FliZ Is a Posttranslational Activator of FlhD 4 C 2 -Dependent Flagellar Gene Expression

Transcription

1 JOURNAL OF BACTERIOLOGY, July 2008, p Vol. 190, No /08/$ doi: /jb Copyright 2008, American Society for Microbiology. All Rights Reserved. FliZ Is a Posttranslational Activator of FlhD 4 C 2 -Dependent Flagellar Gene Expression Supreet Saini, 1 Jonathon D. Brown, 2,3 Phillip D. Aldridge, 2,3 * and Christopher V. Rao 1 * Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, Illinois ; Centre for Bacterial Cell Biology, Newcastle University, Framlington Place, Newcastle upon Tyne NE2 4HH, United Kingdom 2 ; and Institute for Cell and Molecular Biosciences, Newcastle University, Framlington Place, Newcastle upon Tyne NE2 4HH, United Kingdom 3 Received 21 December 2007/Accepted 28 April 2008 Flagellar assembly proceeds in a sequential manner, beginning at the base and concluding with the filament. A critical aspect of assembly is that gene expression is coupled to assembly. When cells transition from a nonflagellated to a flagellated state, gene expression is sequential, reflecting the manner in which the flagellum is made. A key mechanism for establishing this temporal hierarchy is the 28 -FlgM checkpoint, which couples the expression of late flagellar (P class3 ) genes to the completion of the hook-basal body. In this work, we investigated the role of FliZ in coupling middle flagellar (P class2 ) gene expression to assembly in Salmonella enterica serovar Typhimurium. We demonstrate that FliZ is an FlhD 4 C 2 -dependent activator of P class2 /middle gene expression. Our results suggest that FliZ regulates the concentration of FlhD 4 C 2 posttranslationally. We also demonstrate that FliZ functions independently of the flagellum-specific sigma factor 28 and the filament-cap chaperone/flhd 4 C 2 inhibitor FliT. Furthermore, we show that the previously described ability of 28 to activate P class2 /middle gene expression is, in fact, due to FliZ, as both are expressed from the same overlapping P class2 and P class3 promoters at the fliazy locus. We conclude by discussing the role of FliZ regulation with respect to flagellar biosynthesis based on our characterization of gene expression and FliZ s role in swimming and swarming motility. The bacterial flagellum is a rotary motor that enables cells to swim in liquid environments and drift along surfaces (5, 22). In Salmonella enterica serovar Typhimurium, over 50 genes divided among at least 17 operons are involved in motility (9). These genes encode not only the flagellar subunits and chemotaxis proteins but also a number of regulators that synchronize gene expression with the assembly process. The flagellum consists of three structural elements: the basal body, the hook, and the filament (42). The basal body, embedded in the membrane, anchors the flagellum to the cell. It also houses the rotary motor necessary for swimming and the type III secretion apparatus, which is involved in assembly. The hook, a flexible joint, transmits torque produced by the motor to the filament, a rigid helical structure approximately 5 to 15 m in length that functions as the propeller (61). In S. enterica serovar Typhimurium, there are approximately four to six flagella per cell (32). When the motors spin counterclockwise, the filaments form a helical bundle that propels the cell forward in a corkscrew-like manner. Flagellar assembly proceeds in a sequential manner beginning at the base along the inner membrane and concluding with the filament (43). The type III secretion apparatus, located at the cytoplasmic interface of the flagellum, delivers the * Corresponding author. Mailing address for Christopher V. Rao: Department of Chemical and Biomolecular Engineering. University of Illinois at Urbana-Champaign, 600 S. Mathews Ave., Urbana, IL Phone: (217) Fax: (217) chris@scs.uiuc.edu. Mailing address for Phillip D. Aldridge: Centre for Bacterial Cell Biology, Newcastle University, Framlington Place, Newcastle upon Tyne NE2 4HH, United Kingdom. Phone: Fax: p.d.aldridge@ncl.ac.uk. Published ahead of print on 9 May majority of the protein subunits through a central channel within the growing flagellar structure. The process concludes with the nucleation and elongation of the flagellar filament, driven by the secretion of flagellin monomers into the hollow interior of the filament and subsequent incorporation at the distal tip (3, 23, 47, 60). A critical feature of flagellar biogenesis is that gene expression is coupled to assembly. Upon initiation, where cells transition from a nonflagellated to a flagellated state, gene expression proceeds in a sequential manner: first, genes encoding the basal body and hook proteins are expressed, and then, only after these structures are assembled, the late genes encoding the filament, motor, and chemotaxis proteins are expressed (29, 31). If hook or basal body assembly is unsuccessful, then the late genes are not expressed. This checkpoint enables cells to coordinate assembly and is the main regulatory mechanism observed during initiation. The way that cells enforce this checkpoint is to use late protein secretion as a proxy signal for hook-basal body (HBB) completion (24). The flagellar promoters can be divided into three classes (9). A single P class1 promoter controls the expression of the flhdc master operon involved in initiating assembly. This promoter integrates environmental signals through the combinatorial action of multiple global transcriptional regulators, thus allowing cells to determine whether or not to be motile (51, 58). When motility is induced, the FlhD 4 C 2 complex activates the P class2 promoters (25, 55). These promoters control the expression of the genes encoding the HBB and two regulatory proteins, 28 and FlgM. The 28 alternate sigma factor, also known as FliA, is required for activating the P class3 promoters, which control the expression of the late genes (50). However, prior to HBB completion, FlgM binds to 28 and prevents it from activating 4979

