Regulation of Swarming Motility and flhDCSm Expression by RssABSignaling 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 Lai1*Department of Medical Biotechnology and Laboratory Science1 and Department of Physiology,2 Chang Gung University,
259 Wen-Hwa 1st Road, Kwei-Shan, Tao-Yuan, 333, Department of Clinical Laboratory Sciences andMedical Biotechnology, National Taiwan University College of Medicine, Taipei,3 and Graduate School of
Biotechnology and Bioengineering, Yuan Ze University, 135 Yuan-Tung Road,Chung-Li 320,4 Taiwan, Republic of China
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 offlhDCSm 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 theunderlying mechanism, in the present study, we report that expression of flhDCSm and synthesis of flagella aresignificantly increased in the rssA mutant strain at 37°C. Primer extension analysis for determination of thetranscriptional start site(s) of flhDCSm revealed two transcriptional start sites, P1 and P2, in S. marcescensCH-1. Characterization of the phosphorylated RssB (RssB�P) binding site by an electrophoretic mobility shiftassay showed direct interaction of RssB�P, but not unphosphorylated RssB [RssB(D51E)], with the P2promoter region. A DNase I footprinting assay using a capillary electrophoresis approach further determinedthat the RssB�P binding site is located between base pair positions �341 and �364 from the translation startcodon ATG in the flhDCSm promoter region. The binding site overlaps with the P2 “�35” promoter region. Amodified chromatin immunoprecipitation assay was subsequently performed to confirm that RssB�P binds tothe flhDCSm promoter region in vivo. In conclusion, our results indicated that activated RssA-RssB signalingdirectly 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.
Swarming is a bacterial population surface translocation be-havior demonstrated in a wide range of diverse bacterial gen-era and species (2, 12, 14). In Serratia spp., swarming requiresclose interactions between the environment and the bacterialcells, as well as among the cells, in order to develop a highdegree of complex cell coordination within the swarming col-ony (2, 9, 13, 26, 33, 35). Previous studies on the regulation ofswarming showed that bacterial flagellar, quorum-sensing, andtwo-component systems are important for swarming (3, 7, 33).Among these, flagellar motility, which is one of the essentialfactors for bacterial swarming, is controlled by the flagellarsystem, comprising large and complex regulons (4, 9). Studieswith the flagellar systems of Escherichia coli and Salmonellaenterica serovar Typhimurium have identified around 50 genesorganized into three hierarchical transcriptional classes. At thetop of the hierarchical cascade is the class I master operonflhDC (4). The FlhD2C2 complex is a transcriptional activatorof �70-dependent transcription from class II promoters (4).Thus, activation of the whole set of flagellar motility genesdepends 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 discovered a group of S. marcescens mutant strains thatdemonstrated precocious swarming behavior not only at 30°Cbut also at 37°C (15). A pair of bacterial two-component signaltransduction proteins, RssA and RssB, were subsequentlyidentified as negative regulators for S. marcescens swarming at37°C (15, 36). Although RssA-RssB His-Asp phosphorelay andsignaling had already been proven in vitro (36), the underlyingmechanism of the RssA-RssB signaling effect on the inhibitionof swarming at 37°C remained undetermined. Previously wehad also shown that expression of flhDCSm and hagSm (theflagellin structural gene in S. marcescens) was reduced whenthe incubation temperature was increased from 30 to 37°C in S.marcescens (18). In this study, we further report that RssA-RssB negatively regulates flhDCSm expression and flagellumproduction through its signaling status. This regulation isachieved through direct binding of RssB�P with the flhDCSm
P2 promoter region, leading to a reduction in the level offlhDCSm mRNA transcription. The RssA-RssB inhibitory ef-fect is much more significant at 37°C than at 30°C, which mightat least in part explain the underlying mechanism of inhibitionof S. marcescens swarming at 37°C.
MATERIALS AND METHODS
Bacterial strains, mutants, and culture conditions. S. marcescens CH-1 andthe rssA mutant strain S. marcescens CH-1�A, in which rssA is interrupted by aHindIII-digested Smr gene cassette, were from a previous study (36). Escherichiacoli DH5� (Invitrogen) was used as a host strain for the maintenance of recom-binant DNA plasmids. E. coli BL21(DE3)pLysS (Novagen, Germany) was usedfor oversynthesis of recombinant proteins. All bacteria used in this study were
* Corresponding author. Present address: Department of MedicalBiotechnology and Laboratory Science, Chang-Gung University, 259Wen-Hua First Road, Kweishan, Taoyuan, 333 Taiwan, Republic ofChina. Phone: 886 3 2118800, ext. 3585. Fax: 886 3 2118700. E-mail:[email protected].
† P.-C.S. and Y.-T.H. contributed equally to this work.� Published ahead of print on 25 January 2008.
grown in Luria-Bertani (LB) medium at 37°C (36) supplemented with adequateantibiotics when necessary, unless other conditions are specifically mentioned inthe text.
