The kinase Chk2 and tumor suppressor p53 participate in an ill defined regulatory interaction in mammalian cells. The abundance of Chk2 mRNA and protein has now been shown to be decreased by the induction of p53 in Saos2 cells. Ionizing radiation also triggered the phosphorylation and subsequent down-regulation of Chk2 in human colorectal HCT116 (p53 ؉/؉ ) cancer cells; irradiation of its isogenic mutant HCT116 (p53 ؊/؊ ) cells, which lack functional p53, induced Chk2 phosphorylation but not its down-regulation. In addition, HCT116 (p53 ؉/؉ ) cells constitutively expressing a dominant negative p53 (V143A) failed to suppress Chk2 expression after irradiation. Reporter gene assays in HCT116 (p53 ؉/؉ ) cells revealed that wild-type p53 repressed, whereas a dominant negative p53 mutant increased, the activity of the human Chk2 gene promoter. Mutational analysis showed that a CCAAT box located between nucleotides ؊152 and ؊138 of the promoter was responsible for its negative regulation by p53. Electrophoretic mobility shift assays demonstrated that the transcription factor NF-Y binds to this CCAAT sequence. A dominant negative mutant of NF-YA abolished the effect of p53 on Chk2 promoter activity. These results suggest that p53 negatively regulates Chk2 gene transcription through modulation of NF-Y function and that this regulation may be important for reentry of cells into the cell cycle after DNA damage is repaired.

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The type III export apparatus of the Salmonella flagellum consists of six transmembrane proteins (FlhA, FlhB, FliO, FliP, FliQ, and FliR) and three soluble proteins (FliH, FliI, and FliJ). Deletion of the fliO gene creates a mutant strain that is poorly motile; however, suppressor mutations in the fliP gene can partially rescue motility. To further understand the mechanism of suppression of a fliO deletion mutation, we isolated new suppressor mutant strains with partially rescued motility. Whole-genome sequence analysis of these strains found a missense mutation that localized to the clpP gene [clpP (

The Type III flagellar protein export apparatus of bacteria consists of five or six membrane proteins, notably FlhA, which controls the export of other proteins and is homologous to the large family of FHIPEP export proteins. FHIPEP proteins contain a highly-conserved cytoplasmic domain. We mutagenized the cloned Salmonella flhA gene for the 692 amino acid FlhA, changing a single, conserved amino acid in the 68-amino acid FHIPEP region. Fifty-two mutations at 30 positions mostly led to loss of motility and total disappearance of microscopically visible flagella, also Western blot protein/protein hybridization showed no detectable export of hook protein and flagellin. There were two exceptions: a D199A mutant strain, which produced short-stubby flagella; and a V151L mutant strain, which did not produce flagella and excreted mainly un-polymerized hook protein. The V151L mutant strain also exported a reduced amount of hook-cap protein FlgD, but when grown with exogenous FlgD it produced polyhooks and polyhook-filaments. A suppressor mutant in the cytoplasmic domain of the export apparatus membrane protein FlhB rescued export of hook-length control protein FliK and facilitated growth of full-length flagella. These results suggested that the FHIPEP region is part of the gate regulating substrate entry into the export apparatus pore.

Campylobacter jejuni cells have bipolar flagella. Both flagella have similar lengths of about one helical turn, or 3.53±0.52 µm. The flagellar filament is composed of two homologous flagellins: FlaA and FlaB. Mutant strains that express either FlaA or FlaB alone produce filaments that are shorter than those of the wild-type. It is reported that the flaG gene could affect filament length in some species of bacteria, but its function remains unknown. We introduced a flaG-deletion mutation into the C. jejuni wild-type strain and flaA- or flaB-deletion mutant strains, and observed their flagella by microscopy. The ΔflaG mutant cells produced long filaments of two helical turns in the wild-type background. The ΔflaAG double mutant cells produced very short FlaB filaments. On the other hand, ΔflaBG double mutant cells produced long FlaA filaments and their morphology was not helical but straight. Furthermore, FlaG was secreted, and a pulldown assay showed that sigma factor 28 was co-precipitated with purified polyhistidine-tagged FlaG. We conclude that FlaG controls flagella length by negatively regulating FlaA filament assembly and discuss the role of FlaA and FlaB flagellins in C. jejuni flagella formation.