Article Figures & Data
Figures
- FIG. 1.
Construction of the galE mutant and complementation of the galE mutation. (A) Strategy for construction of the galE mutant by allelic exchange. The galE gene interrupted by a 2.2-kb ermF-ermAM cassette was introduced into P. gingivalis by electroporation. ermF-ermAM conferred Ermr in P. gingivalis and E. coli. (B) Construction of the pGQG vector used for complementation of the galE strain. A 2.7-kb tetQ fragment obtained from pKD375 was ligated into the pUC19 vector, resulting in pUC19Q. A 1.3-kb fragment containing the complete sequence of the galE gene and its downstream region was coamplified from the chromosomal DNA of P. gingivalis and cloned between the BamHI and XbaI sites of pUC19Q, resulting in pQG. Finally, a 0.8-kb upstream fragment of the galE gene was amplified from the chromosomal DNA of P. gingivalis and cloned between the KpnI and SmaI sites of pQG, resulting in pGQG. (C) The 4.8-kb fragment (upstream-tetQ-galE-downstream) was retrieved from pGQG and electrotransformed into the P. gingivalis galE strain. A reciprocal recombination event occurred between the areas of homology, which are represented by two slashed bars (0.8-kb upstream and 0.3-kb downstream regions of the galE gene) and by the latter half of the white bar (galE gene) on the chromosome of the galE strain. The tetQ gene conferred Tetr in P. gingivalis.
- FIG.2.
Growth assays and quantitation of intracellular polysaccharide in the absence or presence of galactose. (A) P. gingivalis wild-type and mutant strains were grown in BHI-HM broth, and OD660 absorbance was measured at different time points. Data shown are representative of three independent experiments. The results are expressed as the mean ± standard deviation (SD) from a triplicate assay. Similar results were obtained in three independent experiments. (B) The strains were also grown on BHI-HM agar plates supplemented with 1% galactose. (C) P. gingivalis wild-type and galE strains were grown in BHI-HM broth supplemented with or without galactose (0%, 0.01%, 0.05%, and 0.1%). Quantities of intracellular polysaccharides, which were extracted from every 50 mg of lyophilized cells, were determined by a phenol-sulfuric acid colorimetric method with glucose as the control. Total intracellular carbohydrates are expressed as mg per 50 mg of dry weight. The results are expressed as the mean ± SD of a triplicate assay. Similar results were obtained in two independent experiments.
- FIG. 3.
LPS profiles of P. gingivalis wild-type and mutant strains. LPS samples extracted from the P. gingivalis wild-type and mutant strains were analyzed by sodium dodecyl sulfate-PAGE and silver staining. Each lane contains 5 μg of LPS. (A) Lanes: 1, wild type; 2, the htpG strain; 3, the wecA strain; 4, the wbaP strain; 5, the galE strain; and 6, the wzt strain. M: molecular mass marker. The areas shown with ovals represent a minor shift of the O-antigen ladder (*1) in the wecA strain and a decrease of the high molecular band of O antigen (*2) and an increase of the low molecular band of O antigen (*3) in the galE strain. (B) The band densities of the O-antigen ladders after silver staining in the wild-type, galE, and galE-c strains were evaluated using densitometric scanning. The y axis of the histogram indicates the relative position from 0 point, whose position, denoted in the inset, was determined to be approximately 16 kDa.
- FIG. 4.
Biofilm formation by P. gingivalis wild-type and mutant strains. Biofilm formation was examined after 34 (A) and 48 (B) hours of culturing in BHI-HM broth. OD492/620 absorbance was measured to determine the biofilm mass. Data shown are representative of three independent assays. The results are expressed as the mean ± SD of a triplicate assay. Similar results were obtained in three independent experiments.
- FIG. 5.
SEM images of P. gingivalis wild-type and galE strains. P. gingivalis wild-type (A) and galE (B) strains were grown on plastic sheets for 12 h, after which attached cells were investigated using SEM. Scale bars are shown at the lower right of each electron micrograph.
Tables
- TABLE 1.