Enzymes, chemicals, and primers. DNA restriction and modification enzymeswere purchased from Roche (Germany). Pfu polymerase and PCR-related prod-ucts were from Stratagene and Perkin-Elmer. Other laboratory-grade chemicalswere purchased from Sigma and Merck (Germany). The primers used in thisstudy and summarized in Table 1 were purchased from MD Bio (Taiwan).
Swarming motility assay. A swarming assay was performed on LB mediumsolidified with 0.8% Eiken agar (Eiken, Japan) by inoculating 3-�l portions of anovernight LB broth culture onto the centers of agar plate surfaces and incubatingat 37°C.
Detection of luxCDABE reporter luciferase activity. The Autolumat LB 953luminometer (EG & G, Germany) with the program “replicates” was used forbioluminescence measurement. All procedures followed the protocols suppliedby the manufacturer.
Gel mobility shift assay. Promoter DNA fragments for gel mobility shift assayswere amplified by PCR using primers that were 5� end labeled with digoxigenin(DIG) (MWG Biotech, Germany). Reaction mixtures for the binding assaycomprised 2 �M RssB protein with acetylphosphate treatment and 0.5 ng DIG-labeled promoter DNA fragments. For the serial dilution experiments, phosphor-ylated or unphosphorylated RssB proteins were serially diluted in binding reac-tion buffer (14 mM Tris-HCl [pH 7.4], 6.9 mM MgCl2, 69 mM KCl, and 10 mMEDTA). The binding reaction was performed in binding reaction buffer supple-
mented with 30 �g/ml poly(dI-dC) and 1 �g/�l bovine serum albumin. Thereaction mixtures were incubated for 20 min at room temperature before beingloaded onto 7% nondenaturing polyacrylamide gels containing 0.5� Tris-borate-EDTA buffer. Electrophoresis was performed at 100 V for 1 h. The DNA-proteincomplexes were then electroblotted onto a positively charged Hybond-N nylonmembrane (Amersham, United Kingdom) and detected by alkaline phosphatase-conjugated anti-DIG antibodies (Roche, Germany). CSPD was added as thesubstrate as described by the manufacturer (Roche, Germany). Membranes wereexposed to X-ray film at room temperature for 2 to 30 min.
RT-PCR assay. Total bacterial RNA was extracted using a Trizol kit (Invitro-gen). The relative amounts of transcripts from the flhDCSm gene were evaluatedby the reverse transcription-PCR (RT-PCR) assay. RNA was isolated fromstrains CH-1 and CH-1�A grown aerobically on 0.8% LB agar plates for 2 or 3 h(early-logarithmic phase) and then reverse transcribed into cDNA with a Super-Script III first-strand synthesis system kit (Invitrogen). Equal amounts of totalRNA (5 �g) were used to generate cDNA with random hexamer primers ac-cording to the manufacturer’s protocol. The products were amplified by PCRwith the primer pair flhD-SF–flhD-SR (Table 1). The cycle conditions for thePCR were as follows: 1 min at 96°C, 1 min at 60°C, and 50 s at 72°C for 15 or 25cycles. The number of cycles was decided according to the comparison per-formed in the linear range of amplification. The RT-PCR products were ana-lyzed by electrophoresis on 2% agarose gels, and then the amount of transcriptwas quantified by densitometry using the Scion Imager. 16S rRNA was used asthe internal control to confirm that equal amounts of total RNA were used ineach reaction (11).
Identification of the transcriptional start site(s) in flhDCSm and quantificationof promoter activity. The transcriptional start sites were identified by a primerextension assay using a 6-carboxyfluorescein (FAM)-labeled primer and theGeneScan analysis system (Applied Biosystems) described by Lloyd et al. (19)with some modifications. Briefly, purified RNA was reverse transcribed intocDNA with ImProm-II reverse transcriptase (Promega) by use of primerFlhD-PE to form FAM-labeled cDNA (Table 1). Samples containing 1 �g ofRNA and 1 �M primers were heated at 70°C for 5 min before being placed onice for 1 min. After addition of 0.5 mM deoxynucleoside triphosphates, 25 mMMgCl2, 1 �l reverse transcriptase, and 1� RT buffer (supplied by the manufac-turer) in a 20-�l final volume adjusted with diethylpyrocarbonate water, sampleswere incubated at 55°C for another 60 min. The reaction was stopped by additionof 100 �l of H2O and 100 �l of phenol-chloroform-isoamyl alcohol (25:24:1).After centrifugation at 12,000 � g for 4 min, the supernatant containing cDNAwas precipitated with 0.1 volume of solution III (60% sodium acetate, 11.5%glacial acetic acid [pH 5.2]) and 2.5 volumes of absolute ethanol. After incuba-tion at 70°C for more than 30 min, cDNA was centrifuged at 12,000 � g for 15min. The pellet was washed with 70% ethanol and dissolved in 4 �l of deionizedH2O, followed by addition of 5 �l of deionized formamide containing 0.5 �l ofthe ROX-500 molecular size standard (Applied Biosystems). Samples were de-natured at 95°C for 5 min and then chilled quickly on ice. Electrophoresis wasperformed on 6% polyacrylamide–8 M urea gels by using the ABI Prism 3100capillary DNA genetic analyzer equipped with Avant Genetic Analyzer DataCollection, version 2.0 (both from Applied Biosystems). The data were analyzedusing GeneMapper software, version 3.5 (Applied Biosystems). The cDNAs ofthe flhDCSm transcripts were reverse transcribed with a FAM-labeled primer.The length of the FAM-labeled cDNA primer extension product was then ana-lyzed by using an ABI 3100 automated sequencer and GeneScan software (bothfrom Applied Biosystems).
