Fig 1.
Schematic representation of peptidoglycan and LPS biosynthesis pathway in P. aeruginosa.
The targeted genes are colored in red.
Table 1.
Sequences of primers used for amplification of flanking regions of target genes, pEX18Ap vector backbone and Gm-gfp cassette.
Fig 2.
General scheme of construction of suicide vectors (A) and replacement of target genes (B).
A: For construction of suicide vectors for replacement of target genes, regions of approximately 400 bp flanking the target up- and downstream, respectively, were amplified using primers with integrated restriction sites allowing directed insertion into mobilisable vector pEX18Ap. PCR-products of upstream regions (1) were cleaved with EcoRI and BglII and downstream regions (2) with HindIII and BglII (only in case of PA1793 BamHI instead of BglII). The gentamycin-GFP cassette (3) of pPS858 was excised using BamHI and vector pEX18Ap was linearised with EcoRI and HindIII. Cleaved fragments 1, 2, and 3 and linearised vector pEX18Ap (4) were then combined in ligation mixture. The gentamycin-GFP cassette ligation to up and downstream fragments aided by the compatible cohesive ends between BamHI and BglII. Then E. coli donor strain ST18 was transformed with this mixture. Afterwards the suicide constructs were transferred from E. coli ST18 into P.aeruginosa PAO1 by conjugation. B: After conjugational transfer of suicide constructs recombinational replacement of the target genes and loss of vector backbone was forced by sucrose counter selection and gentamycin selection, respectively. Total DNA was isolated from knock out mutants and correct gene replacement was confirmed by sequencing extended PCR-products of the flanking regions.
Fig 3.
Growth characteristics of P. aeruginosa PAO1 wild-type and cell wall targets mutants.
(A) Growth curves in M9 minimal medium with glucose as the sole carbon source. (B) Growth curves in M9 minimal medium with glycerol as sole carbon source. The standard errors of the mean for three independent experiments are shown.
Fig 4.
Morphological and colonial architectures changes in the knockout strains.
(A) Colony morphology on LB (higher panel) and on M9 supplemented with glucose (Lower panel). (B) FESEM analysis of P. aeruginosa wild-type and cell wall mutants, all strains cultivated on LB (higher panel) and on M9 supplemented with glucose (Lower panel). Scale bar is indicated at the bottom of each image. The scale bars always represent 2 μm.
Fig 5.
Cell wall genes dependent release of extracellular DNA.
PAO1wild-type and cell wall targets mutants were grown in LB medium. Supernatant samples were collected after the respected time. DNA release was delayed and was much lower in the PAO1 rmlA mutants. Standard errors represent the mean of three independent experiments.
Fig 6.
Macrophage-mediated bactericidal assay.
Deletion of cell wall biosynthesis genes resulted in a significantly decreased survival of the bacteria. Standard errors of mean of 3 experimental points are shown.
Fig 7.
Assessment of the effect of cell wall targets gene deletion in vivo.
Quantification of P. aeruginosa colonies grown in the lung of mice intratracheally infected with agarose beads loaded with the P. aeruginosa mutants in comparison to PAO1 wild-type (WT).
Fig 8.
Plant (lettuce) virulence assay with the P. aeruginosa PAO1 and cell wall biosynthesis genes mutants.
The figure represents lettuce midribs after 5 days of infection. Infection by PAO1 wild-type and murA mutant shows necrosis and tissue maceration. Three independent experiments gave similar results.