Evolutionary dynamics of insertion sequences in Helicobacter pylori

Prokaryotic insertion sequence (IS) elements behave like parasites in terms of their ability to invade and proliferate in microbial gene pools and like symbionts when they coevolve with their bacterial hosts. Here we investigated the evolutionary history of IS605 and IS607 of Helicobacter pylori, a genetically diverse gastric pathogen. These elements contain unrelated transposase genes (orfA) and also a homolog of the Salmonella virulence gene gipA (orfB). A total of 488 East Asian, Indian, Peruvian, and Spanish isolates were screened, and 18 and 14% of them harbored IS605 and IS607, respectively. IS605 nucleotide sequence analysis (n = 42) revealed geographic subdivisions similar to those of H. pylori; the geographic subdivision was blurred, however, due in part to homologous recombination, as indicated by split decomposition and homoplasy tests (homoplasy ratio, 0.56). In contrast, the IS607 populations (n = 44) showed strong geographic subdivisions with less homologous recombination (homoplasy ratio, 0.2). Diversifying selection (ratio of nonsynonymous change to synonymous change, >1) was evident in approximately 15% of the IS605 orfA codons analyzed but not in the IS607 orfA codons. Diversifying selection was also evident in approximately 2% of the IS605 orfB and approximately 10% of the IS607 orfB codons analyzed. We suggest that the evolution of these elements reflects selection for optimal transposition activity in the case of IS605 orfA and for interactions between the OrfB proteins and other cellular constituents that potentially contribute to bacterial fitness. Taken together, similarities in IS elements and H. pylori population genetic structures and evidence of adaptive evolution in IS elements suggest that there is coevolution between these elements and their bacterial hosts.     

Transposable Element ISHp608 of Helicobacter pylori: Nonrandom Geographic Distribution, Functional Organization, and Insertion Specificity

A new member of the IS605 transposable element family, designated ISHp608, was found by subtractive hybridization in Helicobacter pylori. Like the three other insertion sequences (ISs) known in this gastric pathogen, it contains two open reading frames (orfA and orfB), each related to putative transposase genes of simpler (one-gene) elements in other prokaryotes; orfB is also related to the Salmonella virulence gene gipA. PCR and hybridization tests showed that ISHp608 is nonrandomly distributed geographically: it was found in 21% of 194 European and African strains, 14% of 175 Bengali strains, 43% of 131 strains from native Peruvians and Alaska natives, but just 1% of 223 East Asian strains. ISHp608 also seemed more abundant in Peruvian gastric cancer strains than gastritis strains (9 of 14 versus 15 of 45, respectively; P = 0.04). Two ISHp608 types differing by approximately 11% in DNA sequence were identified: one was widely distributed geographically, and the other was found only in Peruvian and Alaskan strains. Isolates of a given type differed by < or = 2% in DNA sequence, but several recombinant elements were also found. ISHp608 marked with a resistance gene was found to (i) transpose in Escherichia coli; (ii) generate simple insertions during transposition, not cointegrates; (iii) insert downstream of the motif 5"-TTAC without duplicating target sequences; and (iv) require orfA but not orfB for its transposition. ISHp608 represents a widespread family of novel chimeric mobile DNA elements whose further analysis should provide new insights into transposition mechanisms and into microbial population genetic structure and genome evolution.     

Programmed translational frameshifting and initiation at an AUU codon in gene expression of bacterial insertion sequence IS911.

The proteins expressed by insertion sequence IS911, a member of the widespread IS3 family of elements, have been analyzed. The results indicate that three major species are produced from two consecutive reading frames. A protein of Mr 11,500, ORFA, is synthesized from an upstream reading frame. A larger protein, ORFAB, uses the same initiation codon and is produced by a -1 programmed translational frameshift between orfA and a downstream frame, orfB, whose amino acid sequence shows significant homology with retroviral integrase proteins. The orfB frame is also expressed independently in two alternative forms: the first uses a rare AUU initiation codon in the orfB phase whereas the second appears to initiate in the orfA phase and is produced by a -1 frameshift mechanism similar to that used in ORFAB expression. A specific IS911 integration reaction using a minimal active junction composed of 51 base-pairs of the right inverted repeat and a flanking phase lambda sequence resembling a second end in inverted orientation has been developed to analyze the functions of these proteins by transcomplementation in vivo. The orfA and orfB frames are shown to be essential and production of ORFAB is shown to stimulate integration in this system, suggesting that this fusion protein is the IS911 transposase.     

