Impact of Nutritional Factors on the Proteome of Intestinal Escherichia coli: Induction of OxyR-Dependent Proteins AhpF and Dps by a Lactose-Rich Diet

Abstract

<jats:title>ABSTRACT</jats:title><jats:p>To study the impact of nutritional factors on protein expression of intestinal bacteria, gnotobiotic mice monoassociated with<jats:named-content content-type="genus-species">Escherichia coli</jats:named-content>K-12 were fed three different diets: a diet rich in starch, a diet rich in nondigestible lactose, and a diet rich in casein. Two-dimensional gel electrophoresis and electrospray-tandem mass spectrometry were used to identify differentially expressed proteins of bacteria recovered from small intestine and cecum. Oxidative stress response proteins such as AhpF, Dps, and Fur, all of which belong to the oxyR regulon, were upregulated in<jats:named-content content-type="genus-species">E. coli</jats:named-content>isolates from mice fed the lactose-rich diet. Luciferase reporter gene assays demonstrated that osmotic stress caused by carbohydrates led to the expression of<jats:italic>ahpCF</jats:italic>and<jats:italic>dps</jats:italic>, which was not observed in an<jats:named-content content-type="genus-species">E. coli</jats:named-content>Δ<jats:italic>oxyR</jats:italic>mutant. Growth of<jats:italic>ahpCF</jats:italic>and<jats:italic>oxyR</jats:italic>deletion mutants was strongly impaired when nondigestible sucrose was present in the medium. The wild-type phenotype could be restored by complementation of the deletions with plasmids containing the corresponding genes and promoters. The results indicate that some OxyR-dependent proteins play a major role in the adaptation of<jats:named-content content-type="genus-species">E. coli</jats:named-content>to osmotic stress. We conclude that there is an overlap of osmotic and oxidative stress responses. Mice fed the lactose-rich diet possibly had a higher intestinal osmolality, leading to the upregulation of OxyR-dependent proteins, which enable intestinal<jats:named-content content-type="genus-species">E. coli</jats:named-content>to better cope with diet-induced osmotic stress.</jats:p>

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

“…The key finding in this study was the upregulation of OxyR-dependent proteins in intestinal E. coli from mice fed a lactose-rich diet and their essential role in bacterial adaptation to lactose-mediated osmotic stress [7]. In addition, we observed the diet-dependent expression of two E. coli proteins that have been identified [8], [9], [10] but whose roles in the E. coli metabolism are still obscure: namely, the 2-deoxy-D-gluconate 3-dehydrogenase (KduD), which was 2.4-fold upregulated in E. coli of mice on the lactose diet and 4.0-fold downregulated in E. coli of mice on the casein diet (both versus control [starch] diet), and the 5-keto 4-deoxyuronate isomerase (KduI), which was 8.3-fold lower in E. coli of mice fed the casein diet [7].…”

“…Each mouse was inoculated with 1×10 7 E. coli K-12 MG1655 cells by gastric gavage and killed 21 days after inoculation by cervical dislocation. The small intestinal, caecal, and colonic contents were collected and prepared for two-dimensional difference gel electrophoresis as described previously [7]. Contaminations were excluded by amplification and sequencing of the bacterial 16S rRNA genes of representative caecal samples with the primers 27-f (5′-AGA GTT TGA TCC TGG CTC AG-3′) and 1492-r (5′-TAC CTT GTT ACG ACT T-3′) [7], [15].…”

“…The small intestinal, caecal, and colonic contents were collected and prepared for two-dimensional difference gel electrophoresis as described previously [7]. Contaminations were excluded by amplification and sequencing of the bacterial 16S rRNA genes of representative caecal samples with the primers 27-f (5′-AGA GTT TGA TCC TGG CTC AG-3′) and 1492-r (5′-TAC CTT GTT ACG ACT T-3′) [7], [15]. In this animal experiment we compared the proteomes of E. coli obtained from mice fed the lactose diet or the casein diet with those of mice fed the starch diet and identified differentially expressed proteins by electrospray-tandem mass spectrometry.…”

“…Microbiome analysis revealed an increase in genes encoding bacterial enzymes involved in amino acid degradation, carbohydrate catabolism, vitamin biosynthesis, and bile salt metabolism in response to a protein-rich host diet [6]. Using a gnotobiotic mouse model, we previously demonstrated that intestinal Escherichia coli K-12 (MG1655) modulates its metabolism in response to various diets [7].…”

“…In addition, we observed the diet-dependent expression of two E. coli proteins that have been identified [8], [9], [10] but whose roles in the E. coli metabolism are still obscure: namely, the 2-deoxy-D-gluconate 3-dehydrogenase (KduD), which was 2.4-fold upregulated in E. coli of mice on the lactose diet and 4.0-fold downregulated in E. coli of mice on the casein diet (both versus control [starch] diet), and the 5-keto 4-deoxyuronate isomerase (KduI), which was 8.3-fold lower in E. coli of mice fed the casein diet [7]. In the plant pathogen Erwinia chrysanthemi these enzymes are involved in pectin degradation [11], [12].…”