Advertisement

Microbial Ecology

, Volume 59, Issue 2, pp 390–399 | Cite as

Microbial Protein in Soil: Influence of Extraction Method and C Amendment on Extraction and Recovery

  • Erin B. Taylor
  • Mark A. WilliamsEmail author
Soil Microbiology

Abstract

The capacity to study the content and resolve the dynamics of the proteome of diverse microbial communities would help to revolutionize the way microbiologists study the function and activity of microorganisms in soil. To better understand the limitations of a proteomic approach to studying soil microbial communities, we characterized extractable soil microbial proteins using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Two methods were utilized to extract proteins from microorganisms residing in a Quitman and Benfield soil: (1) direct extraction of bulk protein from soil and (2) separation of the microorganisms from soil using density gradient centrifugation and subsequent extraction (DGC–EXT) of microbial protein. In addition, glucose and toluene amendments to soil were used to stimulate the growth of a subset of the microbial community. A bacterial culture and bovine serum albumin (BSA) were added to the soil to qualitatively assess their recovery following extraction. Direct extraction and resolution of microbial proteins using SDS-PAGE generally resulted in smeared and unresolved banding patterns on gels. DGC–EXT of microbial protein from soil followed by separation using SDS-PAGE, however, did resolve six to 10 bands in the Benfield but not the Quitman soil. DGC–EXT of microbial protein, but not direct extraction following the addition of glucose and toluene, markedly increased the number of bands (~40) on the gels in both Benfield and Quitman soils. Low recoveries of added culture and BSA proteins using the direct extraction method suggest that proteins either bind to soil organic matter and mineral particles or that partial degradation takes place during extraction. Interestingly, DGC may have been preferentially selected for actively growing cells, as gauged by the 10–100× lower cy19:0/18:1ω7 ratio of the fatty acid methyl esters in the isolated community compared to that for the whole soil. DGC can be used to isolate soil communities and provide microbial protein that can be characterized using PAGE.

