title: Purification and characterization of two distinct metalloproteases secreted by the entomopathogenic bacterium Photorhabdus sp. strain Az29.
abstract: Photorhabdus sp. strain Az29 is symbiotic with an Azorean nematode of the genus Heterorhabditis in a complex that is highly virulent to insects even at low temperatures. The virulence of the bacteria is mainly attributed to toxins and bacterial enzymes secreted during parasitism. The bacteria secrete proteases during growth, with a peak at the end of the exponential growth phase. Protease secretion was higher in cultures growing at lower temperatures. At 10 degrees C the activity was highest and remained constant for over 7 days, whereas at 23 and 28 degrees C it showed a steady decrease. Two proteases, PrtA and PrtS, that are produced in the growth medium were purified by liquid chromatography. PrtA was inhibited by 1,10-phenantroline and by EDTA and had a molecular mass of 56 kDa and an optimal activity at pH 9 and 50 degrees C. Sequences of three peptides of PrtA showed strong homologies with alkaline metalloproteases from Photorhabdus temperata K122 and Photorhabdus luminescens W14. Peptide PrtA-36 contained the residues characteristic of metzincins, known to be involved in bacterial virulence. In vitro, PrtA inhibited antibacterial factors of inoculated Lepidoptera and of cecropins A and B. PrtS had a molecular mass of 38 kDa and was inhibited by 1,10-phenanthroline but not by EDTA. Its activity ranged between 10 and 80 degrees C and was optimal at pH 7 and 50 degrees C. PrtS also destroyed insect antibacterial factors. Three fragments of PrtS showed homology with a putative metalloprotease of P. luminescens TTO1. Polyclonal antibody raised against PrtA did not recognize PrtS, showing they are distinct molecules.
authors.affiliation: CIRN and Departamento de Biologia, Universidade dos Açores, 9501-801 Ponta Delgada, Azores, Portugal.
authors.forename: C M
authors.initials: CM
authors.lastname: Cabral
authors.forename: A
authors.initials: A
authors.lastname: Cherqui
authors.forename: A
authors.initials: A
authors.lastname: Pereira
authors.forename: N
authors.initials: N
authors.lastname: Simões
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D023181:Antimicrobial Cationic Peptides / Q000037:antagonists & inhibitors; D006458:Hemolymph / Q000502:physiology; D008666:Metalloendopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D008969:Molecular Sequence Data; D020637:Photorhabdus / Q000201:enzymology* / Q000254:growth & development; D013696:Temperature
chemical_list: D023181:Antimicrobial Cationic Peptides; C031161:cecropin A; D008666:Metalloendopeptidases; C079508:PrtA metalloprotease