Biosilo de residuos de merluza y harina de cebada fermentados con bacterias ácido lácticas seleccionadas

Se estudió la capacidad como inoculantes de biosilo de Lactococcus lactis Tw34 y  Lactobacillus plantarum Lb7. Los experimentos se llevaron a cabo con una mezcla de subproductos de merluza (Merluccius hubbsi) y harina de cebada, fermentada durante 7 días. Como control se utilizó una muestra acidificada con ácido láctico. En las  mezclas biológicas, el pH disminuyó por debajo de 5,0 después de 2 días de fermentación y permaneció estable hasta el final de la experiencia. La población máxima (>109 UFC/g) se alcanzó después de 5 días de incubación a 18°C. La concentración de péptidos solubles en agua aumentó durante los 7 días de incubación y no hubo diferencias significativas (p 0,05). Después de 7 días, las concentraciones de fósforo alcanzadas fueron 2,26 y 2,42 g /100 g en los biosilos fermentados con Lc. lactis Tw34 y Lb. plantarum Lb7 respectivamente, mientras que en el control los valores permanecieron casi estables (1,61 g/100 g). Al final de la experiencia, la actividad inhibitoria de tripsina fue suprimida en ambos biosilos mientras que, en el control los factores antinutricionales seguían siendo activos. Los resultados indican la factibilidad del uso de la mezcla seleccionada como sustrato para la producción de biosilo y la eficacia de Lc. lactis Tw34 y Lb. plantarum Lb7 como inoculantes

Coupling of Fatty Acid and Phospholipid Synthesis in Bacillus subtilis▿

plsX (acyl-acyl carrier protein [ACP]:phosphate acyltransferase), plsY (yneS) (acyl-phosphate:glycerol-phosphate acyltransferase), and plsC (yhdO) (acyl-ACP:1-acylglycerol-phosphate acyltransferase) function in phosphatidic acid formation, the precursor to membrane phospholipids. The physiological functions of these genes was inferred from their in vitro biochemical activities, and this study investigated their roles in gram-positive phospholipid metabolism through the analysis of conditional knockout strains in the Bacillus subtilis model system. The depletion of PlsX led to the cessation of both fatty acid synthesis and phospholipid synthesis. The inactivation of PlsY also blocked phospholipid synthesis, but fatty acid formation continued due to the appearance of acylphosphate intermediates and fatty acids arising from their hydrolysis. Phospholipid synthesis ceased following PlsC depletion, but fatty acid synthesis continued at a high rate, leading to the accumulation of fatty acids arising from the dephosphorylation of 1-acylglycerol-3-P followed by the deacylation of monoacylglycerol. Analysis of glycerol 3-P acylation in B. subtilis membranes showed that PlsY was an acylphosphate-specific acyltransferase, whereas PlsC used only acyl-ACP as an acyl donor. PlsX was found in the soluble fraction of disrupted cells but was associated with the cell membrane in intact organisms. These data establish that PlsX is a key enzyme that coordinates the production of fatty acids and membrane phospholipids in B. subtilis

FapR, a Bacterial Transcription Factor Involved in Global Regulation of Membrane Lipid Biosynthesis

Bacterial cells exert exquisite control over the biosynthesis of their membrane lipids, but the mechanisms are obscure. We describe the identification and purification from Bacillus subtilis of a transcription factor, FapR, that controls the expression of many genes involved in fatty acid and phospholipid metabolism (the fap regulon). Expression of this fap regulon is influenced by antibiotics that specifically inhibit the fatty acid biosynthetic pathway. We show that FapR negatively regulates fap expression and that the effects of antibiotics on fap expression are mediated by FapR. We further show that decreasing the cellular levels of malonyl-CoA, an essential molecule for fatty acid elongation, inhibits expression of the fap regulon and that this effect is FapR dependent. Our results indicate that control of FapR by the cellular pools of malonyl-CoA provides a mechanism for sensing the status of fatty acid biosynthesis and to adjust the expression of the fap regulon accordingly.National Institutes of Health (U.S.) (Public Health Services Grant GM50895

Structural Basis for Feed-Forward Transcriptional Regulation of Membrane Lipid Homeostasis in Staphylococcus aureus

