Overexpression of Methanococcus voltae flagellin subunits in Escherichia coli and Pseudomonas aeruginosa: a source of archaeal preflagellin.
Methanococcus voltae is a flagellated member of the Archaea. Four highly similar flagellin genes have previously been cloned and sequenced, and the presence of leader peptides has been demonstrated. While the flagellins of M. voltae are predicted from their gene sequences to be approximately 22 to 25 kDa, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of purified flagella revealed flagellin subunits with apparent molecular masses of 31 and 33 kDa. Here we describe the expression of a M. voltae flagellin in the bacteria Escherichia coli and Pseudomonas aeruginosa. Both of these systems successfully generated a specific expression product with an apparently uncleaved leader peptide migrating at approximately 26.5 kDa. This source of preflagellin was used to detect the presence of preflagellin peptidase activity in the membranes of M. voltae. In addition to the native flagellin, a hybrid flagellin gene containing the sequence encoding the M. voltae FlaB2 mature protein fused to the P. aeruginosa pilin (PilA) leader peptide was constructed and transformed into both wild-type P. aeruginosa and a prepilin peptidase (pilD) mutant of P. aeruginosa. Based on migration in SDS-PAGE, the leader peptide appeared to be cleaved in the wild-type cells. However, the archaeal flagellin could not be detected by immunoblotting when expressed in the pilD mutant, indicating a role of the peptidase in the ultimate stability of the fusion product. When the +5 position of the mature flagellin portion of the pilin-flagellin fusion was changed from glycine to glutamic acid (as in the P. aeruginosa pilin) and expressed in both wild-type and pilD mutant P. aeruginosa, the product detected by immunoblotting migrated slightly more slowly in the pilD mutant, indicating that the fusion was likely processed by the prepilin peptidase present in the wild type. Potential assembly of the cleaved fusion product by the type IV pilin assembly system in a P. aeruginosa PilA-deficient strain was tested, but no filaments were noted on the cell surface by electron microscopy.
Department of Microbiology and Immunology, Queen's University, Kingston, Ontario, Canada K7L 3N6.
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Bayley
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Jarrell
D000595:Amino Acid Sequence; D019843:Archaeal Proteins; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D019641:DNA, Archaeal / Q000032:analysis; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D036781:Fimbriae Proteins; D005408:Flagellin / Q000235:genetics / Q000378:metabolism*; D015151:Immunoblotting; D017017:Methanococcus / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D011498:Protein Precursors / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA
D019843:Archaeal Proteins; D001426:Bacterial Proteins; D019641:DNA, Archaeal; D004268:DNA-Binding Proteins; C489684:FlaB2 protein, archaeal; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D005408:Flagellin; D036781:Fimbriae Proteins; D010450:Endopeptidases