2 4980 SAINI ET AL. J. BACTERIOL. the P class3 promoters (6, 7, 17). This inhibition, however, is relieved when the HBB is assembled, as the completed structure can secrete FlgM along with other late proteins involved in assembly (24). Thus, the cell is able to use protein secretion as a cue for HBB completion. In addition to FlhD 4 C 2, 28, and FlgM, the flagellar proteins FlgN, FliT, and FliZ have been shown to regulate the assembly process. FlgN, the secretion chaperone for the hook associate proteins FlgK and FlgL, enhances translation of FlgM from class 3 transcripts (2, 4, 15, 31). FliT, the secretion chaperone for the filament cap protein FliD, binds the FlhD 4 C 2 complex and prevents it from activating P class2 promoters (37, 57). FliZ, encoded in the fliazy operon, is a positive activator of P class2 / middle gene expression (37, 48). This protein also regulates the expression of genes encoding the Salmonella pathogenicity island 1 (SPI1) needle complex, which is involved in the invasion of intestinal epithelial cells (27, 40). In addition to FliZ, 28 has also been proposed to activate P class2 promoters (28). However, unlike the case for the 28 -FlgM checkpoint involved in coupling P class3 /late gene expression with HBB completion, the roles of the regulatory circuits dictated by these other proteins are still unclear. In this paper, we investigate the role of FliZ in regulating flagellar assembly. We demonstrate that FliZ is an FlhD 4 C 2 - dependent activator of P class2 /middle gene expression. Furthermore, our data suggest that FliZ regulates FlhD 4 C 2 levels posttranslationally. We demonstrate that 28 is unable to activate P class2 promoters on its own or in conjunction with FlhD 4 C 2. Rather, our data suggest that the effect of 28 on P class2 /middle gene expression is through FliZ. Based on these results, we speculate that FliZ couples P class2 /middle gene expression to FlgM secretion, committing cells to aggressively building multiple flagella once the first few are complete. This model suggests that assembly is far more complex than the sequential one proposed and also suggests that cells tune both P class2 /middle and P class3 /late gene expression in response to FlgM secretion rates. MATERIALS AND METHODS General techniques and growth conditions. All culture experiments were performed in Luria-Bertani (LB) broth at 37 C unless noted otherwise. Motility agar contained 0.3% Bacto agar, 1% tryptone, and 0.8% NaCl. Swarming plates contained either LB broth with 0.6% Difco agar and 0.5% glucose (34) or 0.6% Bacto agar, 1% tryptone, 0.8% NaCl, and 0.02% Tween 80 (49). Antibiotics were used at the following concentrations: ampicillin at 100 g/ml, chloramphenicol at 20 g/ml, kanamycin at 40 g/ml, and tetracycline at 15 g/ml. All experiments involving the growth of strains containing plasmid pkd46 were performed at 30 C as previously described (10). Loss of pkd46 from strains was achieved by growth at 42 C after electroporation of the PCR products. Removal of the antibiotic from the FRT-Cm/Kan-FRT insert was achieved by passing pcp20 through the isolated mutants (8). Enzymes were purchased from Fermentas or New England Biolabs and used according to the manufacturer s recommendations. Primers were purchased from IDT Inc. Strain and plasmid construction. Bacterial strains and plasmids are described in Tables 1 and 2, respectively. All S. enterica serovar Typhimurium strains are isogenic derivatives of strain (American Type Culture Collection). The generalized transducing phage of S. enterica serovar Typhimurium P22 HT105/1 int-201 was used in all transductional crosses (11). Standard scarred FLP recombination target (FRT) mutants were produced as previously described using either pkd3 or pkd4 as the PCR template (10). The fliz mutant was made using primers SS007F (CAC GTT TCA CCA ACA CGA CTC TGC TAC ATC TTA TGC TTT TGT GTA GGC TGG AGC TGC TTC) and SS007R (TGA TCG CAC CCG AAA AGT GCC GCA CAA CGT ATA GAC TAC CCA TAT GAA TAT CCT CCT TAG). The flia mutant was TABLE 1. Strains used during this study Strain Genotype a Source or reference b Wild-type serovar Typhimurium ATCC c CR201 fliz::frt CR202 fliaz::frt CR203 flia::frt CR204 flit::frt CR205 fliz::frt flit::frt CR206 flia::frt flit::frt CR207 fliaz::frt flit::frt CR208 P flhdc ::tetra CR209 P flhdc ::tetra fliz::frt CR210 P flhdc ::tetra flia::frt CR211 P flhdc ::tetra fliaz::frt CR212 P flhdc ::tetra flit::frt CR213 P flhdc ::tetra fliz::frt flit::frt CR214 P flhdc ::tetra flia::frt flit::frt CR215 P flhdc ::tetra fliaz::frt flit::frt JS481 (invh-avra)2916::cm (called SPI1) 12 CR216 SPI1 rtsab::frt CR217 SPI1 rtsab::frt fliz::frt CR218 flhc::3 FLAG Kan r CR219 fliz::frt flhc::3 FLAG Kan r CR220 P flhdc ::tetra flhc::3 FLAG Kan r CR221 P flhdc ::tetra fliz::frt flhc::3 FLAG Kan r CR222 flhdc::frt a All Salmonella strains are isogenic derivatives of serovar Typhimurium strain b Strains are from this study unless specified otherwise. c ATCC, American Type Culture Collection. made using primers SS013F (ATA CGT TGT GCG GCA CTT TTC GGG TGC GAT CAT GCG CGA CGT GTA GGC TGG AGC TGC TTC) and SS013R (TCT GTA GAA ACG GAT AAT CAT GCC GAT AAC TCA TTT AAC GCA TAT GAA TAT CCT CCT TAG). The flhdc mutant was made using primers SS058F (GCT GCT ATG CAT TTG ACC TTT TTG CTT CTT TTA CCG GGC CCA TAT GAA TAT CCT CCT TAG) and SS058R (TAC AGC CTG ATG AGG CGA CCA CGG CTG AGC TGT GTT TCG CGT GTA GGC TGG AGC TGC TTC). The flit mutant was made using primers SS078F (AAA GCA TCT TTC CAG GAG TCT CGT TAA TGA CCT CAA CCG TGT GTA GGC TGG AGC TGC TTC) and SS078R (TCT GGA GTA TGG AAG AAT TTT CAT ACG AGA CGG GAA AAT ACA TAT GAA TAT CCT CCT TAG). The fliaz mutant was made using primers SS013F and SS007R. Deletions of the SPI1 regulator rtsa (STM4315) and flhdc repressor rtsb (STM4314) were made using primers SS120F (CTG AAG ATG ATA TCC AGA GTT GCC TTG CCT ACC ACT CTA CGT GTA GGC TGG AGC TGC TTC) and SS120R (ATA TAG CTA TAT ATT AGT TTT CTT TTG AAA TAT TTT TCA GCA TAT GAA TAT CCT CCT TAG). All mutations were checked using primers that had target sequences outside the deleted region. Prior to removal of the antibiotic resistance marker, the constructs resulting from this procedure were moved into a clean wild-type background (14028) by P22 transduction. The P flhdc ::tetra strain was made by chromosomal replacement of the flhdc promoter with the tetra element from transposon Tn10 using -Red recombination in cells carrying pkd46 as described by Datsenko and Wanner (10). The tetra element was PCR amplified from TH8094 as described by Karlinsey (30) using the primers SS100F (AAA CAA AAA AGA ATT TGG TGT TGA CGT ACC CCT ATT CAG CAG AGT AGG GAA CTG CCA) and SS100R (GTG CGA CGT AGC CGC ACC CCG TGA TGT CGC CGG GAA GGC CCT AAG CAC TTG TCT CCT G). This arrangement put flhdc expression under the control of the teta promoter. In the absence of tetracycline, the teta promoter is repressed by TetR. In the presence of tetracycline, TetR repression is relieved and flhdc is transcribed from the teta promoter. Strains with the FlhC-3 FLAG tag epitope for Western blotting were made by amplifying the 3 FLAG tag and kanamycin resistance marker from the template psub11 (54) using the primers SS125F (TAT TCC ACA ACT GCT GGA TGA ACA GAT CGA ACA GGC TGT TGA CTA CAA AGA CCA TGA CGG) and SS125R (GGG CAA AAA AAA GCA GCG GTA CGT CGT TAC CGC TGC TGG ACA TAT GAA TAT CCT CCT TAG). The PCR product was then electroporated into wild-type cells carrying pkd46, and cells