DNase I footprinting. The DNase I footprinting protocol was modified fromthat described by Yindeeyoungyeon and Schell (39). Briefly, the 500-bp flhDCSm
promoter region was PCR amplified from genomic DNA. The primer pairs usedfor PCR were Fb-F and Fb-R. Depending on which strand was analyzed, oneprimer was labeled with FAM (MD Bio, Taiwan) at the 5� end and the other wasnot. The F�-labeled DNA fragment was incubated with 28 �M phosphorylatedRssB in a 50-�l solution containing 11 mM Tris-HCl (pH 7.4), 0.1 mM EDTA,5.5 mM MgCl2, 20 �g/ml of poly(dI-dC) (Pharmacia, Sweden), and 0.2 mg/ml ofbovine serum albumin. After 20 min of incubation at room temperature, 50 �l ofDNase I (1 � 103 U/�l, freshly prepared by diluting the stock [Promega] in Dbuffer, consisting of 10 mM Tris-HCl [pH 7.5], 10 mM MgCl2, and 5 mM CaCl2)was added, and the mixture was further incubated at 26°C for 3 or 5 min. Thedigestion was stopped by adding 100 �l of stop solution containing 0.2 M NaCl,40 mM EDTA, 1% sodium dodecyl sulfate, and 125 �g/ml of tRNA. Afterincubation at 37°C for 20 or 30 min, samples were extracted with a phenol-chloroform-isoamyl alcohol solution (25:24:1), precipitated with absolute etha-nol, washed with 70% ethanol, and dissolved in 10 �l deionized formamide. Afteraddition of 0.5 �l of the ROX-500 molecular size standard (Applied Biosystems),samples were denatured at 95°C for 5 min and then quickly chilled on ice.
TABLE 1. Bacterial strains, plasmids, and primers usedin this study
Electrophoresis using the ABI Prism 3100 capillary DNA genetic analyzer anddata analysis with GeneMapper software, version 3.5 (both from Applied Bio-systems), were performed according to the instructions supplied by the manu-facturer.
Modified chromatin immunoprecipitation (ChIP) assay. The immunoprecipi-tation assay described by Shin and Groisman (23) was used with modifications.Briefly, cultures of S. marcescens were treated with 1 M sodium phosphate (finalconcentration, 10 mM) and 37% formaldehyde (final concentration, 1%). After15 min, cross-linking was quenched by the addition of glycine (final concentra-tion, 125 mM). Cultures of 10 ml were collected by centrifugation and washedtwice with 10 ml of phosphate-buffered saline. Cells were lysed in 0.6 ml of lysissolution (20 mM Tris [pH 8.0], 150 mM NaCl, 1 mM EDTA [pH 8.0], 1 mMphenylmethylsulfonyl fluoride, 1% Triton X-100, 10 mg/ml of lysozyme), and 0.6ml of 2� IP solution (100 mM Tris [pH 8.0], 300 mM NaCl, 2% Triton X-100)was then added. Cell extracts were then sonicated to produce DNA fragmentswith an average size of 500 to 1,000 bp. The extract (200 �l) was removed fortotal-DNA preparation. For pull-down of glutathione S-transferase (GST)-RssB-cross-linked DNA, a portion of the extracts (600 �l) was incubated with 100 �lglutathione-Sepharose 4B beads (Amersham, United Kingdom) at 4°C for 1 h.GST-RssB-cross-linked DNA was then pulled down with the glutathione-Sepha-rose beads by centrifugation. The beads were washed twice with 1� IP solutionand then twice with a LiCl-detergent solution (10 mM Tris [pH 8.0], 250 mMLiCl, 1 mM EDTA, 0.5% Nonidet P-40, and 0.5% sodium deoxycholate). Thebeads were then resuspended in a 200-�l solution of 50 mM Tris-HCl (pH 8.0),1 mM EDTA, and 0.67% sodium dodecyl sulfate. This was followed by incuba-tion at 65°C for 16 h to reverse the cross-links. DNA samples were purified usingphenol extraction, precipitated with absolute ethanol, and resuspended in TEbuffer (10 mM Tris, 1 mM EDTA [pH 8.0]).
