Mutational and transcriptional analysis of the Campylobacter jejuni flagellar biosynthesis gene flhB

Claudia Matza,1, Arnoud H. M. van Vliet2,3, Julian M. Ketley3 and Charles W. Penn1

School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK1
Department of Gastroenterology and Hepatology, Academic Hospital Dijkzigt, Dr Molewaterplein 40, 3015 GD Rotterdam, The Netherlands2
Department of Genetics, University of Leicester, University Road, Leicester LE1 7RH, UK3

Author for correspondence: Charles W. Penn. Tel: +44 21 146562. Fax: +44 121 145925. e-mail: c.w.penn{at}bham.ac.uk


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
A Campylobacter jejuni gene encoding a homologue of the flagellar biosynthesis gene flhB was identified downstream of the peroxide stress defence gene ahpC. Insertional mutagenesis of the flhB gene rendered C. jejuni non-motile, with most cells aflagellate, although a small number expressed truncated flagella. The absence of FlhB also appeared to affect cell shape, as the majority of cells were straight rather than curved rods. Transcription of the flagellin gene flaA was significantly reduced in the C. jejuni flhB mutants, which also did not express significant amounts of flagellin proteins, indicating that FlhB is an essential protein for subsequent expression of flagellar genes. The transcription start site of the flhB gene, as determined by primer extension, was located 91 bp upstream of the flhB start codon, but no recognizable promoter sequence could be identified immediately upstream of this transcription start site. Transcriptional flhB::lacZ reporter gene fusions confirmed that the flhB gene has its own promoter region, is expressed at very low levels and is transcribed independently of ahpC, and that its transcription is not regulated by iron or growth phase.

Keywords: Campylobacter, motility, transcription

a Present address: School of Pharmaceutical Sciences, University of Nottingham, Nottingham NG7 2RD, UK.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The human pathogen Campylobacter jejuni is recognized as one of the major causes of bacterial gastroenteritis, and is considered to be an important public health and economic burden (van Vliet & Ketley, 2001 ). The high level of motility conferred by flagella, together with the spiral, curved shape, allow C. jejuni to penetrate the mucus layer overlying the intestinal epithelium (Szymanski et al., 1995 ). Flagella and motility have also been implicated in adhesion and invasion of C. jejuni (Wassenaar et al., 1991 ), and are required for intestinal colonization in chicken colonization models (Nachamkin et al., 1993 ; Wassenaar et al., 1993 ).

The determination of the complete genome sequence of C. jejuni has allowed the identification of putative flagellar genes in C. jejuni (Parkhill et al., 2000 ). Comparison with flagellar genes of the Enterobacteriaceae indicated that regulation and biosynthesis of flagella in C. jejuni is likely to be different from that of the Enterobacteriaceae (Chilcott & Hughes, 2000 ; Macnab, 1996 ; Parkhill et al., 2000 ), more closely resembling the organization predicted for the related pathogen Helicobacter pylori (O’Toole et al., 2000 ; Tomb et al., 1997 ). C. jejuni contains the flagellar sigma factors {sigma}28 and {sigma}54 (Jagannathan et al., 2001 ; Wösten et al., 1998a ) and a homologue of the H. pylori flgR gene, which encodes a {sigma}54-associated activator of transcription of the flagellar hook, basal body and FlaB flagellar filament genes (Jagannathan et al., 2001 ; Spohn & Scarlato, 1999 ).

So far, relatively few C. jejuni genes involved in flagellar biosynthesis have been investigated. In recent studies using random transposon mutagenesis or site-directed mutagenesis, it was demonstrated that mutations in at least 19 genes led to altered or absent motility of C. jejuni (Colegio et al., 2001 ; Hendrixson et al., 2001 ; Jagannathan et al., 2001 ). One of these C. jejuni genes is flhB (Hendrixson et al., 2001 ), which encodes a protein that in Salmonella enterica serovar Typhimurium is involved in the flagellar protein export pathway, and in the hook-length-dependent switch in export specificity from basal-body-hook proteins to filament and associated proteins (Kutsukake et al., 1994 ; Kutsukake, 1997 ; Minamino et al., 1994 ; Minamino & Macnab, 1999 , 2000 ; Williams et al., 1996 ). As in C. jejuni, mutation of flhB in H. pylori resulted in a non-motile phenotype (Foynes et al., 1999 ). Synthesis of the flagellar filament proteins FlaA and FlaB, and the hook protein FlgE, was almost completely abolished in the H. pylori flhB mutant, and transcription of the flaA, flaB and flgE genes was significantly reduced (Allan et al., 2000 ).

