Identification of Genes Associated with Natural Competence in Helicobacter pylori by Transposon Shuttle Random Mutagenesis

Biochemical and Biophysical Research Communications 288, 961–968 (2001) doi:10.1006/bbrc.2001.5877, available online at http://www.idealibrary.com on

Identification of Genes Associated with Natural Competence in Helicobacter pylori by Transposon Shuttle Random Mutagenesis Kai-Chih Chang,* Yu-Ching Yeh,* Tzu-Lung Lin,* and Jin-Town Wang* ,† ,1 *Graduate Institute of Microbiology, National Taiwan University, College of Medicine, Taipei, Taiwan; and †Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan

Received October 1, 2001

To identify genes involved in DNA transformation, we generated 1500 insertion mutants of a Helicobacter pylori strain by transposon shuttle mutagenesis. All mutant strains were screened for their frequency of natural transformation. A total of 20 mutant strains were found to exhibit a significantly decreased transformation frequency. DNA sequencing revealed seven genetic loci, including the reported comB locus, HP0017 (a putative virB4 homologue) and five loci without database match (HP0015, HP1089, HP1326, HP1424, and HP1473) from the 20 mutants. Reknockout of HP1326 revealed no impairment in natural transformation, while the other 5 mutants showed the same defective in natural transformation. Mutation of HP0017 severely impaired natural transformation both chromosome and plasmid DNA. Slot blot analysis revealed that some noncompetent strains had decreased virB4 RNA expression levels compared with competent strains. Nineteen ORFs had decreased expression levels in virB4 knockout mutant by microarray. Therefore, our data indicate that HP0017 is a virB4 homologue and is essential in the natural competence of H. pylori. HP0015, HP1089, HP1424, and HP1473 genes could be also involved in natural transformation. © 2001 Academic Press Key Words: DNA transformation; mutagenesis; Helicobacter pylori.

Helicobacter pylori, a spiral, gram-negative bacterium, was first isolated in 1982 from the gastric mucosa of a patient inflicted with gastritis and peptic ulceration (1). The bacterium is an important human pathogen because it is now recognize as being responsible for type B gastritis and also peptic ulcer, and is a 1 To whom correspondence and reprint requests should be addressed at Graduate Institute of Microbiology, College of Medicine, National Taiwan University, 1, Section 1, Jen-Ai Road, Taipei, Taiwan. Fax: 886-2-23948718 or 886-2-23778111. E-mail: [email protected].

risk factor for gastric adenocarcinoma and mucosaassociated lymphoid tissue lymphoma (MALToma) of the stomach in humans (2–5). Although the full genome sequence for the bacterium has been determined (6, 7) and several important virulence factors have been described for the bacterium’s colonization of and survival in the gastric environment (6, 7), the biological function of many genes remain largely unknown. Natural transformation in bacteria is a complex process involving DNA binding, uptake/translocation, and recombination (8). Many H. pylori strains are known to be naturally competent for transformation in vitro (9, 10) although the mechanisms for transformation of DNA have not been closely studied for H. pylori. Thus far, only recA (11, 12) the comB locus (13) the dprA (14, 15) and the comH (16) have been identified as having a role in H. pylori transformation. Further recognition of the mechanisms involved in genetic exchange may help us to understand the adaptation of H. pylori to changing environments and could shed light on clinically important issues of virulence and the development of antibiotic resistance (17–19). Transposons are well known genetic tools that generate mutations by disrupting the linear continuity of a specific gene and, consequently, affect its expression. Unfortunately, the direct transposon mutagenesis method has not yet been found to be functional for H. pylori (20). Here, we modified a previously described genetic approach that overcomes this handicap by using transposon shuttle mutagenesis (21–23). Genes associated with natural competence for H. pylori were identified by screening mutants with decreased transformation frequency. Microarrays are able to analyze the expression of hundreds of genes in a single hybridization experiment. Recently, microarray has been adopted for monitoring gene expressions in bacterial (24). Here, we adopted the same method to explore the changes of gene expression of virB4 knockout mutant H. pylori in genomic scale (24, 25).

