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Lytic enzyme discovery through multigenomic sequence analysis in Clostridium perfringens

  • Biotechnologically Relevant Enzymes and Proteins
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Abstract

With their ability to lyse Gram-positive bacteria, phage lytic enzymes (or lysins) have received a great deal of attention as novel anti-infective agents. The number of known genes encoding these peptidoglycan hydrolases has increased markedly in recent years, due in large part to advances in DNA sequencing technology. As the genomes of more and more bacterial species/strains are sequenced, lysin-encoding open reading frames (ORFs) can be readily identified in lysogenized prophage regions. In the current study, we sought to assess lysin diversity for the medically relevant pathogen Clostridium perfringens. The sequenced genomes of nine C. perfringens strains were computationally mined for prophage lysins and lysin-like ORFs, revealing several dozen proteins of various enzymatic classes. Of these lysins, a muramidase from strain ATCC 13124 (termed PlyCM) was chosen for recombinant analysis based on its dissimilarity to previously characterized C. perfringens lysins. Following expression and purification, various biochemical properties of PlyCM were determined in vitro, including pH/salt-dependence and temperature stability. The enzyme exhibited activity at low μg/ml concentrations, a typical value for phage lysins. It was active against 23 of 24 strains of C. perfringens tested, with virtually no activity against other clostridial or non-clostridial species. Overall, PlyCM shows potential for development as an enzybiotic agent, demonstrating how expanding genomic databases can serve as rich pools for biotechnologically relevant proteins.

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Notes

  1. ORFs that encoded an extended C-terminal region without a pfam-recognized binding domain were not excluded on this fact alone. There exist a number of Gram-positive phage lysins for which this is indeed the case. Most likely, these C-termini do possess a cell-wall binding functions, albeit ones that have not yet been characterized and organized into conserved protein families.

  2. It should be emphasized that l-arginine, in itself, had no affect on bacterial turbidity or viability when cells were exposed to the amino acid for a short time in buffered solution (as in previous experiments).

  3. This is the same BCN5 mentioned previously in the text. It demonstrated sequence homology to the C-terminus of another C. perfringens lysin considered here (NP_562054, a type 2 alanine–amidase of evident proviral origin).

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Acknowledgments

This research was funded by NIH/NIAID grants AI057472 and AI11822 to VAF. JES acknowledges the kind support of the NIH MSTP program (Weill Cornell/Rockefeller/Sloan-Kettering grant GM 07739). The authors would like to thank Dr. Davise Larone of New York Hospital for providing clostridial strains. We also acknowledge Prof. Ezio Bottarelli of the University of Parma for his valued insights, as well as Ms. Cinzia Reverberi and Mr. Roberto Lurisi for technical assistance. Electron micrographs were acquired by Ms. Eleana Sphicas of the Rockefeller University Electron Microscopy Core Facility.

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  1. Laboratory of Bacterial Pathogenesis and Immunology, The Rockefeller University, 1230 York Avenue, Box 172, New York, NY, 10065, USA

    Jonathan E. Schmitz, Kareem R. Rumah & Vincent A. Fischetti

  2. Department of Animal Health, Microbiology and Immunology Section, University of Parma, Faculty of Veterinary Medicine, Via del Taglio, 10, 43100, Parma, Italy

    Maria Cristina Ossiprandi

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  1. Jonathan E. Schmitz
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  2. Maria Cristina Ossiprandi
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  3. Kareem R. Rumah
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Correspondence to Jonathan E. Schmitz.

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Schmitz, J.E., Ossiprandi, M.C., Rumah, K.R. et al. Lytic enzyme discovery through multigenomic sequence analysis in Clostridium perfringens . Appl Microbiol Biotechnol 89, 1783–1795 (2011). https://doi.org/10.1007/s00253-010-2982-8

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