Molecular Microbiology (2001) 39(6), 1623±1637

The crescentus flagellar gene, fliX, encodes a novel trans-acting factor that couples flagellar assembly to transcription

Rachel E. Muir, Tina M. O'Brien and James W. Gober* Introduction Department of Chemistry and Biochemistry and the, The dimorphic bacterium, Caulobacter crescentus, under- Molecular Biology Institute, University of California, goes an asymmetric division every , resulting in Los Angeles, CA 90095-1569, USA. the formation of dissimilar progeny cells: a motile, -bearing swarmer cell and a sessile stalked Summary cell. The biogenesis of the polar flagellum is the best- studied aspect of this programme of cell differentiation The first flagellar assembly checkpoint of Caulobacter (reviewed in Brun et al., 1994; Gober and Marques, 1995; crescentus couples assembly of the early class II Wu and Newton, 1997; Gober and England, 2000). The components of the basal body complex to the temporal synthesis of the flagellum is subject to a complex expression of class III and IV genes, which encode regulatory hierarchy that functions to co-ordinate the extracytoplasmic structures of the flagellum. The expression of approximately 50 flagellar genes with transcription of class III/IV flagellar genes is activated progression through the cell cycle and the developmental by the response regulator factor, FlbD. Gain of function state of the flagellum. Caulbacter crescentus flagellar mutations in flbD,termedbfa, can bypass the tran- genes have been grouped, according to epistasis data, scriptional requirement for the assembly of class II into a regulatory hierarchy of four classes (Bryan et al., flagellar structures. Here we show that the class II 1984; Ohta et al., 1985, 1991; Champer et al., 1987; flagellar gene fliX encodes a trans-acting factor that Minnich and Newton, 1987; Newton et al., 1989; Xu et al., couples flagellar assembly to FlbD-dependent tran- 1989; Ramakrishnan et al., 1994; Mangan et al., 1995). scription. We show that the overexpression of fliX can Class I of the hierarchy is occupied by ctrA which encodes suppress class III/IV gene expression in both wild-type an essential transcription factor that couples the expres- and flbD-bfa cells. Introduction of a bfa allele of flbD sion of early flagellar genes (class II) to the initiation of into cells possessing a deletion in fliX restores motility DNA replication (Quon et al., 1996; Reisenauer et al., indicating that FliX is not a structural component of the 1999). These early flagellar genes encode the first flagellum, but rather a trans-acting factor. Furthermore, assembled components of the basal body including the extragenic motile suppressors which arise in DfliX MS ring (fliF), switch (fliM, fliN) and the flagellum-specific cells map to the flbD locus. These results indicate that secretory system (flhA, fliI, fliJ, fliO, fliP, fliQ, fliR) FlbD functions downstream of FliX in activating class (reviewed in Brun et al., 1994; Gober and Marques, III/IV transcription. b-Lactamase fusions to FliX and 1995; Wu and Newton, 1997; Gober and England, 2000) analysis of cellular fractions demonstrate that FliX is a (see Fig. 7), as well as a transcription factor encoded by cytosolic protein that demonstrates some peripheral flbD (Ramakrishnan and Newton, 1990; Wingrove et al., association with the cytoplasmic membrane. In 1993; Wingrove and Gober, 1994; Benson et al., 1994a, addition, we have isolated a mutant allele of fliX that b; Mullin et al., 1994). Class II flagellar genes share a exhibits a bfa-like phenotype, restoring flbD-dependent conserved promoter sequence that serves as a binding class III/IV transcription in strains that contain muta- site for CtrA, which is phosphorylated at a specific time in tions in class II flagellar structural genes. Taken the cell cycle just after the initiation of DNA replication together, these results indicated both a positive and (Domian et al., 1997). negative regulatory function for FliX in coupling the The expression of class III flagellar genes which encode assembly of class II basal body components to gene the rod (flgF, flgG) and outer rings of the basal body (flgH, expression. flgI, flaD), the hook (flgE) and hook-associated proteins (flgK, flgL) is influenced both by the progression of the cell-division cycle and the assembly of early flagellar structures (Newton et al., 1989; Xu et al., 1989; Mangan Accepted 8 January, 2001. *For correspondence. E-mail gober@ et al., 1995) (see Fig. 7). The translation of class IV genes chem.ucla.edu; Tel. (11) 310 206 9440; Fax (11) 310 206 5213. which encode the flagellins (fljK, fljL, fljM, fljN, fljO), in

Q 2001 Blackwell Science Ltd 1624 R. E. Muir, T. M. O'Brien and J. W. Gober turn, requires the assembly of the hook structure relaying the assembly status of the early basal body (Anderson and Newton, 1997; Mangan et al., 1999; structure to the signal transduction pathway regulating Anderson and Gober, 2000). The transcription of both FlbD-dependent class III and IV flagellar gene expression. class III and a subset of IV genes (fljK, fljL) requires s54- containing RNA polymerase, integration host factor and the response-regulator, transcriptional activator FlbD Results (reviewed in Brun et al., 1994; Gober and Marques, Overexpression of fliX suppresses class III/IV flagellar 1995; Wu and Newton, 1997; Gober and England, 2000). gene expression in a C. crescentus bfa mutant The cell cycle-regulated phosphorylation of FlbD directs the timed transcription of class III and class IV genes Previously, we isolated mutant strains (bfa) in which class (Wingrove et al., 1993) and is thought to be influenced, at III/IV flagellar gene transcription no longer required early least in part, by a cell-cycle event, possibly related to the class II flagellar structures (Mangan et al., 1995). We initiation of cell division. The expression and successful reasoned that bfa was either a null mutation in a repressor assembly of class II flagellar structural gene products is or a gain of function mutant resulting in constitutive also required for transcription of class III and some class activation of class III/IV gene expression. We have IV genes (fljL) (Newton et al., 1989; Xu et al., 1989; recently found that the bfa mutant strains contain a gain Mangan et al., 1995; Anderson and Newton, 1997). For of function mutation in the transcription factor, FlbD (R. A. example, epistasis experiments have demonstrated that Muir and J. W. Gober, in preparation). During the course strains bearing a mutation in any one of the early class II of experiments designed to identify bfa, we constructed a structural genes do not transcribe FlbD-dependent, class random library of wild-type C. crescentus DNA containing III genes. We have isolated mutants, designated bfa, for an average fragment length of 5 kb in a multicopy vector bypass of flagellar assembly that permit the expression of (see Experimental procedures). This library was intro- class III flagellar genes in strains bearing class II duced into strain UC1040, which contains a deletion in the mutations (Mangan et al., 1995). We have recently class II flagellar genes fliQ and fliR, the bfa-1204 allele, found that the bfa mutant strains contain gain-of-function and a class IV fljL±lacZ reporter fusion. The presence of mutations in the gene encoding the transcription factor, the wild-type bfa gene in a clone would be expected to FlbD (R. A. Muir and J. W. Gober, in preparation). The bfa result in a decrease in b-galactosidase generated from mutant strains have defects in the cell cycle timing of fljL promoter activity. class III gene transcription characterized by a delay in the Transformants were selected for the presence of both cessation of transcription at the end of the cell cycle the reporter and library plasmids and then screened for (Mangan et al., 1995). This result indicates that the b-galactosidase activity using an agar overlay method progression of flagellar assembly, at least in part, (Mangan et al., 1995). Colonies displaying lower levels influences temporal transcription. of b-galactosidase activity were removed from the over- In this paper, we identify the fliX gene product as a lay, grown on selective agar medium overnight and component of the signal transduction pathway that rescreened the next day using the same method. Four couples the assembly of class II-encoded structures to potential transformants, from over 10 000 screened, class III/IV flagellar gene transcription. During preliminary possessed lower levels of b-galactosidase activity. Of efforts to identify bfa, we attempted to complement the these, only one generated significantly lower levels of mutation in a class II/bfa double mutant with a wild-type b-galactosidase activity in a quantitative measurement on genomic library. We found that overexpression of the cells grown in liquid culture (1049 units vs. 10 376 units in class II flagellar gene, fliX, on one clone could suppress cells not containing the plasmid) (Fig. 1). Note that the class III/IV flagellar gene expression in bfa mutant strains. fljL-lacZ reporter generates 240 units of b-galactosidase In addition, we show that the original flbD bfa alleles can in the parent DfliQR strain (SC508) containing a wild-type restore motility in a strain bearing a deletion in fliX, allele of flbD. The library construct, pX10, harbored by the indicating that fliX encodes a trans-acting factor rather transformant of interest was isolated, reintroduced than an essential structural component of the flagellum. through bacterial conjugation into UC1040 containing Analysis of subcellular fractionated cells revealed that fliX the fljL±lacZ fusion, and verified to be responsible for encodes a cytosolic protein that exhibits some association the decrease in reporter activity displayed by the original with the cytoplasmic membrane. We also have isolated a transformant. mutant allele of fliX that possesses a bfa-like phenotype The positive clone was subjected to restriction digest and is able to restore class III/IV flagellar gene expression and 803 and 663 bp PstI fragments were used to map its in strains containing mutations in early class II flagellar physical position on the C. crescentus genome using a structural genes. These results indicate that FliX functions pulse-field gel blot of wild-type DNA. Unexpectedly, the as both a positive and negative regulator responsible for clone did not map to the position previously determined

