A flagellar gene cluster from the oral spirochaete Treponema maltophilum

Klaus Heuner1, Karin Große1, Rüdiger Schade2 and Ulf B. Göbel1

Institut für Mikrobiologie und Hygiene1 and Institut für Pharmakologie und Toxikologie2, Universitätsklinikum Charité, Humboldt-Universität zu Berlin, Dorotheenstr. 96, 10117 Berlin, Germany

Author for correspondence: Ulf B. Göbel. Tel: +49 3020934715. Fax: +49 3020934703. e-mail: ulf.goebel{at}charite.de


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
A flagellar gene cluster from the oral spirochaete Treponema maltophilum ATCC 51939T was cloned. Sequence analysis revealed six putative ORFs, two of which encode the flagellar subunit proteins FlaB2 (286 aa) and FlaB3 (285 aa). Northern blot analysis revealed two flagellin transcripts with the expected size of monocistronic mRNAs. Sequence analysis and primer extension experiments indicated that the transcription of the flaB2 gene is directed by a {sigma}28-like FliA factor. Using fliA and fliA+ Escherichia coli K-12 strains, it was shown that flaB2 expression in E. coli required the {sigma}28 factor using an initiation site identical to that in Treponema maltophilum. Primer extension analysis revealed two transcriptional start sites 5' of the flaB3 gene, a strong promoter with a {sigma}28-like -10 promoter element and a weak promoter with a putative {sigma}54 promoter consensus sequence. Downstream of flaB3, a putative fliD homologue was found, probably encoding the flagellar cap protein of Treponema maltophilum. Flagellin-gene-specific DNA probes hybridized to all 13 Treponema strains investigated, whereas a fliD-specific DNA probe only hybridized to Treponema maltophilum, other treponemal group IV isolates and Treponema brennaborense.

Keywords: treponemes, Treponema maltophilum, flagellar filament, fliD

Abbreviations: CBP, calmodulin-binding protein; IP, isoelectric point; UAS, upstream activator sequence

The GenBank accession number for the sequence reported in this paper is Y18889.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Periodontitis is a mixed bacterial infection leading to progressive destruction of connective gingival tissue (Listgarten, 1987 ; Saglie et al., 1982 ). The possible aetiologic role of oral treponemes in this process is based on the presence of elevated numbers of these organisms in periodontal lesions (Fiehn, 1989 ; Listgarten & Levin, 1981 ; Penn, 1991 ). The interaction of periodontal bacteria with the host tissue is complex, involving motility, adherence, invasion of deeper tissues and modulation of the immune response (Listgarten, 1987 ; Loesche, 1993 ; Saglie et al., 1982 ). Treponemes possess a variety of putative virulence factors (for a review see Fenno & McBride, 1998 ), including proteases, haemolysins and adhesins (Dawson & Ellen, 1990 ; Fenno et al., 1996 ; Grenier, 1991 ; Haapasalo et al., 1992 ; Que & Kuramitsu, 1990 ; Reijntjens et al., 1986 ). Motility may also contribute significantly to spirochaete pathogenicity as it has been shown that some treponemes are able to invade tissues (Moter et al., 1998 ). Motility of treponemes is due to the rotation of the periplasmic flagella between the sheath and the cell cylinder (Berg, 1976 ; Berg & Turner, 1979 ; Charon et al., 1992 ), leading to directional movements within viscous environments (Berg & Turner, 1979 ; Klitorinos et al., 1993 ). The flagellum of Treponema pallidum is composed of three core proteins (FlaB1–3) and one sheath protein (FlaA) (Champion et al., 1990 ; Isaacs & Radolf, 1990 ; Pallesen & Hindersson, 1989 ). In most flagellated bacteria, the cap protein (FliD) is involved in flagella assembly and is often located near the flagellin-encoding genes (Arora et al., 1998 ; Chen & Helmann, 1994 ; McCarter, 1995 ). However, although fliD homologous sequences have been found in the published sequences of the T. pallidum and Borrelia burgdorferi genomes (TIGR database), no treponeme fliD gene has been cloned from this or other species.

Little is known about the regulation of flagellin gene expression in treponemes. In ‘Treponema phagedenis’ a {sigma}28-like promoter consensus sequence was identified upstream of the flaB2 gene (Limberger et al., 1992 ). Similar putative {sigma}28 consensus sequences were found next to the flaB1 and flaB2 genes of T. pallidum (Champion et al., 1990 ; Pallesen & Hindersson, 1989 ) as well as in the flgB and fliK operons of Treponema denticola, ‘T. phagedenis’ and T. pallidum (Heinzerling et al., 1997 ; Limberger et al., 1996 ). Although the effect of temperature and viscosity on motility of T. denticola was studied recently, little is known about the influence of environmental factors on flagellin gene expression in treponemes (Klitorinos et al., 1993 ; Ruby & Charon, 1998 ).

