Journal of Bacteriology, December 2002, p . 6424-6433, Vol . 184, No . 23

Despite a long record of being safe, there is increasing public concern about the potential pathogenicity of B . thuringiensis to humans . This is due to the sporadic but sometimes severe infections caused by this organism, which include diarrheal food poisoning (34), corneal ulcer (52), cellulitis (57), and burn (13) as well as war wound (28) infections . Moreover, the pathogenicity of B . thuringiensis has been demonstrated in an animal model of infection by administering spores to mice via nasal inoculation (27, 50) . Similar to Bacillus cereus, B . thuringiensis produces a variety of virulence factors, which include phosphatidylcholine-preferring phospholipase C (PC-PLC) and phosphatidylinositol-specific phospholipase C (38, 40), hemolysins, and enterotoxins (1, 18, 21) . Heierson et al . (24) isolated an avirulent pleiotropic mutant of B . thuringiensis that was unable to express both hemolytic activities and flagella and suggested that flagella and motility in this organism played a role in parasite-host interactions . Such a finding, moreover, was thought to indicate that the regulation of genes encoding flagellar components and virulence factors could be coordinated in B . thuringiensis (60) .

Several lines of evidence convincingly support the idea that virulence and motility are intimately linked in many pathogenic bacteria (reviewed in reference 49); however, little information is available on their genetic or structural relationships, most likely because of the complexity of both the assembly of the flagellar/chemotactic apparatus and the molecular mechanisms involved in the secretion of virulence factors . To the best of our knowledge, nothing is known as to whether common pathways governing expression of flagella and secretion of virulence factors occur in B . thuringiensis .

The flagellar components are known to be sequentially assembled from the cytoplasmic membrane outward (55) . The ordered assembly process arises, at least in part, from the hierarchical nature of flagellar gene expression, flagellin being the last and major flagellar protein expressed . Flagellar genes are organized into several classes in both the enteric bacteria and Bacillus subtilis, and the function of each gene product in a given class is required for the expression of genes in a lower class (44, 45) . While class II genes encode structural and accessory proteins needed for the assembly of the flagellar hook-basal body (HBB) complex, class III genes, the expression of which depends on the late-flagellar sigma factor (²⁸ in the enterics and ^D in B . subtilis), encode proteins required for the maturation of the flagellum and the chemosensory system . Coupling of class III gene expression to the HBB complex is achieved through the action of FlgM, an anti-sigma factor, which inactivates ²⁸ (or ^D) until a functional HBB is complete (44, 48) . FlgM is then secreted from the cell by the flagellar-dedicated secretory apparatus, which is localized within the flagellar basal body and contains a membrane-associated and specialized pore for protein export (46) . Indeed, with the P- and L-ring subunits as the only exception (30, 32), none of the external proteins of the flagellum possess a signal peptide sequence at their amino termini (29, 31) and, therefore, cannot be exported by the bacterial general secretory pathway . The flagellar export apparatus encompasses six integral proteins (FlhA, FlhB, FliO, FliP, FliQ, and FliR in Salmonella) (46) that share substantial homology with components of the type III virulence secretion pathway found in gram-negative bacteria (reviewed in reference 33) . Interestingly, it has been demonstrated that the virulence-associated protein YplA of Yersinia enterocolitica, although not involved in motility, is secreted by the flagellar export apparatus (58) . This finding supports the idea that this export system and the type III virulence secretion pathway may have had a common evolutionary origin and share overlapping functions .

A relationship between virulence and motility is also supported by the demonstration that, in some microorganisms, a substantial increase in the secretion of virulence factors is associated with a specialized form of flagellum-driven motility, the swarming motility that enables bacterial cells to collectively move across solid surfaces (3, 14, 43, 54) . The ability to swarm depends on a complex surface-induced differentiation process which is characterized by the production of elongated and aseptate cells that exhibit a remarkable increase in the number of flagella in comparison with that of the short oligoflagellated swimmer cells (reviewed in reference 22) . A link between swarming motility and the type III virulence secretion pathway has been further suggested by the finding that null mutations in genes encoding subunits of the flagellum type III export machinery abolish swarming migration in Proteus mirabilis, Y . enterocolitica, and Salmonella (20, 59) . On the other hand, the decreased expression of the hpmA hemolysin gene in an flhA null mutant of P . mirabilis indicates that a motility gene could have a regulatory role in the production of virulence factors (20) . The relationship between swarming and virulence has been investigated mostly in gram-negative bacteria (2, 3, 14), although several species belonging to the Bacillus and Clostridium genera have been described to be active swarmers (26, 43, 54) . Swarming differentiation has never been described for B . thuringiensis, and nothing is known as to whether molecular components coupling motility and virulence do exist in this organism .

