What are the products of enzymatic cleavage of organic N?

doi:10.1016/j.soilbio.2021.108152

Soil Biology and Biochemistry

Volume 154, March 2021, 108152

https://doi.org/10.1016/j.soilbio.2021.108152 Get rights and content

Highlights

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Chloroform fumigation was coupled with mass spectrometry identification of enzymatic products.
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Peptides were the dominant depolymerisation product when extra substrate was added.
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Enzymatic cleavage of endogenous SOM produced small peptides and amino acids in equal amounts.
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Peptides in soil extracts and produced by depolymerisation were mostly less than 500 Da.
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Nucleobases + nucleosides and headgroups of membrane lipids were produced at significant rates.

Abstract

Nitrogen commonly limits productivity, yet most soils contain a substantial pool of N in organic forms that are too large for rapid direct uptake. These larger forms of organic N generally become available only after they have been cleaved into smaller units by extracellular enzymes. Enzymatic cleavage of organic N is often equated with complete depolymerisation of protein to amino acids, yet it is likely that small peptides are also products of depolymerisation and there is a suite of other N-containing compounds that could be produced by enzymatic cleavage. The aims of this study were to a) characterize in a range of soils the pools of organic N that represent the substrates and products of enzymatic cleavage, viz., hydrolysable N and dissolved organic nitrogen (DON); b) develop a workflow for the untargeted identification and quantification of the products of enzymatic cleavage of organic N, and c) build a picture of the quantitative significance of different compounds for N cycling by determining their rates of production by enzymatic cleavage. Among a range of soils the pool of <3 kDa DON was quantitatively dominated by protein amino acids (69% of <3 kDa DON in H₂O and K₂SO₄ extracts) and small peptides (15% of DON in H₂O extracts and 26% in K₂SO₄ extracts). Only a small fraction of total hydrolysable N and DON was accounted for by compounds that originate from microbial cell walls (viz. hexosamines, muramic acid, diaminopimelic acid). To determine the products of enzymatic cleavage, soils were vacuum infiltrated with chloroform gas for five days to halt microbial metabolism, then enzymatic cleavage was assessed by incubating with water + casein (potential protein depolymerisation) or water alone (enzymatic cleavage of endogenous organic matter). Reaction products were identified and quantified using mass spectrometry, with rates of production determined from the increase between 30 min and 24 h of incubation. When extra substrate was added in the form of casein, small peptides were the primary products of depolymerisation, suggesting that assays of potential protease should be interpreted in terms of partial depolymerisation to peptides. Enzymatic cleavage of endogenous organic matter resulted in production of small peptides (generally < 500 Da) and amino acids in approximately equal amounts. Hexosamines, muramic acid, and diaminopimelic acid accounted for less than 1% of the products of enzymatic cleavage, while at least 2% was accounted for by nucleobases + nucleosides and headgroups of membrane lipids. We conclude that the workflow described here coupling chloroform gas infiltration with untargeted mass spectrometry of reaction products can reveal the diversity of compounds produced during enzymatic cleavage of organic matter. Our analysis confirmed that amino acids and peptides quantitatively dominate depolymerisation, and that decomposition of nucleic acids and lipids may supply significant amounts of N and ought to be the subject of further study.

Graphical abstract

Introduction

The majority of nitrogen (N) in soils is high molecular weight polymeric compounds (Leinweber et al., 2013) derived primarily from plant and microbial residues (Simpson et al., 2007; Schmidt et al., 2011). Proteins constitute the largest pool of organic N in microbial and plant cells, and typically constitute the single largest pool of organic N in soils (Sowden et al., 1977). For example, polymers of amino acids (i.e. proteins) have been shown to account for 50% or more of organic N in soils -- whether determined by chemical analyses of soil hydrolysates (Yu et al., 2002; Andersson and Berggren, 2005; Farrell et al., 2011; Hill et al., 2011, 2012) or nuclear magnetic resonance (Michalzik and Matzner, 1999; Kögel-Knabner, 2006). A smaller fraction of N in soils is accounted for by other polymeric compound classes derived from microbes, with much of the focus falling on peptidoglycan derived from bacterial cell walls and chitin from fungal cell walls (Liang et al., 2019; Hu et al., 2020). Peptidoglycan composition varies among bacterial species (Schleifer and Kandler, 1972) but typically involves polymer strands of N-acetylglucosamine and N-acetylmuramic acid cross-linked by short peptide chain of 3–5 amino acids attached to N-acetylmuramic acid (Vollmer et al., 2008). The peptide chain often includes not just L-enantiomers of protein amino acids but also D-enantiomers of amino acids and diaminopimelic acid (Vollmer et al., 2008). Chitin is polymeric chains of N-acetylglucosamine (Bowman and Free, 2006) and comprises cell walls of fungi, although the concentration of chitin in fungal cell walls varies among species and may be quite small (Plassard et al., 1982; Ekblad et al., 1998; Fernandez and Koide, 2012). Nucleic acid and other forms of N are usually thought to account for only a small fraction of the total pool (Stevenson, 1982; Kögel-Knabner, 2006). Monomeric compounds such as the membrane lipids of microbes are not high molecular weight (typical molecular weight is 650–800 Da, Warren, 2018) or viewed as significant pools of N, yet headgroups of most membrane lipids contain N and thus could contribute to N nutrition after enzymatic cleavage. More generally, one might expect the substrate pool for enzymatic cleavage to be chemically diverse given that the bulk of organic matter is derived from microbes (Simpson et al., 2007; Liang et al., 2019) and microbes synthesize a broad suite of organic compounds (Kallenbach et al., 2016).

