In summary, polyP has numerous and varied biological functions

in bacteria that have been discovered mainly by studying its deficiency. To better understand the function of polyP we used broad-host-range constitutive and regulated vectors to deplete cellular polyP and found new functional and structural changes. In addition, it is generally accepted that energy supply of the cells could be severely compromised in the absence of polyP. We confirmed this evidence by using differential proteomic studies and suggested that during polyP scarcity energy metabolism and particularly nucleoside triphosphate (NTP) formation were affected, generating a general stress condition. We propose that bacterial cells prevail by increasing the flux of energy-generating metabolic pathways such as tricarboxilic acid (TCA) Staurosporine clinical trial cycle and β-oxidation and by reducing energy-consuming ones such as active transporters and amino acid biosynthesis. Methods Bacterial strains and growth

conditions Pseudomonas sp. B4 wt, control (pMLS7) and polyP-deficient (pS7PPX1) recombinant strains were previously obtained [21] and grown aerobically BIBW2992 research buy at 37°C on Luria-Bertani (LB) rich medium supplemented with trimetropim (50 μg/ml). When required, LB plates (1,5% (w/v) of Bacto-agar) were used for obtaining cells from the colonies after 48 h of growth. Optical and Electron Selleckchem ACY-1215 Microscopy Unstained cells from the different cultures were routinely examined for the presence of polyP granules Mannose-binding protein-associated serine protease by transmission electron microscopy [43]. Cells were mixed and dispersed in distilled water and added onto carbon-coated nickel grids. The drops

containing the microorganisms were drained off with filter paper and air dried during 30-50 s. Electron microscopy was performed with a Philips Tecnai 12 electron microscope using 80 kV accelerating voltage (Electron Microscopy Laboratory, Pontificia Universidad Católica de Chile). Optical microscopy was routinely performed in an Olympus BX50 microscope (Olympus Corporation, Japan). LPS analysis Culture samples were adjusted to an optical density at 600 nm of 2.0 in a final volume of 100 μl. Then, proteinase K-digested whole-cell lysates were prepared as described previously [44], and LPS was separated on 14% acrylamide gels using a Tricine-sodium dodecyl sulfate (SDS) buffer system [45]. Gel loadings were normalized so that each sample represented the same number of cells. Gels were silver stained by a modification of the procedure of Tsai and Frasch [46, 47]. Samples preparation and 2D-PAGE Cells (200 mg) were harvested by centrifugatio n (7,000 × g for 15 min at 25°C) from liquid cultures or were collected with an inoculation loop from agar plates. Pellets were washed four times in sonication buffer (40 mM Tris pH 8.15; 1 mM PMSF).