Biochimica et Biophysica Acta 1579 (2002) 55 – 63 www.bba-direct.com Characterization of the flgG operon of Rhodobacter sphaeroides WS8 and its role in flagellum biosynthesis Bertha González-Pedrajo a,1, Javier de la Mora a, Teresa Ballado a, Laura Camarena b, Georges Dreyfus a,* a Departamento de Genética Molecular, Instituto de Fisiologı́a Celular, Universidad Nacional Autónoma de México, Ap. Postal 70-243, 04510 México, D.F., Mexico b Departamento de Biologı́a Molecular, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Ap. Postal 70-228, 04510 México, D.F., Mexico Received 16 May 2002; received in revised form 26 July 2002; accepted 2 September 2002 Abstract In this work, we show evidence regarding the functionality of a large cluster of flagellar genes in Rhodobacter sphaeroides. The genes of this cluster, flgGHIJKL and orf-1, are mainly involved in the formation of the basal body, and flgK and flgL encode the hook-associated proteins HAP1 and HAP3. In general, these genes showed a good similarity as compared with those reported for Salmonella enterica. However, flgJ and flgK showed particular features that make them unique among the flagellar sequences already reported. flgJ is only a third of the size reported for flgJ from Salmonella; whereas flgK is about three times larger than any other flgK sequence previously known. Our results indicate that both genes are functional, and their products are essential for flagellar assembly. In contrast, the interruption of orf-1, did not affect motility suggesting that this sequence, if functional, is not indispensable for flagellar assembly. Finally, we present genetic evidence suggesting that the flgGHIJKL genes are expressed as a single transcriptional unit depending on the sigma-54 factor. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Rhodobacter sphaeroides; Bacterial flagellum; Bacterial motility; Hook – basal body complex 1. Introduction Swimming and swarming motility in many species of bacteria is achieved by rotating flagella (for recent reviews, see Refs. [1,2]). The flagellum is a complex propulsive organelle composed of three main substructures: a long helical filament, hook, and basal body. Between the hook and the filament are two junction proteins, FlgK and FlgL, also known as HAPs (for hook-associated proteins), which act as structural adapters for connecting these functionally different hook and filament homopolymers [3,4]. The basal body is a complex structure to which many different functions are associated (e.g. protein export as well as * Corresponding author. Tel.: +52-55-5622-5618; fax: +52-55-56225611. E-mail address: gdreyfus@ifisiol.unam.mx (G. Dreyfus). 1 Present address: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114, USA. motor rotation and switching), and is composed of many proteins organized into a set of rings and a central rod. The inner MS ring, formed by subunits of the FliF protein [5], is localized in the cytoplasmic membrane, and serves as a mounting plate for components involved in motor rotation and torque generation. The C ring is a bell-like structure that projects into the cytoplasmic surface of the MS ring, and it contains the three motor/switch proteins responsible for determining the direction of flagellar rotation [6]. The rod is formed by four proteins: FlgB, FlgC and FlgF in the proximal portion, while FlgG constitutes the distal rod. The function of the rod is to allow transmission of torque to the external structures [1]. The L ring (FlgH) lies in the plane of the outer lipopolysaccharide membrane, and the P ring (FlgI) is in the plane of the peptidoglycan layer [7,8]. These rings are connected forming a stable cylindrical structure, which is believed to act as a molecular bushing for the central rod [9,10]. FlgH, FlgI, and FlgA are the only flagellar proteins in which a typical N-terminal signal sequence is present; consequently, they are thought to be 0167-4781/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 7 8 1 ( 0 2 ) 0 0 5 0 4 - 3 56 B. González-Pedrajo et al. / Biochimica et Biophysica Acta 