Cloning of an Aeromonas hydrophila type IV pilus biogenesis gene cluster: complementation of pilus assembly functions and characterization of a type IV leader peptidase/N-methyltransferase required for extracellular protein secretion.
Aeromonas hydrophila secretes several extracellular proteins that are associated with virulence including an enterotoxin, a protease, and the hole-forming toxin, aerolysin. These degradative enzymes and toxins are exported by a conserved pathway found in many Gram-negative bacteria. In Pseudomonas aeruginosa this export pathway and type IV pilus biogenesis are dependent on the product of the pilD gene. PilD is a bifunctional enzyme that processes components of the extracellular secretory pathway as well as a type IV prepilin. An A. hydrophila genomic library was transferred into a P. aeruginosa pilD mutant that is defective for type IV pilus biogenesis. The A. hydrophila pilD homologue, tapD, was identified by its ability to complement the pilD mutation in P. aeruginosa. Transconjugants containing tapD were sensitive to the type IV pilus-specific phage, PO4. Sequence data revealed that tapD is part of a cluster of genes (tapABCD) that are homologous to P. aeruginosa type IV pilus biogenesis genes (pilABCD). We showed that TapB and TapC are functionally homologous to P. aeruginosa PilB and PilC, the first such functional complementation of pilus assembly demonstrated between bacteria that express type IV pili. In vitro studies revealed that TapD has both endopeptidase and N-methyltransferase activities using P. aeruginosa prepilin as substrate. Furthermore, we show that tapD is required for extracellular secretion of aerolysin and protease, indicating that tapD may play an important role in the virulence of A. hydrophila.
Pepe
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US Department of Commerce, NOAA, Seattle, Washington 98112-2097, USA.
Eklund
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Strom
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D016980:Aeromonas hydrophila / Q000235:genetics*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001427:Bacterial Toxins / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D010450:Endopeptidases / Q000235:genetics*; D036781:Fimbriae Proteins; D010861:Fimbriae, Bacterial / Q000235:genetics*; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D008780:Methyltransferases / Q000235:genetics*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics*; D005810:Multigene Family; D009154:Mutation; D010088:Oxidoreductases; D052899:Pore Forming Cytotoxic Proteins; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D009097:Multienzyme Complexes; D052899:Pore Forming Cytotoxic Proteins; C079208:pilC protein, Neisseria gonorrhoeae; D036781:Fimbriae Proteins; C032676:aerolysin; D010088:Oxidoreductases; C068255:pilB protein, Bacteria; D008780:Methyltransferases; D010450:Endopeptidases; C067127:prepilin peptidase protein, Bacteria; C099395:TapD protein, Aeromonas