Involvement of the C-terminal part of Pseudomonas fluorescens OprF in the modulation of its pore-forming properties.
The major outer-membrane protein, OprF, from the psychrotrophic bacterium Pseudomonas fluorescens undergoes a reduction of its conductance value (from 250 pS to 80 pS) when the growth temperature is shifted from 28 degrees C to 8 degrees C. The involvement of changes in tertiary or quaternary structure in this behaviour, was implied by enzymatic digestion experiments in which OprFs purified from 8 degrees C and 28 degrees C cultures showed different accessibility to pronase. Resistant proteolytic fragments of 19 kDa, obtained from both OprF preparations, were identified as the N-terminal half of the native protein. These 19 kDa fragments induced ion channels in planar lipid bilayers with similar conductance values of 65-75 pS in 1 M NaCl, in contrast to the native proteins. Thus, the C-terminal part of the protein is required for the growth temperature-dependent modulation of OprF channel-forming properties. LPS was not detected on the proteolytic fragments while it was found in similar amounts on the native OprFs. These results suggest the LPS/porin association occurs through the C-terminal part of the porin. Radiolabelling experiments showed different phosphorylation levels of LPS for 8 degrees C and 28 degrees C cultures. Thus, in response to growth temperature, the structural modification of the LPS could be associated to the modulation of OprF pore size.
El Hamel
C
C
UMR 6522, CNRS, IFRMP 23, Faculté des Sciences, Mont-Sanit-Aignan, France.
Freulet
M A
MA
Jaquinod
M
M
Dé
E
E
Molle
G
G
Orange
N
N
D001345:Autoradiography; D019075:Electrophoresis, Capillary; D004591:Electrophoresis, Polyacrylamide Gel; D008070:Lipopolysaccharides / Q000737:chemistry / Q000302:isolation & purification; D010766:Phosphorylation; D018272:Porins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D011402:Pronase; D011551:Pseudomonas fluorescens / Q000737:chemistry / Q000235:genetics* / Q000254:growth & development; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature
D008070:Lipopolysaccharides; D018272:Porins; D011402:Pronase