Terminal-restriction fragment length polymorphism (T-RFLP) screening of a marine archaeal clone library to determine the different phylotypes

M. M. Moeseneder*, C. Winter, Jesus Arrieta, G. J. Herndl

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

79 Scopus citations

Abstract

T-RFLP clone characterization (screening) was optimized for a fast and basepair-accurate characterization of clones from marine Archaea collected from the Eastern Mediterranean Sea. Because of the high sensitivity of T-RFLP fingerprinting, a protocol was developed where 10 initial PCR cycles gave detectable terminal fragments from clones. Additionally, forward and reverse primers for PCR were individually labeled and detected simultaneously to assess the suitability of the forward and reverse fragments for T-RFLP screening. Based on independent restriction digests with the tetrameric restriction enzymes HhaI, RsaI and HaeIII to characterize the 49 archaeal clones in our library, the clones were grouped into 13 T-RFLP operational taxonomic units (OTUs). Reverse fragments generally gave less heterogeneous fragments in size. The accuracy of T-RFLP screening was evaluated by sequencing representative clones. Closely related clones (≈97% similarity) could only be resolved with multiple restriction digests where forward and reverse fragments were included in the analysis. All fragments from the clone library were detected in the T-RFLP fingerprint from the complex archaeal community. We found representatives of marine group I, II and III Archaea. Thus, the recently discovered low abundant marine group III Archaea could be clearly differentiated from the other clones in our library and comprised a considerable fraction of the clone library (≈12%). Therefore, our T-RFLP screening approach proved successful in characterizing novel archaeal sequences from the marine environment.

Original languageEnglish (US)
Pages (from-to)159-172
Number of pages14
JournalJournal of Microbiological Methods
Volume44
Issue number2
DOIs
StatePublished - Mar 1 2001

Keywords

  • Clone library
  • Planktonic Archaea
  • T-RFLP fingerprinting

ASJC Scopus subject areas

  • Biotechnology
  • Applied Microbiology and Biotechnology
  • Microbiology

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