High molecular weight forms of organic N are not generally biologically available and must be broken down prior to uptake by microbes or plants. There is some evidence showing direct uptake of intact proteins and microorganisms by plants (Paungfoo-Lonhienne et al., 2008, 2010), but uptake rates are slow and depolymerisation is the main pathway by which high molecular weight N becomes available by conversion into small peptides and amino acids that can be rapidly taken up (Näsholm et al., 2009; Hill et al., 2012; Farrell et al., 2013; Hu et al., 2018, 2020; Warren, 2019). The central role of depolymerisation in producing bio-available forms of N has led to suggestions that depolymerisation may be the rate-limiting step in the N cycle (Schimel and Bennett, 2004) and depolymerisation is related to N availability (Simpson et al., 2017). Depolymerisation of proteins is facilitated by extracellular proteases that catalyse the cleavage of peptide bonds (Ladd and Butler, 1972; Jan et al., 2009; Vranova et al., 2013; Nguyen et al., 2019a). Proteases are subdivided into two major groups -- exopeptidases and endopeptidases – based on their mode of action. The exopeptidases cleave peptide bonds proximal to the amino or carboxy termini of polypeptide chains, whereas endopeptidases cleave the peptide bonds in the inner regions of the polypeptide chain away from the N and C termini (Hartley, 1960; Rao et al., 1998). Soil microbial communities contain a broad suite of exo- and endopeptidases (Watanabe et al., 2003; Nguyen et al., 2019a, 2019b) as do individual species such as ectomycorrhizal fungi (Shah et al., 2013, 2016). It has been speculated that the synergistic action of a combination of exo- and endo-peptidases is necessary for the hydrolysis of proteins into the small peptides and amino acids that can be taken up by membrane transporters (Chalot and Brun, 1998).

We know that soils contain a broad suite of enzymes, and in vitro assays can characterize the function of those enzymes, yet surprisingly little is known about the products of enzymatic cleavage. Early biochemical studies established proteins can be completely hydrolysed to amino acids if the right mixture of enzymes is present (Hill and Schmidt, 1962) and perhaps based on this knowledge, enzymatic cleavage of soil organic N is often (implicitly or explicitly) equated with production of amino acids. The focus on amino acids is hard to justify because no studies to date have determined if the main products of protein depolymerisation are small peptides or amino acids. The products of depolymerisation of organic N cannot be predicted reliably from enzyme data owing to the complex mixture of different peptidases in soil, and because the presence of an enzyme does not directly translate to activity. Similarly, studies probing activity using simple substrates with a range of amino acid and peptide chemistries (Ladd and Butler, 1972; Hayano, 1993; Steen et al., 2019), or inhibitors (e.g. Nguyen et al., 2019a), have built up a picture of the mode of action and activity of different groups of peptidases – yet do not indicate the chemical profile of products of enzymatic cleavage.

Studies using isotope pool dilution have made good progress in determining gross fluxes of specific depolymerisation pathways. For example, rates of gross production of protein amino acids (i.e. due to complete depolymerisation of protein) are often an order of magnitude faster than gross production of D-amino acids, hexosamines, and diaminopimelic acid (Wanek et al., 2010; Hu et al., 2017, 2018). Most recently the isotope pool dilution methodology was extended by adding ¹⁵N labelled cell wall products and following decomposition to yield a partial molecular picture of decomposition of microbial necromass. It was suggested that muropeptides, rather than free muramic acid, were the major decomposition products of peptidoglycan. The authors reported that gross production of protein amino acids was the major contributor to total soil organic N fluxes (Hu et al., 2020), but fluxes of protein to peptide were not investigated. In general, past studies have specifically focused on the products of enzymatic cleavage of proteins and cell wall products of microbes, yet microbes and the microbial necromass are comprised of more than proteins and cell walls (Warren, 2015; Kallenbach et al., 2016; Liang et al., 2020). Much remains to be learnt about the diversity of compounds that could be produced by enzymatic cleavage of organic N.

