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Regulation of Swarming Motility and flhDC_Sm Expression by RssAB Signaling in Serratia marcescens

Po-Chi Soo,^4, Yu-Tze Horng,^3, Jun-Rong Wei,³ Jwu-Ching Shu,¹ Chia-Chen Lu,² and Hsin-Chih Lai^1*

Department of Medical Biotechnology and Laboratory Science,¹ Department of Physiology, Chang Gung University, 259 Wen-Hwa 1st Road, Kwei-Shan, Tao-Yuan, 333,² Department of Clinical Laboratory Sciences and Medical Biotechnology, National Taiwan University College of Medicine, Taipei,³ Graduate School of Biotechnology and Bioengineering, Yuan Ze University, 135 Yuan-Tung Road, Chung-Li 320, Taiwan, Republic of China⁴

Received 16 October 2007/ Accepted 8 January 2008

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 a temperature upshift from 30 to 37°C reduced the expression levels of flhDC_Sm and hag_Sm in S. marcescens CH-1. Mutation in rssA or rssB, cognate genes that comprise a 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 flagella are significantly increased in the rssA mutant strain at 37°C. Primer extension analysis for determination of the transcriptional start site(s) of flhDC_Sm revealed two transcriptional start sites, P1 and P2, in S. marcescens CH-1. Characterization of the phosphorylated RssB (RssBP) binding site by an electrophoretic mobility shift assay showed direct interaction of RssBP, but not unphosphorylated RssB [RssB(D51E)], with the P2 promoter region. A DNase I footprinting assay using a capillary electrophoresis approach further determined that the RssBP binding site is located between base pair positions –341 and –364 from the translation start codon ATG in the flhDC_Sm promoter region. The binding site overlaps with the P2 "–35" promoter region. A modified chromatin immunoprecipitation 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 flhDC_Sm promoter activity at 37°C. This inhibitory effect was comparatively alleviated at 30°C. This finding might explain, at least in part, the phenomenon of inhibition of S. marcescens swarming at 37°C.

Published ahead of print on 25 January 2008.

P.-C.S. and Y.-T.H. contributed equally to this work.