Functional analysis of a chromosomal arsenic resistance operon in Pseudomonas fluorescens strain MSP3

Abstract

We reported earlier about the detection of a chromosomally located arsenic operon (arsRBC) in a gram-negative bacterium Pseudomonas fluorescens strain MSP3, which showed resistance to elevated levels of sodium arsenate and sodium arsenite. The genes for arsenic resistance were cloned into the HindIII site of pBluescript vector producing three clones MSA1, MSA2 and MSI3 conferring resistance to sodium arsenate and arsenite salts. They were further sub-cloned to delineate the insert size and the sub-clones were designated as MSA11, MSA12 and MSI13. The sub-clone pMSA12 (2.6 kb) fragment was further packaged into EcoRI-PstI site of M13mp19 and sequenced. Nucleotide sequencing revealed the presence of three open reading frames homologous to the arsR, arsB and arsC genes of arsenic resistance. Three cistrons of the ars operon encoded polypeptides ArsR, ArsB and ArsC with molecular weights ranging approximately 12, 37and 24 kDa, respectively. These polypeptides were visualized on SDS-PAGE stained with Coomassie blue and measured in a densitometer. The arsenic resistance operon (arsRBC) of strain MSP3 plasmid pMSA12 consists of 3 genes namely, arsR – encoding a repressor regulatory protein, arsB – the determinant of the membrane efflux protein that confers resistance by pumping arsenic from the cells and arsC – a small cytoplasmic polypeptide required for arsenate resistance only, not for arsenite resistance. ArsB protein is believed to use the cell membrane potential to drive the efflux of intracellular arsenite ions. ArsC encodes for the enzyme arsenate reductase which reduces intracellular As(V) (arsenate) to more toxic As(III) (arsenite) and is subsequently extruded from the cell. The arsenate reductase activity was present in the soluble cytoplasmic fraction in E. coli clones. In the context of specified function of the arsenic operon encoded proteins, uptake and efflux mechanisms were studied in the wild strain and the arsenate/arsenite clones. The cell free filtrates of the arsenate clones (MSA11 and MSA12) obtained from P. fluorescens containing the arsC gene showed that arsenate reduction requires glutathione reductase, glutathione (GSH), glutaredoxin and ArsC protein. The protein was purified in an active form and a spectrophotometric assay was developed in which the oxidation of NADPH was coupled to reduction of arsenate. The molecular weights and the location of the polypeptides were obtained from Coomassie stained SDS-PAGE of extracellular and intracellular fractions of the cells.