A polyphasic approach to the study of chitinolytic bacteria in soil
Johnson-Rollings, Ashley S. (2012) A polyphasic approach to the study of chitinolytic bacteria in soil. PhD thesis, University of Warwick.
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Official URL: http://webcat.warwick.ac.uk/record=b2585260~S1
Chitin is the most abundant nitrogen-containing polymer in nature, with >1x10^10 tonnes
produced annually in terrestrial and marine habitats. Chitinolytic bacteria are able to
degrade this recalcitrant substrate through a multiplicity of chitinases. A polyphasic
approach was taken to studying these organisms within three diverse soil communities.
Fluorometric assays employing 4-methylumbelliferyl-labelled chitinooligosaccharides were
used to estimate basal soil chitinase activity as well as its chitinolytic potential in response
to a- and b-chitin amendment. A molecular approach was adopted to profile the bacterial
community and functional chi gene diversity within the soils. Finally, a method of exploring
the metaexoproteome, enabling investigation of the dominant chitin degraders at
a functional level, was developed and implemented. The metaexoproteome and metaproteome,
extracted with an existing method, were compared and used to infer the functional
dominance of chitinolytic phyla.
The basal chitinase activity in all soils was found to be low, yet chitin amendment rapidly
induced chitinases in all soils although intersite differences were seen. b-chitin amendment
induced more chitinolytic activity in Cayo Blanco (CB) compared to Sourhope (SH). The
Test Soil (TS), a site biannually amended with carapaces, retained higher chitinolytic
potential many months after chitin had been consumed.
Next-generation pyrosequencing enabled >50% of the potential OTUs present in the soil
to be recovered. The 16S rRNA gene analysis of SH revealed dominant phyla to be Proteobacteria,
Actinobacteria, and Acidobacteria with little change between amendments. The
TS was dominated by the same phyla but saw a proliferation of Actinobacteria with chitin
amendment. CB experienced the inverse response to the Test Soil, initially dominated by
Actinobacteria only for Proteobacteria to dominate with amendment. Firmicutes were also
prevalent with b-chitin amendment.
Functional chi gene analysis found Streptomyces-like GH19 chi genes to dominate in both
SH and CB. A rare Actinomycete Planobispora dominated chitin-amended TS. This organism
is usually found in extremely arid soil. It was not found in the 16S rRNA gene analysis or the metaproteome; further analysis is required to confirm its presence. Streptomyces-
like GH18 chi genes only dominated CB with amendment and were absent in SH.
A large number of OTUs were identified as uncultured organisms suggesting a large pool
of uncharacterized GH18 chi genes.
Metaproteomics is the functional analysis of complex communities at a given point in time.
The heterogeneity of soil, associated microbial communities, and presence of interfering
compounds make the extraction of protein from soil a technical challenge. Chitinases
are extracellular and so the metaexoproteome was targeted after development of a novel
method that biased extraction towards exoproteins. The protocol successfully extracted the
largest soil metaproteome to date. Actinobacterial chitinases were found to be functionally
dominant in the Test Soil, especially in response to b-chitin amendment.
|Item Type:||Thesis or Dissertation (PhD)|
|Subjects:||Q Science > QR Microbiology|
|Library of Congress Subject Headings (LCSH):||Soil microbiology, Chitin, Bacteria|
|Official Date:||March 2012|
|Institution:||University of Warwick|
|Theses Department:||School of Life Sciences|
|Supervisor(s)/Advisor:||Wellington, E. M. H. (Elizabeth M. H.), 1954-|
|Sponsors:||Natural Environment Research Council (Great Britain) (NERC) ; Society for General Microbiology (SGM) ; Colorado State University|
Supplementary information on disc stored with hard copy - please see Official URL for details of how to access
|Extent:||xxiv, 257 leaves : illustrations, charts|
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