1579 (2002) 55–63 translocated by the general Sec pathway [11 – 14]. Flagellin, and the rest of the secreted flagellar proteins, are exported through a specialized flagellar export apparatus lying within a patch of membrane in the center of the MS ring [15,16]. Over 50 genes, organized into operons, are required for flagellar formation and function [17]. In Escherichia coli and Salmonella enterica serovar Typhimurium, it has been shown that expression of the flagellar genes is tightly controlled in a transcriptional hierarchy of three classes [18]. The purple non-sulfur photosynthetic bacterium Rhodobacter sphaeroides has a single subpolar flagellum that propels the cell very efficiently [19]. Unlike E. coli and Salmonella, R. sphaeroides has a unidirectionally rotating flagellar motor; and instead of swimming and tumbling, it swims and stops randomly re-orienting during the stop periods [19]. Several genes involved in flagellum biogenesis have been identified in this bacterium, for example, flgBCDEF, fliHIJKLMNOPQR, flhB [20 – 23] and the molecular mechanisms that control their expression have just begun to be elucidated. Recently, the nearly completed sequence of R. sphaeroides 2.4.1 genome was reported [24]; however, it has not yet been fully annotated and evidence regarding the function of most of the genes predicted from the reported sequence is still lacking. In this study, we characterized several mutants that allowed us to identify a large gene cluster involved in flagellar biogenesis. Transcriptional organization of these genes was inferred from genetic data. Finally, in accordance with previous results, we show evidence for the existence of a negative control mechanism that down-regulates the expression of flagellin in the absence of a functional hook – basal body. Table 1 Bacterial strains and plasmids used in this work Strain or plasmid Relevant characteristics Source or reference [26] S17-1 hsdR4 D(lac-pro) F’traD36 proAB lacI q ZDM15 recA endA thi hsdR RP4-2-TcDMuDTn7 R. sphaeroides WS8 RsgI RsgI-np RsgJ-np Rsorf1-np wild-type, spontaneous Nalr WS8 TnphoA derivative, flgIDTnphoA WS8 flgIDaadA; Spcr WS8 flgJDaadA; Spcr WS8 orf-1DaadA; Spcr [49] This This This This Suicide vector used for gene replacement cloning vector; pUC derivative; Apr pRK404 derivative; used for expression in R. sphaeroides under lac promoter; lacZ mob+; Tcr pSUP203 derivative carrying TnphoA; Cmr Tcr Kanr pUC derivative carrying the omega-Spc cassette 4.7 kb SalI fragment cloned into pTZ18R, carrying 4.6 kb of TnphoA plus 125 bp of RsgI DNA flanking the site of transposon insertion 1.6 kb SalI – PstI fragment from pBG401 3.8 kb BamHI fragment from WS8 cloned in pTZ19R 3.9 kb PstI fragment from WS8 cloned into pTZ19R 3.9 kb PstI fragment from pBG401 subcloned in pRK415/reversed direction 5.9 kb SalI fragment from WS8 cloned into pTZ19R 8.2 kb fragment obtained after joining the inserts from pBG401 and pBG506 through the HindIII site 8.2 kb fragment from pBG811 subcloned into pRK415 [50] Pharmacia [51] E. coli JM103 Plasmids pJQ200mp18 pTZ18/19R pRK415 pU1800 pWM5 pTngI pBG105 pBG313 pBG401 pBG402A/B pBG506 pBG811 2. Materials and methods 2.1. Bacterial strains and plasmids pBG812 [48] work work work work [52] [53] This work This work This work This work This work This work This work This work The bacterial strains and plasmids used in this work are described in Table 1. 2.3. Recombinant DNA techniques 2.2. Media and growth conditions R. sphaeroides cell cultures were grown photosynthetically in Sistrom’s liquid or solid medium [25] under continuous illumination at 30 jC. Aerobic growth conditions were achieved in the dark with strong shaking. Motility plates were prepared using 1% tryptone, 0.7% NaCl, and 0.3% Bacto-Agar. Strains of E. coli were grown in LuriaBertani medium. When needed, antibiotics were added at the following concentrations: spectinomycin, 10 Ag/ml; gentamicin, 30 Ag/ml; kanamycin, 25 Ag/ml; and tetracyclin, 1 Ag/ml. For E. coli, the following antibiotics were used: ampicillin, 100 Ag/ml; tetracyclin, 15 Ag/ml; gentamycin, 