Genome Walking Method Research Articles

Isolation of a Novel Lipase Gene from Serratia liquefaciens S33 DB-1, Functional Expression in Pichia pastoris and its Properties

A new lipase gene designated as SlLipA was isolated from Serratia liquefaciens S33 DB-1 by the genomic-walking method. The cloned gene contained an open reading frame (ORF) of 1,845 bp encoding 615 amino acids with a conserved GXSXG motif. Genome sequence analysis showed that an aldo/keto reductase gene closed to the SlLipA gene. The lipase gene was cloned into the expression vector pPICZalphaA and successfully integrated into the heterologous host, methylotrophic yeast Pichia pastoris GS115. Five transformants could be expressed as secreted recombinant proteins with the high activity on Triglyceride-Agarose plate and as candidates to produce the recombinant enzyme. A C-terminal His tag was used for its purification. The lipase activity of different transformants against substrate para-nitrophenyl laurate (p-NPL) varied from 14 to 16 U ml(-1). For the substrates para-nitrophenyl caprate (p-NPC), p-NPL, para-nitrophenyl myristate (p-NPM), para-nitrophenyl palmitate (p-NPP), and para-nitrophenyl stearate (p-NPS), the specific activity was shown to be preferred to long acyl chain length of p-NPS.

Acquisition of uncharacterized sequences from Candidatus Liberibacter, an unculturable bacterium, using an improved genomic walking method

An effective PCR-based genomic walking approach is described to discover previously unknown flanking genomic DNA sequences from Candidatus Liberibacter asiaticus, an unculturable, phloem-limited bacterium. Using this technique, 8564 bp of new DNA sequences were obtained from three genomic loci; tufB-secE-nusG-rplKAJL-rpoBC gene cluster, omp gene (outer membrane protein, Omp) and 16/23S rRNA gene in Ca. L. asiaticus. These, together with publicly available Ca. Liberibacter sequences, are clustered into five contigs and two singlets representing 24,477 non-redundant base pairs. BLAST annotation predicts 12 full-length genes, two partial genes and one pseudogene among these sequences. The sequences obtained in this study provide new genome information about Ca. Liberibacter that will facilitate development of new genome-based detection tools. The technique described here can also be employed to acquire new genomic information for other unculturable or fastidious organisms for which available sequences are limited or for filling sequence gaps between known flanking genomic DNA sequences.

A modified universal fast walking method for single-tube transposon mapping

An enhanced universal fast walking (UFW) method adapted for the mapping of transposons is described. This protocol combines the original UFW method with the use of agarase to unravel composite nucleotide sequence, thereby forgoing molecular cloning steps and the use of restriction enzymes and ligases necessary in other available genome walking methods such as the prominent inverse PCR. The minuscule automatable chemistry of UFW is completed within one reaction vessel using a constant enzyme buffer, and the intrinsic DNA fingerprints, from which amplicons may be quantitatively recovered, offer quality assurance. The core steps of the protocol, spanning half a day or less, comprise first-strand synthesis, primer destruction, random-ended-primer annealing, distal branched-end repair, second-primer destruction, lariat formation and final amplification. Distinctively, no starting or intermediate templates are wasted during the reaction series, thus achieving yields comparable to direct PCR. Ultimate per-reaction walk-lengths are schematically illimitable and sequence-ready amplicons can be produced immediately from prevalent single-copy genomic walk origins. The core UFW protocol may be applied, as described here, to expedited transposon boundary retrieval, but is also applicable to general genome walking and cDNA walking, as well as viral and other insertional element mapping.

Promoter analysis of the Catharanthus roseus geraniol 10-hydroxylase gene involved in terpenoid indole alkaloid biosynthesis

