Skip to main content
SpringerLink
Log in

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

  • Original Paper
  • Published:
Archives of Microbiology Aims and scope Submit manuscript

An Erratum to this article was published on 20 February 2014

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Adeola O, Cowieson AJ (2011) Board-invited review: opportunities and challenges in using exogenous enzymes to improve nonruminant animal production. J Anim Sci 89:3189–3218

    Article  CAS  PubMed  Google Scholar 

  • Camiade E, Peltier J, Bourgeois I, Couture-Tosi E, Courtin P, Antunes A, Chapot-Chartier MP, Dupuy B, Pons JL (2010) Characterization of Acp, a peptidoglycan hydrolase of Clostridium perfringens with N-acetylglucosaminidase activity that is implicated in cell separation and stress-induced autolysis. J Bacteriol 192:2373–2384

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Cooper KK, Bueschel DM, Songer JG (2013) Presence of Clostridium perfringens in retail chicken livers. Anaerobe 21:67–68

    Article  PubMed  Google Scholar 

  • Crowe J, Döbeli H, Gentz R, Hochuli E, Stüber D, Henco K (1994) 6xHis–Ni-NTA chromatography as a superior technique in recombinant protein expression/purification. Methods Mol Biol 31:371–387

    CAS  PubMed  Google Scholar 

  • Donovan DM, Dong S, Garrett W, Rousseau GM, Moineau S, Pritchard DG (2006) Peptidoglycan hydrolase fusions maintain their parental specificities. Appl Environ Microbiol 72:2988–2996

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Grass JE, Gould LH, Mahon BE (2013) Epidemiology of foodborne disease outbreaks caused by Clostridium perfringens, United States, 1998–2010. Foodborne Pathog Dis 10:131–136

    Article  PubMed  Google Scholar 

  • Gyles CL (2008) Antimicrobial resistance in selected bacteria from poultry. Anim Health Res Rev 9:149–158

    Article  PubMed  Google Scholar 

  • Hames BD (1990) One-dimensional polyacrylamide gel electrophoresis. In: Hames BD, Rickwood D (eds) Gel electrophoresis of proteins: a practical approach, 2nd edn. Oxford University Press, New York, pp 1–147

    Google Scholar 

  • Kane JF (1995) Effects of rare codon clusters on high-level expression of heterologous proteins in Escherichia coli. Curr Opin Biotechnol 6:494–500

    Article  CAS  PubMed  Google Scholar 

  • Koluman A, Dikici A (2013) Antimicrobial resistance of emerging foodborne pathogens: status quo and global trends. Crit Rev Microbiol 39:57–69

    Article  CAS  PubMed  Google Scholar 

  • Kristensen T, Voss H, Schwager C, Stegemann J, Sproat B, Ansorge W (1998) T7 DNA polymerase in automated dideoxy sequencing. Nucleic Acids Res 16:3487–3496

    Article  Google Scholar 

  • Kumazawa T, Masayama A, Fukuoka S, Makino S, Yoshimura T, Moriyama R (2007) Mode of action of a germination-specific cortex-lytic enzyme, SleC, of Clostridium perfringens S40. Biosci Biotechnol Biochem 71:884–892

    Article  CAS  PubMed  Google Scholar 

  • Lin YT, Labbe R (2003) Enterotoxigenicity and genetic relatedness of Clostridium perfringens isolates from retail foods in the United States. Appl Environ Microbiol 69:1642–1646

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Masayama A, Hamasaki K, Urakami K, Shimamoto S, Kato S, Makino S, Yoshimura T, Moriyama M, Moriyama R (2006) Expression of germination-related enzymes, CspA, CspB, CspC, SleC, and SleM, of Clostridium perfringens S40 in the mother cell compartment of sporulating cells. Genes Genet Syst 81:227–234

    Article  CAS  PubMed  Google Scholar 

  • Millet S, Maertens L (2011) The European ban on antibiotic growth promoters in animal feed: from challenges to opportunities. Vet J 187:143–144

    Article  PubMed  Google Scholar 

  • Miyata S, Moriyama R, Miyahara N, Makino S (1995a) A gene (sleC) encoding a spore-cortex-lytic enzyme from Clostridium perfringens S40 spores; cloning, sequence analysis and molecular characterization. Microbiology 141:2643–2650

    Article  CAS  PubMed  Google Scholar 

  • Miyata S, Moriyama R, Sugimoto K, Makino S (1995b) Purification and partial characterization of a spore cortex-lytic enzyme of Clostridium perfringens S40 spores. Biosci Biotechnol Biochem 59:514–515