30 Ag/ml; and spectinomycin, 100 Ag/ml. The isolation of chromosomal DNA was performed as described elsewhere [26]. Plasmid DNA preparations were carried out with Qiagen Mini or Midi Column Plasmid Purification Kits (Qiagen, Inc., Santa Clarita, CA). DNA amplification was carried out with Pfu DNA polymerase (Stratagene, La Jolla, CA) and 0.5 AM of the appropriate oligonucleotides; the reaction was performed for 30 cycles in a GeneAmp PCR system (Perkin-Elmer, Foster City, CA). Sequencing was carried out using the Thermosequenase kit (Amersham, Piscataway, NJ) on single- or doublestranded clones. DNA hybridization was carried out using the PhotoGene system from Life Technologies (Rockville, MD). B. González-Pedrajo et al. / Biochimica et Biophysica Acta 1579 (2002) 55–63 2.4. Transposon mutagenesis of R. sphaeroides pU1800 plasmid harboring the transposon TnphoA was mobilized into R. sphaeroides WS8 from E. coli S-17 by diparental mating [27]. Transposon mutants were selected on agar plates containing kanamycin. Single independent colonies were tested for loss of motility by using swarm plate assays. Cells were also analyzed by dark-field microscopy. 2.5. DNA analysis Sequences were analyzed and compared with the combined SWISSPROT and TrEMBL protein sequence databases, by using the BLAST server at the National Center for Biotechnology Information (Bethesda, MD). Protein alignments were done using the GAP and PILEUP programs from the Genetics Computer Group software package [28]. 57 and subsequently transferred to R. sphaeroides by conjugation. Transconjugants in which a double recombination event occurred were selected on LB plates in the presence of spectinomycin and 5% sucrose. The correct replacement was confirmed by DNA hybridization. The flgJ and orf-1 mutants were generated using the same procedure. For inactivation of flgJ, the cassette was inserted into the unique BbsI site of plasmid pBG105, located in the central region of the flgJ gene. orf-1 was interrupted at the ClaI site of plasmid pBG313, located also in the central region of the orf-1 gene. 2.10. Immunoblot analysis Bacterial cell suspensions were applied on Formvar-coated grids. Samples were negatively stained with 1% uranyl acetate and observed with a JEM-1200EXII electron microscope (Jeol, Tokyo, Japan). After whole cell extract and cell supernatant samples were run in SDS-polyacrylamide gel (normally 12% acrylamide) electrophoresis (PAGE) [29], they were transferred onto a nitrocellulose membrane (Schleicher & Schull, Keene, NH) with a transblotting apparatus (Hoefer, San Francisco, CA). The membrane was then blocked for 1 h with Tris-buffered saline plus 0.1% Tween 20, and 5% non-fat dry milk and probed with polyclonal anti-flagellin antibody. Immunodetection was performed with an enhanced chemiluminescence immunoblotting detection kit (Amersham International, Little Chalfont, UK). 2.7. Motility assays 2.11. Nucleotide sequence accession number A 5-Al sample of a stationary-phase culture was placed on the surface of swarm plates and incubated aerobically in the dark. The swarming capability was recorded as the ability of bacteria to move away from the inoculation point after 36 – 48 h. The motility of free-swimming bacteria was evaluated in an aliquot from aerobic or anaerobic cultures placed directly between a slide and a cover slip. The samples were observed with an Olympus microscope adapted for highintensity dark-field illumination. The DNA sequences of the flgG, flgH, flgI, flgJ, flgK and flgL genes from R. sphaeroides WS8 have been deposited in GenBank under accession numbers AF205139 and AF317 649. 2.8. Isolation of a P-ring (flgI) mutant A non-motile mutant named RsgI was isolated by random mutagenesis using the Tn5 derivative TnphoA as described in Section 2.4. RsgI strain failed to form a swarm ring as compared to the wild-type strain when inoculated on 2.6. Electron microscopy A non-motile mutant named RsgI was isolated screening a bank of mutants unable to swim. This bank was obtained by transposon mutagenesis of the wild-type WS8 cells using the Tn5 derivative TnphoA as described elsewhere [20]. 