Two frameshift products involved in the transposition of bacterial insertion sequence IS629

IS629 is 1,310 bp in length with a pair of 25-bp imperfect inverted repeats at its termini. Two partially overlapping open reading frames, orfA and orfB, are present in IS629, and two putative translational frameshift signals, TTTTG (T4G) and AAAAT (A4T), are located near the 3'-end of orfA. With the lacZ gene as the reporter, both T4G and A4T motifs are determined to be a -1 frameshift signal. Two peptides representing the two transframe products designated OrfAB' and OrfAB, are identified by a liquid chromatography-tandem mass spectrometric approach. Results of transposition assays show that OrfAB' is the transposase and that OrfAB aids in the transposition of IS629. Pulse-chase experiments and Escherichia coli two-hybrid assays demonstrate that OrfAB binds to and stabilizes OrfAB', thus increasing the transposition activity of IS629. This is the first transposable element in the IS3 family shown to have two functional frameshifted products involved in transposition and to use a transframe product to regulate transposition.     

Detection and characterization of a functional insertion sequence, ISVpa2, in Vibrio parahaemolyticus

PCR analysis of the pandemic strain of Vibrio parahaemolyticus, KX-V237 (total genome sequenced) showed a subculture where the size of the amplicons had increased. The purpose of this study was to analyze the mechanism of this change. We found a 1,243-bp DNA sequence inserted in one of the pandemic marker genes in this strain. The inserted DNA sequence possessed the genetic structures shared by insertion sequences (ISs) of the IS3 family. This IS had 26-bp imperfect terminal inverted repeats (IRs) and two partially overlapping reading frames, orfA and orfB. OrfA codes for a helix-turn-helix, OrfA and OrfAB produced by translational frameshifting code for leucine zipper motifs, and OrfB codes for a DDE motif. orfA and orfB were homologous to those in the IS3 family. This IS was named ISVpa2. Southern blot analysis showed the copy number of ISVpa2 in our stock culture and its subculture of KX-V237 was three and four, respectively; whereas it was only one in the reported genome sequence. Analysis of the flanking sequences for seven ISVpa2 copies showed ISVpa2 is capable of inserting at multiple sites and ISVpa2 causes genetic rearrangements including insertional inactivation of the target gene and adjacent deletion. ISVpa2 created 3-base duplications upon insertion. PCR, hybridization, and nucleotide sequence analyses showed ISVpa2 homologs were detected in all of the 62 other strains of V. parahaemolyticus examined; and in some strains of Vibrio vulnificus (98% identity), Vibrio penaeicida (86% identity), and Vibrio splendidus (87% identity); but was not in 25 other species in the genus Vibrio. The data demonstrate that ISVpa2 is a transpositionally active IS discovered for the first time in V. parahaemolyticus and suggest that ISVpa2 may be transferred among the species of the genus Vibrio.     

2-萘酸加单氧酶基因及NADH:黄素还原酶基因的克隆与分析

伯克霍尔德氏菌(Burkholderiasp.)JT1500对2-萘酸(2-naphthoate)生物降解的关键步骤之一是通过2-萘酸加单氧酶羟化2-萘酸生成1-羟基-2-萘酸(1-hydroxy-2-naphthoate)。在已确定2-萘酸加单氧酶基因及其功能的基础上对含有该基因的一个4.8kb长度的基因簇进行了克隆测序。该序列上含有4个可能的阅读框orfB、orfC、orfD、orfA。序列比对发现,orfA序列与JaponicumUSDA110和RalstoniaeutrophaHF39中的加单氧酶基因同源性较高,orfB序列与BordetllapertussisTohamaI、RalstoniasolanacearumGMI1000和BordetellabronchisepticaRB50等菌中的黄素还原酶基因有一定的同源性。酶活分析发现只含基因orfA的重组大肠杆菌SA细胞提取液有很低的加氧活性,含基因orfB的重组子SB细胞提取液没有加氧活性,但在反应体系中同时加入SA和SB的细胞提取液后,其加氧活性显著增强,包含片段orfB orfA的重组子SB A在黄素(FMN、FAD)存在的情况下也表现出很强的加氧活性;在厌氧条件下,能检测出SB细胞提取液的黄素还原活性。基于以上信息,认为2-萘酸加单氧酶基因簇含有两个重要的组分黄素还原酶基因(nmoB)和加单氧酶基因(nmoA)。2-萘酸加单氧酶Nmo羟化2-萘酸的过程为先由黄素还原酶(NmoB)在NADH存在的条件下将黄素(FMN、FAD)还原为还原型黄素(FMNH2、FADH2),然后加单氧酶(NmoA)利用还原型黄素和O2羟化底物2-萘酸,生成1-羟基-2-萘酸。NmoB是NmoA的偶联蛋白。     

Cloning of insertion sequence IS1485 from Enterococcus species.

