Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, University of London, Keppel Street, London WC1E 7HT, UK1
Department of Neuroimmunology, Division of Clinical Neurosciences, KCL, Guys Campus, London SE1 1UL, UK2
Author for correspondence: Brendan W. Wren. Tel: +44 207 927 2288. Fax: +44 207 637 4314. e-mail: brendan.wren{at}lshtm.ac.uk
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ABSTRACT |
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Keywords: C. jejuni, flagellar biosynthesis, phase variation, slipped-strand mispairing, genetic instability
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INTRODUCTION |
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The flagellar biogenesis apparatus in bacteria is complex and, despite extensive studies, functions of some flagella-related genes remain unknown (Macnab, 1996 ). Analysis of the C. jejuni genome sequence (Parkhill et al., 2000
) revealed a number of putative flagella-related genes similar to genes of Escherichia coli and other bacteria. In addition, genes involved in Campylobacter flagellin glycosylation were found (Guerry et al., 1996
;Linton et al., 2000b
). The genes involved in flagellin glycosylation are part of a large cluster also containing genes thought to be involved in sugar biosynthesis and transport, as well as seven closely related genes of unknown function (Parkhill et al., 2000
). We have termed these genes (Fig. 1
) the motility accessory factor (maf) family of flagellin-associated proteins. Two maf genes (maf1 and maf4) appear to be identical, both containing homopolymeric G tracts. In this study we demonstrate that the genes of the maf family are involved in the variation of motility via the slipped-strand mispairing mechanism.
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METHODS |
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Construction of mutants.
In order to mutate the maf5gene, a 2 kb fragment of this gene was amplified using primers AK100 (GCT TTA AGC GGT TTT GAG TAT AAC AAC TTA CGC) and AK101 (CGT TCG TGT GCA TAA ACC CAA GC) (Fig. 2) and cloned into the pGEM-T vector. A unique BsaBI site in the cloned fragment was used for insertion of blunt-ended BamHI kanr cassette (Trieu-Cuot et al., 1985
). The size and origin of the inserts were confirmed using PCR and restriction mapping techniques (data not shown). The construct was used for transformation of C. jejuni via electroporation (Wassenaar et al., 1993
). Mutants with direct orientation of the cassette were selected using PCR with primers DL3 (kanr cassette specific, ACC CAG CGA ACC ATT TGA GG) and DL38 (flaB specific, GTC AGG CTA ATG CAG TGC AG) (Fig. 2
).
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Sequencing of the homopolymeric tracts.
The poly(G)-tract-containing regions of each gene were amplified from chromosomal DNA using primer DL8 (CAT AAT AAT GAC TCA TCA GTT CTA) in combination with a primer derived from an adjacent gene (neuB3 primer DL2, GCA AGA AGC TTA TAT GCA AGT AAG G, for the maf1 gene and maf3 primer AK93, GCT ATT TAT TTT CAT AAC GAA TGC G, for the maf4 gene) and the PCR products were sequenced directly in both directions (Fig. 2).
PCR conditions.
Crude cell lysates were amplified using Gibco-BRL Taq polymerase in 20 µl volumes containing 0·1 µg primers and DNA: 94 °C for 1 min, 25 cycles of 94 °C 45 s, 50 °C 45 s, 72 °C 2 min, followed by 7 min extension at 72 °C. The PCR products were purified using S300 microspin columns (Bio-Rad) and/or analysed on agarose gels.
Western blotting.
Polyacrylamide gels were Western blotted onto PVDF membrane (Millipore) using a semi-dry electroblotting apparatus (Hoefer Scientific Instruments). Blots were blocked overnight at 4 °C in Tris-buffered saline containing 0·01% Tween 20 (TBST) and 3% skimmed milk. After blocking, blots were incubated for 1 h with anti-flagellin serum at a dilution of 1:100 in TBST containing 1% bovine serum albumin (TBST-BSA) and washed three times in TBST, followed by incubation with peroxidase-labelled anti-rabbit IgG (Sigma) diluted 1:1000 in TBST-BSA for a further 1 h. Following washing as above, blots were developed using the DAB staining kit with nickel enhancement according to the manufacturers instructions (Vector Laboratories).
Electron microscopy.
C. jejuni colonies from a 2 d culture grown on Columbia agar containing 7% horse blood were gently resuspended in phosphate-buffered saline. Copper grids were placed Formvar-coated side down onto a drop of bacterial suspension for 1 min and then onto a drop of 3% ammonium molybdate for a further 1 min. Grids were left to dry and viewed by transmission electron microscopy.
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RESULTS |
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Closer inspection of the maf genes revealed orthologues in three other species: Helicobacter pylori, Clostridium acetobutylicum and Bacillus stearothermophilus (Fig. 1BD
). In all cases the maf-like genes are linked to either flagellar biosynthesis genes or/and genes involved in sugar biosynthesis and transport. For example, in H. pylori an orthologue HP0114 is present in the same operon and immediately downstream from the flaB gene encoding flagellin (Fig. 1D
). Although the genes present in the operon containing paralogous gene HP0465 are hypothetical, the product of one of them (HP0466) is predicted to interact with FlgB and may be a part of the flagellar biosynthesis apparatus (Fig. 1D
). Four paralogous genes similar to the maf family (CAC2168, CAC2196, CAC2200 and CAC2202) are present in Cl. acetobutylicum and are associated with the genes related to both sugar and flagellar biosynthesis. Similarly, both flagellar and sugar/biosynthesis-related genes are associated with a single maf orthologue present in B. stearothermophilus (Fig. 1C
). All Maf-like proteins share extensive local regions of high similarity, particularly in the N-terminal and central regions. One such region is presented in Fig. 1(E)
. Despite the different organization of the regions containing maf-like genes, the striking similarity is that they are always associated with genes involved in biosynthesis/transport of sugars and flagella.
