We previously reported that a lactose-rich diet fed to gnotobiotic mice not only induced OxyR-dependent stress-response proteins in intestinal E. coli
but also the poorly characterized protein KduD 
. This was an unexpected finding because KduD and KduI are involved in pectin degradation in Erwinia chrysanthemi
. In this organism, gene expression of kduI
is induced by galacturonate and polygalacturonate 
. In contrast, E. coli
is not able to utilize pectin, poly- and oligogalacturonates 
. This suggests that the induction of KduD in intestinal E. coli
in response to a lactose-rich diet must have different reasons.
Reporter gene experiments identified galacturonate and glucuronate as inducers of the kduI
gene expression. A role of KduI in the conversion of galacturonate and glucuronate in E. coli
was demonstrated by comparing extracts of cells that did or did not overexpress KduI, KduD, or both. However, in which way KduI and/or KduD facilitate the degradation of these hexuronates is unclear. Possible explanations include: i) KduI and KduD stabilize UxaC and/or UxaB or UxuB, ii) they co-induce transporters that facilitate hexuronate uptake, iii) they activate as yet unknown hexuronate degrading pathways, or iv) they are directly involved in galacturonate and glucuronate conversion. Our experiments do no support the first and second explanation, since in the absence of hexuronates, the expression of UxaABC and UxuAB is repressed () 
. Therefore, stabilization of UxaABC and UxuAB by KduI or KduD cannot take place. Since the KduI- and KduD-dependent hexuronate conversion was determined in cell-free extracts, the observed decrease of galacturonate and glucuronate in these extracts cannot be explained by KduI- and KduD-dependent induction of alternative hexuronate transporters. However, we cannot rule out that KduI activates alternative hexuronate degrading pathways, thereby facilitating the conversion of galacturonate and glucuronate. We consider a catabolic role of KduI and KduD in the utilization of galacturonate and glucuronate the most likely explanation.
In E. coli
, galacturonate and glucuronate enter the cell by the aldohexuronate transporter (ExuT) 
. In the classical pathway, they subsequently undergo isomerization to tagaturonate or fructuronate by uronate isomerase (UxaC) (). In the next step altronate oxidoreductase (UxaB) or mannonate oxidoreductase (UxuB) catalyze the NADH-dependent reduction of tagaturonate or fructuronate to altronate or mannonate, which are further converted by altronate dehydratase (UxaA) or mannonate dehydratase (UxuA) to 2-oxo-3-deoxygluconate 
. Based on sequence similarity to a 5-keto-4-deoxyuronate isomerase in E. chrysanthemi
, which catalyzes the conversion of 5-keto-4-deoxyuronate to 2,5-dioxo-3-deoxygluconate, E. coli
’s KduI was predicted to catalyze the same reaction 
. Based on this and on the structural similarities of galacturonate and glucuronate with 5-keto-4-deoxyuronate we hypothesize that KduI catalyzes the isomerization of these hexuronates to tagaturonate and fructuronate in E. coli
. If this assumption is true, KduI may compensate for the function of UxaC under osmotic stress conditions, because the uxaC
transcription is repressed under such conditions in an OxyR-dependent manner. The OxyR transcriptional regulator not only acts as a sensor for oxidative stress 
but also as a sensor for osmotic stress 
Proposed mechanism of how hexuronates may be converted by KduI and KduD in E. coli.
In E. chrysanthemi, KduD catalyzes the NADH-dependent reduction of 2,5-dioxo-3-deoxygluconate to 2-oxo-3-deoxygluconate. Since KduD was found to be upregulated in intestinal E. coli of mice fed the lactose-rich diet and reporter gene assays revealed that hexuronates induced kduD gene expression, this enzyme is possibly involved in catalyzing a later step in hexuronate conversion although overexpression of KduD alone did not decrease the hexuronate concentration in cell-free extracts. Therefore, KduD may compensate for the function of UxaB or UxuB, whose encoding genes, as mentioned above for uxaC, were repressed by osmotic stress. Based on the proposed function of KduI, this reaction could not occur in the absence of KduI because the KduD-substrates tagaturonate and fructuronate would be missing (). Which protein takes over the function of UxaA or UxuA at high osmolality and subsequently converts altronate or mannonate to 2-oxo-3-deoxygluconate is presently unknown. We therefore postulate the presence of another dehydratase. Nevertheless, the proposed pathway needs to be verified by further experiments and more detailed characterization of KduI and KduD.
