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Journal of Bacteriology, April 2008, p. 2496-2504, Vol. 190, No. 7
0021-9193/08/$08.00+0     doi:10.1128/JB.01670-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Regulation of Swarming Motility and flhDCSm Expression by RssAB Signaling in Serratia marcescens{triangledown}

Po-Chi Soo,4,{dagger} Yu-Tze Horng,3,{dagger} Jun-Rong Wei,3 Jwu-Ching Shu,1 Chia-Chen Lu,2 and Hsin-Chih Lai1*

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 flhDCSm and hagSm 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 flhDCSm 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 flhDCSm revealed two transcriptional start sites, P1 and P2, in S. marcescens CH-1. Characterization of the phosphorylated RssB (RssB~P) binding site by an electrophoretic mobility shift assay showed direct interaction of RssB~P, 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 RssB~P binding site is located between base pair positions –341 and –364 from the translation start codon ATG in the flhDCSm promoter region. The binding site overlaps with the P2 "–35" promoter region. A modified chromatin immunoprecipitation assay was subsequently performed to confirm that RssB~P binds to the flhDCSm promoter region in vivo. In conclusion, our results indicated that activated RssA-RssB signaling directly inhibits flhDCSm 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.

* Corresponding author. Present address: Department of Medical Biotechnology and Laboratory Science, Chang-Gung University, 259 Wen-Hua First Road, Kweishan, Taoyuan, 333 Taiwan, Republic of China. Phone: 886 3 2118800, ext. 3585. Fax: 886 3 2118700. E-mail: hclai{at}mail.cgu.edu.tw

{triangledown} Published ahead of print on 25 January 2008.

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

Journal of Bacteriology, April 2008, p. 2496-2504, Vol. 190, No. 7
0021-9193/08/$08.00+0     doi:10.1128/JB.01670-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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