3 VOL. 190, 2008 FliZ REGULATION OF FLAGELLAR GENE EXPRESSION 4981 TABLE 2. Plasmids used during this study Plasmid Relevant characteristics Source or reference a pkd46 bla P BAD gam beto exo psc101 orits 10 pcp20 bla cat ci857 PRflp psc101 orits 8 pkd3 bla FRT cm FRT orir6k 10 pkd4 bla FRT kan FRT orir6k 10 pprobe GFP tagless kan GFP tagless ori p15a 46 pss001 (P flga -GFP) kan P flga -GFP tagless ori p15a pss002 (P flgb -GFP) kan P flgb -GFP tagless ori p15a pss003 (P flhb -GFP) kan P flhb -GFP tagless ori p15a pss004 (P flie -GFP) kan P flie -GFP tagless ori p15a pss005 (P flid -GFP) kan P flid -GFP tagless ori p15a pss006 (P flgk -GFP) kan P flgk -GFP tagless ori p15a pss007 (P flic -GFP) kan P flic -GFP tagless ori p15a pss008 (P flhd -GFP) kan P flhd -GFP tagless ori p15a pss009 (pprobe luxcdabe) kan luxcdabe ori p15a pss010 (P flga -luxcdabe) kan P flga -luxcdabe ori p15a pss011 (P flic -luxcdabe) kan P flic -luxcdabe ori p15a pprotet.e cm P LtetO-1 ori ColE1 Stratagene pss012 (pprotet.e tetr) cm P LtetO-1 tetr ori ColE1 pss013 (pfliz) cm P LtetO-1 fliz ori ColE1 pss014 (pfliz-tetr) cm P LtetO-1 fliz tetr ori ColE1 pss015 (pfliz-native) cm P flia fliz ori ColE1 pss016 (pflia-tetr) cm P LtetO-1 flia tetr ori ColE1 pss017 (pflhdc-tetr) cm P LtetO-1 flhdc tetr ori ColE1 psub11 bla 3 FLAG FRT kan FRT orir6k 54 pss018 (P flhdc flhdc-lacz) kan P flhdc flhdc-lacz ori p15a pss019 (P LtetO-1 flhc-lacz) cm P LtetO-1 flhc-lacz ori ColE1 pss019 (P LtetO-1 flhc-lacz) cm P LtetO-1 flhc-lacz ori ColE1 pss020 (P LtetO-1 flhdc-lacz) cm P LtetO-1 flhdc-lacz ori ColE1 a Plasmids are from this study unless specified otherwise. were then selected for kanamycin resistance. This arrangement resulted in expression of FlhC protein with a 3 FLAG tag at its carboxy terminus. Green fluorescent protein (GFP) transcriptional fusions were made by amplifying the promoter region for the respective gene using PCR and then cloning the PCR product into the pprobe-gfp[tagless] vector (46). The flga transcriptional fusion was made using primers SS008F (CAT AGG TAC CTT ACT GGC CTA ATG TCA GCG) and SS008R (GGG AGA ATT CTG TGA AGC AAG CAT CAA CGC). The flgb transcription fusion was made using primers SS033F (GCT GGT ACC CGC GCA AAC TGA CGG CAT CC) and SS033R (GGG GAA TTC AGC CTG TCG AGC ATA TCT CC). The flhb promoter was made using primers SS068F (GGG GGT ACC GAC GGC GCA GGC GGC GGA AC) and SS068R (GGG GAA TTC TCG TCG TCG CTC TCT TCT GC). The flie promoter was made using primers SS069F (GGG AAG CTT GCG GGC GCT TTT TAC CGG CC) and SS069R (GGC GGT ACC CAA TCC CCT GTA TTG CTG CC). The flgk promoter was made using primers SS076F (GGG GGT ACC ACC AAC TGA AAG CGA AAG CGG GC) and SS076R (GCG GAA TTC GGC GTG ATT AAT CAA GCT GG). The flid promoter was made using primers SS071F (TTT GGT ACC GTC AGA CCT TTG ATG TTC GC) and SS071R (GTC GAA TTC ATG ATG AAA TTG AAG CCA TG). The flic promoter was made using primers SS030F (ATC GGT ACC AGT GGT GCT GGA CGC CAC GG) and SS030R (ATC GAA TTC TTT GTA TTA ATG ACT TGT GC). The flhd promoter was made using primers SS077F (CCC GGT ACC TTT GTT CAA TCG GAT AAT CC) and SS077R (CCC GAA TTC GAC ATT GTG ACG TAT AAC GC). The PCR fragments were digested with KpnI and EcoRI (sequences are underlined) and then cloned into the multiple cloning site of the pprobe-gfp[tagless] vector. In the case of the flie promoter, the fragment was cloned in pprobe-gfp[tagless] using HindIII and KpnI, as there is an internal EcoRI site. All constructs were sequenced prior to transformations into the wild-type and mutant strains. In order to construct the luciferase reporters, PCR was used to amplify the Photorhabdus luminescens luxcdabe operon from the plasmid prg19 (18) using primers SS082F (GGC GAA TTC CTT TAT AAG GAG GAA AAA CAT ATG ACT AAA AAA ATT TCA TT) and SS082R (AAA TCT AGA TTA TCA ACT ATT AAA TGC TTG GT). The PCR fragment was then cloned into the multiple cloning site of the plasmid pprotet.e. The pprotet.e plasmid was then digested with the enzymes EcoRI and AvrII, in order to include the TI terminator, and then subcloned into the EcoRI and NheI sites of pprobegfp[tagless], yielding pprobe-luxcdabe. Both AvrII and NheI yield compatible digested fragments. luxcdabe transcriptional fusions were then made by cloning the promoter fragments described above into the KpnI and EcoRI (and HindIII and KpnI for flie) restrictions sites of pprobe-luxcdabe. The expression plasmids for flia, fliz, and flhdc were made by cloning the respective gene(s) into the EcoRI and HindIII or PstI sites (sequences are underlined) of pprotet.e under the control of the strong promoter P LtetO-1 (41), resulting in the plasmids pflia, pfliz, and pflhdc, respectively. The plasmid pflia was made using primers SS053F (AGT GGT ACC TGC CGA TAA CTC ATT TAA CG) and SS053R (GGT AAG CTT CGC GAC CTA TAA CTT ACC CA). The plasmid pfliz was made using primers SS046F (ATG GAA TTC GCC GCA CAA CGT ATA GAC TA) and SS046R (GGC AAG CTT TTA ATA TAT ATC AGA ACT GG). The plasmid pflhdc was made using primers LC186 (ATA GAA TTC GTG CGG CTA CGT CGC ACA AA) and LC198 (ATA CTG CAG CGG TTA AAC AGC CTG TTC G). In the absence of TetR, the P LtetO-1 promoter is constitutively active. In order regulate the promoter activity, the tetr gene from transposon Tn10 was cloned using primers SS052F (GGT CTG CAG TGT CAA CAA AAA TTA GGA AT) and SS052R (GCT GCG GCC GC CGG AAA AAG GTT ATG CTG CT) into the PstI and NotI sites of pprotet.e. In the absence of the inducer anhydrotetracycline (atc), P LtetO-1 expression is inhibited