Sequence analysis of percent G+C fraction libraries of human faecal bacterial DNA reveals a high number of Actinobacteria

Lotta Krogius-Kurikka,Anna Kassinen,Jarno Tuimala,Lars Paulin,Harri Mäkivuokko,Jukka Corander,Airi Palva

doi:10.1186/1471-2180-9-68

Abstract

BackgroundThe human gastrointestinal (GI) tract microbiota is characterised by an abundance of uncultured bacteria most often assigned in phyla Firmicutes and Bacteroidetes. Diversity of this microbiota, even though approached with culture independent techniques in several studies, still requires more elucidation. The main purpose of this work was to study whether the genomic percent guanine and cytosine (%G+C) -based profiling and fractioning prior to 16S rRNA gene sequence analysis reveal higher microbiota diversity, especially with high G+C bacteria suggested to be underrepresented in previous studies.ResultsA phylogenetic analysis of the composition of the human GI microbiota of 23 healthy adult subjects was performed from a pooled faecal bacterial DNA sample by combining genomic %G+C -based profiling and fractioning with 16S rRNA gene cloning and sequencing. A total of 3199 partial 16S rRNA genes were sequenced. For comparison, 459 clones were sequenced from a comparable unfractioned sample. The most important finding was that the proportional amount of sequences affiliating with the phylum Actinobacteria was 26.6% in the %G+C fractioned sample but only 3.5% in the unfractioned sample. The orders Coriobacteriales, Bifidobacteriales and Actinomycetales constituted the 65 actinobacterial phylotypes in the fractioned sample, accounting for 50%, 47% and 3% of sequences within the phylum, respectively.ConclusionThis study shows that the %G+C profiling and fractioning prior to cloning and sequencing can reveal a significantly larger proportion of high G+C content bacteria within the clones recovered, compared with the unfractioned sample in the human GI tract. Especially the order Coriobacteriales within the phylum Actinobacteria was found to be more abundant than previously estimated with conventional sequencing studies.

Highlights

The human gastrointestinal (GI) tract microbiota is characterised by an abundance of uncultured bacteria most often assigned in phyla Firmicutes and Bacteroidetes
The previously published 976 sequences from three %G+C fractions (%G+C 25–30, 40–45 and 55–60) [21] were combined with the 2223 new sequences cloned in this study (%G+C fractions 30–35, 35–40, 45–50, 50–55, 60–65, 65–70 and 70–75) for phylogenetic and statistical analyses of the complete %G+C profile ranging from 25% G+C to 75% G+C (Figure 1, Table 1)
Order Coriobacteriales abundant within Actinobacteria We observed that several clones in the high %G+C fractions (60–70% G+C content) were tricky to sequence due to extremely G+C rich regions

Summary

Introduction

The human gastrointestinal (GI) tract microbiota is characterised by an abundance of uncultured bacteria most often assigned in phyla Firmicutes and Bacteroidetes. Diversity of this microbiota, even though approached with culture independent techniques in several studies, still requires more elucidation. The gastrointestinal (GI) microbiota is considered to play an important role in human health and disease via essential metabolic, trophic and protective functions in the host [1]. The bacterial groups most often detected in molecular studies of the healthy human GI tract are phyla Firmicutes (especially Clostridium clusters XIVa and IV), Bacteroidetes, Proteobacteria, Actinobacteria, Fusobacteria and Verrucomicrobia [3]. As a reference to altered conditions, knowledge of the characteristics of a healthy intestinal microbiota is essential

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