Genetic relationship between the 53- and 49-kilodalton forms of exoenzyme S from Pseudomonas aeruginosa

T L Yahr; J T Barbieri; D W Frank

doi:10.1128/jb.178.5.1412-1419.1996

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Genetic relationship between the 53- and 49-kilodalton forms of exoenzyme S from Pseudomonas aeruginosa

Journal article

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Genetic relationship between the 53- and 49-kilodalton forms of exoenzyme S from Pseudomonas aeruginosa

T L Yahr, J T Barbieri and D W Frank

Journal of bacteriology, Vol.178(5), pp.1412-1419

03/1996

DOI: 10.1128/jb.178.5.1412-1419.1996

PMCID: PMC177816

PMID: 8631719

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Abstract

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.

Recombinant Proteins - metabolism

Amino Acid Sequence

Blotting, Southern

Genes, Bacterial

Isoenzymes - genetics

Molecular Sequence Data

Sequence Analysis, DNA

Sequence Homology, Amino Acid

Poly(ADP-ribose) Polymerases - genetics

Pseudomonas aeruginosa - genetics

Bacterial Toxins

Escherichia coli - genetics

Isoenzymes - metabolism

Pseudomonas aeruginosa - enzymology

ADP Ribose Transferases

Base Sequence

Cloning, Molecular

Mutation

Details

Title: Subtitle: Genetic relationship between the 53- and 49-kilodalton forms of exoenzyme S from Pseudomonas aeruginosa
Creators: T L Yahr - Department of Microbiology, Medical College of Wisconsin, Milwaukee 53226, USA
J T Barbieri
D W Frank
Resource Type: Journal article
Publication Details: Journal of bacteriology, Vol.178(5), pp.1412-1419
Publisher: United States
DOI: 10.1128/jb.178.5.1412-1419.1996
PMID: 8631719
PMCID: PMC177816
ISSN: 0021-9193
eISSN: 1098-5530
Grant note: R29-AI31665 / NIAID NIH HHS R01 AI31062 / NIAID NIH HHS KO4-AI01289 / NIAID NIH HHS
Language: English
Date published: 03/1996
Academic Unit: Microbiology and Immunology
Record Identifier: 9984002394302771

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