This report describes the swarming behavior exhibited by B . thuringiensis in response to surface sensing and the isolation of a nonswarming mutant with a transposon insertion in flhA, a flagellar class II gene involved in the type III export of flagellar components (46) . The flhA mutant of B . thuringiensis was defective in both swimming and swarming motility because it lacked flagella . The impairment in the flagellar filament assembly was due to defective export of flagellin, as both flagellin gene transcription and flagellin production could be detected inside the cell . The mutant was also defective in the secretion of the virulence factors hemolysin BL (HBL) (9) and PC-PLC, the intracellular production of which was demonstrated to occur in the flhA mutant as well as in the parental strain . The requirement of flhA for flagellin export, swarming differentiation, and secretion of virulence-associated proteins was confirmed by complementing the flhA mutant with a plasmid harboring the flhA gene . These findings suggest that a putative hierarchy in the expression of B . thuringiensis motility genes may differ from that described for B . subtilis and the enteric bacteria and indicate that a coordinate secretion for flagellin and virulence-associated proteins does exist in B . thuringiensis .

Motility assays. Swimming motility was evaluated on semisolid (0.25% agar) LB medium at 30°C and was confirmed by microscopic examination of bacteria under a phase-contrast microscope . Phenotypic assays for swarming were initiated by spotting 2 µl of an overnight culture at the center of tryptone-NaCl or LB plates containing 0.45 to 1.5% agar . Plates were analyzed after growth at up to 24 to 48 h of incubation at 30°C . Bacteria were Gram stained for microscopy to evaluate the presence of elongated swarm cells (54) . Flagella staining was performed as described by Harshey and Matsuyama (23) .

Mutant screening, sequencing, and complementation of flhA. The thermosensitive plasmid pIC333, carrying the mini-Tn10 insertion that contains a spectinomycin resistance gene (56), was introduced by electroporation (39) into strain IP2 to produce a library of insertional mutants as previously described (19) . Spectinomycin-resistant mutants were isolated on 1% agar LB plates containing spectinomycin and were screened a second time on semisolid LB medium to isolate nonmotile mutants . Chromosomal DNA from the nonmotile strain MP02 was cut with EcoRI or HindIII and was ligated . The ligation mixture was used to transform E . coli, and transformants were screened for resistance to spectinomycin (60 µg ml^-1) . Plasmid DNA was prepared from E . coli transformants, and restriction maps of the plasmid were determined to verify the presence of the mini-Tn10 insertion (a 2.2-kb BamHI fragment is characteristic of the mini-Tn10 insertion) . The junction fragments between the ends of the mini-Tn10 insertion and chromosomal DNA were sequenced by using the oligonucleotides 5'-CGTTGGCCGATTCATTAATGC-3' and 5'-CGATATTCACGGTTTACCCAC-3' that matched the ends of the mini-Tn10 insertion . DNA sequencing was performed by using the ALFexpress AutoRead Sequencing Kit (Pharmacia Biotech, Uppsala, Sweden) and the ALFexpress DNA sequencer (Pharmacia) . Sequences were analyzed by GCG software, and database searching was performed with the BLAST algorithm . Theoretical protein molecular weight and pI values were calculated by the Compute pI/M_w program . Predicted transmembrane segments were identified by the TMpred program at the EMBnet node .

flhA was amplified from strain IP2 by using primers (flhAU, 5'-CAGACGAATGAACTT-3'; flhAL, 5'-CTTTCCATTACTTCACC-3') designed on the basis of sequences external to the coding region and was cloned into the pGEM-T vector (Promega, Madison, Wis.) . flhA was excised from the plasmid by using SphI and SpeI and was cloned into pHT304 (4) . pHT304flhA was introduced into strain MP02 by electroporation, and recombinant clones were screened on LB agar containing erythromycin .