The biosynthesis of membrane lipids is an essential pathway for virtually all bacteria. Despite its potential importance for the development of novel antibiotics, little is known about the underlying signaling mechanisms that allow bacteria to control their membrane lipid composition within narrow limits. Recent studies disclosed an elaborate feed-forward system that senses the levels of malonyl-CoA and modulates the transcription of genes that mediate fatty acid and phospholipid synthesis in many Gram-positive bacteria including several human pathogens. A key component of this network is FapR, a transcriptional regulator that binds malonyl-CoA, but whose mode of action remains enigmatic. We report here the crystal structures of FapR from Staphylococcus aureus (SaFapR) in three relevant states of its regulation cycle. The repressor-DNA complex reveals that the operator binds two SaFapR homodimers with different affinities, involving sequence-specific contacts from the helix-turn-helix motifs to the major and minor grooves of DNA. In contrast with the elongated conformation observed for the DNA-bound FapR homodimer, binding of malonyl-CoA stabilizes a different, more compact, quaternary arrangement of the repressor, in which the two DNA-binding domains are attached to either side of the central thioesterase-like domain, resulting in a non-productive overall conformation that precludes DNA binding. The structural transition between the DNA-bound and malonyl-CoA-bound states of SaFapR involves substantial changes and larg

Revisiting the coupling of fatty acid to phospholipid synthesis in bacteria with FapR regulation

A key aspect in membrane biogenesis is the coordination of fatty acid to phospholipid synthesis rates. In most bacteria, PlsX is the first enzyme of the phosphatidic acid synthesis pathway, the common precursor of all phospholipids. Previously, we proposed that PlsX is a key regulatory point that synchronizes the fatty acid synthase II with phospholipid synthesis in Bacillus subtilis. However, understanding the basis of such coordination mechanism remained a challenge in Gram-positive bacteria. Here, we show that the inhibition of fatty acid and phospholipid synthesis caused by PlsX depletion leads to the accumulation of long-chain acyl-ACPs, the end products of the fatty acid synthase II. Hydrolysis of the acyl-ACP pool by heterologous expression of a cytosolic thioesterase relieves the inhibition of fatty acid synthesis, indicating that acyl-ACPs are feedback inhibitors of this metabolic route. Unexpectedly, inactivation of PlsX triggers a large increase of malonyl-CoA leading to induction of the fap regulon. This finding discards the hypothesis, proposed for B. subtilis and extended to other Gram-positive bacteria, that acyl-ACPs are feedback inhibitors of the acetyl-CoA carboxylase. Finally, we propose that the continuous production of malonyl-CoA during phospholipid synthesis inhibition provides an additional mechanism for fine-tuning the coupling between phospholipid and fatty acid production in bacteria with FapR regulation.Fil: Machinandiarena, Federico. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Nakamatsu, Leandro. Ingeniería Metabólica Sa; ArgentinaFil: Schujman, Gustavo Enrique. Ingeniería Metabólica Sa; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: de Mendoza, Diego. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Albanesi, Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentin

Overall structure of the <i>Sa</i>FapR-operator complex.

<p>(<b>A</b>) Surface representation of the DNA operator (in red) with two bound FapR homodimers looking down the non-crystallographic two-fold symmetry axis. For one homodimeric repressor (in yellow and orange) the DNA-binding domains (DBDs), the linker helix α_L and the dimeric effector-binding domain (DBD) are indicated. (<b>B</b>) ITC study of <i>Sa</i>FapR binding to the P<i>fapR</i> operator at 25°C. The top panel shows the raw heat signal for 6 µl injections of a 68 µM solution of <i>Sa</i>FapR dimer into a 4 µM solution of the 40 bp DNA oligonucleotide (curve 1 obtained by subtraction of the <i>Sa</i>FapR dilution energy curve 3 from the raw titration curve 2). The bottom panel shows the integrated injection heats after normalization fitted with a sequential binding model. Two <i>Sa</i>FapR dimers bind the operator, with parameters (<i>K_d</i>_,I = 0.5±0.1 nM, <i>ΔH</i>°_I = −22.5±0.2 kcal/mol, <i>TΔS</i>°_I = −9.8±0.2, kcal/mol) and (<i>K_d</i>_,II = 51±8 nM, <i>ΔH</i>°_II = −6.95±0.2 kcal/mol, <i>TΔS</i>°_II = 3.0±0.3 kcal/mol).</p

Overall structures of the malonyl-CoA-bound forms of <i>Sa</i>FapR.

<p>(<b>A</b>) Cartoon showing the structure of the <i>Sa</i>FapR-malonyl-CoA homodimer in two different views. The first protomer is shown in green; the second protomer is shown in blue (the helix-turn-helix motif - in dark blue - was partially visible in the electron density map but was not included in the final model due to high protein mobility). Bound malonyl-CoA is shown in surface representation. (<b>B</b>) Closer view of the interactions between the central hot-dog fold (electrostatic surface representation) and the linker helix (green). Hydrophobic side chains involved in inter-domain interactions are labeled. (<b>C</b>) Electron density map of malonyl-CoA and protein-ligand interactions. Hydrogen bonds are indicated by dashed lines and protein residues from each protomer are colored green and yellow respectively.</p

Data collection, phasing and refinement statistics.

<p>Values in parentheses are for highest-resolution shell.</p>1<p>According to the MolProbity server <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003108#ppat.1003108-Davis1" target="_blank">[53]</a>.</p