Examination of the nucleotide sequence downstream of the peroxide stress defence gene ahpC (Baillon et al., 1999 ) revealed the presence of the motility-associated genes flhB and motB (Fig. 1). In this study, we have characterized the transcriptional organization and regulation of flhB, and show that mutation of flhB affects C. jejuni motility and flagellin expression.



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Fig. 1. Schematic diagram representing the genomic region containing the flhB gene. The position and orientation of the cat cassette inserted into the BclI site of flhB and the resulting mutants are shown. Promoters of flhB and ahpC are indicated by arrows, and a putative terminator structure is indicated by a stem–loop symbol.

 

   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains and growth conditions.
The C. jejuni strains used in this study are 81116 (NCTC 11828), 480 (NCTC 12744) (National Collection of Type Cultures) and the C. jejuni 81116 flagellar mutants R2 (FlaA- FlaB-), R3 (FlaA+ FlaB-) and R4 (FlaA- FlaB+) (Wassenaar et al., 1991 ). C. jejuni strains were cultured in Mueller–Hinton (MH) media (Oxoid) at 37 °C under microaerobic conditions (5% O2, 5% CO2, 90% N2). Antibiotics were used at the following final concentrations: 10 µg vancomycin ml-1, 50 µg kanamycin ml-1, and 20 µg chloramphenicol ml-1, when appropriate. Iron-restricted growth conditions were achieved by supplementing MH medium with Desferal (Sigma) to a final concentration of 20 µM (van Vliet et al., 1998b ). For motility tests, C. jejuni grown overnight in MH broth at 37 °C under microaerobic conditions was diluted in MH broth to approximately 100 cells ml-1, and, subsequently, 100 µl of this cell suspension was added to 5 ml soft agar [MH broth containing 0·3% (w/v) agar, cooled to 45 °C], poured onto soft agar plates [MH broth containing 0·4% (w/v) agar], and incubated for 2 days microaerobically at 37 °C.

Recombinant DNA techniques.
Plasmid preparations, restriction endonuclease digestions, ligations and transformations of Escherichia coli were performed according to standard procedures (Sambrook et al., 1989 ). All restriction and modifying enzymes were purchased from Gibco-BRL and used according to the manufacturer’s instructions. Genomic DNA was isolated by the IsoQuick nucleic acid extraction kit (ORCA Research). A PCR was performed using Vent polymerase (New England Biolabs) or Taq polymerase (Life Technology) according to the manufacturer’s instructions. Allelic exchange mutagenesis of flhB was performed by insertion of the chloramphenicol-resistance (cat) cassette of plasmid pAV35 (van Vliet et al., 1998b ) in both transcriptional orientations into the unique BclI restriction site of the flhB gene (Fig. 1). The resulting plasmids, pAV107 and pAV108, were introduced into C. jejuni 81116 using electroporation (van Vliet et al., 1998a ), and yielded C. jejuni strains AV33 and AV34 (flhB::CmR) (Fig. 1). Correct replacement of the wild-type flhB gene by the flhB::cat gene was verified by Southern blotting (not shown).

RNA isolation and analysis.
Total RNA was isolated from C. jejuni overnight cultures as described previously (Oelmuller et al., 1990 ). Primer extension was performed using primers PE1 and PE2 (5'-CTATCTTTTTGGACGTGGGTTC-3' and 5'-CTGCCGCATCTTGAGACTTTGG-3') essentially as described previously (Baillon et al., 1999 ). For Northern hybridization, RNA was transferred, using standard protocols (Sambrook et al., 1989 ), to Amersham Hybond-N+ nylon membranes and hybridized with PCR-amplified flaA- and flhB-specific probes. The flaA and flhB probes consisted of parts of the flaA gene (positions 1269232–1270532 on the C. jejuni genome; Parkhill et al., 2000 ) and the flhB gene (positions 303149–304169 on the C. jejuni genome; Parkhill et al., 2000 ), respectively. All probes were labelled with [{alpha}-32P]dATP by using the Radprime DNA labelling kit and random hexanucleotide primers (Life Technology).