As shown by epidemiologic analysis of patients suffering from rapidly progressive periodontitis, group IV treponemes have the highest prevalence compared to all other cultivable and uncultivable treponemes investigated (Moter et al., 1998 ). Motility may be a virulence factor of treponemes, enabling active tissue invasion. Recently, we described a novel treponeme, Treponema maltophilum, a representative of phylogenetic group IV treponemes (for phylogenetic group determination see Choi et al., 1994 ). T. maltophilum is a small, oral, serum-sensitive spirochaete with two flagella subterminally inserted at each cell pole and was isolated from a patient with rapidly progressive periodontitis (Wyss et al., 1996 ). Here, we report the cloning of a T. maltophilum flagellar gene cluster containing two flagellin subunit genes and the fliD gene. In addition, we report the first characterization of T. maltophilum flagellar gene regulation at the molecular level.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains, media and plasmids.
Bacterial strains are listed in the legend to Fig. 4. Treponema strains were maintained as described previously (Wyss et al., 1996 ). Cultures were examined by dark field microscopy for motility and typical strain morphology.



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Fig. 4. Western blot analysis using the anti-FlaB3 antibody. Equal amounts of whole-cell extracts of different treponemal strains were loaded onto the polyacrylamide gel. Lanes: 1, molecular mass standard (Gibco-BRL); 2, ‘T. vincentii’ ATCC 35580; 3, ‘T. vincentii’ RitzA; 4, ‘T. phagedenis’ biotype Reiter (provided by B. Wilske, Ludwigs-Maximilians-Universität München, Germany); 5, T. denticola CD-1; 6, T. denticola ATCC 35405; 7, T. maltophilum ATCC 51939T; 8, T. lecithinolyticum ATCC 70032T; 9, T. brennaborense DSM 12168T (Schrank et al., 1999 ); 10, T. socranskii subsp. socranskii ATCC 35536T; 11, T. socranskii subsp. buccale ATCC 35534T; 12, T. pectinovorum ATCC 33768T; 13, T. maltophilum ATCC 51940; 14, T. maltophilum ATCC 51941. All strains investigated were oral isolates except ‘T. phagedenis’ and T. brennaborense.

 
Escherichia coli XL-1 Blue (Stratagene) and E. coli DH5 were used for propagation of recombinant plasmid DNA. Plasmid vector pUC19 (Stratagene) was used for subcloning and DNA sequencing. E. coli strains were grown in LB medium. Antibiotics used for selection in E. coli were ampicillin (100 µg ml-1), chloramphenicol (34–50 µg ml-1), tetracycline (12·5 µg ml-1) and kanamycin (50 µg ml-1).

Recombinant FlaB3 protein was overexpressed in E. coli strain BL21 (DE3)/pLys (Stratagene). E. coli strain BL21 (DE3)/pLys harbouring plasmid pKH113 was inoculated into LB broth containing chloramphenicol (25 µg ml-1) and tetracycline (30 µg ml-1). The induction of target protein expression was done according to the manufacturer’s instructions (Affinity Protein Expression and Purification System; Stratagene). Reporter gene expression studies were done with E. coli K-12 strains YK410 and YK4104 (Chen & Helmann, 1992 ). Plasmids used in this study are listed in Fig. 1.



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Fig. 1. Restriction map of the T. maltophilum flaBfliD gene cluster. Coding regions corresponding to ORF1, flaB2, flaB3, ORF111, fliD and ORF2 are indicated by large arrows. Small arrows represent the primers used for PCR. P1, P2 and P3; PCR-generated DNA amplicons used as flaB2-, flaB3- and fliD-specific probes, respectively. Restriction endonuclease sites: Bm, BamHI; Ec, EcoRI; Hd, HindIII; Kp, KpnI; Pt, PstI; Sl, SalI; Sc, SacII; Sm, SmaI.

 
Chemicals.
Enzymes were purchased from MBI Fermentas, Gibco-BRL, Boehringer Mannheim, Pharmacia LKB and Amersham. Chemicals and oligonucleotides were purchased from Sigma, Roth and MWG-Biotech.

Preparation of recombinant flagellin proteins of T. maltophilum.
For isolation of recombinant FlaB3, E. coli strain BL21 (DE3)/pLys harbouring plasmid pKH113 (containing T. maltophilum flaB3 gene) was induced and purified according to the manufacturer’s instructions (Affinity Protein Expression and Purification System; Stratagene). Cells were lysed by sonication and the recombinant protein was purified by a calmodulin affinity column. Samples of eluted protein fractions were checked for purity by SDS-PAGE and recombinant proteins were detected by the Affinity CBP (calmodulin-binding protein) Fusion Protein Detection Kit (Stratagene) (data not shown). Fractions 2 and 3 were denatured with 15% TCA and pelleted by centrifugation (14000 r.p.m. for 10 min in a Labofuge 400R; Hereus). The pellet was resuspended in 200 µl PBS, pH 7·2, and stored at -20 °C.

