Pathways of nitrogen utilization by soil microorganisms - Agricultural ...

Soil Biology & Biochemistry 42 (2010) 2058e2067

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Review

Pathways of nitrogen utilization by soil microorganisms e A review Daniel Geisseler a, c, *, William R. Horwath a, Rainer Georg Joergensen b, Bernard Ludwig c a

Department of Land, Air and Water Resources, University of California, Davis, 1 Shields Avenue, Davis, CA 95616, USA Department of Soil Biology and Plant Nutrition, University of Kassel, Nordbahnhofstr. 1a, 37213 Witzenhausen, Germany c Department of Environmental Chemistry, University of Kassel, Nordbahnhofstr. 1a, 37213 Witzenhausen, Germany b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 19 March 2010 Received in revised form 10 August 2010 Accepted 18 August 2010 Available online 4 September 2010

Microorganisms are able to utilize nitrogen (N) from a wide range of organic and mineral compounds. In this paper, we review the current knowledge about the regulation of the enzyme systems involved in the acquisition of N and propose a conceptual model on the factors affecting the relative importance of organic and mineral N uptake. Most of the N input into soil is in the form of polymers, which first have to be broken down into smaller units by extracellular enzymes. The small organic molecules released by the enzymes can then be taken up directly or degraded further and the N taken up as ammonium (NHþ 4 ). When NHþ 4 is available at high concentrations, the utilization of alternative N sources, such as nitrate þ (NO 3 ) and organic molecules, is generally repressed. In contrast, when the NH4 availability is low, enzyme systems for the acquisition of alternative N sources are de-repressed and the presence of a substrate can induce their synthesis. These mechanisms are known as N regulation. It is often assumed that most organic N is mineralized to NHþ 4 before uptake in soil. This pathway is generally known as the mineralization-immobilization-turnover (MIT) route. An advantage of the MIT route is that only one transporter system for N uptake is required. However, organic N uptake has the advantage that, in addition to N, it supplies energy and carbon (C) to sustain growth. Recent studies have shown that the direct uptake of organic molecules can significantly contribute to the N nutrition of soil microorganisms. We hypothesize that the relative importance of the direct and MIT route during the decomposition of residues is determined by three factors, namely the form of N available, the source of C, and the availability of N relative to C. The regulation system of soil microorganisms controls key steps in the soil N cycle and is central to determining the outcome of the competition for N between soil microorganisms and plants. More research is needed to determine the relative importance of the direct and MIT route in soil as well as the factors affecting the enzyme systems required for these two pathways. Ó 2010 Elsevier Ltd. All rights reserved.

Keywords: Microbial N uptake Direct route Mineralization-immobilization-turnover N regulation Extracellular enzymes

1. Introduction Nitrogen (N) is required by all organisms as an essential nutrient. In terrestrial ecosystems, N is generally of limiting availability to plants, resulting in strong competition between microorganisms and plants (Vitousek and Howarth, 1991). Microorganisms have developed different mechanisms for uptake and assimilation of mineral and organic forms of N, enabling them to utilize a wide range of organic and mineral compounds (see reviews by Merrick and Edwards, 1995; Marzluf, 1997). In terrestrial ecosystems, plant and microbial residues are the two main sources of organic input, while the relative contribution of * Corresponding author at: Department of Environmental Chemistry, University of Kassel, Nordbahnhofstr. 1a, 37213 Witzenhausen, Germany. Tel.: þ49 (0) 5542 98 1635; fax: þ49 (0) 5542 98 1633. E-mail address: [email protected] (D. Geisseler). 0038-0717/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2010.08.021

animal residues is small (Kögel-Knabner, 2002). In residues, the quantitatively most important N containing molecules are proteins, chitin and peptidoglycan. Proteins alone comprise 60% or more of the N in plant and microbial cells (Cochrane, 1958; Christias et al., 1975; Fuchs, 1999; Sinha, 2004). Mineral forms of N, such as ammonium (NHþ 4 ) and nitrate (NO3 ), usually account for a small proportion of the total N in cells. Most of the N input into soil is therefore in the form of polymers, which first have to be broken down into smaller units by extracellular enzymes (Schimel and Bennett, 2004). The small organic molecules released by the enzymes can then be taken up directly or degraded further and the N taken up as NHþ 4 (Fig. 1). Extracellular depolymerases therefore constitute the first step in the degradation of most organic N input to soil. Ammonium is considered the preferred source of N for bacteria and fungi (Merrick and Edwards, 1995; Marzluf, 1997). In addition to NHþ 4 , glutamate and glutamine, which serve as the key N donors for biosynthetic reactions in virtually all cells, are also used as preferred

D. Geisseler et al. / Soil Biology & Biochemistry 42 (2010) 2058e2067

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2. Extracellular depolymerases

Fig. 1. Main steps of the microbial N utilization from organic substrates.

