Hook-associated Proteins Research Articles

NMR structure of protein YvyC from Bacillus subtilis reveals unexpected structural similarity between two PFAM families

109-residue protein YvyC (MW=13 kDa) from B. subtilis (gi|580862, SwissProt/TrEMBL ID YVYC_BACSU, accession number {type:entrez-protein,attrs:{text:P39737,term_id:732320,term_text:P39737}}P39737) was selected as a target of the Protein Structure Initiative 2 and assigned to the Northeast Structural Genomics consortium (NESG; http://www.nesg.org) for structure determination (NESG Target ID SR482). YvyC belongs to the Pfam1 protein family FlaG (PF03646) and is required for proper assembly of bacterial flagella. The FlaG family contains 215 members (for sequence alignment of the FlaG family seeds see Fig. S1 in the Supporting Information). The NMR structure of YvyC presented here is the first atomic resolution structure available for this family. Self-assembly of bacterial flagella has been studied extensively in recent decades and comprehensive reviews are available.2-4 The gene encoding YvyC is a part of the fliD operon comprising the genes yvyC, fliD, fliS and fliT.5 This operon is located immediately downstream of the gene hag (also called fliC) encoding flagellin – the major protein required for assembly of the flagellar filaments. During filament elongation, flagellin monomers are exported through the central channel of a flagellum and oligomerize at its distal end.2,4 FliD (also named ‘hook-associated protein 2’, or HAP26) forms a pentameric cap at the distal end of a flagellum and is essential for oligomerization of flagellin.2,4,7 FliS and FliT serve as flagellin-specific8 and FliD-specific9,10 chaperones, respectively. It was demonstrated that mutations in yvyC gene orthologs in P. fluorescens (flaG) and V. anguillarum (ORF3) lead to phenotypes with unusually long filaments,11,12 but the specific role of YvyC remains unknown.

Roles of Flagellar Hook-Associated Proteins in Vibrio vulnificus Motility and Virulence

The bacterial flagellar structure can be divided into the basal body, the hook and the filament. Three minor components called hook associated proteins (HAP1, HAP2 and HAP3) form a junction between the hook and the filament (HAP1 and HAP3) and a capping structure at the distal end of flagellar filament (HAP2). Vibrio vulnificus is a halophilic pathogenic bacterium that is locomotive by means of a polar flagellum. From a V. vulnificus genome sequencing project, we obtained sequences of V. vulnificus flgK (Vv-flgK), flgL (Vv-flgL), and flaH (Vv-flaH) genes that encode HAP1, HAP3, and HAP2, respectively. To investigate roles of the HAP proteins, deletion mutants of the Vv-flgK, Vv-flgL and Vv-flaH were constructed. Electron microscopic analysis showed that the Vv-flgK or Vv-flgL mutant did not produce an intact polar flagellum while the Vv-flaH mutant produced a fragile flagellar structure. Western blot analysis against a major polar flagellin proposed that the null HAP1 and HAP3 mutations resulted in a failure of normal flagellar assembly since flagellins produced by the mutants were secreted out in the culture supernatants without long flagellar filaments. Motility was completely abolished by a single mutation in HAP1 or HAP3, and the HAP2 mutant showed a decreased motility. Also each of the mutants showed an impaired cytotoxicity and adherence to HeLa cell compared with the isogenic wild type strain. LD?? increased by 10- and 11-fold in the V. vulnificus HAP3 and HAP2 mutant, respectively. These results suggest that the HAP proteins play important roles in polar flagellation and the virulence of V. vulnificus.

Identification of the flagellar chaperone FlgN in the phytopathogen Xanthomonas axonopodis pathovar citri by its interaction with hook-associated FlgK

Genome annotation of the plant pathogen Xanthomonas axonopodis pv. citri (Xac), identified flagellar genes in a 15.7 kb gene cluster. However, FlgN, a secretion chaperone for hook-associated proteins FlgK and FlgL, was not identified. We performed extensive screening of the X. axonopodis pv. citri genome with the yeast two-hybrid system to identify a protein with the characteristics of the flagellar chaperone FlgN. We found a candidate (XAC1990) encoded by an operon for components of the flagellum apparatus that interacted with FlgK. In order to further support this finding, Xac FlgK and XAC1990 were cloned, expressed, and purified. The recombinant proteins were characterized by spectroscopic methods and their interaction in vitro confirmed by pull-down assays. We, therefore, conclude that XAC1990 and its homologs in other Xanthomonas species are, in fact, FlgN proteins. These observations extend the sequence diversity covered by this family of proteins.