due to TetR. However, in the presence of atc at 200 ng/ml, P LtetO-1 expression is activated as TetR is inhibited. FliZ was put under control of its native promoter by cloning the fliaz operon with its promoter using primers SS123F (GGG CTC GAG AGT TTT CGC GCC CAA ATA CC) and SS123R (AAT AAG CTT TTA ATA TAT ATC AGA ACT GG). The PCR product and the plasmid pprotet.e were digested with XhoI and HindIII, resulting in plasmid pfliaz with the fliaz genes under control of their native promoter. To remove the flia gene, the plasmid pfliaz was amplified using primers SS124F (CGT CTC TAT AGA CTA CCA GGA GTT CTC) and SS124R (CGT CTC GCT ATG ATA AAC AGC CCT GCG TTA A). The PCR product was then digested with BsmBI and ligated onto itself such the fliz gene now was solely under control of the native P fliaz promoter. The resulting plasmid was called pfliz-native. Three different types of flhc translational fusions were made. In the first, the fusion was made to the otherwise intact flhdc operon under the control of its native promoter. In this construct, the lacz gene was fused in frame to the first 30 base pairs of the flhc gene. In order to the build this fusion, the region 812

4 4982 SAINI ET AL. J. BACTERIOL. bases upstream of the flhd start codon and 30 base pairs into the flhc gene was amplified using primers SS128F (GGG GCA TGC GGC GAC AAG AAT ATG GGT GT) and SS128RIII (GGG GGT CTC ATC ATA GCT TCC TGA ACA ATG CTT T) and then digested using SphI and BsaI. The lacz gene was amplified using SS127F (GGG GGT CTC TAT GAC CAT GAT TAC GGA TTC) and LC096F (ACT TAA CGG CTG ACA TGG) using pah125 as the template and digested with BsaI and NheI (20). The plasmid pprobe-gfp[tagless] was digested with SphI and NheI and ligated with the flhdc and lacz DNA fragments, thus replacing the GFP gene with the flhdc-lacz translational fusion. This plasmid was called P flhdc flhdc-lacz. In the second translational fusion, the lacz gene was fused 30 bases into the flhc gene. The resulting fusion was then placed under the control of the constitutively active P LtetO-1 promoter. The flhc gene fragment, consisting of 20 bases upstream of the flhc start codon and 30 base pairs into the flhc gene, was amplified using primers SS129FI (GGG GAA TTC CGA TAC GGC GCG TAA GAA AA) and SS128RIII and the resulting product digested using EcoRI and BsaI. The lacz gene was amplified using SS127F and 127R and digested with BsaI and NheI. The digested products were then ligated with pprotet.e digested with EcoRI and AvrII. The resulting flhc-lacz translational fusion plasmid was called P LtetO-1 flhc-lacz. The third translational fusion was made by placing both the flhd gene and the flhc-lacz fusion under the control of the constitutively active P LtetO-1 promoter. The region, 20 bases upstream of the flhd start codon and 30 bases into the flhc gene, was amplified using primers SS129FII (GGG GAA TTC AAT AAA GTT GGT TAT TCT GG) and SS128RIII and digested with EcoRI and BsaI. The lacz gene was amplified using primers SS127F and SS127R and digested with BsaI and NheI. The digested products were then ligated with pprotet.e digested with EcoRI and AvrII. The plasmid was called P LtetO-1 flhdc-lacz. We also made an equivalent set of plasmids where the first 90 bases of the flhc gene were fused to lacz. However, the results were the same and, therefore, are not reported. Fluorescence and luminescence assays. End point and dynamic measurements of the reporter systems (GFP and luxcdabe) were made using a Tecan Safire2 microplate reader. For fluorescence end point measurements, 1 ml of culture was grown for 12 h at 37 C and then diluted 1:1,000 into fresh medium and grown for 6 h at 37 C. One hundred microliters was then transferred to a 96-well microplate, and the relative fluorescence and optical density at 600 nm (OD 600 ) were measured. The fluorescence readings, given as relative fluorescence units (RFU), were normalized with the OD 600 absorbance to account for cell density. For time course measurements using the luciferase reporters, a protocol slightly modified from the one developed by Kalir and coworkers was employed (29). One milliliter of culture was grown overnight at 37 C and then diluted by a factor of 1,000 into fresh medium and grown to an OD 600 of 0.2. One hundred microliters of culture was then transferred to a 96-well microplate that was sealed with a Breath-Easy membrane to allow for aeration while also minimizing evaporation. The temperature was maintained at 30 C, and luminescence and OD 600 readings were taken every 10 min in the microplate reader, with shaking in the interval between subsequent readings. The kinetic experiments were performed at 30 C in order to obtain better resolution and temporal ordering between P class2 and P class3 activation. Identical experiments were also performed at 37 C with similar results except that the separation between the two promoter classes was far less pronounced. Fluorescent measurements of swarming cells were performed by gently scraping the swarm with an o-loop and then resuspending the recovered cells in phosphate-buffered saline (PBS). The cells were then diluted to an OD 600 of 0.1 in PBS prior to making the fluorescence measurement. All experiments were done in triplicate, and average values with standard deviations are reported. Immunoblot analysis. Cells from overnight cultures were subcultured 1:1,000 in fresh medium and grown at 37 C for 6 h. Prior to lysis, OD 600 measurements were taken to ensure that there were equivalent numbers of cells between samples. To lyse the cells, cultures were spun down, resuspended 3:1 in 4 sodium dodecyl sulfate solubilizer, and boiled at 95 C for 10 minutes. Lysates were run on a4to20%tris-hcl precast gel (Bio-Rad) for 50 min at 150 V. Transfer to the membrane was done using Immobilon transfer membranes with 0.2- m pore size (Millipore). 