1887

Abstract

In general, prokaryotes are considered to be single-celled organisms that lack internal membrane-bound organelles. However, many bacteria produce proteinaceous microcompartments that serve a similar purpose, i.e. to concentrate specific enzymic reactions together or to shield the wider cytoplasm from toxic metabolic intermediates. In this paper, a synthetic operon encoding the key structural components of a microcompartment was designed based on the genes for the propanediol utilization (Pdu) microcompartment. The genes chosen included , -, -, -, -, - and -, and each was shown to produce protein in an chassis. In parallel, a set of compatible vectors designed to express non-native cargo proteins was also designed and tested. Engineered hexa-His tags allowed isolation of the components of the microcompartments together with co-expressed, untagged, cargo proteins. Finally, an protease accessibility assay suggested that a PduD–GFP fusion could be protected from proteolysis when co-expressed with the synthetic microcompartment operon. This work gives encouragement that it may be possible to harness the genes encoding a non-native microcompartment for future biotechnological applications.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.069922-0
2013-11-01
2020-10-01
Loading full text...

Full text loading...

/deliver/fulltext/micro/159/11/2427.html?itemId=/content/journal/micro/10.1099/mic.0.069922-0&mimeType=html&fmt=ahah

References

  1. Bartolomé B., Jubete Y., Martínez E., de la Cruz F..( 1991;). Construction and properties of a family of pACYC184-derived cloning vectors compatible with pBR322 and its derivatives. Gene102:75–78 [CrossRef][PubMed]
    [Google Scholar]
  2. Blattner F. R., Plunkett G. III, Bloch C. A., Perna N. T., Burland V., Riley M., Collado-Vides J., Glasner J. D., Rode C. K..& other authors ( 1997;). The complete genome sequence of Escherichia coli K-12. Science277:1453–1462 [CrossRef][PubMed]
    [Google Scholar]
  3. Bobik T. A., Havemann G. D., Busch R. J., Williams D. S., Aldrich H. C..( 1999;). The propanediol utilization (pdu) operon of Salmonella enterica serovar Typhimurium LT2 includes genes necessary for formation of polyhedral organelles involved in coenzyme B(12)-dependent 1,2-propanediol degradation. J Bacteriol181:5967–5975[PubMed]
    [Google Scholar]
  4. Cheng S., Liu Y., Crowley C. S., Yeates T. O., Bobik T. A..( 2008;). Bacterial microcompartments: their properties and paradoxes. Bioessays30:1084–1095 [CrossRef][PubMed]
    [Google Scholar]
  5. Coulthurst S. J., Dawson A., Hunter W. N., Sargent F..( 2012;). Conserved signal peptide recognition systems across the prokaryotic domains. Biochemistry51:1678–1686 [CrossRef][PubMed]
    [Google Scholar]
  6. Crowley C. S., Sawaya M. R., Bobik T. A., Yeates T. O..( 2008;). Structure of the PduU shell protein from the Pdu microcompartment of Salmonella. Structure16:1324–1332 [CrossRef][PubMed]
    [Google Scholar]
  7. DeLisa M. P., Samuelson P., Palmer T., Georgiou G..( 2002;). Genetic analysis of the twin arginine translocator secretion pathway in bacteria. J Biol Chem277:29825–29831 [CrossRef][PubMed]
    [Google Scholar]
  8. Fan C., Bobik T. A..( 2011;). The N-terminal region of the medium subunit (PduD) packages adenosylcobalamin-dependent diol dehydratase (PduCDE) into the Pdu microcompartment. J Bacteriol193:5623–5628 [CrossRef][PubMed]
    [Google Scholar]
