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Title: Isolation of Salmonella typhimurium strains that utilize exogenous 3-deoxy-D-manno-Octulosonate for synthesis of lipopolysaccharide

Abstract

Spontaneous mutants of Salmonella typhimurium LT2 were selected for the ability to accumulate exogenous 3-deoxy-D-manno-octulosonate (KDO). Bacteria containing a gene (kdsA) which codes for a temperature-sensitive KDO-8-phosphate synthetase were plated at the restrictive temperature of 42/sup 0/C on medium containing 5 mM KDO. Since bacteria containing the kdsA lesion are unable to grown at 42/sup 0/C due to inhibition of lipopolysaccharide (LPS) synthesis and accumulation of lipid A precursor, this method allowed direct, positive selection of mutants capable of utilizing exogenous KDO for LPS synthesis. Spontaneous mutants, selected at a frequency of about 10/sup -6/, required exogenous KDO for growth at 42/sup 0/C. The growth rate at 42/sup 0/C was nearly normal in the presence of 20 mM KDO and was directly proportional to KDO concentrations below 20 mM. Exogenous KDO also suppressed accumulation of lipid A precursor. The apparent K/sub m/ for KDO accumulation was 23 mM, and the maximum rate of transport was calculated to be 505 pmol of DKO per min per 10/sup 8/ cells. Bacteria incorporated exogenous (/sup 3/H)KDO exclusively into LPS, with less that 10% dilution in specific activity due to residual endogenous KDO synthesis. The mutation giving rise to the ability to accumulate exogenousmore » KDO was extremely useful in the direct screening for new mutations in the kdsA gene after localized mutagenesis. Five mutations in kdsA were isolated, four of which were new alleles as determined by on fine-structure analysis. The ability to introduce labeled (/sup 3/H, /sup 13/C, and /sup 14/C) KDO in vivo should simplify and extend the analysis of this critical metabolic pathway in gram-negative bacteria.« less

Authors:
;
Publication Date:
Research Org.:
Abbott Labs., Abbott Park, IL
OSTI Identifier:
5408314
Resource Type:
Journal Article
Journal Name:
J. Bacteriol.; (United States)
Additional Journal Information:
Journal Volume: 169:11
Country of Publication:
United States
Language:
English
Subject:
62 RADIOLOGY AND NUCLEAR MEDICINE; 59 BASIC BIOLOGICAL SCIENCES; LIGASES; GENETIC MAPPING; LIPOPOLYSACCHARIDES; BIOSYNTHESIS; SALMONELLA TYPHIMURIUM; METABOLISM; BIOLOGICAL PATHWAYS; CARBON 13; CARBON 14 COMPOUNDS; ENZYME ACTIVITY; GROWTH; LABELLED COMPOUNDS; MUTAGENESIS; MUTANTS; TRACER TECHNIQUES; TRITIUM COMPOUNDS; BACTERIA; CARBOHYDRATES; CARBON ISOTOPES; ENZYMES; EVEN-ODD NUCLEI; ISOTOPE APPLICATIONS; ISOTOPES; LIGHT NUCLEI; LIPIDS; MAPPING; MICROORGANISMS; NUCLEI; ORGANIC COMPOUNDS; POLYSACCHARIDES; SACCHARIDES; SALMONELLA; STABLE ISOTOPES; SYNTHESIS; 550601* - Medicine- Unsealed Radionuclides in Diagnostics; 550701 - Microbiology- Tracer Techniques

Citation Formats

Goldman, R C, and Devine, E M. Isolation of Salmonella typhimurium strains that utilize exogenous 3-deoxy-D-manno-Octulosonate for synthesis of lipopolysaccharide. United States: N. p., 1987. Web.
Goldman, R C, & Devine, E M. Isolation of Salmonella typhimurium strains that utilize exogenous 3-deoxy-D-manno-Octulosonate for synthesis of lipopolysaccharide. United States.
Goldman, R C, and Devine, E M. Sun . "Isolation of Salmonella typhimurium strains that utilize exogenous 3-deoxy-D-manno-Octulosonate for synthesis of lipopolysaccharide". United States.
@article{osti_5408314,
title = {Isolation of Salmonella typhimurium strains that utilize exogenous 3-deoxy-D-manno-Octulosonate for synthesis of lipopolysaccharide},
author = {Goldman, R C and Devine, E M},
abstractNote = {Spontaneous mutants of Salmonella typhimurium LT2 were selected for the ability to accumulate exogenous 3-deoxy-D-manno-octulosonate (KDO). Bacteria containing a gene (kdsA) which codes for a temperature-sensitive KDO-8-phosphate synthetase were plated at the restrictive temperature of 42/sup 0/C on medium containing 5 mM KDO. Since bacteria containing the kdsA lesion are unable to grown at 42/sup 0/C due to inhibition of lipopolysaccharide (LPS) synthesis and accumulation of lipid A precursor, this method allowed direct, positive selection of mutants capable of utilizing exogenous KDO for LPS synthesis. Spontaneous mutants, selected at a frequency of about 10/sup -6/, required exogenous KDO for growth at 42/sup 0/C. The growth rate at 42/sup 0/C was nearly normal in the presence of 20 mM KDO and was directly proportional to KDO concentrations below 20 mM. Exogenous KDO also suppressed accumulation of lipid A precursor. The apparent K/sub m/ for KDO accumulation was 23 mM, and the maximum rate of transport was calculated to be 505 pmol of DKO per min per 10/sup 8/ cells. Bacteria incorporated exogenous (/sup 3/H)KDO exclusively into LPS, with less that 10% dilution in specific activity due to residual endogenous KDO synthesis. The mutation giving rise to the ability to accumulate exogenous KDO was extremely useful in the direct screening for new mutations in the kdsA gene after localized mutagenesis. Five mutations in kdsA were isolated, four of which were new alleles as determined by on fine-structure analysis. The ability to introduce labeled (/sup 3/H, /sup 13/C, and /sup 14/C) KDO in vivo should simplify and extend the analysis of this critical metabolic pathway in gram-negative bacteria.},
doi = {},
journal = {J. Bacteriol.; (United States)},
number = ,
volume = 169:11,
place = {United States},
year = {1987},
month = {11}
}