Regulation of Swarming Motility andflhDC_SmExpression by RssAB Signaling inSerratia marcescens

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 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 ...

Serratia marcescens ShlA Pore-Forming Toxin Is Responsible for Early Induction of Autophagy in Host Cells and Is Transcriptionally Regulated by RcsB

“…5C) strongly suggest that FliA is able to activate ShlA expression by direct induction of shlBA transcription. FliA-mediated regulation of ShlA expression also helps to explain the results obtained by Soo et al in an S. marcescens CH-1 pigmented strain (43) showing that the RssAB system could partially repress shlBA in an FlhDC-dependent manner. …”

The RssB/RssA two-component system regulates biosynthesis of the tripyrrole antibiotic, prodigiosin, in Serratia marcescens

“…The class I master operon, flhDC, encodes the FlhD 2 C 2 complex which is an activator of s 70 -dependent transcription from class II promoters (Chilcott and Hughes, 2000). We have previously shown that synthesis of flagella and flhDC expression were significantly increased in the rssA mutant (S. marcescens CH-1 DrssA) (Soo et al, 2008). The RssA sensor kinase phosphorylates the RssB response regulator (Wei et al, 2005a).…”

“…The RssA sensor kinase phosphorylates the RssB response regulator (Wei et al, 2005a). Phosphorylated RssB (RssB-P) binds to the flhDC promoter (Soo et al, 2008). Missense mutation on histidine 248 of RssA and aspartate 51 of RssB led to abnormal swarming phenotype, unphosphotransfer from RssA to RssB, and unbinding to the flhDC and rssB promoters by RssB (Wei et al, 2005a;Soo et al, 2008).…”

RssAB-FlhDC-ShlBA as a Major Pathogenesis Pathway in Serratia marcescens

“…S. marcescens swarms on Luria-Bertani (LB) agar surfaces at 30°C, but not at 37°C (36). Our previous studies showed that activation of a bacterial two-component system, RssAB, comprising a sensor kinase, RssA, and a response regulator, RssB, inhibited swarming and reduced hemolysin production in S. marcescens CH-1 at 37°C (36,57,63). In contrast, loss of rssBA resulted in a precocious swarming phenotype and a significant increase in hemolytic activity (36).…”

“…Recently, we showed that activated RssAB signaling directly represses expression of flhDC and swarming in S. marcescens CH-1 at 37°C (57). FlhDC is known to be a major checkpoint of swarmer cell differentiation in S. marcescens MG1 (formerly Serratia liquefaciens MG1) (13) and is also involved in early stages of biofilm formation in Escherichia coli (48).…”