3 FLAG-tagged FlhC was detected with an anti-flag M2 monoclonal antibody (Sigma) and an anti-mouse horseradish peroxidase-conjugated antibody (Jackson Laboratories) using the ECL Plus Western blotting detection system (Amersham). In order to quantify the relative protein levels, the membrane was scanned using a STORM 840 PhosphorImager (Amersham) and then analyzed using the LabWorks software package (UVP). The relative amount of protein in each lane was estimated by measuring the integrated OD for each band. All measurements were performed in triplicate. FIG. 1. FliZ enhances P class2 and P class3 activity. A comparison of P class2 (flga, flgb, flhb, and flie), P class2/3 (flid), and P class3 (flgk and flic) promoter activities in the wild-type, fliz, and fliz pfliz strains is shown. In the last strain, FliZ is constitutively expressed from the P LtetO-1 promoter on a plasmid. Error bars indicate standard deviations. -Galactosidase assays. Cells were grown in LB medium at 37 C and harvested after 6 h of growth. Quantification of -galactosidase activity was performed as previously described by Miller (45). All measurements were performed in triplicate. RESULTS FliZ is an activator of P class2 /middle gene expression. Reports describing the regulatory function of FliZ are inconsistent. Kutsukake et al. (37) demonstrated that FliZ positively regulates flagellar gene expression. In contrast, Frye and colleagues (16) were unable to detect a significant change in gene expression in a fliz mutant compared to the wild type using microarray analysis, though we note that the strains used in these two studies were different. To better understand the putative role of FliZ as a positive regulator of flagellar gene expression, we measured gene expression from a subset of flagellar promoters in the wild type (strain 14028), an isogenic fliz mutant, and the fliz mutant constitutively expressing fliz from the P LtetO-1 promoter on a plasmid (41). GFP transcriptional fusions were employed as an indirect measure of promoter activities (46). In the fliz mutant, both P class2 and P class3 activities were reduced slightly less than twofold relative to wild-type activities (Fig. 1). However, in the fliz mutant constitutively expressing fliz, both P class2 and P class3 activities were increased at least twofold relative to wild-type levels. These results indicate that FliZ positively regulates both P class2 and P class3 activities, consistent with the observations of Kutsukake et al. (37). We next tested how deleting fliz affected the dynamics of the P class2 flga and P class3 flic promoters. In these experiments, we used transcriptional reporters involving the luciferase operon, luxcdabe, from Photorhabdus luminescens (56). One advantage of using bacterial luciferase instead of GFP is that it is far more sensitive to changes in gene expression dynamics, particularly at low levels of expression (19). In agreement with our static fluorescence experiments, we observed that deleting fliz reduced the maximal expression level for both the P class2 flga reporter and the P class3 flic reporter relative to the wild-

5 VOL. 190, 2008 FliZ REGULATION OF FLAGELLAR GENE EXPRESSION 4983 FIG. 2. Dynamics of flagellar gene expression in wild-type and fliz strains. Gene expression measurements were made every 10 min after induction following dilution of strains in fresh medium (32). Note that strains were grown at 30 C in the kinetic experiments in order to obtain better resolution and temporal ordering between P class2 and P class3 promoter activities. Identical experiments were also performed at 37 C with similar results except that the separation between class 2 and 3 gene expression was far less pronounced (results not shown). Error bars indicate standard deviations. type levels (Fig. 2). However, the dynamics were the same in the two strains; the only difference was in the peak response. FliZ activates P class2 promoters independent of 28. Previously, a number of researchers have observed that 28 is a positive activator of P class2 activity (28, 36, 39). In particular, Kalir and coworkers demonstrated that overexpressing flia leads to increased P class2 activity (28). As FliZ is also a positive regulator of P class2 activity, this means that two positive factors are encoded in the same operon. Therefore, any changes to flia expression will result in a reciprocal change in fliz expression and vice versa, as the fliazy operon is under the control of both P class2 and P class3 promoters (26, 48). In previous studies of 28 activity, assays have always been performed with strains in which the fliz gene and fliazy promoter were intact. We therefore asked whether the observations made by Kalir and coworkers and others are a direct result of 28 or an indirect effect due to FliZ. To isolate the effects of 28 and FliZ on P class2 promoters, we expressed fliz or flia ( 28 ) and tetr from the P LtetO-1 promoter on a plasmid in a fliaz mutant. In the absence of atc, TetR negatively regulates the P LtetO-1 promoter and, as a consequence, FliZ or FliA is weakly expressed. However, in the presence of atc, TetR no longer represses the P LtetO-1 promoter and FliZ or FliA is strongly expressed. In the case of fliz, we observed approximately a twofold increase in P class2 activity upon induction with atc (Fig. 3). However, in the case of flia, we observed no effect on P class2 activity. As a control, we measured P class3 activity in the fliaz mutant strain. As expected, the plasmid expressing fliz upon induction was unable to activate P class3 promoters, whereas the plasmid expressing flia upon induction could. We conclude, therefore, that FliZ is a positive activator of P class2 activity that functions independently of 28. Furthermore, our results suggest that the previously observed effect of 28 on P class2 activity was actually via FliZ, for as FliZ is under the FIG. 3. Relative effects of FliZ and 28 expression on P class2 activity. A comparison of flagellar promoter activities in a fliaz mutant where flia ( 28 )orfliz is expressed from an atc-inducible promoter on a plasmid is shown. Note that tetr is also expressed from this plasmid in order to achieve atc-inducible expression. Error bars indicate standard deviations. control of both P class2 and P class3 promoters, overexpressing 28 increases FliZ expression, which thus leads to increased P class2 activity. As a result of our constitutive expression experiments, we compared P class2 activity in fliz, flia, and fliaz mutants and the wild type. Interestingly, we observed a greater decrease in the flia and fliaz mutants than in the fliz mutants (Fig. 4A). FlhD 4 C 2 is known to negatively regulate its own expression (35). In addition, FliT counteracts this negative regulation (J. D. Brown et