The protein network of bacterial motility

Motility is achieved in most bacterial species by the flagellar apparatus. It consists of dozens of different proteins with thousands of individual subunits. The published literature about bacterial chemotaxis and flagella documented 51 protein–protein interactions (PPIs) so far. We have screened whole genome two-hybrid arrays of Treponema pallidum and Campylobacter jejuni for PPIs involving known flagellar proteins and recovered 176 and 140 high-confidence interactions involving 110 and 133 proteins, respectively. To explore the biological relevance of these interactions, we tested an Escherichia coli gene deletion array for motility defects (using swarming assays) and found 159 gene deletion strains to have reduced or no motility. Comparing our interaction data with motility phenotypes from E. coli, Bacillus subtilis, and Helicobacter pylori, we found 23 hitherto uncharacterized proteins involved in motility. Integration of phylogenetic information with our interaction and phenotyping data reveals a conserved core of motility proteins, which appear to have recruited many additional species-specific components over time. Our interaction data also predict 18 110 interactions for 64 flagellated bacteria.

The type III secretion chaperone FlgN regulates flagellar assembly via a negative feedback loop containing its chaperone substrates FlgK and FlgL.

The type III secretion (TTS) chaperones are small proteins that act either as cytoplasmic bodyguards, protecting their secretion substrates from degradation and aggregation, facilitators of their cognate substrate secretion or both. FlgN has been previously shown to be a TTS chaperone for the hook-associated proteins FlgK and FlgL (FlgKL), and a translational regulator of the anti-sigma28 factor FlgM. Protein stability assays indicate that a flgN mutation leads to a dramatic decrease in the half-life of intracellular FlgK. However, using gene reporter fusions to flgK we show that a flgN mutation does not affect the translation of a flgK-lacZ fusion. Quantification of FlgM protein levels showed that FlgKL inhibit the positive regulation on flgM translation by FlgN when secretion of FlgKL is inhibited. Suppressors of the motility-defective phenotype of a flgN mutant were isolated and mapped to the clpXP and fliDST loci. Overexpression of flgKL on a plasmid also suppressed the motility defect of a flgN null mutant. These results suggest that FlgN is not required for secretion of FlgKL and that FlgN typifies a class of TTS chaperones that allows for the minimal amount of their substrates expression required in the assembly process by protecting the substrate from proteolysis. Our data leads us to propose a model in which the interaction between FlgN and FlgK or FlgL is a sensing mechanism to determine the stage of flagellar assembly. Furthermore, the interaction between FlgN and FlgK or FlgL inhibits the translational regulation of flgM via FlgN in response to the stage of flagellar assembly.

Characterization of the flgG operon of Rhodobacter sphaeroides WS8 and its role in flagellum biosynthesis

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.

Interactions between Bacterial Flagellar Axial Proteins in Their Monomeric State in Solution

The axial structure of the bacterial flagellum is composed of many different proteins, such as hook protein and flagellin, and each protein forms a short or long axial segment one after another in a well-defined order along the axis. Under physiological conditions, most of these proteins are stable in the monomeric state in solution, and spontaneous polymerization appears to be suppressed, as demonstrated clearly for flagellin, probably to avoid undesirable self-assembly in the cytoplasmic space. However, no systematic studies of the possible associations between monomeric axial proteins in solution have been carried out. We therefore studied self and cross-association between hook protein, flagellin and three hook-associated proteins, HAP1, HAP2 and HAP3, in all possible pairs, by gel-filtration and analytical centrifugation, and found interactions in the following two cases only. Flagellin facilitated HAP3 aggregation into β-amyloid-like filaments, but without stable binding between the two. Addition of HAP3 to HAP2 resulted in disassembly of preformed HAP2 decamers and formation of stable HAP2–HAP3 heterodimers. HAP2 missing either of its disordered terminal regions did not form the heterodimer, whereas HAP3 missing either of its disordered terminal regions showed stable heterodimer formation. This polarity in the heterodimer interactions suggests that the interactions between HAP2 and HAP3 in solution are basically the same as those in the flagellar axial structure. We discuss these results in relation to the assembly mechanism of the flagellum.

Extracellular antigens from Salmonella enteritidis induce effective immune response in mice after oral vaccination.