In contrast to uxaCA
, and uxuAB
, our data demonstrate that the expression of kduI
was independent of OxyR and not diminished under osmotic stress conditions. The crucial role of KduI and KduD in hexuronate metabolism under conditions, in which the standard hexuronate degrading enzymes are repressed, was supported by growth experiments under such conditions (). Therefore, it was important to check the origin of galacturonate and glucuronate in the intestine of the gnotobiotic mice. Hexuronates were not present in the diets fed to the mice (Figure S1
in the supplementary material), but they appear as sugar substituents of glycoproteins and glycolipids in the mammalian mucus layer 
and are therefore expected to occur in intestinal contents of mice. Our results demonstrate that the intestinal hexuronate concentrations were increased in mice fed the lactose diet. However, it is presently not known, whether these hexuronates originate from lactose and, if yes, which pathways are used for their formation. Nevertheless, the transient formation of hexuronates in the cytoplasm of exponentially growing E. coli
on lactose but not on glucose suggests that the detected cytoplasmic hexuronates may originate from lactose.
Potential mechanism of how osmotic stress influences the gene expression of hexuronate degrading enzymes.
To clearly identify possible sources or precursors of the detected intracellular hexuronates, experiments using isotopically labelled lactose would be required. At present, we can only speculate about potential mechanisms by which these lactose-derived hexuronates could be generated from lactose. In E. coli
, lactose is cleaved to galactose and glucose by ß-galactosidase 
. While glucose can directly enter glycolysis, galactose is usually converted via the Leloir pathway 
. The high metabolic rate in the exponential growth phase may result in the accumulation of either intracellular UDP-glucose or galactose as observed for lactic acid-producing bacteria 
. The uronate dehydrogenase assay used in our study cannot distinguish between galacturonate and glucuronate 
. Therefore, it has not been possible to clearly identify the source of the detected intracellular hexuronates. Two possibilities are conceivable: 1. Intracellular glucuronate may stem from UDP-glucuronate, which in turn is produced from UDP-glucose as catalyzed by UDP-glucose 6-dehydrogenase (Ugd) 
. 2. Intracellular galacturonate may be produced by oxidation of galactose. However, such a reaction has not been demonstrated in E. coli
The disappearance of intracellular hexuronates during transition from exponential to stationary growth phase of E. coli
indicates utilization of these compounds. In the intestine of mice fed a lactose-rich diet and monoassociated with E. coli
, lactose is readily available in the feeding periods 
, enabling intestinal bacteria to grow exponentially. Exponential growth on lactose would result in the constant formation of intracellular hexuronates, which in turn induce the alternative hexuronate degrading enzymes KduI and KduD.
Export of intracellular glucuronate via the gluconate transporter (GntT) or facilitated diffusion mechanisms 
may be the reason for the elevated intestinal hexuronate concentrations in the intestines of the gnotobiotic mice fed a lactose-rich diet. However, there would be no benefit for E. coli
to release a potential energy source. Moreover, the cytoplasmic galacturonate and glucuronate concentrations of E. coli
grown on lactose-containing minimal medium were similar but not higher than those in the intestinal contents of mice on a lactose-rich diet. Therefore, export of hexuronates is unlikely to happen.
In conclusion, our study suggests that a lactose-rich diet fed to mice monoassociated with E. coli leads to an increased kduI and kduD gene expression in intestinal E. coli by galacturonate and glucuronate, which conversion was facilitated by KduI.