Q 2001 Blackwell Science Ltd, Molecular Microbiology, 39, 1623±1637 Regulation of flagellar biogenesis in Caulobacter 1625

Fig. 1. Effect of overexpressing fliX on fljL/lacZ expression in a class II/bfa double mutant. b-Galactosidase assays were performed on the class II/bfa double mutant strain UC1040 (DfliQ)/bfa-1204) harbouring the flagellar gene reporter pfljL/lacZ/29021 alone and in combination with the pBBR1-MCS plasmid carrying the C. crescentus genomic inserts. The largest clone, pX10 insert depicted at the top right, consists of a 200 bp Sau3A fragment and a 3.2 kb Sau3A fragment encompassing the first 124 bp of the dskA gene, fliX, and the entire flgI operon plus 330 bp of downstream DNA. The 1.4 kb insert of pX09 contains the fliX promoter and the fliX gene as the only full-length ORF on the clone. pDX09 contains an insert deleted for a functional fliX gene. The pDX09 insert, derived from pX09, lacks the internal 243 bp PstI fragment of fliX as illustrated at the bottom right. The effect each construct on fljL/lacZ reporter gene expression in UC1040 is indicated as units of b-galactosidase activity. for bfa (flbD) (Mangan et al., 1995), but instead mapped to additional copy of fliX (data not shown). To confirm that a region of the genome where the divergently transcribed the increased expression of the class II fliX gene was flagellar genes fliX (class II) and the flgI operon (class III) responsible for suppressing the effect of the bfa mutation, were located (Khambaty and Ely, 1992; Mohr et al., 1998) a clone lacking a functional copy of fliX was constructed (data not shown). The 50 and 30 ends of the C. crescentus by removing the internal 243 bp PstI fragment of fliX from DNA in pX10 were sequenced. With the use of data pX09, generating pDX09. The presence of this clone in available in the GenBank database and from the C. UC1040 failed to decrease the expression of the fljL±lacZ crescentus genome sequencing project, sequence data fusion indicating that the overexpression of fliX was obtained from pX10 verified the presence of the entire fliX responsible for suppressing the bfa effect (Fig. 1). gene and flgI operon within the genomic clone (Fig. 1). The signal transduction pathway coupling early class II FliX encodes a novel trans-acting regulatory factor flagellar assembly to gene expression culminates in influencing the activity of FlbD (Newton et al., 1989; Xu Previously, epistasis experiments placed fliX at the level et al., 1989; Mangan et al., 1995). We reasoned that a of class II genes in the trans-acting flagellar regulatory class II gene on pX10 was responsible for modulating the hierarchy (Mohr et al., 1998). Typical of class II mutants, a activity of the flbD-1204 (bfa) mutant, and therefore strain bearing a deletion in fliX, lacks late flagellar gene created a subclone, pX09, which deleted the class III expression, is non-motile and exhibits a class II mutant genes present in pX10 (Fig. 1). This subclone was cell division phenotype consisting of filamentous growth introduced into the UC1040 tester strain and its effect beginning at late log phase (Fig. 2). It had been previously on fljL transcription was determined (Fig. 1). The pres- suggested that FliX might function as a component of the ence of fliX in pX09 reduced b-galactosidase expression flagellum that is required for either assembly or motility in UC1040 (pfljL/lacZ/290) to levels 12% of that generated (Mohr et al., 1998), therefore, accounting for the class II in the same strain lacking an extragenic copy of fliX phenotype displayed by the DfliX strain. We have (Fig. 1). Immunoblot analysis confirmed the increase in demonstrated that overexpression of fliX can partially FliX protein levels of these strains containing the reverse the effect of a bfa mutation in flbD on late flagellar

Q 2001 Blackwell Science Ltd, Molecular Microbiology, 39, 1623±1637 1626 R. E. Muir, T. M. O'Brien and J. W. Gober

