Figure 1.
Fatty acid biosynthesis in P. aeruginosa.
After the initiation of the acyl chain biosynthesis (reactions represented by red arrows), the biosynthetic cycle is repeated until the assembly of C16 or C18 acyl chains (blue arrows). The dotted arrows represent the entry of compounds in the elongation module.
Figure 2.
SigX overexpression alters growth and morphology of P. aeruginosa.
A. Growth curve of sigX overexpressing strain (ALB04) and control (ALB01) in LB with 50 mg/mL gentamycin and 0.2% of arabinose at 37°C and 240 rpm. B. Survival was measured by counting colony forming units (CFU) along the growth curve. C. Phase contrast and fluorescence microscopy. In both panels, the column on the left shows the cells as seen in phase contrast microscopy, while the column on the right depicts fluorescence microscopy of the cultures previously stained with LIVE/DEAD BacLight Bacterial Viability Kit (Invitrogen), where membrane damaged cells (dead) are colored in red. All pictures are at the same scale. D. Flow cytometry analysis of LIVE/DEAD stained cells. The values plotted in A and B are means of three biological replicates and error bars represent standard deviation. Flow cytometry was carried out with biological duplicates in two different occasions and values shown are representative of one experiment.
Figure 3.
SigX levels are controlled at protein and mRNA levels in the overexpression strain ALB04.
A. Immunobloting using a polyclonal anti-SigX antibody and total protein extracts obtained from cultures growing in the presence of arabinose and gentamycin. The Spectra Multicolor Broad Range Protein Ladder (Fermentas) was used as molecular weight marker (MWM). B and C. Quantitative RT-PCR using cDNA made by reverse transcription of total RNA from ALB01 (control), ALB04 (sigX overexpression) or ALB02 (PA14_21550 overexpression) cells growing in inducing conditions and specific sigX (B) or PA14_21550 (C) primers. Asterisks indicate significant differences comparing to the reference sample ALB01 6h, plotted as 1 (p<0.01).
Figure 4.
SigX overexpression effect in FAS genes and their paralogues.
Comprehensive quantitative RT-PCR assay with cDNA synthesized from RNA of the same cultures used for proteomic analysis shows the effect of SigX overexpression on genes coding for fatty acid biosynthesis enzymes (A) and their homologues (B). Results are shown as relative values in comparison to the control strain ALB01 at 3h after arabinose induction, considered as 1 and coinciding with the x axis. Significant differences among the values for each gene are shown with an asterisk.
Figure 5.
Lower levels of overexpression and natural induction of sigX induce target genes.
A. Arabinose was added to ALB04 cultures at exponential phase and samples (+Ara) were collected after 15, 30 and 180 minutes for RNA extraction and qRT-PCR of sigX, accA and fabH3. Control samples were taken at the same time points (-Ara). All values are shown as relative to t=0 for each treatment, set as 1. B. β-galactosidase activity assay of sigX and fabH3-lacZ promoter fusions in the wild-type background (PA14::sigX_lacZ and PA14::fabH3_lacZ) in LB with 171 mM NaCl or without NaCl. The values plotted are averages of biological triplicates and error bars represent standard deviations.
Figure 6.
PA14_21550 is induced by sigX overexpression, but it does not activate FAS genes.
A. Genomic region containing fabH3 and a sketch showing the PfabH3-lacZ fusion. B. qRT-PCR of PA14_21550 in sigX overexpression (ALB04) and control (ALB01) strains. C. β-galactosidase activity assay of fabH3 promoter-lacZ fusion in strains overexpressing PA14_21550 (ALB02::fabH3_lacZ) and sigX (ALB04::fabH3_lacZ). The values plotted are averages of biological triplicates and error bars represent standard deviations. D. qRT-PCR of some sigX overexpression targets in PA14_21550 overexpression strain (ALB02). The values shown in B and D are averages of technical triplicates of at least two independent assays and error bars represent standard deviations.
Figure 7.
Differences in lipid composition, membrane fluidity and response to temperature stresses in sigX overexpressing cells versus control.
A. Gas chromatography/mass spectrometry analysis of fatty acids methyl esters (FAMEs) derived from ALB01 or ALB04 whole cells preparation. B. Anisotropy assay, where the lower value corresponds to a less organized membrane, so to a higher fluidity. C. Cold and heat shock survival. Cultures growing for 3h at 37°C or 30°C were split and a portion of them was shifted to 15°C or 42°C for 2 and 1h, respectively, while another remained at the initial temperature for control. The values are plotted as a ratio of CFU counts of cultures at the stress temperature and the control.