Structural maintenance of chromosome (SMC) proteins are involved in all the aspects of the higher-order chromosome dynamics in organisms ranging from bacteria to human (Hirano, 2005; Nasmyth and Haering, 2005). MukBEF is the bacterial condensin required for correct folding of the Escherichia coli chromosome (Niki et al., 1991; Petrushenko et al., 2006a). In vitro, SMC subunit MukB forms clamps on DNA and MukB clamps further interact with each other to form a scaffold on DNA, thereby controlling the DNA structure (Cui et al., 2008; Petrushenko et al., 2010).
To determine how the MukB cluster is formed, the subcellular localization of MukB and MukE was investigated. We found that MukE-GFP also formed foci at the quarter positions of the cell length but not in cells that lack MukB. Therefore the condensin factory is formed by MukBEF complex, not its individual subunits. Overproduction of MukEF could disrupt MukE foci. Also MukB foci were disrupted by overproduced MukEF. Thus, the condensin factory only can accommodate a limited number of each subunit of MukBEF.
Then the function of MukE was further studied using random mutagenesis. Eight loss-of-function MukE point mutants were constructed. Mutations L54P and L47P P67C resulted in protein misfolding and MukEG96W was expressed at a reduced level. All other mutants had similar expression levels as the wild type MukE. All loss-of-function MukE mutants were unable to form the quarter position foci. Focal localization of MukB was also disrupted by mutations in MukE. Therefore, the condensin factory was disrupted by all of our loss-of-function MukE mutants. These data suggest that MukBEF foci formation is essential for its function.
Five mutant MukEFs (R140C, G188E, P69T, G96W and S141P) were purified using Ni2+-chelate and gel filtration chromatography. Mutation G96W disrupted MukEF complex. Other four mutant MukEF complexes were stable. They were purified and their biochemical activities were studied. All four mutants were able to form MukBEF complexes in vitro. These four purified mutant MukEFs inhibited the DNA binding activities of MukB as efficiently as the wild type MukEF.
Lastly, we found that four (R140C, G188E, P69T and S141P) out of six tested mutants formed MukBEF complex inside the cell. These four MukE mutants have the ability to form MukBEF complexes in vivo and in vitro and they can regulate the interaction between MukB and DNA as efficiently as wild type MukEF. In contrast, all of our loss-of-function MukE mutants are unable to form MukBEF clusters. Therefore, MukBEF complex formation is not sufficient for the MukBEF cluster formation. Binding with DNA is not sufficient for MukBEF cluster formation either. These results suggest that MukE helps MukBEF to form clusters at the quarter positions. Maybe there is an extra-chromosomal factor that is also involved in MukBEF cluster formation.
A new member of the bacterial condensins is discussed in the end. In bacteria, two families of condensins were identified before this study, MukBEF and SMC_ScpAB complexes. Only MukBEF or SMC_ScpAB was found in a given species. Using sequence analysis, we identified a third family of condensins, MksBEF (MukBEF-like SMC proteins), which is broadly present in diverse bacteria. MksBEF often coexists with another condensin. The physiological function of MksBEF protein was studied in Pseudomonas aeruginosa strain PAO1, which encodes SMC_ScpAB and MksBEF complexes. Inactivation of either SMC or MksB led to anucleate cell formation. Increased frequency of anucleate cells was observed when both smc and mksB genes were knocked out. Moreover, MksBEF can complement anucleate cell formation in SMC-deficient cells. Thus, both MksBEF and SMC contribute to chromosome partitioning in Pseudomonas aeruginosa. Several specialized condensins might be involved in organization of bacterial chromosomes. (Abstract shortened by UMI.)
|Advisor:||Rybenkov, Valentin V.|
|Commitee:||Dunn, Anne K., Klebba, Philip E., Schroeder, Susan J., Zgurskaya, Helen I.|
|School:||The University of Oklahoma|
|Department:||Department of Chemistry and Biochemistry|
|School Location:||United States -- Oklahoma|
|Source:||DAI-B 73/02, Dissertation Abstracts International|
|Keywords:||Bacterial condensins, E. coli, Mksbef, Mukbef, Mutations, Pseudomonas aeruginosa, Structural maintenance of chromosome|
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