N sources by many microorganisms (see reviews by Magasanik, 1993; Merrick and Edwards, 1995; Wong et al., 2008). All transformation and N uptake processes are mediated by enzymatic systems that require carbon (C), N and energy for their synthesis and expression. In soil where N and C are often of low availability, it is crucial for organisms to tightly regulate the synthesis and activity of the different enzyme systems. Soil microorganisms, which regulate enzyme production according to their needs and the availability of substrates, will have a competitive advantage (Koch, 1985). In fact, when preferred N sources are available at high concentrations, the transcription of genes encoding for enzymes required for the utilization of alternative N sources, such as nitrate (NO 3 ) and most organic molecules, is repressed. This mechanism is known as N regulation (Magasanik, 1993). It is often assumed that all organic N is first mineralized to NHþ 4 before it is taken up by microorganisms in soil. This pathway, which was largely formulated by Jansson (1958), is generally known as the mineralization-immobilization-turnover (MIT) route. More recent studies however, have shown that the uptake of small organic molecules, such as amino acids, can significantly contribute to the N nutrition of soil microorganisms (Barak et al., 1990; Hadas et al., 1992; Barraclough, 1997; Luxhøi et al., 2006; Geisseler et al., 2009). In this review, the term direct route will be used as a collective term for the different mechanisms responsible for the uptake of organic molecules. The route of N uptake has implications for the competition for N between plants and microorganisms. When the MIT route is dominant, plants and microorganisms compete for mineral N (Manzoni and Porporato, 2007). On the contrary, when the direct route is dominant, microorganisms meet their N requirements with organic N compounds and plants face reduced competition from microorganisms for mineral N. However, a number of studies, recently reviewed by Näsholm et al. (2009), have shown that plants are also capable of acquiring organic N, either directly through root cell membranes or via mycorrhizal fungi. The quantitative contribution of organic N uptake to the N nutrition of plants is likely affected by the N uptake pathway of soil microorganisms. Only a few studies have quantified the contribution of the MIT and direct route to the N nutrition of soil microorganisms and little is known about the factors affecting the importance of the two pathways in soil. The objectives of this present paper are: (i) to summarize the current knowledge about the regulation of the enzyme systems involved in the acquisition of N by soil microorganisms; and (ii) to propose a conceptual model on the factors affecting the relative importance of the MIT and direct route.

Extracellular depolymerases are required to degrade complex organic polymers from plant and microbial residues into soluble subunits that can be taken up by microorganisms. Based on the chemical composition of the main sources of organic residues in soil, the most important extracellular depolymerases involved in the hydrolysis of N containing molecules are proteases, chitinases and peptidoglycan hydrolases. Extracellular proteases hydrolyze large proteins and polypeptides into peptides and amino acids. The ability to produce extracellular proteases is widespread among bacteria and fungi (Ahearn et al., 1968; Gupta et al., 2002). Extracellular proteases usually have wide substrate-specificities and can degrade most non-structural proteins (Kalisz, 1988). Proteases are also synthesized for intracellular use, where they play a key role in the regulation of metabolic processes and in protein turnover. The latter is essential for adaptation of cells to new environmental conditions, especially in response to starvation for nutrients (Kalisz, 1988). Chitin, an unbranched polymer of N-acetyl-D-glucosamine, is widely distributed in nature and one of the most abundant polymers on earth (Duo-Chuan, 2006). Chitinases hydrolyze the links between N-acetyl-D-glucosamine molecules (Felse and Panda, 1999). Chitinases are produced by a wide range of organisms, including bacteria, fungi and plants, but not by archaea (Gooday, 1990a; Duo-Chuan, 2006). Bacteria produce chitinases mainly to degrade chitin for use as a N and C source. In fungi, however, chitinases also play an important role in cell wall development and architecture during active growth (Gooday, 1990a; Adams, 2004; Bhattacharya et al., 2007). Peptidoglycan is composed of different amino sugars and amino acids which are linked together by three different chemical bonds, namely glycosidic, amide, and peptide, to form a two- or threedimensional net-like polymer (Vollmer et al., 2008). The hydrolysis of peptidoglycan therefore requires the activity of different amidases, peptidases and glycosylases, the latter including glucosaminidases and lysozymes (Shockman et al., 1996). Peptidoglycan hydrolases are common among fungi (Grant et al., 1986). In bacteria, peptidoglycan hydrolases play an important role in the modification of the cell wall for growth, cell separation, or sporulation as well as in the turnover and recycling of peptidoglycans (Vollmer et al., 2008). Maintaining the balance between necessary modifications of the cell walls and unwanted weakening of it may therefore be the main purpose of the regulation of peptidoglycan hydrolases in bacteria. 2.1. Regulation of extracellular depolymerases Four major mechanisms, namely (i) substrate induction, (ii) endproduct repression, (iii) de-repression due to insufficient nutrient supply and (iv) constitutive production, have been found to regulate the production (synthesis and secretion) of extracellular hydrolases. Production of extracellular hydrolases is generally induced by the presence of a substrate. Microbial proteases have been found to be induced by the presence of proteins in the medium (Kalisz, 1988; Haab et al., 1990), while chitinases are induced by the presence of chitin, but also by low concentrations of N-acetyl-D-glucosamine (Felse and Panda, 1999; Scigelova and Crout, 1999; Duo-Chuan, 2006). The presence of bacterial cells has been found to induce the production of extracellular muramidase in the basidiomycete Schizophyhm commune and the cultivated mushroom Agaricus bisporus (Grant et al., 1990; Lincoln et al., 1997) In contrast, end-products and easily metabolizable C sources may repress enzyme production (Table 1). Protease production is

