Roles of the Carboxy-Terminal Half of Pseudomonas aeruginosa Major Outer Membrane Protein OprF in Cell Shape, Growth in Low-Osmolarity Medium, and Peptidoglycan Association

Abstract

OprF, the major outer membrane protein of Pseudomonas aeruginosa, is multifunctional in that it can act as a nonspecific porin, plays a role in the maintenance of cell shape, and is required for growth in a low-osmolarity environment. The latter two structural roles of OprF, and OprF’s association with the peptidoglycan, have been proposed to be localized in the carboxy terminus of the protein, based on this region’s similarity to members of the OmpA family of proteins. To determine if this is correct, we constructed a series of C-terminally truncated OprF derivatives and examined their effects on P. aeruginosa cell length and growth in low-osmolarity medium. While the C terminus of OprF was required for wild-type cell length and growth in low-osmolarity medium, expression of the N terminus (first 163 amino acids [aa]) also influenced these phenotypes (compared with OprF deficiency). The first 154 to 164 aa of OprF seemed required for stable protein expression, consistent with the existence of a β-barrel domain in the N terminus of OprF. Greater than 215 aa of the protein were required for strong peptidoglycan association, confirming that residues in the C-terminal end of OprF are required for peptidoglycan binding. OprF deficiency did not affect the in vivo growth of an OprF-deficient strain in a mouse chamber model. Collectively, these data suggest that the C terminus of OprF plays a role in cell length, growth of P. aeruginosa in low-osmolarity media (but not in vivo), and peptidoglycan association, while the N terminus has an influence on the first two characteristics and is additionally important for stable protein expression.

Ion channel formation by N-terminal domain: a common feature of OprFs of Pseudomonas and OmpA of Escherichia coli

“…The similar ion channel activity of the three N-terminal domains observed in the present study suggests that the pores of OprF 175 and OprF 177 are very likely constituted of eight L-strands as OmpA 171 . This would con¢rm the previous structural predictions made for the N-terminal domain of OprF [11,13,20]. Thus, ion channel activity data and structural models would suggest similar function for the amino-terminal ends of OprFs and OmpA.…”

“…Without crystallographic data to date, the functional tertiary structure of OprF is not well established. Nikaido's group agrees with a periplasmic C-terminal part [12], whilst other authors hypothesise that OprF is constituted of 16 L-strands, suggesting that the structure of the C-terminal part is not globular [11,13]. Nevertheless, there is no consensus to propose the number of L-strands necessary for building the ion channel in native OprF.…”

“…A putative structure of OprF from P. aeruginosa would comprise two main domains : a N-terminal part of about 160 residues containing eight antiparallel L-sheets able to form a L-barrel and a C-terminal domain strongly associated with peptidoglycan which is highly conserved with the C-terminal domain of E. coli OmpA [10,11]. The two domains would be separated by a repeating Pro^Ala region and two disul¢de bonds between the 160th and 210th residues.…”

OprF polymorphism as a marker of ecological niche in Pseudomonas

“…It has been suggested that the translocation of the C-terminal domain between the periplasm and the surface can modulate the pore size (Nikaido, 2003;Tamber and Hancock, 2004). Putative selection factors could be related to one or several known functions of the protein: adhesion (de Mot et al, 1994), nutrient uptake (Orange, 1994) or resistance to a variation of osmotic pressure (Rawling et al, 1998). In P. aeruginosa, it has even been reported that the binding of human interferon-γ to OprF results in the expression of the PA-I lectin, a quorum-sensing-dependent virulence determinant (Wu et al, 2005).…”

“…Although OprF has been found also in Azotobacter vinelandii , this species should certainly be classified in Pseudomonas genus (Rediers et al ., 2004). OprF is a pleiotropic protein that plays a role as porin (Orange, 1994), membrane structural protein (Rawling et al ., 1998) and root adhesive (de Mot et al ., 1992). This protein has been studied extensively due to its utility as a vaccine component, its role in antimicrobial drug resistance and its porin function (Orange, 1994;De et al ., 1995;Rawling et al ., 1995;Jaouen et al ., 2004;Worgall et al ., 2005).…”