Preparation of polyclonal monospecific antibodies against T. maltophilum flagellin proteins.
Rabbits, obtained from a commercial breeder (Hardan Winkelmann, Borchen, Germany), were immunized with 0·2 ml antigenic solution (50 µg recombinant T. maltophilum FlaB3 protein; the CBP tag was not removed) mixed with 0·3 ml PBS and 0·5 ml adjuvant (Specol, Institute for Animal Science and Health, Lelystadt, The Netherlands) in a total volume of 1 ml. Rabbits were immunized by injection of the antigen solution into or near the popliteal lymph nodes. After 4 weeks booster injections (1 ml) were given into the skin (i.d.) at multiple sites. To reduce cross-reactivity of the resulting antiserum, the polyclonal monospecific antibody was absorbed with sonicated whole-cell extracts of E. coli XL-1 Blue MRF' harbouring plasmid pCAL-n. The non-binding fraction containing the purified anti-FlaB3 antibody was used for Western blot analysis. Western blot analysis showed that this antiserum detected the recombinant and wild-type FlaB2 and FlaB3 proteins (data not shown).

Cloning of a flagellar gene cluster exhibiting two flagellum subunit genes and the fliD operon of T. maltophilum.
Based upon published gene sequences encoding the conserved N- and C-terminal regions of flagellin proteins we designed a primer pair, flaBU/flaBR, to amplify T. maltophilum flagellin genes directly from the chromosome by PCR. The sequences and positions of all primers used are shown in Table 1 and Fig. 1, respectively. We obtained two PCR products of 0·7 and 1·9 kb. As shown by sequence analysis the 1·9 kb fragment contained the major coding regions of the flaB2 and flaB3 genes (Fig. 1, pKH107). After cloning and sequencing of the PCR products we designed two primer pairs, flaBU/flaBR5 and flaBU1/flaBR1, to amplify the 5' and 3' adjacent regions of the cloned DNA fragment directly from the chromosome by PCR-mediated chromosome walking (rPCR). T. maltophilum chromosomal DNA was cut with SacII and religated prior to PCR. Resulting amplicons were cloned into vector pUC19 giving plasmids pKH108 and pKH135 (Fig. 1). The complete flaB2 and flaB3 genes were amplified using primer pairs flaBU4/flaB4N or flaBU4/flaBR3 and cloned in pUC19, resulting in plasmids pKH123 and pKH112, respectively. This procedure was performed three times in independent experiments. To avoid PCR-generated errors three clones of each PCR product were sequenced. The flaB3 and flaB2 genes were then subcloned in the expression vector pCAL-n (Stratagene), resulting in plasmids pKH124 and pKH113 (Fig. 1), respectively. The same cloning strategy was used to clone the fliD gene of T. maltophilum using primer pairs flaBU6/flaBR6 and flaBU10/flaBR10, resulting in plasmids pKH121 (not shown) and pKH129 (Fig. 1), respectively. The fliD gene was then amplified from chromosomal DNA using primers fliDU1/fliDR1, cloned, sequenced and subcloned into vector pCAL-n, resulting in plasmid pKH137 (Fig. 1).


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Table 1. Oligonucleotide primers

 
SDS-PAGE and Western blotting.
Whole-cell extracts of Treponema spp. and recombinant E. coli strains were analysed by SDS-PAGE and Western blotting. SDS-PAGE and Western blotting were carried out as described elsewhere (Laemmli, 1970 ; Towbin et al., 1979 ; van Die et al., 1984 ). Briefly, treponemal strains were grown anaerobically in OMIZ Pat medium (10 ml) at 37 °C until late exponential phase and harvested by centrifugation. The pellet was then resuspended in 200 µl ddH2O. Equal amounts of cells were mixed with 25 µl loading buffer, incubated at 95 °C for 6 min and loaded onto an SDS (9%)-polyacrylamide gel. Recombinant E. coli K-12 strains were grown either on agar plates or in Luria-Broth medium, each containing 100 µg ampicillin ml-1, at 37 °C overnight. E. coli cells were resuspended in 25 µl loading buffer and loaded onto an SDS (9%)-polyacrylamide gel.

Southern hybridization.
SacII-digested chromosomal DNA from various Treponema strains was electrophoresed in a 1% (w/v) agarose gel. The DNA fragments were transferred to nylon membranes (Boehringer Mannheim) as described by Southern (1975) . The 395 (P1), 822 (P2) and 795 bp (P3) PCR fragments (P1–3; see Fig. 1) were used as T. maltophilum flaB2-, flaB3- and fliD-specific probes, respectively. The probes were labelled and detected by using the non-radioactive enhanced chemiluminescence detection kit (ECL; Amersham). Hybridization was done as described by Heuner et al. (1995) .

