Article Figures & Data
Figures
- Fig. 1.
Schematic representation of the fliDSTregions of E. coli and P. aeruginosa strains PAK and PAO1. Locations and orientations of various primers used in PCR are shown. The deletion of ca. 300 bp in P. aeruginosa PAO1 is represented by the open triangle. The unique SmaI site used to insert the Gmr cassette in the PAO1 fliD gene and BamHI cleavage sites are marked.
- Fig. 2.
Alignment of deduced amino acid sequences of the PAK (upper row) and PAO1 (lower row) fliD genes, using the GAP program of the Genetics and Computer Group package.
- Fig. 3.
Western blot analysis of different P. aeruginosa strains using specific polyclonal antibodies against A- and B-type FliD and a-type FliC proteins. Whole cell extracts ofP. aeruginosa strains in panel A were probed with the PAK (A-type) FliD antibodies. The identical samples in panels B and C were probed with the PAO1 (B-type) FliD antibodies and a-type FliC antibodies, respectively.
- Fig. 4.
Soft L-agar (0.3%) plates showing the motility phenotypes of PAK and its derivatives (A) and of PAO1 and its derivatives (B). (A) 1, wild-type PAK; 2, A-type fliD mutant PAK-D; 3, PAK-D containing vector control pET15BVP; 4, PAK-D complemented with pET15BVPa (A-type fliD); 5, PAK-D containing pET15BVPDb (B-type fliD). (B) 1, wild-type PAO1; 2, B-type fliD mutant PAO-D; 3, PAO-D containing vector control pET15BVP; 4, PAO-D complemented with pET15BVPDa (A-type fliD); 5, PAK-D containing pET15BVPDb (B-type fliD).
- Fig. 5.
Phylogenetic analysis of the FliD sequences. The tree was constructed using P. aeruginosa (Pa) sequences from this work and additional sequences retrieved from GenBank: Bsu,Bacillus subtilis (U56901 ); Hly1 and Hly2,Helicobacter pylori (accession no. U82981 ) and H. pylori J99 (accession no. AE001500 ); Eco, E. coli K-12 MG1655 (accession no. AE000285 ); Tpa, Treponema pallidum(accession no. AE001257 ); Bbu, Borrelia burgdorferi(accession no. U66699 ); PaPAK, P. aeruginosa strain PAK (accession no. L81176 ); Vpa, Vibrio parahaemolyticus(accession no. U52957 ); and Sty, Salmonella enterica serovar Typhimurium (accession no. M33541 ). Bootstrap values represent 100 replications as indicated at branch points.
Tables
- Table 1.
Bacterial strains and plasmids used
Strain or plasmid Relevant characteristics Source or reference Escherichia coli DH5α hsdR recA lacZYA φ80 lacZ ΔM15 GIBCO-BRL BL21(DE3) F−ompT hsdSB(rB− mB−) gal dcm (DE3) Novagen Pseudomonas aeruginosa PAK Laboratory strain, motile D. Bradley PAO1 Laboratory strain, motile M. Vasil PA103 Laboratory strain, nonmotile 17 CS2 Clinical strain, motile This study CS29 Cystic fibrosis strain, motile, nonmucoid This study CS32 Cystic fibrosis strain, motile, nonmucoid This study 1244 Clinical strain MDR Clinical strain, motile This study PAK-D PAKfliD::Gmr 5 PAO-D PAO1 fliD::Gmr This study Plasmids pLysS Plasmid containing the T7 lysozyme gene Novagen pBluescriptKS (+) E. colivector, Ampr Stratagene pET15BVP Expression vector, T7 promoter, His tag coding sequence, Ampr, pBR322 origin, contains broad-host-range origin of replication oriV 4 pET15BVPDb PAO1fliD gene inserted as a PCR product into theNdeI/BamHI sites of pET15BVP This study pET15BVPDa PAK fliD gene inserted as a PCR product into the NdeI/BamHI sites of pET15BVP This study pBS55XBAM 2-kb PCR product containing the complete PAO1 fliD gene and flanking sequence cloned into the EcoRI/BamHI sites of pBluescript This study pUC7G pUC7 plasmid containing a Gmr cassette excisable with restriction enzymes PstI, SalI,BamHI, and EcoRI S. Lory pBS55XBAMG Gmr cassette inserted into the uniqueSmaI site of pBS55XBAM; plasmid used for fliDknockout in PAO1 This study
