Veterinary Laboratories Agency (Weybridge), New Haw, Addlestone, Surrey KT15 3NB, UK1
Molecular Microbiology and Genomics Consultants, Zotzenheim, Germany2
Author for correspondence: Diane G. Newell. Tel: +44 1932 357547. Fax: +44 1932 357595. e-mail: dnewell.cvl.wood{at}gtnet.gov.uk
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ABSTRACT |
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Keywords: subtractive hybridization
Abbreviations: AP, alkaline phosphatase; RM, restrictionmodification
The GenBank accession numbers for the sequences reported in this paper can be found in Table 2.
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INTRODUCTION |
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To date, only a few potential colonization factors, specific for the avian host, have been identified using defined mutants in experimental oral models of chick colonization. A role for the flagellin genes in chicken colonization has been demonstrated (Wassenaar et al., 1993 ; Nachamkin et al., 1993
); however, non-motile and aflagellate bacteria are still able to colonize (Wassenaar et al., 1993
). Other genes apparently important in avian-gut colonization identified so far include sod (Purdy et al., 1999
), racR (Bras et al., 1999
), pglH (A. Karlyshev, unpublished data) and cadF (Ziprin et al., 1999
).
Epidemiological studies on large numbers of C. jejuni isolates have shown a high degree of phenotypic and genotypic diversity (Owen et al., 1997 ; Lindstedt et al., 2000
; Dingle et al., 2001
; Wassenaar & Blaser, 1999
). It seems likely that such diversity is reflected in phenotypic variation in the colonization and disease-causing potentials of strains. Certainly strain variation in virulence properties has been observed in both in vitro (Gorelov et al., 1990
) and in vivo models, though such models may poorly reflect the spectrum of disease seen in humans (Pang et al., 1987
; Black et al., 1988
; Wassenaar & Blaser, 1999
). Understanding the molecular basis and biological consequences of genetic diversity among strains is important for disease control. To this end the recent genome sequencing of C. jejuni NCTC 11168 (Parkhill et al., 2000
) now enables, by DNA array technology (Lan & Reeves, 2000
), the absence of genes in other C. jejuni strains to be detected. However, the identification of the presence of additional genes in other strains is not feasible using this approach.
C. jejuni NCTC 11168 was derived from a case of human campylobacteriosis in 1977 (M.B. Skirrow, personal communication). Preliminary studies indicated that this strain displayed a relatively poor colonization potential in chickens compared to that previously reported for the well-characterized strain 81116 (Cawthraw et al., 1996 ). The inability of chicken passage of strain 11168 to fully restore its colonization potential indicated that this phenotype had a genetic basis. Therefore, the method of subtractive hybridization was used to identify genetic material, potentially associated with colonization, present in C. jejuni strain 81116 but not present in the genome sequence of strain 11168.
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METHODS |
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Media and growth conditions.
C. jejuni strains were routinely grown for 24 h at 42 °C under microaerobic conditions (85% N2, 7·5% CO2, 7·5% O2) on blood agar plates supplemented with 10% sheep blood, 50 µg Actidione ml-1 and selective antibiotics (Oxoid) (Skirrow, 1977 ). Escherichia coli was maintained on LuriaBertani (LB) agar or in LuriaBertani broth (Difco) at 37 °C in atmospheric conditions. When required the media were supplemented with 100 µg ampicillin ml-1 or 50 µg kanamycin ml-1. C. jejuni and E. coli were stored as frozen cultures at -80 °C in 1% (w/v) proteose peptone water (Difco) containing 10% (v/v) glycerol or in Luria broth (LB) containing 50% glycerol (Ausubel et al., 1994
), respectively.
Chicken-colonization model.