We have studied polyacryl starch microparticles as an adjuvant in oral vaccination in mice. Secreted antigens from Salmonella enterica serovar Enteritidis were administered covalently conjugated to microparticles, or as free antigens, orally or intramuscularly and evaluated for their immunogenicity and ability to elicit protective immune response against an oral challenge with live serovar Enteritidis. The highest immunoglobulin M (IgM)-plus-IgG titers were obtained in the groups immunized with antigen-conjugated microparticles. The subclass profile switched to a stronger Th1 influence in the oral groups after booster, while the intramuscular group showed a constant Th1/Th2 profile. A strong specific IgA response was seen in feces in the oral groups, which was further confirmed in an enzyme-linked immunospot assay. The delayed-type hypersensitivity test, as a measure of the cellular response, showed a significant increase in ear thickness in all the immunized groups, except for the group that received free antigen orally, compared to the nonimmunized group. The cytokines released from in vitro-stimulated spleens showed a strong gamma interferon response in all immunized groups. A significant reduction in CFU in liver and spleen was seen in the orally immunized groups compared to the nonimmunized group after oral challenge with serovar Enteritidis. Western blotting analysis with both sera and feces revealed that antibodies against three bands, 53, 56, and 60 kDa, dominated the oral groups, and an electrospray-mass spectroscopy analysis of these bands showed amino acid sequences coinciding with those of phase-1 flagellin and hook-associated protein 2.

Substrate complexes and domain organization of the Salmonella flagellar export chaperones FlgN and FliT.

The flagellar proteins FlgN and FliT have been proposed to act as substrate-specific export chaperones, facilitating incorporation of the enterobacterial hook-associated axial proteins (HAPs) FlgK/FlgL and FliD into the growing flagellum. In Salmonella typhimurium flgN and fliT mutants, the export of target HAPs was reduced, concomitant with loss of unincorporated flagellin into the surrounding medium. Gel filtration chromatography of wild-type S. typhimurium cell extracts identified stable pools of FlgN and FliT homodimers in the cytosol, but no chaperone-substrate complexes were evident. Nevertheless, stable unique complexes were assembled efficiently in vitro by co-incubation of FlgN and FliT with target HAPs purified from recombinant Escherichia coli. The sizes of the chaperone-substrate complexes indicated that, in each case, a chaperone homodimer binds to a substrate monomer. FlgN prevented in vitro aggregation of FlgK monomers, generating a soluble form of the HAP. Recombinant polypeptides spanning the potentially amphipathic C-terminal regions of FlgN or FliT could not complement in trans the chaperone deficiency of the respective flgN and fliT mutants, but efficient flagellar assembly was restored by homodimeric translational fusions of these domains to glutathione S-transferase, which bound FlgK and FlgL like the wild-type FlgN. These data provide further evidence for the substrate-specific chaperone function of FlgN and FliT and indicate that these chaperones comprise common N- and C-terminal domains mediating homodimerization and HAP substrate binding respectively. In support of this view, the flgN mutation was specifically complemented by a hybrid chaperone comprising the N-terminal half of FliT and the C-terminal half of FlgN.

The flagellar hook protein, FlgE, of Salmonella enterica serovar typhimurium is posttranscriptionally regulated in response to the stage of flagellar assembly.

We investigated the posttranscriptional regulation of flgE, a class 2 gene that encodes the hook subunit protein of the flagella. RNase protection assays demonstrated that the flgE gene was transcribed at comparable levels in numerous strains defective in known steps of flagellar assembly. However, Western analyses of these strains demonstrated substantial differences in FlgE protein levels. Although wild-type FlgE levels were observed in strains with deletions of genes encoding components of the switch complex and the flagellum-specific secretion apparatus, no protein was detected in a strain with deletions of the rod, ring, and hook-associated proteins. To determine whether FlgE levels were affected by the stage of hook-basal-body assembly, Western analysis was performed on strains with mutations at individual loci encompassed by the deletion. FlgE protein was undetectable in rod mutants, intermediate in ring mutants, and wild type in hook-associated protein mutants. The lack of negative regulation in switch complex and flagellum-specific secretion apparatus deletion mutants blocked for flagellar construction prior to rod assembly suggests that these structures play a role in the negative regulation of FlgE. Quantitative Western analyses of numerous flagellar mutants indicate that FlgE levels reflect the stage at which flagellar assembly is blocked. These data provide evidence for negative posttranscriptional regulation of FlgE in response to the stage of flagellar assembly.