RNA isolation, cDNA synthesis (RT), and PCR. Total RNA was purified from B . thuringiensis cultures grown in LB broth for 3 h . Briefly, after being washed with diethylpyrocarbonate-treated water, 10⁸ bacterial cells were resuspended in 450 µl of lysis buffer (Rneasy Mini Kit; Qiagen) containing 0.35 g of glass beads (diameter, 0.1 mm) and was vortexed for 15 min to break the cells . Samples were centrifuged for 2 min at 10,000 x g and the aqueous phase was removed . Two hundred-fifty microliters of absolute ethanol was added, and the mixture was applied to an RNeasy mini spin column (Qiagen) . After being digested with 40 Kunitz units of RNase-free DNase (Qiagen) for 20 h, total RNA was eluted from the column by following the instructions of the manufacturer . An aliquot of the RNA was examined on agarose gel to ensure its integrity and lack of DNA contamination . For cDNA synthesis, up to 2 µg of RNA was annealed with 1 µg of random hexamer primers at 70°C for 5 min, chilled on ice for 5 min, and mixed with reverse transcription (RT) buffer (50 mM Tris [pH 8.3], 75 mM KCl, 3 mM MgCl2), 13 mM dithiothereitol, 0.5 mM desoxynucleoside triphosphate, and 200 U of Superscript II reverse transcriptase (Gibco BRL, Life Technologies) . Reactions were carried out at 42°C for 50 min and were stopped by incubation at 72°C for 15 min . For detection of flagellin gene expression, cDNA was amplified with the primers flaBU2 (5'-TAACAGCATGCGTACTCAAGAG-3') and flaBL1 (5'-CATTTGTGGAGTTTGGTTAGC-3') . For detection of flgM the primers flgMF1 (5'-AATCAATTTGGAACACA-3') and flgMR3 (5'-ATTTGCGTCTACTTTGTA-3') were used in PCR to amplify B . thuringiensis or B . subtilis chromosomal DNA . For Southern analysis, B . thuringiensis and B . subtilis genomes were digested with either EcoRI or PstI, electrophoresed, and transferred to positively charged nylon membranes . The amplification product obtained with flgMF1 and flgMR3 on the B . subtilis genome was labeled with the ECL Direct Labeling and Detection kit (Pharmacia) . Filters were hybridized with this probe, and chemiluminescence was detected by Hyperfilm ECL (Pharmacia) .

Preparation of culture supernatants and cell lysates. Bacterial cells were grown in BHIG in a rotary shaker at 200 rpm for 5 h at 30°C and were pelleted by centrifugation at 10,000 x g . Culture supernatants were concentrated with Microcon YM-10 filters (Millipore, Bedford, Mass.) . Cells were washed with cold Tris-buffered saline (TBS; pH 7.4) and lysed in 1 ml of TBS with zirconium beads (diameter, 0.1 mm) by shaking at 4°C for 4 min with a mini-bead beater (Biospec Products, Barltesville, Okla.) . Residual cells and debris were removed from the lysate by centrifugation at 12,000 x g . Concentrated culture supernatants and cell lysates were stored at 20°C and were used within 1 week .

Protein gel and immunoblot analysis. Protein samples were resuspended in sample buffer containing ß-mercaptoethanol (51) and were normalized with respect to the optical density at 600 nm of the culture . After being heated at 95°C for 10 min, proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and gels were either silver stained (11) or were used for protein blotting . Immunoblotting was performed as described previously (10), and membranes were probed with rabbit antisera to the individual HBL components, with an anti-B monoclonal antibody, or with a mouse antiserum to B . thuringiensis strain IP2 flagellin (produced during this investigation) . Isoelectric points were determined on a PhastSystem (Pharmacia) by using Pharmacia isoelectric focusing (IEF) standards on Phastgel IEF gels with a pH range of 9 to 3 .