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Table 1 Ammonium (NHþ 4 ) concentrations found to repress extracellular enzymes involved in microbial N acquisition. Enzyme system/NHþ 4 concentration

Effect on activity (%)

Species

Environment

Reference

Mixed community Beauveria bassiana

Soil Growth medium

Geisseler and Horwath, 2008 Bidochka and Khachatourians, 1988

30 mM NH4eN 30 mM NH4eN

16% to þ7% 49% (in the medium) 6% mg1 biomass 5% 7%

Scleroaum rolfsii Scleroaum bataticola

Growth medium Growth medium

Sayed et al., 1994 Sayed et al., 1994

Glycine aminopeptidase 114 mmol NH4eN added g1 soil

approx. 35%

Mixed community

Soil

Allison and Vitousek, 2005

N-acetyl-D-glucosaminidase 37.5 mM NH4eN

50% mg1 biomass

Beauveria bassiana

Growth medium

Bidochka and Khachatourians, 1993

Urease 5 mmol NH4eN added g1 soil 25 mmol NH4eN added g1 soil 50 mmol NH4eN added g1 soil 110e170 mM NH4eN

13 34 76 71

Mixed community Mixed community Mixed community Proteus rettgeri

Soil Soil Soil Growth medium

McCarty et al., 1992 McCarty et al., 1992 McCarty et al., 1992 Magaña-Plaza and Ruiz-Herrera, 1967

Amino Acid Oxidase 10 mM NH4eN 30 mM NH4eN

22% 30%

Chlumydomonas reinhardtii Chlumydomonas reinhardtii

Enzyme extract Enzyme extract

Vallon et al., 1993 Vallon et al., 1993

Protease 2.9 mmol NH4eN added g1 soil 37.5 mM NH4eN

to to to to

15% 85% 95% 92%

The microorganisms were cultivated in the presence of the substrates for the different enzyme systems and varying concentrations of a Enzymes were extracted from bacteria grown on an N-free medium.

generally repressed by amino acids, NHþ 4 , glucose and other readily available C compounds (Glenn, 1976; Allison and Macfarlane, 1992). High levels of N-acetyl-D-glucosamine, however, repress chitinase synthesis, as do glucose and other easily available C sources (Felse and Panda, 1999; Scigelova and Crout, 1999; Duo-Chuan, 2006). In the entomopathogenic fungus Beauveria basssiana, chitinase was also repressed by NHþ 4 and certain amino acids (Bidochka and Khachatourians, 1993; Table 1). Different effects of end-products on muramidases have been reported. While extracellular muramidases of A. bisporus and S. commune were repressed by glucose (Grant et al., 1990; Lincoln et al., 1997), b-glucosaminidase production by A. bisporus was highest in the presence of glucose and fructose (Lincoln et al., 1997). In addition, Fermor et al. (1991) found that A. bisporus mineralized bacteria even in the presence of glucose and ammonium sulfate. De-repression of protease production has been found to take place when C, N or sulfur (S) were limiting (Kalisz, 1988). Finally, extracellular enzymes may be secreted constitutively at low levels regardless of the availability of substrates or endproduct. Constitutive production has been reported for protease (Kalisz, 1988; Haab et al., 1990), chitinases (Felse and Panda, 1999), and muramidase (Grant et al., 1990; Lincoln et al., 1997). Constitutive production of extracellular enzyme synthesis in situations with limited substrate availability may be sufficient to initiate the degradation of new substrates, resulting in the release of soluble oligomers, which in turn induces enzyme synthesis (Felse and Panda, 1999). 2.2. Extracellular depolymerases in soil The ability to degrade protein, chitin and peptidoglycan is widespread among soil microorganisms (Grant et al., 1986; Gooday, 1990b; Adesina et al., 2007). Compared to studies with cultured microorganisms, less work has been done to systematically study the regulation of extracellular depolymerases by soil microbial communities in their natural environment. Substrate induction has been found to be an important mechanism regulating the production of extracellular depolymerases. In tundra soil, protease activity was increased with the addition of protein (Weintraub and Schimel, 2005). This result is in line with a laboratory incubation carried out by Geisseler and Horwath

