Genetic Determination of Resistance to Acriflavine, Phenethyl Alcohol, and Sodium Dodecyl Sulfate in Escherichia coli

Wild-type strains of Escherichia coli K-12 are resistant to acriflavine. Gene acrA(+) which determines resistance to acriflavine is located near the lac region of the chromosome. This gene determines not only resistance to basic dyes but also resistance to phenethyl alcohol. Acriflavine resistance was transmitted, together with phenethyl alcohol resistance, from a resistant Hfr strain to a sensitive recipient by mating. Reversion of the mutant gene acrA1 (phenotypically acriflavine-sensitive) to acriflavine resistance was accompanied by a change from phenethyl alcohol sensitivity to resistance, and conversely the revertants selected for phenethyl alcohol resistance were resistant to acriflavine. A suppressor mutation, sup-100, closely linked to the acr locus, suppresses the acrA1 gene (phenotypically acriflavine-resistant), but does not determine resistance to phenethyl alcohol and basic dyes other than acriflavine. The genetic change in the locus acrA1 to types resistant to basic dyes and phenethyl alcohol was accompanied by an increase in resistance to sodium dodecyl sulfate, a potent solvent of lipopolysaccharide and lipoprotein. It is suggested that gene acrA determines synthesis of a membrane substance. The system seemed to be affected strongly by the presence of inorganic phosphate.     

Effect of acriflavine on the plasma membrane of Escherichia coli K12

Plasma membranes of acriflavine-sensitive mutant (acrA) and acriflavine-resistant (acrA+, wild-type and true revertant) Escherichia coli K12 strains treated with acriflavine were observed under the electron microscope by means of the freeze-fracture technique. The plasma membrane of the acrA mutant exhibited a complex lamellar structure at the end of the cell when treated with 20 micrograms acriflavine ml-1. However, the membrane of the acrA+ cells also gave the lamellar complex when treated with a very high concentration of acriflavine (100 micrograms ml-1). The size of the intramembranous particles was not affected by the acriflavine treatments.     

Effect of inorganic phosphate on acridine inhibition and plasmid curing in Escherichia coli.

Some mutants and stock strains of Escherichia coli K12 were sensitive to acriflavine in the presence of inorganic phosphate but were resistant to acriflavine in its absence. They mutated spontaneously to resistance to acriflavine plus phosphate. The synergistic effect of phosphate on acriflavine sensitivity was increased at high pH values. Genetic analysis suggested that the mutations occurred in the gene acrA. Electron microscopic observation suggested that the presence of acriflavine plus phosphate affected the structure of the plasma membrane and the cytoplasm under it. This structural alteration was not caused by acriflavine alone. Acridine orange plus phosphate can more effectively eliminate the plasmid F8-gal+ than acridine orange alone.     

Interaction of the expression of two membrane genes, acrA and plsA, in Escherichia coli K-12.

The mutation acrA1, leading to acriflavine sensitivity through disorganization of the plasma membrane, is located between proC and purE on the Escherichia coli K-12 chromosome. Gene plsA has been reported to determine biosynthesis of membrane phospholipid and to be located very near acrA (1). Genes acrA and plsA fall into different cistrons and are arranged in the order proC-acrA-plasA-purE. The genes were shown to interact with each other. Introduction of acrA mutation into a plsA temperature-sensitive mutant mitigated the heat sensitivity. Plasmid (F-gal+) stability in acrA mutants was restored by introduction of the plasA mutation into the acrA cells. When an Hfr plsA donor was conjugated with an acrA recipient, or when reciprocally conjugated, the exogenotes were eliminated at high frequency during subsequent subcultivation in broth. However, the exogenotes were not eliminated in all other allelic combinations of genes acrA and plsA. When an F-gal+ plasmid was introduced into the unstable heterozygotes (acrA+plsA/acrApls1+), the plasmids were stably hosted, whereas the acrA+ plasA exogenotes were spontaneously lost at a high frequency. On the other hand, when the unstable heterozygotes carrying F-gal+ were cultured in acriflavine-containing medium, the F-gal+ plasmids were preferentially eliminated but the acrA+plasA exogenotes were not affected. The results suggest that the organization of the plasma membrane controls the recombination of the exogenotes introduced into zygotes.     