Preparation of a mouse antiserum to B . thuringiensis flagellin. Purified flagellin was prepared with cultures of strain IP2 grown in BHIG for 5 h at 30°C . Bacterial cells were vortexed, and after centrifugation to remove cells flagellar filaments were collected from supernatants by high-speed centrifugation at 100,000 x g for 1 h . Flagellin was analyzed by SDS-PAGE separation as described by Gygi et al . (20) and were electroeluted . BALB/c mice were immunized by intraperitoneal injection of 30 µg of the protein preparation dissolved in Tris-HCl buffer and emulsified in Freund's incomplete adjuvant (1:3) . At day 21 animals received a booster injection (30 µg of the protein in adjuvant), and serum was obtained after 48 h .

Detection of hemolysin and PC-PLC activities. HBL activity was determined on sheep blood agar plates by the formation of a discontinuous zone of hemolysis around colonies (8) . Thuringiolysin O and hemolysin II, III, and IV were detected by IEF sheep blood agar overlay of culture supernatants with or without cholesterol (Sigma Chemical Co., St . Louis, Mo.) . PC-PLC activity was measured in culture supernatants and cell lysates by a gel diffusion assay with a gel containing 1.5 mg (wt/vol) of crude PC (Sigma)/ml in TBS . Different amounts of pure PC-PLC (Sigma) were used to generate a standard curve . Slides were kept at 37°C in a humidified chamber, and after 12 to 18 h the activity was recorded as the diameter of the opaque zone .

 
Isolation and characterization of the nonmotile and nonswarming mutant MP02. Several mutants of B . thuringiensis strain IP2 were obtained by transposon mutagenesis with the mini-Tn10 insertion (56) that contains a spectinomycin resistance gene . Among the mini-Tn10 mutants obtained, we selected a strain that was unable to move on semisolid (0.25% agar) media even after prolonged incubation (24 h) . Microscopic examination of the mutant, referred to as MP02, showed that it was no longer motile, and flagella staining revealed no flagella on the cell surface (Fig . 1E) . When propagated over solid media, strain MP02 was unable to swarm, giving rise to compact colonies (Fig . 1F) in which swarm cell differentiation was never observed . Only short rods (Fig . 1G) never exhibiting flagella (Fig . 1H) were observed both in the center and at the colony rim .

The mini-Tn10 element contains a pUC replicon allowing recovery of the chromosomal DNA sequences flanking the insertion site . Therefore, chromosomal DNA from the mutant MP02 was digested with HindIII or EcoRI (restriction sites for these two enzymes are not present in the mini-Tn10 element) and was treated with T4 DNA ligase . The ligation mixture was used to transform E . coli cells, and transformants were selected for resistance to spectinomycin . The recombinant plasmids isolated from the Spec^R transformants were used as templates to determine the nucleotide sequence of the ends of the mini-Tn10 element and the chromosomal DNA adjacent to the insertion site . Sequencing showed that the transposon had inserted into a 2,082-bp coding sequence sharing similarities (58% identity in 977 overlapping nucleotides) with B . subtilis flhA; therefore, the B . thuringiensis gene was named flhA (GenBank accession number AJ438180) . This gene has a putative ribosome-binding site 9 nucleotides upstream of the first nucleotide and encodes a 688-residue protein having a calculated size of 76.8 kDa and a pI of 5.19 . The predicted amino acid sequence of B . thuringiensis FlhA showed significant homology to the FlhA protein of B . halodurans (37% identity, 57% positives), B . subtilis (35% identity, 56% positives), extremophilic eubacteria (Thermotoga maritima and Aquifex aeolicus), and several other gram-negative species (Helicobacter pylori, E . coli, Campylobacter coli, Salmonella enterica serovar Typhimurium, P . mirabilis, Treponema pallidum, Y . enterocolitica, and Borrelia burgdorferi) . Analysis of the predicted amino acid sequence identified seven hydrophobic regions (putative transmembrane segments) in the N-terminal sequence and a long hydrophilic C-terminal domain . This finding suggests that the protein encoded by B . thuringiensis flhA is bound to the cytoplasmic membrane and possesses a soluble domain protruding into the cytoplasm, as already demonstrated for Salmonella FlhA (46) . Like many other proteins constituting the flagellar export apparatus in gram-negative and -positive organisms, B . thuringiensis FlhA displayed a high degree of relatedness to a family of membrane proteins (LcrD of Yersinia pestis, Yersinia pseudotuberculosis, and Y . enterocolitica; PcrD of Pseudomonas aeruginosa; MxiA of Shigella flexneri; InvA and SsaV of S . enterica serovar Typhimurium; and SctV of Chlamydia spp.) involved in the type III secretion of virulence factors (reviewed in reference 33) . The highest sequence similarity was observed in the hydrophobic N-terminal region, predicted to contain transmembrane segments, while less similarity was found in the putative cytoplasmic C terminus .