    Article  CAS  PubMed  Google Scholar 

  • Myers GS, Rasko DA, Cheung JK, Ravel J, Seshadri R, DeBoy RT, Ren Q, Varga J, Awad MM, Brinkac LM, Daugherty SC, Haft DH, Dodson RJ, Madupu R, Nelson WC, Rosovitz MJ, Sullivan SA, Khouri H, Dimitrov GI, Watkins KL, Mulligan S, Benton J, Radune D, Fisher DJ, Atkins HS, Hiscox T, Jost BH, Billington SJ, Songer JG, McClane BA, Titball RW, Rood JI, Melville SB, Paulsen IT (2006) Skewed genomic variability in strains of the toxigenic bacterial pathogen, Clostridium perfringens. Genome Res 16:1031–1040

    Article  CAS  PubMed  Google Scholar 

  • National Academy of Sciences (2006) Treating infectious diseases in a microbial world: report of two workshops on novel antimicrobial therapeutics. ISBN: 0-309-65490-4, (http://www.nap.edu/catalog.php?record_id=11471)

  • Niall HD (1973) Automated Edman degradation: the protein sequenator. Methods Enzymol 27:942–1010

    Article  CAS  PubMed  Google Scholar 

  • Oakley BB, Talundzic E, Morales CA, Hiett KL, Siragusa GR, Volozhantsev NV, Seal BS (2011) Comparative genomics of four closely related Clostridium perfringens bacteriophages reveals variable evolution among core genes with therapeutic potential. BMC Genomics 12:282

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Paredes CJ, Alsaker KV, Papoutsakis ET (2005) A comparative genomic view of clostridial sporulation and physiology. Nat Rev Microbiol 3:969–978

    Article  CAS  PubMed  Google Scholar 

  • Persoons D, Dewulf J, Smet A, Herman L, Heyndrickx M, Martel A, Catry B, Butaye P, Haesebrouck F (2010) Prevalence and persistence of antimicrobial resistance in broiler indicator bacteria. Microb Drug Resist 16:67–74

    Article  CAS  PubMed  Google Scholar 

  • Prescott JF (2008) Antimicrobial use in food and companion animals. Anim Health Res Rev 9:127–133

    Article  PubMed  Google Scholar 

  • Pritchard DG, Dong S, Baker JR, Engler JA (2004) The bifunctional peptidoglycan lysin of Streptococcus agalactiae bacteriophage B30. Microbiology 150:2079–2087

    Article  CAS  PubMed  Google Scholar 

  • Rosenfeld J, Capdevielle J, Guillemot JC, Ferrara P (1992) In-gel digestion of proteins for internal sequence analysis after one- or two-dimensional gel electrophoresis. Anal Biochem 203:173–179

    Article  CAS  PubMed  Google Scholar 

  • Sawires YS, Songer JG (2006) Clostridium perfringens: insight into virulence evolution and population structure. Anaerobe 12:23–43

    Article  CAS  PubMed  Google Scholar 

  • Scallan E, Hoekstra RM, Angulo FJ, Tauxe RV, Widdowson MA, Roy SL, Jones JL, Griffin PM (2011) Foodborne illness acquired in the United States—major pathogens. Emerg Infect Dis 17:7–15

    Article  PubMed  Google Scholar 

  • Schäffer AA, Aravind L, Madden TL, Shavirin S, Spouge JL, Wolf YI, Koonin EV, Altschul SF (2001) Improving the accuracy of PSI-BLAST protein database searches with composition-based statistics and other refinements. Nucleic Acids Res 29:2994–3005

    Article  PubMed Central  PubMed  Google Scholar 

  • Schmitz JE, Ossiprandi MC, Rumah KR, Fischetti VA (2011) Lytic enzyme discovery through multigenomic sequence analysis in Clostridium perfringens. Appl Microbiol Biotechnol 89:1783–1795

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Shimizu T, Ohtani K, Hirakawa H, Ohshima K, Yamashita A, Shiba T, Ogasawara N, Hattori M, Kuhara S, Hayashi H (2002) Complete genome sequence of Clostridium perfringens, an anaerobic flesh-eater. Proc Natl Acad Sci USA 99:996–1001