3. Results 3.1. Isolation of a P-ring (flgI) mutant 2.9. Construction of flgI, flgJ and ORF1 replacement strains The isolation of non-polar mutants in the flgI, flgJ and orf1 genes was done by insertion of an internal portion of the omega-Spc cassette, from which transcriptional termination signals were removed [20]. For flgI inactivation, the cassette was cloned into the unique BlpI site of pBG401, which is located in the 3V-end region of the flgI gene. The 5.3-kb PstI fragment carrying flgIDaadA was then subcloned into pJQ200, which is unable to replicate in R. sphaeroides. The resulting plasmid was transformed into E. coli S17-1 Fig. 1. (A) Swarming assay of RsgI and wild-type R. sphaeroides WS8. (B) Hybridization of genomic DNA of RsgI digested with SalI and probed against a 4.3-kb DraI – SalI probe from the transposon TnphoA. 58 B. González-Pedrajo et al. / Biochimica et Biophysica Acta 1579 (2002) 55–63 Fig. 2. (A) Cloning strategy for the flgGHIJKLorf1 genes. Plasmid pTngI contains a 4.7-kb DNA fragment (4.6 kb of TnphoA DNA plus 125 bp of chromosomal DNA adjacent to the insertion site, indicated as a black box). Plasmid pBG401 contains a 3.9-kb PstI chromosomal DNA fragment from WS8 cells. pBG506 and pBG313 carrying a SalI and BamHI chromosomal DNA fragment from WS8 cells, respectively. The pBG811 plasmid was obtained after joining the fragments present in pBG401 and pBG506 through the HindIII site present in both fragments. Some of the relevant restriction sites are indicated: S, SalI; D, DraI; P, PstI; B, BamHI; H, HindIII. The plasmid pTZ19R was used as vector in all these constructions. The insert present in pBG811 was also cloned in pRK415; this plasmid was named pBG812 (not shown). (B) Organization of the flg genes characterized in this work, each gene is represented as an arrow. The sequences identified as similar to the consensus sigma-54 and sigma-28 promoters are shown. motility plates (Fig. 1A). Further analysis of mutant RsgI by dark-field microscopy confirmed the lack of motility, and observation by electron microscopy revealed the absence of flagella in this strain. A DNA hybridization experiment confirmed that a single transposition event occurred in RsgI cells (data not shown). To identify the insertion point of the transposon, chromosomal DNA isolated from mutant RsgI was digested with SalI. A single band of 4.7 kb was detected in a DNA hybridization experiment, using a 4.3-kb DraI– SalI fragment from TnphoA as a probe (Fig. 1B). The identified fragment was then cloned into pTZ18R generating plasmid pTngI; the sequence of this fragment revealed the presence of 125 bp of chromosomal DNA adjacent to the insertion site (Fig. 2A). This sequence showed homol- ogy to the flgI gene of different flagellated bacteria when compared against the database from the National Center for Biotechnology Information. 3.2. Cloning of the wild-type flgI gene and identification of the flagellar cluster flgGHIJKL and orf-1 To clone the complete wild-type flgI gene, a 379-bp SalI– DraI fragment from pTngI plasmid (see Fig. 2A) was used as a probe against chromosomal DNA digested with PstI. A 3.9-kb fragment clearly detected by DNA hybridization (not shown) was cloned into pTZ19R generating plasmid pBG401 (Fig. 2A). DNA sequence analysis revealed pBG401 to contain four open reading frames (ORF) that Table 2 Comparison of encoded proteins of R. sphaeroides WS8 flagellar genes with those of other bacterial species Percentage homology (identity/similarity) Gene Function flgG flgH flgI flgJ Distal rod L ring P ring Putative rod capping protein HAP1 HAP3 flgK flgL Size product (amino acids) Salmonella enterica Thermotoga maritima Aquifex aeolicus Helicobacter pylori Caulobacter crescentus Sinorhizobium meliloti Agrobacterium tumefaciens 262 222 371 100 50.6/60.1 30.9/39.9 44/54 21.8/30.7 47.5/57.8 21.5/31.3 41.8/52.6 – 35.8/44.5 23.8/34.1 44.7/56.9 – 26.8/34.1 17.5/25.2 34.2/49.4 – 43.3/53.6 33.9/37.1 43.8/54.1 – 45.9/57.8 26.7/31.7 42.2/53 – 26.8/34.1 23.4/30.2 