NHþ 4;

a a

except.

(2008), where protease activity increased when casein, zein or gluten were added to soil. Allison and Vitousek (2005) also found increased glycine aminopeptidase activity in soil samples amended with collagen. Similarly, chitinase activity in soil has been found to be induced by the presence of chitin (Rodriguez-Kabana et al., 1983; Abdel-Fattah and Mohamedin, 2000; Ueno and Miyashita, 2000). Protease activity is often well correlated with the microbial biomass and a certain level of residual activity of extracellular depolymerases is generally found in soil (Doran, 1980; Geisseler and Horwath, 2008). These findings suggest constitutive synthesis. However, as soil organic matter and residues are constantly decomposed, and enzymes may be protected by soil colloids, as discussed below, it is challenging to determine whether the enzymes are produced at a constitutive level or are induced by the presence of substrates. Similarly, microbial biomass C and N as well as b-glucosaminidase increased in a field study at intermediate poultry litter rates greater than 6.7 Mg ha1 compared to sites with no applied litter (Acosta-Martínez and Harmel, 2006). In this study, the specific b-glucosaminidase activity (per unit microbial C and N) increased in general with increasing poultry litter application in cultivated soil, while it remained relatively constant in the soil under pasture. Based on these studies it cannot be clearly distinguished between substrate induced and constitutive enzyme synthesis at low levels of activity. De-repression of protease synthesis under N and S limiting conditions has been reported by Sims and Wander (2002) in sand cultures. Not only total, but also specific (i.e. per unit biomass) proteolytic activity was increased under these conditions. In contrast, Asmar et al. (1992) found that the addition of glucose to an agar containing medium increased protease activity. In contrast to cultured species, where NHþ 4 generally represses extracellular depolymerase activity, the effect of NHþ 4 on the activity of these enzymes is less consistent in soil. While Allison and Vitousek (2005) found that glycine aminopeptidase was decreased by about one third after 28 days in response to NHþ 4 addition, Geisseler and Horwath (2008) found that the NHþ 4 concentration in soil had no effect on protease activity. However, the amount of NH4eN added was much lower in the latter study compared to the former (Table 1). In a study by Jezierska-Tys and Frac (2009), increased protease and urease activity were observed together with elevated NHþ 4 concentrations over a period of several weeks after


