pubmed.ncbi.nlm.nih.gov

Internal transcribed spacer 1 secondary structure analysis reveals a common core throughout the anaerobic fungi (Neocallimastigomycota) - PubMed

  • ️Wed Jan 01 2014

Internal transcribed spacer 1 secondary structure analysis reveals a common core throughout the anaerobic fungi (Neocallimastigomycota)

Christian Koetschan et al. PLoS One. 2014.

Abstract

The internal transcribed spacer (ITS) is a popular barcode marker for fungi and in particular the ITS1 has been widely used for the anaerobic fungi (phylum Neocallimastigomycota). A good number of validated reference sequences of isolates as well as a large number of environmental sequences are available in public databases. Its highly variable nature predisposes the ITS1 for low level phylogenetics; however, it complicates the establishment of reproducible alignments and the reconstruction of stable phylogenetic trees at higher taxonomic levels (genus and above). Here, we overcame these problems by proposing a common core secondary structure of the ITS1 of the anaerobic fungi employing a Hidden Markov Model-based ITS1 sequence annotation and a helix-wise folding approach. We integrated the additional structural information into phylogenetic analyses and present for the first time an automated sequence-structure-based taxonomy of the ITS1 of the anaerobic fungi. The methodology developed is transferable to the ITS1 of other fungal groups, and the robust taxonomy will facilitate and improve high-throughput anaerobic fungal community structure analysis of samples from various environments.

PubMed Disclaimer

Conflict of interest statement

Competing Interests: Four of the authors are from AgResearch Ltd., which is a Crown Research Institute, and is funded by the Pastoral Greenhouse Gas Research Consortium (PGgRc) to develop means of mitigating ruminant methane emissions. PGgRc's members are AgResearch, Fonterra, Fert Research, PGG Wrightson, DairyNZ, Deer Research, Beef+Lamb New Zealand, Landcorp, NIWA, the Ministry for Primary Industries, and the Ministry of Science and Innovation (formerly Foundation for Research, Science and Technology). The publication of the data reported here is at the discretion of the PGgRc. The PGgRc did not control which data were presented or how these data were interpreted within this paper. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials. The other authors have declared that no competing interests exist. There are no patents, products in development or marketed products to declare.

Figures

Figure 1
Figure 1. Visualization of secondary structure, sequence motifs and sequence annotation.

(A) The secondary structure of the ITS1 region of JF423742 (selected as an example) illustrates the common core shape observed in all analysed neocallimastigomycete sequences. It folds into a three helix conformation, with two short helices (I and III) and a longer one in between (II). Additionally, four very conserved sequence motifs are highlighted in the structure. (B) The black bar shows a nucleotide string of rDNA. On the left is the terminus of the 18S rRNA gene, with its 18S HMM. Together with the 5.8S HMM motif on the right, both enable an exact delineation of the ITS1 region.

Figure 2
Figure 2. Secondary structures of the ITS1 region from three different sequence types.

ITS1 secondary structures from (A) EU414759, (B) HQ832485 and (C) JF423612. The illustrated structures did not fold into the typical three helix conformation when folded initially with UNAFold (upper images). By breaking the sequences into three parts based on the known location of sequence motifs, each part could be folded individually. The concatenated helices (bottom images) result in the typical three-helix common core structure predicted by the direct folding of the majority of all annotated sequences.

Figure 3
Figure 3. CBC visualization of neocallimastigomycete ITS1 regions.

(A) Parts of the consensus secondary ITS1 structure of GQ850303 and JF423532 from helix II. Marked in yellow are compensatory base changes between the two sequences. (B) Partial consensus secondary ITS1 structure of JF423714 and JX184822. CBCs occurring in helix I are highlighted in yellow.

Figure 4
Figure 4. Profile Neighbor Joining tree of neocallimastigomycete ITS1 sequences.

Profile Neighbor Joining tree calculated using sequence and structure data from 1120 (575 unique) complete neocallimastigomycete ITS1 sequences with 1000 bootstrap replicates in 29 iterations. Open circles indicate bootstrap values in a range of 50 to 90, closed circles indicate a bootstrap value above 90, and the scale bar indicates the distance. The tree contains the six known genera Anaeromyces, Caecomyces, Cyllamyces, Neocallimastix, Orpinomyces and Piromyces. and the unclassified sequences GQ850325, AF170206 and AF170205. Other monophyletic groups are highlighted and named according to Kittelmann et al. . A total of ten sequences did not cluster into any of the defined groups and were named according to their accession numbers only (JF423517, JF423484, JF423882, GU055516, JX184570, JF423626, JF423625, GQ850325, AF170205, and AF170206). Sequences, accession numbers and taxonomic classifications (including definitions of subclusters of the genera Cyllamyces, Orpinomyces and Piromyces) are available from the taxonomy file (File S2). Subcluster Orpinomyces 3 is not represented in this tree due to the lack of full-length ITS1 sequences for this group. A comparison of bootstrap values from this study to the sequence–only analysis of Kittelmann et al. is given in Table S1. YE505  =  Anaeromyces mucronatus YE505.

Similar articles

Cited by

References

    1. Hibbett DS, Binder M, Bischoff JF, Blackwell M, Cannon PF, et al. (2007) A higher-level phylogenetic classification of the Fungi. Mycological Research 111: 509–547. - PubMed
    1. Ebersberger I, De Matos Simoes R, Kupczok A, Gube M, Kothe E, et al. (2012) A consistent phylogenetic backbone for the fungi. Molecular Biology and Evolution 29: 1319–1334. - PMC - PubMed
    1. Orpin CG (1975) Studies on the rumen flagellate Neocallimastix frontalis. Journal of General Microbiology 91: 249–262. - PubMed
    1. Schoch CL, Seifert KA, Huhndorf S, Robert V, Spouge JL, et al. (2012) Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi. Proceedings of the National Academy of Sciences 109: 1–6. - PMC - PubMed
    1. Nilsson RH, Ryberg M, Abarenkov K, Sjökvist E, Kristiansson E (2009) The ITS region as a target for characterization of fungal communities using emerging sequencing technologies. FEMS Microbiology Letters 296: 97–101. - PubMed

Publication types

MeSH terms

Substances

Grants and funding

This work was supported by and research was carried out under contract to the Pastoral Greenhouse Gas Research Consortium (PGgRc; www.pggrc.co.nz), and the New Zealand Ministry for Primary Industries (MPI; www.mpi.govt.nz) as part of its support for the Global Research Alliance on Agricultural Greenhouse Gases (GRA; www.globalresearchalliance.org). The funders had no role in study design, data collection and analysis, or preparation of the manuscript. The authors required the funders' approval to publish.

LinkOut - more resources