Article Figures & Data
Figures
- FIG. 1.
Identification of novel type IV-related genes of the comB gene cluster. (A) Schematic representation of the A. tumefaciens virB locus with the number and location of putative TM domains in the VirB2 and VirB3 proteins, as predicted by ConPred II. (B) Overview of the complete H. pylori comB gene cluster and depiction of the comB region hp0014 to hp0018, with a comparison of the ComB2 (HP0015) and ComB3 (HP0016) TM domains, as predicted by ConPred II. Amino acid (aa) similarities and identities of the VirB and ComB homologues are shown between the corresponding ORFs.
- FIG. 2.
Construction of precise deletions of comB2 (hp0015) or comB3 (hp0016) and genetic complementation. (A) Precise deletions of the hp0015, hp0016, or hp0015 to hp0017 genes were constructed by replacing the corresponding gene sequences with a chloramphenicol acetyltransferase (cat) or kanamycin (aphA-3) resistance gene cassette. (B) Complementation of the corresponding genes was obtained by cloning the comB4, comB3-comB4, or comB2 to comB4 gene(s) in the shuttle vector pHel2 behind the H. pylori flaA promoter (P) of plasmid pDH80 (pAK20 to pAK22) and transfer of the shuttle plasmid into P1Δhp0015-hp0017::aphA-3. The competence for genetic transformation of the corresponding strains is indicated (+ or −). For quantitative data of transformation experiments, see Table 3.
- FIG. 3.
RT-PCR analysis of comB mRNA expression in H. pylori 26695 wt and ΔcomB mutant strains. Lanes 1 through 9, H. pylori 26695 wt; lanes 10 through 12, 26695Δhp0015. RT-PCR was performed with H. pylori 26695 wt cDNA (RT) (see Materials and Methods) using primer pairs AK05/CH81 (lane 3), AK05/CH77 (lane 6), and CH82/CH83 (lane 9) and with H. pylori 26695Δhp0015 cDNA using primer pair CH78/CH79 (lane 12). As positive controls, analogous PCRs with chromosomal DNA (C) of H. pylori 26695 wt (lanes 1, 4, and 7) and H. pylori 26695Δhp0015 (lane 10) were performed. No amplification products were detected in the negative controls (lanes 2, 5, 8, and 11), where RNA only, without cDNA synthesis (R), served as a template for PCR. Lane M, DNA size marker.
- FIG. 4.
Essential role of comB6 for transformation competence as revealed by deletion and complementation studies. (A) Precise deletions of comB6 (hp0037) or the region comprising comB6 to comB10 (hp0037 to hp0042) were constructed by replacing the corresponding gene sequences with a cat or a kanamycin (aphA-3) resistance gene cassette, respectively. (B) Complementation of the corresponding genes was obtained by cloning the comB6 to comB10 or the comB7 to comB10 genes in the shuttle vector pHel2 behind the H. pylori flaA promoter (plasmid pDH80) to result in pAK24 or pDHO46 (25), respectively. The shuttle plasmids were transferred into strain P228 (P1ΔcomB6-comB10::aphA-3). The competence for genetic transformation of the corresponding strains is indicated at right (+ or −). For quantitative data of transformation experiments, see Table 3.
- FIG. 5.
Topology analyses of the ComB6 subunit of the H. pylori transformation competence-mediating type IV transport system. (A) The computer programs listed at left predicted the transmembrane segments shown as solid rectangles with residue numbers relative to the N terminus. Solid rectangles identify transmembrane segments supported by results of reporter protein fusion studies; shaded rectangles are not supported by experimental data. *, amino acid (aa) positions of the PhoA or GFP fusions represented in panel B. Kyte-Doolittle hydrophobicity blot of HP0037: SP, signal peptide; TMS1 to TMS5, transmembrane segments 1 to 5; CP, cytoplasmic location; PP, periplasmic location. (B) Reporter protein activity levels of E. coli CC118 (phoA) or XL1-Blue (gfp) expressing six defined fragments of comB6 translationally fused to phoA (above the x axis) or gfp (below). Numbers at the bottom refer to the fusion site of the corresponding ComB fusion protein tested. Reporter protein activities represent the average of at least three experiments, with standard deviations indicated.
- FIG. 6.