the application of dairy sewage sludge to soil. Across a chronosequence, chitinase activity decreased significantly with increasing soil age and N availability. In addition, N fertilization repressed chitinase activity only at the N limited young site. These results suggest that N supply in older soil reached levels high enough to trigger a negative feedback and inhibit enzyme production (Olander and Vitousek, 2000). In contrast, in samples of the organic horizon from forest floors, Michel and Matzner (2003) found that b-glucosaminidase activity responded positively to mineral N additions, particularly in samples with low internal N concentrations. They attributed the lack of negative effects to the fact that b-glucosaminidase is related to the actively growing biomass in soil. Increasing the N supply in these soils may have increased fungal biomass. This is in line with a cropping system comparison carried out by Ekenler and Tabatabai (2002) who found that b-glucosaminidase activity was generally increased in plots with þ NHþ 4 applications compared to plots without NH4 additions. 2.3. Regulatory enzyme mechanisms in soils versus culture media Studies with cultured microorganisms are very helpful in determining the regulatory mechanisms for different enzyme systems. However, the conditions in soil differ from those in culture media in many ways, which may affect the observed response to specific treatments. The studies discussed above suggest that extracellular depolymerases are less tightly regulated in soil than in culture media. One reason why soil microorganisms do not down-regulate their enzyme synthesis as readily as cultured microbes may be the fact that soil microbes live in a nutrient poor environment. Oligotroph organisms may have adapted over evolutionary time to be very efficient in growing under low levels of nutrients and the achievement of high efficiency under low-substrate conditions may have crippled the cell to not being effective when there are high concentrations of nutrients. Most organisms in a nutrient-poor environment cannot be selective and cannot choose to use only certain organic substrates (Koch, 2005). In fact, it appears that substrate induction has a stronger effect on enzyme production in soil than repression by end-products and easily metabolizable C sources (Asmar et al., 1992; Geisseler and Horwath, 2008). In contrast to soil microorganisms, cultured microorganisms often belong to species important for industrial applications. These species were selected for their capacity to maximize their metabolic activity under constant conditions with a high nutrient availability. In addition, the microbial population in soil is very diverse with most of these species being involved in organic matter turnover. This genetic diversity results in a wide variety of metabolic processes and interactions between organisms. Another reason of the slow response of soil microorganisms to an increase in NHþ 4 availability may be that extracellular enzymes are beyond the control of microorganisms. Extracellular enzymes have been found to be associated with soil colloids, which may increase their half-life (Burns, 1982). Several studies found this to be true for proteases. Asmar et al. (1992) found that in soils amended with King’s agar and nutrients, the activity of soluble extracellular protease was about 30% of the total protease activity. Total protease activity persisted longer than the activity of its soluble extracellular fraction in these soils. Using different substrates, Bonmatí et al. (1998, 2009) found that the proteases induced by these substrates were predominantly associated with soil organic matter fractions. Only casein-hydrolyzing proteases were present, at least partially, as glyco-proteins not associated to humus (Bonmatí et al., 2009). In contrast to culture media, soil is a spatially and temporally heterogeneous environment. The spatial arrangement of soil

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particles creates a large number of microenvironments differing in their physical, chemical and biological properties (Ranjard and Richaume, 2001). In addition, the input of organic material in soil is highly heterogeneous in space and time, and temporal changes in the soil moisture content affect solute diffusion and nutrient fluxes (Or et al., 2007). In unsaturated soil, water films are thin and pores may be disconnected, resulting in restricted diffusion of substrates towards microorganisms and of waste products and extracellular enzymes away from cells. Compared to culture media, molecules must follow a more tortuous path to diffuse from one point to another, reducing the substrate flux to the cell surface (Griffin, 1981; Moldrup et al., 2001). Due to these differences between culture media and soil, the response of soil microbial communities may not be as pronounced as found with cultured species and processes, which occur separately in culture media, may take place concurrently in soil. It is therefore important to verify results from cultured species in soil. The differences between culture media and soil may also affect the regulation of extracellular N mineralizing enzymes, which will be discussed in the following section. 3. Extracellular N mineralization The small organic molecules released by extracellular depolymerases can be taken up directly or be subjected to extracellular mineralization. The MIT route includes the mineralization of organic molecules, followed by the uptake and assimilation of the released NHþ 4 . With the exception of urease, which has been extensively studied due to the importance of urea as fertilizer, the enzymes responsible for the extracellular ammonification of organic compounds in soil and their regulation have received little attention. 3.1. Urease Urease catalyzes the hydrolysis of urea to yield NH3 and carbamate. The latter compound spontaneously decomposes to yield another molecule of ammonia and carbonic acid (Mobley et al., 1995). Urea is continuously released into the environment through biological processes such as urine excretion from mammals. In addition, urea is a product of the degradation of the amino acid arginine, of uric acid, which is excreted by birds, reptiles, and most terrestrial insects as their primary detoxification product, as well as of purines and pyrimidines, which are building blocks of nucleic acids (Cunin et al., 1986; Mobley and Hausinger, 1989; Vogels and van der Drift, 1976). Urease can be produced by bacteria, yeasts, filamentous fungi, and algae (Mobley and Hausinger, 1989), as well as plants (Follmer, 2008). Urease may be synthesized constitutively in some organisms; however, most commonly urease expression is under N regulation (Mobley et al., 1995). Urease synthesis is repressed when the cells are grown in the presence of a preferred N source such as NHþ 4 (Table 1). In contrast, urease synthesis is activated in the presence of urea and alternative N sources, as well as under conditions of N starvation (Mobley et al., 1995). Considerable evidence indicates that urease is cytoplasmatic in both yeasts and bacteria (Mobley and Hausinger, 1989). However, a large proportion of the urease activity in soil has been found to be extracellular and associated with soil particles. Pettit et al. (1976) calculated that in the soil examined approximately 60% of the total urease activity was extracellular-bound and that the remainder was composed of intracellular and extracellularunbound enzyme. Klose and Tabatabai (1999) estimated the extracellular proportion of urease activity to be 46%. Soil urease has been found to be primarily associated with soil organic matter and

