Single amino acid substitutions in either YhjD or MsbA confer viability to 3-deoxy-d- manno -oct-2-ulosonic acid-depleted Escherichia coli

The Escherichia coli K-12 strain KPM22, defective in synthesis of 3-deoxy-d- manno -oct-2-ulosonic acid (Kdo), is viable with an outer membrane (OM) composed predominantly of lipid IV A , a precursor of lipopolysaccharide (LPS) biosynthesis that lacks any glycosylation. To sustain viability, the presence of a second-site suppressor was proposed for transport of lipid IV A from the inner membrane (IM), thus relieving toxic side-effects of lipid IV A accumulation and providing sufficient amounts of LPS precursors to support OM biogenesis. We now report the identification of an arginine to cysteine substitution at position 134 of the conserved IM protein YhjD in KPM22 that acts as a compensatory suppressor mutation of the lethal δKdo phenotype. Further, the yhjD400 suppressor allele renders the LPS transporter MsbA dispensable for lipid IV A transmembrane trafficking. The independent derivation of a series of non-conditional KPM22-like mutants from the Kdo-dependent parent strain TCM15 revealed a second class of suppressor mutations localized to MsbA. Proline to serine substitutions at either residue 18 or 50 of MsbA relieved the Kdo growth dependence observed in the isogenic wild-type strain. The possible impact of these suppressor mutations on structure and function are discussed by means of a computationally derived threading model of MsbA.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/75126/1/MMI_6074_sm_Figure_S1.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/75126/2/j.1365-2958.2007.06074.x.pd

A Complete Pathway Model for Lipid A Biosynthesis in Escherichia coli.

Lipid A is a highly conserved component of lipopolysaccharide (LPS), itself a major component of the outer membrane of Gram-negative bacteria. Lipid A is essential to cells and elicits a strong immune response from humans and other animals. We developed a quantitative model of the nine enzyme-catalyzed steps of Escherichia coli lipid A biosynthesis, drawing parameters from the experimental literature. This model accounts for biosynthesis regulation, which occurs through regulated degradation of the LpxC and WaaA (also called KdtA) enzymes. The LpxC degradation signal appears to arise from the lipid A disaccharide concentration, which we deduced from prior results, model results, and new LpxK overexpression results. The model agrees reasonably well with many experimental findings, including the lipid A production rate, the behaviors of mutants with defective LpxA enzymes, correlations between LpxC half-lives and cell generation times, and the effects of LpxK overexpression on LpxC concentrations. Its predictions also differ from some experimental results, which suggest modifications to the current understanding of the lipid A pathway, such as the possibility that LpxD can replace LpxA and that there may be metabolic channeling between LpxH and LpxB. The model shows that WaaA regulation may serve to regulate the lipid A production rate when the 3-deoxy-D-manno-oct-2-ulosonic acid (KDO) concentration is low and/or to control the number of KDO residues that get attached to lipid A. Computation of flux control coefficients showed that LpxC is the rate-limiting enzyme if pathway regulation is ignored, but that LpxK is the rate-limiting enzyme if pathway regulation is present, as it is in real cells. Control also shifts to other enzymes if the pathway substrate concentrations are not in excess. Based on these results, we suggest that LpxK may be a much better drug target than LpxC, which has been pursued most often

Comparing CYP2S1 and CYP102A1: Factors that influence the chemistry of CYP2S1

Cytochromes P450s have been investigated as drug targets for anticancer prodrugs (to be activated by P450 only in cancer cells). Current investigation involves mutating a well-studied P450 BM-3 to mimic the chemistry of CYP2S1. We tested different structured substrates and saw the enzymatic activity is affected when an acidic residue in the oxygen binding pocket is mutated to a methionine

Regulation of phosphatidylinositol kinase activity in Saccharomyces cerevisiae.

The effects of growth phase and carbon source on membrane-associated phosphatidylinositol kinase in cell extracts of Saccharomyces cerevisiae were examined. Phosphatidylinositol kinase activity increased 2- and 2.5-fold in glucose- and glycerol-grown cells, respectively, in the stationary phase as compared with the exponential phase of growth. The increase in phosphatidylinositol kinase activity in the stationary phase of growth correlated with an increase in the relative amounts of phosphatidylinositol 4-phosphate, the product of the reaction. The increase in phosphatidylinositol kinase activity was not due to the presence of water-soluble effector molecules in cell extracts as indicated by mixing experiments. Phosphatidylinositol kinase activity decreased in cell extracts of exponential-phase cells preincubated under phosphorylation conditions which favor cyclic AMP-dependent protein kinase activity. Phosphatidylinositol kinase activity was not affected in cell extracts of stationary-phase cells preincubated under phosphorylation conditions

Secondary Acylation of Vibrio cholerae Lipopolysaccharide Requires Phosphorylation of Kdo*

The lipopolysaccharide of Vibrio cholerae has been reported to contain a single 3-deoxy-d-manno-octulosonic acid (Kdo) residue that is phosphorylated. The phosphorylated Kdo sugar further links the hexa-acylated V. cholerae lipid A domain to the core oliogosaccharide and O-antigen. In this report, we confirm that V. cholerae possesses the enzymatic machinery to synthesize a phosphorylated Kdo residue. Further, we have determined that the presence of the phosphate group on the Kdo residue is necessary for secondary acylation in V. cholerae. The requirement for a secondary substituent on the Kdo residue (either an additional Kdo sugar or a phosphate group) was also found to be critical for secondary acylation catalyzed by LpxL proteins from Bordetella pertussis, Escherichia coli, and Haemophilus influenzae. Although three putative late acyltransferase orthologs have been identified in the V. cholerae genome (Vc0212, Vc0213, and Vc1577), only Vc0213 appears to be functional. Vc0213 functions as a myristoyl transferase acylating lipid A at the 2′-position of the glucosamine disaccharide. Generally acyl-ACPs serve as fatty acyl donors for the acyltransferases required for lipopolysaccharide biosynthesis; however, in vitro assays indicate that Vc0213 preferentially utilizes myristoyl-CoA as an acyl donor. This is the first report to biochemically characterize enzymes involved in the biosynthesis of the V. cholerae Kdo-lipid A domain

Lipopolysaccharide biosynthesis genes in koala type I Chlamydia: Cloning and characterization

We showed in 1988 that there are two strains of Chlamydia psittaci which infect the koala (Phascolarctos cinereus). In order to further investigate the role of these chlamydial strains in pathogenesis, we have attempted to identify genes of koala type I strain chlamydial which are involved in the immunogenic response, Transformation of Escherichia coli with a plasmid containing a 6.3-kb fragment (pKOC-10) of C. psittaci DNA caused the appearance of a specific chlamydial lipopolysaccharide (LPS) epitope on the host strain. The smallest DNA fragment capable of inducing the expression of chlamydial LPS was an Xbal fragment, 2.4 kb in size (pKOC-5). DNA sequence analysis of the complete fragment revealed regions of high identity, at the amino acid level, to the gseA genes of C. pneomoniae, C. psittaci 6BC and C. trachomatis, and the kdtA gene of E. coli which code for transferases catalysing the addition of 3-deoxy-D-manno-octulosonic acid (Kdo) residues to lipid A. Two open reading frames (ORFs) of 1,314 and 501 nucleotides in size, within the 2.4-kb fragment, were evident, and mRNA species corresponding to these ORFs were detected by Northern analysis. Both ORF1 and ORF2 are required for the appearance of chlamydia-specific LPS on the surface of recombinant E. coli