Reporter gene assays.
The C. jejuni promoter probe vector pMW10 contains a promoterless lacZ gene, and has previously been used to quantify promoter activity in C. jejuni (Baillon et al., 1999 ; van Vliet et al., 2000 ; Wösten et al., 1998b ). Overlapping fragments containing the flhB start codon and various parts of the upstream region were amplified by PCR, checked for the absence of PCR incorporation errors by DNA sequencing, and subsequently cloned into the BamHI site of pMW10 (Table 1). As positive controls, the C. jejuni 81116 ahpC (Baillon et al., 1999 ) and flaA promoters were used (Table 1). The constructs were transformed into C. jejuni strain 480, and ß-galactosidase activity was measured as described previously (Baillon et al., 1999 ; van Vliet et al., 2000 ; Wösten et al., 1998b ), after approximately 7·5 h growth, at an OD600 of approximately 0·4. The ß-galactosidase activities were expressed in Miller units (Sambrook et al., 1989 ) and were derived from three independent experiments.


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Table 1. Promoter activity of flhB promoter–lacZ fusions

 
Protein analysis.
C. jejuni wild-type and flhB mutants were grown on MH agar for 24 h, fractionated into periplasmic, cytoplasmic and membrane-bound proteins (van Vliet et al., 1998b ) and subjected to SDS-PAGE (Sambrook et al., 1989 ). Gels were either stained with PAGE blue 83 (Fluka) or immunoblotted and subsequently incubated with mouse monoclonal antibodies P3B2 and O2D4, which recognize both Campylobacter flagellin A and flagellin B (Constantinidou et al., 1996 ; Jagannathan et al., 2001 ).

Electron microscopy.
C. jejuni wild-type and flhB mutants were grown on MH agar for 24 h, negatively stained in 1·25% (w/v) sodium phosphotungstate (pH 7·0), and examined with a Jeol JEM1200EX transmission electron microscope.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Identification and mutation of a C. jejuni flhB homologue
Examination of the sequence downstream of the C. jejuni 81116 ahpC oxidative stress defence gene had previously revealed the presence of a gene encoding an FlhB homologue (Baillon et al., 1999 ). The predicted C. jejuni FlhB protein is 37% identical, and 56% similar, to the FlhB protein of H. pylori and shows strong conservation of hydrophobic and hydrophilic domains when compared with the FlhB protein of S. enterica serovar Typhimurium (not shown).

The flhB gene of C. jejuni strain 81116 was mutated by insertion of a cat cassette in both transcriptional orientations into the unique BclI restriction site of flhB (Fig. 1), followed by allelic replacement of the wild-type flhB gene with the interrupted gene. This resulted in the isolation of C. jejuni strains AV33 and AV34 (81116 flhB::CmR) (Fig. 1). Correct replacement of the wild-type flhB gene with its interrupted counterpart was confirmed by Southern blot analysis (data not shown). Mutants AV33 and AV34, which had the cat cassette in opposite orientations, gave identical flagellar phenotypes in all tests (not shown). This indicates there was no polar effect on flagellar gene expression from the insertion of the cat cassette.

Mutation of flhB affects C. jejuni motility and cell shape
The relative motility of flhB mutants was compared with that of C. jejuni 81116 wild-type and the aflagellate, non-motile mutant R2 (FlaA- FlaB-) (Wassenaar et al., 1991 ). C. jejuni 81116 had spread to the edge of soft-agar plates after 3 days incubation, whilst the R2 mutant had produced small, distinct colonies. Like the R2 mutant, the flhB mutants were non-motile, although the colonies were slightly larger than those of R2 (data not shown). Electron microscopy of the C. jejuni flhB mutants (Fig. 2) showed that the flhB mutants lacked flagella and often lost the spirally curved morphology typical of wild-type C. jejuni 81116. Occasionally, cells with truncated flagella and a spirally curved morphology were observed among flhB mutants (Fig. 2b), but we were unable to identify these as colonies showing motility on motility plates and hence could not characterize them further (data not shown).



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Fig. 2. Electron microscopy of C. jejuni 81116 and flhB mutant AV34. Cells were grown for 24 h and then negatively stained with potassium phosphotungstate (pH 7·0). (a) C. jejuni wild-type 81116. Most cells have a spirally curved morphology and full-length flagella. (b) C. jejuni flhB mutant AV34. Most cells are elongated, straight, and lack flagella. One cell exhibits a spirally curved morphology and truncated flagella (indicated by arrowheads). Bars, 0·5 µm.