A quantitative oral chick-colonization assay was performed as previously described (Wassenaar et al., 1993 ). Briefly, groups of 10 specific pathogen-free chickens (SPAFAS, Charles River), housed in isolators, were dosed at day 1 of age by oral gavage. Doses ranged from 102 to 109 c.f.u. (100 µl 0·1 M PBS, pH 7·2)-1 of C. jejuni NCTC 11168, administered in 100 µl 0·1 M PBS (0·0234 M disodium hydrogen phosphate; 0·0066 M potassium dihydrogen orthophosphate; 0·07 M NaCl; pH 7·2). Doses were prepared by harvesting bacteria, grown overnight on blood agar plates at 42 °C, into sterile PBS. At 5 days post-challenge, the levels of colonization were determined by plating out dilutions of caecal contents. Chick-colonization levels were given as c.f.u. (g caecal contents)-1 for individual birds. The limit of detection of colonization was 100 c.f.u. (g caecal contents)-1. The dose response for the colonization potential of C. jejuni 81116 has been previously described (Wassenaar et al., 1993
) and has been consistent in over 20 experiments.
Subtractive hybridization.
A schematic diagram of the subtractive-hybridization technique is shown in Fig. 1. The DNA sequences of adapters and primers used in this study are shown in Table 1
. Initially, genomic DNA preparations from C. jejuni 81116 (tester) and strain 11168 (driver) were isolated using the CTAB method for DNA isolation (Ausubel et al., 1994
). Each preparation was digested with AluI and DraI, according to the methods described in the PCR-Select Bacterial Subtraction kit (Clonetech), to give a range of blunt-ended fragments of between 0·1 and 2·0 kbp, as determined by gel electrophoresis. The blunt-ended DNA fragments from C. jejuni 81116 were subdivided into two aliquots and each aliquot was ligated to a different adapter (1 or 2R). This resulted in double-stranded DNA fragments with an extended, single-stranded adapter sequence on the 5' end of each complementary strand. Both aliquots were denatured and combined with an excess of denatured driver DNA and the mixture was incubated at 63 °C for 1·5 h (Fig. 1
, hybridization one). In the second hybridization (Fig. 1
, hybridization two) the two aliquots were combined and incubated at 63 °C overnight with more fresh denatured driver DNA. During this stage tester-specific DNA (ligated to adapter 1) annealed to tester-specific DNA (ligated to adapter 2R). The recessed ends of the adapters were filled and the primary PCR was carried out to exponentially amplify tester-specific DNA (type f molecules). In the secondary PCR, nested primers to adapters 1 and 2R were used to further enrich the tester-specific DNA (type f molecules). The first genomic subtraction was carried out with a hybridization temperature of 63 °C, as recommended by the manufacturer. A second round of subtractive hybridization was performed with 10 µl of the secondary PCR products, but this time a hybridization temperature of 61 °C was used.
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Dot-blot and Southern-blot hybridizations.
For the dot blots, 50 ng of each PCR product was denatured by incubating in a boiling water bath for 5 min and spotted onto a Hybond-N+ membrane (Amersham). DNA was cross-linked to the membrane by UV light. For probe labelling, 200 ng of genomic DNA digested with AluI and DraI was labelled using alkaline phosphatase (AP) according to AlkPhosDirect labelling kit guidelines (Amersham). The blots were prehybridized in AlkPhos Direct Hybe buffer for 1 h at 60 °C and then hybridized overnight at 60 °C with 200 ng of the AP probe. For Southern-blot analysis, 10 µg of each C. jejuni genomic DNA was digested with either HindIII or HinfI, electrophoresed through a 1% (w/v) agarose gel and transferred onto a Hybond-N+ membrane. Each DNA insert (50 ng) of interest was labelled with AP; hybridizations and stringency washes were carried out at 60 °C as before.
Sequencing and analysis.
Strain-81116-specific clones were cultured in 1 ml LB broth with kanamycin selection, purified using a QIAprep spin miniprep kit (Qiagen) and sequenced by using the CEQ DTCS sequencing system (with 3·2 pmol M13 primer µl-1) on a CEQ 2000 DNA sequencer (Beckman Coulter). The DNA sequences were edited using the EDITSEQ program (DNAstar, Lasergene) and homology searches were performed using the BLAST suite of programs (Altschul et al., 1997 ).