Acriflavine-binding capacity controlled by the acrA gene of Escherichia coli

The acrA mutation in Escherichia coli led to a substantial increase of the acriflavine-binding capacity of the cell, whereas the related mutations acrB (gyrB) and arcC did not. Metal ions such as Na+, K+, Mg2+, Ca2+ and Al3+ effectively released the bound acriflavine, in proportion to their ionic strengths. The presence of cations, in fact, increased the survival fraction of the cells in the acriflavine-containing medium. Polymyxin B, an antibiotic which binds to membrane phospholipid, competed with acriflavine for binding sites. Cell wall digestion by treatment with lysozyme and EDTA slightly decreased the acriflavine-binding capacity. Almost no difference was observed in acriflavine-binding capacity between intact cells and cells from which lipopolysaccharide has been extracted (46.9% removed from the acrA cells and 47.4% from the acrA+ cells). Acriflavine bound to the cells was most effectively extracted by ethanol containing 1% HCl or by 2% (w/v) SDS. The difference in the acriflavine-binding capacity between the acrA and acrA+ cells was also observed in the spheroplasts. These facts indicate a relationship between the acrA gene product and the acriflavine-binding capacity of the cells.     

The gene that determines resistance to tioconazole and to acridine derivatives in Aspergillus nidulans may have a corresponding gene in Trichophyton rubrum

Understanding the genetic mechanisms involved in resistance to antifungal agents is important in the fight against pathogenic fungi. In the present investigation we studied a strain of the model fungus Aspergillus nidulans which presents resistance to tioconazole and behaves as the wild strain in the presence of other azole derivatives. Genetic analysis revealed that this resistance is due to a mutation in a single gene located on chromosome II, closely linked to the allele responsible for resistance to acriflavine and other acridine derivatives, i.e., acrA1. This result suggests that a multidrug resistance (MDR)-type mechanism may be involved. Two tioconazole-resistant strains of the pathogenic fungus Trichophyton rubrum obtained after mutagenic treatment also became simultaneously resistant to acriflavine and ethidium bromide, suggesting the existence of a resistance mechanism similar to that observed with the acrA1 mutation in A. nidulans. This revised version was published online in June 2006 with corrections to the Cover Date.     

Sensitivity of Escherichia coli acrA mutants to psoralen plus near-ultraviolet radiation

The sensitivity to psoralen plus near-ultraviolet radiation (PUVA) was compared in a pair of E. coli strains differing at the acrA locus. Survival was determined for both bacteria and phage lambda. AcrA mutant cells were 40 times more sensitive than wild type to the lethal effect of PUVA. Free lambda phage exposed to PUVA survived as well when plated on acrA mutants as on wild type. In contrast, prophage lambda CI857 ind carried in lysogenic acrA strains was hypersensitive to PUVA. The enhanced sensitivity of bacterial and lambda DNA, when inside acrA cells, was paralleled by an increased photobinding of radiolabelled psoralens in the mutant. Binding was increased specifically to DNA rather than to nucleic acids in general. The difference in psoralen-binding ability determined by the acrA gene persisted after permeabilizing treatment of the cells. The results suggest that the acrA mutation causes an alteration specifically in the environment of the cellular DNA so as to allow increased intercalation and photobinding of psoralens.     

Effect of mutation, electric membrane potential, and metabolic inhibitors on the accessibility of nucleic acids to ethidium bromide in Escherichia coli cells

The uptake of ethidium bromide by Escherichia coli K 12 cells has been studied by using 14C-labeled ethidium and spectrofluorometry on three E. coli strains: the first one (AB1157) has an ethidium-resistant phenotype; the second one derives from the first one after a single mutation (at 10 min on the E. coli genetic map) and has an ethidium-sensitive (Ebs) phenotype; the third one is the acrA strain which appeared to have the same phenotype as the Ebs strain. When the cells are in exponential growth, no ethidium enters wild-type cells, and a very limited amount of ethidium enters Ebs and acrA cells. Massive quantities of ethidium enter AB1157, Ebs, and acrA cells treated by uncouplers and respiring Ebs cells treated by the membrane ATPase-inhibitor dicyclohexylcarbodiimide. A small amount of ethidium enters cells treated in M9 succinate medium by metabolic inhibitors such as KCN or cells starved with oxygen in the same M9 medium. The amount of ethidium and ethidium dimer retained at equilibrium by either type of cell, and by cells infected by T5 phage, as well as the kinetics of influx and efflux, has been measured under a variety of situations (membrane energized or not, and/or membrane ATPase inhibited or not). Furthermore, it was shown that ethidium binds to both RNA and DNA when it enters CCCP-treated wild-type E. coli cells, whereas it binds mainly to DNA when it enters Ebs and acrA cells in exponential growth. As it will be discussed, it is difficult to account for the EthBr uptake by invoking only membrane functions and active transport. Therefore, it is proposed that the variations of the nucleic acid accessibility in E. coli cells might play a role in the control of this uptake. Accordingly, in ethidium-sensitive cells, the mutation would have caused a significant part of the chromosomal DNA (10-20%) to become accessible to ethidium. Hansen [Hansen M. T. (1982) Mutat. Res. 106, 209-216], after a study of the photobinding of psoralen to nucleic acids in the acrA mutant, also suggested that DNA environment was modified in acrA cells.     

