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
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ABSTRACT |
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Keywords: Campylobacter, motility, transcription
a Present address: School of Pharmaceutical Sciences, University of Nottingham, Nottingham NG7 2RD, UK.
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INTRODUCTION |
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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 (OToole et al., 2000
; Tomb et al., 1997
). C. jejuni contains the flagellar sigma factors
28 and
54 (Jagannathan et al., 2001
; Wösten et al., 1998a
) and a homologue of the H. pylori flgR gene, which encodes a
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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METHODS |
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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 manufacturers 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 manufacturers 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 12692321270532 on the C. jejuni genome; Parkhill et al., 2000
) and the flhB gene (positions 303149304169 on the C. jejuni genome; Parkhill et al., 2000
), respectively. All probes were labelled with [
-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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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.
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RESULTS |
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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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DISCUSSION |
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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
; OToole 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
; OToole 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.
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ACKNOWLEDGEMENTS |
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Received 7 January 2002;
accepted 15 February 2002.