Structural and functional studies indicate that Shigella VirA is not a protease and does not directly destabilize microtubules

VirA, an essential virulence factor in Shigella disease pathogenesis, is involved in the uptake, motility, and cell-to-cell spread of Shigella organisms within the human host. These functions have been attributed to a VirA protease activity and a mechanism of microtubule destruction via tubulin degradation [Yoshida, S., et al. (2006) Science 314, 985-989]. We report functional and crystallographic data indicating a novel VirA structure that lacks these activities but highlights the homology to the EspG virulence factor of pathogenic Escherichia coli.     

Construction of a Low-Temperature Protein Expression System Using a Cold-Adapted Bacterium, Shewanella sp. Strain Ac10, as the Host

A recombinant protein expression system working at low temperatures is expected to be useful for the production of thermolabile proteins. We constructed a low-temperature expression system using an Antarctic cold-adapted bacterium, Shewanella sp. strain Ac10, as the host. We evaluated the promoters for proteins abundantly produced at 4°C in this bacterium to express foreign proteins. We used 27 promoters and a broad-host-range vector, pJRD215, to produce β-lactamase in Shewanella sp. strain Ac10. The maximum yield was obtained when the promoter for putative alkyl hydroperoxide reductase (AhpC) was used and the recombinant cells were grown to late stationary phase. The yield was 91 mg/liter of culture at 4°C and 139 mg/liter of culture at 18°C. We used this system to produce putative peptidases, PepF, LAP, and PepQ, and a putative glucosidase, BglA, from a psychrophilic bacterium, Desulfotalea psychrophila DSM12343. We obtained 48, 7.1, 28, and 5.4 mg/liter of culture of these proteins, respectively, in a soluble fraction. The amounts of PepF and PepQ produced by this system were greater than those produced by the Escherichia coli T7 promoter system.     

Isoelectric focusing of the inbred mouse antibody to bacterial alpha-amylase

In the IgG antibody response to bacterial alpha-amylase (B alpha A) assayed by the enzymatic procedure, C3H/He (C3) mice were high and C57BL/6 (B6) mice were low responders. High responsiveness was inherited as a dominant characteristic in (B6XC3)F1 hybrid mice. In these strains, the primary antibody response was analyzed for heterogeneity by isoelectric focusing (IEF). The IEF spectra were visualized with the use of the capacity of antibody to inhibit the amylase activity of antigen. Increases in the antigen dose and in the time interval between immunization and bleeding resulted in increases in antibody titers accompanied by strong staining of focused antibodies and by the expansion of the pH range where antibodies were focused. High responsiveness in C3 and F1 hybrid mice was also associated with the increase in intensity of stain and the rapid expansion of pH range of focused antibodies. Another strain difference was noted in the isoelectric point (pI) values of antibodies taken early in the primary response. B6 antisera contained those fractions of antibodies focusing over a more alkaline area than C3 antibodies. A similar strain difference in the pI values of antibodies occurred in the response to an irrelevant antigen, Taka-amylase A (TAA), suggesting that the hypervariable regions of antibody molecules play no major part in the strain difference observed. Antisera from F1 hybrid mice displayed bands covering the combined pH ranges of B6 and C3 spectrotypes.     

Cdk1 phosphorylation of the kinetochore protein Nsk1 prevents error-prone chromosome segregation

Cdk1 controls many aspects of mitotic chromosome behavior and spindle microtubule (MT) dynamics to ensure accurate chromosome segregation. In this paper, we characterize a new kinetochore substrate of fission yeast Cdk1, Nsk1, which promotes proper kinetochore-MT (k-MT) interactions and chromosome movements in a phosphoregulated manner. Cdk1 phosphorylation of Nsk1 antagonizes Nsk1 kinetochore and spindle localization during early mitosis. A nonphosphorylatable Nsk1 mutant binds prematurely to kinetochores and spindle, cementing improper k-MT attachments and leading to high rates of lagging chromosomes that missegregate. Accordingly, cells lacking nsk1 exhibit synthetic growth defects with mutations that disturb MT dynamics and/or kinetochore structure, and lack of proper phosphoregulation leads to even more severe defects. Intriguingly, Nsk1 is stabilized by binding directly to the dynein light chain Dlc1 independently of the dynein motor, and Nsk1-Dlc1 forms chainlike structures in vitro. Our findings establish new roles for Cdk1 and the Nsk1-Dlc1 complex in regulating the k-MT interface and chromosome segregation.