Geraniol 10-hydroxylase (G10H) is an important enzyme in the biosynthetic pathway of monoterpenoid alkaloids found in diverse plant species. The Catharanthus roseus G10H controls the first committed step in biosynthesis of terpenoid indole alkaloids (TIA). The C. roseus G10H promoter sequence was isolated by a PCR-based genome walking method. Sequence analysis revealed that the G10H promoter contains several potential eukaryotic regulatory elements involved in regulation of gene expression. The major transcription start site of the promoter was mapped to an adenine 31 bp downstream of the TATA-box. For functional characterization, transcriptional fusions between the G10H promoter fragments with 5′ or 3′ deletions and the GUS reporter gene were generated and their expressions were analyzed in a tobacco protoplast transient expression assay. Deletion of the promoter down to − 318 bp had little effect on GUS activity. However, further deletion of the promoter to position − 103 resulted in approximately 5-fold reduction of GUS activity. Gain-of-function experiments revealed the presence of three potential transcriptional enhancers located in regions between − 191 and − 147, − 266 and − 188, and − 318 and − 266, respectively. The G10H promoter was capable of conferring stable GUS expression in transgenic tobacco plants and C. roseus hairy roots. In transgenic tobacco seedlings GUS expression was tissue-specific, restricted to leaf and actively growing cells around the root tip, and not detected in the hypocotyls, root cap and older developing areas of the root. The GUS expression in both transgenic C. roseus hairy roots and tobacco seedlings were responsive to fungal elicitor and methyljasmonate. Compared to other known promoters of TIA pathway genes, the G10H promoter contains unique binding sites for several transcription factors, suggesting that the G10H promoter may be regulated by a different transcriptional cascade.

Structural and functional characterization of microcystin detoxification-related liver genes in a phytoplanktivorous fish, Nile tilapia ( Oreochromis niloticus)

Liver genes related to phase I and phase II detoxification, as well as inhibition of reactive oxygen species (ROS) production, were cloned, and their response to microcystin-LR (MC-LR) and lipopolysaccharide (LPS) exposure via intraperitoneal injection, was determined in a phytoplanktivorous fish, Nile tilapia ( Oreochromis niloticus). The cloned full-length cDNA of tilapia soluble glutathione S-transferase (sGST) was classified as alpha-class GST based on their amino acid sequence identity with other species. The tilapia sGST clone was 861 bp in length, and contained a 25 bp 5′-UTR, a 167 bp 3′-UTR and an open reading frame of 669 bp, encoding a polypeptide of 222 amino acids. Using genome walker method, a 366 bp 5′-flanking sequence of tilapia sGST gene was further obtained, and the possible regulatory elements were identified. Partial cDNA sequences of glutathione peroxidase (GPX) and uncoupling protein 2 (UCP2) were also obtained by PCR using degenerate primers from tilapia liver. To study the transcriptional response of liver genes to microcystin treatment, tilapia were respectively exposed to a single 50 μg kg − 1 body weight (bwt) dose of pure MC-LR, a single 2 mg kg − 1 bwt dose of LPS and a co-exposure MC-LR and LPS (50 μg kg − 1 bwt + 2 mg kg − 1 bwt), and were then sacrificed at 24 h post-exposure. Using beta-actin as external control, a significant increase (about 80%) in sGST mRNA expression was found in response to the MC-LR exposure after 24 h ( P < 0.05), indicating the importance of sGST in microcystin detoxification. A slight decrease of sGST mRNA expression was observed in the liver of tilapia, exposed to LPS and MC-LR + LPS. It seems that the LPS response element (LPSRE), identified in the promoter region of tilapia sGST gene, may be functional at a rather low level. In contrast, the levels of cytochrome P450 1A (CYP1A) mRNA expression were found to keep unchanged to either MC-LR, or LPS, or MC-LR + LPS treatment, indicating that unlike the phase II enzyme (sGST), the phase I enzyme (CYP1A) might not play an important role in the detoxification process of microcystins. Although not significant, the mRNA expression level of GPX tended to increase in the liver of tilapia exposed to both MC-LR and LPS ( P > 0.05). In addition, a significant increase in UCP2 mRNA expression was observed in the liver of tilapia exposed to LPS ( P < 0.05), as well as an obvious but not significant increase in MC-LR exposure group. We suggest that phase II detoxification enzyme, instead of phase I detoxification enzyme, might be responsible for the strong tolerance of the phytoplanktivorous fish to microcystins, and hepatocyte proteins coping with oxidative stress (GPX and UCP2), might also have some auxiliary effect. In addition, the rather low and insignificant response of tilapia sGST gene to the inhibitory effect of LPS exposure, might possibly be critical to the phytoplanktivorous fish to utilize toxic blue-green algae.