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RESULTS |
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DISCUSSION |
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The low percentage of genuine strain-81116-specific sequences, obtained from the initial subtractive-hybridization experiment, may have been due to the inadequate annealing of homologous DNA sequences from strain 11168 to 81116. Decreasing the temperature from 63 °C (optimal for bacteria with a GC ratio of 4051 mol%) to 61 °C improved this efficiency by allowing the preferential amplification of 81116-specific sequences. Improved optimization might be achieved by decreasing the hybridization temperature further. The presence of a number of chimeric clones also confounded the overall efficiency, as these were detected as positive clones. Nonetheless, a total of 23 DNA sequences present in strain 81116 and absent from strain 11168 have been identified, confirming that intra-species variation exists between strains of C. jejuni (Dorrell et al., 2001 ). Of the 23 strain-81116-specific DNA inserts identified six sequences had no significant similarities to any entry in the databases searched. In several cases, two or more of the strain-81116-specific inserts appeared to have been derived from the same genomic location, as indicated by Southern blotting, suggesting that such strain-specific genes may be clustered; for example, the HinfI profiles of inserts 60, 243 and 246 were identical.
The appearance of identical banding profiles on Southern blots for the two C. jejuni strains 99/312 and 99/322, when probed with inserts 50, 68, 135 and 202, suggests that the genetic arrangement of these strains is similar. Indeed, PFGE profiles of these two strains are also identical, indicating that they are clonally related (A.M. Ridley, personal communication).
Although subtractive hybridization was developed to screen for the presence or absence of genes, it can also be used to detect polymorphisms between two strains. This was apparent for inserts 205 and 141. These inserts have similarities to dTDP-glucose 4,6-dehydratase (rmlB) from Legionella pneumophila, an enzyme involved in the glycosylation of the cell wall of pathogenic bacteria (Allard et al., 2000 ), and a putative glycosyltransferase from C. jejuni 11168, respectively. Both inserts had identical HinfI Southern-blot profiles and both were either present or absent in the C. jejuni strains tested. This suggests that the functions of these two genes, involved in polysaccharide biosynthesis, are coordinated. That inserts were identified that do not hybridize to strain 11168, despite significant amino acid similarity as in the case of insert 141, suggests that (at least some) polymorphic genes can be detected by subtractive hybridization. These results confirm DNA microarray observations indicating that strain 81116 differs from strain 11168 within this genetic region (Dorrell et al., 2001
). Thus, subtractive hybridization not only complements DNA microarray technology but provides the additional benefit of detecting novel sequences in test strains.
It has been reported that sequences within the flagellar modification region and the capsule locus vary between strains. In particular, it has been reported that a region between Cj1318 and Cj1336 of the flagella modification locus has been lost from strain 81116 (Dorrell et al., 2001 ). However, subtractive hybridization identified insert 209 to have similarity to Cj1333. One explanation of this anomaly would be a genetic rearrangement in strain 81116, so that Cj1333 was no longer located where expected relative to strain 11168. However, the identification of a second insert (149) with similarity to Cj1337, and the identical Southern-blot profiles using inserts 149 and 209 as probes suggests that these two DNA fragments are in close proximity on the 81116 genome.
A number of DNA inserts were found to have similarities to restrictionmodification (RM) genes. Similarities to both type I and II RM systems were identified in inserts 60, 68, 135, 136 (all type I from Haemophilus influenzae Rd, Salmonella enterica, S. enterica and Streptococcus thermophilus, respectively) and 202 (type II from Helicobacter pylori J99) (Table 2). Southern-blot profiles of inserts 68 and 135 were identical with both HindIII and HinfI, suggesting that they are closely located in all strains tested. Analysis of these two inserts revealed a substantial amount of overlapping DNA sequence, suggesting that they might be part of the same RM gene. There is increasing evidence for the existence of multiple RM systems in C. jejuni (Miller, 2001
). The functions of those RM genes found in strain 81116 are currently under investigation. Interestingly, the chromosomal DNA of this strain does not cut with SmaI (G. Manning, personal communication), even though SmaI sites have been shown to be present (I.H. Ahmed, unpublished data). It is therefore interesting to speculate that the RM genes detected have a role in this enzyme restriction.
