F exclusion of bacteriophage T7
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T 7 fails to productively infect E. coli cells containing the plasmid F, a phenomenon now referred to as F exclusion. The F protein PifA and the T7 proteins gp10 and gp1.2 are known to be responsible for exclusion. Coexpression of pifA and gene 1.2 or pifA and gene 10 is known to be lethal to E. coli; selecting for survivors of a lethal challenge pifA and gene 1.2 resulted in the isolation of a strain containing the fxsAp109 mutation. This mutation leads to overproduction of the inner membrane protein FxsA. The same mutation had been isolated previously by selecting survivors of pifA and gene 10 co-expression, suggesting that both combinations, PifA plus gp10, and PifA plus gp1.2, attack the same or similar cellular function. FxsA is not this function, rather, overexpression of fxsA serves a protective role. PifA has been shown to interact with gp10 (Kd=0.3µM) and gp1.2 (Kd=1µM), whereas mutant gp10 and gp1.2, synthesized by mutant phages that escape F exclusion, bind with dramatically reduced affinity. FxsA also interacts with PifA, gp10A and gp1.2. It was shown that gp1.2 and FxsA bind to the of Cterminal 254 residues of PifA while gp10A interacts with the region between residues 181 and 463. PifA fractionates with the cytoplasmic membrane of E. coli but may only be membrane-associated, whereas FxsA was shown to possess four transmembrane segments. Both the N- and C-termini of FxsA are cytoplasmic but the hydrophilic C-terminus is not required for FxsA function; conversely, the fourth transmembrane segment of FxsA is essential for function. As gp10A and gp1.2 are localized in the cytoplasm, these proteins likely interact with FxsA and PifA at the cytoplasmic membrane. This membrane is therefore the site within E.coli where F exclusion of T7 is initiated.