  9. Fan C., Cheng S., Liu Y., Escobar C. M., Crowley C. S., Jefferson R. E., Yeates T. O., Bobik T. A..( 2010;). Short N-terminal sequences package proteins into bacterial microcompartments. Proc Natl Acad Sci U S A107:7509–7514 [CrossRef][PubMed]
    [Google Scholar]
  10. Fan C., Cheng S., Sinha S., Bobik T. A..( 2012;). Interactions between the termini of lumen enzymes and shell proteins mediate enzyme encapsulation into bacterial microcompartments. Proc Natl Acad Sci U S A109:14995–15000 [CrossRef][PubMed]
    [Google Scholar]
  11. Frank S., Lawrence A. D., Prentice M. B., Warren M. J..( 2013;). Bacterial microcompartments moving into a synthetic biological world. J Biotechnol163:273–279 [CrossRef][PubMed]
    [Google Scholar]
  12. Havemann G. D., Bobik T. A..( 2003;). Protein content of polyhedral organelles involved in coenzyme B12-dependent degradation of 1,2-propanediol in Salmonella enterica serovar Typhimurium LT2. J Bacteriol185:5086–5095 [CrossRef][PubMed]
    [Google Scholar]
  13. Iancu C. V., Ding H. J., Morris D. M., Dias D. P., Gonzales A. D., Martino A., Jensen G. J..( 2007;). The structure of isolated Synechococcus strain WH8102 carboxysomes as revealed by electron cryotomography. J Mol Biol372:764–773 [CrossRef][PubMed]
    [Google Scholar]
  14. Jack R. L., Buchanan G., Dubini A., Hatzixanthis K., Palmer T., Sargent F..( 2004;). Coordinating assembly and export of complex bacterial proteins. EMBO J23:3962–3972 [CrossRef][PubMed]
    [Google Scholar]
  15. Karzai A. W., Roche E. D., Sauer R. T..( 2000;). The SsrA-SmpB system for protein tagging, directed degradation and ribosome rescue. Nat Struct Biol7:449–455 [CrossRef][PubMed]
    [Google Scholar]
  16. Keasling J. D..( 2008;). Synthetic biology for synthetic chemistry. ACS Chem Biol3:64–76 [CrossRef][PubMed]
    [Google Scholar]
  17. Keiler K. C., Waller P. R., Sauer R. T..( 1996;). Role of a peptide tagging system in degradation of proteins synthesized from damaged messenger RNA. Science271:990–993 [CrossRef][PubMed]
    [Google Scholar]
  18. Kerfeld C. A., Sawaya M. R., Tanaka S., Nguyen C. V., Phillips M., Beeby M., Yeates T. O..( 2005;). Protein structures forming the shell of primitive bacterial organelles. Science309:936–938 [CrossRef][PubMed]
    [Google Scholar]
  19. Kerfeld C. A., Heinhorst S., Cannon G. C..( 2010;). Bacterial microcompartments. Annu Rev Microbiol64:391–408 [CrossRef][PubMed]
    [Google Scholar]
  20. Kofoid E., Rappleye C., Stojiljkovic I., Roth J..( 1999;). The 17-gene ethanolamine (eut) operon of Salmonella typhimurium encodes five homologues of carboxysome shell proteins. J Bacteriol181:5317–5329[PubMed]
    [Google Scholar]
  21. Komine Y., Kitabatake M., Yokogawa T., Nishikawa K., Inokuchi H..( 1994;). A tRNA-like structure is present in 10Sa RNA, a small stable RNA from Escherichia coli. Proc Natl Acad Sci U S A91:9223–9227 [CrossRef][PubMed]
    [Google Scholar]
  22. Laemmli U. K..( 1970;). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature227:680–685 [CrossRef][PubMed]
    [Google Scholar]
  23. Lee S. K., Chou H., Ham T. S., Lee T. S., Keasling J. D..( 2008;). Metabolic engineering of microorganisms for biofuels production: from bugs to synthetic biology to fuels. Curr Opin Biotechnol19:556–563 [CrossRef][PubMed]
    [Google Scholar]
  24. Lyons L. B., Zinder N. D..( 1972;). The genetic map of the filamentous bacteriophage f1. Virology49:45–60 [CrossRef][PubMed]
