Molecular characterization of the essential response regulator protein YycF in Bacillus subtilis.
The response regulator YycF is essential for cell growth in gram-positive bacteria including Bacillus subtilis, Staphylococcus aureus and Streptococcus pneumoniae. To study the function of YycF in the essential process, we characterized a YycF (H215P) mutation that caused temperature-sensitive growth in B. subtilis. The response regulators YycF and YycF (H215P) were analyzed using circular dichroism spectroscopy, whose T(m) values were 56.0 and 45.9 degrees C, respectively, suggesting that YycF (H215P) significantly affects the protein structure with an increase in temperature. Furthermore, using the gel mobility shift assay and DNase I footprinting, we investigated the effect of YycF (H215P) on binding to the YycF box of ftsAZ operon of B. subtilis. The replacement of the histidine 215 with proline resulted in a decrease of the DNA-binding ability of YycF in vitro. In vivo, using Escherichia coli two-hybrid and homodimerization assays, we clarified that His 215 of YycF plays a crucial role in the homodimerization of the protein. Thus the essential genes involved in growth of B. subtilis appear to be regulated by the homodimer of YycF. These results suggest that the YycF dimerization is an excellent target for the discovery of novel antibiotics.
Watanabe
Takafumi
T
Department of Bioscience and Biotechnology, Graduate School of Agriculture, Kinki University, Nara, Japan.
Hashimoto
Yoshiki
Y
Umemoto
Yoshihito
Y
Tatebe
Daisuke
D
Furuta
Eiji
E
Fukamizo
Tamo
T
Yamamoto
Kaneyoshi
K
Utsumi
Ryutaro
R
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000235:genetics / Q000254:growth & development / Q000502:physiology*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002942:Circular Dichroism; D004269:DNA, Bacterial / Q000378:metabolism; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D020043:Genes, Essential; D008969:Molecular Sequence Data; D009154:Mutation; D009876:Operon; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013696:Temperature; D020798:Two-Hybrid System Techniques
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C065103:FtsA protein, Bacteria; C489310:FtsA protein, E coli; C119939:YycF protein, Bacteria