Eight Gram-negative bacteria are 10,000 times more sensitive to cationic detergents than to anionic detergents

In liquid culture, eight typical Gram-negative bacteria were ca. 10,000-fold more sensitive to cationic detergents than to the anionic detergent sodium dodecyl sulfate. Cetyltrimethylammonium bromide (CTAB) was inhibitory at concentrations ranging from 0.0006% to 0.01%. Four pseudomonads able to form biofilms were ca. 1000-fold more resistant to CTAB on Luria–Bertani agar plates than they were in liquid culture. A lasI mutant of Pseudomonas aeruginosa was only able to tolerate 0.1% CTAB on Luria–Bertani agar plates but could tolerate 5% CTAB when supplemented with homoserine lactone containing culture supernatants. En culture liquide, huit bactéries Gram négatif typiques étaient ca. 10,000 fois plus sensible aux détergents cationiques qu\u27au détergent anionique dodécyl sulfate de sodium. Le bromure de cétyltriméthylammonium (CTAB) était inhibiteur à des concentrations allant de 0,0006% à 0,01%. Quatre pseudomonades capables de former des biofilms étaient ca. 1000 fois plus résistant au CTAB sur des plaques de gélose Luria – Bertani qu\u27en culture liquide. Un mutant lasI de Pseudomonas aeruginosa ne pouvait tolérer que 0,1% de CTAB sur des plaques de gélose Luria-Bertani, mais pouvait tolérer 5% de CTAB lorsqu\u27il était complété par des surnageants de culture contenant de l\u27homosérine lactone

Sodium Dodecyl Sulfate Hypersensitivity of \u3ci\u3eclpP\u3c/i\u3e and \u3ci\u3eclpB\u3c/i\u3e Mutants of \u3ci\u3eEscherichia coli\u3c/i\u3e

We studied the hypersensitivity of clpP and clpB mutants of Escherichia coli to sodium dodecyl sulfate (SDS). Both wild-type E. coli MC4100 and lon mutants grew in the presence of 10% SDS, whereas isogenic clpP and clpB single mutants could not grow above 0.5% SDS and clpA and clpX single mutants could not grow above 5.0% SDS. For wild-type E. coli, cellular ClpP levels as determined by Western immunoblot analysis increased ca. sixfold as the levels of added SDS increased from 0 to 2%. Capsular colanic acid, measured as uronic acid, increased ca. sixfold as the levels of added SDS increased from 2 to 10%. Based on these findings, 3 of the 19 previously identified SDS shock proteins (M. Adamowicz, P. M. Kelley, and K. W. Nickerson, J. Bacteriol. 173:229-233, 1991) are tentatively identified as ClpP, ClpX, and ClpB

Mechanism of detergent resistance in Escherichia coli and related bacteria

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 \u3e2%. 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

Eight Gram-negative bacteria are 10,000 times more sensitive to cationic detergents than to anionic detergents

In liquid culture, eight typical Gram-negative bacteria were ca. 10,000-fold more sensitive to cationic detergents than to the anionic detergent sodium dodecyl sulfate. Cetyltrimethylammonium bromide (CTAB) was inhibitory at concentrations ranging from 0.0006% to 0.01%. Four pseudomonads able to form biofilms were ca. 1000-fold more resistant to CTAB on Luria–Bertani agar plates than they were in liquid culture. A lasI mutant of Pseudomonas aeruginosa was only able to tolerate 0.1% CTAB on Luria–Bertani agar plates but could tolerate 5% CTAB when supplemented with homoserine lactone containing culture supernatants. En culture liquide, huit bactéries Gram négatif typiques étaient ca. 10,000 fois plus sensible aux détergents cationiques qu\u27au détergent anionique dodécyl sulfate de sodium. Le bromure de cétyltriméthylammonium (CTAB) était inhibiteur à des concentrations allant de 0,0006% à 0,01%. Quatre pseudomonades capables de former des biofilms étaient ca. 1000 fois plus résistant au CTAB sur des plaques de gélose Luria – Bertani qu\u27en culture liquide. Un mutant lasI de Pseudomonas aeruginosa ne pouvait tolérer que 0,1% de CTAB sur des plaques de gélose Luria-Bertani, mais pouvait tolérer 5% de CTAB lorsqu\u27il était complété par des surnageants de culture contenant de l\u27homosérine lactone