Molecular cloning and characterization of alpha‐class glutathione S‐transferase gene from the liver of silver carp, bighead carp, and other major chinese freshwater fishes

Two full-length cDNAs encoding glutathione S-transferase (GST) were cloned and sequenced from the hepatopancreas of planktivorous silver carp (Hypophthalmichthys molitrix) and bighead carp (Aristichthys nobilis). The silver carp and bighead carp GST cDNA were 920 and 978 bp in length, respectively, and both contained an open reading frame that encoding 223 amino acids. Partial GST cDNA sequences were also obtained from the liver of grass carp (Ctenopharyngodon idellus), crucian carp (Carassius auratu), mud carp (Cirrhinus molitorella), and tilapia (Oreochromis nilotica). All these GSTs could be classified as alpha-class GSTs on the basis of their amino acid sequence identity with other species. The three-dimensional structure of the silver carp GST was predicted using a computer program, and was found to fit the classical two-domain GST structure. Using the genome walker method, a 875-bp 5'-flanking region of the silver carp GST gene was obtained, and several lipopolysaccharide (LPS) response elements were identified in the promoter region of the phytoplanktivorous fish GST gene, indicating that the GST gene expression of this fish might be regulated by LPS, released from the toxic blue-green algae producing microcystins. To compare the constitutive expression level of the liver GST gene among the six freshwater fishes with completely different tolerance to microcystins, beta-actin was used as control and the ratio GST/beta-actin mRNA (%) was determined as 130.7 +/- 6.6 (grass carp), 103.1 +/- 8.9 (bighead carp), 92.6 +/- 15.0 (crucian carp), 72.3 +/- 7.8 (mud carp), 58.8 +/- 11.5 (silver carp), and 33.6 +/- 13.7 (tilapia). The constitutive expression level of the liver GST gene clearly shows that all the six freshwater fishes had a negative relationship with their tolerance to microcystins: high-resistant fishes (phytoplanktivorous silver carp and tilapia) had the lowest tolerance to microcystins and the high-sensitive fish (herbivorous grass carp) had the highest tolerance to microcystins. Taken together with the reciprocal relationship of constitutive and inducible liver GST expression level in some of the tested fish species to microcystin exposure, a molecular mechanism for different microcystin detoxification abilities of the warm freshwater fishes was discussed.

A genomic walking method for screening sequence length polymorphism

AbstractWe adapted a recently developed nonrestrictional, nonligational genome walking method, Universal Fast Walking (UFW), for detection of length polymorphism in the proximal promoter region of genes. We demonstrate its efficacy at discovering naturally occurring transposition into heat‐shock genes of wild Drosophila and show that it surmounts limitations of simple polymerase chain reaction (PCR) approaches. We further present modifications to the standard UFW protocol and provide some guidelines to improve specificity. Although the resultant banding pattern of a standard UFW can be regarded as a DNA fingerprint, many amplicons result from false priming and not real polymorphisms. We describe ways to distinguish between UFW amplicons and false priming products in a high‐throughput assay.

Characterization of two novel lipase genes isolated directly from environmental sample

Two novel lipase genes (lipJ02, lipJ03) were isolated directly from environmental DNA via genome-walking method. Lipase gene lipJ02 contained an open reading frame (ORF) of 1,425 bp and encoded a 474-amino acids lipase protein, while lipase gene lipJ03 contained an ORF of 1,413 bp and encoded a 470-amino acids lipase protein. The lipase genes were cloned into expression vector pPIC9K and successfully integrated into a heterologous fungal host, Pichia pastoris KM71, and the recombinant P. pastoris were screened via a high-throughput method. The recombinants were induced by methanol to secrete active lipases into cultural medium. The recombinant lipases were also purified and characterized. The optimum temperature for the purified lipase LipJ02 and LipJ03 was 30 and 35 degrees C, respectively, at pH 8.0. They exhibited similar thermostability, but LipJ02 exhibited better pH stability than LipJ03.

Isolation of a cotton reversibly glycosylated polypeptide ( GhRGP1) promoter and its expression activity in transgenic tobacco