al., submitted for publication), presumably by binding FlhD 4 C 2 and preventing it from inhibiting flhdc expression (37). As flit, located in the flidst operon, is under the control of both P class2 and P class3 promoters, its expression is responsive to flia ( 28 ) expression (59). Therefore, in a flia mutant, flit expression is reduced (5, RFU/OD [wild type] versus 3, RFU/OD [ flia mutant]). As FliT is thought to prevent FlhD 4 C 2 from inhibiting its own expression, we hypothesize that reduced flit expression reduces flhdc expression. The net result is reduced class 2 gene expression. We therefore compared P class2 activity in fliz and fliaz mutants to that in fliz flit and fliaz flit mutants. Consistent with our hypothesis, there was no further decrease in P class2 activity when flit was introduced into the fliz, flia, and fliaz backgrounds (Fig. 4A). Note that P class2 activity is slightly elevated in the flia flit relative to the fliz flit and fliaz flit mutants, as fliz is still being expressed. To confirm that the observed changes in P class2 activity are a result of the modulation of FlhD 4 C 2 autoregulation by FliT, we replaced the flhdc promoter with the tetra element from Tn10 ( P flhdc ::tetra). This arrangement constitutively expresses flhdc from its chromosomal locus in the presence of tetracycline. Using this construct, we observed that the decrease in P class2 activity was similar for the fliz, flia, and fliaz mutants in the P flhdc ::tetra background (Fig. 4B). Likewise, there was no appreciable difference between the fliz and fliaz mutants in the P flhdc ::tetra flit back-

6 4984 SAINI ET AL. J. BACTERIOL. FIG. 5. FliZ s effect on flagellar promoter activity is independent of SPI1, the SPI1 activator, rtsa, and the P flhdc repressor, rtsb. A comparison of flagellar promoter activities in SPI1 fliz and SPI1 fliz rtsab mutants is shown. Error bars indicate standard deviations. FIG. 4. Comparison of flagellar promoter activities for wild-type, fliz, flia, fliaz, flit, fliz flit, flia flit, and fliaz flit strains in a P flhdc (A) or a P flhdc ::tetra (B) background. P class2 (flhb) and P class3 (flic) activities were measured. Error bars indicate standard deviations. ground. Therefore, we conclude that the decrease in P class2 activity observed in the flia and fliaz mutants is due to a combination of reduced fliz and flhdc expression, the latter being a direct result of the concomitant reduction in flit expression. However, in the absence of FlhD 4 C 2 autoregulation, the decrease is due solely to FliZ. FliZ operates independently of FliT. The above results also allowed us to test whether FliZ and FliT interact with one another. One potential mechanism from this hypothesis is that FliZ binds to FliT and prevents it from inhibiting FlhD 4 C 2.To test the hypothesis that FliZ may operate through FliT, we compared P class2 activity in fliz, flit, and fliz flit mutants in the P flhdc ::tetra background to control for flhdc autoregulation (Fig. 4B). If FliZ operates through FliT, then the fliz flit mutant should behave similar to the flit mutant. Contrary to this prediction, we observed roughly a 50% decrease in P class2 activity in the fliz flit mutant relative to the wild type. The decrease is slightly less than the decrease observed in the fliz mutant. As a comparison, we observed roughly a 25% increase in P class2 activity in the flit mutant relative to the wild type. We note that similar experiments were performed by Kutsukake et al. (37), and our results are consistent with theirs. Therefore, we conclude that FliZ and FliT operate independently, though we cannot discount the possibility that their actions are competitive. FliZ activation is dependent upon FlhD 4 C 2. FlhD 4 C 2 is the master transcriptional regulator in the flagellar system. In the case of P class3 promoters, 28 can activate these promoters independent of FlhD 4 C 2. In the case of P class2 promoters, 70 can activate these promoters only when aided by FlhD 4 C 2.As FliZ has a positive effect on P class2 activity, one possibility is that it can interact with 70 to activate P class2 promoters independent of FlhD 4 C 2. Using the atc-inducible expression system, we observed that FliZ is unable to increase the activity of P class2 promoters in the flhdc mutant (results not shown). As a control, we also expressed flhdc from the P LtetO-1 promoter using the atc-inducible expression system. In this case, the addition of atc resulted in the strong activation of P class2 promoters (flga, RFU/OD [uninduced] versus 12,742 1,301 RFU/OD [induced]). These results demonstrate that FliZ activation of P class2 requires FlhD 4 C 2. FliZ s effect on flagellar gene expression is independent of SPI1. In addition to being a flagellar regulator, FliZ is known to also enhance the expression of SPI1 genes (27, 40). Likewise, a number of SPI1 transcriptional regulators such as HilA and RtsB are known to affect flagellar gene expression (12, 13, 53). Due to this cross talk, we wanted to determine whether FliZ s effect on flagellar gene expression was through its effect on SPI1 gene expression. To test this possibility, we compared P class2 and P class3 activity in SPI1 rtsab and SPI1 rtsab fliz mutants. In the SPI1 rtsab mutant, flagellar gene expression was similar to that in the wild type. However, in the SPI1 rtsab fliz mutant, P class2 and P class3 activity was reduced approximately 50% compared to that in the SPI1 rtsab mutant, similar to the decrease observed in the fliz mutant relative to the wild type (Fig. 5). Therefore, we conclude that the regulations of flagellar gene expression by FliZ and SPI1 are independent of each other. FliZ acts at the level of flhdc translation. The results described above suggest that FliZ acts through FlhD 4 C 2. One possibility is that FliZ regulates flhdc transcription, resulting in an indirect effect on P class2 activity due to changes in FlhD 4 C 2 expression. However, our P flhdc ::tetra data suggest regulation at a posttranscriptional level. To confirm that FliZ

