CHARACTERIZATION AND CRYSTALLIZATION OF PROTEINS
INVOLVED IN CONTROL OF CELL DIVISION AND CHROMOSOME SEGREGATION IN Bacillus subtilis
N. Pavlendová, K. Muchová, I. Barák
Institute of Molecular Biology, Slovak Academy of Sciences, 845 51 Bratislava
Gram-positive bacterium Bacillus subtilis is the one of the most commonly studied model organism. Its life cycle represents a complex biological process with a machinery of proteins involved. The mechanisms that ensure the correct positioning of the septum and correct chromosome segregation during both vegetative growth and sporulation are relatively complicated and still intensively studied. DivIVA, MinCD, RacA, Spo0J and Soj are the key proteins involved in control of cell division and in chromosome segregation.
DivIVA is a 19, 5 kDa tropomyosin-like a-helical coiled-coil protein. It assembles in vitro into a “doggy-bone”-like oligomers, which can build higher structures like strings, wires and 2D nets . DivIVA is a bifunctional protein involved in both vegetative growth and in sporulation. It localizes to the division site and to the cell poles. For polar targeting of DivIVA are responsible amino acid residues at N-terminal domain. Therefore, mutations at these sites may mimic conformational change that probably occurs and enables DivIVA its dual function . During vegetative growth, DivIVA acts as a controller of the mid-cell division site positioning. DivIVA protein arrests an inhibition complex of two proteins: MinC and MinD at cell poles and thus releases the cell center for septum formation . During sporulation, DivIVA participates in chromosome segregation . It interacts directly or indirectly with RacA, which serves as a “bridge” connecting ori regions of chromosome with cell poles . Besides DivIVA and RacA protein, also Soj, Spo0J are involved in chromosome segregation during sporulation. Spo0J protein localizes around the ori region and together with dynamically behaving Soj protein ensures precise anchoring of ori regions to opposite cell poles [6, 7].
We have cloned genes divIVAwt, divIVAR18C, racA, minD, spo0J and soj into pET vectors for expression and purification of all corresponding proteins. They will be used for crystallization and/or protein-protein interaction assays.
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The work in author’s laboratory is supported by grant 2/1004/21 from the Slovak Academy of Sciences and by grant APVT-51-027804 from Ministry of Education of Slovak Republic.