Evidence for repair of ultraviolet-induced damage in Cystobacter species (Myxobacterales).

Cystobacter species strain CK 1 does not grow with more than 0.2 microgram/ml acriflavine. Spontaneous two-step mutants growing with 2 microgram acriflavine per ml have been selected. One mutant (strain CK3) was used to investigate the effect of repair inhibitors. Both strains exhibit pronounced shoulders in their UV dose curves of inactivation. Acriflavine (AF), coumarin (CU), and caffeine (CA) when incorporated in the post-irradiation plating medium decreased survival of irradiated cells. Post-treatment with 2 microgram acriflavine/ml abolished the shoulder of the curve. Caffeine (1600 microgram/ml) and coumarin (350 microgram/ml) reduced it only to about 40%. It is concluded that probably two repair mechanisms are present. Pre-treatment of the cells with 2 microgram acriflavine/ml for two hours before UV-irradiation resulted in a constant dose enhancement factor of 1.9. The protective effect is increased with the time of treatment with acriflavine. This may indicate that pyrimidine dimers are responsible for UV-inactivation.     

The pma1 and pma2 H(+)-ATPases from Schizosaccharomyces pombe are functionally interchangeable.

The pma2 gene of Schizosaccharomyces pombe codes for a polypeptide having a predicted Mr of 110,126 and which is 79% identical to the plasma membrane H(+)-ATPase encoded by the pma1 gene. The pma2 gene, unlike pma1, is weakly expressed and not essential to mitotic growth. By constructing yeast strains in which the chromosomal pma2 gene is under control of the adh promoter, it has been possible to identify the overproduced ATPase in plasma membrane via formation of a phosphoenzyme. In a pma1-1 mutant strain whose ATPase activity is insensitive to vanadate, the overexpressed pma2 gene restores vanadate sensitivity. It also rescues a pma1 null mutant from lethality. These results demonstrate that the two H(+)-ATPases are functionally interchangeable in vivo but differently expressed.     

Use of polymyxin B, levallorphan, and tetracaine to isolate novel envelope mutants of Escherichia coli.

Mutants of Escherichia coli were isolated by their resistance to the bacteriocidal effects of the membrane-active drugs polymyxin B, levallorphan, and tetracaine. The mutants were examined for additional changes in cellular physiology evoked by the lesions; many polymyxin-resistant strains had a concomitant increased sensitivity to anionic detergents, and several strains of each type had concomitant alterations in generation time and morphology. Mutants of each class (polymyxin resistant, tetracaine resistant, and levallorphan resistant) were transduced into recipient strains. The levallorphan resistance site (lev) was located at approximately 9 min on the E. coli chromosome. Polymyxin (pmx) and tetracaine (tec) resistance loci were also transduced. The lev and tec strains had a slight prolongation of generation time, in contrast with their isogenic wild-type strains. The tec transductant produced long filaments in the absence of tetracaine and had an altered colonial morphology, it reverted at high frequency, with the morphological abnormalities reverting along with the tetracaine resistance. The pmx transductant had an increased sensitivity to levallorphan and to anionic detergents. In contrast, both lev and tec mutants were more resistant to acriflavine than was the wild type or the pmx transductant. The pmx, lev, and tec loci differed in sensitivity to mitomycin C; the lev strain was more resistant, the tec strain was more sensitive, and the pmx strain was much more sensitive than the wild type. There was no difference in sensitivity to several other dyes and detergents, colicins, or T bacteriophage between the transductant and isogenic wild-type strains. Thus, lev, tec, and pmx loci confer more subtle alterations in the permeability barrier than do lipopolysaccharide-deficient mutants previously studied.     

The enhancement of the mutagenic effect of ultraviolet radiation inEscherichia coli by caffeine and acriflavine

The mutational synergism of caffeine and acriflavine was studied in five types ofEscherichia coli mutants induced by u. v.-radiation. The following types of mutations were compared: streptomycinrresistance (strain B/r), streptomycin-independence (strain Sd-4), and reversions to prototrophy (strains WP-14 pro⁻, WP-2 try⁻, and WP-2 try⁻ hcr⁻). In all hcr⁺ strains tested the presence of caffeine or acriflavine in a post-irradiation plate medium slightly decreases the survival of u.v.-irradiated cells and increases considerably the frequency of induced mutations. The mutational synergism of caffeine and acriflavine in the str-r and str-i mutants is observed only within the range of low doses. The abovementioned dose-dependence of the synergistic effect is discussed from the point of view of qualitative difference between the premutational damage caused by low and high doses. The post-irradiation treatment by caffeine slightly increases the frequency of induced prototrophs also in the WP-2 hcr⁻ strain. This finding is explained by the inhibition of the residual HCR-activity of the strain. The post-irradiation mutational synergism of acriflavine was not found in the WP-2 hcr⁻ strain.     

