The functions of the mechanosensitive channels from Lactococcus lactis were determined by biochemical, physiological, and electrophysiological methods. Patch-clamp studies showed that the genes yncB and mscL encode MscS and MscL-like channels, respectively, when expressed in Escherichia coli or if the gene products were purified and reconstituted in proteoliposomes. However, unless yncB was expressed in trans, wild type membranes of L. lactis displayed only MscL activity. Membranes prepared from an mscL disruption mutant did not show any mechanosensitive channel activity, irrespective of whether the cells had been grown on low or high osmolarity medium. In osmotic downshift assays, wild type cells survived and retained 20% of the glycine betaine internalized under external high salt conditions. On the other hand, the mscL disruption mutant retained 40% of internalized glycine betaine and was significantly compromised in its survival upon osmotic downshifts. The data strongly suggest that L. lactis uses MscL as the main mechanosensitive solute release system to protect the cells under conditions of osmotic downshift.
"e also represented ( ⨀ ) . attB A was found downstream of a tmRNA sequence , which often represents a target for mobile DNAs ; attB C and attB B were downstream of genes encoding s catabolite control protein A homologue ( ccpA ) , and a large mechanosensitive channel ( mcsl ) which possibly contributes to protection against extreme turgor in LAB ( Folgering et al . , 2005 ) , respectively . Lastly , attB D was located between genes encoding a NADPH - quinone reductase and a ribosomal protein ."
[Show abstract][Hide abstract] ABSTRACT: Temperate bacteriophages are a contributor of the genetic diversity in the lactic acid bacterium Oenococcus oeni. We used a classification scheme for oenococcal prophages based on integrase gene polymorphism, to analyze a collection of Oenococcus strains mostly isolated in the area of Bordeaux, which represented the major lineages identified through MLST schemes in the species. Genome sequences of oenococcal prophages were clustered into four integrase groups (A to D) which were related to the chromosomal integration site. The prevalence of each group was determined and we could show that members of the intB- and intC-prophage groups were rare in our panel of strains. Our study focused on the so far uncharacterized members of the intD-group. Various intD viruses could be easily isolated from wine samples, while intD lysogens could be induced to produce phages active against two permissive O. oeni isolates. These data support the role of this prophage group in the biology of O. oeni. Global alignment of three relevant intD-prophages revealed significant conservation and highlighted a number of unique ORFs that may contribute to phage and lysogen fitness.
International journal of food microbiology 07/2013; 166(2):331-340. DOI:10.1016/j.ijfoodmicro.2013.06.032 · 3.08 Impact Factor
"Homologs of MscS have been identified throughout bacterial and archaeal kingdoms. Many provide MS channel activity and/or function in osmotic stress protection , , , , , , though others do not , , , . MscS homologs have also been identified in the genomes of a subset of eukaryotic lineages, including several fungal and all plant genomes examined to date , , , . "
[Show abstract][Hide abstract] ABSTRACT: The Mechanosensitive channel of Small conductance (MscS) of Escherichia coli has become an excellent model system for the structural, biophysical, and functional study of mechanosensitive ion channels. MscS, a complex channel with multiple states, contributes to protection against lysis upon osmotic downshock. MscS homologs are widely and abundantly dispersed among the bacterial and plant lineages, but are not found in animals. Investigation into the eukaryotic branch of the MscS family is in the beginning stages, and it remains unclear how much MscS homologs from eukaryotes resemble E. coli MscS with respect to structure, function, and regulation. Here we test the effect of mutating three conserved motifs on the function of MscS-Like (MSL)2, a MscS homolog localized to the plastids of Arabidopsis thaliana. We show that 1) a motif at the top of the cytoplasmic domain, referred to here as the PN(X)(9)N motif, is essential for MSL2 function and for its proper intraplastidic localization; 2) substituting polar residues for two large hydrophobic residues located in the predicted pore-lining transmembrane helix of MSL2 produces a likely gain-of-function allele, as previously shown for MscS; and 3) mis-expression of this allele causes severe defects in leaf growth, loss of chloroplast integrity, and abnormal starch accumulation. Thus, two of the three conserved motifs we analyzed are critical for MSL2 function, consistent with the conservation of structure and function between MscS family members in bacteria and plants. These results underscore the importance of plastidic mechanosensitive channels in the maintenance of normal plastid and leaf morphology.
PLoS ONE 06/2012; 7(6):e40336. DOI:10.1371/journal.pone.0040336 · 3.23 Impact Factor
"Experiments were performed as described previously (Blount et al. 1999; Folgering et al. 2005). Samples of 1–5 mL of spheroplast (0.2 -0.8 mg/mL total protein) were transferred to a sample chamber containing a ground electrode and 300 mL of patch clamp buffer: 5 mM HEPES (pH 7.2), 200 mM KCl, 90 mM MgCl 2 , plus 10 mM CaCl 2 . "
[Show abstract][Hide abstract] ABSTRACT: To obtain a gene construct for making single substitutions per channel and to determine the quaternary structure of the mechanosensitive channel MscL from Escherichia coli, covalent oligomers (monomer to hexamer) were engineered by gene fusion; up to six copies of the mscL gene were fused in tandem. All the multimeric tandem constructs yielded functional channels with wild-type conductance and dwell times. Importantly, only the covalent pentamer opened at the same relative pressure (compared to the pressure required to open MscS) as the wild-type MscL channel. The in vivo data strongly suggest that pentameric MscL represents the functional state of the channel.
Protein Science 01/2006; 14(12):2947-54. DOI:10.1110/ps.051679005 · 2.85 Impact Factor
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