Proposed mechanism of how hexuronates may be converted by KduI and KduD in <i>E. coli</i>.

<p>In the classical pathway, galacturonate and glucuronate enter the bacterial cell by the aldohexuronate transporter (ExuT) and subsequently undergo isomerization to tagaturonate or fructuronate by uronate isomerase (UxaC). The altronate oxidoreductase (UxaB) or mannonate oxidoreductase (UxuB) catalyze their NADH-dependent reduction to altronate or mannonate, which are further converted by altronate dehydratase (UxaA) or mannonate dehydratase (UxuA) to 2-oxo-3-deoxygluconate. Our results indicate that the 5-keto 4-deoxyuronate isomerase (KduI) and the 2-deoxy-D gluconate 3-dehydrogenase (KduD) may compensate for reduced levels of UxaC, UxaB, and UxuB under osmotic stress conditions. The arrows indicate established, the broken arrows indicate hypothetical reactions.</p

Primers used for generation of deletion mutants, complementing plasmids, and pGEM-T Easy vectors.

a<p>forward primer,</p>b<p>reverse primer.</p

Conversion of galacturonate and glucuronate by cell-free extracts of <i>E. coli</i> overexpressing KduI and KduD.

<p>Conversion of galacturonate (A, B) and glucuronate (C, D) by cell-free extracts from <i>E. coli</i> overexpressing KduI (<i>E. coli</i> KRX pGEM-T-<i>kduI</i>), KduD (<i>E. coli</i> KRX pGEM-T-<i>kduD</i>), or both (<i>E. coli</i> JM109 pGEM-T-<i>kduID</i>) was investigated. <i>E. coli</i> strains were selected according to the orientation of the cloned gene: expression in <i>E. coli</i> KRX was controlled by the T7-RNA-polymerase and induced by rhamnose (0.1%). Expression in <i>E. coli</i> JM109 was controlled by the lacZ-promoter and induced by addition of IPTG (1 mM). <i>E. coli</i> containing the empty vector served as a control. The reactions were started by addition of 10 mM galacturonate or glucuronate and incubation at 37°C. Concentration of hexuronates was measured enzymatically. Broken black line, <i>E. coli</i> JM109 pGEM-T (A, C), <i>E. coli</i> KRX pGEM-T (B, D) (negative controls); black line, <i>E. coli</i> JM109 pGEM-T-<i>kduID</i> (A, C); gray line, <i>E. coli</i> KRX pGEM-T-<i>kduI</i>; broken gray line, <i>E. coli</i> KRX pGEM-T-<i>kduD</i> (B, D). Data are expressed as median and 25% and 75% percentile (n = 11–12). The Mann-Whitney test was used for calculations. *, P<0.05; **, P<0.01; ***, P<0.001.</p

Potential mechanism of how osmotic stress influences the gene expression of hexuronate degrading enzymes.

<p>High carbohydrate concentrations caused by lactose <i>in vivo</i> or sucrose <i>in vitro</i> lead to an osmotic shock (<b>1</b>), which is proposed to activate the OxyR transcriptional regulator by a closer contact of normally separated protein domains or an unknown osmosensitive transducing component <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056906#pone.0056906-Rothe1" target="_blank">[7]</a> (<b>2</b>). The genes of the standard hexuronate degrading enzymes UxaABC and UxuAB are repressed in an OxyR-dependent manner (<b>3</b>) and lead to diminished hexuronate utilization in the presence of osmotic stress. In contrast, expression of <i>kduI</i> and <i>kduD</i>, which is inducible by galacturonate and glucuronate, is neither osmosensitive nor OxyR-dependent (<b>4</b>). Since KduI and KduD facilitate the conversion of galacturonate and glucuronate, these enzymes are proposed to replace UxaC, UxaB, and/or UxuB under conditions of high osmolality (<b>5</b>). The arrows indicate experimentally proven reactions; the broken arrows indicate hypothetical functions.</p

Induction of the <i>kduD</i> and <i>kduI</i> promoter in <i>E. coli</i> MG1655 by mouse diets, carbohydrates, casaminoacids, or hexuronates under aerobic and anaerobic growth conditions.

a<p>Relative luminescence data for <i>E. coli</i> MG1655 carrying p<i>kduDp::luxAB</i> or p<i>kduIp::luxAB</i> are shown. For both aerobic and anaerobic growth conditions, luciferase activity was normalized to values determined for cells grown on glucose (50 mM). Data are expressed as medians and 25% and 75% percentile (aerobic, n = 5–7; anaerobic, n = 4–7).</p>b<p>Cultures were grown on M9 minimal medium containing pulverized mouse diets or dietary components under aerobic or anaerobic conditions and shaken at 220 and 120 rpm, respectively, at 37°C for 16 h.</p>c,d<p>Kruskal-Wallis one-way analysis of variance and Dunn’s multiple-comparison test were used for calculations. <i>^c</i>, P<0.05; <i>^d</i>, P<0.01; <i>^e</i>, P<0.001.</p

