Cloning and functional expression of an MscL ortholog from Rhizobium etli: characterization of a mechanosensitive channel.
Rhizobium etli is equipped with several systems to handle both hyper- and hypo-osmotic stress. For adaptation to hypo-osmotic stress, R. etli possesses a single gene with clear homology to MscS, four MscS-like channels and one ortholog of MscL (ReMscL, identity approximately 44% compared to Escherichia coli MscL). We subcloned and expressed the ReMscL channel ortholog from R. etli in E. coli to examine its activity by patch clamp in giant spheroplasts and characterized it at the single-channel level. We obtained evidence that ReMscL prevents the lysis of E. coli null mutant log-phase cells upon a rapid, osmotic downshock and identified a slight pH dependence for ReMscL activation. Here, we describe the facilitation of ReMscL activation by arachidonic acid (AA) and a reversible inhibitory effect of Gd(3+). The results obtained in these experiments suggest a stabilizing effect of micromolar AA and traces of Gd(3+) ions in the partially expanded conformation of the protein. Finally, we discuss a possible correlation between the number of gene paralogs for MS channels and the habitats of several microorganisms. Taken together, our data show that ReMscL may play an important role in free-living rhizobacteria during hypo-osmotic shock in the rhizosphere.
Balleza
Daniel
D
Laboratory of Molecular Biology, University of Wisconsin, Madison, WI, 53706, USA. dballeza.uwisc@gmail.com
Gómez-Lagunas
Froylan
F
Quinto
Carmen
C
D000595:Amino Acid Sequence; D016718:Arachidonic Acid / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D005682:Gadolinium / Q000494:pharmacology; D015640:Ion Channel Gating / Q000187:drug effects; D007473:Ion Channels / Q000235:genetics* / Q000378:metabolism; D040542:Mechanotransduction, Cellular; D008969:Molecular Sequence Data; D009997:Osmotic Pressure; D018408:Patch-Clamp Techniques; D042163:Rhizobium etli / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D007473:Ion Channels; C086596:MscL protein, E coli; D016718:Arachidonic Acid; D005682:Gadolinium