RESULTS
RssA negatively regulates the flagellar system on LB swarm-ing plates. Previously we had shown that inactivation in S.marcescens CH-1 of rssA or rssB, a sensor kinase gene and acognate response regulator gene, respectively, making up abacterial two-component system, resulted in precociousswarming behavior on a 0.8% LB swarming agar plate at 37°C(Fig. 1A) (15). To further characterize the underlying mecha-nism of this precocious swarming behavior, the amounts offlagellin synthesized by S. marcescens strain CH-1 and the rssAmutant strain S. marcescens CH-1�A seeded on LB swarmingplates at 37°C were first quantified by Western blot analysisafter 3 h of bacterial growth (early-logarithmic phase). Theamounts of flagellin synthesized by CH-1 and CH-1�A differedsignificantly (Fig. 1A); relative intensities were determined tobe 20 � 5 and 78 � 10 arbitrary units, respectively (a 3.9-foldincrease for CH-1�A) (Fig. 1A). These results suggest thatCH-1�A produces many more flagella than CH-1 at 37°C,indicating derepression of the flagellar synthesis system as apotential underlying mechanism for the precocious swarmingphenotype observed in S. marcescens CH-1�A.
To compare the transcriptional levels of flhDCSm in CH-1and CH-1�A at 37°C, the recombinant plasmid pBG300(PflhDCSm::luxCDABE) containing luxCDABE reporter genestranscriptionally fused with the potential flhDCSm promoterregion [a 655-bp DNA region upstream of the A(�1)TG trans-lational start site (see Fig. 5A)] was constructed for real-timemonitoring of flhDCSm promoter activity. The flhDCSm pro-moter activity in CH-1�A was ca. 3.6-fold elevated over that inCH-1 on LB plates seeded and incubated for 2 h at 37°C (forbacterial growth to the early-logarithmic phase) (Fig. 1B). Forfurther confirmation of the difference in flhDCSm expressionlevels, RT-PCR using total cellular RNA as the template wasperformed for quantification. As bacterial cells were grown tothe early-logarithmic phase (2 h) on LB plates at 37°C, CH-
1�A cells showed a 53% increase in the flhDCSm expressionlevel over that of CH-1 cells (72 � 18 versus 47 � 16 relativeintensity units) (Fig. 1C). These findings suggested that theinhibition of swarming by RssA at 37°C in S. marcescens CH-1was correlated with the repression of flhDCSm expression andflagellum production by RssA.
FIG. 1. Swarming behavior, flagellum production, and transcrip-tional activity of flhDCSm in S. marcescens CH-1 and CH-1�A on LBswarming plates at 37°C. (A) (i) Swarming assay and determination ofthe amount of flagellin 3 h after inoculation onto a swarming plate byWestern blotting of whole-cell lysates with an antiflagellin monoclonalantibody. (ii) Relative intensities of flagellin synthesized were mea-sured using Scion Imaging software. (B) Transcriptional activities offlhDCSm (pBG300) in CH-1�A (f) and CH-1 (Œ) measured by aluxCDABE luciferase reporter activity assay after seeding onto aswarming plate. (C) Determination by RT-PCR of flhDCSm mRNAand 16S rRNA levels in S. marcescens cells incubated for 2 h on aseeding plate. Hatched bar, CH-1; open bar, CH-1�A; �, P 0.01.Results are means � standard deviations from three independentexperiments.
Characterization of P1 and P2 promoters in flhDCSm. Thecomplexity of the regulation of flhDC expression has been wellstudied in many bacterial species, including E. coli and Salmo-nella serovar Typhimurium (4, 28). Compared to the charac-terized flhDC upstream promoter region in Salmonella serovarTyphimurium (28, 38), potential multiple transcriptional startsites were identified in S. marcescens CH-1 flhDCSm. To iden-tify the transcriptional start site(s) of flhDCSm in S. marcescensat 37°C, primer extension assays were performed. Total RNAwas extracted from S. marcescens seeded onto LB swarmingplates and incubated for 2 h after inoculation at 37°C. TheflhDCSm transcripts were first reverse transcribed into cDNAby primer extension using a FAM-labeled DNA primer de-signed from 9 bp downstream of the ATG translational initi-ation site. This was followed by length analysis of synthesizedsingle-stranded cDNAs by using the ABI 3100 automatedDNA sequencer. Two major flhDCSm transcripts were identi-fied in S. marcescens CH-1 and S. marcescens CH-1�A (Fig. 2).The start sites of these two transcripts were mapped 68 bpand 306 bp upstream of the flhDCSm ATG initiation codon,and the transcripts were designated P1 and P2, respectively(Fig. 2 and 3).
