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    Characterization of YisK, a Cell Shape Modifier and Enzyme in Bacillus subtilis

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    Date
    2017-12-08
    Author
    Sperber, Anthony Michael
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    Abstract
    Bacterial growth and division requires the careful coordination of peptidoglycan (PG) synthesis and PG hydrolysis, allowing the insertion of new cell wall material at sites of active growth. In many rod-shaped bacteria, the bacterial actin homolog MreB is thought to coordinate this balance of synthesis and hydrolysis, particularly during cell elongation, and the current model is that MreB-like proteins act as a scaffold, directing the PG synthesis machinery to sites of active growth. Despite their importance, very little is known about how MreB-like proteins in prokaryotes are regulated. Using a Bacillus subtilis misexpression screen, we identified yisK and yodL, which cause a loss of cell shape and viability when misexpressed. Suppressors resistant to YisK’s killing activity primarily occur in mbl (the structural gene for an MreB paralog in B. subtilis), while suppressors resistant to YodL’s activity primarily occur in MreB. Consistent with the idea that YisK targets Mbl activity and YodL targets MreB activity, deletion of mbl confers resistance to YisK, while deletion of MreB confers resistance to YodL. In an mbl deletion background, YisK expressing cells also become 20% shorter, suggesting that YisK activity affects at least one other target integral to cell shape. Using a bacterial 2-hybrid assay, we detected an interaction between YisK and FtsE (the ATPase of the ABC Transporter FtsEX). Interestingly, published data indicates that FtsEX, which is important for regulating the activity of the D,L-endopeptidase CwlO, appears to act in the same pathway as Mbl, and both ftsE and cwlO mutants exhibit short-cell phenotypes. Our data suggest that ftsE is required for YisK-dependent cell shortening, but not cell widening. YisK shows ~40% amino acid identity to an FAH from Mycobacterium abcsessus, and we have obtained a preliminary crystal structure for YisK, with a dicarboxylic acid, most likely L-tartrate, bound in the active site. Surprisingly, introducing mutations in YisK’s active site has no effect on its ability to perturb cell shape. Our current model is that YisK is an enzyme, possibly involved in the dicarboxylate pathway, that utilizes interactions with Mbl and possibly FtsE to localize its enzymatic activity to specific regions within the cell.
    URI
    https://hdl.handle.net/1969.1/173245
    Subject
    MreB
    Mbl
    YodL
    YisK
    peptidoglycan
    cell division
    PBSX
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    • Electronic Theses, Dissertations, and Records of Study (2002– )
    Citation
    Sperber, Anthony Michael (2017). Characterization of YisK, a Cell Shape Modifier and Enzyme in Bacillus subtilis. Doctoral dissertation, Texas A & M University. Available electronically from https : / /hdl .handle .net /1969 .1 /173245.

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