B . thuringiensis flhA is necessary for the export of flagellin but not for its synthesis. Enteric bacteria and B . subtilis carrying mutations in flhA exhibit a nonflagellated phenotype, and the mutation in flhA has been demonstrated to be the only factor responsible for the lack of flagellar assembly in the mutant strains (12, 16, 20, 35) . Indeed, flhA mutants are completely defective in the export of the anti-sigma factor FlgM, which downregulates the expression of flagellar class III genes, flagellin included (44) . Furthermore, it is generally assumed that the protein encoded by flhA is an essential component for the export of structural flagellar proteins outside the cell . In Salmonella, FlhA is associated with the flagellar basal body, physically interacts with the MS ring, and strongly binds to flagellin, giving rise to a complex that is necessary for promoting the export of flagellin outside the cell (47) .

The complete lack of flagellar-filament assembly observed in the flhA mutant of B . thuringiensis prompted us to investigate whether the flhA mutation in B . thuringiensis affected both flagellin gene transcription and flagellin export . Flagellin mRNA was searched for in both strains MP02 and IP2 by the use of RT-PCR by using primers designed on the basis of the available flagellin gene sequence of B . thuringiensis subsp . alesti (41) . The flagellin transcript was detected in the wild type as well as in the flhA mutant (Fig . 2) . The ability of the flhA mutant of B . thuringiensis to transcribe a purported class III gene product, flagellin, suggested that B . thuringiensis flhA cannot be considered a flagellar class II gene .

 
To evaluate whether a gene similar to flgM could be found in B . thuringiensis, chromosomal DNAs from strain IP2, the mutant MP02, and B . subtilis ATCC 6633 (as the positive control) were subjected to amplification with the primers flgMF1 and flgMR3, constructed on the basis of the B . subtilis flgM sequence (http://genolist.pasteur.fr) . Although a variety of PCR conditions were used, no amplification product was ever obtained with both strains IP2 and MP02 of B . thuringiensis, while amplification was achieved with B . subtilis ATCC 6633 . Moreover, a flgM-specific probe prepared from B . subtilis always gave a complete lack of hybridization when used with B . thuringiensis (Fig . 3) . The lack of PCR amplification and DNA-DNA hybridization might be due to the distant relationship between B . thuringiensis and B . subtilis . However, B . thuringiensis is closely related to B . cereus and Bacillus anthracis (25) . Thus, a search of sequences similar to that of flgM was performed with the available genome sequences of B . anthracis (http://www.tigr.org) and B . cereus (http://www.integratedgenomics.com) . No significant similarity was found, thus supporting the hypothesis that the lack of flgM could be a common feature of species belonging to the B . cereus group .

 
The question of whether flagellin was synthesized and not exported by strain MP02 was addressed by performing immunoblot assays on cell lysates and culture supernatants from strains MP02 and IP2 . As shown in Fig . 4, flagellin was synthesized by both strains but the protein was not exported by strain MP02 . Moreover, the amount of flagellin that accumulated intracellularly in the mutant appeared to be higher than that in the parental strain . This finding clearly shows that, in contrast to B . subtilis (12), the flhA mutation in B . thuringiensis does not influence flagellin production . Therefore, the lack of functional flagellar export machinery due to the flhA mutation appears to be the only factor responsible for the lack of flagellar assembly in this species .

