Mutational Analysis of Bacterial Condensins

Abstract

Genetic information is carried on chromosomes in all organisms and chromosome is at the center of all life processes. The chromosome must be properly folded to fit into the cells. Moreover, replication and segregation of chromosomes to daughter cells must be precisely controlled and coordinated during the cell cycle. Any failure in chromosome dynamics could lead to developmental diseases and cancer. The mechanism of chromosome organization remains unsolved. 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). Because SMC proteins are so important, studying SMC proteins will not only help us better understand chromosome organization, but also understand mechanisms of related human diseases.

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).

MukF is the kleisin subunit which recruits MukE to MukB. MukF forms complex with MukE, and the MukEF complex can further form a complex with MukB (Petrushenko et al., 2006b). MukEF also modulates the assembly of MukBEF macromolecular structure (Woo et al., 2009). MukEF disrupts MukB-DNA interactions in vitro (Petrushenko et al., 2006b). Therefore, MukEF regulates the interaction between MukB and DNA. Since these activities are mainly contributed by MukF, the role of MukE has long been unclear.

MukB forms clusters at the quarter positions along the cell length. The MukB cluster can be observed when MukB is tagged with GFP. MukEF is required for the assembly of MukB clusters (Ohsumi et al., 2001). The quarter position also is the new home of replicated DNA. These data suggest that in vivo, MukEF helps MukB to form a scaffold at the quarter positions of the cell length. According to this model, this MukBEF scaffold, "a condensin factory", controls the global architecture of the chromosome.

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.