7 VOL. 190, 2008 FliZ REGULATION OF FLAGELLAR GENE EXPRESSION 4985 A B C D E FIG. 6. FliZ is responsible for increased levels of FlhC protein independent of flhdc transcription. The amount of FlhC protein was measured by Western blotting using anti-flag antibody targeting chromosomally 3 FLAG-tagged FlhC protein. Lanes: A, wild type; B, fliz::frt; C, P flhdc ::tetra; D,P flhdc ::tetra fliz::frt; E, wild type without 3 FLAG tag. acts posttranscriptionally, we measured the expression of the P class1 flhdc promoter in the wild type and a fliz mutant. As predicted, only a slight change in flhdc expression in the fliz mutant (8, RFU/OD) relative to the wild type (10, RFU/OD) was observed. This suggests the possibility that FliZ affects flhdc translation. To test this model, we fused a 3 FLAG tag to flhc at its native chromosomal locus in a manner similar to that described by Kato and coworkers (33). Note that this strain is still motile, as it forms rings of similar size as those of the wild type on motility plates (results not shown). Using this construct, we observed approximately a 50% decrease ( absorbance units [AU] [wild type] versus AU [ fliz mutant]) in the amount of FlhC-3 FLAG protein in a fliz mutant relative to the wild type (Fig. 6). We also performed similar experiments in a P flhdc ::tetra background and observed a similar decrease in FlhC-3 FLAG protein in the fliz mutant ( AU [wild type]) versus AU [ fliz mutant]). Based on these results, we conclude that FliZ operates primarily at the level of the FlhD 4 C 2 protein. The above analysis indicates that FliZ s effect on FlhD 4 C 2 is the result of either translational regulation or protein stability. We therefore explored whether FliZ directly affects flhc translation by utilizing translational reporter fusions. We constructed three different flhc-lacz translation fusions on plasmids and then measured -galactosidase activity in the wild type and a fliz mutant. In the first, which we called P flhdc flhdc-lacz, the lacz gene was fused to the flhdc operon under the control of its native promoter. In the second, which we called P LtetO-1 flhc-lacz, the lacz gene was fused to flhc under the control of the constitutive P LtetO-1 promoter. In the third, which we called P LtetO-1 flhdc-lacz, the lacz gene was fused to the flhdc operon again under the control of the constitutive P LtetO-1 promoter. In all three cases, the fusions were made on plasmids (see Materials and Methods for construction details). Comparing the results using these three translational fusions in the wild type and the fliz mutant, we observed no significant change in -galactosidase activity (P flhdc flhdc-lacz, 1, [wild type] versus 1, [ fliz mutant]; P LtetO-1 flhc-lacz, 1, [wild type] versus 1, [ fliz mutant]; P LtetO-1 flhdc-lacz, 1, [wild type] versus 1, [ fliz mutant]). Based on these results, we conclude that FliZ is a posttranslational regulator of FlhD 4 C 2 protein levels. FliZ enhances both swimming and swarming motility. The goal of the flagellar regulatory network is to make sure that bacterial cells committed to moving have sufficient flagella to efficiently reach their desired destination. Therefore, to understand the role that FliZ regulation plays in motility, we tested FIG. 7. FliZ enhances both swimming and swarming motilities. (A) Swimming on tryptone broth plates with 0.3% agar. (B) Swarming on LB plates with 0.6% agar and 0.5% glucose. (C) Swarming on tryptone broth plates with 0.6% agar and 0.02% Tween 80. The first column shows the results for the wild type, the second for the fliz mutant, the third for the fliz mutant complemented with the fliz gene under the control of its native promoter on a plasmid, and the fourth the fliz mutant complemented with the fliz gene under the control of a constitutive promoter on a plasmid. For all motility and swarming experiments, the plates were incubated at 37C for 8 h. the ability of fliz mutants to swim through liquids and swarm on surfaces. In the case of liquids, we observed that the fliz mutant formed smaller rings than the wild type on motility plates (Fig. 7A). While there is a reduction in the ability to form rings, consistent with reduced flagellar gene expression, the fliz mutant is still motile. These results indicate that FliZ is not essential for swimming motility, though deleting it causes reduced swimming efficiency as determined by motility plates. Complementing this mutant with fliz constitutively expressed from a plasmid, however, did not restore ring size. In addition, the ring morphology changes, with a diffuse ring phenotype observed in the fliz pfliz strain compared to the sharp rings observed in the fliz mutant and the wild type. In contrast, we were able to partially restore the ring size when fliz was expressed using its native fliazy promoter on a plasmid, in the process recapitulating a similar result by Ikebe and coworkers (26). These results imply that the timing and regulation of fliz expression play an important role in motility. Apart from swimming in liquid viscous media, S. enterica serovar Typhimurium also employs flagella to swarm on solid surfaces (21). We therefore tested the swarming phenotype of a fliz mutant using two different medium recipes. In the first case, where we used LB medium with 0.6% agar and 0.5% glucose (34), we observed reduced swarm diameters in the fliz mutant relative to the wild type (Fig. 7B). We were able to restore the swarm diameter by expressing fliz from both a constitutive promoter and its native one on a plasmid. In the second case, where we used TB medium with 0.6% agar and 0.02% Tween 80 (49), we observed that the fliz mutant strain was unable to swarm whereas the wild type could (Fig. 7C). When the fliz mutant was complemented with fliz expressed from either a constitutive promoter or its native one on a plasmid, the swarming phenotype was partially restored. As a

