A polyphasic approach to the study of chitinolytic bacteria in soil

Chitin is the most abundant nitrogen-containing polymer in nature, with >1x10^10 tonnes
\nproduced annually in terrestrial and marine habitats. Chitinolytic bacteria are able to
\ndegrade this recalcitrant substrate through a multiplicity of chitinases. A polyphasic
\napproach was taken to studying these organisms within three diverse soil communities.
\nFluorometric assays employing 4-methylumbelliferyl-labelled chitinooligosaccharides were
\nused to estimate basal soil chitinase activity as well as its chitinolytic potential in response
\nto a- and b-chitin amendment. A molecular approach was adopted to profile the bacterial
\ncommunity and functional chi gene diversity within the soils. Finally, a method of exploring
\nthe metaexoproteome, enabling investigation of the dominant chitin degraders at
\na functional level, was developed and implemented. The metaexoproteome and metaproteome,
\nextracted with an existing method, were compared and used to infer the functional
\ndominance of chitinolytic phyla.
\nThe basal chitinase activity in all soils was found to be low, yet chitin amendment rapidly
\ninduced chitinases in all soils although intersite differences were seen. b-chitin amendment
\ninduced more chitinolytic activity in Cayo Blanco (CB) compared to Sourhope (SH). The
\nTest Soil (TS), a site biannually amended with carapaces, retained higher chitinolytic
\npotential many months after chitin had been consumed.
\nNext-generation pyrosequencing enabled >50% of the potential OTUs present in the soil
\nto be recovered. The 16S rRNA gene analysis of SH revealed dominant phyla to be Proteobacteria,
\nActinobacteria, and Acidobacteria with little change between amendments. The
\nTS was dominated by the same phyla but saw a proliferation of Actinobacteria with chitin
\namendment. CB experienced the inverse response to the Test Soil, initially dominated by
\nActinobacteria only for Proteobacteria to dominate with amendment. Firmicutes were also
\nprevalent with b-chitin amendment.
\nFunctional chi gene analysis found Streptomyces-like GH19 chi genes to dominate in both
\nSH and CB. A rare Actinomycete Planobispora dominated chitin-amended TS. This organism
\nis 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-
\nlike GH18 chi genes only dominated CB with amendment and were absent in SH.
\nA large number of OTUs were identified as uncultured organisms suggesting a large pool
\nof uncharacterized GH18 chi genes.
\nMetaproteomics is the functional analysis of complex communities at a given point in time.
\nThe heterogeneity of soil, associated microbial communities, and presence of interfering
\ncompounds make the extraction of protein from soil a technical challenge. Chitinases
\nare extracellular and so the metaexoproteome was targeted after development of a novel
\nmethod that biased extraction towards exoproteins. The protocol successfully extracted the
\nlargest soil metaproteome to date. Actinobacterial chitinases were found to be functionally
\ndominant in the Test Soil, especially in response to b-chitin amendment.