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

Involvement of an N-Acetylglucosaminidase in Autolysis of Propionibacterium freudenreichii CNRZ 725

Propionibacterium freudenreichii plays an important role in Swiss cheese ripening (it produces propionic acid, acetic acid, and CO(2)). Moreover, autolysis of this organism certainly contributes to proteolysis and lipolysis of the curd because intracellular enzymes are released. By varying external factors, we determined the following conditions which promoted autolysis of both whole cells and isolated cell walls of P. freudenreichii CNRZ 725: (i) 0.1 M potassium phosphate buffer (pH 5.8) at 40°C and (ii) 0.05 to 0.1 M KCl at 40°C. We found that early-exponential-phase cells possessed the highest autolytic activity. It should be emphasized that the pH of Swiss cheese curd (pH 5.5 to 5.7) is near the optimal pH which we determined. Ultrastructural observations by electron microscopy revealed a 16-nm-thick homogeneous cell wall, as well as degradation of the cell wall that occurred concomitantly with cell autolysis. In the presence of 0.05 M potassium chloride, there was a great deal of isolated cell wall autolysis (the optical density at 650 nm decreased 77.5% ± 7.3% in 3 h), and one-half of the peptidoglycan material was released. Finally, the main autolytic activity was due to an N-acetylglucosaminidase activity

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

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 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 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

Discovery of intracellular heme-binding protein HrtR, which controls heme efflux by the conserved HrtB-HrtA transporter in Lactococcus lactis

International audienceMost commensal and food bacteria lack heme biosynthesis genes. For several of them, the capture of environmental heme is a means of activating aerobic respiration metabolism. Our previous studies in the Gram-positive bacterium Lactococcus lactis showed that heme exposure strongly induced expression of a single operon, called here hrtRBA, encoding an ortholog of the conserved membrane heme-regulated transporter (hrt) and a unique transcriptional regulator we named HrtR. We show that HrtR expressed as a fusion protein is a heme-binding protein. Heme iron interaction with HrtR is non-covalent, hexacoordinated and involves two histidines, His-72 and His-149. HrtR specifically binds a 15 nt palindromic sequence in the hrtRBA promoter region, which is needed for hrtRBA repression. HrtR-DNA binding is abolished by heme addition, which activates expression of the HrtB-HrtA (HrtBA) transporter in vitro and in vivo. The use of HrtR as an intracellular heme sensor appears to be conserved among numerous commensal bacteria, in contrast with numerous Gram-positive pathogens that use an extracellular heme-sensing system, HssRS, to regulate hrt. Finally, we show for the first time that HrtBA permease controls heme toxicity by its direct and specific efflux. The use of an intracellular heme sensor to control heme efflux constitutes a novel paradigm for bacterial heme homeostasis