DNA techniques, PCR and nucleotide sequencing analysis.
Purification of plasmid DNA, DNA cloning and hybridization procedures were performed according to standard protocols (Sambrook et al., 1989 ; Thuring et al., 1975 ). PCR was carried out by the method of Saiki et al. (1988) , using a Thermocycler TRIO-Thermoblock (Biometra) and AmpliTaq polymerase (Perkin Elmer). A standard amplification protocol was used: initial denaturation step of 3 min at 95 °C followed by 30 cycles of 95 °C for 60 s, 55 °C for 75 s and 72 °C for 60 s and a final step of 5 min at 72 °C. The annealing temperature was modified for each primer pair. PCR products were cloned in pUC19, using the Sure Clone Ligation Kit (Pharmacia). Transformation was done by electroporation using a Bio-Rad gene pulser. Electroporation of E. coli strains was carried out at 2·0 kV, 200 and 25 µF.

Sequencing of plasmid DNA was done on both strands with IR-dye-labelled primers as described by Choi et al. (1994) and Sanger et al. (1977) . Sequences were analysed using the program Husar 4.0 (Deutsches Krebsforschungszentrum, Heidelberg, Germany). Homology searches were conducted against the GenBank, EMBL, EMPRO and SWISS-PROT databases, using the FASTA program. Sequence similarities and homologies were calculated by the GAP program [Genetics Computer Group (GCG) package, Husar 4.0]. Multiple alignments were accomplished by using the CLUSTAL program (GCG package, Husar 4.0).

RNA isolation and Northern (RNA) blot analysis.
T. maltophilum total RNA was purified using an RNA isolation kit (Boehringer Mannheim). Bacteria were grown anaerobically in OMIZ-PAT at 37 °C for 22, 30, 52 or 76 h (OD600 0·036, 0·045, 0·2 and 0·22, respectively), representing the exponential, late exponential, stationary and late stationary growth phases, respectively. Bacteria were harvested by centrifugation and RNA was isolated as described by the manufacturer. RNA was stored at -80 °C. Total RNA (10 µg) was mixed with formaldehyde loading buffer and electrophoresed in 1% (w/v) agarose/formaldehyde gels. The RNA was then transferred to nylon membranes by capillary blotting. DNA probes were labelled by using the non-radioactive enhanced chemiluminescence detection kit (ECL; Amersham). Hybridization with specific DNA probes was done as described by the manufacturer. The hybridization temperature was 42 °C.

Primer extension.
Primer extension analysis was carried out with IR-dye-labelled primers flaBU7 and flaB3PE on an automated DNA sequencer (LI-COR-DNA 4000; MWG-Biotech). Each primer (4 pmol) was annealed in a thermocycler to 10 µg RNA in a volume of 10 µl H2O (RNase-free) by heating at 90 °C for 2 min and subsequent cooling to 30 °C within a period of 30 min. Extension was done in a total volume of 37 µl at 42 °C for 90 min as described by Ausubel et al. (1987) . Nucleic acids were precipitated by ethanol. The pellet was dissolved in equal volumes (5 µl each) of H2O and formamide loading buffer. Samples were boiled for 2 min and aliquots (3 or 6 µl) were applied to sequencing gels. Sequencing reactions were done using the same primers used for the primer extension analysis.

Construction of pflaBlacZ fusions and ß-galactosidase activity assay.
Plasmid pKH14 was constructed by cloning the 3·6 kb BamHI–SalI DNA fragment containing the promoterless lacZ gene of pDN19lac (Totten & Lory, 1990 ) into the low-copy-number vector pMMB207 (Morales et al., 1991 ). This plasmid was used to generate T. maltophilum flagellin promoter (pflaB)–lacZ gene fusions in a similar way to that described by Heuner et al. (1997 , 1999 ). Therefore, the flaB2 and flaB3 promoter-containing regions were amplified by PCR using primer pairs flaBU7/flaBR7 (pflaB2, 335 bp) and flaB3PU/flaB3PR (pflaB3, 354 bp). Resulting amplicons were first cloned into plasmid pUC19 to confirm the promoter sequence by DNA sequencing. PCR-generated SphI and BamHI restriction sites were then used to clone the flagellin promoters upstream of the promoterless lacZ gene of pKH14, resulting in plasmids pKH126 (pflaB2lacZ fusion) and pKH128 (pflaB3lacZ fusion). The {sigma}28 consensus sequence of the flaA gene of Legionella pneumophila is recognized by the {sigma}28 factor of E. coli strain YK410 (Heuner et al., 1997 ). Therefore, plasmid pKH12 containing the flagellin promoter of L. pneumophila fused to the lacZ gene of pKH14 (Heuner et al., 1999 ) was used as a positive control in ß-galactosidase activity assays.