39.8/49.3 – 1363 409 26.9/32.8 29.2/40.1 24.6/34.4 23/35.3 23.6/31.8 18.8/30.1 28.7/37.2 32.3/39.2 25.1/30.5 – 23.8/29.9 29.2/36.1 27.2/33.4 20.5/26.9 B. González-Pedrajo et al. / Biochimica et Biophysica Acta 1579 (2002) 55–63 were similar to the flgG, flgH, flgI, flgJ genes, and to the 5Vend of flgK, which have been previously identified in other bacterial species (Fig. 2B). The restriction fragments downstream of this region were identified by chromosomal walking on overlapping SalI and BamHI fragments. These fragments were then cloned into pTZ19R generating plasmids pBG506 and pBG313 (Fig. 2A). From the sequence of 59 these two clones, the ORF corresponding to flgK was completed, flgL was identified downstream of flgK, and an ORF of 797 bp that initiates only six base pairs downstream of the stop codon of flgL was also detected. A BLAST search conducted on the sequence of this ORF showed a fair similarity to hypothetical proteins from Rickettsia prowazekii, Sinorhizobium meliloti, and Agrobacterium tumefaciens. Fig. 3. (A) Sequence alignment of the FlgJ proteins from various bacterial species. R. sphaeroides (RsFlgJ), S. enterica serovar Typhimurium (StFlgJ), Yersinia pestis (YpFlgJ), Nitrosomonas europaea (NeFlgJ), Vibrio cholera (VcFlgJ), Thermotoga maritima (TmFlgJ). Identical residues are shaded. (B) Sequence alignment of the FlgK proteins from R. sphaeroides (RsFlgK) and S. enterica serovar Typhimurium (StFlgK). Identical residues are shaded. 60 B. González-Pedrajo et al. / Biochimica et Biophysica Acta 1579 (2002) 55–63 The flgGHIJKL genes have been shown to be conserved in many other flagellated species, in which they encode for the distal rod protein (FlgG), the outer pair of rings: L ring (FlgH) and P ring (FlgI), a recently described muramidase (FlgJ), and two of the HAP proteins: HAP1 (FlgK) and HAP3 (FlgL). Table 2 shows a comparison of the encoded proteins of R. sphaeroides with those of other bacterial species. FlgJ and FlgK show interesting features. An alignment of FlgJ from different bacterial species is shown in Fig. 3A. The muramidase domain is missing in FlgJ from R. sphaeroides; however, there are many conserved residues in the N-terminal region which in S. enterica plays the role of the rod capping protein [30,31]. In turn, FlgK is about three times larger in R. sphaeroides than its homolog in S. enterica. An alignment between these two sequences is shown in Fig. 3B. The N- and C-terminal regions are conserved, the additional sequence, which accounts for the increase in length of the R. sphaeroides FlgK, is located in the central region of the protein. The gene organization of this 9-kb flagellar cluster is shown in Fig. 2B. Upstream of the flgG gene, we identified the 3V-end of flgF, which is the last gene of the flgBCDEF operon recently described [20]. At the intercistronic region between the flgF and flgG genes, a sequence similar to that of the j54-consensus promoter was identified (Fig. 2B). Fig. 4. (A) Swarming assay of wild-type WS8, RsgI strain, and RsgI carrying pBG402A and pBG402B plasmids. Negatively stained electron micrographs of hook structures isolated from RsgI cells carrying pBG402A (B), and pBG402B (C). Fig. 5. Swarming assay of wild-type WS8, RsgI-np, and RsgI-np carrying pBG402A and pBG402B. 3.3. Complementation of the RsgI mutant For complementation experiments, the 3.9-kb PstI fragment from pBG401 was subcloned into pRK415 plasmid. The resultant plasmids, pBG402A and B, have the two possible orientations with respect to the pRK415 promoter. Both of these clones were unable to restore motility to RsgI cells (Fig. 4A). However, electron microscopic analysis of RsgI cells carrying pBG402A or pBG402B revealed the presence of hook-like structures. In fact, material sheared from these cells contained hook structures (Fig. 4B and C). As control, the same procedure was carried out for the RsgI mutant from which no hook structures were isolated. Given that FlgI is necessary for hook assembly, the presence of hooks in these cells indicates that the flgI gene must be expressed from