    Article  CAS  PubMed  Google Scholar 

  • Simmons M, Donovan DM, Siragusa GR, Seal BS (2010) Recombinant expression of two bacteriophage proteins that lyse Clostridium perfringens and share identical sequences in the C-terminal cell wall binding domain of the molecules but are dissimilar in their N-terminal active domains. J Agric Food Chem 58:10330–10337

    Article  CAS  PubMed  Google Scholar 

  • Simmons M, Morales CA, Oakley BB, Seal BS (2012) Recombinant expression of a putative amidase cloned from the genome of Listeria monocytogenes that lyses the bacterium and its monolayer in conjunction with a protease. Probiotics Antimicrob Proteins 4:1–10

    Article  CAS  Google Scholar 

  • Siragusa GR, Danyluk MD, Hiett KL, Wise MG, Craven SE (2006) Molecular subtyping of poultry-associated type A Clostridium perfringens isolates by repetitive-element PCR. J Clin Microbiol 44:1065–1073

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Smedley JG 3rd, Fisher DJ, Sayeed S, Chakrabarti G, McClane BA (2004) The enteric toxins of Clostridium perfringens. Rev Physiol Biochem Pharmacol 152:183–204

    Article  CAS  PubMed  Google Scholar 

  • Studier FW, Moffatt BA (1986) Use of bacteriophage T7 RNA polymerase to direct selective high level expression of cloned genes. J Mol Biol 189:113–130

    Article  CAS  PubMed  Google Scholar 

  • Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599

    Article  CAS  PubMed  Google Scholar 

  • Tang SS, Labbé RG (1987) Mode of action of Clostridium perfringens initiation protein (spore-lytic enzyme). Ann Inst Pasteur Microbiol 138:597–608

    Article  CAS  PubMed  Google Scholar 

  • Uehara T, Bernhardt TG (2011) More than just lysins: peptidoglycan hydrolases tailor the cell wall. Curr Opin Microbiol 14:698–703

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Vollmer W, Joris B, Charlier P, Foster S (2008) Bacterial peptidoglycan (murein) hydrolases. FEMS Microbiol Rev 32:259–286

    Article  CAS  PubMed  Google Scholar 

  • Wen Q, McClane BA (2004) Detection of enterotoxigenic Clostridium perfringens type A isolates in American retail foods. Appl Environ Microbiol 70:2685–2691

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Zimmer M, Vukov N, Scherer S, Loessner MJ (2002) The murein hydrolase of the bacteriophage phi3626 dual lysis system is active against all tested Clostridium perfringens strains. Appl Environ Microbiol 68:5311–5317

    Article  CAS  PubMed Central  PubMed  Google Scholar 

Download references

Acknowledgments

Support for the research was provided by the US Department of Agriculture, Agricultural Research Service (ARS CRIS project #6612-32000-060). The authors acknowledge primary amino acid sequencing and mass spectrometry analyses of the recombinant protein by Ms. Rebekah Woolsey and Dr. Kathleen Schegg at the Nevada Proteomics Center which operates under the auspices of grants from the National Center for Research Resources (5P20RR016464-11) and the National Institute of General Medical Sciences (8 P20 GM103440-11) from the National Institutes of Health.

Conflict of interest

None.

Author information

Author notes

  1. Glenn E. Tillman & Mustafa Simmons

    Present address: Outbreak Section of the Eastern Laboratory, Food Safety and Inspection Service, Richard B. Russell Agricultural Research Center, USDA, 950 College Station Road, Athens, GA, 30605, USA

Authors and Affiliations

  1. Poultry Microbiological Safety Research Unit, Richard B. Russell Agricultural Research Center, Agricultural Research Service, USDA, Athens, GA, 30605, USA

    Glenn E. Tillman, Mustafa Simmons, Johnna K. Garrish & Bruce S. Seal

Authors
  1. Glenn E. Tillman
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Mustafa Simmons
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Johnna K. Garrish
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Bruce S. Seal
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Bruce S. Seal.

Additional information

Communicated by Erko Stackebrandt.

Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the ARS, USDA.

Electronic supplementary material

Rights and permissions

Reprints and permissions

About this article

Cite this article

Tillman, G.E., Simmons, M., Garrish, J.K. et al. Expression of a Clostridium perfringens genome-encoded putative N-acetylmuramoyl–l-alanine amidase as a potential antimicrobial to control the bacterium. Arch Microbiol 195, 675–681 (2013). https://doi.org/10.1007/s00203-013-0916-4

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00203-013-0916-4

Keywords

Search

Navigation