EXOENZYME-S OF PSEUDOMONAS-AERUGINOSA IS SECRETED BY A TYPE-III PATHWAY

Exoenzyme S is an extracellular ADP-ribosyltransferase of Pseudomonas aeruginosa. Transposon mutagenesis of P. aeruginosa 388 was used to id entify genes required for exoenzyme S production. Five Tn5Tc insertion mutants were isolated which exhibited an exoenzyme S-deficient phenot ype (388::Tn5Tc 469, 550, 3453, 4885, and 5590), Mapping experiments d emonstrated that 388::Tn5Tc 3453, 4885, and 5590 possessed insertions within a 5.0 kb EcoRI fragment that is not contiguous with the exoenzy me S trans-regulatory operon, 388::Tn5Tc 469 and 550 mapped to a regio n downstream of the trans-regulatory operon which has been previously shown to contain a promoter region that is co-ordinately regulated wit h exoenzyme S synthesis, Nucleotide sequence analysis of a 7.2 kb regi on flanking the 388::Tn5Tc 469 and 550 insertions, identified 12 conti guous open reading frames (ORFs), Database searches indicated that the first ORF, ExsD, is unique. The other 11 ORFs demonstrated high homol ogy to the YscB-L proteins of the yersiniae Yop type III export appara tus, RNase-protection analysis of wild-type and mutant strains indicat ed that exsD and pscB-L form an operon. To determine whether ExoS was exported by a type III mechanism, derivatives consisting of internal d eletions or lacking amino- or carboxy-terminal residues were expressed in P. aeruginosa, Deletion analyses indicated that the amino-terminal nine residues are required for ExoS export, Combined data from mutage nesis, regulatory, expression, and sequence analyses provide strong ev idence that P. aeroginosa possesses a type III secretion apparatus whi ch is required for the export of exoenzyme S and potentially other co- ordinately regulated proteins.