 
Mutation of flhB affects flagellin expression and transcription
Expression of the flagellar proteins FlaA and FlaB was absent in the flhB mutants, as assessed using monoclonal antibodies (Fig. 3a). This indicates that the non-motile phenotype of the flhB mutants is caused, at least in part, by blocked expression of flagellin protein. Repression of flagellin expression appeared to be mediated at the transcriptional level, since flagellin mRNA levels were significantly reduced in the flhB mutants (Fig. 3b), though not absent as in the flagellar mutants R2 and R4 (Fig. 3b). Since the flaA probe used shows a certain degree of cross-reactivity with the highly similar flaB gene (Nuijten et al., 1990 ), it is likely that the mRNA hybridizing to the flaA probe represents low levels of flaB expression. In contrast, transcription of the flagellar motor gene motB was not significantly affected by any of the mutations (Fig. 3b).



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Fig. 3. Analysis of (a) Fla protein levels and (b) flaA and motB transcript levels in C. jejuni wild-type, flhB mutants AV33 and AV34, and flagellar mutants R2, R3 and R4. (a) Western immunoblot of C. jejuni whole-cell protein probed with the flagellum-specific monoclonal antibody P2D3. Marker sizes are given in kDa on the right, while on the left the FlaA protein is indicated. The monoclonal antibody or conjugate used displays a low level of cross-reactivity with the major outer-membrane protein (MOMP), as indicated on the left. (b) Northern hybridization of RNA isolated from C. jejuni cells in exponential growth phase, probed with flaA-specific (top panel) and motB-specific (lower panel) probes. Lanes: 1, C. jejuni 81116; 2, C. jejuni AV33; 3, C. jejuni AV34; 4, C. jejuni R2; 5, C. jejuni R3; 6, C. jejuni R4.

 
Identification of the flhB promoter
The transcription start point for flhB was determined by primer extension. This gave a weak but reproducible primer extension signal located 91 bp upstream of the flhB ATG codon (Fig. 4). Typical {sigma}70 or {sigma}28 promoter sequences were not obvious in the sequence directly upstream of the flhB transcription start point, but 39 bp upstream of the transcription start point a typical {sigma}54 promoter sequence, GG-N10-GC, was identified, as reported for the Campylobacter coli flaB and flgE genes (Guerry et al., 1992 ; Kinsella et al., 1997 ). This promoter-like sequence, however, is too far upstream from the transcription start for it to be involved in this activity (Buck et al., 2000 ). The weak primer extension signal indicated that flhB is transcribed at a very low level, or that the transcript is unstable, and this was confirmed by the failure to detect an flhB transcript on Northern hybridization (not shown).



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Fig. 4. Characterization of the flhB promoter region. (a) Primer extension analysis of C. jejuni mRNA with the flhB-specific oligonucleotide PE1. The lane marked PE contains the primer extension product, whereas the lanes marked A, C, G and T contain a DNA sequencing reaction initiated with primer PE1 used for the primer extension reaction. (b) Sequence and annotation of the ahpCflhB intergenic region. The transcription start site is indicated as +1 in bold typeface and is underlined. The stop codon of ahpC and the start codon of flhB are shown in bold typeface; DNA sequences resembling the C. jejuni {sigma}54 consensus sequence are underlined, highlighted, and labelled as -12 and -24. (c) Strategy for determining the promoter of flhB. Overlapping fragments of the C. jejuni ahpCflhB region were transcriptionally fused to the promoterless lacZ gene of pMW10 (Table 1).

 
The pMW10-based lacZ reporter gene system (Wösten et al., 1998b ) was used to identify the flhB promoter and to quantify transcription of flhB, including any co-transcription with the upstream, iron-repressed ahpC gene. Overlapping fragments were generated, containing either the direct upstream region of flhB, or, in addition to this, the upstream ahpC gene with and without its iron-regulated promoter (Fig. 4c, Table 1). The flaA promoter and the ahpC promoter were used as positive controls (Table 1). Expression of the flhB::lacZ fusions was only slightly, though reproducibly, higher than background levels, and was up to 100-fold lower than activity from the ahpC::lacZ fusion and 25-fold lower than the flaA::lacZ fusion (Table 1). The inclusion of the ahpC gene alone (pCM2) or the ahpC gene and promoter (pCM1) upstream of the putative flhB promoter region did not significantly affect expression of the flhB::lacZ fusion when compared with pCM3, indicating that transcriptional readthrough from the ahpC promoter does not seem to play a role in transcription of flhB. Conversely, removal of the sequences upstream of the transcription start point (pCM4) lowered ß-galactosidase activity to background levels. Growth in iron-restricted media only affected ß-galactosidase activity of the ahpC::lacZ fusion (Baillon et al., 1999 ), but not that of any of the flhB::lacZ or the flaA::lacZ fusions (Table 1), again confirming that there was no influence of the iron-regulated ahpC promoter on flhB transcription.


