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 (ϳ74, ϳ55, ϳ54, and ϳ37 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 ( -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.

Transcriptional Analysis of a Photorhabdus sp. Variant Reveals Transcriptional Control of Phenotypic Variation and Multifactorial Pathogenicity in Insects

“…Finally, extracellular activities, such as hemolysis and lipase and protease activities, were found to be weaker in the phenotypic variants (5). The activities of two metalloproteases, PrtA and PrtS, were attenuated in the supernatant of the P. temperata K122 phenotypic variant (9,49). Our data suggest that the transcription of prtA (plu0655) is not modified but that the prtS gene (plu1382) and its downstream gene (plu1381), which probably belongs to the same transcription unit, are less strongly transcribed in VAR* than in TT01␣.…”

A Metalloprotease Secreted by the Insect Pathogen Photorhabdus luminescens Induces Melanization

“…When the protease inhibitor was removed, the protein underwent an apparent autocatalytic event, clipping off the N-terminal 48 amino acids. We determined the N-terminal sequence of the active rPrtS to be SSDDS, which corresponds to the N-terminal sequence determined for this protein by Marokhazi et al (13). The apparent molecular weight of the cleaved rPrtS correlated with that of the native PrtS found in culture supernatants; we are confident that this result reflects the actual N-terminal sequence of the active protein.…”

“…We use the PrtS designation here. The levels of PrtS produced by Photorhabdus strains vary (13), and for undetermined reasons this protein is made in abundance by TT01.…”

“…Many potential bacterial products are implicated in this killing, including a toxin termed "makes caterpillars floppy," hemolysins, RTX (repeats in toxin) family members, and large toxin complexes (Tc) (1-3, 5, 7). Photorhabdus also secretes numerous proteases and lipases and is able to inhibit competitor bacteria by synthesizing various antibiotics and bacteriocins (7)(8)(9)13).…”

Phospholipases C from Pseudomonas aeruginosa and Bacillus cereus isolates, chromosome-mediated enzymes with roles in virulence

“…Protease activity was detected by the gelatin hydrolysis assay on plates as described by Marokhazi et al (26). Gelatin nutrient agar plates were spot-inoculated with 20 µL of partially purified enzyme preparations.…”