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Mechanism of detergent resistance in Escherichia coli and related bacteria

Soumitra Rajagopal, University of Nebraska - Lincoln

Abstract

The mechanism of detergent resistance of gram negative bacteria was investigated in this study. Using Escherichia coli as the model organism and sodium dodecyl sulfate (SDS) as the model detergent, it was observed that all five compartments of the cell were required for detergent resistance. Moving progressively from inside out these are (i) the Clp proteases in the cytoplasm, (ii) the cytoplasmic membrane which is the site for numerous efflux pumps, (iii) the membrane derived oligosaccharides (MDOs) in the periplasm, (iv) the outer membrane as a selective barrier, and (v) the capsule. The cytoplasmic and periplasmic compartments were investigated for their involvement in SDS resistance. The ClpP and ClpB proteases in the cytoplasm are required for SDS resistance. Mutants of clpP and clpB in E. coli were unable to grow in 0.5% SDS. Western blot analysis of ClpP protein in wild type E. coli cells grown in 0–2% added SDS increased ca. six fold and then decreased to two fold at SDS concentrations >2%. A corresponding increase in the levels of colanic acid (capsule) was observed for cells grown in 2–10% added SDS indicating an alternate mechanism for SDS resistance. Additionally, the ClpA and ClpX chaperones were not found to be involved in SDS resistance. A ClpAX double mutant was found to be resistant to 5% SDS, however a clpAclpBclpX triple mutant was found to be sensitive to SDS, indicating the association of a different factor with ClpP in SDS resistance. The role of membrane derived oligosaccharides (MDOs) in the periplasm was also investigated. Wild type E. coli MC4100 grew in the presence of 10% SDS, whereas isogenic mdoA and mdoB mutants could not grow above 0.5% SDS. Similarly, E. coli DF214, a glucose negative (pgi, zwf ) mutant, exhibited conditional sensitivity to SDS in that it grew in gluconate and glucose or galactose but not in gluconate and mannose or sorbose. DF214 requires both gluconate and glucose/galactose because the gluconate is used for energy production, while glucose/galactose is used for MDO synthesis. Finally, the fate of E. coli cells subjected to SDS shock during growth or when used as an inoculum is dependent on the presence or absence of sufficient MDOs. In both cases, cells grown under high osmolarity (low MDO) conditions were rapidly lysed by 5% SDS. ^ The mechanism of resistance of gram negative bacteria to cationic detergents involved formation of biofilms. In liquid culture eight typical Gram negative bacteria were ca. thousand fold sensitive to cationic detergents than they were to SDS. Four strains of Pseudomonas able to form biofilms were ca. thousand fold more resistant to hexadecyltrimethylammoniumbromide (HDT) on LB agar plates than they were to liquid culture. A lasI mutant of P. aeruginosa was only able to tolerate 0.1% HDT on LB agar plates but could tolerate 5% HDT when supplemented with homoserine lactone containing supernatants. ^

Subject Area

Biology, Microbiology

Recommended Citation

Rajagopal, Soumitra, "Mechanism of detergent resistance in Escherichia coli and related bacteria" (2003). ETD collection for University of Nebraska - Lincoln. AAI3085740.
http://digitalcommons.unl.edu/dissertations/AAI3085740

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