Plasmid-mediated resistance to arsenite and arsenate in Escherichia coli

Although arsenic compounds are prevalent in the environment, the most toxic forms are added intentionally in vast quantities as pesticides and herbicide~, or liberated as by-products of industry. The quantity of the trivalent inorganic form (arsenite) introduced 2 to the environment poses a serious health hazard because the toxicity of this form is explicable in terms of its ability to form stable bonds with cellular constituents. It is accumulated in tissues and it is known to be mutagenic. In the environment, arsenite is transformed into other arsenic compounds, mostly arsenate (the pentavalent inorganic form). Arsenate is also highly toxic, but less so than arsenite. Bacterial strains resistant to arsenite, arsenate (or both) are readily isolated from nature. The resistance, in many cases, can be shown to be determined by conjugal plasmids. The mechanisms by which these plasmids confer resistance to arseni~ compounds are entirely unknown. This study was undertaken to shed light on some of the physiological and genetic aspects of this resistance using plasmidbearing strains of Escherichia coli which are highly resistant to both arsenite and arsenate. Most studies were carried out using E. coli J53 (R773) or the plasmid-free strain J53. A minimal, defined medium was used in most aspects of this study. A system for separating and detecting arsenite, arsenate and phosphate, by thin-layer electrophoresis, was developed and used to show that plasmid-mediated resistance to arsenite is not due to oxidation to arsenate. Nor is it due to excretion of a detoxifying thiol reagent, as shown by growth experiments. The tolerance of plasmid-free and plasmid-bearing strains of£. coli to arsenite and arsenate was examined by determining the minimal inhibitory concentration (MIC) of these compounds. Growth responses to arsenite and arsenate, as well as MIC determinations, indicate that a probable target site for arsenite toxicity lies within respiratory metabolism. Growth 3 inhibition by arsenate toxicity is shown to be dependent on arsenate accumulation by phosphate uptake systems. Studies on the reversibility of arsenite and arsenate toxicity using growth experiments show that these compounds are bacteriostatic. Growth experiments were used to examine for plasmid-mediated protection of the phosphate uptake systems (Pst, Pit, GlpT and UhpT) from arsenate accumulation. The constitutive Pit system was shown to be functional in J53 (R773), and along with GlpT and UhpT, shown to be protected by R773. Growth experiments were used to study the induction and regulation of the arsenic resistance system in R773. Arsenite was shown to induce arsenite resistance alone, while arsenate was shown to induce both arsenate and arsenite resistance. Non-inhibiting levels of arsenite were shown to induce arsenite resistance to high levels within 15 minutes. The process was shown, by amino acid starvation experiments, to require protein systhesis. Finally, arsenic sensitive variants of R773 and R45 were isolated by transposon insertion mutagenesis. The phenotypes of the variants isolated indicate that the arsenite and arsenate resistance systems are genetically separate in these plasmids. A model