We have cloned and identified an insertion sequence, IS1485, that was present in several members of the genus Enterococcus. IS1485 exists in varying copy numbers with at least 12 copies in E. durans (ATCC 11576), 3 copies in E. faecium (ATCC 19434), and one copy each in E. faecalis (ATCC 19433) and E. avium (ATCC 14024). It was also detected in clinical strains of E. gallinarum, E. casseliflavus, and E. saccharolyticus. IS1485 is 1366 bp in length, it has imperfect terminal inverted repeats with 25 of the terminal 39 residues matched, and it contains three open reading frames exceeding 50 codons, designated orfA, orfB, and orfC. The largest, orfB, was located 36 bp downstream and in the -1 reading frame relative to orfA; orfC is oriented in the opposite direction and overlaps orfA. The genetic organization of IS1485 resembles that of members of the IS3 family of transposable elements. Sequence homology exists with several members of the IS3 family especially with IS199 from Streptococcus mutans.     

Identification and characterization of IS1381, a new insertion sequence in Streptococcus pneumoniae.

A new insertion sequence (IS1381) was identified in the genome of Streptococcus pneumoniae R6 as an 846-bp segment containing 20-bp terminal inverted repeats and flanked by 7-bp direct repeats. The three sequenced copies of this element have two overlapping open reading frame (ORF) genes named orfA and orfB. However, significant variations between individual copies were found, suggesting that inactivating mutations have occurred in an original single ORF. Accordingly, the consensus IS1381 element derived from the comparison of the three available copies should contain a single ORF sufficient to encode a basic protein of 267 amino acids which exhibited high similarity to the putative transposases of ISL2 from Lactobacillus helveticus and of IS702 from the cyanobacterium Calothrix sp. strain PCC 7601. A minimum of five to seven copies were detected by hybridization experiments in the R6 genome. In remarkable contrast with the two previously reported pneumococcal insertion sequences, several copies of IS1381 have been detected in all of the clinical isolates tested so far. Interestingly, Streptococcus oralis NCTC 11427 (type strain), a close relative of pneumococcus, does not contain this element, but its occurrence in the type strain of Streptococcus mitis (NCTC 12261) suggests that this species has exchanged DNA with S. pneumoniae directly or through an intermediate species.     

Characterization of a novel insertion sequence,IS<Emphasis Type="Italic">Bp1</Emphasis>, in<Emphasis Type="Italic"> Burkholderia pseudomallei</Emphasis>

During screening for antigenic proteins in Burkholderia pseudomallei, a novel insertion sequence, IS Bp1, was found by sequence similarity searches. IS Bp1 contains two overlapping ORFs of 261 bp ( orfA) and 852 bp ( orfB), encoding 87 and 284 amino acid residues, respectively, and an imperfect inverted repeat. The putative protein encoded by orfA (OrfA) is similar to the OrfA in insertion sequences of the IS 3 family in other bacteria, showing 49% and 76% amino acid identity and similarity, respectively, with the transposase encoded by IS D1 of Desulfovibrio vulgaris vulgaris. The putative protein encoded by orfB (OrfB) is similar to the OrfB in insertion sequences of the IS 3 family in other bacteria, showing 43% and 62% amino acid identity and similarity, respectively, with the transposase encoded by IS 1222 of Enterobacter agglomerans. Sequence analysis of OrfA showed the presence of an alpha-helix-turn-alpha-helix motif, as well as the putative leucine zipper at its 3' end, for possible DNA binding to the terminal inverted repeats. Sequence analysis of OrfB showed the presence of a DDE motif of aspartic acid, aspartic acid, and glutamic acid, a highly conserved motif present in OrfB of other members of the IS 3 family. Furthermore, several other conserved amino acid residues, including the arginine residue located seven amino acids downstream from the glutamic acid residue, were observed. PCR amplification of the IS Bp1 gene showed a specific band in 65% of the 26 B. pseudomallei strains tested. Southern blot hybridization after XhoI or SacI digestion showed nine different patterns of hybridization. The number of copies of IS Bp1 in those strains that possessed the insertion sequence ranged from three to 12. Using several insertion sequences and a combination of insertion-sequence-based and non-insertion-sequence-based methods such as ribotyping will probably increase the discriminatory power of molecular typing in B. pseudomallei.     