both plasmids. In contrast, the lack of motility may be explained in terms of the polar effect exerted by the TnphoA transposon over genes lying downstream of flgI that belong to the same transcriptional unit. The fact that pBG402B plasmid was able to express flgI, independently of the vector promoter, strongly suggests that this 3.9-kb PstI fragment carries a functional flagellar promoter responsible for the expression of flgI. This promoter activity may be ascribed to the sequence resembling the j54-consensus promoter previously shown (Fig. 2B). Interestingly, the fact that pBG402 (flgGHIJ+) and pBG812 (flgGHIJK+) were incapable of restoring motility of RsgI cells, but allowed the assembly of the hook, suggests that the expression of flgK and flgL is negatively affected by the flgIDTnphoA allele, indicating that these genes are likely expressed from the sigma-54 promoter located upstream of flgG. To determine the actual phenotype of a flgI mutant avoiding the polar effect exerted by the transposon, a strain carrying a non-polar mutation in flgI was isolated as described in Section 2.9. The strain, RsgI-np (flgIDaadA) showed a Fla phenotype and was unable to form a swarm ring on motility plates (Fig. 5). Plasmids pBG402A or pBG402B, which had failed to restore motility to the RsgI mutant, were able to complement RsgI-np. The observed complementation was independent of the pRK415 promoters (see Fig. 5, pBG402B), further supporting the pres- B. González-Pedrajo et al. / Biochimica et Biophysica Acta 1579 (2002) 55–63 ence of a promoter responsible for flgI expression in this clone. 3.4. Flagellin is properly expressed and exported in RsgI cells carrying pBG402 plasmid To test the possibility that the fliC gene expression or FliC export was affected by the flgIDTnphoA allele, the levels of flagellin were determined by immunoblotting in the mutant RsgI, and in RsgI carrying pBG402. As shown in Fig. 6, RsgI cells produced only basal levels of flagellin, of which none was detected in the supernatant. However, when the plasmid pBG402A was introduced into RsgI cells, the levels of flagellin were considerably increased, and the protein could be exported into the culture medium (Fig. 6). This result suggests the existence of a negative control mechanism that down-regulates the expression of late genes (i.e. flagellin) in the absence of a functional hook – basal body structure. The presence of basal levels of flagellin in RsgI strain is consistent with our previous finding that in this bacterium, the control exerted over the expression of the late genes is not tight, and consequently, the expression of fliC is not absolutely dependent on the presence of a functional hook –basal body structure [32]. On the other hand, the fact that RsgI cells carrying pBG402A export a large amount of flagellin into the culture medium indicates that in these cells, the export apparatus is fully competent to export flagellin-type substrates [33]; therefore, the failure to assemble a filament in these cells must be ascribed to the lack of HAP1 and HAP3 proteins. This explanation implies that the flgK and flgL genes are mainly transcribed from the j54 promoter located upstream of the flgG gene, as suggested above. 3.5. Isolation of mutant strains with lesions at either flgJ or orf-1 Since the putative flgJ gene is highly unusual as compared with its counterparts found in other flagellated bacteria, we Fig. 6. Immunoblotting of RsgI and RsgI/pBG402A using a polyclonal antiFliC antibody against whole cell (C) and supernatant (S). An aliquot of a mid-log culture was treated as described elsewhere. 61 decided to isolate a mutant in this gene, as well as in orf-1, which presumably is part of this transcriptional unit. The cells carrying the flgJDaad mutation, showed a Fla phenotype suggesting that this atypical flgJ gene is required for flagellar assembly, probably to assemble the flagellar rod. In agreement with our complementation studies of RsgI and RsgI-np cells, pBG402 A or B plasmids were both able to restore motility in the cells carrying the flgJDaad allele (data not shown). On the other hand, the mutant carrying the orf1Daad allele was motile and did not show any apparent difference with the wild-type cells. Therefore, this ORF is not required for flagellar function. 