Fig. 2. The bfa-1204 allele of flbD suppresses the class II cell division phenotype and restores late flagellar gene expression in cells containing a fliX deletion. (A) Phase contrast micrographs of wild-type (LS107), (B) DfliX and (C) DfliX/bfa-1204 cells in late log phase (OD600 ˆ 1.3± 1.5). (D) Comparison of class II and III/IV flagellar gene expression in the C. crescentus strains shown in (A±C). Transcription was assayed using the class II (fliF±lacZ and fliX±lacZ) and class III and IV (flbG±lacZ, flgI±lacZ and fljL±lacZ) reporter fusions. Promoter activity is reflected as units of b-galactosidase activity. gene expression in a class II/bfa double mutant (Fig. 1). for at least 4 days along with wild-type and DfliX cells for These results raised the possibility that the fliX gene comparison (Fig. 3A). Interestingly, motile suppressors to product may influence the regulation of flagellar gene the DfliX strain readily arose when the strain was expression. Consistent with previous observations (Mohr inoculated into the motility agar (Fig. 3B±D). These motile et al., 1998), a strain bearing a deletion in fliX had cells termed msx (for motile suppressor of fliX) were markedly decreased activity of flbG, flgI and fljL class III/ isolated, reinoculated into semisolid medium and like the IV flagellar promoters (Fig. 2). In addition, this strain DfliX/bfa double mutants were able to generate swarms expressed the class II fliF and fliX promoters at levels smaller than wild-type cells (Fig. 3A) and contained motile exceeding wild type, a phenomenon characteristic of cells as assayed by microscopy. Both PCR and immuno- class II flagellar mutant strains (R. A. Muir and J. W. blot analysis showed that the original fliX deletion was Gober, in preparation) (Fig. 2). We next tested the effect present in both the motile DfliX/bfa and DfliX/msx strains of a flbD-1204 (bfa) mutation on class III/IV flagellar gene (data not shown), indicating that these extragenic muta- expression in a strain containing a fliX deletion (Fig. 2). tions could restore motility to DfliX cells. One of these The bfa mutation could restore late flagellar gene mutant msx alleles (msx-3) was genetically linked to the expression to the DfliX mutant (Fig. 2). In addition, class transposon Tn5 and its location on the C. crescentus II promoter activity decreased to a level less than that of a genetic map was determined using transduction and wild-type strain (Fig. 2). Also, as seen in a number of pulse-field gel electrophoresis. The msx-3 mutation class II/bfa double mutants (R. A. Muir and J. W. Gober, in mapped to the same chromosomal region as the original preparation), a normal pattern of cell division was restored bfa alleles of flbD, suggesting that msx-3 is allelic to flbD to the DfliX/bfa double mutant strain (Fig. 2). (data not shown). We have confirmed, by sequence Most surprisingly, in contrast to other class II/bfa double analysis, that the strain bearing the msx-3 allele, like the mutants, the DfliX/bfa cells were motile. This result original bfa strains, contains a mutation in flbD altering indicates that fliX does not encode an essential structural codon 451 from encoding a valine to a glycine. This component of the flagellum, but rather is a trans-acting mutant flbD allele could also restore motility to a DfliX regulatory factor. In order to demonstrate the restoration strain when introduced into the genome on a plasmid of motility to DfliX mutant cells by the bfa mutation, DfliX/ (R. A. Muir and J. W. Gober, in preparation). In addition, bfa double mutants were grown in semisolid motility agar this mutation was introduced by transduction into several

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Fig. 3. The flbD bfa mutation bypasses the requirement of fliX for motility. A. C. crescentus cells from LS107 (wild type; w.t.), JG1172 (DfliX), JG1177 (DfliX/bfa-1204) and JG1173 (DfliX/msx3) were inoculated into motility agar and grown for 4±6 days. Motile cells swarming away from the points of inoculation are visualized as halos. Only strain JG1172 (DfliX) lacks the concentric ring of swarming cells that is indicative of a motile inoculant. Both the bfa-1204 and msx3 mutations restored motility to strain JG1172 cells. (B±D) Magnified images of the JG1172 (DfliX) colony from (A) after 4,5 and 6 days of growth. B. JG1172 (DfliX) colony after 4 days in motility agar. The motile suppressor mutants are readily visible as a motile flare of cells. C. The same colony of JG1172 (DfliX) cells after 5 days in motility agar. D. The same colony of JG1172 (DfliX) cells after 6 days in motility agar. The flares of motile cells were clearly visible after 5±6 days. Strain JG1173 (DfliX/msx3) seen in (A) was isolated from a motile flare generated in this manner. class II strains harbouring the pfljL/lacZ/290 reporter class II/bfa double mutants. Initially, membrane and plasmid and determined to have the same effect on late combined periplasmic/cytosolic fractions were prepared flagellar gene expression as the original bfa mutations from wild-type cells. Surprisingly, these preliminary (data not shown) (Mangan et al., 1995). These results experiments with fractionated wild-type cells revealed suggest that FliX functions as a trans-acting factor, that the majority of FliX resided in the cytosolic fraction upstream of FlbD, in a signal transduction pathway with a lesser, but significant, amount present in the coupling flagellar assembly to gene expression. membrane fraction (Fig. 4). To test whether the portion of FliX in the membrane fraction was integral, membrane samples were washed FliX is a cytosolic protein that demonstrates peripheral with buffer or buffer containing either 6 M urea, 0.5% association with the cytoplasmic membrane Triton X-100, or 0.2% Sarkosyl (Fig. 4). Treatment with It had been previously reported that the amino terminus of buffer alone removed some of the FliX associated with the FliX contained a putative cleavable signal sequence and membrane fraction suggesting FliX functions as a at least one transmembrane domain suggesting that FliX peripherally associated membrane protein. FliX was might function extracytoplasmically (Mohr et al., 1998). completely removed by urea (Fig. 4), which preferentially Furthermore, it was demonstrated that FliX associated solubilizes peripherally associated membrane proteins, and with the membrane of fractionated cells with some protein Sarkosyl, which preferentially solubilzes inner membrane present in the cytosolic fraction (Mohr et al., 1998). proteins (Filip et al., 1973). Triton X-100 was also able to Overexpression of FliX partially reversed the effect of bfa solubilize most of the FliX present in the membrane fraction mutations on flagellar gene expression (Fig. 1). It is (Fig. 4). These results suggest that FliX is a cytosolic possible that this is a consequence of the missorting of protein that is peripherally associated with the membrane. FliX to the cytosol. In order to test this idea, we wanted to The fractionation procedures that we employed do not determine the subcellular localization of FliX in class II vs. completely rule out the possibility that FliX is associated

Q 2001 Blackwell Science Ltd, Molecular Microbiology, 39, 1623±1637 1628 R. E. Muir, T. M. O'Brien and J. W. Gober

