Cloning, high yield overexpression, purification, and characterization of AlgH, a regulator of alginate biosynthesis in Pseudomonas aeruginosa.

The most common cause of mortality among cystic fibrosis sufferers is infection by antibiotic resistant strains of Pseudomonas aeruginosa. Means to control these strains continue to be an important goal. An integral component of the ability of many of these strains to defy antibiotic therapies is the protection afforded by the mucoexopolysaccharide alginate. Production of alginate by P. aeruginosa is tightly regulated at the transcriptional level. AlgH, a putative transcriptional regulator, is involved in regulating alginate biosynthesis as well as nucleoside diphosphate kinase activity and succinyl coenzyme A synthetase activity in P. aeruginosa. Sequence homologues are found in many bacterial species. Here, we describe a method for high level overexpression and high yield/high purity production of AlgH for biophysical and functional studies. The algH gene was cloned and AlgH was overexpressed in Escherichia coli using a commercially available vector with an inducible T7 promoter. We purified the recombinantly produced protein using a rapid classical purification scheme. The yield of purified protein, either isotopically labeled for NMR studies or unlabeled, is excellent (30-37 mg of purified protein per liter of minimal media culture), as is the purity (>95% pure). Analysis of the secondary structure using circular dichroism and NMR indicates that the protein is comprised of both beta-sheet and alpha-helical secondary structural elements. Heteronuclear NMR spectra indicate that AlgH is a monodisperse, folded globular protein. This rapid, high yield, and high purity method for AlgH production will permit further biophysical characterization of this protein including high resolution structural studies.

Aurora B phosphorylates centromeric MCAK and regulates its localization and microtubule depolymerization activity.

BACKGROUND: Sister kinetochores must bind microtubules in a bipolar fashion to equally segregate chromosomes during mitosis. The molecular mechanisms underlying this process remain unclear. Aurora B likely promotes chromosome biorientation by regulating kinetochore-microtubule attachments. MCAK (mitotic centromere-associated kinesin) is a Kin I kinesin that can depolymerize microtubules. These two proteins both localize to mitotic centromeres and have overlapping mitotic functions, including regulation of microtubule dynamics, proper chromosome congression, and correction of improper kinetochore-microtubule attachments. RESULTS: We show that Aurora B phosphorylates and regulates MCAK both in vitro and in vivo. Specifically, we mapped six Aurora B phosphorylation sites on MCAK in both the centromere-targeting domain and the neck region. Aurora B activity was required to localize MCAK to centromeres, but not to spindle poles. Aurora B phosphorylation of serine 196 in the neck region of MCAK inhibited its microtubule depolymerization activity. We found that this key site was phosphorylated at centromeres and anaphase spindle midzones in vivo. However, within the inner centromere there were pockets of both phosphorylated and unphosphorylated MCAK protein, suggesting that phosphate turnover is crucial in the regulation of MCAK activity. Addition of alpha-p-S196 antibodies to Xenopus egg extracts or injection of alpha-p-S196 antibodies into cells caused defects in chromosome positioning and/or segregation. CONCLUSIONS: We have established a direct link between the microtubule depolymerase MCAK and Aurora B kinase. Our data suggest that Aurora B both positively and negatively regulates MCAK during mitosis. We propose that Aurora B biorients chromosomes by directing MCAK to depolymerize incorrectly oriented kinetochore microtubules.