4. Discussion In this work, we identified a 9-kb flagellar cluster that contains flgGHIJKL and orf-1 genes. The functionality of these genes was evaluated by the isolation and characterization of four different mutant strains carrying the alleles: flgIDTnphoA, flgIDaadA, flgJDaadA or orf-1DaadA. Three of these strains showed a Fla phenotype indicating that with exception of orf-1, each of these genes is essential for flagellar biogenesis. This conclusion is particularly relevant since flgJ and flgK genes show unique features that make them different from their counterparts found in other bacteria. Analyzing the sequence of the predicted polypeptides from these genes, we observed that the N-terminal sequences for FlgH and FlgI conformed well to the consensus for signal peptides [34], consistent to what has been reported for S. enterica in which these proteins are presumed to be exported via the Sec secretory pathway [11,12]. Although this pathway has not been characterized in R. sphaeroides, the presence of the N-terminal signal sequence suggests that these proteins might be exported in a Sec-dependent fashion. On the other hand, the FlgJRs protein showed to be much smaller than FlgJSe (about one third). A comparison between these proteins revealed that the similarity resides only at the N-terminal region. Recently, it has been suggested that the N-terminus of FlgJSe is involved in the assembly of the rod, whereas the C-terminus has peptidoglycan hydrolyzing activity [30,31]. Therefore, it could be possible that in R. sphaeroides, the FlgJ protein does not posses the peptidoglycan hydrolyzing activity (in fact, the residues proposed to form the active center are missing in FlgJRs). However, the possibility that this activity might reside in a different protein remains to be elucidated. Database analysis of finished bacterial genomes did not show any homologous gene to flgJ in which the region responsible of the muramidase activity was absent. It will be interesting to examine if other examples of FlgJ without the C-terminal region are found in other bacterial genomes not yet sequenced; this will allow to examine if this change occurred in a particular phylogenetic 62 B. González-Pedrajo et al. / Biochimica et Biophysica Acta 1579 (2002) 55–63 group or if it was a single event in R. sphaeroides. Interestingly, the FlgJ protein of Thermotoga maritima has only the muramidase domain (see Fig. 3A) [35]. In contrast, the FlgKRs protein is three times larger than FlgKSe. Both terminal domains of the FlgKRs protein are well conserved, but there is an additional central region that shows no similarity with any other FlgK protein reported so far. Noteworthy, the larger size of FlgK could explain that the interface connecting the hook structure with the filament is clearly distinguished in preparations of isolated flagella from R. sphaeroides [36]; in contrast with what is observed in S. enterica (as example, see Ref. [37]). It has been suggested that HAP3 prevents torsion-induced transformation in E. coli filaments [38]; however, the flagellar filament of R. sphaeroides shows less control over conformational changes induced by rotation [39]. Since HAP3 may be responsible for this difference, we are currently investigating the relationship between size and function of FlgKRs (D. Castillo, T. Ballado, F. de la Mora, L. Camarena, and G. Dreyfus, unpublished data). Genetic evidence presented in this work indicates that flgGHIJKL genes conform a single transcriptional unit, as suggested from the complementation analysis of RsgI and RsgI-np cells. This operon lies downstream of the 3V-end of flgF, which is the last gene of another flagellar operon that we have recently described [20]. At the intercistronic region between flgF and flgG, we found a sequence that strongly resembles the j54 consensus promoter; presumably, this