Molecular cloning and characterization of the Helicobacter pylori fliD gene, an essential factor in flagellar structure and motility.

Helicobacter pylori colonizes the human stomach and can cause gastroduodenal disease. Flagellar motility is regarded as a major factor in the colonizing ability of H. pylori. The functional roles of flagellar structural proteins other than FlaA, FlaB, and FlgE are not well understood. The fliD operon of H. pylori consists of flaG, fliD, and fliS genes, in the order stated, under the control of a sigma(28)-dependent promoter. In an effort to elucidate the function of the FliD protein, a hook-associated protein 2 homologue, in flagellar morphogenesis and motility, the fliD gene (2,058 bp) was cloned and isogenic mutants were constructed by disruption of the fliD gene with a kanamycin resistance cassette and electroporation-mediated allelic-exchange mutagenesis. In the fliD mutant, morphologically abnormal flagellar appendages in which very little filament elongation was apparent were observed. The fliD mutant strain was completely nonmotile, indicating that these abnormal flagella were functionally defective. Furthermore, the isogenic fliD mutant of H. pylori SS1, a mouse-adapted strain, was not able to colonize the gastric mucosae of host mice. These results suggest that H. pylori FliD is an essential element in the assembly of the functional flagella that are required for colonization of the gastric mucosa.

Effect of hook subunit concentration on assembly and control of length of the flagellar hook of Salmonella.

The flagellar hook of Salmonella is a filamentous polymer made up of subunits of the protein FlgE. Hook assembly is terminated when the length reaches about 55 nm. After our recent study of the effect of cellular levels of the hook length control protein FliK, we have now analyzed the effect of cellular levels of FlgE itself. When FlgE was overproduced in a wild-type strain, a fliC (flagellin) mutant, or a fliD (hook-associated protein 2 [HAP2], filament capping protein) mutant, the hooks remained at the wild-type length. In a fliK (hook length control protein) mutant, which produces long hooks (polyhooks), the overproduction of FlgE resulted in extraordinarily long hooks (superpolyhooks). In a flgK (HAP1, first hook-filament junction protein) mutant or a flgL (HAP3, second hook-filament junction protein) mutant, the overproduction of FlgE also resulted in longer than normal hooks. Thus, at elevated hook protein levels not only FliK but also FlgK and FlgL are necessary for the proper termination of hook elongation. When FlgE was severely underproduced, basal bodies without hooks were often observed. However, those hooks that were seen were of wild-type length, demonstrating that FlgE underproduction decreases the probability of the initiation of hook assembly but not the extent of hook elongation.

Substrate-specific binding of hook-associated proteins by FlgN and FliT, putative chaperones for flagellum assembly.

During flagellum assembly by motile enterobacteria, flagellar axial proteins destined for polymerization into the cell surface structure are thought to be exported through the 25-30 A flagellum central channel as partially unfolded monomers. How are premature folding and oligomerization in the cytosol prevented? We have shown previously using hyperflagellated Proteus mirabilis and a motile but non-swarming flgN transposon mutant that the apparently cytosolic 16. 5 kDa flagellar protein FlgN facilitates efficient flagellum filament assembly. Here, we investigate further whether FlgN, predicted to contain a C-terminal amphipathic helix typical of type III export chaperones, acts as a chaperone for axial proteins. Incubation of soluble radiolabelled FlgN from Salmonella typhimurium with nitrocellulose-immobilized cell lysates of wild-type S. typhimurium and a non-flagellate class 1 flhDC mutant indicated that FlgN binds to flagellar proteins. Identical affinity blot analysis of culture supernatants from the wild-type and flhDC, flgI, flgK, flgL, fliC or fliD flagellar mutants showed that FlgN binds to the flagellar hook-associated proteins (HAPs) FlgK and FlgL. This was confirmed by blotting artificially expressed individual HAPs in Escherichia coli. Analysis of axial proteins secreted into the culture medium by the original P. mirabilis flgN mutant demonstrated that export of FlgK and FlgL was specifically reduced, with concomitant increased release of unpolymerized flagellin (FliC), the immediately distal component of the flagellum. These data suggest that FlgN functions as an export chaperone for FlgK and FlgL. Parallel experiments showed that FliT, a similarly small (14 kDa), potentially helical flagellar protein, binds specifically to the flagellar filament cap protein, FliD (HAP2), indicating that it too might be an export chaperone. Flagellar axial proteins all contain amphipathic helices at their termini. Removal of the HAP C-terminal helical domains abolished binding by FlgN and FliT in each case, and polypeptides comprising each of the HAP C-termini were specifically bound by FlgN and FliT. We suggest that FlgN and FliT are substrate-specific flagellar chaperones that prevent oligomerization of the HAPs by binding to their helical domains before export.