E. coli strains grown to late exponential phase were adjusted to an OD600 of 1·5. ß-Galactosidase measurements were done as described by Miller (1972) .


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cloning of the T. maltophilum flagellar filament genes and the fliD gene cluster
T. maltophilum flaB2 and flaB3 genes were cloned directly from the chromosome of T. maltophilum by PCR as described above. The complete chromosomal region of T. maltophilum encoding the flaB2, flaB3 and fliD region was obtained by rPCR. The flaB2-, flaB3- and fliD-encoding DNA sequences were subcloned in vector pCAL-n, resulting in plasmids pKH113, pKH124 and pKH137, respectively (Fig. 1). Purified recombinant FlaB3 protein was used to generate a monospecific polyclonal rabbit anti-FlaB3 antiserum. Western blot analysis showed that this antiserum detected the recombinant and wild-type FlaB2 and FlaB3 proteins (data not shown).

Southern blot analysis of T. maltophilum chromosomal DNA digested with various restriction enzymes using flaB2-, flaB3- and fliD-specific DNA probes (see Fig. 1, P1–3) indicated that flaB2 cross-hybridizes with the flaB3 gene and that T. maltophilum may contain a third flaB gene located approximately 3–5 kb upstream of flaB2. These experiments suggest that T. maltophilum contains only one copy of the fliD gene (data not shown).

Nucleotide and amino acid sequence analysis of the chromosomal flagellar gene cluster of T. maltophilum
The complete inserts of plasmids pKH108, pKH129, pKH135, pKH124, pKH113 and pKH137 were sequenced. Six putative ORFs were identified (Fig. 1). The 5' region of the DNA cloned suggested it encoded the N-terminal region of a putative ORF (named ORF1). The deduced amino acid sequence (231 aa) showed no significant similarity to any protein present in current databases. The second and third ORFs, of 861 and 858 bp, encode the T. maltophilum FlaB2 (286 aa) and FlaB3 (285 aa) proteins, respectively. Both ORFs are separated by 390 nt. A putative Shine–Dalgarno (SD) consensus sequence (GGAGGA) is located 5 bp upstream of the ATG start codon of flaB2. Downstream of the TAG stop codon the sequence shows a potential {rho}-independent transcriptional termination signal (nt 1363–1397). A potential promoter sequence (-35) TAAA-N16-GCCGATAT (-10), which is almost identical to the consensus sequence of a {sigma}28 promoter, was identified 164 nt upstream the initiation codon of flaB2. No potential promoter consensus sequence was obvious upstream of the start codon of flaB3. A putative SD sequence (GGAGGG) of flaB3 is located 6 nt upstream of the ATG start codon. Downstream of the TGA stop codon the sequence shows a putative strong stem–loop with a T tail, representing a {rho}-independent transcriptional termination signal (data not shown).

The flaB2 and flaB3 genes encode proteins with a theoretical molecular mass of 31·3 and 30·7 kDa, respectively. This is in reasonable agreement with the size (about 32–35 kDa) of the T. maltophilum wild-type proteins determined by SDS-PAGE and Western blot analysis (see Fig. 4, lane 7). FlaB2 has a theoretical isoelectric point (IP) of 6·98, whereas the theoretical IP of FlaB3 is 4·75. This corresponds to the IP values of T. pallidum flagellins exhibiting IP values of 7·9 and 4·9. T. maltophilum FlaB2 exhibited 79·5% similarity to its own FlaB3 protein, but 82% similarity to T. pallidum FlaB3, whereas FlaB2 exhibited 86 and 84% similarity to T. pallidum FlaB2 and FlaB1, respectively, as summarized in Table 2. Based upon these similarities, the T. maltophilum flagellar filament genes were named flaB2 and flaB3.


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Table 2. Flagellin gene sequence identity

 
Downstream of flaB3, additional ORFs are present, one of which encodes a protein of 111 aa (ORF111) exhibiting 32 and 30% similarity to the ORF99 and FlaG proteins of Bacillus subtilis and Pseudomonas aeruginosa, respectively. The putative protein has a theoretical mass of 12 kDa and an IP of 9·78, containing no cysteine, histidine or tryptophan residues.

Downstream of ORF111 another ORF encodes a T. maltophilum FliD (putative flagellar cap protein) homologue, possessing 673 aa with a theoretical mass of 73·8 kDa. It showed the highest similarity (54%) throughout the sequence to the FliD protein of T. pallidum (data not shown) and 48, 46 and 44% similarity to the FliD proteins of Borrelia burgdorferi, Bacillus subtilis and E. coli, respectively. Southern hybridization using a fliD-specific probe (P3; see Fig. 1) revealed fliD homologue genes in other group IV treponemes and Treponema brennaborense, but in no other treponeme investigated so far (data not shown).

