Genetic relationship between the 53- and 49-kilodalton forms of exoenzyme S from Pseudomonas aeruginosa.
Exoenzyme S is an ADP-ribosylating extracellular protein of Pseudomonas aeruginosa that is produced as two immunologically related forms, a 49-kDa enzymatically active form and a 53-kDa inactive form. The postulated relationship between the two proteins involves a carboxy-terminal proteolytic cleavage of the 53-kDa precursor to produce an enzymatically active 49-kDa protein. To determine the genetic relationship between the two forms of exoenzyme S, exoS (encoding the 49-kDa form) was used as a probe in Southern blot analyses of P. aeruginosa chromosomal digests. Cross-hybridizing bands were detected in chromosomal digests of a strain of P. aeruginosa in which exoS had been deleted by allelic exchange. A chromosomal bank was prepared from the exoS deletion strain, 388deltaexoS::TC, and screened with a probe internal to exoS. Thirteen clones that cross-hybridized with the exoS probe were identified. One representative clone contained the open reading frame exoT; this open reading frame encoded a protein of 457 amino acids which showed 75% amino acid identity to ExoS. The exoT open reading frame, cloned into a T7 expression system, produced a 53-kDa protein in Escherichia coli, termed Exo53, which reacted to antisera against exoenzyme S. A histidine-tagged derivative of recombinant Exo53 possessed approximately 0.2% of the ADP-ribosyltransferase activity of recombinant ExoS. Inactivation of exoT in an allelic-replacement strain resulted in an Exo53-deficient phenotype without modifying the expression of ExoS. These studies prove that the 53- and 49-kDa forms of exoenzyme S are encoded by separate genes. In addition, this is the first report of the factor-activating-exoenzyme-S-dependent ADP-ribosyltransferase activity of the 53-kDa form of exoenzyme S.
Yahr
T L
TL
Department of Microbiology, Medical College of Wisconsin, Milwaukee 53226, USA.
Barbieri
J T
JT
Frank
D W
DW
D036002:ADP Ribose Transferases; D000595:Amino Acid Sequence; D001427:Bacterial Toxins; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D007527:Isoenzymes / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D011065:Poly(ADP-ribose) Polymerases / Q000235:genetics; D011550:Pseudomonas aeruginosa / Q000201:enzymology / Q000235:genetics*; D011994:Recombinant Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D001427:Bacterial Toxins; D007527:Isoenzymes; D011994:Recombinant Proteins; D036002:ADP Ribose Transferases; D011065:Poly(ADP-ribose) Polymerases; C444820:exoenzyme S