The type III secretion determinants of the flagellar anti-transcription factor, FlgM, extend from the amino-terminus into the anti-sigma28 domain.

The flagellar-specific anti-sigma factor, FIgM, inhibits the expression of late flagellar genes until the hook-basal body structure is assembled and competent for export of the flagellins and hook-associated proteins (flagellar late proteins). FIgM monitors this assembly checkpoint by being a substrate for export via the hook-basal body structure, which includes a type III protein secretion complex. Amino acid sequence alignment of late-secreted flagellar proteins identified a region of homology present in the amino-terminus of FIgM and the other late flagellar proteins, but not in flagellar proteins secreted earlier during flagellar biosynthesis. Single amino acid substitutions at specific positions within this motif decreased the export of FIgM. Deletion of this region (S3-P11) resulted in lower intracellular FIgM levels, but did not prevent recognition and export by the flagellar-specific secretion system. Mutations were isolated in a second region of FIgM spanning residues K27 to A65 that exhibited increased anti-sigma28 activity. These FIgM 'hyperinhibitor' mutants were secreted less than wild-type FIgM. Mutations that interfere with the secretion of FIgM without abolishing anti-sigma28 activity have a negative effect upon the secretion of a His-tagged FIgM mutant that lacks anti-sigma28 activity. Models are proposed to explain the dominant negative phenotype of the FIgM secretion mutants reported in this study.

Cloning, sequencing and expression of the flagellin core protein and other genes encoding structural proteins of the Vibrio cholerae flagellum

Vibrio cholerae is a Gram-negative bacterium with a single polar flagellum. Motility is an important virulence factor for this non-invasive pathogen. We cloned and sequenced a locus in V. cholerae V86 (El Tor, Inaba) that contained five different structural genes of the flagellum. The cloned genes and their products were assigned names and functions based on homology with sequences of similar genes and their products from other related bacteria. All of these genes of V. cholerae V86, namely, flgI, J, M, L and flaA, were transcribed in the same direction. These genes respectively encoded the P- and L-ring proteins, the hook-associated proteins 1 and 3 and the flagellin core protein of the flagellum. Our data indicated the presence of more than one flagellar locus in V. cholerae which could provide a means of immunoavoidance during infection. When compared with homologs in other bacteria, the flagellin core protein of V. cholerae exhibited conservation in the N- and C-termini, but had diverged in the central region.

Cloning, sequencing and expression of the flagellin core protein and other genes encoding structural proteins of the Vibrio cholerae flagellum.

Vibrio cholerae is a Gram-negative bacterium with a single polar flagellum. Motility is an important virulence factor for this non-invasive pathogen. We cloned and sequenced a locus in V. cholerae V86 (El Tor, Inaba) that contained five different structural genes of the flagellum. The cloned genes and their products were assigned names and functions based on homology with sequences of similar genes and their products from other related bacteria. All of these genes of V. cholerae V86, namely, flgI, J, M, L and flaA, were transcribed in the same direction. These genes respectively encoded the P- and L-ring proteins, the hook-associated proteins 1 and 3 and the flagellin core protein of the flagellum. Our data indicated the presence of more than one flagellar locus in V. cholerae which could provide a means of immunoavoidance during infection. When compared with homologs in other bacteria, the flagellin core protein of V. cholerae exhibited conservation in the N- and C-termini, but had diverged in the central region.