Two more inserts (31 and 186), which were absent from the strain 11168 genome, have similarities with genes involved in arsenic resistance in several bacteria. Insert 31 has 64% amino acid identity to ArsB from Sinorhizobium sp. As4, an integral membrane protein needed for the extrusion of arsenite. Insert 186 has 35% amino acid identity to ArsC from Aquifex aeolicus. This gene encodes arsenate reductase, which reduces intracellular arsenate to arsenite. The Southern-blot profiles of inserts 31 and 186 were different (data not shown), suggesting that these two genes do not form part of an operon. However, insert 186 had identical HindIII and HinfI profiles (data not shown) to those of insert 212, which has similarity to Cj1560 ( a putative membrane protein). This suggests that ArsB and Cj1560 are co-located in the genome of all strains tested. Generally, in Gram-negative bacteria the arsenic-efflux pump comprises ArsA, the ATPase subunit, ArsB, which anchors the former to the membrane, and ArsC in the cytosol (Cervantes et al., 1994 ). The observation of arsenite-metabolizing genes in strain 81116 and not in strain 11168 is interesting especially since phenylarsonic compounds have been used, in the past, in poultry feed for controlling caecal coccidiosis and to promote growth (Pergantis et al., 1997
). The use of such compounds may have led to the contamination of some agricultural lands on which poultry litter has been used as manure (Gupta & Charles, 1999
). The ars operon is widespread among the Enterobacteriaceae and arsenic has also been shown to induce the synthesis of heat-shock proteins, indicative of the stress response triggered due to exposure to this toxic chemical (Chang et al., 1989
). Thus, the biological role of these genes in the colonization of chickens by campylobacters requires further investigation.
Insert 236 has 72% amino acid identity to the H. pylori -glutamyl transpeptidase (ggt) gene. Southern-blot analysis of insert 236 revealed an identical HinfI, but a different HindIII pattern, to that of insert 65, for which no significant protein similarities could be identified. This suggests that these inserts are located in close proximity in the genome of strain 81116 and that they may be linked. As well as functions in the antioxidant glutathione pathway (Carmel-Harel & Storz, 2000
), GGT appears to have a role in the colonization of the gastric mucosa by H. pylori (Chevalier et al., 1999
; McGovern et al., 2001
). Whether this gene product has a similar role in colonization of the chicken intestinal tract by C. jejuni will require further investigation.
The successful application of subtractive hybridization, to genetically compare two strains of C. jejuni with differing colonization potentials, has revealed a number of strain-specific differences, some of which may be potential colonization factors. It should, however, be noted that the sequences identified in this study are only fragments of much larger genes and that expression of the corresponding genes has not yet been proven. Moreover, future work involving the mutagenesis of the 81116-specific sequences in tandem with further chick-colonization experiments will be necessary to determine the importance of such genes in colonization. Insert 50 is particularly interesting since it was universally present in the other C. jejuni strains tested (apart from strain 11168) and for one strain (94/194) was the only insert hybridizing. As this strain colonizes the chick model to the same extent as freshly isolated strains (K.J. Clow, personal communication), insert 50 may be especially important in chicken colonization.
In conclusion, the 23 novel DNA segments identified in this study represent a selection of genes present in strain 81116 and absent in strain 11168, and they indicate extensive genetic variation even among a small number of C. jejuni strains. These results imply that we have, to date, found only a portion of the total gene pool existing within the C. jejuni species. While C. jejuni genome sequence data are limited techniques like subtractive hybridization can be used to identify novel genes, which can then be used to construct more comprehensive DNA arrays to accurately determine genomic diversity.
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ACKNOWLEDGEMENTS |
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Received 31 August 2001;
revised 5 November 2001;
accepted 22 November 2001.