A major outer membrane protein of Rahnella aquatilis functions as a porin and root adhesin

Abstract

A 38-kDa major outer membrane protein (OMP) was isolated from the nitrogen-fixing enterobacterium Rahnella aquatilis CF3. This protein exists as a stable trimer in the presence of 2% sodium dodecyl sulfate at temperatures below 60 degrees C. Single channel experiments showed that this major OMP of R. aquatilis CF3 is able to form pores in the planar lipid membrane. Two oligonucleotides encoding the N-terminal portion of the 38-kDa OMP and C-terminal portion of OmpC were used to amplify the 38-kDa gene by PCR. The deduced amino acid sequence showed a strong homology with Escherichia coli, Klebsiella pneumoniae, Salmonella typhi, and Serratia marcescens OmpC sequences, except loops L6 and L7, which are postulated to be cell surface exposed. On the basis of the OmpF-PhoE three-dimensional structure, it seems likely that this 38-kDa organizes three 16-strand beta-barrel subunits. The relationship between the structure and the double functionality of this protein as porin and as a root adhesin is discussed.

FIG. 1

Coomassie blue-stained gels, resolved by SDS-PAGE, of OMPs of R. aquatilis CF3 (lane 1) and E. coli HB101 (lane 2) (a) and Zw 3-16 partially purified 38-kDa OMP solubilized at various temperatures (b). Solubilization temperatures, from left to right, were 20, 37, 55, 65, 70, 80, and 96°C. For both panels, arrows show the position of the 38-kDa monomer and molecular mass position markers are given on the left.

FIG. 2

Single-channel current records of POPC-DOPE bilayers formed in 1 M NaCl at 25°C in the presence of 0.5 μg of the 38-kDa protein of R. aquatilis per ml. Data were recorded at 2,000 Hz with a 300-Hz filter. The applied membrane potential was 55 mV. The lower trace shows an expanded recording of the box marked on the plot above it.

FIG. 3

Single-channel records of porin under same conditions as those described for Fig. 2, but with a protein concentration of 1 μg/ml. The trace was recorded at 500 Hz and nonfiltered after 30 min of standby. The applied potential was 30 mV.

FIG. 4

Multiple alignment of the amino acid sequence deduced from the 1-kbp PCR product encoding the 38-kDa OMP from R. aquatilis (Raq) with the corresponding sequences of mature porins of E. coli (Eco), S. typhi (Sty), and S. marcescens (Sma) OmpC. b strands, β-strands; a helix, α-helix.

FIG. 5

Theoretical folding pattern of the 38-kDa OMP. Rectangles indicate residues forming α-helices, diamonds indicate those making up β-strands (in boldface print if their side chains are external), and circles indicate those constituting hydrogen-bonded reverse turns and loops.