Outer Penetration Barrier of Escherichia coli K-12: Kinetics of the Uptake of Gentian Violet by Wild Type and Envelope Mutants

Abstract

Wild-type strains of Escherichia coli K-12 adsorb gentian violet to the cell surface, but the dye is not transported into the cytoplasm. However, when some mutants that have an altered outer membrane are exposed to gentian violet, the dye is also found in the ribosomal fraction. The transport into the cytoplasm is inhibited at 0 C and requires that the concentration of gentian violet exceeds a threshold value. The initial rate of uptake as well as the amount of gentian violet found in the cytoplasm increases with the concentration of the dye in the medium. The rate of transport of the dye into the cytoplasm is much lower for stationary mutant cells than for exponentially growing cells. The rate of uptake into the cytoplasm increases with increasing deficiency of carbohydrate in the lipopolysaccharide (carbohydrate content lpsB > IpsA > galT). However, other components are also responsible for the barrier since an envA mutant which is not altered in the lipopolysaccharide carbohydrates show an extremely rapid uptake of the dye. The rate of uptake for the envA mutant was the highest found and the same as that of spheroplasts. Growth in the presence of agents affecting the murein sacculus, e.g., lysozyme and sublethal concentrations of penicillin, increased the rate of uptake of gentian violet. Brief treatments with tris(hydroxymethyl)aminomethane-ethylenediaminetetraacetic acid drastically impaired the barrier function. Inhibition of protein synthesis by chloramphenicol also opened the barrier to gentian violet. In conclusion, the outer part of the bacterial envelope is a penetration barrier for gentian violet and probably also for other substances. The lipopolysaccharide, the murein and also other components are important for the function of this barrier. Resistance to gentian violet was found to be inversely correlated to the rate of penetration of the dye into the cytoplasm.

Role of the receptor for bacteriophage lambda in the functioning of the maltose chemoreceptor of Escherichia coli

“…Thus, the outer membrane protein can be grouped with the vitamin B12-E colicins receptor (5) and the iron bacteriophage Ti receptor (6) as a cell surface protein that is part of a transport system and also the receptor for an invading particle. The outer membrane has been characterized as a barrier to medium and large-size molecules (8,14,18,22). The discovery that in that membrane there are several apparent transport systems for small molecules implies that, at least in certain cases, the outer membrane also limits the passage of such molecules.…”

Gentian Violet: a 19th century drug re‐emerges in the 21st century

“…The exact mechanism of action of GV is unknown . Multiple hypothesis exist for an explanation of its antimicrobial effects : an alteration in redox potential by the dye , inhibition of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidases , free radical formation , formation of an unionized complex of bacteria with the dye , inhibition of protein synthesis , inhibition of glutamine synthesis , uncoupling of oxidative phosphorylation or inhibition of formation of the bacterial cell wall . Recently, our group discovered that the closely related dye brilliant green formed covalent adducts with thioredoxin reductase 2, a protein conserved from bacteria to humans, with an essential function for cellular activity (Fig.…”

Binding of Progesterone to Neisseria gonorrhoeae and Other Gram-Negative Bacteria

“…N. gonorrhoeae CS-7 exhibited significant crystal violet uptake and inhibition and was sensitive to progesterone. P. aeruginosa and S. typhimurium DB-21 showed no inhibition by crystal violet and bound only 30% of the added dye, which has been shown to remain bound to the cell surface (9). Both of these organisms were insensitive to progesterone.…”

“…The resistance of gram-negative organisms to many antibiotics and dyes such as crystal violet has been attributed to the outer membrane permeability barrier (6). Outer membrane mutants with abbreviated lipopolysaccharide chains (rough strains) often have increased permeability to these agents (1,9,10). A comparison between crystal violet uptake and inhibition and progesterone binding and inhibition for N. gonorrhoeae and other gram-negative bacteria is shown in Table 1.…”