Characterization of two Lactococcus lactis zinc membrane proteins, Llmg_0524 and Llmg_0526, and role of Llmg_0524 in cell wall integrity

International audienceBackground: Due to its extraordinary chemical properties, the cysteine amino acid residue is often involved in protein folding, electron driving, sensing stress, and binding metals such as iron or zinc. Lactococcus lactis, a Gram-positive bacterium, houses around one hundred cysteine-rich proteins (with the CX2C motif) in the cytoplasm, but only a few in the membrane. Results: In order to understand the role played by this motif we focused our work on two membrane proteins of unknown function: Llmg-0524 and Llmg-0526. Each of these proteins has two CX2C motifs separated by ten amino-acid residues (CX2CX10CX2C). Together with a short intervening gene (llmg-0525), the genes of these two proteins form an operon, which is induced only during the early log growth phase. In both proteins, we found that the CX2CX10CX2C motif chelated a zinc ion via its cysteine residues, but the sphere of coordination was remarkably different in each case. In the case of Llmg-0524, two of the four cysteines were ligands of a zinc ion whereas in Llmg-0526, all four residues were involved in binding zinc. In both proteins, the cysteine-zinc complex was very stable at 37 °C or in the presence of oxidative agents, suggesting a probable role in protein stability. We found that the complete deletion of llmg-0524 increased the sensitivity of the mutant to cumene hydroperoxide whereas the deletion of the cysteine motif in Llmg-0524 resulted in a growth defect. The latter mutant was much more resistant to lysozyme than other strains. Conclusions: Our data suggest that the CX2CX10CX2C motif is used to chelate a zinc ion but we cannot predict the number of cysteine residue involved as ligand of metal. Although no other motif is present in sequence to identify roles played by these proteins, our results indicate that Llmg-0524 contributes to the cell wall integrity

Additional file 2: Figure S2. of Characterization of two Lactococcus lactis zinc membrane proteins, Llmg_0524 and Llmg_0526, and role of Llmg_0524 in cell wall integrity

Deletion of llmg_0524 or llmg_0526 decreases modestly operon expression. The plasmid P0524-pTCV-lac is established in mutant ∆llmg_0524 and ∆llmg_0526. Cells were grown in M17Glu0.5 up to OD600= 0.1 for β-galactosidase determination. Results, plus standard deviation, are means of three independent experiments. They are expressed in percentage of values of wild type strain. (PDF 171 kb

Additional file 3: Figure S3. of Characterization of two Lactococcus lactis zinc membrane proteins, Llmg_0524 and Llmg_0526, and role of Llmg_0524 in cell wall integrity

Determination of PhoA activity of different fusion proteins. Data are the means of results, ± standard deviations, from three independent experiments. PhoA1 contains only the Nter extremity whereas PhoA2 contains the Nter extremity and the predicted transmembrane domain. (PDF 178 kb

Additional file 4: Figure S4. of Characterization of two Lactococcus lactis zinc membrane proteins, Llmg_0524 and Llmg_0526, and role of Llmg_0524 in cell wall integrity

UV-visible spectra of protein fusions. 20 μM of proteins were used. Analysis was performed in 50 mM Tris–HCl buffer, pH 7.4, at room temperature with a Libra S22 spectrophotometer. (PDF 193 kb

Additional file 5: Table S2. of Characterization of two Lactococcus lactis zinc membrane proteins, Llmg_0524 and Llmg_0526, and role of Llmg_0524 in cell wall integrity

Proteins containing the CX2CX10CX2C motif in various bacterial species. (PDF 512 kb

Additional file 1: Figure S1. of Characterization of two Lactococcus lactis zinc membrane proteins, Llmg_0524 and Llmg_0526, and role of Llmg_0524 in cell wall integrity

Amino acid sequence of Llmg_0524 and Llmg_0526. Llmg_0524 has 200 amino-acid residues, including four cysteines in the Nter region and two transmembrane domains (TMDs). Llmg_0526 has 421 amino-acid residues, including four cysteines in the Nter region and a transmembrane domain. The cysteine residues cluster in a CX2CX10CX2C motif in both proteins. Cysteine amino-acid residues are in bold red; predicted membrane helices are in bold black. (PDF 304 kb

Green strategies to control redox potential in the fermented food industry

International audienceLactic acid bacteria (LAB) are important microorganisms in the food industry as functional starters for the manufacture of fermented food products and as probiotics. Redox potential (Eh) is a parameter of the physicochemical environment of foods that influences key oxidation-reduction reactions involved in process performances and product quality. Eh can be modified by different methods, using redox molecules, catalytic activity of enzymes or LAB themselves, technological treatments like electroreduction or heating, and finally gases. Nowadays new applications for food manufacture must undertake green process innovation. This paper presents the strategies for Eh modification in a sustainable manner for production of LAB biomass (starters, probiotics) and fermented food products (fermented milks, cheeses and others). While the use of chemical or enzymes may be subject to controversy, the use of gases offers new opportunities, in combination with LAB. Protection against food-borne microorganisms, an increasing growth and viability of LAB, and a positive impact on food flavour are expected