HIV protease inhibitors attenuate adherence of Candida albicans to epithelial cells in vitro

The cagA gene is a key marker for the Helicobacter pylori cag pathogenicity island (PAI), which may vary in composition in different strains with insertion sequence mediated interruptions and deletions of genes. While presence of cagA has been associated with increased risk for peptic ulcer disease and gastric cancer, the precise link with virulence is controversial. We investigated H. pylori from dyspeptics in one location in England (mid-Essex) with reference to the prevalence and distribution by age cohort of different cag PAI forms to determine if presence of the insertion element IS605 had a modifying effect on the severity of associated disease. H. pylori isolated from gastric biopsies over a 4-year period were screened by specific PCR assays for the presence of cagA, cagD, cagE and virD4 genes in the cag PAI, and for the presence of IS605 in the PAI and elsewhere in the genome. Most (68%) of the 166 isolates of H. pylori contained a PAI based on detection of cagA whereas 29% had no detectable PAI using multiple loci. The cagA+ genotype frequencies were similar in the peptic ulcer and non-ulcer dyspepsia-gastritis groups (79% vs. 74%) whereas frequencies in the NUD-oesophagitis and normal mucosa groups were lower (58%) but not significantly different (P>0.41). Genomic IS605 inserts were present at an overall frequency of 32% and were widely distributed with respect to patient age and disease severity. The combined cagA+/IS- strain genotype was common but not significantly associated with PUD compared to endoscopically normal mucosa (P> or =0.807). We concluded that presence of the IS605 element, whether in cagA+ or cagA- strains of H. pylori, did not systematically modify the severity of associated disease in the study population.     

Characterisation of IS153, an IS3-family insertion sequence isolated from Lactobacillus sanfranciscensis and its use for strain differentiation

An insertion sequence has been identified in the genome of Lactobacillus sanfranciscensis DSM 20451T as segment of 1351 nucleotides containing 37-bp imperfect terminal inverted repeats. The sequence of this element encodes two out of phase, overlapping open reading frames, orfA and orfB, from which three putative proteins are produced. OrfAB is a transframe protein produced by -1 translational frame shifting between orf A and orf B that is presumed to be the transposase. The large orfAB of this element encodes a 342 amino acid protein that displays similarities with transposases encoded by bacterial insertion sequences belonging to the IS3 family. In L. sanfranciscensis type strain DSM 20451T multiple truncated IS elements were identified. Inverse PCR was used to analyze target sites of four of these elements, but except of their highly AT rich character not any sequence specificity was identified so far. Moreover, no flanking direct repeats were identified. Multiple copies of IS153 were detected by hybridization in other strains of L. sanfranciscensis. Resulting hybridization patterns were shown to differentiate between organisms at strain level rather than a probe targeted against the 16S rDNA. With a PCR based approach IS153 or highly similar sequences were detected in L. acidophilus, L. casei, L. malefermentans, L. plantarum, L. hilgardii, L. collinoides L. farciminis L. sakei and L. salivarius, L. reuteri as well as in Enterococcus faecium, Pediococcus acidilactici and P. pentosaceus.     

The interplay of mRNA stimulatory signals required for AUU-mediated initiation and programmed -1 ribosomal frameshifting in decoding of transposable element IS911

The IS911 bacterial transposable element uses -1 programmed translational frameshifting to generate the protein required for its mobility: translation initiated in one gene (orfA) shifts to the -1 frame and continues in a second overlapping gene (orfB), thus generating the OrfAB transposase. The A-AAA-AAG frameshift site of IS911 is flanked by two stimulatory elements, an upstream Shine-Dalgarno sequence and a downstream stem-loop. We show here that, while they can act independently, these stimulators have a synergistic effect when combined. Mutagenic analyses revealed features of the complex stem-loop that make it a low-efficiency stimulator. They also revealed the dual role of the upstream Shine-Dalgarno sequence as (i) a stimulator of frameshifting, by itself more potent than the stem-loop, and (ii) a mandatory determinant of initiation of OrfB protein synthesis on an AUU codon directly preceding the A6G motif. Both roles rely on transient base pairing of the Shine-Dalgarno sequence with the 3' end of 16S rRNA. Because of its effect on frameshifting, the Shine-Dalgarno sequence is an important determinant of the level of transposase in IS911-containing cells, and hence of the frequency of transposition.     

