Biochemical characterization of RssA-RssB, a two-component signal transduction system regulating swarming behavior in Serratia marcescens.
Our previous study had identified a pair of potential two-component signal transduction proteins, RssA-RssB, involved in the regulation of Serratia marcescens swarming. When mutated, both rssA and rssB mutants showed precocious swarming phenotypes on LB swarming agar, whereby swarming not only occurred at 37 degrees C but also initiated on a surface of higher agar concentration and more rapidly than did the parent strain at 30 degrees C. In this study, we further show that the predicted sensor kinase RssA and the response regulator RssB bear characteristics of components of the phosphorelay signaling system. In vitro phosphorylation and site-directed mutagenesis assays showed that phosphorylated RssA transfers the phosphate group to RssB and that histidine 248 and aspartate 51 are essential amino acid residues involved in the phosphotransfer reactions in RssA and RssB, respectively. Accordingly, while wild-type rssA could, the mutated rssA(H248A) in trans could not complement the precocious swarming phenotype of the rssA mutant. Although RssA-RssB regulates expressions of shlA and ygfF of S. marcescens (ygfF(Sm)), in vitro DNA-binding assays showed that the phosphorylated RssB did not bind directly to the promoter regions of these two genes but bound to its own rssB promoter. Subsequent assays located the RssB binding site within a 63-bp rssB promoter DNA region and confirmed a direct negative autoregulation of the RssA-RssB signaling pathway. These results suggest that when activated, RssA-RssB acts as a negative regulator for controlling the initiation of S. marcescens swarming.
Wei
Jun-Rong
JR
Department of Clinical Laboratory Sciences and Medical Biotechnology, National Taiwan University College of Medicine, No. 1. Chang-Der Street, Taipei 100, Taiwan, Republic of China.
Tsai
Yu-Huan
YH
Soo
Po-Chi
PC
Horng
Yu-Tze
YT
Hsieh
Shang-Chen
SC
Ho
Shen-Wu
SW
Lai
Hsin-Chih
HC
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009068:Movement; D016296:Mutagenesis; D010766:Phosphorylation; D011401:Promoter Regions, Genetic / Q000502:physiology; D012706:Serratia marcescens / Q000235:genetics* / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D014157:Transcription Factors