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Vol. 288, No. 4, 2001

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS extracted by Puregene DNA Isolation Kits (Gentra Systems, Minneapolis, MN). Partial Sau3A restriction of chromosome DNA and ligation of the genomic fragments (range between 3 and 5 kb in size) into the pILL570 vector were performed as Dr. Labigne A described previously (22, 23). After the genomic library had been constructed, the transposon MiniTnKm was transposed into the cloned H. pylori fragments in Escherichia coli HB101. Ninety-six independent recombinant plasmids could be mutagenized in parallel, the random mutagenesis being achieved by two consecutive conjugation steps as described previously (22, 23). The E. coli strains HB101, NS2114, DH1, and plasmids pILL570, pTCA, pILL553 (Table 1) were kindly donated by Dr. Labigne A., Pasteur Institute.

TABLE 1

Bacterial Strains and Plasmids Used in This Study Helicobacter pylori Strains

Competence

NTUH-C1 NTUH-C2 NTUH-C3 NTUH-C4 NTUH-C5 NTUH-C6 NTUH-C7 NTUH-N1 NTUH-N2 NTUH-N3 NTUH-N4 NTUH-N5 ATCC49503

Competent Competent Competent Competent Competent Competent Competent Noncompetent Noncompetent Noncompetent Noncompetent Noncompetent Noncompetent E. coli

Strains

Relevant properties

Reference

HB101*

Hsd R hsdM recA supE44 lacZ4 leuB6 proA2 thi-1Sm RecA, Sm100, contains a ␭-cre prophage, rifampicin R F-supE44 recA1 endA1 gurA96 thi-1 hsdR17T [r k⫺m k⫹]relA1

Boyer & Roulland, 1969

NS2114*

DH1*

Seifert et al., 1985; Labigne et al., 1992 Bachmann, 1996

Plasmids Plasmid pILL570 pTCA pILL553 pHel2

Phenotypic characteristics Rep pBR322, mob, Sp Rep pACYC184, Tc, TnpA, ImmTn3 pox38::TnKm E. coli-H. pylori shuttle, cat GC resistance

Size

Reference

5.3 kb 7.0 kb

Labigne et al., 1992 Seifert et al., 1985

⬎50 kb 5.1 kb

Labigne et al., 1992 Heuermann & Hass, 1998

* E. coli strains HB101, NS2114, DH1, were kindly donated by Dr. Labigne A., Pasteur Institute.

MATERIALS AND METHODS Bacterial strains and culture conditions. Clinical isolates were obtained at the National Taiwan University Hospital (NTUH) as described previously (26). All bacterial strains and plasmids used in this study are listed in Table 1. H. pylori strains were grown on the Columbia agar (Oxoid Unipath Ltd., Hampshire, England) plates containing 5% defibrinogen sheep blood (Difco Laboratories, Detroit, MI) and chloramphenicol (Sigma Chemical Co.) (20 ␮g/ml) or kanamycin (Sigma) (10 ␮g/ml), and incubated for two to three days under microaerophilic conditions (5% O 2, 10% CO 2, 85% N 2) at 37°C (27). The strains were stored at ⫺80°C after successful culture. E. coli strains were grown on LB agar (Gibco BRL, Inc., NY) plates or LB broth (Gibco) containing appropriate antibiotics (23). Construction of a genomic library in the pILL570 vector and mutagenesis of 96 independent DNA inserts. Genomic DNA of a naturally competent clinical isolate of H. pylori NTUH-C1 (Table 1) was

