Article Figures & Data
Figures
- FIG. 1.
DNA-MTase activity of 1 U Dam (New England Biolabs) or 1 μM Cj1461-His6 was measured using in vitro reaction mixtures containing 100 μM SAM, with a substrate of 500 ng of chromosomal DNA (from E. coli JM110 dam or C. jejuni Δcj1461 cells, respectively). Both DAM and Cj1461 showed MTase activity.
- FIG. 2.
Organization of cj1461, its surrounding genes, and complementation constructs in the cj0046 pseudogene. Gene designations are based on the C. jejuni NCTC 11168 genome (32); however, the organization of these genes is similar in C. jejuni 81-176. Arrows represent open reading frames and the direction of transcription. Arrows representing genes are not to scale for clarity of the figure. The solid arrow represents the cj1461 gene, and the dashed arrow indicates the cj0046 pseudogene. Triangles indicate the points of insertion of a kanamycin resistance transposon (aphA) in cj1461 and complementation constructs in cj0046. DNA included in the two complementation constructs is indicated by thick black lines below the gene map. Bent arrows represent promoters. The shaded box indicates the location of a putative σ54 enhancer for flgI. flgI encodes a flagellar structural component, the P ring. cj1459, cj1460, and cj1463 encode hypothetical proteins.
- FIG. 3.
Relative motility (percent of the value for the wild type) at 37°C of the complemented and uncomplemented Δcj1461 mutant cells. The cj1461 mutant showed impaired motility (*, P < 0.01), and complementation with cj1461 significantly restored motility (*, P < 0.01). In contrast, complementation with flgI and cj1463 did not restore motility. Data are the averages from at least three independent experiments. Bars represent standard deviation.
- FIG. 4.
Transmission electron micrographs reveal that the Δcj1461 mutant has normal flagellar length (A), but a large number of cells show altered flagellar structure (B). Fifty Campylobacter cells from two independent samples of each strain were analyzed. wt, wild type.
- FIG. 5.
Adherence to (A) and invasion of (B) INT-407 cells by wild-type (WT), Δcj1461 mutant, and complemented mutant strains. Mutation of cj1461 resulted in sevenfold increased adherence compared to the wild type (A). In contrast, the Δcj1461 mutant was 50-fold less invasive than the wild type (B). For both adherence and invasion, complementation with cj1461 restored wild-type phenotypes. Data are the averages from two independent experiments, each performed in triplicate. Bars represent standard deviations.
Tables
- TABLE 1.
Strains and plasmids used in this study
Strain or plasmid Description Resistancea Source Strains C. jejuni 81-176 Wild type 21 C. jejuni 81-176 Δcj1461 cj1461 mutant of 81-176 Kan This study C. jejuni 81-176 Δcj1461+cj1461 Δcj1461 mutant complemented with cj1461 Kan Cm This study C. jejuni 81-176 Δcj1461+flgI-cj1463 Δcj1461 mutant complemented with flgI-cj1463 Kan Cm This study E. coli JM110 dam strain New England Biolabs Plasmids pRY111 C. jejuni-E. coli shuttle vector Cm 36 pBluescript SKII(−) E. coli cloning vector Amp Stratagene pBS-cj1461 cj1461 gene in pBluescript SKII(−) Amp This study pBS-cj1461K pBS-cj1461 with transposon insertion in cj1461 Amp Kan This study pBS-cj0046 Pseudogene cj0046 in pBluescript SKII(−) Amp This study pSAT1000 pBS-cj0046 with cat insertion Amp Cm This study pSAT1001 pSAT1000 containing cj1461 gene Amp Cm This study ↵ a Amp, ampicillin; Kan, kanamycin; Cm, chloramphenicol.
- TABLE 2.
PCR primers used in this study
Primer Sequence (5′→3′)a Restriction site cj1461-F1 TTG GAATTC AAACAAACTCGTAAAAGCAC EcoRI cj1461-R1 CCA GAATTC TTAAGAAGAGTAAAAACTTA EcoRI If313-F CACTACCCAATTGAAAATCTAAG NAb If314-R GTGAGTTAATGCCATCATAAC NA If320-IV-F GAG ATGCAT GATCTTATAGCACAAGCTAC NsiI If319-IV-R GAG ATGCAT GAGCTGTAGCTGTAATAAAC NsiI If317-F TGCTCGGCGGTGTTCCTTTCCAAG NA If318-R TGCGCCCTTTAGTTCCTAAAGGGT NA JL15 TGAGCTCAATGATGAATTTATAAGCGTTAAAG SacI JL16 GAGCTC TTAACCCCTTTCTTTTAAAAAATC SacI
Supplemental material
Files in this Data Supplement:
- Supplemental file 1 -
Fig. S1, phylogenetic analysis of representative DNA MTases; Fig. S2, digestion of 81-176 chromosomal DNA using restriction
endonucleases recognizing GATC sequences.
PDF file, 198K.
- Supplemental file 1 -
Fig. S1, phylogenetic analysis of representative DNA MTases; Fig. S2, digestion of 81-176 chromosomal DNA using restriction
endonucleases recognizing GATC sequences.
- Top
- Article
- ABSTRACT
- Cj1461 is a DNA MTase but is not a Dam ortholog.
- Generation of cj1461 mutant and complemented strains.
- Mutation of cj1461 causes a motility defect.
- Mutation of cj1461 causes a defect in flagellar appearance.
- cj1461 mutation causes hyperadherence to but decreased invasion of INT-407 cells.
- Conclusion.
- ACKNOWLEDGMENTS
- FOOTNOTES
- REFERENCES
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