Keywords

Microbial Community Microbial Biomass Fatty Acid Methyl Ester Soil Microbial Community Methanotrophs 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 1.
    Aebersold R, Goodlett DR (2001) Mass spectrometry in proteomics. Chem Rev 101:269–296CrossRefPubMedGoogle Scholar
  2. 2.
    Aebersold R, Mann M (2003) Mass spectrometry-based proteomics. Nature 422:198–207CrossRefPubMedGoogle Scholar
  3. 3.
    Amaral JA, Ren T, Knowles R (1998) Atmospheric methane consumption by forest soils and extracted bacteria at different pH values. Appl Environ Microbiol 64:2397–2402PubMedGoogle Scholar
  4. 4.
    Benndorf D, Balcke GU, Harms H, von Bergen M (2007) Functional metaproteome analysis of protein extracts from contaminated soil and groundwater. ISME J 1:224–234CrossRefPubMedGoogle Scholar
  5. 5.
    Chevallier T, Muchaonyerwa P, Chenu C (2003) Microbial utilisation of two proteins adsorbed to a vertisol clay fraction: toxin from Bacillus thuringiensis subsp. tenebrionis and bovine serum albumin. Soil Biol Biochem 35:1211–1220CrossRefGoogle Scholar
  6. 6.
    Ehlers K, Bünemann EK, Oberson A, Frossard E, Frostegård Å, Yuejian M, Bakken LR (2008) Extraction of soil bacteria from a ferralsol. Soil Biol Biochem 40:1940–1946CrossRefGoogle Scholar
  7. 7.
    Feng Y, Motta AC, Reeves DW, Burmester CH, van Santen E, Osborne JA (2003) Soil microbial communities under conventional-till and no-till continuous cotton systems. Soil Biol Biochem 35:1693–1703CrossRefGoogle Scholar
  8. 8.
    Frostegard A, Tunlid A, Baath E (1993) Phospholipid fatty acid composition, biomass, and activity of microbial communities from two soil types experimentally exposed to different heavy metals. Appl Environ Microbiol 59:3605–3617PubMedGoogle Scholar
  9. 9.
    Griffin TJ, Gygi SP, Ideker T, Rist B, Eng J, Hood L, Aebersold R (2002) Complementary profiling of gene expression at the transcriptome and proteome levels in Saccharomyces cerevisiae. Mol Cell Proteomics 1:323–333CrossRefPubMedGoogle Scholar
  10. 10.
    Guckert JB, Antworth CP, Nichols PD, White DC (1985) Phospholipid, ester-linked fatty acid profiles as reproducible assays for changes in prokaryotic community structure of estuarine sediments. FEMS Microbiol Lett 31:147–158Google Scholar
  11. 11.
    Gygi SP, Rochon Y, Franza BR, Aebersold R (1999) Correlation between protein and mRNA abundance in yeast. Mol Cell Biol 19:1720–1730PubMedGoogle Scholar
  12. 12.
    Hanson JR, Macalady JL (1999) Linking toluene degradation with specific microbial populations in soil. Appl Environ Microbiol 65:5403–5408PubMedGoogle Scholar
  13. 13.
    Hurkman WJ, Tanaka CK (1986) Solubilization of plant membrane proteins for analysis by two-dimensional gel electrophoresis. Plant Physiol 81:802–806CrossRefPubMedGoogle Scholar
  14. 14.
    Kieft TL, Ringelberg DB, White DC (1994) Changes in ester-linked phospholipid fatty acid profiles of subsurface bacteria during starvation and desiccation in a porous medium. Appl Environ Microbiol 60:3292–3299PubMedGoogle Scholar
  15. 15.
    Kieft TL, Wilch E, O’Connor K, Ringelberg DB, White DC (1997) Survival and phospholipid fatty acid profiles of surface and subsurface bacteria in natural sediment microcosms. Appl Environ Microbiol 63:1531–1542PubMedGoogle Scholar
  16. 16.
    Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685CrossRefPubMedGoogle Scholar
  17. 17.
    Lindahl V, Bakken LR (1995) Evaluation of methods for extraction of bacteria from soil. FEMS Microbiol Ecol 16:135–142CrossRefGoogle Scholar
  18. 18.
    Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the folin phenol reagent. J Biol Chem 193:265–275PubMedGoogle Scholar
  19. 19.
    Maron PA, Mougel C, Siblot S, Abbas H, Lemanceau P, Ranjard L (2007) Protein extraction and fingerprinting optimization of bacterial communities in natural environment. Microb Ecol 53:426–434CrossRefGoogle Scholar
  20. 20.
    Maron PA, Ranjard L, Mougel C, Lemanceau P (2007) Metaproteomics: a new approach for studying functional microbial ecology. Microb Ecol 53:486–493CrossRefPubMedGoogle Scholar
  21. 21.
    Maron PA, Schimann H, Ranjard L, Brothier E, Domenach AM, Lensi R, Nazaret S (2006) Evaluation of quantitative and qualitative recovery of bacterial communities from different soil types by density gradient centrifugation. Eur J Soil Biol 42:65–73CrossRefGoogle Scholar
  22. 22.
    Nie L, Wu G, Zhang W (2006) Correlation between mRNA and protein abundance in Desulfovibrio vulgaris: A multiple regression to identify sources of variations. Biochem and Biophys Res Comm 339:603–610CrossRefGoogle Scholar
  23. 23.
    Ogunseitan OA (1997) Direct extraction of catalytic proteins from natural microbial communities. J Microbiol Meth 28:55–63CrossRefGoogle Scholar
  24. 24.
    Ram RJ, VerBerkmoes NC, Thelen MP, Tyson GW, Baker BJ, Blake RC, Shah M, Hettich RL, Banfield JF (2005) Community proteomics of a natural microbial biofilm. Science 308:1915–1920CrossRefPubMedGoogle Scholar
  25. 25.
    Rigou P, Rezaei H, Grosclaude J, Staunton S, Quiquampoix H (2006) Fate of prions in soil: adsorption and extraction by electroelution of recombinant bovine prion protein from montmorillonite and natural soils. Environ Sci Tech 40:1497–1503CrossRefGoogle Scholar
  26. 26.
    Sasser M (1990) Identification of bacteria by gas chromatography of cellular fatty acids: identification of bacteria by gas chromatography of cellular fatty acids. Microbial ID, Newark DEGoogle Scholar
  27. 27.
    Schulze WX, Gleixner G, Kaiser K, Guggenberger G, Mann M, Schulze ED (2005) A proteomic fingerprint of dissolved organic carbon and of soil particles. Oecologia 142:335–343CrossRefPubMedGoogle Scholar
  28. 28.
    Singleton I, Merrington G, Colvan S, Delahunty JS (2003) The potential of soil protein-based methods to indicate metal contamination. Appl Soil Ecol 23:25–32CrossRefGoogle Scholar
  29. 29.
    Courtois S, Fostegard A, Goransson P, Depret G, Jeannin P, Simonet P (2001) Quantification of bacterial subgroups in soil: comparison of DNA extracted directly from soil or from cells previously released by density gradient centrifugation. Environ Microbiol 3:431–439CrossRefPubMedGoogle Scholar
  30. 30.
    Wang H, Ye Q, Gan J, Wu J (2008) Adsorption of Cry1Ab protein isolated from Bt transgenic rice on bentone, daolin, humic acids, and soils. J Agric Food Chem 56:4659–4664CrossRefPubMedGoogle Scholar
  31. 31.
    White DC, Flemming CA, Leung KT, Macnaughton SJ (1998) In situ microbial ecology for quantitative appraisal, monitoring, and risk assessment of pollution remediation in soils, the subsurface, the rhizosphere and in biofilms. J Microbiol Meth 32:93–105CrossRefGoogle Scholar
  32. 32.
    Williams MA, Rice CW (2007) Seven years of enhanced water availability influences the physiological, structural, and functional attributes of a soil microbial community. Appl Soil Ecol 35:535–545CrossRefGoogle Scholar
  33. 33.
    Wilmes P, Bond PL (2006) Metaproteomics: studying functional gene expression in microbial ecosystems. Trends Microbiol 14:92–97CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2009

Authors and Affiliations

  1. 1.Plant and Soil SciencesMississippi State UniversityStarkvilleUSA

Personalised recommendations