Fig. 4. Association of FliX with the cytoplasmic membrane. Individual isolated cellular fractions were suspended in sample loading buffer and subjected to SDS±PAGE and then immunoblot analysis using antiserum to C. crescentus FliX. Equal protein concentrations of the cytosolic/ periplasmic and membrane samples were applied to the gel. Caulobacter crescentus LS107 whole cell extract (lane 1), combined cytosolic/ periplasmic fractions (lane 2) and membrane fraction (lane 3). FliX was present in both the cytosolic/periplasmic and membrane fractions with the majority of FliX appearing in the cytosolic/periplasmic fraction. A portion of the membrane fraction was treated with buffer alone (lanes 4 and 5) or with buffer containing 6.0 M Urea (lanes 6 and 7), 0.5% Triton X-100 (lanes 8 and 9), or 2% Sarkosyl (lanes 10 and 11). After treatment, soluble (lanes 4, 6, 8, and 10) and insoluble (lanes 5, 7, 9, 11) protein fractions were isolated. Purity of the membrane and cytosolic fractions was determined by immunoblot analysis utilizing anti-Mcp antibody (membrane control) (lanes 12 and 13) and anti-ParB antibody (cytosolic control) (lanes 14 and 15). with the cytoplasmic membrane within the periplasmic containing these fusions were unable to grow in media space. In order to test this, amino-terminal portions of FliX containing as little as 1.5 mgml21 ampicillin, a result were fused to b-lactamase lacking a periplasmic targeting consistent with previously published findings of b-lacta- sequence and resistance to ampicillin was determined mase fused to the cytoplasmically exposed residues in (Fig. 5). The sorting of b-lactamase to the periplasm FliF (Jenal and Shapiro, 1996). These results, in addition confers resistance to ampicillin. This technique has been to the lack of any signal sequence predicted for FliX by the utilized in C. crescentus to map the amino acid residues PSORT program (http://psort.nibb.ac.jp) (Nakai and Kane- exposed to the periplasm in the integral flagellar hisa, 1991), indicates that FliX is not probably targeted membrane protein, FliF (Jenal and Shapiro, 1996). An to the periplasm by a cleavable signal sequence and amino-terminal fragment of FliX consisting of the first 26 strengthen the argument of FliX functioning as a cytosolic amino acids of the protein containing the putative protein that demonstrates some peripheral association cleavable signal sequence, responsible for targeting the with the cytoplasmic membrane. protein to the periplasm by the secA-dependent pathway, was fused to b-lactamase (Fig. 5A). b-Lactamase was Isolation of a fliX mutant that exhibits a bfa phenotype also fused to an amino-terminal fragment containing the first 76 amino acids of FliX, where it would follow the first The class II phenotype displayed by the DfliX strain and putative transmembrane domain (Fig. 5A). In addition, a the negative influence of overexpression of fliX on class third fusion of full-length FliX was constructed (Fig. 5A). III/IV gene expression suggested that FliX functions as The b-lactamase fusion constructs were introduced into both a positive and negative regulator of gene expression. C. crescentus strain LS107, which contains a deletion in Furthermore, our experiments indicated that FliX may the gene encoding b-lactamase (Dbla). The expression of function upstream in a signal transduction pathway each of the fusions was then determined using antib- coupling flagellum assembly to class III/IV gene expres- lactamase and anti-FliX antibodies (Fig. 5B). The 1- to 26- sion. If this is the case, then it should be possible to isolate amino-acid fusion migrated with an apparent molecular mutant alleles of fliX that exhibit a bfa phenotype (i.e. weight of 35 kd. The 1- to 76-amino-acid fusion had an restore class III/IV gene expression in class II flagellar apparent molecular weight of 41 kd, and the full-length mutant strains). In order to test this, the fliX containing fusion of 45 kd (Fig. 5B). The antib-lactamase probed blot plasmid, pX09, was subjected to mutagenesis (see of protein from cells expressing the 1- to 26-amino-acid Experimental procedures) by propagation in the Escheri- fusion revealed no visible band at the 31 kd position chia coli mutator strain, XL1-red. The mutated plasmids predicted to be occupied by b-lactamase after cleavage of were pooled and transformed into strain JG1189 which the putative signal sequence. None of the b-lactamase contains a deletion in fliX,aTn5 insertion in the class II fusions could confer ampicillin resistance to Dbla strains flagellar gene, fliP and a fljL±lacZ reporter fusion. compared with that displayed by the naturally ampicillin- Transformants were screened for the restoration of fljL resistant C. crescentus strain NA1000 (Fig. 5C). Cell promoter activity using an Xgal agar overlay and potential

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Fig. 5. Amino-terminal fragments of FliX fused to b-lactamase indicate that FliX is a cytosolic protein. A. Diagram illustrating the amino-terminal fragments of FliX used in the construction of FliX::b-lactamase fusions. A Kyte Doolittle hydropathy plot of FliX is shown above to illustrate the regions of hydrophobicity and putative transmembrane domains, contained in the N-terminal fragments of FliX. Cross bars below represent the fragments chosen to fuse to b-lactamase for determining cellular localization of FliX function. B. An immunoblot of protein extracts using antib-lactamase antibody. Lane 1, prestained molecular weight standards; Lane 2, strain JG1172 (DfliX/bla-6); Lane 3, strain JG1172 expressing a FliX::b-lactamase fusion protein containing the first 26 amino acids of FliX (1±26 aa); Lane 4, strain JG1172 expressing a FliX::b-lactamase fusion protein containing the first 76 amino acids of FliX (1±76 aa); Lane 5, strain JG1172 expressing a FliX::b-lactamase fusion protein containing full-length FliX (1±44 aa). Apparent molecular weights of the fusion proteins are noted. The 28 kd band is possibly a degradation product common to only those cells expressing FliX::b-lactamase fusions; it is recognized by antibodies to both FliX and b-lactamase and is absent from the DfliX strain without a fusion. C. Counter clockwise starting with the top right, the C. crescentus strains NA1000 (ampicillin resistant), DfliX (ampicillin sensitive), and DfliX containing the 1±26 aa. FliX-b-lactamase fusion, the 1±76 aa FliX-b-lactamase fusion, and the full-length (1±144 aa) FliX-b-lactamase fusion in pMR4 are shown after 4 days of growth on PYE agar containing tetracycline (top) and ampicillin (bottom). All fusions were expressed in the DfliX strain from pMR4, which confers tetracycline resistance to the DfliX strain as shown at the top. None of the fusions were found to confer ampicillin resistance (bottom) to the DfliX strain compared with that of the naturally ampicillin-resistant NA1000 strain. positives were isolated, grown in liquid culture and Table 1. The mutant fliX1 allele exhibits bfa-like activity. subjected to a quantitative b-galactosidase assay. Two b-Galactosidase activity (U)a mutagenized pX09 clones, from approximately 5000 screened, exhibited an increase in b-galactosidase fljL/lacZ expression from the fljL±lacZ reporter fusion compared with the same strain containing wild-type fliX on a plasmid Strain Vector Wild-type fliX fliX1

(8976 vs. 283 units) (Table 1). Sequence analysis Wild type 9981 7452 8425 revealed that the two mutagenized pX09 clones contained flbD 256 333 255 identical fliX mutant alleles. fliP 262 203 3133 fliLM 303 296 4977 The plasmid containing the bfa-like mutant fliX allele bfa 20671 11383 19338 (fliX1) was then introduced into several class II mutant fliX 296 9888 11812 strains. In order to prevent recombination between the fliX flbD 331 287 335 fliX fliP 224 283 8976 fliX1 allele on the plasmid with the fliX locus on the fliX fliLM 407 320 8355 chromosome, a recA mutation was introduced into each fliX bfa 22497 16858 19399 of these strains, and the effect on fljL/lacZ expression was determined (Table 1). The presence of fliX1 was incap- a. The reported values are the mean of assays performed in triplicate on three independently grown cultures. The mean standard deviation able of restoring fljL expression in a strain bearing a Tn5 for all reported values was less than 5%.