Determining the products of enzymatic cleavage of organic N has proved challenging for several reasons. The ID and quantification of reaction products is difficult because enzymatic cleavage of organic N produces a complex mixtures of hydrophilic compounds that includes not only many hundreds of small peptides (Warren, 2013a) but also amino sugars, non-protein amino acids and muropeptides (Hu et al., 2018, 2020), all of which are present at low concentrations. The challenges are for the most part solvable, with studies showing a single analytical platform – capillary electrophoresis-mass spectrometry (CE-MS) – can provide good separation of a broad range of small monomeric organic N compounds (Warren, 2013b) and small peptides (Warren, 2013a), and has most recently shown to be capable of analysing muropeptides (Boulanger et al., 2019). The more intractable challenge is that in a living soil the products of enzymatic cleavage are consumed by microbes, such that pools of products (e.g. amino acids) do not accumulate over time but instead remain at low steady-state concentrations. One approach is to use isotope pool dilution to determine gross fluxes (i.e. rates of production) of individual compounds. However, applying isotope pool dilution to determine gross production of small peptides is impractical because of the need to add a mix of labelled isotopologues matching the molecular composition of the target pool – which in the case of soil comprises hundreds of small peptides at sub-micromolar concentrations (Warren, 2013a). More generally isotope pool dilution is limited to situations in which the product pool is well characterized, and thus cannot be applied where the products and their concentrations are unknown. An alternative approach is to disable living cells while leaving exoenzymes functional by vacuum infiltrating soil with chloroform gas (Blankinship et al., 2014). The logic behind this approach is that vacuum infiltration of soil with chloroform gas largely inactivates soil microbes but has little effect on exoenzyme activity. Consequently, the products of enzymatic cleavage ought to build up over time, thereby revealing the rate of production of individual compounds.

The aims of this study were to a) characterize the pools of organic N that represent the substrates and products of enzymatic cleavage, viz., hydrolysable N and DON; b) develop a workflow for the untargeted identification and quantification of the chemical products of enzymatic cleavage of organic N, and c) build a picture of the quantitative significance of different compounds for N cycling by determining their rates of production by enzymatic cleavage. To obtain a broad range in N availability we examined pools of <3 kDa and >3 kDa DON of soils from 19 sites encompassing a range of geologies and vegetation types. For a subset of 12 of these sites we characterized the total pool of (hydrolysable) organic N by subjecting soils to acid hydrolysis then analysing the released monomers of D- and L-amino acids, muramic acid, diaminopimelic acid and hexosamines. In five soils the products of enzymatic cleavage of organic N were chemically characterized and quantified by vacuum infiltrating soils with chloroform gas for five days, then using mass spectrometry to identify and quantify the reaction products during incubation of soils with water + casein (potential protein depolymerisation) or water alone (enzymatic cleavage of endogenous organic matter).

Section snippets

Site characteristics and soil sampling

The overall aim was to generalize among multiple soils rather than test for differences between soils, and thus with our finite time and resources we prioritized the number of sites, depth of chemical analysis and extent of data curation. Soils were collected from 19 undisturbed sites along the eastern seaboard of Australia (Table 1). The 19 sites included six chronosequence sites at Cooloola (Thompson, 1992), two sites on different geologies in Royal National Park (Beadle, 1954), five sites

Pools of DON and total hydrolysable N

Among sites CE-MS identified between 22 and 34 monomeric organic N compounds in the < 3 kDa fraction of H₂O extracts and K₂SO₄ extracts. The <3 kDa DON pool varied among sites from 5 to 118 nmol g⁻¹ in H₂O extracts and from 51 to 190 nmol g⁻¹ in K₂SO₄ extracts (Fig. 2) generally in accordance with differences in total N (Table 1). Protein amino acids were the most abundant compound class and comprised an average of 69% of <3 kDa DON in H₂O extracts and 49% in K₂SO₄ extracts (Supplementary Fig 2

Pools of dissolved and hydrolysable organic N

Pools representing the ultimate (total hydrolysable N) and proximate (>3 kDa DON) substrates for enzymatic cleavage were dominated by polymers of amino acids, with only a small fraction accounted for by compounds derived from the peptidoglycan (muramic acid and diaminopimelic acid) and chitin of microbial cell walls (hexosamines). Studies have consistently shown that polymers of amino acids account for >50% of total hydrolysable N (Stevenson, 1982; Kögel-Knabner, 2006), and absolute and

Conclusions

The inhibition of microbial uptake by chloroform fumigation coupled with mass spectrometry analysis of reaction products permits the identification and quantification of the products of enzymatic cleavage of organic N. Our analysis used CE-MS because it affords good coverage of N-containing compounds, but the same protocol could be extended to a more general view of organic matter breakdown by adding complementary analysis methods (e.g. GC-MS and LC-MS) that afford coverage of other compound

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

The University of Sydney is thanked for financial support via the major equipment scheme. Access to sample soils was provided by Queensland Parks and Wildlife Service, NSW National Parks and Wildlife Service, and The Tasmanian Department of Primary Industries, Parks, Water & Environment. Federico Maggi is thanked for providing useful feedback on the manuscript.