Reversibly glycosylated polypeptides (RGPs) are thought to be involved in polysaccharide metabolism. A cDNA of the cotton (Gossypium hirsutum) RGP gene, designated GhRGP1, has previously been characterized, and is preferentially expressed in fiber cells. In order to investigate its temporal and spatial control, we isolated a 624bp fragment upstream of the GhRGP1 coding sequence using a polymerase chain reaction (PCR)-based genomic walking method, transcriptionally fused the 624bp promoter sequence to the beta-glucuronidase (GUS) gene, and analyzed the stable gene expression in tobacco (Nicotiana tabacum). In 4-week-old transgenic tobacco plants, the highest expression level was observed in roots, and the GUS activity was 1.13- and 6.65-fold higher than that in stems and leaves, respectively. In the reproductive growth stage, the GUS expression level was highest in the pistils and the GUS activity in the stigmas and styles were 17.6-fold higher than that in the ovaries. High GUS activity was also detected in the anthers. In addition, histochemical staining for GUS activity on transgenic tobacco plants further indicated a higher expression in the trichomes, seeds and vascular tissues of stems. Abiotic stress treatments on transgenic tobacco plants showed that wounding and dehydration induced GUS expression. These results demonstrated the spatial and temporal regulation of a cotton RGP promoter in a model plant, and provided an important insight into the factors that control the fiber development and stress responses of the gene.

Isolation of promoter for N-methyltransferase gene associated with caffeine biosynthesis in Coffea canephora

N-Methyltransferases (NMTs) catalyze the three SAM dependent sequential methylation of xanthosine, producing caffeine in Coffea species. In the present work, a PCR based genome walking method was adopted to isolate and clone the promoter for the NMT gene. Inspection of the promoter sequence revealed the presence of several motifs important for the regulation of the gene expression. The whole fragment was fused to the β-glucuronidase ( gus) reporter gene and used in Agrobacterium tumefaciens mediated transformation of Nicotiana tabacum. GUS assays proved that the isolated promoter was able to direct the expression of the reporter gene in transgenic tobacco. Based on the promoter sequence, primer was designed and the genomic fragment comprising the promoter and its corresponding gene was amplified and cloned. Sequencing of one of the genomic clones revealed the presence of four exons and three introns in NMT gene. The differences in the restriction pattern among the genomic clones were studied using PCR-RFLP. This is the first report of cloning of the promoter for a gene involved in caffeine biosynthetic pathway and it opens up the possibility of studying the molecular mechanisms that regulate the production of caffeine.

Cloning and Expression of a Novel Lipase Gene From &lt;I&gt;Pseudomonas fluorescens&lt;/I&gt; B52

A novel lipase gene (lipB52) was isolated directly from the genomic DNA of Pseudomonas fluorescens B52 with the genome-walking method, an effective method for isolating lipase gene from bacteria. There was an open reading frame (ORF) of 1854 bp, which encoded 617 amino acids. The lipase gene (lipB52) was cloned into expression vector pPIC9K and successfully integrated into a heterologous fungal host, Pichia pastoris KM71, and the recombinant Pichia pastoris were screened with a high throughput method. The recombinant was induced by methanol to secrete active lipase into the culture medium. The recombinant lipase LipB52 was also purified and characterized. The optimum temperature for the purified lipase LipB52 was 40 degrees C at pH 8.0. It exhibited better thermostability and pH stability than its homologs.

RpoB gene sequencing for identification of Corynebacterium species.

The genus Corynebacterium is a heterogeneous group of species comprising human and animal pathogens and environmental bacteria. It is defined on the basis of several phenotypic characters and the results of DNA-DNA relatedness and, more recently, 16S rRNA gene sequencing. However, the 16S rRNA gene is not polymorphic enough to ensure reliable phylogenetic studies and needs to be completely sequenced for accurate identification. The almost complete rpoB sequences of 56 Corynebacterium species were determined by both PCR and genome walking methods. In all cases the percent similarities between different species were lower than those observed by 16S rRNA gene sequencing, even for those species with degrees of high similarity. Several clusters supported by high bootstrap values were identified. In order to propose a method for strain identification which does not require sequencing of the complete rpoB sequence (approximately 3,500 bp), we identified an area with a high degree of polymorphism, bordered by conserved sequences that can be used as universal primers for PCR amplification and sequencing. The sequence of this fragment (434 to 452 bp) allows accurate species identification and may be used in the future for routine sequence-based identification of Corynebacterium species.

Genome walking of large fragments: an improved method

The PCR-based genome walking method has been commonly used to isolate upstream regions from known cDNA sequences. The limitation of this technique is based on the location of the restriction site upstream to the gene-specific primer in the genome; hence, different restriction enzymes have to be used to isolate larger upstream fragments. In this paper, we present the advantageous use of partial and size-selected DNA as templates for genome walking, in isolating larger upstream fragments. We have successfully tested this approach to isolate larger upstream fragments using the FailSafe™ PCR System. Use of partial digestion and size selection can provide better chances in obtaining larger flanking regions of known DNA sequence, when compared to use of total digested DNA.