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clay minerals. In surface soil samples of cultivated field, Reynolds et al. (1985) found a strong positive correlation between urease activity and soil organic C and total soil N, while the correlation between urease activity and clay content was weaker. In addition, Kandeler et al. (1999) reported that organic amendments increased the capacity of a soil to protect urease in the clay-sized fraction. This association protects the enzyme from attack by proteolytic enzymes, does not prevent urease-urea activity and allows diffusion of substrate molecules to, and product molecules from sites of active enzymes (Burns et al., 1972). The bonds between soil particles and urease appear to be strong. Zanuta and Bremner (1977) found that leaching a wide range of soils with water, drying for 24 h at temperatures ranging from 30 to 60 C, storage for six months at temperatures from 20 to 40 C, and incubation under aerobic or waterlogged conditions did not decrease urease activity significantly. Therefore, extracellular urease activity in soil seems to be mainly the result of its release from decaying microbial and plant cells and subsequent protection from proteolysis, rather than excretion by living cells. In contrast to studies with cultured microorganisms, several studies in soil found that addition of urea did not increase urease activity (Lloyd and Sheaffe, 1973; Zanuta and Bremner, 1976), which may be due to the high proportion of protected extracellular urease. However, repression by NHþ 4 may also be responsible for these results. In fact, when C in the form of glucose or other readily available C sources was added together with urea to soil, urease activity increased. These results suggest that the addition of a C source increased the N demand of microorganisms above its availability, so that N became limiting (Lloyd and Sheaffe, 1973; Zanuta and Bremner, 1976). In response, the genes responsible for urease synthesis were de-repressed resulting in an increase of urease activity. This is in line with McCarty et al. (1992) who found evidence that microbial production of urease in soil was repressed 1 soil resulted by NHþ 4 and NO3 . The addition of 50 mmol NH4eN g in a decrease in urease activity of 76e95%, while the same addition of NO3eN decreased urease activity by about two thirds (McCarty et al., 1992).

subsequent intracellular deamination when C sources other than amino acids are available. Why would an organism utilize a cell-surface amino acid oxidase rather than take up the amino acid? An advantage of the latter, and therefore of the direct route, is that amino acids not only supply N, but also C. In addition, the ability to incorporate the amino acid directly into protein saves the energy required to synthesize a C skeleton. However, the composition of the soil amino acid pool may not match the needs of the microorganisms. Furthermore, it might require several different amino acid transporters to take up the different amino acids available while NHþ 4 transporters are most likely already operative (see below). Secretion of a broad-specificity L-amino acid oxidase may provide a means to release NHþ 4 from diverse amino acids while the toxic by-products are produced outside the cell (Davis et al., 2005). In addition, the production of H2O2 may also suppress competing species (Tong et al., 2008). Several ectomycorrhizal basidiomycetes and ascomycetes as well as saprotrophic fungal and bacterial species, some of them isolated from soil, have been found to produce amino acid oxidases (Braun et al., 1992; Davis et al., 2005; Nuutinen and Timonen, 2008). The amino acid oxidases of soil microorganisms seem to have predominantly broad substrate specificities (Nuutinen and Timonen, 2008). Assays specific to amino acid oxidase activity are not yet available for soil samples. However, assays for enzymes deaminating glutamine, asparagine, hystidine, aspartate have been developed for soil (Frankenberger and Johanson, 1981; Frankenberger and Tabatabai, 1991a,b; Senwo and Tabatabai, 1996). As these assays measure enzyme activity based on the release of NHþ 4 , amino acid oxidases may contribute to the activity measured. Relatively little is known about the location and regulation of these deaminases in soil. The activities of asparagines, glutamine and histidine deaminating enzymes in soil were found to originate predominantly from intracellular enzymes (Badalucco et al., 1996; Deng et al., 2006). However, enzymes protected by soil colloids may also contribute to the overall activity (Klose and Tabatabai, 2002). 4. Nitrogen uptake mechanisms by microbial cells