To further confirm the potential P1 and P2 flhDCSm pro-moter activities, the P1 (bp 60 to 210) and P2 (bp 299 to423) promoter regions were fused with the luxCDABE genesin the pACYC184 vector to form recombinant plasmidspBG301 and pBG302, respectively (Fig. 4A). These two plas-
mids were then transformed into S. marcescens CH-1 and CH-1�A for measurement of P1 and P2 promoter activities. BothP1 and P2 displayed significant promoter activities in S. marc-escens CH-1 and CH-1�A compared with the pBG vectorcontrol (promoterless luxCDABE) (data not shown). However,the activities of the two promoters were lower in S. marcescensCH-1 than in CH-1�A, although a more significant reductionwas observed for P2 than for P1 (Fig. 4B and C). To sum up,two flhDCSm promoters were identified in S. marcescens CH-1,and the transcriptional level of the P2 promoter was moresignificantly inhibited by RssA.
Phosphorylated RssB interacts with the flhDCSm upstreampromoter region. RssA and RssB form a cognate two-com-ponent sensing system in S. marcescens CH-1. Once acti-vated, phosphorelay signaling from RssA is transferred toRssB (15, 36). The possibility that RssB in its phosphory-lated form (RssB�P) binds directly to the flhDCSm pro-moter region was evaluated. Electrophoretic mobility shiftassays (EMSA) were performed by using purified RssB�P(36) mixed with either the F0 or the Fa flhDCSm promoterfragment. The 291-bp F0 DNA fragment, spanning thetranslational start site, ATG, and its upstream 291-bp re-gion, contained the P1 promoter (Fig. 3 and 5A) (GenBankaccession number AF077334) (18). For comparison, the pro-moter regions of ygfFSm (370-bp promoter DNA fragment)and rssB (104-bp promoter DNA fragment) were used asnegative and positive controls, respectively (36). No sign ofa DNA shift was observed (Fig. 5B), indicating no RssB�Pbinding onto the F0 flhDCSm promoter fragment. Subse-quently, the 378-bp (bp 278 to 655 from ATG) Fa DNA
FIG. 2. Characterization of transcriptional initiation sites offlhDCSm in S. marcescens CH-1 (A) and CH-1�A (B). Primer extensionanalysis of in vivo transcripts was performed with primer FlhD-PE.RNA was isolated from S. marcescens CH-1 and the rssA mutant strain(CH-1�A) grown on swarming plates at 37°C for 2 h. The fluorescenceintensities of the DNA fragments (ordinate) were plotted against thesequence lengths of the fragments (abscissa). P1 and P2 indicate thepotential transcription starts that map 68 and 306 bases upstream ofthe translational initiation codon of the flhD gene, respectively.
FIG. 3. Transcriptional start sites and RssB�P binding site in theupstream promoter region of the flhDCSm operon in S. marcescensCH-1. Open arrowheads and boldfaced letters indicate the transcrip-tional start sites P1 and P2. The RssB�P binding region (see Fig. 6) isboxed. The potential Shine-Dalgarno sequence is underlined; thetranslational start site of flhDSm is double underlined. Wavy underlin-ing indicates the promoter consensus sequences. Nucleotides are num-bered by labeling the translation initiation site of the flhDSm gene as�1. The flhDCSm promoter sequence was deposited in GenBank underaccession number AF077334 (18).
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fragment, upstream of F0 and containing the P2 promoter(Fig. 5A), was PCR amplified for EMSA. A clear DNA shiftwas observed when RssB�P (2 �M) and Fa (0.5 ng) wereused (Fig. 5B). Furthermore, a competition assay throughaddition of unlabeled Fa DNA fragments (0.5 ng to 50 ng)inhibited the DNA shift and verified the specific binding ofRssB�P to Fa (Fig. 5C). These results suggested specificinteraction between RssB�P and the Fa flhDCSm promoterregion. To confirm that phosphorylation of RssB was nec-essary for binding to the Fa region, RssB(D51E), whereRssB cannot be phosphorylated (36), was used in theEMSA. No DNA shift was observed (Fig. 5D), indicatingthat phosphorylation was essential for the binding of RssB
to the Fa fragment. Thus, RssB�P specifically binds to theupstream promoter region of flhDCSm, and the potentialbinding site is located between bp 278 and 655. Theseresults suggested that the binding site of phosphorylatedRssB is located in the Fa fragment, which contains P2, butnot in F0, containing the P1 region.
