Identification and characterization of PorH, a new cell wall channel of Corynebacterium glutamicum.
The cell wall of Corynebacterium glutamicum contains the cation-selective channel (porin) PorA(C.glut) and the anion-selective channel PorB(C.glut) for the passage of hydrophilic solutes. Lipid bilayer experiments with organic solvent extracts of whole C. glutamicum cells cultivated in minimal medium suggested that also another cation-selective channel-forming protein, named PorH(C.glut), is present in C. glutamicum. The protein was purified to homogeneity by fast-protein liquid chromatography across a HiTrap-Q column. The pure protein had an apparent molecular mass of about 12 kDa on SDS-PAGE. Western blot analysis suggested that the cell wall channel is presumably formed by protein oligomers. The purified protein forms cation-selective channels with an average single-channel conductance of about 2.5 nS in 1 M KCl in the lipid bilayer assay. The PorH(C.glut) protein was partially sequenced, and based on the resulting amino acid sequence, the corresponding gene, designated as porH(C.glut), was identified in the published genome sequence of C. glutamicum ATCC13032. PorH(C.glut) contains only the inducer methionine but no N-terminal extension, which suggests that the export and assembly of the protein follow a yet unknown pathway. PorH(C.glut) is coded in the bacterial chromosome by a gene that is localized in the vicinity of porA(C.glut), within a putative operon of 13 genes. RT-PCR revealed that both porins are cotranscribed. They coexist according to immunological detection experiments in the cell wall of C. glutamicum together with PorB(C.glut) and PorC(C.glut).
Hünten
Peter
P
Lehrstuhl für Biotechnologie, Biozentrum der Universität Würzburg, Am Hubland, D-97074 Würzburg, Germany.
Costa-Riu
Noelia
N
Palm
Dieter
D
Lottspeich
Friedrich
F
Benz
Roland
R
D000595:Amino Acid Sequence; D015153:Blotting, Western; D002473:Cell Wall / Q000737:chemistry; D002853:Chromatography, Liquid; D048230:Corynebacterium glutamicum / Q000378:metabolism*; D008051:Lipid Bilayers / Q000737:chemistry; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D018272:Porins / Q000737:chemistry* / Q000302:isolation & purification*; D016415:Sequence Alignment
D008051:Lipid Bilayers; D018272:Porins