Q 2001 Blackwell Science Ltd, Molecular Microbiology, 39, 1623±1637 1630 R. E. Muir, T. M. O'Brien and J. W. Gober insertion in flbD indicating that FliX functions upstream of expression in a strain containing a bfa allele of flbD.To FlbD in the activation of class III/IV gene expression. The test whether the fliX1 mutant retained this capacity we mutant fliX allele could, however, restore fljL expression in introduced it into a bfa mutant strain containing the fljL± fliM and fliP mutant strains (Table 1), as well as in several lacZ reporter fusion. The presence of fliX1 had little effect other strains bearing mutations in class II flagellar in suppressing fljL expression (Table 1) in the bfa mutant structural genes (data not shown). The data presented indicating that the mutation had abolished this negative here indicate that the fliX gene product has both positive regulatory aspect of FliX function. and negative regulatory outputs: suppressing class III/IV The fliX mutant allele, fliX1, contained three different gene expression in the absence of flagellar assembly and point mutations and a one basepair deletion (Fig. 6). It activating gene expression when other class II gene possessed a transversion of a cytosine to a guanine at the products are present. If this is the case then the wild-type first base of the sixteenth codon changing the encoded allele of fliX in the fliM and fliP class II mutant strains amino acid from an arginine to a glycine, and a deletion of would be expected to be suppressing fljL expression. To a cytosine in codon 141 resulting in a silent mutation in test this idea, we introduced the plasmid containing the that codon, but generating a frameshift that extended the fliX1 allele into strains containing both a fliX deletion on open reading frame (ORF) of the fliX1 allele from 144 to the chromosome and a class II Tn5 mutation (fliP or fliM) 213 codons. The frameshift extended the carboxyl and assayed the effect on fljL/lacZ expression (Table 1). terminus of the fliX1 gene product by 67 amino acids; In both cases the expression of the fljL±lacZ fusion fliX encodes a 14.5 kd polypeptide and fliX1 encoded a increased significantly in the fliX/class II double mutant 23 kd polypeptide as determined by immunoblot analysis strain. For the fliX/fliP double mutant, b-galactosidase (data not shown). In addition, two transition mutations, levels were 2.9 times higher than the strain bearing only a which altered the predicted amino acid sequence, were mutation in fliP (Table 1), and for the fliX/fliM mutant, also found within this extended ORF. Immunoblot analysis levels were 1.7 times higher than the single fliM mutant. also revealed that the mutant fliX on a plasmid generated These results suggest that fliX actively suppresses class protein in amounts equal to that of the wild-type allele III/IV gene expression in the absence of class II flagellar present in single copy in the genome. To identify which gene products. We have also shown here that wild-type mutations in fliX1 were responsible for the bfa-like effect, fliX on a multicopy plasmid can suppress class III/IV gene hybrid gene constructs of the wild-type and fliX1 alleles

Fig. 6. Summary of the effect of mutant alleles of fliX on motility, suppression of flbD (bfa-1204) activity, and restoration of class III gene expression in class II mutants. A schematic of fliX alleles is depicted. Numbers indicate amino acid residues. Motility indicates the ability to complement the motility defect in strain JG1172 (DfliX). bfa suppression indicates the ability to suppress class III/IV gene expression in strains bearing a the flbD bfa-1204 mutation. bfa phenotype indicates the ability of the fliX allele to restore class III/IV gene expression in a class II flagellar mutant. Data for this summary is shown in Tables 1 and 2. Wild-type fliX (w.t.) complemented motility in a DfliX strain and suppressed FlbD-dependent gene expression in a bfa mutant strain (also see Fig. 1). The mutant fliX1 complemented motility in a DfliX strain, but was unable to suppress FlbD-dependent transcription in a bfa mutant strain when overexpressed. This mutation could, however, restore class III/IV gene expression in a class II flagellar mutant (Table 1). The fliX1 mutant contains a mutated codon in the amino terminus of the coding sequence (R16G) as well as a mutation at position 141 which creates a 21 bp frameshift extending the ORF of fliX by 67-amino-acid residues. In order to determine which of these mutations was responsible for conferring a bfa phenotype, mutant alleles of fliX were created that contained differing combinations of these mutations (see Results and Table 2). The mutant fliX1mutN which contains only the R16G mutation could complement motility in a DfliX strain, suppress class III/IV gene expression, but did not confer a bfa-like phenotype. The mutant derivative fliX1DC, which contains the R16G mutation and the 21 bp frameshift at codon 141 with an introduced stop codon, which retains the wild-type length of the fliX gene product, likewise, could complement motility in a DfliX strain, suppress class III gene expression, but did not confer a bfa-like phenotype. The mutant allele fliX1mutC possessed the 21 bp frameshift at codon 141 with the extended ORF containing two additional mutations (see text for details). This mutant could also complement motility in a DfliX strain but was unable to suppress FlbD- dependent gene expression. This mutation, however, was capable of restoring class III/IV transcription in a class II flagellar mutant (Table 2).

Q 2001 Blackwell Science Ltd, Molecular Microbiology, 39, 1623±1637 Regulation of flagellar biogenesis in Caulobacter 1631

Table 2. Effect of mutant fliX alleles on fljL promoter activity Discussion

b-Galactosidase activity (U)a Flagellar biogenesis in C. crescentus is regulated both by fljL/lacZ the cell cycle and the progress of flagellum assembly (reviewed in Brun et al., 1994; Gober and Marques, 1995; Strain fliX1 mutN fliX1 DC fliX1 mutC Wu and Newton, 1997; Gober and England, 2000). In this Wild type 5331 3983 7732 report we provide evidence that the product of the fliX flbD 318 274 254 gene encodes a novel trans-acting factor that couples the fliP 210 278 4922 assembly of early, class II flagellar structures (MS-ring/ fliLM 278 272 4241 bfa 5845 7728 18447 switch complex, secretion system) to the transcription of fliX 6295 6172 12509 class III and IV genes which encode the rod and external fliX flbD 323 312 301 rings of the basal body, the hook structure and one fliX fliP 271 294 6520 fliX fliLM 252 239 6103 flagellin gene (fljL). This conclusion is largely based on fliX bfa 4941 6763 18236 three experimental observations: (i) overexpression of fliX can suppress transcription of class III flagellar genes in a. The reported values are the mean of assays performed in triplicate on three independently grown cultures. The mean standard deviation both wild-type and flbD-bfa strains; (ii) introduction of for all reported values was less than 5%. a constitutive bfa allele of flbD into a strain bearing a deletion in fliX completely restores motility; and (iii) were created (Fig. 6) and their effects on fljL expression a frameshift mutation in the DNA encoding the carboxyl were assayed in the same class II mutant strains terminus of FliX exhibits a bfa phenotype, restoring class (Table 2). To determine whether the amino-terminal III/IV transcription in class II flagellar mutants. mutation of the fliX1 gene product alone was the cause The transcription of class III/IV flagellar genes requires of the bfa-like effect, a fliX allele containing only the R16G the phosphorylation of the response regulator domain of mutation (fliX1mutN) was constructed using PCR (Fig. 6). FlbD (Wingrove et al., 1993; Benson et al., 1994a). The To determine whether the altered amino acid sequence at activation of FlbD has been hypothesized to require that the C terminus of FliX1 was responsible for the bfa-like the C. crescentus pre-divisional cell progress to a certain phenotype, fliX1DC was constructed by reintroducing a stage in the cell cycle (Wingrove and Gober, 1996). The translational stop after codon 144 of the mutant allele fliX1 results presented here indicate that FliX, and by exten- (Fig. 6). To determine whether the extended ORF in fliX1 sion, the assembly status of the flagellum also influences was solely responsible for the bfa phenotype a mutant this signal transduction pathway. Because fliX is both containing an analogous single basepair deletion in codon required for class III/IV transcription and can suppress 144 was created via PCR generating fliX1mutC (Fig. 6). transcription when overexpressed, we envisaged that FliX These mutant versions of fliX were introduced into C. functions as both a positive and negative regulator of late crescentus and their effect on motility, the ability to flagellar gene expression. Although cellular fractionation suppress bfa, as well as restore fljL gene expression in experiments show FliX to be mostly cytosolic, a significant class II mutants was assayed (summarized in Fig. 6). Like amount of the protein was found to associate peripherally the original fliX1 allele, all these mutants could restore with the membrane fraction. It is possible, based upon the motility in a strain bearing a deletion in fliX (data not demonstrated association with the cytoplasmic mem- shown) (Fig. 6). Mutants, fliX1mutN and fliX1DC, had the brane, that FliX may play a direct role in sensing the same effect on gene expression as wild-type fliX. These assembly status of the early basal body structure. We mutants were unable to restore fljL/lacZ expression in propose that FliX exists in two states: functioning as a either fliP or fliM mutant strains (Table 2). In addition, they repressor of the FlbD signal transduction pathway in the were equally capable of suppressing fljL/lacZ expression absence of flagellar assembly and an activator when in bfa mutant strains (Table 2). In contrast, fliX1mutC, assembly is completed (Fig. 7). Although it is possible encoding the extended carboxyl terminus resulted in the that FliX may regulate FlbD activity through a direct restoration of fljL/lacZ expression in class II mutants, interaction, there is little precedent for modulating the generating similar levels of b-galactosidase (Table 2); as activity of response regulator proteins in this fashion. expected, this effect was also dependent on flbD. As was Rather, we favour a model where FliX regulates a sensor the case with the original fliX1 allele, fliX1mutC could not histidine kinase in the FlbD signal transduction pathway suppress the levels of fljL/lacZ reporter gene expression (Fig. 7). In this case, FliX may function to stimulate a in the bfa mutant strain (Table 2). These results show that phosphatase activity in the sensor kinase that phosphor- the bfa phenotype of the original fliX1 allele is solely ylates FlbD. This would be analogous to the regulation of attributable to the extended amino acid sequence of the the sensor histidine kinase NtrB in enteric encoded protein. (reviewed in Ninfa et al., 2000). The sensor kinase/

