Archives of Microbiology

, Volume 195, Issue 10–11, pp 675–681

Expression of a Clostridium perfringens genome-encoded putative N-acetylmuramoyl–l-alanine amidase as a potential antimicrobial to control the bacterium

  • Glenn E. Tillman
  • Mustafa Simmons
  • Johnna K. Garrish
  • Bruce S. Seal
Original Paper

Abstract

Clostridium perfringens is a gram-positive, spore-forming anaerobic bacterium that plays a substantial role in non-foodborne human, animal, and avian diseases as well as human foodborne disease. Previously discovered C. perfringens bacteriophage lytic enzyme amino acid sequences were utilized to identify putative prophage lysins or autolysins by BLAST analyses encoded by the genomes of C. perfringens isolates. A predicted N-acetylmuramoyl–l-alanine amidase or MurNAc–LAA (also known as peptidoglycan aminohydrolase, NAMLA amidase, NAMLAA, amidase 3, and peptidoglycan amidase; EC 3.5.1.28) was identified that would hydrolyze the amide bond between N-acetylmuramoyl and l-amino acids in certain cell wall glycopeptides. The gene encoding this protein was subsequently cloned from genomic DNA of a C. perfringens isolate by polymerase chain reaction, and the gene product (PlyCpAmi) was expressed to determine if it could be utilized as an antimicrobial to control the bacterium. By spot assay, lytic zones were observed for the purified amidase and the E. coli expression host cellular lysate containing the amidase gene. Turbidity reduction and plate counts of C. perfringens cultures were significantly reduced by the expressed protein and observed morphologies for cells treated with the amidase appeared vacuolated, non-intact, and injured compared to the untreated cells. Among a variety of C. perfringens strains, there was little gene sequence heterogeneity that varied from 1 to 21 nucleotide differences. The results further demonstrate that it is possible to discover lytic proteins encoded in the genomes of bacteria that could be utilized to control bacterial pathogens.

Keywords

Enzybiotic Alternative antimicrobial Bacteriophage Prophage Autolysin Animal/human health 

Supplementary material

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Copyright information

© Springer-Verlag Berlin Heidelberg (outside the USA) 2013

Authors and Affiliations

  • Glenn E. Tillman
    • 1
    • 2
  • Mustafa Simmons
    • 1
    • 2
  • Johnna K. Garrish
    • 1
  • Bruce S. Seal
    • 1
  1. 1.Poultry Microbiological Safety Research Unit, Richard B. Russell Agricultural Research Center, Agricultural Research ServiceUSDAAthensUSA
  2. 2.Outbreak Section of the Eastern Laboratory, Food Safety and Inspection Service, Richard B. Russell Agricultural Research CenterUSDAAthensUSA

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