The 3' end of an additional ORF located downstream of flaB3 (named ORF2), is missing. The truncated gene encodes a hypothetical protein with 43% similarity to the hypothetical protein TP0873 (200 aa) of T. pallidum (sequence from the TIGR database).

Transcriptional analysis
The size of the flaB transcripts were determined by Northern blot analysis with total RNA prepared from T. maltophilum cells grown to exponential, late exponential, stationary and late stationary growth phases. By using probes P1 (385 bp, flaB2) and P2 (822 bp, flaB3) corresponding to the FlaB2 and FlaB3 coding regions, two specific transcripts of about 1·2 and 1·4 kb were detected (Fig. 2a, lane 2). While the flaB3-specific DNA probe predominantly detected the 1·2 kb transcript (Fig. 2a, lane 4), probe P1 recognized both transcripts (Fig. 2a, lane 2). The length of the 1·2 kb transcript corresponds to the coding regions of flaB2 and flaB3, indicating that flaB genes are transcribed as monocistronic units. Northern blot analysis revealed that the flaB2/3 genes are transcribed during exponential growth (Fig. 2b, lanes 2 and 3), but significantly fewer transcripts are detectable in the late stationary growth phase (Fig. 2b, lane 5).



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Fig. 2. Northern blot analysis of flaB gene transcription. (a) Northern blot analysis showing the presence of different flaB transcripts. (b) Northern blot analysis showing growth-phase-dependent flaB transcription. RNA was extracted from T. maltophilum grown anaerobically for 30 (a, lanes 2 and 4; b, lane 3), 22 (b, lane 2), 52 (b, lane 4) or 76 h (b, lane 5) at 37 °C. PCR-generated flaB2, flaB3 or flaB2flaB3 fragments, corresponding to flaB2, flaB3 or flaB2flaB3 coding regions, respectively, were used as probes. PCR-generated DNA probes were used as positive controls in Northern blot analysis (a, lanes 1/3, flaB2-specific probe; b, lane 1, 1·9 kb flaB2flaB3-specific probe).

 
To determine the promoters of T. maltophilum flaB2 and flaB3 genes, we mapped the transcriptional start site of the chromosomal flagellin genes by primer extension (Fig. 3). These experiments revealed an adenine residue as the transcriptional start site of flaB2, located 8 nt downstream of the -10 region of a {sigma}28 promoter consensus sequence (Fig. 3c). This indicates that a {sigma}28-like promoter element of the flaB2 gene acts as a promoter in T. maltophilum. Primer extension products were identified in the exponential and late exponential growth phase (Fig. 3a, lanes 1 and 2). Two transcriptional start sites were found upstream of the flaB3 gene (Fig. 3b). The major start site, a thymidine residue (transcript P1), 118 nt upstream of the start codon, and a minor start site, a guanine residue (transcript P2) 168 nt upstream of the start codon of flaB3 (Fig. 3c), were identified. A putative -10 {sigma}28-like element (GCCGTAAC) is found 4 nt upstream of P1. However no obvious E. coli -35 element could be observed. A putative {sigma}54-promoter-like consensus sequence (GG-N10-GC) is located 7 nt upstream of P2 (Fig. 3c). Up- and downstream of this putative {sigma}54 promoter two more GG-N10-GC invariants and one putative upstream activator sequence (UAS, TGT-N7-ACA) are found near the -100 region of the promoter (data not shown). Primer extension products were identified in the early and late exponential growth phases (Fig. 3b, lanes 1 and 2).



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Fig. 3. Primer extension experiments to map the transcriptional start site of the T. maltophilum flaB2 (a, using primer flaBR7) and flaB3 (b, using primer flaB3) genes. Transcription start sites are indicated by arrows. Total RNA was isolated from T. maltophilum cultures grown to exponential (lane 1), late exponential (2) and stationary phase (3). Total RNA from E. coli strains YK410 (wild-type) and YK4104 (fliA mutant) harbouring plasmid pKH126 (pflaB2lacZ fusion) was isolated (lanes 4 and 5). Lanes designated G, A, T and C represent DNA sequencing ladders. (c) Positions of the transcription start sites deduced from primer extension analysis. Putative promoter elements are underlined and transcriptional start sites are indicated by arrows.

