1887

Microbiology Society logo

Volume 154, Issue 5

The lytic cassette of mycobacteriophage Ms6 encodes an enzyme with lipolytic activity Free

Abstract

dsDNA bacteriophages use the dual system endolysin–holin to achieve lysis of their bacterial host. In addition to these two essential genes, some bacteriophages encode additional proteins within their lysis module. In this report, we describe the activity of a protein encoded by gene from the mycobacteriophage Ms6. is localized within the lysis cassette, between the endolysin gene () and the holin gene (). Analysis of the deduced amino acid sequence of LysB revealed the presence of a conserved motif (Gly-Tyr-Ser-Gln-Gly) characteristic of enzymes with lipolytic activity. A search within the sequences of protein databases revealed significant similarities to other putative proteins that are encoded by mycobacteriophages only, indicating that LysB and those proteins may be specific to their mycobacterial hosts. A screening for His-LysB activity on esterase and lipase substrates confirmed the lipolytic activity. Examination of the kinetic parameters of recombinant His-LysB for the hydrolysis of -nitrophenyl esters indicated that although this protein could use a wide range of chain length substrates (C–C), it presents a higher affinity for -nitrophenyl esters of longer chain length (C and C). Using -nitrophenyl butyrate as a substrate, the enzyme showed optimal activity at 23 °C and pH 7.5–8.0. Activity was increased in the presence of Ca and Mn. To the best of our knowledge, this is the first description of a protein with lipolytic activity encoded within a bacteriophage.

  • Received:
  • Accepted:
  • Revised:
  • Published Online:
Keyword(s): DEPC, diethylpyrocarbonate , pNP, p-nitrophenyl , pNPB, p-nitrophenyl butyrate , pNPC, p-nitrophenyl caprylate , pNPL, p-nitrophenyl laurate , pNPM, p-nitrophenyl myristate , pNPP, p-nitrophenyl palmitate and pNPS, p-nitrophenyl stearate
SGM
Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.2007/014621-0
2008-05-01
2024-04-16
Loading full text...

Full text loading...

/deliver/fulltext/micro/154/5/1364.html?itemId=/content/journal/micro/10.1099/mic.0.2007/014621-0&mimeType=html&fmt=ahah

