In a search for genes responsible for the accumulation of antimonite in Escherichia coli, TnphoA was used to create a pool of random insertional mutants, from which one antimonite-resistant mutant was isolated. Sequence analysis showed that the TnphoA insertion was located in the glpF gene, coding for the glycerol facilitator GlpF. The mutant was shown to be defective in polyol transport by GlpF. These results suggest that in solution Sb(III) is recognized as a polyol by the glycerol facilitator.Resistance to antimonite [Sb(III)], arsenite [As(III)], and arsenate [As(V)] is encoded by both plasmid-borne and chromosomal arsenical resistance (ars) operons (2, 3, 10). These operons encode transport systems that extrude the toxic metalloids, thus lowering the intracellular concentration and producing resistance (2,8,13). Arsenate is accumulated by both the Pit and Pst phosphate transport systems, although the Pit system may be responsible for the majority of uptake (13). The pathways for antimonite and arsenite accumulation have not been defined. In an attempt to identify the cellular transporters for the metalloid salts, Escherichia coli AW3110 (2) was subjected to random TnphoA mutagenesis (7).Random TnphoA-mediated mutagenesis to obtain Sb(III)-resistant mutant. Strains, plasmids, and phage used in this study are given in Table 1. E. coli AW3110, which lacks the chromosomal ars operon (2), was infected with b221 rex::TnphoA cI857 (7). Cells were plated on Luria-Bertani (LB) agar containing 1 mM either potassium antimonyl tartrate or sodium arsenite plus 35 g of kanamycin/ml. One mutant was obtained on antimonite-containing agar. No arsenite-resistant mutants were isolated. Colonies of the antimoniteresistant strain, OSBR1, were white on plates containing 20 g of 5-bromo-4-chloro-3-indolyl phosphate (XP)/ml. This could indicate an intracellular localization of the alkaline phosphatase portion of the moiety, an out-of-frame fusion, or fusion with the reading frames in the opposite orientation.Antimonite resistance is due to a single TnphoA insertion. To determine whether the mutant strain carried the TnphoA insertion in a single locus, the kanamycin resistance phenotype was transduced back into strain AW3110 by generalized transduction with P1 phage. All transductants were Sb(III) resistant. Southern blot hybridization was performed with BamHIdigested genomic DNA of OSBR1, with DNA from AW3110 as a control, by using a 485-bp TnphoA-specific probe. The result of the Southern blot confirmed the existence of only a single TnphoA insertion (data not shown).The TnphoA insertion is located in the glpF gene. Since there is no BamHI site between the site of fusion in TnphoA and the kanamycin phosphotransferase gene, and there is a BamHI site immediately following the 3Ј end of the kanamycin phosphotransferase gene (5), chromosomal DNA of OSBR1 was digested with BamHI. The portion of DNA proximal to the fusion junction was cloned into the unique BamHI site of pUC18; the transformed colonies were screened for Km r . The resu...

The arsenical resistance (ars) operon of the conjugative R-factor R773 confers resistance to arsenical and antimonial compounds in Escherichia coli, where resistance results from active extrusion of arsenite catalyzed by the products of the arsA and arsB genes. Previous in vivo studies on the energetics of arsenite extrusion showed that expression of both genes produced an ATP-coupled arsenite extrusion system that was independent of the electrochemical proton gradient. In contrast, in cells expressing only the arsB gene, arsenite extrusion was coupled to electrochemical energy and independent of ATP, suggesting that the Ars transport system exhibits a dual mode of energy coupling depending on the subunit composition. In vitro the ArsA-ArsB complex has been shown to catalyze ATP-coupled uptake of 73AsO2(-1) in everted membrane vesicles. However, transport catalyzed by ArsB alone has not previously been observed in vitro. In this study we demonstrate everted membrane vesicles prepared from cells expressing only arsB exhibit uptake of 73AsO2(-1) coupled to electrochemical energy.