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Flagellar biogenesis has been extensively studied in S. enterica serovar Typhimurium and E. coli (Chilcott & Hughes, 2000 ; Macnab, 1996 ), and it has been shown that the FlhB protein plays a central part therein. FlhB is involved in hook-length control and switch of export substrate as part of the flagellar export apparatus (Kutsukake et al., 1994 ; Kutsukake, 1997 ; Minamino et al., 1994 ; Minamino & Macnab, 1999 , 2000 ; Williams et al., 1996 ). However, experimental evidence as well as the availability of complete genome sequences from several bacteria have indicated that models developed for S. enterica serovar Typhimurium do not always correspond to those of other bacteria (O’Toole et al., 2000 ; Parkhill et al., 2000 ; Tomb et al., 1997 ). Here, we have initiated characterization of flagellar biogenesis in C. jejuni by studying the role and transcription of its flhB gene.

The FlhB protein seems to play a central role in flagellar biogenesis of C. jejuni, since the flhB mutants used in this study did not assemble flagella. Similar observations were made with H. pylori flhB mutants, which were also non-motile and lacked flagella (Foynes et al., 1999 ). The flhB mutants of C. jejuni strain 81116 also generally exhibited a change in cell shape, from spiral cells with a relatively short aspect ratio to a straighter and apparently longer form. No explanation is apparent for this, but in polar flagellate bacteria where a new pole requires a new flagellum, there clearly must be co-ordination between flagellar insertion and cell division. Furthermore, it has been shown in the Enterobacteriaceae that cell-division events are affected by mutations in the flhD flagellar master-regulator, and it is possible that a cell-division gene(s), with implications for determination of aspects of cell shape, might be affected in aflagellate mutants (Chilcott & Hughes, 2000 ; Prüß & Matsumura, 1997 ).

Flagellin expression was significantly decreased in the C. jejuni flhB mutant, and this was mediated at the transcriptional level. This repression of flagellin transcription and expression has also been described in mutants lacking other predicted components of the flagellar export apparatus in either H. pylori, C. coli or C. jejuni (Allan et al., 2000 ; Miller et al., 1993 ; O’Toole et al., 2000 ; Park et al., 2000 ). In contrast, mutants in the flagellar hook-encoding gene flgE in Helicobacter mustelae, H. pylori and C. coli are also non-motile and aflagellate, but express flagellin at normal levels so that it accumulates intracellularly (Kinsella et al., 1997 ; O’Toole et al., 1994 , 2000 ). This suggests that full activation of flagellin genes in Campylobacter requires the proper assembly of the flagellar-specific export apparatus.

The levels of flhB in C. jejuni were too low to allow detection in Northern hybridization experiments, and therefore we utilized reporter gene analysis to identify promoter sequences driving flhB transcription. The reporter gene experiments confirmed that levels of flhB transcripts are very low, since the ß-galactosidase activities of flhB::lacZ fusions were only slightly higher than background values. Transcription of flhB is not affected by the presence or absence of the upstream ahpC gene and promoter, thus demonstrating that the flhB gene is transcribed at a low level from its own promoter, and indicating there is an (as yet unidentified) transcriptional stop between the flhB and ahpC genes. Taken together, these results demonstrate that flhB is transcribed at a low level, that the flhB promoter is located in the ahpCflhB intergenic region, that transcription starts at the experimentally determined transcription start point, and that it is independent of the iron-repressed promoter of the upstream-located aphC gene. In conclusion, the flhB gene is essential for flagellar biogenesis in C. jejuni, and is likely to function as a component of the flagellar export apparatus.


   ACKNOWLEDGEMENTS
 
We thank Ben van der Zeijst for C. jejuni mutants R2, R3 and R4, and Marc Wösten for plasmid pMW10. This work was supported by grants from the School of Biosciences, University of Birmingham, to C.M., from the Wellcome Trust, to J.M.K., and from the Nederlandse Organizatie voor Wetenschappelijk Onderzoek (NWO 901-14-206), to A.H.M.v.V.


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ABSTRACT
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RESULTS
DISCUSSION
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Received 7 January 2002; accepted 15 February 2002.