Metagenomic analysis of two important, but difficult to culture soil borne bacterial phyla, the Acidobacteria and the Verrucomicrobia

A.M. Kielak

Onderzoeksoutput: PhD Thesis (Proefschrift)PhD thesis (Proefschrift)


Based on phylogenetic marker genes, such as 16S rRNA genes, it is clear that numerous
bacterial lineages exist that appear to be quite common in the environment, yet poorly
characterized and underrepresented in culture. Two of the most common bacterial phyla in
soils that fall into this category are the Acidobacteria and Verrucomicrobiales. Although
knowledge of these two groups, and of their roles in global biogeochemical cycles is very
limited, their abundance and diversity suggest that they may be ecologically important.
The main objective of this thesis was to gain access to the genomic potential of soil-borne
Acidobacteria and Verrucomicrobiales using culture-independent approaches, namely
metagenomics. This objective was pursued via the following approaches:
- Studying the diversity and structure of microbial communities in relation to the
different experimental plant diversity treatments and across different soil
compartments using a combination of 16S rRNA gene-based methods (PCR-DGGE,
Q-PCR 16S rRNA gene library analysis) (Chapter 3).
- Examining the diversity of 16S rRNA genes affiliated with the phylum
Acidobacteria via three independent approaches: 1) screening of a fosmid
metagenome library for inserts containing Acidobacteria-like 16S rRNA genes; 2)
PCR-cloning using general bacterial primers; and 3) PCR-cloning with primers
specific for acidobacterial 16S rRNA gene sequences (Chapter 4)
- Screening of metagenomic libraries for genomic fragments affiliated with the
Acidobacteria and Verrucomicrobia, full-length inserts sequencing of positive clones
followed by subsequent gene annotation and evolutionary analyses (Chapter 5 and 6).
Acidobacteria were determined to be relatively more abundant in bulk soil as compared to
the rhizosphere, whereas Verrucomicrobia followed an opposite pattern, and plant species
composition appeared to have no significant effects on the structure of these two bacteria
phyla. Also, distinct differences were observed in the diversity of acidobacterial sequences
recovered via the different cloning strategies employed, partly due to observed biases of the
primer commonly use to specifically target 16S rRNA genes of the phylum Acidobacteria.
PCR-based screening methods were developed and implemented to analyze a fosmid
metagenomic library (28,800 clones) for the detection of clones containing Acidobacteriaand
Verrucomicrobia-like 16S rRNA genes. The resulting 17 confirmed clones of
acidobacterial and 4 of verrucomicrobial origin, representing a broad phylogenetic range,
were fully sequenced and annotated. These partial genome analyses provided a glimpse into
the functional capabilities within soil-borne members of the bacterial phyla Acidobacteria
and Verrucomicrobia, and yielded a novel dataset for comparative genomic analyses.
Originele taal-2Engels
KwalificatieDoctor of Philosophy
Toekennende instantie
  • Vrije Universiteit (VU) Amsterdam
  • van Veen, J.A., Promotor
  • Kowalchuk, G.A., Promotor
Datum van toekenning18 jan. 2010
Plaats van publicatieAmsterdam
StatusGepubliceerd - 2010


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