Nature of the Penetration Barrier inEscherichia coliK-12: Effect of Macromolecular Inhibition on Penetrability in Strains Containing theenvAGene

Abstract

The envA mutation in Escherichia coli K-12, which maps at 1.5 min, was previously shown to mediate sensitivity to gentian violet as well as to several antibiotics. Moreover, strains containing the envA gene were recently found to be lysed by lysozyme in the absence of ethylenediaminetetraacetate. It is here reported that the envA mutation mediates an increased uptake of gentian violet. The uptake of the dye was markedly affected by growth with different antibiotics interfering with macromolecular synthesis. Amino acid starvation of a strain containing envA with a stringent control of ribonucleic acid (RNA) synthesis resulted in a decreased uptake of gentian violet. However, no decrease in dye uptake was found during starvation in an envA transductant with a relaxed control of RNA synthesis. Inhibition of deoxyribonucleic acid (DNA) synthesis by nalidixic acid decreased the uptake of gentian violet of envA cells and, in addition, rendered the cells insensitive to the lytic action of lysozyme. Chloramphenicol treatment increased penetrability in wild-type and starved envA cells. In most instances, this effect of chloramphenicol was prevented by selectively interfering with DNA or RNA synthesis. A coordinate regulation of nucleic acid synthesis and penetrability is suggested.Gentian violet (GV) and other basic dyes have a high affinity for nucleic acids (9, 25). Resistance and sensitivity to GV and other basic dyes have been attributed to a number of genes located close to the marker for lactose utilization (13,14,22). In a report by Nakamura (13), it was also concluded that resistant strains had a lower capacity to bind basic dyes. However, Kushner and Khan (10) found that proflavinesensitive and -resistant strains bound the same amount of the dye and that binding of proflavine was a passive process. A release of bound dye caused the increased tolerance of the resistant cells.It was previously found that the envA mutation increased sensitivity to GV as well as to several antibiotics and that it mediated an unspecific increase in permeability through the outer layers of the cell envelope (16). It is reported here that sensitivity to GV is correlated to an increased uptake of the dye. The uptake of GV was markedly decreased during amino acid starvation in envA strains with stringent but not with relaxed ribonucleic acid (RNA) control. Treatment with antibiotics affecting macromolecular synthesis markedly affected uptake of GV and the response to lysozyme in both wild-type and envA strains. 45 MATERIALS AND METHODSOrganisms. Strain D21 of Escherichia coli K-12 was described by Boman et al. (3). Strain D22 is an ethyl methane sulfonate-induced mutant from strain D2 1 (19). The altered gene envA at 1.5 min on the chromosomal map mediates chain formation and an increased penetrability to antibacterial agents (16). The isogenic transductant pair E64-113 (wild type) and E64-120 (envA) and the spontaneous revertant D22S1 were described by Normark (16). Strain CP791 is an envA transductant of the rel strain CP79 (5).Med...

Phenethyl Alcohol as a Suppressor of theEnvAPhenotype Associated with theenvAGene inEscherichia coliK-12

“…It was previously found that the envA mutation caused a considerable increase in the uptake of the basic dye GV. The increased uptake of the dye was correlated to a decreased tolerance to the agent (21). When the envA mutant D22 and its parental strain D21 were grown in LB medium and treated with various concentrations of PEA for a period of 60 min, it was found that low concentrations of PEA decreased the uptake of dye in both of the strains.…”

“…All portions were harvested after 50 min, chilled, and centrifuged. Uptake of G V was tested as described by Normark (21). considerable change in colony morphology occurred.…”

“…Strain D22S1 is a spontaneous ampicillin-resistant revertant of strain D22 (18). Strain CP791 is an envA transductant of strain CP79 (21).…”

Genetic and physiological classification of periplasmic-leaky mutants of Salmonella typhimurium

“…Egan and Russell (5) described temperaturesensitive, osmotic-remedial mutants that both leak RNase and show hypersensitivity to inhibitors. Normark et al (12)(13)(14) have reported mutants that are presumably envelope mutants and that show hypersensitivity to inhibitors. Thus, a large number of enteric bacterial mutants have been described that may have in common an increased membrane permeability.…”

Relation of cell growth and colicin tolerance to vitamin B12 uptake in Escherichia coli

“…The rate of excretion of this complex was markedly increased upon the inhibition of protein synthesis by amino acid starvation or antibiotic treatment. It has subsequently been shown that growth inhibition can lead to alterations in surface properties, such as sensitization to actinomycin D (16) or other changes in outer membrane permeability (19) or morphology (12). Thus, the profound changes in B12 uptake could be merely a reflection of these general alterations on the integrity or function of the cell surface, rather than a specific alteration of the B12 uptake mechanism in addition to these general changes.…”