Characterization of PitA and PitB from Escherichia coli
AUTOR(ES)
Harris, Robyn M.
FONTE
American Society for Microbiology
RESUMO
Escherichia coli contains two major systems for transporting inorganic phosphate (Pi). The low-affinity Pi transporter (pitA) is expressed constitutively and is dependent on the proton motive force, while the high-affinity Pst system (pstSCAB) is induced at low external Pi concentrations by the pho regulon and is an ABC transporter. We isolated a third putative Pi transport gene, pitB, from E. coli K-12 and present evidence that pitB encodes a functional Pi transporter that may be repressed at low Pi levels by the pho regulon. While a pitB+ cosmid clone allowed growth on medium containing 500 μM Pi, E. coli with wild-type genomic pitB (pitA ΔpstC345 double mutant) was unable to grow under these conditions, making it indistinguishable from a pitA pitB ΔpstC345 triple mutant. The mutation ΔpstC345 constitutively activates the pho regulon, which is normally induced by phosphate starvation. Removal of pho regulation by deleting the phoB-phoR operon allowed the pitB+ pitA ΔpstC345 strain to utilize Pi, with Pi uptake rates significantly higher than background levels. In addition, the apparent Km of PitB decreased with increased levels of protein expression, suggesting that there is also regulation of the PitB protein. Strain K-10 contains a nonfunctional pitA gene and lacks Pit activity when the Pst system is mutated. The pitA mutation was identified as a single base change, causing an aspartic acid to replace glycine 220. This mutation greatly decreased the amount of PitA protein present in cell membranes, indicating that the aspartic acid substitution disrupts protein structure.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=95376Documentos Relacionados
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