Subclonal variation in motility of C. jejuni NCTC 11168
We found that motility of the original strain NCTC 11168 was significantly lower than that of fresh clinical isolates (data not shown). However, a small fraction of the clonal population of this strain revealed variable motility. Variants with motility ranging from almost non-motile to hypermotile, occurring with a frequency of approximately 10-3, can be isolated from the wild-type strain (Fig. 3). A hypermotile variant (11168H) was selected for mutagenesis along with the wild-type strain NCTC 11168 (see below).
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Gene maf5 is involved in motility
During analysis of naturally occurring non-motile mutants we also investigated the integrity of other maf genes and found that some of these mutants contained deletions, resulting from recombination between homologous regions of the adjacent paralogous genes. In one such case the region was PCR amplified and sequenced, confirming recombination between adjoining maf4 and maf5 genes (Fig. 4). These preliminary studies suggested that some genes of the maf family might be involved in motility. However, as these changes may be coincidental with independent mutations in other genes, defined mutants were required. We selected one of the genes, maf5, for further studies.
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The maf5::kanr mutation affects flagellar biosynthesis
Electron microscopy revealed the complete lack of the flagella in the maf5::kanrmutant, whereas the hook organelle could be seen (Fig. 6B). Analysis of cell lysates demonstrated reduced intensity of a band corresponding to flagellin in the maf5::kanr mutant both on the polyacrylamide gel (Fig. 7A
) and on a Western blot with anti-flagellin antibodies (Fig. 7B
).
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DISCUSSION |
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Over 40 genes are involved in flagellar biogenesis of E. coli and Salmonella typhimurium (Macnab, 1996 ). Since Maf proteins do not have similarity in the non-redundant amino acid sequence database, their exact role in flagellar biogenesis remains an enigma. However, based on their position in the C. jejuni genome, and, more importantly, partial overlap with sugar biosynthesis (neuB for maf1) or flagellar modification (ptmA for maf2) genes, one can hypothesize that the maf genes may also be involved in flagellin modification. In C. coli VC167, motility is modulated by flagellin modification (Guerry et al., 1992
). We found that, similarly to NCTC 11168, in a number of other C. jejuni strains the representatives of the maf family are linked to either the neuB3 gene or the ptmA gene (data not shown). Both neuB3 and ptmA are known to be involved in flagellin modification (Doig et al., 1996
;Linton et al., 2000b
). Flagellin modification is thought to occur in the cytoplasm (Doig et al., 1996
). ProSite analysis of the putative maf gene products suggests that they are likely to be cytoplasmic or inner membrane bound. Our Western blot results demonstrate decreased intensity of the flagellin band in the maf5::kanr mutant. It seems likely that the maf genes are involved in post-translational processing of the flagellin and/or assembly of the flagella. The lack of modification or transport may result in proteolytic degradation of the flagellin.
Reversible expression of flagella in C. coli resulting from a slipped-strand mispairing mechanism of phase variation in flhA gene has recently been demonstrated (Park et al., 2000 ). In contrast to C. coli, C. jejuni does not have a homopolymeric tract in this gene. The results presented here indicate the presence of alternative mechanisms of variable flagellar expression in C. jejuni. One mechanism involves slipped-strand mispairing involving the contingency genes maf1 and maf4, whereas the other involves recombination between homologous genes. As demonstrated in this report, homologous recombination between adjacent genes can also be involved in variation. Further evidence of homologous recombination between maf genes is suggested by the absence of genes between maf1 and maf5 in C. jejuni strains 81116 and 81-176 (unpublished observation). The presence in strain NCTC 11168 of four maf genes, organized in a tandem fashion, indicates a potential for gene deletion/duplication.
The formation of non-motile deletion derivatives in the natural C. jejuni population may confer flexibility upon bacteria in adapting to changing environmental conditions when flagellar expression and motility are undesirable. The presence of the remaining homologous gene copies might potentially allow restoration of the missing copy through gene duplication. The need for flexibility in the expression of the flagellum could be explained by its possible dual function. As an adhesin, the glycosylated flagellum might be required for initial attachment to the host cells (Nuijten et al., 1992 ). Once colonization is established, flagella may not be required and their formation may be switched off. When the nutrients at the infection site become limiting, a fraction of bacteria expressing flagella may acquire an advantage, as they can move towards a new colonization site. In addition, reversible expression of flagella may be beneficial in evading the host immune response (Nuijten et al., 1995
). For an organism with a relatively small genome size (1·64 Mb for C. jejuni NCTC 11168), the availability of such a large number of genes dedicated to flagellar biosynthesis, modification and phase variability seems extraordinary. The presence of a complex mechanism for flagellar expression may provide C. jejuni with a selective advantage in the ecological niches it occupies.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 18 June 2001;
revised 1 October 2001;
accepted 8 October 2001.