 
The activity of flhA is necessary for the export of nonflagellar proteins. Mutations in flhA have been reported to affect the expression of the hemolysin gene hpmA in P . mirabilis (20) and the secretion of the virulence-associated phospholipase YplA in Y . enterocolitica (58) . Since the flhA mutant of B . thuringiensis was found to be completely defective in the export of flagellin, preliminary investigations were focused on establishing whether the lack of functional flagellar export machinery could also affect the secretion of nonflagellar proteins in this species . SDS-PAGE of culture supernatants from strains MPO2 and IP2 revealed that the mutant was defective in the secretion of several proteins possessed by the wild type (Fig . 5) . Moreover, in contrast to strain IP2, the flhA mutant did not produce the typical discontinuous pattern of hemolysis caused by the secretion of the enterotoxic tripartite HBL (8, 9, 10) on sheep blood agar plates (Fig . 6) . None of the HBL components (B, L1, or L2) could be detected in culture supernatants (100-fold concentrated) from the mutant strain when subjected to immunoblotting with polyclonal antisera specific to the individual components (data not shown) . As expected, immunoblot analysis performed with a monoclonal antibody to the B protein showed that the wild-type strain yielded a positive signal in both culture supernatant and cell lysate (Fig . 7) . However, while only one protein, with a predicted molecular size of 37.5 kDa (corresponding to that estimated for the secreted B component [9]), was detectable in the supernatant, more than one reactive band was observed in the cell lysate . On the basis of these results we hypothesized that the 40-kDa band corresponded to the unprocessed B component still containing the signal peptide, the 37.5-kDa band corresponded to the mature protein, and the lower band (about 36 kDa) corresponded to a degraded form of the protein . No signal was obtained from the culture supernatant of the mutant, while one reactive band (about 36 kDa) was observed in the cell lysate (Fig . 7) . In the mutant, the mature form of B, which was not released into the culture supernatant, could be subjected to rapid hydrolysis, thus accounting for the substantial increase in the intracellular concentration of the purported B degradation product . These observations suggested that in strain MP02 the B component of HBL was synthesized, blocked in its secretion, and intracellularly degraded .

 
To investigate whether the flhA mutation influenced the secretion of other B . thuringiensis hemolysins, culture supernatants from strains IP2 and MP02 were subjected to IEF sheep blood agar overlay assays (Fig . 8) . Zymograms of the mutant were similar to those of the wild type, since both strains were able to secrete thuringiolysin O (pI values of about 6.2, 6.5, and 6.8), which was identified through the inhibition of its activity by cholesterol (36), hemolysin II (6), and/or hemolysin III (5), which have similar isoelectric points (pI values of about 8.2), and hemolysin IV (pI 5.5) (7, 42) .

 
PC-PLC activity was evaluated in culture supernatants and cell lysates from strains IP2 and MP02 by a gel diffusion assay with PC as the substrate . Intracellular PC-PLC activity was 2.7 times lower in strain MP02 than in strain IP2 (0.021 U/ml versus 0.056 U/ml); however, the mutant showed a significant reduction in the secretion of PC-PLC compared to that secreted from the wild type (0.05 U/ml versus 3.46 U/ml) . This result indicates that the mutation in flhA, although weakly reducing PC-PLC synthesis, dramatically reduces its secretion .

Complementation of the flhA mutant. Complementation of the flhA mutant with a plasmid harboring the flhA gene was performed to establish if the defect in flhA was the only one responsible for the phenotype exhibited by strain MP02 . flhA was subcloned into the SphI/SpeI sites of pHT304, an E . coli-B . thuringiensis shuttle and expression vector (4) . pHT304flhA was introduced into strain MP02, generating strain MP03 that displayed flagella on the cell surface and, as in the parental strain, exhibited a swimming or swarming behavior depending on whether it was grown in liquid or solid medium (Fig . 9) . The ability to produce HBL and PC-PLC was regained, although the level of both toxins was lower than that produced by the wild type . Weak HBL activity was observed when MP03 was propagated on sheep-blood agar plates, and all the HBL components were detected by immunoblot analysis . Moreover, extra- and intracellular PC-PLC (0.64 and 0.048 U/ml, respectively) were 12.8 and 2.3 times higher than the corresponding activities measured with the mutant .