Transformation and selection of randomly mutagenized H. pylori mutants. After all independent recombinant plasmids were mutagenized, the pILL570-derivative⬋TnKm plasmids from pooled NS2114 transconjugants were extracted and transformed NTUH-C1 strain by natural transformation. To obtain a more representative library we tested different numbers of independent pILL570derivative⬋TnKm plasmids pooled for each transformation. Finally, we found that 96 independent pILL570-derivative⬋TnKm plasmids pooled for a natural transformation yielded the largest number of diverse mutants in terms of cost and effect. Subsequently, each pool of plasmid was transformed into NTUH-C1 by natural transformation as previously described (9, 26). The selection of kanamycinresistant H. pylori transformants was performed on Columbia blood agar plates containing 10 ␮g/ml kanamycin. After 25 pools of disrupted hybrid were transformed, all mutant strains were collected and stored at ⫺80°C until testing. Restriction pattern and sequence analysis of the H. pylori mutant strains. Chromosomal DNA was isolated from 14 random H. pylori mutant strains by using Puregene DNA Isolation Kits (Gentra Systems), and completely cleaved with the restriction endonuclease HindIII (Roche Molecular Biochemicals, Mannheim, Germany). The DNA was fractionated on a 0.8% agarose gel, and transferred to a nylon membrane (Oncor, Gaithersburg, MD). The membrane was then hybridized with the kanamycin gene probe, which was labeled with enhanced chemiluminescence kit (Amersham, Buckinghamshire, UK) according to the manufacturer’s instructions. Subsequently, Southern hybridization was performed as described previously (28), the film was finally exposed for one minute after treating with the substrate. To estimate the redundancy of this mutant library, inverse PCR and DNA sequence procedures were preformed as later description. Two pools of H. pylori mutants were randomly selected for sequences. One of them yielded less than 96 mutant strains, and the other one yielded more than 96 mutant strains. In addition, the pool that contained redundant HP0017 (a putative virB4 homologue) knockout mutants was chosen for sequence. Screening of mutant strains that decreased the extent of natural competence. To select genes involved in DNA transformation competence, we determined the competence of all 1500 mutants by natural transformation as described by Wang et al. (9). A chloramphenicol acetyltransferase (CAT) expression cassette (gift from Dr. D. E. Taylor, University of Alberta) inserted within the H. pylori gene yxjD (HP0691), a 23S rRNA gene from a clarithromycinresistant strain (with A to G mutation at 2143) in the pCR2.1 vector (Invitrogen), and a E. coli–Helicobacter pylori shuttle vector pHel2 (29) that carries the Cat GC chloramphenicol resistance gene (gift from Dr. R. Hass, Max-Planck-Institute fu¨r Biologie, Tu¨bingen, Germany) were used as DNA donors (26, 29, 30). After successful transformation with CAT or 23S rRNA gene or plasmid pHel2, the H. pylori clones were able to express chloramphenicol-resistance or clarithromycin-resistance phenotypes. To relate the mutants to the wild-type, the frequencies were depicted as a percentage of the corresponding wild-type’s transformation frequency conform the method of Hofreuter et al. (13). Growth of the mutants on the nonselective media was observed as control.

