Nucleotide sequence and characterization of cdrA, a cell division-related gene of Helicobacter pylori

Abstract

We identified cell division-related gene cdrA in Helicobacter pylori HPK5. The putative gene product, CdrA, is a 367-amino-acid polypeptide that exhibited a high level of homology to conserved hypothetical ATP-binding protein HP0066 of H. pylori 26695, except in the N-terminal region, and showed some similarity to the FtsK/SpoIIIE family proteins. We isolated a cdrA-disrupted mutant by allelic exchange mutagenesis. Because of the low transformation frequency, the possibility that a suppressing mutation would be found in the obtained cdrA mutant was discussed. A repressive role for CdrA on cell division was suggested by the observations that the wild-type strain formed filamentous cells in a high-salt level medium at early stationary phase, while a cdrA-disrupted mutant did not show such an abnormality. In addition, the wild-type strain adopted coccoid forms in the stationary phase, whereas the cdrA-disrupted mutant remained mostly as short rods. Furthermore, the cdrA-disrupted mutant regained the filamentation phenotype when the intact cdrA gene was introduced by allelic exchange. Taken together, these observations show that the cdrA gene plays an important role in the cell growth of H. pylori.

FIG. 1

Restriction maps of the urease gene cluster and the region downstream of this cluster of H. pylori HPK5. Plasmid pHPT177 carries a 12.8-kb SacI-BamHI fragment containing the urease operon (ureA to ureH) and cdrA; pS4-1 carries a 3,539-bp BamHI-EcoRI fragment containing cdrA; and pTA10 contains cdrA disrupted by a 3.13-kb xylE-kan fragment inserted at the Tth111I site. Arrows above the maps show the direction of transcription. Numbers represent the nucleotide positions starting from the right BamHI site. S, SacI; M, MulI; V, EcoRV; B, BamHI; G, BglII; E, EcoRI; T, Tth111I; H, HindIII.

FIG. 2

(A) Nucleotide sequence of the cdrA gene. The deduced amino acid sequence is indicated under the nucleotide sequence, and an asterisk marks the stop codon. Both −35 and −10 promoter consensus sequences and a putative Shine-Dalgarno (SD) sequence are indicated. An inverted repeat at the end of the ORF is indicated below the sequence (boldface arrows). The arrows above the sequence show the PCR primers (P1, P3, and P4). (B) Alignment of amino acid sequences of H. pylori CdrA [CdrA(Hp)] and hypothetical ATP-binding protein HP0066 (26). The C-terminal amino acid sequence of HP0066 is abbreviated. (C) Alignment of partial amino acid sequences of H. pylori CdrA [CdrA(Hp)], C. burnetii FtsK/SpoIIIE [SpoIIIE(Cb)] (22), B. subtilis SpoIIIE [SpoIIIE(Bs)] (29), H. influenzae FtsK [FtsK(Hi)] (11), and E. coli FtsK [FtsK(Ec)] (2). ATP binding motifs are boxed. In panels B and C, identical amino acids and conserved changes are indicated by asterisks and by dots, respectively; dashes indicate gaps included to optimize the alignment.

FIG. 3

PCR analysis of the cdrA regions of plasmid pS4-1 and H. pylori HPK5, CPY3401, CPY2052, and CPY1113. The PCR products obtained with the P3-P4 primer pair (lanes 1, 3, 5, 7, and 9) and with the P1-P3 primer pair (lanes 2, 4, 6, 8, and 10) are shown. The 100-bp ladder markers with 2 kb as the longest are indicated in the left lane.

FIG. 4

Growth curves and CFUs of HPK5 (A) and HPKT510 (B). Bacteria were grown in brucella medium at 37°C under microaerobic conditions, and the OD₅₉₀ values and CFUs were measured. ⧫, HPK5 in 0.5% NaCl; ◊, HPK5 in 1% NaCl; ▴, HPKT510 in 0.5% NaCl; ▵, HPKT510 in 1% NaCl.

FIG. 5

Morphology of HPK5 and HPKT510 from the cultures represented in Fig. 4 at 44 h (A) and 88 h (B) as determined by DAPI staining. Arrows indicate coccoid forms, and arrowheads indicate short rods. For both panels, segments are as follows: a, HPK5 in 0.5% NaCl; b, HPKT510 in 0.5% NaCl; c, HPK5 in 1% NaCl; d, HPKT510 in 1% NaCl. Bars, 10 μm.