A polyphasic approach to the study of chitinolytic bacteria in soil

Doctoral thesis English OPEN
Johnson-Rollings, Ashley S.
  • Subject: QR
    mesheuropmc: complex mixtures | bacteria | fungi

Chitin is the most abundant nitrogen-containing polymer in nature, with >1x10^10 tonnes\ud produced annually in terrestrial and marine habitats. Chitinolytic bacteria are able to\ud degrade this recalcitrant substrate through a multiplicity of chitinases. A polyphasic\ud approach was taken to studying these organisms within three diverse soil communities.\ud Fluorometric assays employing 4-methylumbelliferyl-labelled chitinooligosaccharides were\ud used to estimate basal soil chitinase activity as well as its chitinolytic potential in response\ud to a- and b-chitin amendment. A molecular approach was adopted to profile the bacterial\ud community and functional chi gene diversity within the soils. Finally, a method of exploring\ud the metaexoproteome, enabling investigation of the dominant chitin degraders at\ud a functional level, was developed and implemented. The metaexoproteome and metaproteome,\ud extracted with an existing method, were compared and used to infer the functional\ud dominance of chitinolytic phyla.\ud The basal chitinase activity in all soils was found to be low, yet chitin amendment rapidly\ud induced chitinases in all soils although intersite differences were seen. b-chitin amendment\ud induced more chitinolytic activity in Cayo Blanco (CB) compared to Sourhope (SH). The\ud Test Soil (TS), a site biannually amended with carapaces, retained higher chitinolytic\ud potential many months after chitin had been consumed.\ud Next-generation pyrosequencing enabled >50% of the potential OTUs present in the soil\ud to be recovered. The 16S rRNA gene analysis of SH revealed dominant phyla to be Proteobacteria,\ud Actinobacteria, and Acidobacteria with little change between amendments. The\ud TS was dominated by the same phyla but saw a proliferation of Actinobacteria with chitin\ud amendment. CB experienced the inverse response to the Test Soil, initially dominated by\ud Actinobacteria only for Proteobacteria to dominate with amendment. Firmicutes were also\ud prevalent with b-chitin amendment.\ud Functional chi gene analysis found Streptomyces-like GH19 chi genes to dominate in both\ud SH and CB. A rare Actinomycete Planobispora dominated chitin-amended TS. This organism\ud 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-\ud like GH18 chi genes only dominated CB with amendment and were absent in SH.\ud A large number of OTUs were identified as uncultured organisms suggesting a large pool\ud of uncharacterized GH18 chi genes.\ud Metaproteomics is the functional analysis of complex communities at a given point in time.\ud The heterogeneity of soil, associated microbial communities, and presence of interfering\ud compounds make the extraction of protein from soil a technical challenge. Chitinases\ud are extracellular and so the metaexoproteome was targeted after development of a novel\ud method that biased extraction towards exoproteins. The protocol successfully extracted the\ud largest soil metaproteome to date. Actinobacterial chitinases were found to be functionally\ud dominant in the Test Soil, especially in response to b-chitin amendment.
  • References (173)
    173 references, page 1 of 18

    1 Introduction 1 1.1 Chitin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1.1.1 Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1.1.2 Presentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 1.1.3 Uses of chitin and chitosan . . . . . . . . . . . . . . . . . . . . . . . 3 1.1.4 Degradation of chitin . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 1.1.4.1 Two families of chitinases . . . . . . . . . . . . . . . . . . . 5 1.1.4.2 Two mechanisms, exo- and endo- acting . . . . . . . . . . . 7 1.1.5 Multiplicity in the chitinolytic system . . . . . . . . . . . . . . . . . 8 1.1.6 Chitin as a nitrogen source . . . . . . . . . . . . . . . . . . . . . . . 9 1.2 Molecular approach to studying microbial diversity . . . . . . . . . . . . . . 11 1.3 Surveying the functionally dominant chitin degraders . . . . . . . . . . . . . 14 1.4 Hypotheses and aims . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

    2 Materials and General Methods 18 2.1 Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 2.2 Materials and Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 2.3 Strains and Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 2.4 Field sites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 2.4.1 Sourhope, Scotland, UK . . . . . . . . . . . . . . . . . . . . . . . . . 24 2.4.2 Cayo Blanco, Cuba . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 2.4.3 Test Soil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 2.4.4 Soil Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 2.4.5 Sampling methods for soil . . . . . . . . . . . . . . . . . . . . . . . . 30 2.4.5.1 Sourhope . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 2.4.5.2 Cayo Blanco . . . . . . . . . . . . . . . . . . . . . . . . . . 30 2.4.5.3 Test Soil . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 2.5 The preparation of the microcosms . . . . . . . . . . . . . . . . . . . . . . . 31 2.5.1 Calculating water content . . . . . . . . . . . . . . . . . . . . . . . . 31 2.5.2 General microcosm preparation . . . . . . . . . . . . . . . . . . . . 31 2.5.3 Degradation of carapace waste microcosm preparation . . . . . . . . 32 6 General Discussion 203 6.1 Future work . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206

    7 Appendix 209 7.1 Soil Texture Triangle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209 7.2 Supplementary metaproteomic analysis information . . . . . . . . . . . . . 210 7.2.1 Sampling 1D SDS-PAGE gel . . . . . . . . . . . . . . . . . . . . . . 210 7.2.2 Distribution of same-set hits . . . . . . . . . . . . . . . . . . . . . . . 210 7.2.3 Summary of pyrosequencing data . . . . . . . . . . . . . . . . . . . . 211 7.2.4 Calculating the coefficient of determination . . . . . . . . . . . . . . 211 7.2.5 Distribution of COGs between TS a TP and TS b TP . . . . . . . . 212 7.2.6 Calculation of Spearman's rank correlation coefficient . . . . . . . . 212 7.3 Programmes and packages used . . . . . . . . . . . . . . . . . . . . . . . . . 213 Lee, H.-J., Han, S.-I., and Whang, K.-S., (2011), Catenulispora graminis sp. nov., rhizobacterium from bamboo (Phyllostachys nigro var. henonis) rhizosphere soil, International Journal of Systematic and Evolutionary Microbiology, p. [In Press].

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