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Description:
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Most bacteriophage escape the confines of the host bacterium by
compromising the integrity of its cell wall , an event that results in rupture (lysis )
of the cell . The lysis strategy of bacteriophage Qβ is inhibition of cell wall
biosynthesis while the cell is growing . To elicit lysis , the maturation protein (A2 )
of Qβ inhibits the catalytic activity of MurA , an essential , induced fit enzyme in
the cell wall biosynthetic pathway . Consequent lysis releases progeny phage
into the environment .
The research in this dissertation addresses how lysis timing is integrated
into Qβ’s life cycle and discerns the molecular basis of the lytic event . Working
off the notion that , as displayed by the mature virion , A2 inhibits MurA , we
developed an in vivo bioassay to resolve the amount of inhibitory A2 during
infection . We found that the amount of free A2 is vastly greater than the amount
of virion -associated A2 and that both forms inhibit MurA . Additionally , the
amount of A2 correlates to lysis time and the burst size , as mutant Qβ with
upregulated expression of A2 (Qβpor ) elicit host cell lysis faster and release fewer
mature virions than with the wildtype level of A2 . This further suggests that
protection from Qβ lysis afforded by MurAL138Q is due to perturbed affinity between A2 and MurA . Yeast two -hybrid analysis supports that A2 and
MurAL138Q interact with weaker affinity by rendering small colonies compared to
yeast containing interacting A2 -MurAwt . Scanning mutagenesis of MurA’s
surface near L138 identified residues that may be important for A2 contact in the
inhibitory complex . Potentially important residues map to a contiguous area on
the surface of MurA that spans both lobes on the flexible loop face of the
enzyme , suggesting that A2 prevents the induced fit mechanism of MurA in an
uncompetitive manner . Subsequent truncation analysis reveals that the aminoterminal
half of A2 is sufficient to mediate host cell lysis . Together , these
findings insinuate a model in which the amino -terminus of free A2 interacts with ,
and inhibits MurA . Then , when the infected cell initiates division , septal
catastrophe ensues causing the cell to lyse and liberate progeny bacteriophage
Qβ . |