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Electroporation. Bacterial cells grown on two Columbia blood agar plates for 48 h were harvested and suspended into 40 ml of cold WEB solution (10% glycerol in water). For each electroporation, bacteria were washed three times in 40 ml cold WEB, and suspended in a final volume of 1 ml WEB. The 100 ␮l of competent cell of H. pylori were put into prechilled 0.2-cm electroporation curettes; 1 ␮g chloramphenicol acetyltransferase (CAT) expression cassette was then added. Electroporation was done at 2.5 kV and 246 ohm and 25 ␮F by an Electro cell manipulator (BTX Electroporation System, San Diego, CA). After electroporation, 100 ␮l of SOC medium (BRL) was added to each sample. The 200-␮l aliquots were plated onto nonselective agar plates. Following 2 days expression period, the bacteria was removed and resuspended in 1 ml Brucella broth. For selection, 10 9 resuspended bacteria were planted on Columbia blood agar containing 20 ␮g/ml of chloramphenicol. The plates were incubated for 4 days to allow for outgrowth of resistant colonies. Inverse PCR and DNA sequencing. Chromosomal DNA from mutant strains with decreased natural transformation frequency were extracted by Puregene DNA Isolation Kits (Gentra Systems). Subsequently, these chromosomal DNA species were completely digested with the restriction endonuclease HindIII (Roche), and ligated with themselves by T4 DNA ligase (Roche). Oligonucleotide primers were synthesized according to the kanamycin gene originating from Campylobacter coli (31) (sense: KmCF 5⬘-ATATCYCGRGGATAAACCCAGCGAACCATT-3⬘; antisense: KmCR 5⬘-TATACYCGRGCTCGACATACTGTTCTTCCC-3⬘). PCR was performed by denaturing DNA for 5 min at 96°C, after 30 amplification cycles (elongation 72°C for 1 21 min, denaturation at 96°C for one minute, annealing at 55°C for 1 min). The PCR products were purified by Gene GelPure Kit (Watson Biotech). DNA sequences were determined in PCR products, and Km-seq3 primer (5⬘-TGGTAACTGTCAGACCAAGTTTACTC-3⬘) was used for sequencing. For the sequencing reaction, the purified PCR products were used as a template for direct sequencing using a cycle-sequencing protocol and reagents supplied with the Taq Dye Terminator Cycle Sequencing Kit (Perkin–Elmer, Foster City, CA). The thermal-cycling conditions for sequencing were specified according to the manufacturer’s instructions. Finally, electrophoresis was performed on an ABI 377 automated sequencer (ABI, Foster City, CA). The identified reading frames were compared with the Gene-Bank databases as well as to a H. pylori genome database (HPDB) using the program BLAST. DNA sequences of putative virB4 in competent and noncompetent H. pylori strains were determined from PCR products, and oligonucleotide primers were synthesized according to the full genomic sequences. Amino acid sequences and alignment were conducted using the MacVector 6.5 program (Oxford Molecular Group PLC, London, England). Reknockout of HP0017 (putative virB4 homologue), HP0015, HP1089, HP1326, HP1424, and HP1473. Genomic DNA of mutant strains M10-75, M17-84, M1-38, M9-4, M16-3, and M19-44 were extracted by Puregene DNA Isolation Kits (Gentra Systems). Oligonucleotide primers were synthesized according to the full genomic sequences. PCR was performed after 30 amplification cycles. Knockout mutants were made by transformed individual PCR products that contained a kanamycin resistance marker into the naturally competent clinical isolate H. pylori NTUH-C1 and other naturally competent isolates. After successful transformation with six individual MiniTnKm insertion knockout genes, the six different H. pylori mutant strains were able to express kanamycin-resistance phenotypes. And the correct integration of the transposon into the bacterial chromosome was verified by PCR. Then, transformation frequencies of these six reknockout mutants were determined by natural transformation and electroporation as described above. Construction of HP0018 or HP0441 knockout mutants. HP0018 (the immediate ORF down stream of virB4 homologue) and HP0441 (the other putative virB4 in H. pylori) were cloned by PCR using primers HP0018-F: 5⬘-TTGAAAATATTCGTTCTGTTG-3⬘;HP0018R:5⬘-TTTTAAACGACTCAAAACAAAC-3⬘; HP0441-F: 5⬘-ATGCTTGAGAAAATTTTTAATTCTTTATGCTCG-3⬘; and HP0441-R: 5⬘-CTC-

CTTTGTCAAGTAAATTTCTTTATAATCC-3⬘ derived from strain 26695. Transposon shuttle mutagenesis was adopted for knockout of both genes. The HP0018 and HP0441 knockout mutants were confirmed by PCR and DNA sequencing (not shown). RNA slot blots and hybridization with RNA probes. Bacterial cells were grown for 48 h on Columbia agar (Oxoid) plates, harvested, washed with TE buffer (pH 7.4) and pelleted. Cell pellets were then resuspended and lysed in boiled 2% SDS–TES buffer (TES is 50 mM Tris– hydrochloride [pH 8.0], 1 mM EDTA and 50 mM NaCl) for 5 min, subjected to saturated phenol (pH 4.5) extraction at 65°C for 5 min, extracted twice with phenol:chloroform:isopropanol solution (25:24:1), precipitated with an equal volume of isopropanol and then stored at ⫺70°C until needed for use. Ten micrograms of total RNA from each H. pylori strain was transferred onto a nylon membrane (Roche) using a slot-blot system (Hoefer Pharmacia Biotech Inc., San Francisco, CA). The membranes were prehybridized with a hybridization buffer (Roche) at 55°C for 3 h, and hybridized with RNA probes at 55°C for 18 h. Antisense 23S rRNA, antisense putative virB4 (HP0017) RNA, and sense putative virB4 RNA probes were labeled using a DIG RNA labeling kit (SP6/T7; Roche) using the above-described genes cloning in the pCRII-TOPO vector (Invitrogen) as appropriate templates. The H. pylori putative virB4 gene was cloned by PCR amplification from genomic DNA of NTUH-C1 and ligated to a TA vector. Detection was performed utilizing DIG Luminescent Detection Kit (Roche) according to the manufacturer’s instructions, and densitometry was analyzed with the NIH Image 1.62 program using 23s rRNA as internal standard. Only data from slots negative by sense virB4 RNA probe and positive by antisense rRNA probe were used. Microarray hybridization. To understand the RNA expression changes of virB4 knockout mutant in genomic scale, a microarray membrane containing 1,534 open reading frames (ORFs) from strain 26695 was used (24). Total RNAs of H. pylori wild type strain NTUH-C1 and virB4 knockout mutant M10-30 were extracted, reverse transcribed into cDNA, and labeled with biotin. Subsequently, Southern hybridization was performed as described previously (24). Each microarray membrane was hybridized with cDNA probe from wild type or virB4 mutant H. pylori strains and developed by a catalyzed reporter deposition method. Gene expression of all ORFs was measured by densitometry (24).

