- Soil nitrogen exists largely as organic matter, including plant liter, dead animal matter, and microbial necromass. About 90% of soil organic nitrogen is proteinaceous material that is too large for plants and microorganisms to assimilate directly. Protein depolymerization therefore plays a critical role in mobilizing this organic source of nitrogen, producing lower molecular weight molecules that are bioavailable for both microorganisms and plants. The decomposition of proteins in soils serves as the rate-limiting step of the nitrogen cycle. The ability of microorganisms to access and break down proteinaceous material depends largely on their production of extracellular peptidases, but it involves a trade-off with the energetic cost of producing and secreting these enzymes into the environment, including the risk that other microorganisms can compete with the peptidase-producing organisms for the products released through depolymerization. Consequently, in order to optimize this energy investment, there might be a tight connection between soil environmental conditions and microbial proteolytic activity. Despite its ecological importance, there is a lack of understanding about the diversity of these extracellular peptidases and their activity as an important factor influencing the protein degradability in soils.
In this dissertation, I first assessed the genetic potential for microorganisms to produce extracellular enzymes, and second, I developed and applied a novel approach to measure the activities of different classes of peptidases in soil. In my first two chapters, I evaluated the abundance and diversity of microbial extracellular peptidases, their evolutionary conservation, and distribution as a function of environmental habitat and lifestyle. Chapter 2 focuses on the secreted peptidases of prokaryotes (Archaea, Bacteria); chapter 3 focuses on Fungi, the dominant soil eukaryote. In both chapters, I analyzed secreted peptidases across microbial lineages using their genomic information and corresponding annotated protein sequences assembled from several databases, including MEROPS, Silva, JGI Genome Portal, and MycoCosm. Peptidase gene sequences of 147 archaeal, 2,191 bacterial and 612 fungal genomes were screened for secretion signals, resulting in 55,072 prokaryotic and 31,668 eukaryotic genes coding for secreted peptidases. I found that Archaea, Bacteria, and Fungi possess unique complements of secreted peptidases and there are differences in the number of secreted peptidases per genome, indicating potential differential abilities for organic nitrogen acquisition. The majority of secreted peptidase families not only follow the phylogenetic evolutionary distribution, but also segregate based on the microbial lifestyles and microbial habitats. This suggests that microorganisms optimize their secreted peptidases to match their surrounding environments.
In Chapter 4, I incorporated the use of selective inhibitors to block the activity of different classes of peptidases. I designed a protocol with these peptidase inhibitors to use directly in natural soils. I validated and optimized this protocol with pure enzymes and peptidase-supplemented soils. This research revealed that the profile of extracellular peptidase activities belonging to different catalytic types varies among soils and correlates with both soil chemical and microbial properties. This is in line with our assumption that soil microorganisms respond to their environmental conditions by investing in peptidases that can optimize their activity.
Collectively, this work provides a comprehensive and foundational understanding about the contribution of different catalytic types of microbial extracellular peptidases to organic nitrogen turnover in soils.