Skip to main content

Advertisement

Log in

A new approach to retrieve full lengths of functional genes from soil by PCR-DGGE and metagenome walking

  • Methods
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

Metagenomes are a vast genetic resource, and various approaches have been developed to explore them. Here, we present a new approach to retrieve full lengths of functional genes from soil DNA using PCR-denaturing gradient gel electrophoresis (DGGE) followed by metagenome walking. Partial fragments of benzoate 1,2-dioxygenase alpha subunit gene (benA) were detected from a 3-chlorobenzoate (3CB)-dosed soil by PCR-DGGE, and one DGGE band induced by 3CB was used as a target fragment for metagenome walking. The walking retrieved the flanking regions of the target fragment from the soil DNA, resulting in recovery of the full length of benA and also downstream gene (benB). The same strategy retrieved another gene, tfdC, and a complete tfdC and two downstream genes were obtained from the same soil. PCR-DGGE allows screening for target genes based on their potential for degrading contaminants in the environment. This feature provides an advantage over other existing metagenomic approaches.

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

Access this article

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or Ebook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

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

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  • Amann RI, Ludwig W, Schleifer KH (1995) Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol Rev 59:143–169

    CAS  PubMed  PubMed Central  Google Scholar 

  • Bell PJ, Sunna A, Gibbs MD, Curach NC, Nevalainen H, Bergquist PL (2002) Prospecting for novel lipase genes using PCR. Microbiology 148:2283–2291

    Article  CAS  PubMed  Google Scholar 

  • Butler CS, Mason JR (1997) Structure–function analysis of the bacterial aromatic ring-hydroxylating dioxygenases. Adv Microb Physiol 38:47–84

    Article  CAS  PubMed  Google Scholar 

  • Cowan D, Meyer Q, Stafford W, Muyanga S, Cameron R, Wittwer P (2005) Metagenomic gene discovery: past, present and future. Trends Biotech 23:321–329

    Article  CAS  Google Scholar 

  • Daniel R (2004) The soil metagenome—a rich resource for the discovery of novel natural products. Curr Opin Biotechnol 15:199–204

    Article  CAS  PubMed  Google Scholar 

  • Dar SA, Yao L, van Dongen U, Kuenen JG, Muyzer G (2007) Analysis of diversity and activity of sulfate-reducing bacterial communities in sulfidogenic bioreactors using 16S rRNA and dsrB genes as molecular markers. Appl Environ Microbiol 73:594–604

    Article  CAS  PubMed  Google Scholar 

  • Eschenfeldt WH, Stols L, Rosenbaum H, Khambatta ZS, Quaite-Randall E, Wu S, Kilgore DC, Trent JD, Donnelly MI (2001) DNA from uncultured organisms as a source of 2, 5-diketo-D-gluconic acid reductases. Appl Environ Microbiol 67:4206–4214

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ferraroni M, Solyanikova IP, Kolomytseva MP, Scozzafava A, Golovleva L, Briganti F (2004) Crystal structure of 4-chlorocatechol 1, 2-dioxygenase from the chlorophenol-utilizing gram-positive Rhodococcus opacus 1CP. J Biol Chem 279:27646–27655

    Article  CAS  PubMed  Google Scholar 

  • Fuchu G, Ohtsubo Y, Ito M, Miyazaki R, Ono A, Nagata Y, Tsuda M (2008) Insertion sequence-based cassette PCR: cultivation-independent isolation of gamma-hexachlorocyclohexane-degrading genes from soil DNA. Appl Microbiol Biotechnol 79:627–632

    Article  CAS  PubMed  Google Scholar 

  • Kubota M, Nodate M, Yasumoto-Hirose M, Uchiyama T, Kagami O, Shizuri Y, Misawa N (2005) Isolation and functional analysis of cytochrome P450 CYP153A genes from various environments. Biosci Biotechnol Biochem 69:2421–2430

    Article  CAS  PubMed  Google Scholar 

  • Leander M, Vallaeys T, Fulthorpe R (1998) Amplification of putative chlorocatechol dioxygenase gene fragments from alpha- and beta-proteobacteria. Can J Microbiol 44:482–486

    CAS  PubMed  Google Scholar 

  • Liu S, Ogawa N, Senda T, Hasebe A, Miyashita K (2005) Amino acids in positions 48, 52, and 73 differentiate the substrate specificities of the highly homologous chlorocatechol 1, 2-dioxygenases CbnA and TcbC. J Bacteriol 187:5427–5436

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lorenz P, Liebeton K, Niehaus F, Eck J (2002) Screening for novel enzymes for biocatalytic processes: accessing the metagenome as a resource of novel functional sequence space. Curr Opin Biotechnol 13:572–577

    Article  CAS  PubMed  Google Scholar 

  • Morimoto S, Togami K, Ogawa N, Hasebe A, Fujii T (2005) Analysis of a bacterial community in 3-chlorobenzoate-contaminated soil by PCR-DGGE targeting the 16S rRNA gene and benzoate 1, 2-dioxygenase gene (benA). Microbes Environ 20:151–159

    Article  Google Scholar 

  • Morimoto S, Ogawa N, Hasebe A, Fujii T (2008) Isolation of effective 3-chlorobenzoate-degraders in soil using community analyses by PCR-DGGE. Microbes Environ 23:285–292