Q 2001 Blackwell Science Ltd, Molecular Microbiology, 39, 1623±1637 1632 R. E. Muir, T. M. O'Brien and J. W. Gober

Fig. 7. Model depicting FliX function in the signal-transduction pathway coupling flagellar assembly to gene expression in C. crescentus.We have demonstrated a role for FliX as a trans-acting factor that functions upstream of the transcription factor, FlbD. The experimental results indicate that FliX functions as both a positive and negative regulator of FlbD-dependent transcription. A schematic diagram of the relevant stages of flagellar assembly is depicted (OM: outer membrane; IM: inner membrane). In the absence of assembly we envisaged FliX interacting with the cognate kinase for FlbD (Y), forming an inactive complex. Assembly of a class II flagellar structure may transform FliX into an activator of the kinase. The kinase, in turn phosphorylates FlbD leading to class III/IV transcription (see text for details). response regulator pair, NtrB and NtrC, regulate the flagellar regulatory hierarchies contains a master regu- transcription of genes involved in nitrogen acquisition. The lator transcription factor: CtrA in C. crescentus (Quon et al. pool of available nitrogen in the cell is sensed by the PII 1996), and the FlhCD complex of S. typhimurium regulatory protein. When 2-ketoglutarate levels are (Komeda, 1986; Kutsukake et al., 1990), both of which relatively high in the cell, under conditions of low nitrogen, function with s70-containing RNA polymerase in the PII which can bind 2-ketoglutarate, does not interact with activation class II flagellar gene expression. In C. NtrB, and the kinase phosphorylates NtrC (Jiang and crescentus, the class II genes encode the MS-ring, switch Ninfa, 1999). When 2-ketoglutarate levels are relatively complex and secretory system. In contrast, in S. low (e.g. high nitrogen), PII interacts with NtrB and typhimurium, FlhCD activates the transcription of genes stimulates dephosphorylation of NtrC. A similar regulatory encoding these structures as well as the rods, outer rings scheme may be operating in the FlbD signal transduction of the basal body and the hook. In both C. crescentus and pathway. Here FliX may be activating the FlbD kinase in enteric bacteria, the master regulators also activate the the presence of an assembled flagellar structure and transcription of essential transcription factors: fliA stimulating phosphatase activity when the flagellum is not encodes the transcription factor s28 in enterics and flbD assembled (Fig. 7). We hypothesize that the decrease in encodes a s54-dependent response regulator, transcrip- class III/IV flagellar gene transcription evident in cells tion factor in C. crescentus. overexpressing FliX is a consequence of the presence of In C. crescentus, the regulatory hierarchy consists of a higher concentration of FliX in the cell that is not two flagellar assembly checkpoints that function to activated by flagellar assembly. Conversely, we propose regulate late flagellar gene expression during flagellum that the bfa-like allele, fliX1, is locked in a constitutively synthesis (reviewed in Brun et al., 1994; Gober and activated state. Identification of the FlbD kinase will permit Marques, 1995; Wu and Newton, 1997; Gober and a direct test of this idea. [Note that, we have previously England, 2000). One is the assembly of the MS-ring/ reported that the product of the flbE gene may be the switch/secretion system complex, which is required for cognate kinase for FlbD (Wingrove and Gober, 1996). We the expression of the outer rings, rods and the hook. The now have data that FlbE does not function as the FlbD other is the assembly of the hook, which is required for the kinase (Muir and Gober, in preparation).] expression of flagellins. The enteric regulatory hierarchy, The role of FliX in coupling the assembly of early class however, is limited to only one assembly checkpoint, II basal body structures to later class III and IV flagellar requiring the achievement of a single structural inter- gene expression, differs significantly from the mode of mediate (the hook) in order to activate expression of the regulation exercised in Salmonella typhimurium (reviewed final class of flagellar genes. The antisigma factor, FlgM, in Aizawa, 1996). S almonella typhimurium contains three of enteric bacteria functions to inhibit the s28-dependent levels within its flagellar regulatory hierarchy. The class I expression of class III flagellin genes, encoding the last level in both the C. crescentus and the S. typhimurium assembled component of the flagellum (Gillen and

Q 2001 Blackwell Science Ltd, Molecular Microbiology, 39, 1623±1637 Regulation of flagellar biogenesis in Caulobacter 1633