Identification of DNA sequences flanking T-DNA insertions by PCR-walking

In recent years, concerns over genetic modification issues have resulted in regulatory authorities requiring comprehensive analysis of transgene insertion events in the plants that are to be commercialized. Determining that plants are devoid of vector backbone sequences is a trivial task that is best achieved by Southern blot analysis; however, identifying the DNA sequences flanking the T-DNA insertions can be arduous. In this paper, we present a robust method of characterizing this insertion event. We have applied and modified a genomic walking method that combines vectorette and suppression PCR walking.

Citrate synthase gene sequence: a new tool for phylogenetic analysis and identification of Ehrlichia.

The sequence of the citrate synthase gene (gltA) of 13 ehrlichial species (Ehrlichia chaffeensis, Ehrlichia canis, Ehrlichia muris, an Ehrlichia species recently detected from Ixodes ovatus, Cowdria ruminantium, Ehrlichia phagocytophila, Ehrlichia equi, the human granulocytic ehrlichiosis [HGE] agent, Anaplasma marginale, Anaplasma centrale, Ehrlichia sennetsu, Ehrlichia risticii, and Neorickettsia helminthoeca) have been determined by degenerate PCR and the Genome Walker method. The ehrlichial gltA genes are 1,197 bp (E. sennetsu and E. risticii) to 1,254 bp (A. marginale and A. centrale) long, and GC contents of the gene vary from 30.5% (Ehrlichia sp. detected from I. ovatus) to 51.0% (A. centrale). The percent identities of the gltA nucleotide sequences among ehrlichial species were 49.7% (E. risticii versus A. centrale) to 99.8% (HGE agent versus E. equi). The percent identities of deduced amino acid sequences were 44.4% (E. sennetsu versus E. muris) to 99.5% (HGE agent versus E. equi), whereas the homology range of 16S rRNA genes was 83.5% (E. risticii versus the Ehrlichia sp. detected from I. ovatus) to 99.9% (HGE agent, E. equi, and E. phagocytophila). The architecture of the phylogenetic trees constructed by gltA nucleotide sequences or amino acid sequences was similar to that derived from the 16S rRNA gene sequences but showed more-significant bootstrap values. Based upon the alignment analysis of the ehrlichial gltA sequences, two sets of primers were designed to amplify tick-borne Ehrlichia and Neorickettsia genogroup Ehrlichia (N. helminthoeca, E. sennetsu, and E. risticii), respectively. Tick-borne Ehrlichia species were specifically identified by restriction fragment length polymorphism (RFLP) patterns of AcsI and XhoI with the exception of E. muris and the very closely related ehrlichia derived from I. ovatus for which sequence analysis of the PCR product is needed. Similarly, Neorickettsia genogroup Ehrlichia species were specifically identified by RFLP patterns of RcaI digestion. If confirmed this technique will be useful in rapidly identifying Ehrlichia spp.

Cloning of a calcitonin gene-related peptide from genomic DNA and its mRNA expression in flounder, Paralichthys olivaceus

A part of genomic DNA including the calcitonin gene-related peptide (CGRP) gene was cloned from flounder by the genome-walking method. The intron/exon boundary was predicted to occur exactly at the same position as in salmon. The 37-amino acid molecule coded by the region from the intron/exon boundary to the stop codon was preceded by a typical Lys-Arg cleavage signal and included a cleavage/amidation site common to the CGRP of other vertebrates. The predicted amino acid sequence of flounder CGRP had 78%, 78%, 78%, 81%, and 73–78% identity to that of salmon, cod, frog, chicken, and mammalian CGRPs, respectively. Among vertebrates, CGRP is more conserved than calcitonin (CT) because the identity of flounder CT to mammalian CTs is 31–50%. Expression analysis indicated that this hormone is synthesized in the brain, heart, intestine, testis, and ovary. Since we have previously shown that the CGRP receptor is expressed in these tissues, it is suggested that CGRP secreted from each tissue functions in a paracrine or autocrine manner.

