title: Comparison of proteolytic activities produced by entomopathogenic Photorhabdus bacteria: strain- and phase-dependent heterogeneity in composition and activity of four enzymes.
abstract: Twenty strains (including eight phase variant pairs) of nematode-symbiotic and insect-pathogenic Photorhabdus bacteria were examined for the production of proteolytic enzymes by using a combination of several methods, including gelatin liquefaction, zymography coupled to native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and activity measurement with two chromogen substrate types. Four protease activities (approximately 74, approximately 55, approximately 54, and approximately 37 kDa) could be separated. The N-terminal sequences of three of the proteases were determined, and a comparison with sequences in databases allowed identification of these proteases as HEXXH metallopeptidases. Thus, the 74-kDa protease (described formerly as Php-B [J. Marokhazi, G. Koczan, F. Hudecz, L. Graf, A. Fodor, and I. Venekei, Biochem. J. 379:633-640, 2004) is an ortholog of OpdA, a member the thimet oligopeptidase family, and the 55-kDa protease is an ortholog of PrtA, a HEXXH+H peptidase in clan MB (metzincins), while the 37-kDa protease (Php-C) belongs to the HEXXH+E peptidases in clan MA. The 54-kDa protease (Php-D) is a nonmetalloenzyme. PrtA and Php-C were zymographically detected, and they occurred in several smaller forms as well. OpdA could not be detected by zymography. PrtA, Php-C, and Php-D were secreted proteases; OpdA, in contrast, was an intracellular enzyme. OpdA activity was found in every strain tested, while Php-D was detected only in the Brecon/1 strain. There was significant strain variation in the secretion of PrtA and Php-C activities, but reduced activity or a lack of activity was not specific to secondary-phase variants. The presence of PrtA, OpdA, and Php-C activities could be detected in the hemolymph of Galleria melonella larvae 20 to 40 h postinfection. These proteases appear not to be directly involved in the pathogenicity of Photorhabdus, since strains or phase variants lacking any of these proteases do not show reduced virulence when they are injected into G. melonella larvae.
authors.affiliation: Department of Biochemistry, Eötvös Loránd University, Budapest, Hungary.
authors.forename: Judit
authors.initials: J
authors.lastname: Marokházi
authors.forename: Katalin
authors.initials: K
authors.lastname: Lengyel
authors.forename: Szilvia
authors.initials: S
authors.lastname: Pekár
authors.forename: Gabriella
authors.initials: G
authors.lastname: Felföldi
authors.forename: András
authors.initials: A
authors.lastname: Patthy
authors.forename: László
authors.initials: L
authors.lastname: Gráf
authors.forename: András
authors.initials: A
authors.lastname: Fodor
authors.forename: István
authors.initials: I
authors.lastname: Venekei
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D005780:Gelatin / Q000378:metabolism; D006458:Hemolymph / Q000382:microbiology; D007814:Larva / Q000382:microbiology; D008969:Molecular Sequence Data; D009036:Moths / Q000382:microbiology*; D010447:Peptide Hydrolases / Q000737:chemistry / Q000378:metabolism*; D020637:Photorhabdus / Q000145:classification / Q000201:enzymology* / Q000254:growth & development / Q000472:pathogenicity; D017172:Rhabditoidea / Q000382:microbiology; D013379:Substrate Specificity; D013559:Symbiosis; D014774:Virulence
chemical_list: D005780:Gelatin; D010447:Peptide Hydrolases