RESULTS Construction of the Mutant Library After ligation of DNA inserts into pILL570, 20 strains were randomly selected and the insert size (range between 3 and 5 kb) was confirmed by restriction enzyme digestion patterns (not shown). Two thousand and four hundred transposed plasmids were obtained after 2 conjugation steps and were assembled into 25 separate 96-well microtiter plates. Plasmids from 96 independent clones were pooled for each natural transformation of H. pylori. Each independent natural transformation procedure generated 30 to 200 kanamycin resistant colonies. All mutants were collected if a transformation procedure yielded less than 96 strains. If more than 96 mutant strains were found in particular transformation, only 96 mutant strains were randomly selected and retained. After 25 independent transformation in NTUH-C1, a total of 1500 H. pylori transposon insertion strains were obtained. Southern hybridization analysis of genomic DNA from H. pylori mutant strains using the kanamycin gene

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FIG. 1. Southern hybridization analysis of H. pylori mutant strains genomic DNA using the Kanamycin gene as the probe. Lane M: molecular size markers, 1-kb DNA ladder; Lanes 1– 4: Chromosomal DNA of the mutant strains M6-1–M6-4; Lane 5– 8: Chromosomal DNA of the mutant strains M7-1–M7-4; Lanes 9 –12: Chromosomal DNA of the mutant strains M8-1–M8-4; Lanes 13 and 14: Chromosomal DNA of the mutant strains M9-1 and M9-2; Lane 15: PCR Kanamycin cassette were used as positive control; Lane16: Chromosomal DNA of the wild-type strain were used as negative control.

(22, 32) as a probe suggested that distinct genes had been interrupted (Fig. 1). To estimate the redundancy of this mutant library, inverse PCR and sequences were performed. Of 8 mutants randomly selected from

pool 16 (less than 96 mutants in a single transformation), all insertions were different. Of 9 randomly selected mutants from pool 15 (more than 96 mutants in a single transformation), sequencing revealed 8 independent insertion loci. In contrast, 13 strains from pool that contained the redundant virB4-disrupted clones revealed only 7 independent insertions. According to the sequence results, we estimated there could be 23.3% redundant clones. Therefore, approximately 1150 independent loci would be covered by the 1500 mutants. Identification and Analysis of Mutant Strains with Decreased Natural Competence Of the 1500 H. pylori mutant strains, 20 revealed a markedly loss of natural transformation frequency using both donor DNAs (23S rRNA and CAT cassette) compared with the wild type strain (Table 2). The growth rate and morphology of these mutants were the same as the parent strain NTUH-C1. the MiniTnKm insertion site for each of the mutants was determined and compared with the GenBank databases as well as to a H. pylori genome database (HPDB). Seven genetic

TABLE 2

Frequency of DNA Transformation by Natural Transformation, and Capability of Different H. pylori Mutants Transformation frequencies of the mutants e Mutant number M17-84 M10-4 M10-30 M10-32 M10-75 M22-1 M22-70 M22-8 M22-19 M22-58 M22-27 M22-42 M22-74 M22-79 M12-21 M8-23 M1-38 M9-4 M16-3 M19-44 Wild type HP1326 Reknockout

