

<p>The lines represent traces of H₂ concentration as a function of time. Before time 0, the cultures were sparged with 100% N₂ in order to eliminate any residual H₂ in the culture. Anaerobic cultures of W3110 (<b>A</b>), Δ<i>hybC</i> (<b>B</b>), and Δ<i>hycE</i> (<b>C</b>) were grown to log phase at pH 5.5, pH 6, and pH 6.5 and assayed for H₂ production as stated in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010132#s4" target="_blank">Materials and Methods</a>. Lines are representative samples of n = 3.</p

<i>E. coli</i> survives at pH lower than pH 2.0.

<p>Overnight cultures of <i>E. coli</i> K-12 strain W3110 were grown in LBK 100 mM MES pH 5.0. These cultures were exposed to medium at pH 1.2; 1.6;and 2.0, respectively, for two hours. Dilutions from exposed cells were completed as in <b>Fig. 1</b>. Strains were exposed in autoclave-sterilized medium (light bars) and in filtered medium (dark bars). Error bars indicate SEM (n = 5 or 6).</p

Effect of pH on the H₂ consumption by W3110 and Δ<i>hycE</i>.

<p>The lines represent traces of H₂ concentration as a function of time. Before time 0 the cultures were sparged with 20% H₂/80% N₂ in order to saturate the culture with H₂. Anaerobic cultures of W3110 (<b>A</b>) and Δ<i>hycE</i> (<b>B</b>) were grown to log phase at pH 5.5, pH 7, and pH 8 and assayed for H₂ consumption as stated in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010132#s4" target="_blank">Materials and Methods</a>. A sample of LBK saturated with H₂ was assayed as a control to assess residual H₂ loss. Lines are representative samples of n = 3.</p

Gene expression affected by <i>fnr</i> during growth at pH 5.5.

<p>RNA was isolated from anaerobic cultures of JLS1115 (W3110 <i>fnr</i>) (gray bars) grown to stationary phase at pH 5.5 in buffered LBK. qRT-PCR was used to measure the differential expression of mRNA levels for <i>cfa</i>, <i>gadB</i>, <i>hdeA katE</i>, <i>sdhC</i>, and <i>frdB</i> in the <i>fnr</i> mutant compared to the wild-type using primers listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056796#pone-0056796-t002" target="_blank">Table 2</a>. Positive values denote higher expression in the <i>fnr</i> mutant than in the wild-type and vice-versa. Error bars represent SEM, n = 3 (RNA from independent cultures). The expression profile for each gene was verified in triplicate.</p