Molecular cloning, sequencing, and expression of omp-40, the gene coding for the major outer membrane protein from the acidophilic bacterium Thiobacillus ferrooxidans

Abstract

Thiobacillus ferrooxidans is one of the chemolithoautotrophic bacteria important in industrial biomining operations. Some of the surface components of this microorganism are probably involved in adaptation to their acidic environment and in bacterium-mineral interactions. We have isolated and characterized omp40, the gene coding for the major outer membrane protein from T. ferrooxidans. The deduced amino acid sequence of the Omp40 protein has 382 amino acids and a calculated molecular weight of 40,095.7. Omp40 forms an oligomeric structure of about 120 kDa that dissociates into the monomer (40 kDa) by heating in the presence of sodium dodecyl sulfate. The degree of identity of Omp40 amino acid sequence to porins from enterobacteria was only 22%. Nevertheless, multiple alignments of this sequence with those from several OmpC porins showed several important features conserved in the T. ferrooxidans surface protein, such as the approximate locations of 16 transmembrane beta strands, eight loops, including a large external L3 loop, and eight turns which allowed us to propose a putative 16-stranded beta-barrel porin structure for the protein. These results together with the previously known capacity of Omp40 to form ion channels in planar lipid bilayers strongly support its role as a porin in this chemolithoautotrophic acidophilic microorganism. Some characteristics of the Omp40 protein, such as the presence of a putative L3 loop with an estimated isoelectric point of 7.21 allow us to speculate that this can be the result of an adaptation of the acidophilic T. ferrooxidans to prevent free movement of protons across its outer membrane.

FIG. 1

Solubilization of Omp40 at different temperatures. The purified outer membrane fraction from T. ferrooxidans was solubilized in Laemmli buffer at 20°C (lanes a), 37°C (lanes b), 56°C (lanes c), 75°C (lanes d), and 100°C (lanes e). The proteins were then resolved by SDS-PAGE and were stained with Coomassie blue (A) or were transferred to a PVDF membrane, followed by reaction with antiserum against Omp40 and colorimetric development as described in Materials and Methods (B). Numbers to the left indicate molecular mass markers in kilodaltons. The migrating position of Omp40 is indicated by an arrow, and its trimeric form is indicated by a filled dot.

FIG. 2

Nucleotide and deduced amino acid sequences of the omp-40 gene. The black dot indicates the putative transcription initiation site. The signal peptide sequence recognized by a putative signal peptidase is underlined. The possible ribosomal binding site is shaded.

FIG. 3

Determination of a putative transcription initiation site for omp40. RNA was purified from cells of T. ferrooxidans, and the cDNA was obtained with reverse transcriptase and a primer as described in Materials and Methods. The purified DNA was tailed with dCTP and, after amplification by PCR, was used for nucleotide sequencing. The Omp40-Ext1 and Omp40-Ext2 primers were used in combination with the 5′ RACE anchored primer to generate the sequence. Lanes A, C, G, and T show the sequence ladders generated. The relevant DNA sequence is shown on the right, and the position of the possible start site is indicated with an asterisk.

FIG. 4

Expression of T. ferrooxidans omp-40 gene in E. coli. The omp-40 gene containing the signal peptide coding region (lanes 3, b, and e) or without this fragment (lanes 2, a, and d) were amplified by PCR using Pwo polymerase and a low number of cycles. The amplified fragments were then cloned in the pET21a plasmid which was employed to transform E. coli strain BL21(DE3)/pLysS. Control E. coli cells transformed with pET21a without the insert (lanes 1 and c) were also used. All of the strains were grown for 2 h in the presence (+) or in the absence (−) of 1 mM IPTG added at the half-logarithmic phase of growth as indicated. The total cell proteins (A and B) or the outer membrane fraction (C and D) from each bacterial strain were separated by SDS-PAGE and stained with Coomassie blue (A and C) or were subjected to Western blotting employing antiserum against Omp40 (B and D). Some of the samples (C and D) were denatured before electrophoresis at 45°C (lanes a and b) or at 95°C (lanes c, d, and e). The arrow indicates the monomeric form of Omp40, and the filled dot indicates its trimeric form.

FIG. 5

Multiple amino acid sequence alignment of omp-40 with potential OmpC porin homologues. Organisms are indicated as follows: ST, Salmonella typhimurium (EMBL accession no. AF039309); STY, Salmonella typhi (SwissProt accession no. 052503); EC, E. coli (SwissProt accession no. P06996); KP, Klebsiella pneumoniae (SwissProt accession no. Q48473); RA, Rahnella aquatilis (SwissProt accession no. 033507); SM, Serratia marcescens (SwissProt accession no. Q54471); and TF, T. ferrooxidans. Conserved residues present in at least four of the sequences compared, including Omp40, are in boldface. The β strands, loops, and turns present in the previously known porins are indicated.

FIG. 6

A proposed model for the predicted folding of Omp40 from T. ferrooxidans. The tentative β-strands array was located on the basis of the proposal by Paul and Rosenbusch (26) and by comparison with other known models for porins (8). The conserved amino acid residues indicated in boldface in Fig. 5 are indicated here in boldface and circled.