Two abundant intramolecular transposition products, resulting from reactions initiated at a single end, suggest that IS2 transposes by an unconventional pathway

The Escherichia coli insertion sequence, IS 2 , is a member of the IS 3 family of bacterial transposable elements. Its transposase is a fusion protein, OrfAB, made by a programmed −1 translational frameshift near to the end of orfA and just after the start of orfB . We have characterized two major products of IS 2 intramolecular transposition, which accumulate in cells that express the IS 2 OrfAB fusion protein at elevated levels. The more abundant product is a minicircle composed of the complete IS 2 with just a single basepair (occasionally 2 bp) separating the two IS ends. In all cases, this basepair is derived from the vector sequence immediately adjacent to the left IS 2 end (IRL). The second product is a figure-eight molecule that contains all the IS 2 and vector sequences present in the parental plasmid. One DNA strand contains the parental sequences unrearranged. The other contains a single-stranded version of the minicircle junction — the precise 3' end of IRR has been cleaved and joined to a target just outside the 5' end of IRL; the remaining vector sequences have a free 5' end, derived from cleavage at the 3' end of IRR, and a free 3' end, released upon cleavage of the target site adjacent to IRL. We propose that figure-eight molecules are the precursor to IS 2 minicircles and that the formation of these two products is the initial step in IS 2 intermolecular transposition. This proposed transposition pathway provides a means for a transposase that can cleave only one strand at each IS end to produce simple insertions and avoid forming co-integrates.     

The genes coding for enterocin EJ97 production by Enterococcus faecalis EJ97 are located on a conjugative plasmid

Enterococcus faecalis EJ97 produces a cationic bacteriocin (enterocin EJ97) of low molecular mass (5,327.7 Da). The complete amino acid sequence of enterocin EJ97 was elucidated after automated microsequencing of oligopeptides generated by endoproteinase GluC digestion and cyanogen bromide treatment. Transfer of the 60-kb conjugative plasmid pEJ97 from the bacteriocinogenic strain E. faecalis EJ97 to E. faecalis OG1X conferred bacteriocin production and resistance on the recipient. The genetic determinants of enterocin EJ97 were located in an 11.3-kb EcoRI-BglII DNA fragment of pEJ97. This region was cloned and sequenced. It contains the ej97A structural gene plus three open reading frames (ORFs) (ej97B, ej97C, and ej97D) and three putative ORFs transcribed in the opposite direction (orfA, orfB, and orfC). The gene ej97A translated as a 44-amino-acid residue mature protein lacking a leader peptide with no homology to other bacteriocins described so far. The product of ej97B (Ej97B) shows strong homology in its C-terminal domain to the superfamily of bacterial ATP-binding cassette transporters. The products of ej97C (Ej97C) and ej97D (Ej97D) could be proteins with 71 and 64 residues, respectively, of unknown functions and with no significant similarity to known proteins. There are two additional ORFs (ORF1 and ORF6) flanking the ej97 module, which have been identified as a transposon-like structure (tnp). ORF1 shows similarities to transposase of the Lactococcus lactis element ISS1 and is up to 50% identical to IS1216. This is flanked by two 18-bp inverted repeats (IRs) that are almost identical to those of ISS1 and IS1216. ORF6 (resEJ97) shows strong homology to the resolvase of plasmid pAM373 and up to 40 to 50% homology with the recombinase of several multiresistant plasmids and transposons from Staphylococcus aureus and E. faecalis. These data suggest that EJ97 could represent a new class of bacteriocins with a novel secretion mechanism and that the whole structure could be a composite transposon. Furthermore, two additional gene clusters were found: one cluster is probably related to the region responsible for the replication of plasmid pEJ97, and the second cluster is related to the sex pheromone response. These regions showed a high homology to the corresponding regions of the conjugative plasmids pAM373, pPD1, and pAD1 of E. faecalis, suggesting that they have a common origin.     