 
These findings demonstrate that in B . thuringiensis the flagellum-aided swimming and swarming motility, the export of flagellin, and the secretion of virulence-associated proteins, such as HBL and PC-PLC, depend on the activity of flhA.

B . thuringiensis flhA and motility. The identification of the flhA gene in B . thuringiensis was achieved by sequencing the DNA regions flanking the insertion site of the mini-Tn10 element in a mutant (MP02) that was completely devoid of flagella (Fig . 1E) . The nonflagellated phenotype observed for the mutant suggested that B . thuringiensis flhA could have a role similar to that played by flhA in enteric bacteria and B . subtilis (12, 16, 20, 44, 46), where: (i) flhA has been classified as a flagellar class II gene regulating the expression of flagellar class III genes, flagellin included, and (ii) FlhA is a membrane protein involved in the export of structural flagellar proteins outside the cell . flhA has not been described previously for B . thuringiensis, and there is no report demonstrating that, in this organism, flagellar gene expression is governed by a given hierarchical pathway . Therefore, it was interesting to address the question of whether a B . thuringiensis mutant having a genetic defect in flhA transcribed the flagellin gene, synthesized flagellin, and exported flagellin outside the cell .

The predicted amino acid sequence of B . thuringiensis flhA suggested that FlhA is a membrane protein also in this organism and, by analogy with FlhA of other organisms, was thought to behave as a component of the flagellar export apparatus . Evidence is provided to demonstrate that the flagellin transcript and the intracellular production of flagellin occurred in the wild type as well as in the flhA mutant of B . thuringiensis (Fig . 2 and 4) . In contrast, the export of flagellin outside the cell could be detected only in the parental strain, while an increased amount of intracellular flagellin was observed in the mutant (Fig . 4) . These findings clearly show that the flhA mutation in B . thuringiensis, in contrast to the flhA mutants of B . subtilis and enteric bacteria (12, 20, 35), does not abolish flagellin production . The protein is accumulated in the mutant apparently because it is not exported outside the cell . Therefore, flhA cannot be regarded as a flagellar class II gene in B . thuringiensis, since the expression of a purported class III gene, the flagellin gene, does not depend on its activity . Since the expression of flagellar class III genes is coupled with that of class II genes through the action of FlgM, we hypothesized that a gene similar to flgM could not be involved in the regulation of genes encoding external flagellar proteins in B . thuringiensis . This was supported by three findings: (i) the absence of a PCR amplification product with primers designed on the sequence of B . subtilis flgM, (ii) the complete lack of hybridization of a flgM-specific probe with B . thuringiensis DNA (Fig . 3), and (iii) the absence of a gene related to flgM in the genomic sequences of B . anthracis and B . cereus . Therefore, the lack of functional export machinery due to the flhA mutation was considered to be the only factor responsible for the impairment of flagellar assembly in this species .

The flhA mutant of B . thuringiensis was unable to swarm in response to surface sensing (Fig . 1) . Swarming by B . thuringiensis, previously unobserved in this organism, was found to be very similar to that recently described for its closest relative, B . cereus (54) . B . thuringiensis swarm colonies did not produce regularly spaced rings of swarming migration and consolidation; rather, the macroscopic appearance of swarm colonies was characterized by a central consolidation phase surrounded by a continuous layer of an advancing front movement of swarm cells all around the colony rim (Fig . 1B) . The ability of B . thuringiensis to swarm was documented by the demonstration that swarming motility is brought about by elongated and hyperflagellated swarm cells organized as multicellular masses rapidly and coordinately moving outward to the colony border (Fig . 1B to D) . Swarming differentiation was completely lacking in the flhA mutant, which never produced hyperflagellated or elongated swarm cells (Fig . 1F to H) . Characterization of nonswarming mutants has firmly established that all defects in genes encoding subunits of the flagellar export apparatus completely abolish swarming differentiation (20, 59), since swarming differentiation requires functional flagella . The observation that flhA is required for exhibiting a swarming behavior by B . thuringiensis is in agreement with the involvement of flhA in both the assembly of a functional flagellar filament and the control of cell differentiation, as already reported for a nonswarming flhA mutant of P . mirabilis, for which a negative feedback from the flhA defect to the flhDC master operon has been demonstrated (17) .

