This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Caldas, C.
Right arrow Articles by Simões, N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Caldas, C.
Right arrow Articles by Simões, N.
Agricola
Right arrow Articles by Caldas, C.
Right arrow Articles by Simões, N.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, March 2002, p. 1297-1304, Vol. 68, No. 3
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.3.1297-1304.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Purification and Characterization of an Extracellular Protease from Xenorhabdus nematophila Involved in Insect Immunosuppression

C. Caldas, A. Cherqui,,{dagger} A. Pereira, and N. Simões*

Centro de Investigação de Recursos Naturais and Departamento de Biologia, Universidade dos Açores, 9501-801 Ponta Delgada, Açores, Portugal

Received 1 August 2001/ Accepted 20 December 2001

Xenorhabdus nematophila, a bacterium pathogenic for insects associated with the nematode Steinernema carpocapsae, releases high quantities of proteases, which may participate in the virulence against insects. Zymogram assays and cross-reactions of antibodies suggested that two distinct proteases were present. The major one, protease II, was purified and shown to have a molecular mass of 60 kDa and an estimated isoelectric point of 8.5. Protease II digested the chromogenic substrate N-tosyl-Gly-Pro-Arg-paranitroanilide (pNA) with Vmax and Km values of 0.0551 µM/min and 234 µM, respectively, and the substrate DL-Val-Leu-Arg-pNA with Vmax and Km values of 0.3830 µM/min and 429 µM, respectively. Protease II activity was inhibited 93% by Pefabloc SC and 45% by chymostatin. The optimum pH for protease II was 7, and the optimum temperature was 23°C. Proteolytic activity was reduced by 90% at 60°C for 10 min. Sequence analysis was performed on four internal peptides that resulted from the digestion of protease II. Fragments 29 and 45 are 75 and 68% identical to alkaline metalloproteinase produced by Pseudomonas aeruginosa. Fragment 29 is 79% identical to a metalloprotease of Erwinia amylovora and 75% identical to the protease C precursor of Erwinia chrysanthemi. Protease II showed no toxicity to hemocytes but destroyed antibacterial activity on the hemolymph of inoculated insects' larvae and reduced 97% of the cecropin A bacteriolytic activity.

* Corresponding author. Mailing address: Departamento de Biologia, Universidade dos Açores, 9501-801 Ponta Delgada, Açores, Portugal. Phone: 351 296650119. Fax: 351 296650100. E-mail: simoes{at}notes.uac.pt.

{dagger} Present address: Faculté des Sciences, Laboratoire de Biologie des Entomophages, Université de Picardie Jules Verne, Amiens 80039, France.

Applied and Environmental Microbiology, March 2002, p. 1297-1304, Vol. 68, No. 3
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.3.1297-1304.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Richards, G. R., Herbert, E. E., Park, Y., Goodrich-Blair, H. (2008). Xenorhabdus nematophila lrhA Is Necessary for Motility, Lipase Activity, Toxin Expression, and Virulence in Manduca sexta Insects. J. Bacteriol. 190: 4870-4879 [Abstract] [Full Text]  
  • Hussain, M. B. B. M., Zhang, H.-B., Xu, J.-L., Liu, Q., Jiang, Z., Zhang, L.-H. (2008). The Acyl-Homoserine Lactone-Type Quorum-Sensing System Modulates Cell Motility and Virulence of Erwinia chrysanthemi pv. zeae. J. Bacteriol. 190: 1045-1053 [Abstract] [Full Text]  
  • Herbert, E. E., Cowles, K. N., Goodrich-Blair, H. (2007). CpxRA Regulates Mutualism and Pathogenesis in Xenorhabdus nematophila. Appl. Environ. Microbiol. 73: 7826-7836 [Abstract] [Full Text]  
  • Marokhazi, J., Lengyel, K., Pekar, S., Felfoldi, G., Patthy, A., Graf, L., Fodor, A., Venekei, I. (2004). Comparison of Proteolytic Activities Produced by Entomopathogenic Photorhabdus Bacteria: Strain- and Phase-Dependent Heterogeneity in Composition and Activity of Four Enzymes. Appl. Environ. Microbiol. 70: 7311-7320 [Abstract] [Full Text]  
  • Orchard, S. S., Goodrich-Blair, H. (2004). Identification and Functional Characterization of a Xenorhabdus nematophila Oligopeptide Permease. Appl. Environ. Microbiol. 70: 5621-5627 [Abstract] [Full Text]  
  • Bowen, D. J., Rocheleau, T. A., Grutzmacher, C. K., Meslet, L., Valens, M., Marble, D., Dowling, A., ffrench-Constant, R., Blight, M. A. (2003). Genetic and biochemical characterization of PrtA, an RTX-like metalloprotease from Photorhabdus. Microbiology 149: 1581-1591 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»