sequence may represent the functional promoter of this operon. According to this idea, a 3.9-kb PstI fragment cloned in the opposite direction of the plasmid promoter was able to complement RsgI-np cells, indicating that this fragment carries a functional promoter. Our previous work has shown that mutants in fliR and flgE were complemented when the wild-type genes were expressed from the j54 promoter located upstream of their respective operons (Refs. [20,32], L. Camarena and G. Dreyfus, unpublished data). In agreement with this proposal, it has been shown that in R. sphaeroides, the expression of the flagellar promoters flgBp, fliKp, and fliOp is dependent on the sigma-54 factor [32]. Recently, we have demonstrated that a specific j54 factor is required for the transcription of flagellar genes [40]. In S. enterica, the flgKL operon is transcribed by a class 2 promoter located upstream of flgB, and also by a class 3 promoter located immediately upstream of flgK [41]. Therefore, considering that these genes might also be expressed from a j28 promoter located downstream of flgJ, we analyzed the intercistronic region between the flgJ and flgK genes looking for a sequence resembling the j28-consensus promoter sequence [42,43]. A sequence showing a weak resemblance with the j28-consensus promoter was identified in this region (see Fig. 2B); however, the lack of motility of RsgI cells carrying pBG402 or pBG812 plasmids suggests that this is not a functional promoter; or that the level of the protein expressed from this putative promoter is not sufficient to restore motility. It has been previously observed for the flagellar regulatory hierarchy of E. coli and S. enterica that the expression of a late flagellar gene (i.e. fliC) is repressed when there is a mutation in any of the earlier hook – basal body (HBB) genes. In these enteric bacteria, the negative regulation of late gene expression is achieved by the FliA – FlgM system, in which the export of the anti-sigma factor FlgM can only proceed after the HBB complex has been properly assembled [44,45]. Our results indicate that the assembly checkpoint that avoids the unnecessary synthesis of flagellin when the HBB complex cannot be assembled is conserved in R. sphaeroides (as suggested by the low level of flagellin expression in RsgI mutant). However, no FlgM homolog has been found in the draft genome sequence of R. sphaeroides 2.4.1 [24]. The anti-sigma factor may yet be discovered when the genome sequence project is finished, or R. sphaeroides could have a different regulatory system based on transcriptional or post-transcriptional control mechanisms, rather than on the export of an anti-sigma factor. Interestingly, from the genome sequence of R. sphaeroides 2.4.1, it was noticed that several flagellar genes are duplicated. A copy of the flgG, flgH and flgI genes is located on contig 109 (flgG and flgH) and on contig 124 (flgI). These genes show a moderate identity with the genes reported in this work that are located on contigs 138 and 102. These identity values ranged from 41% for FlgG to 29% for FlgH. From the results presented in this work, we can establish that the flgGHIJKL genes present on contigs 102 and 138 are functional and essential to form the subequatorial flagellum in this bacterium. This is the first report showing experimental evidence regarding the functionality of these genes, and suggesting that the product of the flgI gene cannot be substituted by that synthesized from the flgI gene present on contig 124. It is possible that the second copy of the flagellar genes might be involved in the formation of an alternative flagellar structure, like the lateral flagella induced in some bacteria to achieve surface motility [46] or perhaps related with a putative type III secretion system [47]. Acknowledgements We thank Aurora V. Osorio for technical assistance. We also thank the Molecular Biology Unit and the Microscopy Unit of IFC, for the synthesis of oligonucleotides and electron microscopy, respectively. 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