Abstract
Photorhabdus luminescens is a pathogenic bacterium that produces many toxic proteins. The mono-ADP-ribosyltransferases (mARTs) are an enzyme class produced by numerous pathogenic bacteria and participate in disease in plants and animals, including humans. Herein we report a novel mART from P. luminescens called Photox. This 46-kDa toxin shows high homology to other actin-targeting mARTs in hallmark catalytic regions and a similar core catalytic fold. Furthermore, Photox shows in vivo cytotoxic activity against yeast, with protection occurring when catalytic residues are substituted with alanine. In vitro, enzymatic activity (k(cat), 1680 +/- 75 min(-1)) is higher than that of the related iota toxin, and diminishes by nearly 14,000-fold following substitution of the catalytic Glu (E355A). This toxin specifically ADP-ribosylates monomeric alpha-skeletal actin and nonmuscle beta- and gamma-actin at Arg(177), inhibiting regular polymerization of actin filaments. These results indicate that Photox is indeed an ADP-ribosyltransferase, making it the newest member of the actin-targeting mART family.
Highlights
Among other toxins, Photorhabdus bacteria produce toxin complexes, high molecular weight, multisubunit, insecticidal toxins [4], some of which show oral toxicity in the same range as Bacillus thuringiensis endotoxins [5], as well as the “makes caterpillars floppy” toxin, responsible for insect midgut destruction [6]
Because overall primary sequence identity among mART family members is most often low, identification of new members must rely on a shared core structure (SCOP code d.166.1.1.), sequence identity in several key catalytic regions, and pathogenicity of the organism as a positive indicator
Plu0822 Encodes a Putative mART—The gene plu0822 of P. luminescens strain TT01 was found to encode a protein of 408 residues (45.9 kDa) with high identity to other mART toxins
Summary
Identification and Modeling in Silico—The plu0822 gene was identified as a putative mART in silico after searching the Genomic Threading Data base [23] using SCOP code d.166.1.1 [24]. Lysate was centrifuged for 25 min at 14,000 ϫ g and the pellet was resuspended in 20 ml of inclusion body wash buffer (50 mM Tris-HCl, pH 7.5, 2 mM EDTA, 100 mM NaCl, 0.05% deoxycholate, 0.5 mg/ml of lysozyme). Native PAGE—Actin (10 mM Tris-HCl, 0.2 mM CaCl2, 0.2 mM ATP, 0.2 mM -ME, 0.2 mM ⑀-NADϩ) was incubated for 1 h at ambient temperature in the presence or absence of 1 nM Photox. Actin and ADPr-actin solutions were brought to 25 mM Tris-HCl, pH 8.0, 50 mM KCl, 1 mM EGTA, 2 mM MgCl2, 0.2 mM ATP, 0.2 mM -ME using a 10-fold stock of polymerization salts. Actin samples in buffer G (2 mM Tris-HCl pH, 8.0, 0.2 mM ATP, 0.2 mM CaCl2, 0.2 mM -ME) were diluted and brought to 2.5% pyrene-actin. Samples were treated with 1 ng of Photox and the pyrene fluorescence intensity was measured as described previously. The 4 l of stained cell suspension was visualized with a Nikon Eclipse 6600 epifluorescent microscope
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