Determination of the specific RssB�P binding site withinthe Fa region. To determine the specific binding site(s) ofRssB�P in the Fa region, DNase I footprinting experimentsusing an automated capillary DNA sequencer (ABI Prism3100) were performed. The Fb DNA fragment, whichspanned bp 156 to 655 and was 122 bp longer than theFa fragment (Fig. 5A), was PCR amplified for DNase I
FIG. 4. Effects of RssA on promoter activities of PflhDCSm in S.marcescens CH-1. (A) flhDCSm promoter region. P1 and P2 indicatethe two transcriptional start sites, and each potential promoter wasfused with the luxCDABE reporter genes in the pACYC184 vectorto form pBG301 and pBG302, respectively. (B and C) P1 and P2promoter activities, respectively, at 37°C. Triangles and squares,growth curves in CH-1 and CH-1�A, respectively; solid and openbars, transcription activities of strains CH-1 and CH-1�A, respec-tively. Results are means � standard deviations from three inde-pendent experiments.
FIG. 5. The flhDCSm promoter region and its interaction withRssB. (A) Diagram of each flhDCSm promoter DNA fragment. Nucle-otides are numbered by labeling the translation initiation site of theflhDSm gene as �1. (B) Determination by EMSA of the interactionbetween RssB�P and the Fa fragment, the rssB promoter region(positive control), the ygfFSm promoter region (negative control) (36),or the F0 fragment. (C) Competition assay. A 0.5-ng portion of labeledFa, either without RssB�P or with 4 �M RssB�P, was mixed withunlabeled DNA. , no unlabeled DNA; � Fa, 0.5 ng unlabeled Fa;�� Fa, 50 ng unlabeled Fa; �� ygfF, 50 ng unlabeled ygfFSm promoterfragment. (D) An aspartate residue (D51) is essential for interactionbetween RssB and Fa. EMSA were performed using (from left toright) a labeled Fa fragment either alone, together with RssB�P, ortogether with RssB(D51E), which had lost the ability to be phosphor-ylated.
footprinting assays. Either the “template” or the “nontem-plate” strand of Fb was modified by the fluorescent dyeFAM before the assay. The results, as shown in Fig. 6,revealed that for the “template” and “nontemplate” strands,
the DNA region spanning bp 364 to 341 in the flhDCSm
promoter region was protected by RssB�P. In contrast, noregion within the Fb fragment was protected byRssB(D51E) in the DNase I footprinting assay (data not
FIG. 6. Identification of the RssB�P binding site in the flhDCSm promoter region by a DNase I footprinting assay. (A and B) FAM was usedto label the template (A) or nontemplate (B) strand of the PCR-amplified Fb fragment (see Fig. 5). (i and ii) Each strand was incubated in theabsence or presence of RssB�P before treatment with DNase I. (iii and iv) Expanded views of the RssB�P binding region, selected from panelsi and ii. The fluorescence intensity of the FAM-labeled DNA fragment (ordinate) was plotted against the sequence length of the fragment.(C) Partial sequence of the flhDCSm promoter region. Nucleotide positions are numbered by labeling the translation initiation site of the flhDCSmgene as �1. Nucleotides protected by RssB�P are shadowed.
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shown). These results confirmed that phosphorylated, andnot unphosphorylated, RssB binds the region of the flhDCSm
promoter from bp 364 to 341. Detailed analysis of theflhDCSm P2 promoter sequence revealed that the RssB bind-ing site partially overlaps with the 35 region of P2 (Fig. 3).
In vivo RssAB signaling and binding of RssB�P with theflhDCSm promoter. To determine whether RssAB signalingwas important for the interaction of RssB�P with theflhDCSm promoter in vivo, a modified ChIP assay (23) was used toevaluate the binding of RssB�P to the flhDCSm promoterregion in vivo at 37°C and 30°C. DNA binding proteins inactively growing S. marcescens CH-1 cells (early-logarithmicphase) were cross-linked onto DNAs by formaldehyde, fol-lowed by extraction and shearing of the genomic DNA intofragments of 500 to 1,000 bp. Glutathione-Sepharose was thenused to pull down the complexes comprising recombinant GSTfusion proteins [GST, GST-RssB, and GST-RssB(D51E)] to-gether with the cross-linked DNA fragments. After separationof cross-linked protein-DNA complexes by heat, the presenceof DNA in the pulled-down complexes was analyzed by PCRusing primers designed to amplify the 378-bp Fa fragment. Theresults in Fig. 7A show that while no amplified Fa fragmentwas detected in either the GST-only or the GST-RssB(D51E)group, it was clearly detected in the GST-RssB group. Theseresults indicated that RssB phosphorylation and the aspartateresidue in RssB are essential for in vivo Fa binding. Furthercomparison of the amount of the Fa fragment captured byRssB in S. marcescens CH-1 showed that less Fa fragment wasbound by RssB at 30°C than at 37°C (Fig. 7A).
