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 P seudomonas fluorescens undergoes a reduction of its conductance value (from 250 pS to 80 pS) when the growth temperature is shifted from 28 degreesC t o 8 degreesC. The involvement of changes in tertiary or quaternary structur e in this behaviour, was implied by enzymatic digestion experiments in whic h OprFs purified from 8 degreesC and 28 degreesC cultures showed different accessibility to pronase. Resistant proteolytic fragments of 19 kDa, obtain ed from both OprF preparations, were identified as the N-terminal half of t he native protein. These 19 kDa fragments induced ion channels in planar li pid bilayers with similar conductance values of 65-75 pS in 1 M NaCl, in co ntrast to the native proteins. Thus, the C-terminal part of the protein is required for the growth temperature-dependent modulation of OprF channel-fo rming properties. LPS was not detected on the proteolytic fragments while i t was found in similar amounts on the native OprFs. These results suggest t he LPS/porin association occurs through the C-terminal part of the porin. R adiolabelling experiments showed different phosphorylation levels of LPS fo r 8 degreesC and 28 degreesC cultures. Thus, in response to growth temperat ure, the structural modification of the LPS could be associated to the modu lation of OprF pore size. (C) 2000 Elsevier Science B.V. All rights reserve d.