The requirement of flhA activity for flagellar filament assembly, flagellum-driven motility, and swarming behavior of B . thuringiensis was demonstrated by complementing the flhA mutant with a plasmid harboring an intact flhA gene . The complemented strain regained the ability to export flagellin, swim in liquid media, and differentiate from swimmer cells into the elongated and hyperflagellated swarm cells when propagated over solid culture media (Fig . 9) .

B . thuringiensis flhA and virulence. In addition to the motility defects, the flhA mutant of B . thuringiensis was impaired in the secretion of HBL (Fig . 6 and 7), whereas the secretion of thuringiolysin O and hemolysins II to IV were similar in the wild type and the flhA mutant (Fig . 8) . Our results showed that HBL was synthesized intracellularly, completely blocked in its secretion, and most likely degraded inside the cell in the flhA mutant of B . thuringiensis (Fig . 7) .

The finding that HBL secretion is blocked in a mutant having a defective flagellar export apparatus is of great interest . Secretion of a nonflagellar protein through the flagellar export machinery has already been reported for Y . enterocolitica (58); however, in such a case the protein was exported in a process similar to that of the type III secretion, which implies that the protein is not modified as it crosses the plasma membrane (reviewed in reference 33) . The amino acid sequences of the HBL proteins showed that they all possess a signal peptide sequence at their amino termini, thus indicating their secretion through an S-dependent secretion pathway . However, since the molecular mass of the secreted B component of HBL corresponded to that estimated for the protein lacking the amino-terminal signal sequence, we speculate that signal peptidases could cooperate with the flagellar export apparatus in the secretion of HBL . This hypothesis is supported by the observation that the flagellar P- and L-ring subunits, which do possess a signal peptide sequence before being secreted (30, 32), are exported and assembled in the flagellar structure only when all the components of the flagellar export apparatus are active (44) .

Impairment in HBL secretion was not the only defect we observed in the secretion of virulence-associated proteins in the flhA mutant of B . thuringiensis . The secretion of PC-PLC also appeared to be significantly influenced by the mutation in flhA, although in this case the amount of intracellular PC-PLC activity detected in the mutant was reduced to about one third of the activity measured in the wild type . However, the amount of the enzyme activity secreted by the mutant was about 70 times lower than that by the wild type; indeed, the ratio between the amount of extra- and intracellular PC-PLC activity was higher than 60 and lower than 3 when calculated for the wild type and the mutant, respectively . Such a difference does not merely reflect the reduced amount of PC-PLC synthesized intracellularly in the mutant; rather, the remarkable lowering in the extracellular PC-PLC activity could be explained by assuming that impairment in PC-PLC secretion occurs in the flhA mutant . Therefore, whatever the mechanism of PC-PLC secretion is, this observation strongly suggests that flhA is required for or in some way coupled with the export of PC-PLC outside the cell .

The overall results produced by this investigation provide the first evidence that in B . thuringiensis the activity of a single gene, flhA, is crucial for the assembly of flagellar filament, swimming motility, swarming differentiation, and export of flagellar as well as nonflagellar proteins, such as flagellin and the virulence-associated proteins HBL and PC-PLC . Moreover, the observation that B . thuringiensis flhA cannot be considered a flagellar class II gene is of intrinsic interest, since it opens a new perspective in studying the flagellar gene expression hierarchy of gram-positive bacteria . Finally, the involvement of flhA in the virulence potential of B . thuringiensis could be exploited to produce a B . thuringiensis product that is demonstrably nonpathogenic to humans but still active against insects; this could provide an added margin of safety and allay public concern .

Present address: FBI Academy, Range Rd., Quantico, VA 22135 .

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