Figure 1.
a. Sequence alignment of the EF-hand domains in EfhP (PA4107) and CasA (YP_472788) from Rhizobium etli [24]. The predicted Ca2+-binding loops are underlined. b. The predicted transmembrane (TM) region and two Ca2+-binding loops (1 and 2) are shown. EF-hand domains were predicted by PROSITE. Transmembrane region and cellular localization of the protein were predicted by TMHMM.
Figure 2.
Free [Ca2+]in profiles of PAO1 WT (black line) and efhP mutant strain PAO1043 (grey line).
Cultures were grown in 02+. After the basal level of [Ca2+]in was monitored for 1 min, 1 mM Ca2+ was added (indicated by the arrow) followed by further [Ca2+]in measurement for 20 min.
Figure 3.
Proteomics analysis of efhP mutant strains.
Sections of 2D gels showing selected proteins that are affected by the efhP mutation in FRD1, cultured in tubing biofilms. Circled are protein missing or with reduced abundances in the efhP mutant. Proteins were identified by MALDI-TOF analysis.
Table 1.
Proteins identified by MASCOT-based analysis of MALDI-TOF generated mass spectra.
Figure 4.
Alginate production at no iron in FRD1 and FRD1043.
To deprive the cells of iron, seven passages on no-iron BMM (BMM-NI) agar were performed. The cells were collected using saline. The concentration of alginate was determined using sodium alginate (Spectrum) as a standard and normalized by total cellular protein. Fold difference was calculated between the alginate produced after seventh and first passages. The measurements were obtained for at least three biological replicates, and the mean values with standard deviation are presented.
Figure 5.
Survival of FRD1 and FRD1043 under oxidative stress.
Cells were exposed to 12O2 for 1 h at 37°C. Viable cells were determined as colony forming units. Percent survival was calculated considering that non-treated cells are 100% viable. Data represent the mean and standard deviation of at least three biological replicates.
Figure 6.
Pyocyanin production of FRD1 and FRD1043.
Cells were grown on BMM agar plates at 102 for 24 h, and collected using saline. Percent change was calculated vs. FRD1 cells grown at no added CaCl2. Data represent the mean and standard deviation of at least three biological replicates.
Figure 7.
Lettuce infection assay of P. aeruginosa virulence.
a. Photographs showing representative samples of lettuce midribs after 6 days of infection with FRD1 and PAO1 and their efhP mutant strains FRD1043 and PAO1043. Complementing with efhP in trans on plasmid pMF470 partially restored the zone of infection. b. The areas of disease were measured and the percent change vs. wild types was calculated. The experiments were repeated at least two times, with 3–5 biological replicates each.