Inhibition of exogenous 3-deoxy-D-manno octulosonate incorporation into lipid A precursor of toluene-treated Salmonella typhimurium cells

Journal Article · Tue Sep 01 04:00:00 EDT 1987 · J. Bacteriol.; (United States)

OSTI ID:6062076

Capobianco, J O; Darveau, R P; Goldman, R C; Lartey, P A; Pernet, A G

Analogs of 3-deoxy-D-manno-octulosonate (KDO) were designed to inhibit CTP:CMP-KDO cytidylyltransferase (CMP-KDO synthetase). Since these analogs lacked whole-cell antibacterial activity, a permeabilized-cell method was developed to measure intracellular compound activity directly. The method employed a mutant of Salmonella typhimurium defective in KDO-8-phosphate synthetase (kdsA), which accumulated lipid A precursor at 42/sup 0/C. Cells permeabilized with 1% toluene were used to evaluate inhibitor effect on (/sup 3/H)KDO incorporation into preformed lipid A precursor. KDO incorporation proceeded through the enzymes CMP-DKO synthetase and CMP-KDO:lipid A KDO transferase. Optimum KDO incorporation occurred between pH 8 and 9 and required CTP, prior lipid A precursor accumulation, and a functional kdsB gene product, CMP-KDO synthetase. The apparent K/sub m/ for KDO in this coupled system at pH 7.6 was 1.38 mM. The reaction products isolated and characterized contained 1 and 2 KDO residues per lipid A precursor molecule. Several KDO analogs produced concentration-related reductions of DKO incorporation in toluenized cells with 50% inhibitor concentrations comparable to those obtained in purified CMP-DKO synthetase systems. Two compounds, 8-amino-2-deoxy-KDO (A-60478) and 8-aminomethyl-2-deoxy-KDO (A-60821), competitively inhibited KDO incorporation, displaying K/sub i/s of 4.2 /sup +/M for A=60478 and 2.5 /sup +/M for A-60821.

• Research Organization: Abbott Labs., Abbott Park, IL
• OSTI ID: 6062076
• Journal Information: J. Bacteriol.; (United States), Journal Name: J. Bacteriol.; (United States) Vol. 169:9; ISSN JOBAA
• Country of Publication: United States
• Language: English

Related Subjects

550201 -- Biochemistry-- Tracer Techniques
560300* -- Chemicals Metabolism & Toxicology
59 BASIC BIOLOGICAL SCIENCES
63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.
ALKYLATED AROMATICS
AROMATICS
BACTERIA
BIOLOGICAL EFFECTS
CARBOHYDRATES
CELL CONSTITUENTS
CELL MEMBRANES
ENZYME INHIBITORS
ENZYMES
HYDROCARBONS
INHIBITION
ISOTOPE APPLICATIONS
LABELLED COMPOUNDS
LIGASES
LIPIDS
LIPOPOLYSACCHARIDES
MEMBRANES
MICROORGANISMS
MOLECULAR BIOLOGY
MUTANTS
ORGANIC COMPOUNDS
PERMEABILITY
POLYSACCHARIDES
PRECURSOR
SACCHARIDES
SALMONELLA
SALMONELLA TYPHIMURIUM
TOLUENE
TRACER TECHNIQUES
TRITIUM COMPOUNDS