3.2. Amino acid oxidase More than 60% of the organic N input into soil is in the form of proteinaceous material (Christias et al., 1975; Fuchs, 1999; Sinha, 2004). The deamination of amino acids and subsequent uptake of the released NHþ 4 are therefore key reactions of the MIT route. While amino acid deaminases seem to be mainly intracellular enzymes (Badalucco et al., 1996; Deng et al., 2006), the production of cell surface-bound amino acid oxidases has been reported for fungal and bacterial species (Böhmer et al., 1989; Braun et al., 1992; Davis et al., 2005; Nuutinen and Timonen, 2008). L-amino acid oxidases catalyze the oxidative deamination of L-amino acids to produce the corresponding keto-acids, NH3 and hydrogen peroxide (H2O2). Some amino acid oxidases have been characterized by strict substrate specificity, while others exhibit relatively broad substrate specificity (Braun et al., 1992). Extracellular deamination of amino acids is mainly associated with enzymes bound to the cell surface rather than with enzymes liberated into the environment or with abiotic deamination processes (Pantoja and Lee, 1994). The expression of the genes encoding for amino acid oxidases have been found to be repressed by NHþ 4 (Palenik and Morel, 1990; Vallon et al., 1993; Table 1), de-repressed by N limitation or starvation (Davis et al., 2005) and induced by amino acids in the presence of a C source (Vallon et al., 1993; Davis et al., 2005). The requirement for C may indicate that extracellular deamination is only favorable compared to the direct amino acid uptake and

The products of extracellular depolymerases and N mineralizing enzymes are N sources for the cells. The enzyme systems responsible for the transport across membranes are generally very specific. Enzyme systems for mineral N uptake and assimilation, required for the operation of the MIT route, as well as systems for the uptake of organic molecules, which play a central role for the direct route, will be presented in the following sections. 4.1. Uptake of ammonia and ammonium Ammonia is a gas that can diffuse through biological membranes (Andrade and Einsle, 2007). Most biological membranes seem to exhibit a measurable permeability towards NH3. The rate of unspecific diffusion depends of the concentration gradient across the membrane and the permeability of the membrane (Kleiner, 1981). Microorganisms therefore have limited possibilities to regulate NH3 diffusion into or from cells. However, they possess membrane proteins for the uptake of NHþ 4 and NH3. Several fungal and bacterial species have been found to possess more than one system with distinct uptake properties (Marini et al., 1997; Meier-Wagner et al., 2001; Monahan et al., 2002; Mitsuzawa, 2006). These membrane protein systems are capable of forming a large NHþ 4 gradient across biomembranes. For this reason they were generally believed to be transporters, which actively, at the expense


of energy, transport NHþ 4 into the cytoplasm (Kleiner, 1981). Some recent studies, however, suggested that these proteins may serve as passive NH3 channels (Khademi et al., 2004; Zheng et al., 2004). Andrade and Einsle (2007) proposed a mechanistic model that links the transport of NH3 to a symport of Hþ. According to their model, NHþ 4 is being deprotonated after binding to the transporter, and NH3 and Hþ are taken up following separate, but coupled, pathways. In the presence of a proton motive force (pH in solution below cytoplasmatic pH) the transport would be active, while in its absence passive uptake of NH3 may be observed (Andrade and Einsle, 2007). Therefore, in alkaline environments, less energy may be required for NH3/NHþ 4 uptake, as NH3 diffusion may contribute considerably to the intracellular accumulation of NHþ 4 (Kleiner, 1985). The optimum pH for the active uptake of NH3/NHþ 4 has been found to be between 6 and 7 (Kleiner, 1981). At external NHþ 4 concentrations greater than 10e20 mM, the synthesis of the NH3/NHþ 4 uptake proteins is repressed, presumably because diffusion of NH3 through the membrane is fast enough to support the cell’s demand for N (Kleiner, 1981; Marini et al., 1997; Table 2). However, when the NHþ 4 supply is low and the organisms þ grow on N sources other than NHþ 4 , the synthesis of the NH3/NH4 uptake systems is de-repressed (Kleiner, 1985). 4.2. Assimilation of ammonium Once taken up, NHþ 4 is predominantly assimilated into biological molecules via either the glutamine synthetase/glutamate synthase pathway (GS/GOGAT) or through glutamate dehydrogenase (GDH). The energy requirement for the GS/GOGAT pathway has been estimated to be 18e30% higher than for the GDH pathway (Helling, 1994; Neidhardt et al., 1990). The expenditure of energy is compensated for by the fact that GS has a much higher affinity for þ NHþ 4 than GDH and therefore enables organisms to scavenge NH4 at low concentrations (Merrick and Edwards, 1995; Schmid et al., 2000; Silberbach et al., 2005).

