We have previously shown that the TolA protein is required for the correct surface expression of the Escherichia coli O7 antigen lipopolysaccharide (LPS). In this work, ⌬tolA and ⌬pal mutants of E. coli K-12 W3110 were transformed with pMF19 (encoding a rhamnosyltransferase that reconstitutes the expression of O16-specific LPS), pWQ5 (encoding the Klebsiella pneumoniae O1 LPS gene cluster), or pWQ802 (encoding the genes necessary for the synthesis of Salmonella enterica O:54). Both ⌬tolA and ⌬pal mutants exhibited reduced surface expression of O16 LPS as compared to parental W3110, but no significant differences were observed in the expression of K. pneumoniae O1 LPS and S. enterica O:54 LPS. Therefore, TolA and Pal are required for the correct surface expression of O antigens that are assembled in a wzy (polymerase)-dependent manner (like those of E. coli O7 and O16) but not for O antigens assembled by wzy-independent pathways (like K. pneumoniae O1 and S. enterica O:54). Furthermore, we show that the reduced surface expression of O16 LPS in ⌬tolA and ⌬pal mutants was associated with a partial defect in O-antigen polymerization and it was corrected by complementation with intact tolA and pal genes, respectively. Using derivatives of W3110⌬tolA and W3110⌬pal containing lacZ reporter fusions to fkpA and degP, we also demonstrate that the RpoE-mediated extracytoplasmic stress response is upregulated in these mutants. Moreover, an altered O16 polymerization was also detected under conditions that stimulate RpoE-mediated extracytoplasmic stress responses in tol ؉ and pal ؉ genetic backgrounds. A Wzy derivative with an epitope tag at the C-terminal end of the protein was stable in all the mutants, ruling out stress-mediated proteolysis of Wzy. We conclude that the absence of TolA and Pal elicits a sustained extracytoplasmic stress response that in turn reduces O-antigen polymerization but does not affect the stability of the Wzy O-antigen polymerase.Gram-negative bacteria have a unique envelope, consisting of a plasma membrane surrounded by the cell wall peptidoglycan and an outer membrane. One major component of the outer membrane is the lipopolysaccharide (LPS), which consists of lipid A, core oligosaccharide, and in some bacteria an O-specific polysaccharide that extends from the cell surface. The biogenesis of LPS is a complex, multistep process occurring at both sides of the plasma membrane, which is followed by the translocation of LPS molecules to the outer membrane cell surface (for recent reviews see references 44 and 54). LPS biosynthesis requires the participation of many enzymes and assembly proteins, encoded by more than forty genes (20,21,23,44). The core oligosaccharide is assembled by the sequential transfer of monosaccharides onto preformed lipid A, while the O antigen is assembled onto undecaprenol-phosphate (Und-P), a polyisoprenoid lipid, to which is linked via a phosphodiester bond (44). These two pathways eventually converge by the ligation of the O antigen onto the outer core domain of the lipi...

The adipocyte enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) amplifies local glucocorticoid action by generating active glucocorticoids from inactive metabolites and has emerged as a key player in the pathogenesis of central obesity and metabolic syndrome. However, the regulation of adipocyte 11beta-HSD1 is incompletely understood. Therefore, the present study was designed to investigate the effects of insulin and glucocorticoid as well as their underlying molecular mechanisms on 11beta-HSD1 activity and expression in 3T3-L1 adipocytes and determine whether the in vitro findings could be confirmed in vivo. Our main in vitro findings are 1) insulin stimulated whereas dexamethasone inhibited 11beta-HSD1 activity and expression in a time- and concentration-dependent manner; 2) the effect of dexamethasone was mimicked by both cortisol and corticosterone but blocked by the glucocorticoid receptor antagonist RU486; 3) the p38 MAPK inhibitor SB220025, but not the ERK inhibitor U0126 or the phosphatidylinositol 3-kinase inhibitor LY294002, prevented insulin stimulation of 11beta-HSD1 activity; and 4) although dexamethasone did not alter the half-life of 11beta-HSD1 mRNA, insulin doubled it. Taken together, these in vitro results demonstrate that insulin stimulates adipocyte 11beta-HSD1 through a posttranscriptional mechanism that involves activation of the p38 MAPK signaling pathway, whereas dexamethasone exerts an opposite effect by a glucocorticoid receptor-mediated transcriptional mechanism. In contrast, both insulin and dexamethasone augmented 11beta-HSD1 activity and expression in rat white adipose tissue in vivo, thus confirming the role of insulin but revealing a fundamental difference regarding the role of dexamethasone in regulating adipocyte 11beta-HSD1 between the two model systems.