MiniTn3-Km insertion site/homologue

Cm resistant cassette

Cla resistant cassette

pHel2 plasmid

HP0015 (23) a HP0017 (622)/virB4 HP0017 (622)/virB4 HP0017 (622)/virB4 HP0017 (622)/virB4 HP0037 (339) HP0037 (339) HP0038 (216)/comB1 HP0038 (216)/comB1 HP0038 (216)/comB1 HP0038 (286)/comB1 HP0038 (286)/comB1 HP0038 (300)/comB1 HP0040 (277) HP0042 (329)/comB3 HP0042 (457)/comB3 HP1089 (1431) HP1326 (345) HP1424 (405) HP1473 (426) — mutant d

0 b {⬍0.01%} 0 {⬍0.01%} 0 {⬍0.01%} 0 {⬍0.01%} 0 {⬍0.01%} 0 {⬍0.01%} 0 {⬍0.01%} 0 {⬍0.01%} 0 {⬍0.01%} 0 {⬍0.01%} 0 {⬍0.01%} 0 {⬍0.01%} 0 {⬍0.01%} 0 {⬍0.01%} 0 {⬍0.01%} 0 {⬍0.01%} 7 ⫻ 10 ⫺8 {0.7%} 3 ⫻ 10 ⫺6 {30%} 2 ⫻ 10 ⫺6 {20%} 0 {⬍0.01%} 1 ⫻ 10 ⫺5 {100%} 1 ⫻ 10 ⫺5 {100%}

0 {⬍0.01%} f 0 {⬍0.01%} 0 {⬍0.01%} 0 {⬍0.01%} 0 {⬍0.01%} 0 {⬍0.01%} 0 {⬍0.01%} 0 {⬍0.01%} 0 {⬍0.01%} 0 {⬍0.01%} 0 {⬍0.01%} 0 {⬍0.01%} 0 {⬍0.01%} 0 {⬍0.01%} 0 {⬍0.01%} 0 {⬍0.01%} 1 ⫻ 10 ⫺7 {1.25%} 2 ⫻ 10 ⫺6 {25%} 4 ⫻ 10 ⫺6 {50%} 0 {⬍0.01%} 8 ⫻ 10 ⫺6 {100%} ND

ND c 0 {⬍0.01%} 0 {⬍0.01%} ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 5 ⫻ 10 ⫺6 {100%} ND

a

Numbers in parentheses refer to the position of the MiniTnKm insertion in the ORFs. 0, No transformant was found during each transformation. ND: Not done. d HP0015, HP0017, HP1089, HP1424, and HP1473 reknockout mutants showed the same impairment in natural transformation frequency compared with original mutants from library. e Mean of 3 experiments. f Between brackets the relative transformation frequency of the mutants as a percentage of the wild type. b c

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loci, including the reported comB locus (13), of H. pylori were identified for the 20 mutants with impaired natural competence (Table 2). Mutant strains M8-23, M12-21, M22-1, M22-8, M22-19, M22-27, M22-42, M22-58, M22-70, M22-74, M22-79 contained the MiniTnKm insertion at the comB locus of H. pylori involved in DNA transformation competence (13). Mutant strains M10-4, M10-30, M10-32, and M10-75 contained the MiniTnKm insertion at HP0017, the gene homologous to Agrobacterium tumefaciens virB4, which was reported to be involved in conjugation (33). Sequence analysis of the other five mutant strains demonstrated that they contained insertions into function unknown genes (Table 2). All the later six loci were reknockout in a new NTUH-C1 and other competent isolates. HP1326 reknockout mutants showed no impairment in natural competence while all other 5 revealed the same decreased natural transformation frequency (Table 2). All the reknockout mutants showed a single MiniTnKm insertion by Southern hybridization analysis (not shown). While transformation of chromosome DNA depends on allelic exchange through recombination, transformation with plasmids most often occurs independent of recombination. To investigate whether HP0017 (putative virB4 homologue) also plays a role in transformation with an H. pylori plasmid, the mutant was also tested for transformation with the selfreplicating plasmid pHel2 (29). For HP0017 knockout mutant no major differences were observed between transformation with either chromosomal DNA or plasmids as the DNA source (Table 2). Mutation of HP0017 not only completely blocked natural transformation but also caused quantitative defect in electroporation (not shown). According to the full-length genomic sequence of H. pylori (6, 7), the immediate open-reading frame downstream of putative virB4 is HP0018. Knockout mutant of HP0018 was obtained by the same MiniTnKm shuttle mutagenesis. Determination of natural competence of HP0018 knockout mutant revealed that the frequency of natural transformation was the same as the wild-type strain. Genomic DNA from 7 competent and 6 noncompetent strains (Table 1) were all positive for the putative virB4 (HP0017) by PCR using the same primer pairs that were used for cloning (not shown). There are two other putative virB4 homologues in H. pylori genome sequence (6, 7). Knockout of HP0441 did not affect the transformation frequency in our study. Another locus, HP0459, was mutated and tested by Smeets et al. and showed no effect on natural transformation either (15). DNA sequences from HP0017 (putative virB4 homologue) of naturally-competent clinical isolates NTUH-C1 revealed a 96% similarity in nucleotide sequences compared with the published full-length genomic sequences of strain 26695. Comparisons of amino acid sequences of the putative virB4