Proposed membrane topology model of ComB6. The model is derived from predictions by four widely used computer algorithms and the results of PhoA and GFP reporter protein fusion studies. The experimental data provide support for the five transmembrane segments (TMS) shown. The N-terminal signal sequence is shown as an additional TMS (shaded). A long C-terminal segment is located in the cytoplasm. The filled arrowhead represents a postulated signal sequence cleavage site; open arrowheads mark GFP or PhoA fusion sites. CM, cytoplasmic membrane.
Tables
- TABLE 1.
Bacterial strains and plasmids used in this study
Strain or plasmid Genotype or characteristicsa Reference or source H. pylori strains P1 H. pylori wild type, naturally competent 26 P12 H. pylori wild type, naturally competent 25 J99 Wild-type, genome-sequenced strain 2 26695 Wild-type, genome-sequenced strain 52 P149 Wild-type, Mongolian gerbil-adapted strain 29 P224 P12; deletion of hp0015 (comB2), cat insertion This study P225 P12; deletion of hp0016 (comB3), cat insertion This study P226 P1; deletion of hp0015-hp0017, aphA-3 insertion This study P227 P1; deletion of hp0037 (comB6), cat insertion This study P228 P1; deletion of region spanning hp0037 (comB6)-hp0042 (comB10) This study P229 P1Δhp0015-hp0017 complemented with pHel2 plasmid carrying comB2-comB4 (pAK20) This study P230 P1Δhp0015-hp0017 complemented with pHel2 plasmid carrying comB3-comB4 (pAK21) This study P231 P1Δhp0015-hp0017 complemented with pHel2 plasmid carrying comB4 (pAK22) This study P232 P1Δhp0037-hp0042 complemented with pHel2 plasmid carrying comB6-comB10 (pAK24) This study P233 P1Δhp0037-hp0042 complemented with pHel2 plasmid carrying comB7-comB10 (pDHO46) This study P234 P149[hp1421::TnHK9] Tn insertion at codon 20 29 E. coli strains CC118 Δ(ara-leu)7697 ΔlacX74 ΔphoA20 galE galK thi rpsE rpoB argE(Am) recA1 31 β2155 thrB1004 pro thi strA hsdS lacZΔM15 (F′ lacZΔM15 lacIqtraD36 proA+ proB+) ΔdapA::erm (Ermr) pir::RP4 [::kan (Kmr) from SM10] 17 XL1-Blue recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F′ proAB lacIqZΔM15] Tn10 (Tetr) Stratagene Plasmids pRB67 pMin2 plasmid carrying phoA without start codon This study pAK03 pBA with region hp0014-hp0018, primers AK04-AK05 This study pAK04 pBA with hp0015::cat and flanking regions This study pAK05 pBA with hp0016::cat and flanking regions This study pAK07 pBA with region hp0036-hp0038, AK02-AK03 This study pAK08 pBA with hp0037::cat and flanking regions This study pAK09a pRB67 with hp0037 fragment AK12-AK13 (34 aa) This study pAK09b pRB67 with hp0037 fragment AK12-AK14 (62 aa) This study pAK09c pRB67 with hp0037 fragment AK12-AK15 (115 aa) This study pAK09d pRB67 with hp0037 fragment AK12-AK16 (198 aa) This study pAK09e pRB67 with hp0037 fragment AK12-AK23 (236 aa) This study pAK09f pRB67 with hp0037 fragment AK12-AK18 (296 aa) This study pAK19 pBluescriptIISK comB2-comB4 deletion plasmid, aphA-3 This study pAK20 pDH80, comB2-comB4 This study pAK21 pDH80, comB3-comB4 This study pAK22 pDH80, comB4 This study pAK23 pMin1, comB6-comB10 deletion plasmid, aphA-3 This study pAK24 pDH80, comB6-comB10 This study pDHO36 pMin1, comB7-comB10 deletion plasmid, aphA-3 17 pDHO46 pDH80, comB7-comB10 25 ↵ a aa, amino acids.
- TABLE 2.