Rapid acquisition of entire DNA polymerase gene of a novel herpesvirus from green turtle fibropapilloma by a genomic walking technique

A 4837-bp sequence of a newfound green turtle herpesvirus (GTHV), implicated in the etiology of green turtle fibropapilloma, was obtained from tumor tissues of a green turtle with fibropapilloma using a genomic walking method based on restriction enzyme digestion, self-ligation and inverse polymerase chain reaction (IPCR). The 4837-bp sequence was 56.23% G/C rich and contained three nonoverlapping open reading frames (ORF). The largest ORF (3507-bp) encoded the DNA polymerase gene ( pol gene), which exhibited a high degree of homology at both amino acid and nucleotide levels with the DNA pol genes of human and animal herpesviruses, with a predicted protein of 1169 amino acids and molecular weight of 132.6 kilodaltons. The ATG at 518 to 520 was the first initiation codon in the ORF and was presumed to be the first methionine codon of the pol gene. Phylogenetic analysis, based on the amino acid sequence of the GTHV DNA pol gene and the corresponding regions of other known human and animal herpesviruses, indicated that GTHV belonged to the Alphaherpesvirinae subfamily. The upstream ORF of the pol gene encoded the N-terminal region of the GTHV homologue of the DNA-binding protein gene, whereas the downstream ORF was the C-terminal region of a gene which was homologous to ORFs conserved in human and animal herpesviruses, i.e., herpes simplex virus 1 (HSV1) gene UL31, Epstein-Barr virus (EBV) gene BFLF2, equine herpesvirus 1 (EHV1) gene 29, and alcelaphine herpesvirus 1 (AHV1) hypothetical protein 69 gene. The arrangement of these three genes in GTHV genome was identical to that seen in other alphaherpesviruses. The sequence and location of the DNA pol gene in the GTHV genome should greatly facilitate future studies of the viral life cycle.

A gene encoding Achlya bisexualis β-amylase and its expression in Saccharomyces cerevisiae

Part of a β-amylase genomic DNA sequence from the oomycete, Achlya bisexualis was cloned by polymerase chain reaction (PCR) using degenerate oligonucleotide primers derived from the conserved regions of other known β-amylase sequences. The 5′- and 3′-regions of the β-amylase gene were amplified by genome walking method. The Ach. bisexualis β-amylase gene consisted of a 1338bp open reading frame, encoding a protein of 446 amino acids with a molecular weight of 49 381Da, and was not interrupted by any intron. The deduced amino acid sequence of the β-amylase gene had 67% similarity to the β-amylase of Saprolegnia ferax, followed by 40% similarity to that ofArabidopsis thaliana. The β-amylase gene was expressed in Saccharomyces cerevisiae placing it under the control of the alcohol dehydrogenase gene (ADC1) promoter.

Cloning and promoter analysis of the cotton lipid transfer protein gene Ltp3

A cotton Ltp3 gene and its 5′ and 3′ flanking regions have been cloned with a PCR-based genomic DNA walking method. The amplified 2.6 kb DNA fragment contains sequences corresponding to GH3 cDNA which has been shown to encode a lipid transfer protein (LTP3). The gene has an intron of 80 bp which is located in the region corresponding to the C-terminus of LTP3. The Ltp3 promoter was systematically analyzed in transgenic tobacco plants by employing the Escherichia coli β-glucuronidase gene (GUS) as a reporter. The results of histochemical and fluorogenic GUS assays indicate that the 5′ flanking region of the Ltp3 gene contains cis-elements conferring the trichome specific activity of Ltp3 promoter.

The ClpB protein from Campylobacter jejuni: molecular characterization of the encoding gene and antigenicity of the recombinant protein.

The ClpB heat-shock protein is necessary for the survival of Escherichia coli cells upon sudden increase of temperature. Using a PCR-based genomic walking method, the nucleotide sequence of a clpB homolog from Campylobacter jejuni was determined. The clpB gene encodes a protein of 857 amino acid (aa) residues, with a predicted molecular mass of 95.3kDa. Alignment of the deduced aa sequence with other known bacterial ClpB proteins revealed overall identity from 47% (E. coli) to 61% (Helicobacter pylori). Within the clpB promoter region, as indicated by primer extension analysis, we identified a sequence identical to the E. coli sigma70 consensus promoter. Northern blot analysis confirmed that clpB is heat-inducible in C. jejuni. The ClpB protein, fused to a 6xHis tag, was synthesized in E. coli and purified by metal-affinity and size exclusion chromatography. In ELISA studies, IgA levels reactive to recombinant ClpB were significantly higher in sera of patients with prior C. jejuni infections than in sera obtained from healthy control persons.