 
Promoter expression studies of T. maltophilum flagellin genes were performed in E. coli strain YK410 and strain YK4104, the isogenic {sigma}28 factor (fliA-) mutant strain of strain YK410 (Chen & Helmann, 1992 ). Strains YK410 and YK4104 transformed with pKH126, harbouring the T. maltophilum flaB2 promoter–lacZ gene fusion, or with pKH128, containing the flaB3 promoter–lacZ gene fusion, were used for reporter gene expression studies. Reporter gene activity (ß-galactosidase) was measured in the late exponential growth phase. E. coli strains harbouring pKH12, containing the L. pneumophila {sigma}28 flaA promoter–lacZ gene fusion, and pKH14, containing the promoterless lacZ gene were used as positive and negative controls, respectively. As shown in Table 3 the lacZ gene containing the pflaB2 promoter of T. maltophilum is expressed in the E. coli wild-type strain, but not in the fliA mutant. Primer extension analysis with total RNA isolated during these experiments revealed the same transcriptional start site in E. coli wild-type as in T. maltophilum (Fig. 3a, lane 4). No signal was seen in the fliA mutant strain (Fig. 3a, lane 5), indicating that the T. maltophilum {sigma}28-like element is recognized by the {sigma}28 factor of E. coli. However, one should note that expression is approximately sevenfold lower than for the L. pneumophila pflaAlacZ fusion (Table 3). No significant ß-galactosidase activity was seen in E. coli strains harbouring the pflaB3lacZ gene fusion, indicating that this promoter element is not active in E. coli (Table 3). Accordingly, no primer extension products were detected in these strains (data not shown).


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Table 3. T. maltophilum flaB promoter activity in E. coli

 
Distribution and expression of flagellin genes in various treponeme species
The results of Western blot analysis of whole-cell proteins from various treponemal strains with the T. maltophilum FlaB3-specific antiserum is shown in Fig. 4. Immunoblot analysis of all treponeme isolates investigated so far showed two to three protein bands of approximately 31–35 kDa, indicating the conserved structure of treponeme FlaB proteins. For ‘T. phagedenis and in T. denticola CD-1 Western blot analysis showed only one band reacting with the antiserum (Fig. 4, lanes 4 and 5), but Southern blot analysis revealed the presence of more then one flagellar gene in these strains. Southern blot analysis using T. maltophilum flaB2- and flaB3-specific DNA probes exhibited positive bands in all strains tested (data not shown).


   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The flaB gene cluster
In this paper we have reported the sequence and expression analysis of a flagellar gene cluster cloned by PCR-mediated chromosomal walking of the oral spirochaete T. maltophilum. Six putative ORFs were identified. Two ORFs, 861 and 858 bp in length encode the FlaB2 and FlaB3 proteins, respectively. The apparent sizes in SDS-PAGE analysis seemed too high compared to the calculated molecular masses of both proteins: 31·3 and 30·7 kDa, respectively. This may be explained by post-translational modification of the FlaB proteins, e.g. by glycosylation of T. maltophilum FlaB proteins as reported by Wyss (1998) . Furthermore, flagellar proteins are known to migrate irregularly in SDS-PAGE (Simon et al., 1977 ).

While only two flagellins were detected by immunoblotting, the presence of a third flagellar gene was shown by Southern blot analysis (data not shown). This has also been shown for T. pallidum (Champion et al., 1990 ; Pallesen & Hindersson, 1989 ). The cloned genes were named flaB2 and flaB3 according to their similarity to the respective T. pallidum genes. The similarity of T. maltophilum FlaB2 to T. pallidum FlaB2 is higher (86%) than the similarity to its own FlaB3 protein (79·5%). This is also the case for FlaB proteins of other spirochaetes (Charon et al., 1992 ; Ruby et al., 1997 ).

Little is known about the mechanisms of flagellin gene expression in treponemes, but it has been suggested that differential gene regulation is required for optimal motility in varying environments (Alm et al., 1993 ; Belas et al., 1986 ; Wassenaar et al., 1994 ). Northern blot analysis revealed the presence of monocistronic flaB transcripts (1·2 and 1·4 kb) in T. maltophilum. The 1·4 kb transcript seen in Northern blot analysis may represent the mRNA of the flaB1 gene. In T. pallidum a similar flaB1flaB3 gene arrangement was found (Champion et al., 1990 ) and these authors speculated about the existence of a polycistronic mRNA, but no Northern blot or primer extension analyses were performed.

We showed that the flaB2 gene of T. maltophilum contains a {sigma}28-like promoter sequence and primer extension analysis revealed that it acts as a promoter for flaB2 gene expression. Using fliA/fliA+ E. coli strains we showed that an identical initiation site was used in both E. coli and T. maltophilum. This strongly suggests that the T. maltophilum {sigma}28 promoter is recognized by the {sigma}28 factor of E. coli. The significant reduction of promoter activity compared to the L. pneumophila flaA {sigma}28 promoter may be explained by a variation in length of the spacer region (Dombroski et al., 1996 ; Mazouni et al., 1998 ). The T. maltophilum spacer contains 16 instead of 15 nt typically found in the L. pneumophila flaA promoter or other {sigma}28 consensus sequences (Fig. 5a). {sigma}28-factor-dependent expression was also shown for the ‘T. phagedenisflaB2 gene (Limberger et al., 1992 ) and the flgB and fliK operons of T. denticola and ‘T. phagedenis (Heinzerling et al., 1997 ; Limberger et al., 1996 ). In contrast, motility operons in B. burgdorferi are transcribed from {sigma}70 promoters (Ge et al., 1997 ).