To determine whether intact RssA-RssB signaling is essentialfor the binding of RssB�P to the Fa region, a modified ChIPassay was performed using the pull-down extracts from S. marc-escens CH-1(RssB) (i.e., CH-1 containing pGST-RssB), S. marc-escens CH-1�A(RssB), and S. marcescens CH-1�A[RssB(D51E)][i.e., CH-1�A containing pGST-RssB(D51E)] cells. Only S. marc-escens CH-1(RssB) showed a positive binding result (Fig. 7B).Thus, RssA is essential for RssB�P binding with the Fa fragment,indicating that under in vivo conditions, phosphorylated RssBbinds directly to the flhDCSm promoter region only when itscognate sensor, RssA, is present. Thus, a complete RssA-RssBsignaling pathway is important for RssB�P binding and inhibitionof flhDCSm promoter activity on LB swarming plates at 37°C.
Role of RssA signaling in flhDCSm promoter activity at 30and 37°C. We had previously shown that the promoter activi-ties of PflhDCSm and PhagSm are reduced when the incubationtemperature is changed from 30 to 37°C (18). The amount offlagellin synthesized in S. marcescens CH-1 on LB seedingplates was measured at both 30 and 37°C for comparison. Asignificant, 2.61-fold increase in the amount of flagellin at 30°Cover that at 37°C was observed in CH-1 (Table 2). Furtherquantification by primer extension of flhDCSm promoter activ-ity in S. marcescens CH-1 grown to early-log phase showed thatthe P1 and P2 promoter activities at 30°C were 1.96-fold and2.31-fold higher than those at 37°C, respectively (Table 2). Incontrast, the amount of flagellin synthesized and the level offlhDCSm expression in S. marcescens CH-1�A showed no sta-tistically significant difference when the incubation tempera-ture was changed from 30 to 37°C.
DISCUSSION
In this communication, we characterized the underlyingmechanism of the RssA-RssB effect on the regulation (inhibi-tion) of swarming in S. marcescens CH-1 at 37°C. When rssAwas mutated, significant increases in flagellum production and
FIG. 7. In vivo identification of RssB�P binding to the flhDCSm pro-moter region. (A) Oversynthesis of the GST-tagged fusion proteins in S.marcescens CH-1 grown at 37°C or 30°C was used to capture the flhDCSmpromoter DNA fragment, followed by a modified ChIP assay. The pre-cipitated DNA and total (input) DNA were subjected to PCR usingprimers FlhD-PF and FlhD-PR, specific to the flhDCSm promoter region.(B) The modified ChIP assay was performed against the pull-down ex-tracts from S. marcescens CH-1(RssB) (i.e., CH-1 cells containingpGEX::rssBSm), CH-1�A(RssB), and CH-1�A[RssB(D51E)] [i.e., CH-1�A containing pGEX::rssBSm(D51E)] cells at 37°C or 30°C. S. marc-escens CH-1 is the parent strain, and S. marcescens CH-1�A is the rssAdeletion strain.
flhDCSm promoter activity, and in accompanying precociousswarming behavior, were observed. Further studies showedthat a complete RssA-RssB signaling system is required forinhibition of S. marcescens CH-1 swarming. The underlyingmolecular mechanism of the RssA-RssB effect lies in directinteraction of RssB�P with the flhDCSm promoter region lo-cated between bp 341 and 364 from the translational ini-tiation codon ATG (Fig. 6C). Such interaction reduced thelevels of transcription and expression of flhDCSm. P1 promoteractivity was also affected by RssAB signaling, albeit to a lesserextent (Fig. 4B). To date, however, no evidence has beenobtained showing that RssB binds to the flhDCSm P1 promoterregion. Thus, the possibility exists that P1 promoter activity isindirectly affected by RssAB signaling through a steric hin-drance effect. Although the growth phase-dependent phosphor-ylation (and thus activation) of RssA-RssB signaling in vivoremains to be characterized, our experimental results indicatedthat when S. marcescens is grown to the early-logarithmicphase on LB swarming plates at 37°C, RssB�P shows evidenceof binding to the promoter region of flhDCSm, subsequentlyinhibiting its level of transcription (Table 2; Fig. 7). Theseresults suggested that RssA-RssB signaling modulates flhDCSm
expression and that when this signaling is activated, flhDCSm
expression is inhibited.Synthesis of FlhD and FlhC from flhDC is complicated, and
multiple levels of intracellular regulation exist, including tran-scriptional and posttranscriptional control in E. coli and evenposttranslational control in Proteus mirabilis (5, 28) and Sal-monella serovar Typhimurium (32). The expression of flhDC isboth positively and negatively regulated by the histone-likenucleoid-structuring H-NS protein, and H-NS-binding siteshave been identified upstream and downstream of the pro-moter region in E. coli (27, 29). flhDC expression is also pos-itively regulated by the cAMP-CAP (catabolite gene activatorprotein) complex, and the CAP-binding site is found upstreamof the flhDC promoter in E. coli and Salmonella serovarTyphimurium (1, 25, 27, 40). A LysR-type regulator, LrhA, alsonegatively regulates flhDC expression in E. coli (16). In P. mira-bilis, the flhDC operon is up-regulated by four unlinked genes,
umoA, umoB, umoC, and umoD, encoding putative membrane orperiplasmic proteins (8). Moreover, besides transcriptional activ-ity, the stability of flhDC mRNA is controlled by the RNA bindingregulator, CsrA, in E. coli (21, 34). These results indicate that theregulation of flhDC expression is under complicated and stringentcontrols.