FIG. 2. Slot blot result of HP0017 in competent and incompetent strains. (A) Using labeled antisense 23S rRNA as the probe. (B) Using labeled antisense HP0017 RNA as the probe. RNA expressions are measured by densitometry using ribosomal RNA as the internal control. Lanes 1– 6: RNA from the competent NTUH-C1–C6 H. pylori strains. Lanes 7–11: RNA from the incompetent H. pylori strains NTUH-N1–N5. Lane 12: RNA from the incompetent ATCC49503 H. pylori strain.

gene of the naturally competent clinical isolates NTUH-C1 and the noncompetent clinical isolate NTUH-N1 revealed a 95% similarity to the published genomic sequences of strain 26695. Slot blot analysis result revealed that HP0017 (putative virB4 homologue) RNA expression for noncompetent strains were lower than the competent strains (P ⫽ 0.011, nonparametric test) (Fig. 2). To identify genes of H. pylori that were expressed in virB4 knockout mutant, microarray hybridization signals were compared between RNA from wild-type NTUH-C1 and virB4 knockout mutant M10-30 after standardization with internal rRNA controls. No significant increase of expression level in any locus was noted by microarray (defined as increase 2-fold greater than standard deviation). In contrast, there were 19 ORFs had decreased (defined as decrease greater than 4-fold of standard deviation) expression levels in virB4 knockout mutant M10-30. These 19 ORFs were divided into two groups: (i) 2 ORFs that had no database match, and (ii) 17 ORFs homologous to previously reported sequences but not reported to be related to natural transformation (Table 3). DISCUSSION Shuttle mutagenesis provides a systemic approach to the study of H. pylori genes. Our H. pylori mutant library was consisted of 1500 independent strains. Excluding genes that are vital, our mutant library is estimated to cover over 97% of the H. pylori genome by Poisson distribution if all the 1500 mutants were all different. Sequences from 30 randomly selected mutants revealed that different loci had been inserted (not shown). However, pools that contained the virB4 homologue showed a much higher redundancy. Many comB and virB4 mutants had a same insertion site (Table 2). The exact reason is unknown. There could be clones that had growth advantage over others, or there could be preferential site for this transposon in a small

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H. pylori ORFs That Were Downregulated in virB4 Knockout Mutant Group

ORF number

Putative gene product (gene)

(i)

HP0248 HP0920

Conserved hypothetical protein Conserved hypothetical integral membrane protein

(ii)

HP0254 HP0275 HP0317 HP0570 HP0593 HP0597 HP0601 HP0655 HP0695 HP0751 HP0791 HP0824 HP1132 HP1164 HP1271

Outer membrane protein (omp8) ATP-dependent nuclease (addB) Outer membrane protein (omp9) Aminopeptidase a/i (pepA) Adenine specific DNA methyltransferase (mod) Penicillin-binding protein 1A (PBP-1A) Flagellin A (flaA) Protective surface antigen D15 Hydantoin utilization protein A (hyuA) Polar flagellin (flaG) Cadmium-transporting ATPase, P-type (cadA) Thioredoxin (trxA) ATP synthase F1, subunit ␤ (atpD) Thioredoxin reductase (trxB) NADH-ubiquinone oxidoreductase, NQO12 subunit (NQO12) NADH-ubiquinone oxidoreductase, NQO13 subunit (NQO13) Ribosomal protein L16 (rpl16)