Oligonucleotide sequences used in this study
Use Primer Sequence cDNA synthesis and PCR CH80 5′-TCAAAAGTGATAGAACTAGCG-3′ CH82 5′-GATTAGCGTGTTCTTTGG-3′ PCR AK02 5′-CGGGCTCGAGATCCACTCATTAGCGGAG-3′ AK03 5′-GAAGATCTCGCCCCAATGAGCGAACG-3′ AK04 5′-GCCGCTCGAGGGGGTGTGTAACAATTTC-3′ AK05 5′-GGGGATCCCCCATTAATGTATTCCGC-3′ AK06 5′-GCCGCTCGAGCAAAACCCTTCTGTTTAA-3′ AK07 5′-GCGGATCCAGCGAATTGGTTTATGGG-3′ AK08 5′-GCCGCTCGAGTAAAAAATTCCCATAAAC-3′ AK09 5′-GCGGATCCGTTATAGAGCGTAGAATG-3′ AK12 5′-ACCGCTCGAGCTTTAAGAAGGAGATATACATATGAAAAATGACGCTTATG-3′ AK13 5′-GCGGATCCCGCATGCAAAGTGTAGAG-3′ AK14 5′-GCGGATCCAGCGCTGAAGAAATTCTG-3′ AK15 5′-GCGGATCCAGAAAAGTTACTCAAGCT-3′ AK16 5′-GCGGATCCTAGGCATATAACCACAAC-3′ AK18 5′-GCGGATCCTGGGGTCGTGATGTATTG-3′ AK19 5′-GCCGCTCGAGCAATACTTCAACGGACTT-3′ AK20 5′-GCGGATCCAGGAATTTAATGAGAATT-3′ AK23 5′-GCGGATCCTAAGTCTTGTTTTTCTTG-3′ AK37 5′-CCGCTCGAGTATCGCTTTAGGCTATGCTA-3′ AK38 5′-GCATCGATCAAAACCCTTCTGTTTAATT-3′ AK39 5′-GGAATTCGCACTCCTATTCAGATGGCT-3′ AK40 5′-ACGAGCTCCGGCTCTTTACCATCTTCAA-3′ AK49 5′-ATGCCATATGTCCGCTCATTTTTTAAA-3′ AK50 5′-ATGCCTCGAGAGCCATCTGAATAGGAGTGC-3′ AK51 5′-ATGCCATATGATTATCCTGTCAGCGAG-3′ AK52 5′-CGGCCATATGTTAGAAAAGCTTTTAAG-3′ AK57 5′-CAGCATATGTAAAGTCCGTTGAAGTATTG-3′ AK58 5′-TCTTGCATATGAGCGTCTA-3′ AK59 5′-GGAATTCGAAGAACATCATAAGCGTTT-3′ AK65 5′-CAGAATTCCAATACTTCAACGGACTTT-3′ CH77 5′-CCAAAGAACACGCTAATC-3′ CH78 5′-ATGGAGAAAAAAATCACTGG-3′ CH79 5′-TTCTTCACGCTCGCTG-3′ CH81 5′-TAGTAGGCATGTGCGTTTC-3′ CH83 5′-GCAAGAGTTTTTAGGTTTTG-3′ - TABLE 3.
Transformation frequencies of H. pylori wt and mutant strains
H. pylori strain and mutation Transformation frequency with: Chromosomal H. pylori DNA (str)a Plasmid DNA (pDH29) (erm)b P1 (1.3 ± 0.5) × 10−5 (1.1 ± 0.3) × 10−5 P12 (1.3 ± 0.6) × 10−6 (2.0 ± 0.9) × 10−5 P12ΔcomB2 <10−9 <10−9 P12ΔcomB3 <10−9 <10−9 P1ΔcomB2-comB4 <10−9 <10−9 P1ΔcomB2-comB4[pAK20] (5.2 ± 1.1) × 10−6 (3.4 ± 1.2) × 10−6 P1ΔcomB2-comB4[pAK21] <10−9 <10−9 P1ΔcomB2-comB4[pAK22] <10−9 <10−9 P1ΔcomB6 <10−9 <10−9 P1ΔcomB6-comB10 <10−9 <10−9 P1ΔcomB6-comB10[pAK24] (0.4 ± 0.1) × 10−7 (1.9 ± 0.7) × 10−7 P1ΔcomB6-comB10[pDHO46] <10−9 <10−9 P149 ND (5.4 ± 1.9) × 10−4 P149[hp1421::TnHK9] ND (3.8 ± 1.4) × 10−4 ↵ a Chromosomal DNA from strain P12 (str) or P1 (str) was used for transformation. str, streptomycin resistance gene cassette. ND, not done.
↵ b Plasmid pDH29 was used for transformation. It consists of pBluescript plasmid with the H. pylori recA gene interrupted by an erm (erythromycin resistance) cassette (25).