    [Google Scholar]
  25. McClelland M., Sanderson K. E., Spieth J., Clifton S. W., Latreille P., Courtney L., Porwollik S., Ali J., Dante M..& other authors ( 2001;). Complete genome sequence of Salmonella enterica serovar Typhimurium LT2. Nature413:852–856 [CrossRef][PubMed]
    [Google Scholar]
  26. Ngu T. T., Lee J. A., Rushton M. K., Stillman M. J..( 2009;). Arsenic metalation of seaweed Fucus vesiculosus metallothionein: the importance of the interdomain linker in metallothionein. Biochemistry48:8806–8816 [CrossRef][PubMed]
    [Google Scholar]
  27. Pang A., Liang M., Prentice M. B., Pickersgill R. W..( 2012;). Substrate channels revealed in the trimeric Lactobacillus reuteri bacterial microcompartment shell protein PduB. Acta Crystallogr D Biol Crystallogr68:1642–1652 [CrossRef][PubMed]
    [Google Scholar]
  28. Parsons J. B., Dinesh S. D., Deery E., Leech H. K., Brindley A. A., Heldt D., Frank S., Smales C. M., Lünsdorf H..& other authors ( 2008;). Biochemical and structural insights into bacterial organelle form and biogenesis. J Biol Chem283:14366–14375 [CrossRef][PubMed]
    [Google Scholar]
  29. Parsons J. B., Frank S., Bhella D., Liang M., Prentice M. B., Mulvihill D. P., Warren M. J..( 2010;). Synthesis of empty bacterial microcompartments, directed organelle protein incorporation, and evidence of filament-associated organelle movement. Mol Cell38:305–315 [CrossRef][PubMed]
    [Google Scholar]
  30. Sagermann M., Ohtaki A., Nikolakakis K..( 2009;). Crystal structure of the EutL shell protein of the ethanolamine ammonia lyase microcompartment. Proc Natl Acad Sci U S A106:8883–8887 [CrossRef][PubMed]
    [Google Scholar]
  31. Shively J. M., Ball F., Brown D. H., Saunders R. E..( 1973;). Functional organelles in prokaryotes: polyhedral inclusions (carboxysomes) of Thiobacillus neapolitanus. Science182:584–586 [CrossRef][PubMed]
    [Google Scholar]
  32. Sinha S., Cheng S., Fan C., Bobik T. A..( 2012;). The PduM protein is a structural component of the microcompartments involved in coenzyme B(12)-dependent 1,2-propanediol degradation by Salmonella enterica. J Bacteriol194:1912–1918 [CrossRef][PubMed]
    [Google Scholar]
  33. So A. K., Espie G. S., Williams E. B., Shively J. M., Heinhorst S., Cannon G. C..( 2004;). A novel evolutionary lineage of carbonic anhydrase (epsilon class) is a component of the carboxysome shell. J Bacteriol186:623–630 [CrossRef][PubMed]
    [Google Scholar]
  34. Tabor S., Richardson C. C..( 1985;). A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci U S A82:1074–1078 [CrossRef][PubMed]
    [Google Scholar]
  35. Tanaka S., Kerfeld C. A., Sawaya M. R., Cai F., Heinhorst S., Cannon G. C., Yeates T. O..( 2008;). Atomic-level models of the bacterial carboxysome shell. Science319:1083–1086 [CrossRef][PubMed]
    [Google Scholar]
  36. Towbin H., Staehelin T., Gordon J..( 1979;). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A76:4350–4354 [CrossRef][PubMed]
    [Google Scholar]
  37. Wheatley N. M., Gidaniyan S. D., Liu Y., Cascio D., Yeates T. O..( 2013;). Bacterial microcompartment shells of diverse functional types possess pentameric vertex proteins. Protein Sci22:660–665 [CrossRef][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.069922-0
Loading
/content/journal/micro/10.1099/mic.0.069922-0
Loading

Data & Media loading...

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