Detection of smr and qacA genes in Staphylococcus aureus strains using PCR

The 31 strains of Staphylococcus aureus were examined for the presence of smr and qacA determinants. The smr gene was found in 15 strains. Fourteen of them were MRSA resistant to quaternary ammonium compounds, ethidium bromide, and acriflavine. One was MSSA strain resistant to ethidium bromide and acriflavine. The qacA gene was found in two MRSA strains resistant to quaternary ammonium compounds, ethidium bromide, chlorhexidine and acriflavine. One of these two strains possessed both smr and qacA genes.     

Proton motive force is not obligatory for growth of Escherichia coli.

When 50 microM carbonyl cyanide-m-chlorophenyl hydrazone (CCCP), a protonophore, was added to growth medium containing glucose at pH 7.5, Escherichia coli TK1001 (trkD1 kdpABC5) started exponential growth after 30 min; the generation time was 70 min at 37 degrees C. Strain AS1 (acrA), another strain derived from E. coli K-12, also grew in the presence of 50 microM CCCP under the same conditions, except that the lag period was ca. 3 h. When this strain was grown in the presence of 50 microM CCCP and then transferred to fresh medium containing 50 microM CCCP, cells grew without any lag. Neither a membrane potential nor a pH gradient was detected in strain AS1 cells growing in the presence of CCCP. When either succinate or lactate was substituted for glucose, these strains did not grow in the presence of 50 microM CCCP. Thus, it is suggested that E. coli can grow in the absence of a proton motive force when glucose is used as an energy source at pH 7.5.     

Single point mutations in various domains of a plant plasma membrane H(+)-ATPase expressed in Saccharomyces cerevisiae increase H(+)-pumping and permit yeast growth at low pH.

In plants, the proton pump-ATPase (H(+)-ATPase) of the plasma membrane is encoded by a multigene family. The PMA2 (plasma membrane H(+)-ATPase) isoform from Nicotiana plumbaginifolia was previously shown to be capable of functionally replacing the yeast H(+)-ATPase, provided that the external pH was kept above pH 5.5. In this study, we used a positive selection to isolate 19 single point mutations of PMA2 which permit the growth of yeast cells at pH 4.0. Thirteen mutations were restricted to the C-terminus region, but another six mutations were found in four other regions of the enzyme. Kinetic studies determined on nine mutated PMA2 compared with the wild-type PMA2 revealed an activated enzyme characterized by an alkaline shift of the optimum pH and a slightly higher specific ATPase activity. However, the most striking difference was a 2- to 3-fold increase of H(+)-pumping in both reconstituted vesicles and intact cells. These results indicate that point mutations in various domains of the plant H(+)-ATPase improve the coupling between H(+)-pumping and ATP hydrolysis, resulting in better growth at low pH. Moreover, the yeast cells expressing the mutated PMA2 showed a marked reduction in the frequency of internal membrane proliferation seen with the strain expressing the wild-type PMA2, indicating a relationship between H(+)-ATPase activity and perturbations of the secretory pathway.     

Amiloride-sensitive NaCl taste responses are associated with genetic variation of ENaC alpha-subunit in mice

An epithelial Na(+) channel (ENaC) is expressed in taste cells and may be involved in the salt taste transduction. ENaC activity is blocked by amiloride, which in several mammalian species also inhibits taste responses to NaCl. In mice, lingual application of amiloride inhibits NaCl responses in the chorda tympani (CT) gustatory nerve much stronger in the C57BL/6 (B6) strain than in the 129P3/J (129) strain. We examined whether this strain difference is related to gene sequence variation or mRNA expression of three ENaC subunits (alpha, beta, gamma). Real-time RT-PCR and in situ hybridization detected no significant strain differences in expression of all three ENaC subunits in fungiform papillae. Sequences of the beta- and gammaENaC subunit genes were also similar in the B6 and 129 strains, but alphaENaC gene had three single nucleotide polymorphisms (SNPs). One of these SNPs resulted in a substitution of arginine in the B6 strain to tryptophan in the 129 strain (R616W) in the alphaENaC protein. To examine association of this SNP with amiloride sensitivity of CT responses to NaCl, we produced F(2) hybrids between B6 and 129 strains. Amiloride inhibited CT responses to NaCl in F(2) hybrids with B6/129 and B6/B6 alphaENaC R616W genotypes stronger than in F(2) hybrids with 129/129 genotype. This suggests that the R616W variation in the alphaENaC subunit affects amiloride sensitivity of the ENaC channel and provides evidence that ENaC is involved in amiloride-sensitive salt taste responses in mice.     

