Antimonite is accumulated by the glycerol facilitator GlpF in Escherichia coli

Abstract

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...

Analysis of the Pore of the Unusual Major Intrinsic Protein Channel, Yeast Fps1p

“…Through the regulation of this uptake, the channel has been suggested to have a role in toxic metalloid resistance in yeast (36). In the same study, no equivalent uptake could be demonstrated for GlpF, although GlpF has already been suggested to transport Sb(III), but not As (III), in E. coli (16), thus indicating a specificity difference between these close homologs. This hypothesis awaits a more thorough functional analysis of the transport specificity of Fps1p, which is currently in progress.…”

Arsenite transport by mammalian aquaglyceroporins AQP7 and AQP9

“…Aquaglyceroporins have been shown to facilitate metalloid uptake in both E. coli (13) and S. cerevisiae (18). Without these transporters, cells are unusually resistant to arsenite and͞or antimonite.…”

“…Two microbial aquaglyceroporins have been shown to facilitate metalloid uptake, GlpF in E. coli (13) and Fps1p in S. cerevisiae (18). The most closely related mammalian homologs to the two microbial proteins are AQP7 and AQP9.…”

“…In Escherichia coli, we showed that disruption of the glpF gene led to increased tolerance for antimonite, which is chemically similar to arsenite (13). GlpF facilitates glycerol uptake (14,15) and is a bacterial member of the aquaporins, a family of membrane proteins that includes water-selective pores (aquaporins) and multifunctional channels (aquaglyceroporins), which also transport organic polyols and urea (16).…”

“…With a pKa of 11.8, this protonated form would predominate in neutral solutions. Although the glpF mutant did not exhibit increased arsenite tolerance, we postulated that GlpF also transports As(OH) 3 , the primary ionization state of arsenite in neutral solutions, but that redundancy in arsenic transporters dampens the effect of the glpF mutation (13). Recently the GlpF homolog Fps1p, a S. cerevisiae aquaglyceroporin (17), was clearly shown to be involved in uptake of arsenite (18).…”