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 twocomponent 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 (RssBϳP) binding site by an electrophoretic mobility shift assay showed direct interaction of RssBϳP, 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 RssBϳP 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 RssBϳP 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.Swarming is a bacterial population surface translocation behavior demonstrated in a wide range of diverse bacterial genera and species (2,12,14). In Serratia spp., swarming requires close interactions between the environment and the bacterial cells, as well as among the cells, in order to develop a high degree of complex cell coordination within the swarming colony (2,9,13,26,33,35). Previous studies on the regulation of swarming showed that bacterial flagellar, quorum-sensing, and two-component systems are important for swarming (3,7,33). Among these, flagellar motility, which is one of the essential factors for bacterial swarming, is controlled by the flagellar system, comprising large and complex regulons (4, 9). Studies with the flagellar systems of Escherichia coli and Salmonella enterica serovar Typhimurium have identified around 50 genes organized into three hierarchical transcriptional classes. At the top of the hierarchical cascade is the class I master operon flhDC (4). The FlhD 2 C 2 complex is a transcriptional activator of 70 -dependent transcription from class II promoters (4). Thus, activation of the whole set of flagellar motility genes depends mainly on the expression of flhDC.Serratia marcescens cells swarm at 30°C but not at 37°C (15).In a previous study utilizing a mini-Tn5 mutagenesis approach, we had ...