Hughes, 1991a, b; Onishi et al., 1992). The export of FlgM manipulations were performed essentially as described through the flagellar-specific export apparatus requires (Ausubel et al., 1989). Plasmids were introduced into C. the successful assembly of the hook structure (Hughes crescentus either by bacterial conjugation or electroporation. The fliX deletion strain was generated utilizing the sacB et al., 1993) and the resulting decrease in the intracellular 28 selection method (Schweizer, 1992). Plasmid pDPstIX09/ FlgM concentration relieves the inhibition of s -depen- NPTS139 was conjugated into C. crescentus LS107 (Stephens dent transcription. Analogously, the expression of C. et al., 1997) and transformants selected on PYE agar contain- crescentus class IV flagellar genes, encoding flagellin ing kanamycin and naladixic acid. A 3 ml culture inoculated monomers, also requires a competent basal body-hook with multiple transformants was grown for approximately 8 h in structure that is sensed through the function of FlbT PYE without selection and then plated on PYE agar containing (Mangan et al., 1999; Anderson and Gober, 2000). A 2.25% sucrose. Sucrose-resistant, kanamycin-sensitive colo- nies were isolated and chosen for further analysis. The deletion notable difference between this Caulobacter checkpoint of fliX in these cells was confirmed by PCR and immunoblot and that of the enteric system is that FlbT regulation is analysis using anti-FliX antibody. imposed post-transcriptionally on class IV messages. Motile suppressor mutations (msx) in the DfliX (JG1172) Flagellar biogenesis in enteric bacteria does not strain were generated by inoculating cells into motility agar possess the additional level of regulation within the and allowed to grow at 318C. Motile cells were rescued from Caulobacter flagellar regulatory hierarchy, which governs independent swarms after 4±6 days. The mutation in cells the FliX/FlbD-dependent expression of the class III- isolated from one independent swarm (JG1173) was geneti- cally linked to the Tn5 transposon as previously described encoded external basal body and hook. What is the (Mangan et al., 1995). A C. crescentus strain (JG1175) with regulatory logic in possessing this additional level of approximately 85% linkage between the Tn5 and msx-3 regulation? The most notable difference between flagellar mutation was isolated and used in subsequent transductions. biogenesis in C. crescentus and enteric bacteria is that Sequence analysis revealed the Tn5 insertion to be in the II.1 late flagellar gene transcription in C. crescentus is ORF upstream of the fliF operon. restricted to the swarmer compartment of the pre- In order to isolate a bfa-like allele of fliX, plasmid pX09 divisional cell (Gober et al., 1991; Gober and Shapiro, containing the promoter and full-length ORF of fliX was introduced, by transformation, into the E. coli mutator strain 1992; Wingrove et al., 1993; Wingrove and Gober, 1994, XL1-Red. Randomly mutagenized plasmid from a 2 day old, 1996). This occurs after a barrier, as a consequence of an 200 ml culture was isolated and used to transform strain early cell division event, has formed between the nascent JG1176 containing a class IV fljL±lacZ reporter fusion. swarmer and stalked cell compartments. We hypothesize Transformants were screened for restoration of b-galacto- that the early class II flagellar structure within the swarmer sidase expression using an agar overlay method (Mangan compartment of the pre-divisonal cell is the internal, cell et al., 1995). Mutant clones were sequenced using the type-specific cue for compartmentalized transcription of dideoxy chain termination method (Sanger et al., 1977). In order to construct a multicopy library, C. crescentus late flagellar genes. Because FliX is present in all cell LS107 DNA was subjected to partial digestion with Sau3A types throughout the cell cycle (Mohr et al., 1998), we The digested DNA was applied to a sucrose gradient and a propose that the positive regulatory form would be present fraction consisting primarily of 4±6 kb fragments isolated. A in the swarmer compartment and the negative regulatory portion of these fragments were ligated into the BamHI site of form in the stalked compartment. In this case, the intrinsic pBBR1-MCS (Kovach et al., 1994) then electroporated into asymmetry of the C. crescentus pre-divisional cell may the E. coli strain DH5a and screened by blue/white selection. dictate compartmentalized transcription. Isolated plasmid DNA from approximately 8000 white colonies constituted the library that was used to electroporate strain UC1040 containing the fljL/lacZ reporter plasmid. Reporter gene expression was assayed using an agar- Experimental procedures overlay method and quantitative measurements of b-galacto- sidase activity determined in triplicate, on three different Construction of bacterial strains, plasmids and growth cultures as previously described (Mangan et al., 1995). conditions

Bacterial strains and plasmids used in this work are Subcellular localization of FliX described in Table 3. Transductions were performed as previously described (Ely and Johnson, 1977). Caulobacter For preparation of membrane and combined cytosolic and crescentus strains were grown at 318C in peptone-yeast periplasmic fractions: C. crescentus LS107 membranes were extract (PYE) medium (Poindexter, 1964) supplemented with prepared and treated essentially as described (Hale and de one or more of the following antibiotics: chloramphenicol Boer, 1997). Cells from a 1 l culture (OD600nm ˆ 0.6) were (2.5 mgml21), kanamycin (50 mgml21), tetracycline suspended in 22 ml of cold CBB buffer (20 mM Tris-HCl, (2 mgml21), ampicillin (from 1.5 to 10 mgml21) (gentamicin pH 8.0, 25 mM NaCl, 5 mM EDTA, 3.6 mM 2-mercapto- (5 mgml21), or spectinomycin (50 mgml21). PYE motility ethanol) and lysed by a French pressure cell. Unlysed cells plates contained 0.3% agar and 0.1% glucose. Escherichia were removed from the extract by centrifugation at 20 000 g coli strains were grown at 378C in LB medium. DNA for 20 min at 48C. The membrane and combined cytosolic

Q 2001 Blackwell Science Ltd, Molecular Microbiology, 39, 1623±1637 1634 R. E. Muir, T. M. O'Brien and J. W. Gober

Table 3. Bacterial strains and plasmids used in this study.

Bacterial strain or plasmid Genotype or description Reference or source

C. crescentus PC7070 zzz::Tn5 recA101 Ohta et al. (1990) JG1139 syn-1000 bla-6 zzz::Tn5 recA101 This work LS107 syn-1000, amps derivative of NA1000 Stephens et al. (1997) JG1172 syn-1000 bla-6 DfliX This work JG1173 syn-1000 bla-6 DfliX msx3 This work JG1175 syn-1000 bla-6 DfliX Tn5::II.1msx3 This work SC1042 fliF106::Tn5 proA str-140 Ohta et al. (1984) SC508 flaS153 (DfliQR) Johnson and Ely (1977) SC1032 flbD198::Tn5 Ohta et al. (1984) SC1048 fliP::Tn5 Gober and Marques (1995) SC1131 fliLM196::Tn5 Yu and Shapiro (1992) UC1040 flaS153 (DfliQR) bfa-1204 Mangan et al. (1995) JG1176 syn-1000 bla-6 DfliX fliP::Tn5 This work JG1177 syn-1000 bla-6 DfliX bfa-1204 This work JG1178 syn-1000 bla-6 DfliX flbD198::Tn5 This work JG1179 syn-1000 bla-6 DfliX fliLM196::Tn5 This work JG1180 syn-1000 bla-6 fliP::Tn5 This work JG1181 syn-1000 bla-6 bfa-1204 This work JG1182 syn-1000 bla-6 flbD198::Tn5 This work JG1183 syn-1000 bla-6 fliLM196::Tn5 This work JG1184 syn-1000 bla-6 fliP::Tn5 zzz::Tn5 recA101 This work JG1185 syn-1000 bla-6 bfa-1204 zzz::Tn5 recA101 This work JG1186 syn-1000 bla-6 flbD198::Tn5 zzz::Tn5 recA101 This work JG1187 syn-1000 bla-6 fliLM196::Tn5 zzz::Tn5 recA101 This work JG1188 syn-1000 bla-6 DfliX zzz::Tn5 recA101 This work JG1189 syn-1000 bla-6 DfliX fliP::Tn5 zzz::Tn5 recA101 This work JG1190 syn-1000 bla-6 DfliX bfa-1204 zzz::Tn5 recA101 This work JG1191 syn-1000 bla-6 DfliX flbD198::Tn5 zzz::Tn5 recA101 This work JG1192 syn-1000 bla-6 DfliX fliLM196::Tn5 zzz::Tn5 recA10 This work

E. coli DH5a endA1 hsdR17 supE44 thi-1 recA1 gyrA relA1 Life Technologies D (lacZYA-argF)U169 deoR [f80 dLacD (lac)M15] XL1-Red endA1 gyrA96 thi-1hsdR17 supE44 relA1 mutD5 Stratagene mutS mutT::Tn10 S17-1 Rp4-2, Tc::Mu, Km::Tn7 Simon et al. (1983)

