TgpA, a protein with a eukaryotic-like transglutaminase domain, plays a critical role in the viability of Pseudomonas aeruginosa.
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The purified periplasmic domain (aa 180-544) of PA2873 is active in a colorimetric transglutaminase assay, at about 45% of the guinea-pig reference enzyme, and the activity is not abolished by EDTA, so it is calcium-independent.
"In actual fact, 2.5 µg of TG180–544, equal to reference gpTGase, showed TGase activity that differed significantly from the negative control and was about 45% of gpTGase activity in the absence of EDTA."
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The assay measures incorporation of biotin-cadaverine into immobilised CBZ-Gln-Gly, that is gamma-glutamyl transfer to a primary amine, not protein-to-protein cross-linking.
"Transglutaminase activity (TGase; EC 2.3.2.13) was assayed by a Transglutaminase Colorimetric Microassay Kit (Covalab) which uses immobilized N-carbobenzoxy(CBZ)-Gln-Gly as amine acceptor and biotin-conjugated cadaverine as amine donor."
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TgpA is detected in PAO1 membrane fractions and its periplasmic domain is accessible to trypsin shaving.
"Since all five PA2873 peptides “shaved” by trypsin belonged to the predicted outside membrane domain, this strongly supports the notion that it actually emerges from membrane itself."
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Antisense knockdown and conditional expression show tgpA is required for viability; the authors propose an essential cell-envelope function and note that related TG-domain proteins act as pseudomurein endoisopeptidases or as an LPS O-antigen deamidase.
"Therefore, we suggest that TgpA takes part in an essential function linked to the cell wall."