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Fig. 5. DNA sequence comparison of putative {sigma}28 (a) and {sigma}54 (b) promoter consensus sequences of T. maltophilum (Tm) flaB2 and flaB3 genes with flagellar promoter elements of T. pallidum (Tp), ‘T. phagedenis’ (Tph), L. pneumophila (Lp), Bacillus thuringiensis (Bth), C. coli (Cc) and H. pylori (Hp). P1 and P2, transcription initiation sites 1 and 2.

 
The T. maltophilum flaB3 gene contains two initiation sites (P1 and P2). Upstream of the major initiation site, P1, a {sigma}28-like promoter element lacking an obvious -35 element was found. A putative {sigma}54-like promoter element was identified upstream of P2 (Fig. 5b). Flagellin genes under the control of different sigma factors were also reported for P. aeruginosa, Helicobacter pylori and Campylobacter coli (Alm et al., 1993 ; Guerry et al., 1991 ; Josenhans et al., 1995 ). The T. maltophilum flaB3 promoter contained an upstream TGT-N7-ACA motif and two GG-N10-CC invariants. Comparable sequence elements were described in C. coli and P. aeruginosa upstream of their respective flagellar {sigma}54 promoters (Alm et al., 1993 ; Totten & Lory, 1990 ). In general {sigma}54 promoters are characterized by the presence of a TGT-N10-ACA UAS (Cannon et al., 1991 ; Wu et al., 1995 ). However, the complete promoter region of the T. maltophilum flaB3 gene failed to be active in E. coli strain YK410. Similar results were obtained with other {sigma}54-like consensus sequences (McCarter, 1995 ). If our interpretation holds true, this would be the first report of a putative {sigma}54-like promoter element involved in flagellar gene regulation in spirochaetes.

The fliD gene cluster
In T. maltophilum we identified an ORF encoding a putative filament cap protein (FliD) homologue. The deduced FliD protein exhibited 54% similarity to FliD of T. pallidum and 44–48% similarity to the FliD proteins of Borrelia burgdorferi, Bacillus subtilis and E. coli. However, the fliD gene seems to be less conserved than the flagellin genes as shown by Southern blot analysis. In general the filament cap protein prevents the free release of flagellin and promotes their polymerization onto growing filament tips (Homma et al., 1986 ; Ikeda et al., 1985 ). The fliD operons of P. aeruginosa, Bacillus subtilis and Vibrio parahaemolyticus consist of a small ORF followed by the fliDST genes (Arora et al., 1998 ; Chen & Helmann, 1994 ; McCarter, 1995 ; McGee et al., 1996 ). The fliDST genes are involved in negative regulation of FlgM (anti-{sigma}28 factor) export (Yokoseki et al., 1996 ). In T. maltophilum a similar gene arrangement may be present downstream of the two flaB genes.

Upstream of fliD we identified an ORF of 111 aa exhibiting low similarities (32 and 30%) at the protein level to the ORF99 and FlaG proteins of Bacillus subtilis and P. aeruginosa, respectively, but the function of these proteins is not known. Comparison of the cloned fla operon of T. maltophilum with sequences retrieved from the genomic database (TIGR) of T. pallidum revealed a similar flaBfliD cluster arrangement, except that no ORF111 homologue is present in the T. pallidum strain sequenced. It has yet to be shown whether the proteins encoded by the genes of the putative T. maltophilum fliD operon possess a similar function to the fliDST genes of other bacteria.

Moter et al. (1998) described the invasion of treponemes into deep tissue of inflamed digital dermatitis lesions. It is therefore conceivable that motility constitutes an important virulence factor in these bacteria. Hence, the molecular characterization of treponemal motility, e.g. by generating flaB and fliD mutants, may be of great help in elucidating their role in the pathogenesis of chronic human periodontitis and related infections in cattle.


   ACKNOWLEDGEMENTS
 
We would like to thank Marco Kachler for excellent technical assistance and Dr Ralf Schumann for careful reading of the manuscript. This study was supported by a grant (01KI9318) from the Bundesministerium für Bildung und Forschung to U.B.G. and the Graduiertenkolleg (GRK 325/1-97) of the Deutsche Forschungsgemeinschaft.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 23 July 1999; revised 12 October 1999; accepted 13 October 1999.