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.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:560426 |
| Date | January 2012 |
| Creators | Johnson-Rollings, Ashley S. |
| Publisher | University of Warwick |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://wrap.warwick.ac.uk/51637/ |
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