Plasmids pSUP2021 pBR325-Mob::Tn5 Simon et al. (1983 placZ/290 lacZ reporter vector, tetr Gober and Shapiro (1992) pKSII(1) cloning vector, single stranded phagemid, ampr Stratagene pBR322 cloning vector, ampr,tetr pNPTS228 cloning vector, contains oriT, kanr M.R.K. Alley pNPTS139 cloning vector, contains oriT and sacB, kanr M.R.K. Alley pBBR1-MCS broad-host-range, mulitcopy, chlorr, Kovach et al. (1994) pJAMY31 b-lactamase fusion vector, contains ori V, kanr M.R.K. Alley pMR4 broad-host-range vector, tetr C. Mohr pfliF/lacZ/290 fliF-lacZ transcriptional reporter Wingrove and Gober (1994) pfliX/lacZ/290 678 bp BamHI±HindIII fliX promoter fragment from This work p X-I P/KS1 in placZ/290 pflbG/lacZ/290 flbG-lacZ transcriptional reporter Gober and Shapiro (1992) pflgI/lacZ/290 714 bp KpnI±BamHI flgI promoter fragment from This work pfljL/lacZ/290 p X-I P/KS1 in placZ/290 Mangan et al. (1995) fljL-lacZ transcriptional reporter p X-I P 678 bp (PCR generated) BamHI±HindIII This work fliX promoter fragment in KSII(1) pfljL/lacZ/ 4.5 kb BamHI±SacII fljL promoter-lacZ transcriptional This work NPT228 fusion fragment from pfljL lacZ21 KSII21(1) in pNPT228 pfljL/lacZ/KSII21(1) 4.5 kb BamHI±NotI fljL promoter-lacZ transcriptional This work fusion fragment from pfljLII/lacZ/290 in KSII(1) pfljLII/lacZ/290 842 bp BamHI±PstI fljL promoter fragment from This work pfljL/BR322 in placZ/290 pfljL/BR322 1.2 kb BssHII±BstEII fljL promoter fragment in P. Anderson pBR322

Q 2001 Blackwell Science Ltd, Molecular Microbiology, 39, 1623±1637 Regulation of flagellar biogenesis in Caulobacter 1635

Table 3. continued

Bacterial strain or plasmid Genotype or description Reference or source pDPstIX09/ 819 bp DdeI±XhoI DfliX fragment from pDPstI-X09 This work NPTS139 in EcoRV-SalI of pNPTS139 pX10 3.4 kb insert in pBBR1-MCS containing fliX and This work flgI operon pX09 1.4 kb XhoI±XbaI fliX fragment from pX10 in This work pBBR1-MCS pDPstIX09 pX09 lacking the 243-bp PstI fragment of fliX This work pfliX1 pX09 recovered from XL-1 Red, containing fliX1 This work allele of fliX pfliX1 mutN 879 bp PCR generated, XhoI±XbaI fliX1 mutN This work fragment in pBBR1-MCS pfliX1 DC 879 bp PCR generated, XhoI±XbaI fliX1 DC- This work fragment in pBBR1-MCS pfliX1 mutC 1.4 kb XhoI±XbaI fliX I mutC fragment in pBBR1-MCS This work p1-26X 528 bp fragment containing the first 26 codons of This work fliX in pJAMY31 p1-76X 675 bp fragment containing the first 76 codons This work of fliX in pJAMY31 p1-144X 879 bp fragment containing full-length fliX in This work pJAMY31 p1-26X/MR4 XhoI linearized p1-26X in pMR4 This work p1-76X/MR4 XhoI linearized p1-76X in pMR4 This work p1-144X/MR4 XhoI linearized p1-144X in pMR4 This work

and periplasmic fractions were isolated by subjecting the Draper, J. England and R. Figge for critical reading of the cleared extract to centrifugation at 200 000 g for 1 h at 48C. manuscript. R.E.M. was supported by a USPHS predoctoral The supernatant, containing the cytosolic and periplasmic fellowship GM07104. This work was supported by Public fraction, was poured off and retained. The pellet membrane Health Service Grant GM48417 from the National Institutes of fraction was rinsed with cold CBB buffer then resuspended in Health. 3 ml of cold CBB buffer by sonication. Protein concentration of both fractions was determined by the Bradford assay (Bio-Rad). References In order to assay whether FliX was either an integral, or Aizawa, S.-I. (1996) Flagellar assembly in Salmonella peripherally associated protein, approximately 2.5 mg typhimurium. Mol Microbiol 19: 1±5. (0.375 ml) of the membrane fraction was treated with cold Anderson, D., and Newton, A. (1997) Posttranscriptional CBB alone or with cold CBB containing either 6.0 M urea, regulation of Caulobacter flagellin genes by a late flagellum 0.5% Triton X-100, or 0.2% Sarkosyl. Membrane samples assembly checkpoint. J Bacteriol 179: 2281±2288. were treated on ice for 30 min with gentle mixing periodically Anderson, P.E., and Gober, J.W. (2000) FlbT, the post- then subjected to centrifugation again at 200 000 g for 1 h. transcriptional regulator of flagellin synthesis in Caulo- Supernatant, soluble fractions, were poured off and retained. bacter crescentus, interacts with the 50 untranslated region Pellets were rinsed with cold CBB and resuspended in 0.3 ml of flagellin mRNA. Mol Microbiol 38: 41±53. of cold CBB by sonication. Protein from an equivalent portion Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., of soluble and insoluble fractions was precipitated with Seidman, J.G., Smith, J.A., and Struhl, K. (1989) Current trichloroacetic acid (Pohl, 1990). The resulting pellets were Protocols in Molecular Biology. New York: John Wiley and resuspended in water and equal volumes separated on Sons, Inc. SDS±PAGE gels and used in immunoblot analysis (Towbin et al., 1979). In order to address the possibility of fliX Benson, A.K., Wu, J., and Newton, A. (1994a) The role of encoding a periplasmic functioning protein, full-length fliX and FlbD in regulation of flagellar gene transcription in amino-terminal fragments of the fliX-coding region were Caulobacter crescentus. Res Microbiol 145: 420±430. fused to b-lactamase and resistance to ampicillin was Benson, A.K., Ramakrishnan, G., Ohta, N., Feng, J., Ninfa, assessed. Expression of the fusions was determined by A.J., and Newton, A. (1994b) The Caulobacter crescentus immunoblot analyses utilizing antibodies generated to both FlbD protein acts at ftr sequence elements both to activate FliX (Mohr et al., 1998) and to b-lactamase (50 to 30). and to repress transcription of cell cycle-regulated flagellar genes. Proc Natl Acad Sci USA 91: 2369±2373. Brun, Y., Marczynski, G., and Shapiro, L. (1994) The Acknowledgements expression of asymmetry during cell differentiation. Ann Rev Biochem 63: 419±450. We are grateful to A. Reisenauer and L. Shapiro for the Bryan, R., Purucker, M., Gomes, S.L., Alexander, W., and generous gift of anti-FliX antibody. We also thank G. C. Shapiro, L. (1984) Analysis of the pleiotropic regulation of

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