Characterization and distribution of IS8301 in the radioresistant bacterium Deinococcus radiodurans

The insertion sequence element IS8301 isolated from the radiation resistant bacterium Deinococcus radiodurans strain KD8301 was characterized. IS8301 is comprised of 1,736-bp, lacks terminal inverted repeats and does not duplicate target DNA upon its insertion. The amino acid sequence homology of two open reading frames encoded in IS8301 indicates that this insertion sequence element belongs to the IS200/IS605 group. There were seven loci completely identical with the IS8301 sequence in the published D. radiodurans R(1) genome sequence. The genome distribution profiles of IS8301 in strain KD8301 as well as in the three different laboratory isolates (KR(1), MR(1), and R(1)) of wild-type D. radiodurans were investigated using genomic hybridization analysis. At least 21 strong hybridization signals were detected in strain KD8301 while only one hybridization signal was detected in strain KR(1), the parent strain of KD8301. In strain MR1, a different wild-type isolate, six strong hybridization signals were detected. In spite of the identification of seven copies of IS8301 in the published D. radiodurans R(1) genome sequence, only one hybridization signal was detected in strain R(1) purchased from American Type Culture Collection. Using inverse PCR and sequencing analyses, total 13 different insertion loci of IS8301 in the D. radiodurans genome were identified. Sequence comparison of the flanking region of insertion sites indicated that the sequence 5'-TTGAT-3' preceded the left end of IS8301 in all cases.     

Structure and host recognition of serotype 13 glycopeptidolipid from Mycobacterium intracellulare

The Mycobacterium avium-M. intracellulare complex (MAIC) is divided into 28 serotypes by a species-specific glycopeptidolipid (GPL). Previously, we clarified the structures of serotype 7 GPL and two methyltransferase genes (orfA and orfB) in serotype 12 GPL. This study elucidated the chemical structure, biosynthesis gene, and host innate immune response of serotype 13 GPL. The oligosaccharide (OSE) structure of serotype 13 GPL was determined to be 4-2'-hydroxypropanoyl-amido-4,6-dideoxy-β-hexose-(1 → 3)-4-O-methyl-α-L-rhamnose-(1 → 3)-α-L-rhamnose-(1 → 3)-α-L-rhamnose-(1 → 2)-α-L-6-deoxy-talose by using chromatography, mass spectrometry, and nuclear magnetic resonance (NMR) analyses. The structure of the serotype 13 GPL was different from those of serotype 7 and 12 GPLs only in O-methylations. We found a relationship between the structure and biosynthesis gene cluster. M. intracellulare serotypes 12 and 13 have a 1.95-kb orfA-orfB gene responsible for 3-O-methylation at the terminal hexose, orfB, and 4-O-methylation at the rhamnose next to the terminal hexose, orfA. The serotype 13 orfB had a nonfunctional one-base missense mutation that modifies serotype 12 GPL to serotype 13 GPL. Moreover, the native serotype 13 GPL was multiacetylated and recognized via Toll-like receptor 2. The findings presented here imply that serotypes 7, 12, and 13 are phylogenetically related and confirm that acetylation of the GPL is necessary for host recognition. This study will promote better understanding of the structure-function relationships of GPLs and may open a new avenue for the prevention of MAIC infections.     

Frame-shift mutations in NAD(P)H flavin oxidoreductase encoding gene (frxA) from metronidazole resistant Helicobacter pylori ATCC43504 and its involvement in metronidazole resistance

Metronidazole is a critical ingredient for combination therapies of Helicobacter pylori infection, the major cause of peptic ulcer and gastric cancer. It has been recently reported that metronidazole resistance from H. pylori ATCC43504 is caused by the insertion of a mini-IS605 sequence and deletion of sequences in an oxygen insensitive NAD(P)H nitroreductase encoding gene (rdxA). We also found that an additional gene (frxA) encoding NAD(P)H flavin oxidoreductase in the same strain was truncated by frame-shift mutations. To assess whether the frxA truncation is also involved in metronidazole resistance, metronidazole sensitive H. pylori strains ATCC43629 and SS1 were transformed by the truncated frxA gene cloned from strain ATCC43504. All transformed cells grew on agar plates containing 16 microg ml(-1) of metronidazole. The involvement of the frxA gene in metronidazole resistance was also confirmed by insertion inactivation of frxA and/or rdxA genes from strain ATCC43629 and one metronidazole sensitive clinical isolate H. pylori 2600. In addition, the frxA gene cloned from the H. pylori 2600 showed metronidazole nitroreductase activity in Escherichia coli and rendered ordinary metronidazole resistant E. coli to metronidazole sensitive cell. These results indicate that the frxA gene may also be involved in metronidazole resistance among clinical H. pylori isolates.