Novel Insights into <em>E. coli</em>’s Hexuronate Metabolism: KduI Facilitates the Conversion of Galacturonate and Glucuronate under Osmotic Stress Conditions

<div><p>Using a gnotobiotic mouse model, we previously observed the upregulation of 2-deoxy-D-gluconate 3-dehydrogenase (KduD) in intestinal <i>E. coli</i> of mice fed a lactose-rich diet and the downregulation of this enzyme and of 5-keto 4-deoxyuronate isomerase (KduI) on a casein-rich diet. The present study aimed to define the role of the so far poorly characterized <i>E. coli</i> proteins KduD and KduI <i>in vitro</i>. Galacturonate and glucuronate induced <i>kduD</i> and <i>kduI</i> gene expression 3-fold and 7 to 11-fold, respectively, under aerobic conditions as well as 9 to 20-fold and 19 to 54-fold, respectively, under anaerobic conditions. KduI facilitated the breakdown of these hexuronates. In <i>E. coli</i>, galacturonate and glucuronate are normally degraded by UxaABC and UxuAB. However, osmotic stress represses the expression of the corresponding genes in an OxyR-dependent manner. When grown in the presence of galacturonate or glucuronate, <i>kduID</i>-deficient <i>E. coli</i> had a 30% to 80% lower maximal cell density and 1.5 to 2-fold longer doubling times under osmotic stress conditions than wild type <i>E. coli</i>. Growth on lactose promoted the intracellular formation of hexuronates, which possibly explain the induction of KduD on a lactose-rich diet. These results indicate a novel function of KduI and KduD in <i>E. coli</i> and demonstrate the crucial influence of osmotic stress on the gene expression of hexuronate degrading enzymes.</p> </div

Growth of <i>E. coli</i> MG1655 and <i>E. coli</i> Δ<i>kduID</i> under aerobic and anaerobic conditions ^a.

a<p>Data are expressed as medians and minima versus maxima (aerobic, n = 6; anaerobic, n = 5).</p>b<p>Cultures were incubated in M9 minimal medium containing 50 mM galacturonate or glucuronate with or without sucrose.</p>c,d<p>Data represent comparisons of the results obtained with <i>E. coli</i> MG1655 versus <i>E. coli</i> Δ<i>kduID</i> that included use of the same medium (Mann-Whitney test; <i>^c</i>, P<0.05; <i>^d</i>, P<0.01).</p

Formation of intracellular galacturonate and glucuronate during growth of <i>E. coli</i> on lactose.

<p>Intracellular galacturonate and glucuronate concentration was monitored during growth of <i>E. coli</i> MG1655 on M9 minimal medium containing 25 mM lactose or 50 mM glucose under aerobic conditions. Cell densities were determined at 600 nm. OD₆₀₀ on glucose medium (dashed gray line), and on lactose medium (black line); intracellular galacturonate concentration on glucose medium (gray dots), and on lactose medium (black dots). Data are expressed as medians (n = 6). The Mann-Whitney test was applied. <i>a</i>, P<0.05; <i>b</i>, P<0.01.</p

Effect of osmotic stress on hexuronate-induced <i>kduI</i> and <i>kduD</i> expression in wild type and Δ<i>oxyR</i> cells.

<p>Expression of <i>kduD</i> (A, B) and <i>kduI</i> (C, D) in <i>E. coli</i> MG1655 (gray bars) and <i>E. coli</i> Δ<i>oxyR</i> (white bars) in response to galacturonate or glucuronate with and without osmotic stress caused by non-fermentable sucrose under aerobic (A, C) or anaerobic (B, D) conditions after 90 min of incubation was investigated. Cultures were grown on M9 minimal medium containing 50 mM galacturonate or glucuronate in the presence or absence of 400 mM sucrose. Relative luminescence data for <i>E. coli</i> MG1655 and <i>E. coli</i> Δ<i>oxyR</i> carrying p<i>kduDp::luxAB or</i> p<i>kduIp::luxAB</i> are shown. Luciferase activity was normalized to values determined for cells grown on 50 mM glucose. Data are expressed as medians (aerobic: n = 6–7, anaerobic: n = 4–7). Kruskal-Wallis one-way analysis of variance and Dunn’s multiple-comparison test were used for calculations. *, P<0.05; **, P<0.01.</p