Regulation of swarming motility and flhDC_Sm expression by RssAB signaling in Serratia marcescens

Department of Medical Biotechnology and Laboratory Science, and Department of Physiology, Chang Gung University, 259 Wen-Hwa 1st Road, Kwei-Shan, Tao-Yuan, 333, Taiwan, R.O.C.; Department of Clinical Laboratory Sciences and Medical Biotechnology, National Taiwan University College of Medicine, Taipei, Taiwan, R.O.C.; Graduate School of Biotechnology and Bioengineering, Yuan Ze University, 135 Yuan-Tung Road, Chung-Li 320, Taiwan, R.O.C.

^* To whom correspondence should be addressed. Email: hclai{at}mail.cgu.edu.tw.

	Abstract

Serratia marcescens cells swarm at 30°C but not at 37°C, and the underlying mechanism is not characterized. Our previous studies had shown that temperature upshift from 30 to 37°C reduced expression level of flhDC_Sm and hag_Sm in S. marcescens CH-1. Mutation in rssA or rssB, a pair of two-component system, also resulted in precocious swarming phenotypes at 37°C. To further characterize the underlying mechanism, in the present study, we report that expression of flhDC_Sm and synthesis of flagellum is significantly increased in the rssA mutant strain at 37°C. Primer extension analysis for determination of transcriptional start site(s) of flhDC_Sm revealed two transcriptional start sites, P1 and P2 in S. marcescens CH-1. Characterization of phosphorylated RssB (RssBP) binding site by electrophoretic mobility shift assay showed direct interaction of RssBP, but not unphosphorylated RssB (RssB_D51E), with the P2 promoter region. DNase I footprinting assay using a capillary electrophoresis approach further determined the RssBP binding site to be located between base pair positions -341 to -364 from the translation start codon ATG in the flhDC_Sm promoter region. The binding site overlaps with P2 "-35" promoter region. Modified chromatin immunoprecipitation (ChIP) assay was subsequently performed to confirm that RssBP binds to the flhDC_Sm promoter region in vivo. In conclusion, our results indicated that activated RssA-RssB signaling directly inhibits the flhDC_Sm promoter activity at 37°C. Such inhibitory effect was comparatively alleviated at 30°C. This might explain, at least in part, the phenomenon of inhibition of S. marcescens swarming at 37 °C.