8 4986 SAINI ET AL. J. BACTERIOL. comparison, we note that FliT has no effect on swarming (results not shown). We also measured P class2 and P class3 activity in swarming cells using the LB medium recipe. Consistent with our results for swimming cells, we observed a decrease in gene expression in the fliz mutant relative to the wild type (P flga, [wild type] versus [ fliz mutant]; P flic, [wild type] versus [ fliz mutant]). Similar results were also obtained in a P flhdc ::tetra background (data not shown). The simplest interpretation regarding the swarm results is that the cells make insufficient flagella when FliZ is not expressed. Depending on the medium/environment, these changes can have either a minor or a profound effect. Finally, we note that we also measured swarming in S. enterica serovar Typhimurium LT2. In this strain, we observed only a minor decrease in the swarm diameter in the fliz mutant irrespective of the medium. Likewise, FliZ s effect on gene expression in LT2 was also less pronounced (results not shown). DISCUSSION We have shown that FliZ is an FlhD 4 C 2 -dependent activator of P class2 activity that acts, at least in part, at the level of FlhD 4 C 2 protein. Deleting fliz causes approximately a 50% decrease in P class2 activity and FlhC protein levels. Likewise, overexpressing FliZ roughly doubles P class2 activity. Furthermore, we have shown that FliZ functions independently of 28 and FliT. In the case of 28, we have shown that it does not directly activate P class2 promoters. Rather, the ability of 28 to influence P class2 activity is indirect through FliZ, as the two are under the control of both P class2 and P class3 promoters. This indicates that fliz expression is partly responsive to 28 activity and, as a consequence, late protein secretion. Finally, an interesting finding was that FliZ had a strong effect on swarming motility for one specific medium recipe. In particular, we found that a fliz mutant was unable to swarm on TB medium supplemented with Tween 80. However, only a moderate reduction in swarming was observed when a more traditional recipe was used. These results potentially imply that the specific environment places differential demands on noncanonical flagellar regulators such FliZ. We still do not know how FliZ increases FlhD 4 C 2 protein levels. Our data suggest that FliZ does not directly increase protein translation rates but rather enhances the stability of FlhD 4 C 2. Note that as we measured only FlhC protein levels and translation rates, it is not yet clear whether FliZ is also affecting FlhD. As for a possible mechanism, sequence analysis indicates that FliZ shares no similarity to any other known regulator. However, FliZ does possess a SAM-like phage integrase domain (PFAM PF02988) (14, 52). The presence of this domain suggests that FliZ is a DNA-binding protein. In the related insect pathogen Xenorhabdus nematophila, FliZ was recently shown to bind to the flhdc promoter and increase the rate of transcription (38). As the FliZ proteins from S. enterica serovar Typhimurium and X. nematophila share 56% sequence identify, these results suggest that FliZ is also a transcription factor in S. enterica serovar Typhimurium. However, transcriptional regulation of the flagellar genes in S. enterica serovar Typhimurium and X. nematophila is different despite the fact that the organisms share common regulators. For example, transcription of the fliaz operon in X. nematophila is FlhD 4 C 2 dependent, whereas the equivalent fliazy operon is under independent control of both P class2 and P class3 promoters in S. enterica serovar Typhimurium (26, 48). Likewise, our data show that FliZ is not a transcriptional regulator of the flhdc operon in S. enterica serovar Typhimurium, whereas it is in X. nematophila. Furthermore, our data suggest that FliZ regulates the FlhD 4 C 2 complex posttranslationally. Based on the results from X. nematophila regarding FliZ being a DNA-binding protein, the most likely mechanism in S. enterica serovar Typhimurium is that FliZ increases the expression of some protein that stabilizes FlhD 4 C 2 or, alternatively, inhibits the expression of a protease such as ClpXP that degrades it. Furthermore, such promiscuity in FliZ DNA binding between the organisms is not altogether unreasonable, as the greatest difference between the two FliZ orthologs is at the C terminus, the region predicted to bind DNA based on the homology to the SAM-like phage integrase domain (38). In the overall context of assembly, our data suggest the following model for FliZ-dependent regulation. Prior to completion of the HBB, FlhD 4 C 2 can only partially activate P class2 promoters due to low protein levels arising from weak FliZ expression. However, when the first few HBBs are completed and FlgM is secreted at a sufficient rate such that 28 is active, FliZ expression then increases. Note that FliZ is under the control of both P class2 and P class3 promoters, where the latter is the dominant promoter. Increased FliZ expression then leads to increased stability of FlhD 4 C 2 and, as a consequence, further activation of the P class2 promoters. This mechanism suggests that P class2 activity is coupled to assembly. Unlike the case for P class3 promoters, where the coupling results in a strict binary checkpoint, P class2 activity is tuned in response to assembly. In other words, the cell increases P class2 activity once the first few HBBs are complete. In support of this model, we have recently shown that in HBB mutants P class2 expression rates are reduced, consistent with reduced FliZ regulation prior to HBB completion (Brown et al., submitted). The current model for flagellar gene regulation focuses predominantly on initiation, when cells transition from a nonflagellated to a flagellated state (1, 9, 44). Key to regulating initiation is the 28 -FlgM checkpoint. Once cells already possess flagella, however, this checkpoint is no longer relevant, as FlgM is being continuously secreted from the cell. The fact that cells already possess flagella does not mean that the assembly process no longer needs to be regulated. Rather, different regulatory tasks and their associated mechanisms become necessary. In particular, cells need to ensure that they have sufficient flagella, not too many or too few. Furthermore, every time the cell divides, the progeny need to reinitiate the assembly process to ensure that they have sufficient flagella for efficient motility. This regulation means that cells need to continually monitor assembly and adjust gene expression accordingly. We speculate that FliZ is one element of this regulation, providing cells with a mechanism for increasing gene expression in response to assembly. Note that FliT has the reciprocal effect of FliZ. Like fliz expression, flit expression is under the control of both P class2 and P class3 promoters, where again the latter is dominant. This means that flit is maximally expressed when its P class3 promoter is active, concomitant with the completion of the first