Interaction between tobramycin and CSA-13 on clinical isolates of Pseudomonas aeruginosa in a model of young and mature biofilms

The bactericidal activity of a cholic acid antimicrobial derivative, CSA-13, was tested against eight strains of Pseudomonas aeruginosa (both reference and clinical strains) and compared with the response to tobramycin. In planktonic cultures, the minimal inhibitory and minimal bactericidal concentrations of CSA-13 and tobramycin were in the 1–25 mg/L range except for one mucoid clinical strain which was much less sensitive to tobramycin (minimal bactericidal concentration, 65–125 mg/L). In young (24 h) biofilms, the sensitivity to CSA-13 was reduced (half-maximal concentration CSA-13 averaged 88 mg/L) and varied among the eight strains. The sensitivity to tobramycin was also very variable among the strains and some were fully resistant to the aminoglycoside. The combination of tobramycin with CSA-13 was synergistic in five strains. Only one strain showed antagonism between the two drugs at low concentrations of CSA-13. One reference and five clinical strains were tested in mature (12 days) biofilms. The effect of CSA-13 was delayed, some strains requiring 9 days exposure to the drug to observe a bactericidal effect. All the strains were tolerant to tobramycin but the addition of CSA-13 with tobramycin was synergistic in three strains. CSA-13 permeabilized the outer membrane of the bacteria (half-maximal concentration, 4.4 mg/L). At concentrations higher than 20 mg/L, it also permeabilized the plasma membrane of human umbilical vein endothelial cells. In conclusion, CSA-13 has bactericidal activity against P. aeruginosa even in mature biofilms and cationic steroid antibiotics can thus be considered as potential candidates for the treatment of chronic pulmonary infections of patients with cystic fibrosis. Considering its interaction with the plasma membrane of eukaryotic cells, less toxic derivatives of CSA-13 should be developed.     

Strain differences in heat-induced neural tube defects in mice

Neural tube defects are common congenital anomalies affecting approximately 0.1% of liveborn infants. It is widely accepted that these disorders are of a multifactorial origin, having both a genetic and an environmental component to their development. In a study designed to elucidate the genetic factors involved in a mouse model of hyperthermia-induced neural tube defects, it is apparent that a hierarchy of susceptibility exists among various inbred mouse strains. Female SWV mice were extremely sensitive to a 10-minute hyperthermic treatment on day 8.5 of gestation, with 44.3% of their offspring having exencephaly. The other strains used in these studies (LM/Bc, SWR/J, C57BL/6J, and DBA/2J) all had less than 14% affected offspring. In experimental situations where the environment is held constant and the only difference between the strains is their genotype, it is assumed that the difference in response to a teratogen is genetically mediated. To test the hypothesis that several genes are involved, reciprocal crosses were made between strains of high, moderate, and low sensitivity. When this was done, the high sensitivity of the SWV strain was lost in the F1 hybrid, implying not only that multiple genes are involved, but that it is the embryo's genotype and not the maternal genotype that is the major factor in determining susceptibility to heat-induced neural tube defects.     

Molecular cloning and characterization of acrA and acrE genes of Escherichia coli.

The DNA fragment containing the acrA locus of the Escherichia coli chromosome has been cloned by using a complementation test. The nucleotide sequence indicates the presence of two open reading frames (ORFs). Sequence analysis suggests that the first ORF encodes a 397-residue lipoprotein with a 24-amino-acid signal peptide at its N terminus. One inactive allele of acrA from strain N43 was shown to contain an IS2 element inserted into this ORF. Therefore, this ORF was designated acrA. The second downstream ORF is predicted to encode a transmembrane protein of 1,049 amino acids and is named acrE. Genes acrA and acrE are probably located on the same operon, and both of their products are likely to affect drug susceptibilities observed in wild-type cells. The cellular localizations of these polypeptides have been analyzed by making acrA::TnphoA and acrE::TnphoA fusion proteins. Interestingly, AcrA and AcrE share 65 and 77% amino acid identity with two other E. coli polypeptides, EnvC and EnvD, respectively. Drug susceptibilities in one acrA mutant (N43) and one envCD mutant (PM61) have been determined and compared. Finally, the possible functions of these proteins are discussed.