    Article  PubMed  Google Scholar 

  • Muyzer G, De Waal EC, Uitterlinden AG (1993) Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Appl Environ Microbiol 59:695–700

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Neidle EL, Hartnett C, Ornston LN, Bairoch A, Rekik M, Harayama S (1991) Nucleotide sequences of the Acinetobacter calcoaceticus benABC genes for benzoate 1, 2-dioxygenase reveal evolutionary relationships among multicomponent oxygenases. J Bacteriol 173:5385–5395

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Nicolaisen MH, Ramsing NB (2002) Denaturing gradient gel electrophoresis (DGGE) approaches to study the diversity of ammonia-oxidizing bacteria. J Microbiol Methods 50:189–203

    Article  CAS  PubMed  Google Scholar 

  • Ogawa N, Miyashita K, Chakrabarty AM (2003) Microbial genes and enzymes in the degradation of chlorinated compounds. Chem Rec 3:158–171

    Article  CAS  PubMed  Google Scholar 

  • Okuta A, Ohnishi K, Harayama S (1998) PCR isolation of catechol 2, 3-dioxygenase gene fragments from environmental samples and their assembly into functional genes. Gene 212:221–228

    Article  CAS  PubMed  Google Scholar 

  • Perkins EJ, Gordon MP, Caceres O, Lurquin PF (1990) Organization and sequence analysis of the 2, 4-dichlorophenol hydroxylase and dichlorocatechol oxidative operons of plasmid pJP4. J Bacteriol 172:2351–2359

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Reineke W (1998) Development of hybrid strains for the mineralization of chloroaromatics by patchwork assembly. Annu Rev Microbiol 52:287–331

    Article  CAS  PubMed  Google Scholar 

  • Retallack DM, Thomas TC, Shao Y, Haney KL, Resnick SM, Lee VD, Squires CH (2006) Identification of anthranilate and benzoate metabolic operons of Pseudomonas fluorescens and functional characterization of their promoter regions. Microb Cell Fact 5:1

    Article  PubMed  PubMed Central  Google Scholar 

  • Schloss P, Handelsman J (2003) Biotechnological prospects from metagenomics. Curr Opin Biotechnol 14:303–310

    Article  CAS  PubMed  Google Scholar 

  • Shrestha A, Toyota K, Okazaki M, Suga Y, Quevedo MA, Loreto AB, Mariscal AA (2007) Enhancement of nitrogen-fixing activity of Enterobacteriaceae strains isolated from sago palm (Metroxylon sagu) by microbial interaction with non-nitrogen fixers. Microbes Environ 22:59–70

    Article  Google Scholar 

  • Stokes HW, Holmes AJ, Nield BS, Holley MP, Nevalainen KM, Mabbutt BC, Gillings MR (2001) Gene cassette PCR: sequence-independent recovery of entire genes from environmental DNA. Appl Environ Microbiol 67:5240–5246

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Throback IN, Enwall K, Jarvis A, Hallin S (2004) Reassessing PCR primers targeting nirS, nirK and nosZ genes for community surveys of denitrifying bacteria with DGGE. FEMS Microbiol Ecol 49:401–417

    Article  CAS  PubMed  Google Scholar 

  • Uchiyama T, Watanabe K (2006) Improved inverse PCR scheme for metagenome walking. BioTechniques 41:183–188

    Article  CAS  PubMed  Google Scholar 

  • Uchiyama T, Abe T, Ikemura T, Watanabe K (2005) Substrate-induced gene-expression screening of environmental metagenome libraries for isolation of catabolic genes. Nat Biotechnol 23:88–93

    Article  CAS  PubMed  Google Scholar 

  • Wertz S, Poly F, Le Roux X, Degrange V (2008) Development and application of a PCR-denaturing gradient gel electrophoresis tool to study the diversity of Nitrobacter-like nxrA sequences in soil. FEMS Microbiol Ecol 63:261–271

    Article  CAS  PubMed  Google Scholar 

  • Williamson N, Brian P, Wellington EM (2000) Molecular detection of bacterial and streptomycete chitinases in the environment. Antonie Van Leeuwenhoek 78:315–321

    Article  CAS  PubMed  Google Scholar 

  • Yamada K, Terahara T, Kurata S, Yokomaku T, Tsuneda S, Harayama S (2008) Retrieval of entire genes from environmental DNA by inverse PCR with pre-amplification of target gene using primers containing locked nucleic acids. Environ Microbiol 10:978–987

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Yeates C, Holmes AJ, Gillings MR (2000) Novel forms of ring-hydroxylating dioxygenases are widespread in pristine and contaminated soils. Environ Microbiol 2:644–653

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We would like to thank Naoto Ogawa (Shizuoka University) and Masahito Hayatsu (National Institute for Agro-Environmental Sciences) for their helpful suggestions. This work was supported by a grant-in-aid (Hazardous Chemicals) from the Ministry of Agriculture, Forestry, and Fisheries of Japan (HC-08-2323(1)-2).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Sho Morimoto.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Morimoto, S., Fujii, T. A new approach to retrieve full lengths of functional genes from soil by PCR-DGGE and metagenome walking. Appl Microbiol Biotechnol 83, 389–396 (2009). https://doi.org/10.1007/s00253-009-1992-x

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-009-1992-x

Keywords

Navigation