Cloning and nucleotide sequence of a flagellin encoding genetic locus from Xenorhabdus nematophilus: phase variation leads to differential transcription of two flagellar genes ( fliCD)

The insect-pathogenic bacterium Xenorhabdus undergoes spontaneous phase variation involving a large number of phenotypes. Our previous study indicated that phase I variants were motile, whereas phase II variants of X. nematophilus F1 were nonflagellated cells which did not synthesize flagellin [Givaudan, A., Baghdiguian, S., Lanois, A. and Boemare, N. (1995) Appl. Environ. Microbiol. 61, 1408–1413]. In order to approach the study of the flagellar switching, a locus containing two ORFs from X. nematophilus F1 (phase I) was identified by using functional complementation of flagellin-negative E. coli. The sequence analysis revealed that the first ORF corresponds to the fliC gene coding for flagellin, and showed a high degree of homology between the N-terminal and C-terminal of Xenorhabdus FliC and flagellins from other bacteria. The second identified ORF in the opposite orientation encodes a homologue of the enterobacterial hook-associated protein 2, FliD. Both Xenorhabdus fliCD genes were required for the entire restoration of E. coli motility. A sequence highly homologous to the σ 28 consensus promoter was identified upstream from the coding sequences from both genes. The structure of the fliC gene and its surrounding region was shown to be the same in both phase variants, but Northern blot analysis revealed that fliC and fliD were, respectively, not and weakly transcribed in phase II variants. In addition, complementation experiments showed that motility and flagellin synthesis of phase II cannot be recovered by placing in trans fliCD genes from phase I. These latter results suggest that a gene(s) higher in the transcriptional hierarchy of the flagellar regulon is switched off in Xenorhabdus phase II variants.

Production and identification of recombinant proteins of Salmonella typhimurium and their use in detection of antibodies in experimentally challenged animals

Antibodies to experimental Salmonella typhimurium challenge in cattle and sheep were assessed by 15 recombinant flagellum proteins. The 15 DNA fragments selected for gene expression were derived from external flagellin, hook, hook-associated protein, and basal body gene domains. Our efforts were focused on characterizing the humoral immune response of Salmonella infected and vaccinated animals and identifying immunodominant antigenic determinants. This communication reports that the 159–261 amino acids of external flagellum (FliCi-1), 285–331 amino acids of hook protein (FlgE-2), and 309–391 amino acids and 440–537 amino acids of hook-associated protein (FlgK-1 and -2) appeared to be the most immunoreactive proteins and were recognized by all of the experimental animal sera tested in this study.

Production and identification of recombinant proteins ofSalmonella typhimuriumand their use in detection of antibodies in experimentally challenged animals

Antibodies to experimental Salmonella typhimurium challenge in cattle and sheep were assessed by 15 recombinant flagellum proteins. The 15 DNA fragments selected for gene expression were derived from external flagellin, hook, hook-associated protein, and basal body gene domains. Our efforts were focused on characterizing the humoral immune response of Salmonella infected and vaccinated animals and identifying immunodominant antigenic determinants. This communication reports that the 159-261 amino acids of external flagellum (FliCi-1), 285-331 amino acids of hook protein (FlgE-2), and 309-391 amino acids and 440-537 amino acids of hook-associated protein (FlgK-1 and -2) appeared to be the most immunoreactive proteins and were recognized by all of the experimental animal sera tested in this study.

Transcriptional analysis of the flgK and fliD operons of Salmonella typhimurium which encode flagellar hook-associated proteins.

In Salmonella typhimurium, three hook-associated proteins, HAP1, HAP2 and HAP3, are known to be essential for formation of flagellar filament. HAP1 and HAP2 are encoded by the flgK and flgL genes, respectively, which together constitute an operon, called the flgK operon. HAP3 is encoded by the fliD gene which forms part of the fliD operon together with the fliS and fliT genes. In the flagellar regulon, the operons are divided into three classes, 1, 2 and 3, based on their positions within a transcriptional hierarchy. Transcriptional analysis suggested that the flgK and fliD operons should belong to class 3, whose expression is dependent on the flagellum-specific sigma factor FliA. However, biochemical data indicated that these HAP proteins are detectable even in the hook-basal body structures produced by the fliA mutant. This work was carried out to resolve this discrepancy. More careful examination of transcription revealed that the fliA mutation reduces but does not eliminate the expression of these operons, whereas a mutation in the flhD operon, which encodes activator proteins for the class 2 operons, eliminates their expression. This suggests that the flgK and fliD operons may be transcribed from both class 2 and class 3 promoters. Primer extension analysis indicated that the promoter region of fliD contains both class 2 and class 3 promoters, while that of flgK contains only a class 3 promoter. Transposon insertion into the flgB operon, which belongs to class 2 and lies upstream of the flgK operon, was found to decrease the expression of the flgK operon to the basal level.(ABSTRACT TRUNCATED AT 250 WORDS)