HP1272 HP1312

region. However the transposon seemed to evenly distributed in the entire genome by our sequencing data. Therefore, if a preferential site in some small regions did exit, it would not cause too much bias in our mutant library. Although the exact loci covered by our mutant library is difficult to calculate, we found more mutants lost competence than other reports (13, 16). However, our study missed several reported loci too (15, 16). A previous study employed BlaM-transposon shuttle mutagenesis to construct H. pylori mutants that consisted of strains containing surface-related knockout loci (20). The method of previous study is very efficient but it may be responsible for the loss of genes that are not closely surface related. By their novel adaptation of BlaM transposon shuttle mutagenesis to H. pylori, a single comB gene locus was identified to be involved in DNA transformation competence for H. pylori (13). From our sample of 1500 different H. pylori mutant strains, 20 strains revealed a markedly loss of natural competence. Of these 20 mutants, 11 strains contained a MiniTnKm insertion at the different open-reading frames of the comB locus (Table 2). Although some comB knockout mutants might due to redundancy, comB was reconfirmed as a very important locus for natural transformation for H. pylori. We found six additional loci related to natural competence by our systemic approach preliminarily. Apart from the 11 comB insertion strains, other 4 mutants contained a MiniTnKm insertion at H. pylori

ORF0017, the gene homologous to Agrobacterium tumefaciens virB4, which was reported to be involved in conjugation (33). The complete loss of natural transformation ability (frequency ⬍10 ⫺9) of the putative virB4 gene knockout mutant strains were detected by repeated experiments with different donor DNAs (Table 2). We also tested HP0017 mutant for its ability to take up self-replicating plasmids and found that transformation is equally impaired. However, knockout mutant in HP0017 did not completely block electroporation. From these results we conclude that HP0017 is not involved in chromosomal integration but in earlier stage of the transformation process, e.g., uptake of DNA. In addition to HP0017, there are two other virB4 homologues in H. pylori genome (6, 7). HP0441 was proved not relate to natural transformation by our study. Another locus, HP0459, was mutated and tested by Smeets et al. and showed no effect on natural transformation either (15). Therefore, HP0441 and HP0459 are not true virB4 homologues in function. Slot blot analysis revealed significantly decreased RNA expression in some noncompetent strains. Therefore, virB4 might account for the phenotypic difference between some of these strains. According to the published full-length genomic sequence of H. pylori (6, 7), the open-reading frames flanked by putative virB4 are HP0016 and HP0018. The two open-reading frames were not matched to the current database and they were not related to conjugation or transformation variants (6, 7). Natural competence of HP0018 knockout mutants was not different from the wild-type strain. This result verified that putative virB4 (HP0017) is essential for natural competence of H. pylori, and the lost natural competence of the putative virB4 mutant was not due to polar effect. By reknockout, all the mutant alleles were moved to a fresh NTUH-C1 strain as well as other competent strains and the transformation ability were decreased in all except HP1326 mutant. We therefore concluded that the loss of competence in virB4 (HP0017) and HP0015, HP1089, HP1424, and HP1473 mutants were due to the identified insertion mutation, not due to changes elsewhere in the genome. Nineteen ORFs had decreased expression levels in virB4 knockout mutant by microarray. Six of them, HP0245, HP0317, HP0601, HP0655, HP0751, and HP0920, encoded cell envelope proteins. virB4 knockout may influence the conformation of cell membrane and affect the natural competence of H. pylori. Furthermore, a restriction-modification system associated loci (HP0593) was affect in virB4 knockout mutant. The variation of R-M system may play a role in natural competence of H. pylori (34). Although most of the functions of these ORFs and their roles in natural competence of H. pylori remained unknown, the result of down regulation by virB4 knockout could give a direction for future studies.

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In conclusion, we identify a virB4 homologue (HP0017) essential in the natural competence of H. pylori. The expression level of virB4 seems to decreased in some non-competent strains. The other two virB4 homologues in H. pylori genome, HP0441 and HP0459, do not play a role in natural transformation. ACKNOWLEDGMENT This work is supporting by grants from National Science Council (NSC-89-2315-B-002-042-M58), Taiwan.

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