Bacterial strains and plasmids used in this study
Bacterial strain/plasmid Relevant phenotype, description, and/or selective markera Source or reference Strains Escherichia coli DH5α F′ φ80dlacZ ΔM15 Δ(lacZYA · argF)U169 deoR recA1 endA1 hsdR17(rK− mK+) phoA supE44 λ−thi-1 gyrA96 relA1 Takara Bio, Otsu, Japan Porphyromonas gingivalis ATCC 33277 Wild type American Type Culture Collection ATCC 33277 htpG strain Ermr This study ATCC 33277 wecA strain Ermr This study ATCC 33277 wbaP strain Ermr This study ATCC 33277 galE strain Ermr This study ATCC 33277 galE-c strain Tetr Erms This study ATCC 33277 wzt strain Ermr This study Plasmids pUC19 Cloning vector; Ampr Takara Bio, Otsu, Japan pBR322 Cloning vector; Ampr Takara Bio, Otsu, Japan pHS19 Contains the ermF-ermAM cassette between EcoRI and BamHI sites of pBR322 without bla; Ermr 10 pBR322-erm Contains the ermF-ermAM cassette between EcoRI and BamHI sites of pBR322; Ampr Ermr This study pRN2 Contains PG0045 between KpnI and AvaI sites of pUC19; Ampr This study pRN2-erm Contains the ermF-ermAM cassette in PmaCI site within PG0045 of pRN2; Ampr Ermr This study pRN3 Contains PG0106 between XbaI and BamHI sites of pUC19; Ampr This study pRN3-erm Contains the ermF-ermAM cassette in BglII site within PG0106 of pRN3; Ampr Ermr This study pRN4 Contains PG1964 between EcoRI and XbaI sites of pUC19; Ampr This study pRN4-erm Contains the ermF-ermAM cassette in Bsp1407I site within PG1964 of pRN4; Ampr Ermr This study pRN6 Contains PG0347 between EcoRI and BamHI sites of pUC19; Ampr This study pRN6-erm Contains the ermF-ermAM cassette in BseRI site within PG0347 of pRN6; Ampr Ermr This study pRN7 Contains PG1225 between XbaI and BamHI sites of pUC19; Ampr This study pRN7-erm Contains the ermF-ermAM cassette in ApaI site within PG1225 of pRN7; Ampr Ermr This study pKD375 Contains tetQ cassette in pUC19; Ampr Tetr 25 pUC19Q Contains tetQ cassette between SmaI and BamHI sites in pUC19; Ampr Tetr This study pQG Contains an entire region of galE and the 0.3-kb downstream region between BamHI and XbaI sites of pUC19Q; Ampr Tetr This study pGQG Contains a region of 0.8 kb upstream of galE between KpnI and SmaI sites of pQG; Ampr Tetr This study ↵ a Ampr, ampicillin resistant; Ermr, erythromycin resistant; Erms, erythromycin sensitive; Tetr, tetracycline resistant.
- TABLE 2.
Primer pairs used for gene cloning
Target region Name Sequence (5′-3′)a htpG htpGp2-f GGGGTACCTGAGGAGAATAGCGAGTT htpGp2-r2 CCCCCGGGTTGGGTTTTCAGCACAAC wecA PG0106-D GCTCTAGATGCGACCTCAATCCTTC PG0106-E CGGGATCCCAACCAGTGTTTGTCTCT wbaP PG1964-A GGAATTCGGGATACTACAACAAACC PG1964-C GCTCTAGAAGGCTCATATTCTCGTAG galE PG0347-A GGAATTCGGATCCCATACGACTGTG PG0347-C GCTCTAGAACGACTCCAAAGCTTTCC wzt PG1225-A GCTCTAGAGATCGGACTGCTTCTGGT PG1225-B CGGGATCCTTTCGACGGTCTGCCTTTC tetQ SmaI-tetQ-F TCCCCCGGGCGTTCCATTGGCCCTCAAAC BamHI-tetQ-R CGGGATCCCTCCTGCCATTCATAGAGGC galE and its downstream BamHI-down-F CGGGATCCGAAACCGAAATGAAACAAAAG XbaI-down-R GCTCTAGAAGGACACCGCGCAGCTGGAT Upstream of galE KpnI-up-F GGGGTACCCTCTCCAATGCCAGACTTTG SmaI-up-R TCCCCCGGGCGGTTTCTTATTTCGTAGC ↵ a Restriction enzyme sites incorporated into oligonucleotides for subcloning are italicized.
- TABLE 3.
MICs for P. gingivalis wild-type, galE, and galE-c strains
Antibiotic MIC (μg/ml)a for: 33277 (wild type) galE strain galE-c strain Benzylpenicillin 0.011 ± 0.0023 0.0047 ± 0.0012b 0.0073 ± 0.0012 Oxacillin 0.10 ± 0.020 0.053 ± 0.010b 0.084 ± 0.017 Ampicillin <0.016 <0.016 <0.016 Cefotaxime 0.0067 ± 0.0012 0.002 ± 0.00b 0.006 ± 0.002c Ceftriaxone <0.002 <0.002 <0.002 Imipenem 0.084 ± 0.035 0.024 ± 0.0080b 0.043 ± 0.018 Tetracycline 0.042 ± 0.0087 0.037 ± 0.0087 1.7 ± 0.29d Erythromycin <0.016 >256e <0.016 Clindamycin <0.016 >256e <0.016 Kanamycin >256 >256 >256 Tobramycin >256 >256 >256 Vancomycin 12 ± 4 1.8 ± 0.29b 4 ± 0c a Values are shown as means ± standard deviations.
↵ b The galE strain had significant difference in sensitivity to the indicated antibiotic compared to the wild-type 33277.
↵ c The galE-c strain had significant difference in sensitivity to the indicated antibiotic compared to the ΔgalE strain.
↵ d Resistance was dependent on insertion of tetQ.
↵ e Resistance was dependent on insertion of ermF.