Sodium Dodecyl Sulfate Hypersensitivity of clpP and clpB Mutants of Escherichia coli

We studied the hypersensitivity of clpP and clpB mutants of Escherichia coli to sodium dodecyl sulfate (SDS). Both wild-type E. coli MC4100 and lon mutants grew in the presence of 10% SDS, whereas isogenic clpP and clpB single mutants could not grow above 0.5% SDS and clpA and clpX single mutants could not grow above 5.0% SDS. For wild-type E. coli, cellular ClpP levels as determined by Western immunoblot analysis increased ca. sixfold as the levels of added SDS increased from 0 to 2%. Capsular colanic acid, measured as uronic acid, increased ca. sixfold as the levels of added SDS increased from 2 to 10%. Based on these findings, 3 of the 19 previously identified SDS shock proteins (M. Adamowicz, P. M. Kelley, and K. W. Nickerson, J. Bacteriol. 173:229-233, 1991) are tentatively identified as ClpP, ClpX, and ClpB

Survey of Extreme Solvent Tolerance in Gram-Positive Cocci: Membrane Fatty Acid Changes in \u3ci\u3eStaphylococcus haemolyticus\u3c/i\u3e Grown in Toluene

We exploited the unique ecological niche of oil fly larval guts to isolate a strain of Staphylococcus haemolyticus which may be the most solvent-tolerant gram-positive bacterium yet described. This organism is able to tolerate 100% toluene, benzene, and p-xylene on plate overlays and saturating levels of these solvents in monophasic liquid cultures. A comparison of membrane fatty acids by gas chromatography after growth in liquid media with and without toluene showed that in cells continuously exposed to solvent the proportion of anteiso fatty acids increased from 25.8 to 33.7% while the proportion of 20:0 straight-chain fatty acids decreased from 19.3 to 10.1%. No changes in the membrane phospholipid composition were noted. Thus, S. haemolyticus alters its membrane fluidity via fatty acid composition to become more fluid when it is exposed to solvent. This response is opposite that commonly found in gram-negative bacteria, which change their fatty acids so that the cytoplasmic membrane is less fluid. Extreme solvent tolerance in S. haemolyticus is not accompanied by abnormal resistance to anionic or cationic detergents. Finally, six strains of Staphylococcus aureus and five strains of Staphylococcus epidermidis, which were not obtained by solvent selection, also exhibited exceptional solvent tolerance

Scaffold Proteins IRSp53 and Spinophilin Regulate Localized Rac Activation by T-lymphocyte Invasion and Metastasis Protein 1 (TIAM1)*

The Rac exchange factor Tiam1 is involved in diverse cell functions and signaling pathways through multiple protein interactions, raising the question of how signaling and functional specificity are achieved. We have shown that Tiam1 interactions with different scaffold proteins activate different Rac-dependent pathways by recruiting specific Rac effector proteins, and reasoned that there must be regulatory mechanisms governing each interaction. Fibroblasts express at least two Tiam1-interacting proteins, insulin receptor substrate protein 53 kDa (IRSp53) and spinophilin. We used fluorescent resonance energy transfer (FRET) to measure localized Rac activation associated with IRSp53 and spinophilin complexes in individual fibroblasts to test this hypothesis. Pervanadate or platelet-derived growth factor induced localized Rac activation dependent on Tiam1 and IRSp53. Forskolin or epinephrine induced localized Rac activation dependent on Tiam1 and spinophilin. In spinophilin-deficient cells, Tiam1 co-localized with IRSp53 in response to pervanadate or platelet-derived growth factor. In IRSp53-deficient cells, Tiam1 co-localized with spinophilin in response to forskolin or epinephrine. Total cellular levels of activated Rac were affected only in cells with exogenous Tiam1, and were primarily increased in the membrane fraction. Downstream effects of Rac activation were also stimulus and scaffold-specific. Cell ruffling, spreading, and cell adhesion were dependent on IRSp53, but not spinophilin. Epinephrine decreased IRSp53-dependent adhesion and increased cell migration in a Rac and spinophilin-dependent fashion. These results support the idea that Tiam1 interactions with different scaffold proteins couple distinct upstream signals to localized Rac activation and specific downstream pathways, and suggest that manipulating Tiam1-scaffold interactions can modulate Rac-dependent cellular behaviors