References

  1. Arpigny J. L., Jaeger K. E. 1999; Bacterial lipolytic enzymes: classification and properties. Biochem J 343:177–183
     [Google Scholar]
  2. Bornscheuer U. T. 2002; Microbial carboxyl esterases: classification, properties and application in biocatalysis. FEMS Microbiol Rev 26:73–81
     [Google Scholar]
  3. Bradford M. M. 1976; A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 72:248–254
     [Google Scholar]
  4. Brennan P. J. 2003; Structure, function, and biogenesis of the cell wall of Mycobacterium tuberculosis . Tuberculosis (Edinb) 83:91–97
     [Google Scholar]
  5. Carvalho C. M. L., Aires-Barros M. R., Cabral J. M. S. 1999; Cutinase: from molecular level to bioprocess development. Biotechnol Bioeng 66:17–34
     [Google Scholar]
  6. Chandry P. S., Moore S. C., Boyce J. D., Davidson B. E., Hillier A. J. 1997; Analysis of the DNA sequence, gene expression, origin of replication and modular structure of the Lactococcus lactis lytic bacteriophage sk1. Mol Microbiol 26:49–64
     [Google Scholar]
  7. Chen Z., Franco C. F., Baptista R. P., Cabral J. M. S., Coelho A. V., Rodrigues C. J. Jr, Melo E. P. 2007; Purification and identification of cutinases from Colletotrichum kahawae and Colletotrichum gloeosporioides . Appl Microbiol Biotechnol 73:1306–1313
     [Google Scholar]
  8. Garcia M., Pimentel M., Moniz-Pereira J. 2002; Expression of mycobacteriophage Ms6 lysis genes is driven by two sigma (70)-like promoters and is dependent on a transcription termination signal present in the leader RNA. J Bacteriol 184:3034–3043
     [Google Scholar]
  9. Grundling A., Manson M. D., Young R. 2001; Holins kill without warning. Proc Natl Acad Sci U S A 98:9348–9352
     [Google Scholar]
  10. Gupta R., Gupta N., Rathi P. 2004; Bacterial lipases: an overview of production, purification and biochemical properties. Appl Microbiol Biotechnol 64:763–781
     [Google Scholar]
  11. Hatfull G. F., Pedulla M. L., Jacobs-Sera D., Cichon P. M., Foley A., Ford M. E., Gonda R. M., Houtz J. M., Hryckowian A. J. other authors 2006; Exploring the mycobacteriophage metaproteome: phage genomics as an educational platform. PLoS Genet 2:e92
     [Google Scholar]
  12. Henrich B., Binishofer B., Blasi U. 1995; Primary structure and functional analysis of the lysis genes of Lactobacillus gasseri bacteriophage φ adh. J Bacteriol 177:723–732
     [Google Scholar]
  13. Holmquist M. 2000; Alpha/beta-hydrolase fold enzymes: structures, functions and mechanisms. Curr Protein Pept Sci 1:209–235
     [Google Scholar]
  14. Jaeger K. E., Ransac S., Dijkstra B. W., Colson C., van Heuvel M., Misset O. 1994; Bacterial lipases. FEMS Microbiol Rev 15:29–63
     [Google Scholar]
  15. Jaeger K. E., Dijkstra B. W., Reetz M. T. 1999; Bacterial biocatalysts: molecular biology, three-dimensional structures, and biotechnological applications of lipases. Annu Rev Microbiol 53:315–351
     [Google Scholar]
  16. Kaiser P., Raina C., Parshad R., Johri S., Verma V., Andrabi K. I., Qazi G. N. 2006; A novel esterase from Bacillus subtilis (RRL 1789): purification and characterization of the enzyme. Protein Expr Purif 45:262–268
     [Google Scholar]
  17. Kouker G., Jaeger K. E. 1987; Specific and sensitive plate assay for bacterial lipases. Appl Environ Microbiol 53:211–213
     [Google Scholar]
  18. Longhi S., Cambillau C. 1999; Structure–activity of cutinase, a small lipolytic enzyme. Biochim Biophys Acta 1441:185–196
     [Google Scholar]
  19. Ma J., Zhang Z., Wang B., Kong X., Wang Y., Cao S., Feng Y. 2005; Overexpression and characterization of a lipase from Bacillus subtilis . Protein Expr Purif 45:22–29
     [Google Scholar]
  20. Mannese M. L. M., Cox R. C., Koops B. C., Verheij H. M., de Haas G. H., Egmond M. R., van der Hieden H. T. W. M., de Vlieg J. 1995; Cutinase from Fusarium solani pisi hydrolyzing triglyceride analogues. Effect of acyl chain length and position in the substrate molecule on activity and enantioselectivity. Biochemistry 34:6400–6407
     [Google Scholar]
  21. Minnikin D. E. 1982; Lipids: complex lipids, their chemistry, biosynthesis and roles. In The Biology of Mycobacteria, Physiology, Identification and Classification vol. 1 pp 95–184 Edited by Ratledge C., Stanford J. L. London: Academic Press;
     [Google Scholar]
  22. Nawani N., Khurana J., Kaur J. 2006; A thermostable lipolytic enzyme from a thermophilic Bacillus sp.: Purification and characterization. Mol Cell Biochem 290:17–22
     [Google Scholar]
  23. Nikoleit K., Rosenstein R., Verheij H. M., Gotz F. 1995; Comparative biochemical and molecular analysis of Staphylococcus hyicus, Staphylococcus aureus and a hybrid lipase. Indication for a C-terminal phospholipase domain. Eur J Biochem 228:732–738
     [Google Scholar]
  24. Parker S. K., Curtin K. M., Vasil M. L. 2007; Purification and characterization of a mycobacterial phospholipase A: an activity associated with mycobacterial cutinase. J Bacteriol 189:4153–4160
     [Google Scholar]
  25. Portugal I., Anes E., Moniz-Pereira J. 1989; Temperate mycobacteriophage from M. smegmatis . Acta Leprol 7:243–244
     [Google Scholar]
  26. Sambrook J., Russell D. W. 2001 Molecular Cloning: a Laboratory Manual , 3rd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
     [Google Scholar]
  27. Sayari A., Agrebi N., Jaoua S., Gargouri Y. 2001; Biochemical and molecular characterization of Staphylococcus simulans lipase. Biochimie 83:863–871
     [Google Scholar]
  28. Schmidt J. A., Browning G. F., Markham P. F. 2004; Mycoplasma hyopneumoniae p65 surface lipoprotein is a lipolytic enzyme with a preference for short-chain fatty acids. J Bacteriol 186:5790–5798
     [Google Scholar]
  29. Summer E. J., Berry J., Tran T. A., Niu L., Struck D. K., Young R. 2007; Rz/Rz1 lysis gene equivalents in phages of Gram-negative hosts. J Mol Biol 373:1098–1112
     [Google Scholar]
  30. Teo J. W. P., Zhang L. H., Poh C. L. 2003; Cloning and characterization of a novel lipase from Vibrio harveyi strain AP6. Gene 312:181–188
     [Google Scholar]
  31. Young R. 1992; Bacteriophage lysis: mechanism and regulation. Microbiol Rev 56:430–481
     [Google Scholar]
  32. Young R. 2002; Bacteriophage holins: deadly diversity. J Mol Microbiol Biotechnol 4:21–36
     [Google Scholar]
  33. Young R. 2005; Phage lysis. In Phages: Their Role in Bacterial Pathogenesis and Biotechnology pp 92–127 Edited by Waldor M. K., Friedman D. I., Adhya S. L. Washington, DC: American Society for Microbiology;
     [Google Scholar]
  34. Young R., Wang I.-N., Roof W. D. 2000; Phages will out: strategies of host cell lysis. Trends Microbiol 8:120–128
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.2007/014621-0
Loading
The lytic cassette of mycobacteriophage Ms6 encodes an enzyme with lipolytic activity
Microbiology 154, 1364 (2008); https://doi.org/10.1099/mic.0.2007/014621-0
/content/journal/micro/10.1099/mic.0.2007/014621-0
/content/journal/micro/10.1099/mic.0.2007/014621-0
Loading

Data & Media loading...

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error