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This point was supported by our finding that the higher newly formed MBC was detected under N addition regardless of which forest soil was being used (Fig. S4). Meanwhile, we found that glucose addition (without N addition) caused a stronger N limit, as indicated by the higher enzymatic activity of acquiring N and higher C:N ratio under N addition (Fig. S6; Oulehle et al., 2018; Zheng et al., 2022), which could lead to the utilization of amino sugars to acquire N sources (Cui et al., 2020; Warren, 2021). We also detected that β-N-acetylglucosaminidase (NAG) had a negative effect on newly formed total amino sugars, while soil N availability had a negative effect on NAG (Fig. S2).
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Microbial biomass is increasingly considered to be the main source of organic carbon (C) sequestration in soils. Quantitative information on the contribution of microbial necromass to soil organic carbon (SOC) formation and the factors driving necromass accumulation, decomposition and stabilization during the initial soil formation in biological crusts (biocrusts) is absent. To address this knowledge gap, we investigated the composition of microbial necromass and its contributions to SOC sequestration in a biocrust formation sequence consisting of five stages: bare sand, cyanobacteria stage, cyanobacteria-moss stage, moss-cyanobacteria stage, and moss stage on sandy parent material on the Loess Plateau. The fungal and bacterial necromass C content in soil was analyzed based on amino sugars - the cell wall biomarker. Microbial necromass was an important source of SOC, and was incorporated into the particulate and mineral-associated organic C (MAOC). Because bacteria have smaller and thinner cell wall fragments as well as more proteins than fungi, bacterial necromass mainly contributed to the MAOC pool, while fungal residues remained more in the particulate organic C (POC). MAOC pool was saturated fast with the increase of microbial necromass, and POC more rapid accumulation than MAOC suggests that the clay content was the limiting factor for stable C accumulation in this sandy soil. The necromass exceeding the MAOC stabilization level was stored in the labile POC pool (especially necromass from fungi). Activities of four enzymes (i.e., β-1,4-glucosidase, β-1,4-N-acetyl-glucosaminidase, leucine aminopeptidase, and alkaline phosphatase) increasing with fungal and bacterial necromass suggest that the raised activity of living microorganisms accelerated the turnover and formation of necromass. Microbial N limitation raised the production of N acquisition enzymes (e.g., β-1,4-N-acetyl-glucosaminidase and leucine aminopeptidase) to break down necromass compounds, leading to further increase of the nutrient pool in soil solution. The decrease of microbial N limitation along the biocrusts formation chronosequence is an important factor for the necromass accumulation during initial soil development. High microbial N demands and insufficient clay protection lead to fast necromass reutilization by microorganisms and thus, result in a low necromass accumulation coefficient, that is, the ratio of microbial necromass to living microbial biomass (on average, 9.6). Consequently, microbial necromass contribution to SOC during initial soil formation by biocrust is lower (12–25%) than in fully developed soils (33%–60%, literature data). Nitrogen (N) limitation of microorganisms and an increased ratio between N-acquiring enzyme activities and microbial N, as well as limited clay protection, resulted in a low contribution of microbial necromass to SOC by initial formation of biocrust-covered sandy soil. Summarizing, soil development leads not only to SOC accumulation, but also to increased contribution of microbial necromass to SOC, whereas the plant litter contribution decreases.
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However, we acknowledge that potential microbial differences induced during soil storage may have increased the response we observed when using 5 mM sucrose perfusates, and so it is difficult to determine if a C dose response is apparent in our study. We also acknowledge that organic N compounds (such as amino acids) can also contribute significantly to N fluxes in soils (Inselsbacher and Näsholm, 2012; Brackin et al., 2015; Oyewole et al., 2016) and along with proteins and peptides, are likely products of soil organic matter decomposition (Farrell et al., 2011; Macdonald et al., 2014; Hill et al., 2019; Warren, 2021) and are quickly acquired by soil microbes. As proteolysis forms a bottleneck for N availability in N-limited soil systems such as the one studied here (Schimel and Bennett, 2004), C-rich root exudates may provide a critical ecosystem function by promoting microbial exoenzyme production and, consequently, increasing rates of proteolysis and protein availability.
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