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

Journal Article · Sun Nov 01 04:00:00 EST 1987 · J. Bacteriol.; (United States)

OSTI ID:5408314

Goldman, R C; Devine, E M

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.

• Research Organization: Abbott Labs., Abbott Park, IL
• OSTI ID: 5408314
• Journal Information: J. Bacteriol.; (United States), Journal Name: J. Bacteriol.; (United States) Vol. 169:11; ISSN JOBAA
• Country of Publication: United States
• Language: English

Related Subjects

550601* -- Medicine-- Unsealed Radionuclides in Diagnostics
550701 -- Microbiology-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
62 RADIOLOGY AND NUCLEAR MEDICINE
BACTERIA
BIOLOGICAL PATHWAYS
BIOSYNTHESIS
CARBOHYDRATES
CARBON 13
CARBON 14 COMPOUNDS
CARBON ISOTOPES
ENZYME ACTIVITY
ENZYMES
EVEN-ODD NUCLEI
GENETIC MAPPING
GROWTH
ISOTOPE APPLICATIONS
ISOTOPES
LABELLED COMPOUNDS
LIGASES
LIGHT NUCLEI
LIPIDS
LIPOPOLYSACCHARIDES
MAPPING
METABOLISM
MICROORGANISMS
MUTAGENESIS
MUTANTS
NUCLEI
ORGANIC COMPOUNDS
POLYSACCHARIDES
SACCHARIDES
SALMONELLA
SALMONELLA TYPHIMURIUM
STABLE ISOTOPES
SYNTHESIS
TRACER TECHNIQUES
TRITIUM COMPOUNDS