The regulation of flhDC expression is complicated by influ-ences from numerous environmental signals, such as temper-ature, osmolarity, and pH. The two-component signal trans-duction systems (17, 22, 29) play important roles in mediatingthese regulatory processes (31, 37). High osmolarity inhibitsflhDC expression through the response regulator OmpR, andtwo OmpR-binding sites have been found in the flhDC pro-moter region in E. coli (24). Expression of flhDC in Xenorh-abdus nematophila is also repressed by EnvZ-OmpR in re-sponse to high environmental osmolarity (20). A report bySperandio et al. showed that flhDC expression was positivelyregulated by quorum sensing through QseBC in E. coli (30).QseB binds directly to the flhDC promoter at two sites, thehigh- and low-affinity binding sites, respectively (6). In E. coli,the RcsCDB His-Asp phosphorelay system is a negative regu-lator of the flhDC operon. The site of binding of RcsAB to theflhDC promoter was mapped as the RcsAB box, located down-stream of the promoter in E. coli (10, 20). These results indi-cated that two-component systems either positively or nega-tively regulate the expression of flhDC, depending on theenvironmental signals and binding sites of the response regu-lators in the promoter regions.
Compared with the findings for known regulatory systemsfor flhDC expression, the experimental results of this studyshowed a negative regulatory characteristic of RssAB in S.marcescens as the culture temperature is shifted from 30 to37°C. When S. marcescens was cultured on LB swarming platesat 37°C, the transcriptional level of flhDCSm was reduced fromthat at 30°C. This might be due, at least in part, to interactionbetween RssB�P and the upstream region of the flhDCSm P2promoter (from bp 364 to 341) and subsequent inhibitionof flhDCSm RNA transcription (Fig. 7; Table 2). Based onthese results, it was reasoned that the extents of RssAB sig-naling and phosphorylation of RssB might be decreased whenthe culture temperature is shifted from 37 to 30°C. Indeed,ChIP results confirmed less Fa binding by RssB at 30°C than at37°C in vivo (Fig. 7). Furthermore, such temperature-relatedinhibition of flhDCSm expression activities might be connectedto S. marcescens pathogenesis in humans. Since increases in theexpression of S. marcescens virulence factor genes, such ashemolysin, were observed for precocious-swarming mutants(15), and S. marcescens swarming is inhibited at the humanbody temperature, 37°C, it is reasoned that the expression ofvirulence factors in S. marcescens is reduced during humaninfection. In a rat model of acute pneumonia, S. marcescensstrain CH-1�AB, in which rssA and rssB were deleted, showeda significantly more virulent phenotype and caused a muchhigher mortality rate than its parent strain, S. marcescens CH-1(data not shown). In conclusion, swarming of S. marcescensCH-1 at 37°C is under strict regulation, and activation of theRssA-RssB signaling system and inhibition of the flagellar sys-tem and virulence factor expression contribute to such pheno-type regulation. Whether similar regulatory effects occur at
TABLE 2. Quantification of flhDCSm promoter activity and flagellinsynthesis in S. marcescens CH-1 and CH-1�A at 30 and 37°Ca
a RNA was isolated from S. marcescens CH-1 and the rssA mutant strain(CH-1�A) grown on swarming plates for 2 h. flhDCSm P1 and P2 promoteractivities were evaluated by primer extension using a FAM-labeled primer fol-lowed by electrophoresis on an ABI Prism 3100 capillary DNA genetic analyzer.The promoter activity was scored for relative fluorescence intensity by using thepeak area with GeneMapper software, version 3.5. The amount of flagellin wasevaluated by calculation of the relative intensities of Western blotting resultsusing Scion Imaging software after incubation for 3 h on seeding plates. Thepromoter activities and amounts of flagellin are presented as arbitrary units.Each result is the mean � standard error from three independent experiments,with triplicate samples in each assay. Ratios were calculated as the promoteractivity or flagellin amount of a strain incubated at 30°C divided by that at 37°C.
VOL. 190, 2008 REGULATION OF flhDCSm PROMOTER ACTIVITY BY RssAB 2503
30°C, leading to a density-dependent swarming phenomenonin S. marcescens, is currently under study.
ACKNOWLEDGMENTS
This work was supported by grants from the National Science Coun-cil (NSC-95-2320-B-002-061-MY3 and NSC96-2320-B-155-001) andthe Technology Development Program for Academia, Ministry of Eco-nomical Affairs (91-EC-17-A-10-S1-0013), which were greatly appre-ciated.
We are grateful to Yu-Huan Tsai for excellent technical assistance.
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