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In both, bacteria and fungi, GS activity is repressed by high NHþ 4 concentrations (Ertan, 1992; Baars et al., 1995; Table 2) and its activity is decreased when the organism is stressed for energy (Gräzer-Lampart et al., 1986; Helling, 1998). GS has also been reported to be repressed by several amino acids such as alanine and glycine (Hubbard and Stadtman, 1967; Zofall et al., 1996). 4.3. Uptake and assimilation of nitrate Nitrate can be used by many microorganisms as N source and is taken up by specific transporters (Gonzalez et al., 2006). While NHþ 4 can be assimilated directly, NO 3 has to be reduced first in two steps by assimilatory NO 3 reductase (ANR) and nitrite reductase before it can be assimilated in the form of NHþ 4 (McCarty, 1995). This þ assimilatory reduction of NO 3 to NH4 requires energy. As many other N utilization enzymes systems, NO 3 uptake is under N regulation. The assimilation of NO-3 has been found to be induced by NHþ 4 limitation and by the presence of NO3 and nitrite, while it is in the culture medium (Table 2; repressed by the presence of NHþ 4 Stewart, 1994; Gonzalez et al., 2006). In aerated soil, NHþ 4 is generally rapidly oxidized to NO3, a reaction known as nitrification (Myrold, 1998). Therefore, the NO 3 concentration often exceeds the NHþ 4 concentration in soil. Several studies found a strong inhibition of NO-3 uptake by soil microorganisms in the presence of NHþ 4 in soil solution (Rice and Tiedje, 1989; McCarty and Bremner, 1992). In a study using selective inhibitors, Myrold and Posavatz (2007) found that bacteria had a greater potential for assimilating NO 3 than fungi in the soils used. 4.4. Uptake of organic nitrogen molecules For the direct route to be operative, microorganisms have to be able to take up organic N molecules. In fact, microorganisms possess a wide range of transport enzymes for the uptake of organic N molecules and monomers, such as amino acids and amino sugars.

Table 2 Ammonium (NHþ 4 ) concentrations found to repress microbial enzyme systems for N uptake and assimilation. Enzyme system/NHþ 4 concentration

Effect on activity (%)

Species

Environment

Reference

NHþ 4 /NH3 uptake systems 10 mM NH4eN 10 mM NH4eN 50 mM NH4eN

100% 43% 99%

Rhizobium Ieguminosarum MNF3841 Aspergillus nidulans Aspergillus nidulans

Growth medium Growth medium Growth medium

O’Hara et al., 1985 Pateman et al., 1973 Pateman et al., 1973

Glutamine synthetase 10 mM NH4eN 100 mM NH4eN 20 mM NHþ 4 eN 142 mM NH4eN

14% 72% approx. 70% 83%

Corynebacterium callunae Corynebacterium callunae Agaricus biosporus Klebsiella aerogenes MK53

Growth Growth Growth Growth

Ertan, 1992 Ertan, 1992 Baars et al., 1995 Friedrich and Magasanik, 1977

NO 3 uptake system 8.6 mM NH4eN 714 mM NH4eN 57 mM NH4eN 57 mM NH4eN 57 mM NH4eN

65% 60 to 80% 0% 79% 93%

Mixed community Mixed community Acotobacter vinelandii Saccharomyces cerevisiae Pseudumonas fluorescens

Soil slurries Soil slurries Growth medium Growth medium Growth medium

Rice Rice Rice Rice Rice

Nitrate reductase 48 mM NH4eN

100%

Alcaligenes eutrophus

Growth medium

Warnecke- Eberz and Friedrich, 1993

General amino acid permease 10 mM NH4eN

approx. 90%

Saccharomyces cerevisiae NY13

Growth medium

Springael and André, 1998

Acidic Amino Acid Permease 25 mM NH4eN 10 mM NH4eN

approx. 91% 9%

Aspergillus nidulans (germinating) Aspergillus nidulans (germinated)

Growth medium Growth medium

Robinson et al., 1973b Robinson et al., 1973a

L-Glutamate permease 10 mM NH4eN

93%

Aspergillus nidulans

Growth medium

Pateman et al., 1973

medium medium medium medium

The microorganisms were cultivated in the presence of the substrates for the different enzyme systems and varying concentrations of a An N-free medium served as control.

and and and and and

NHþ 4;

Tiedje, Tiedje, Tiedje, Tiedje, Tiedje,

a a

1989 1989 1989 1989 1989

except:.

a

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Utilization of amino acids as N sources involves transport of the molecules into cells followed by enzymatic removal of the alpha amino N through deamination or transamination (DeBusk and Ogilvie, 1984). The transport of amino acids into cells is catalyzed by functionally specific transport systems that are located in the cytoplasmatic membrane. Up to twelve kinetically-defined transport systems for groups of structurally related amino acids have been found in bacterial cells. Fungi posses fewer amino acid transport systems than bacteria. However, fungal transport systems are less specific, acting on groups of amino acids with similar properties. The transport of amino acids is active (Anraku, 1980; Booth and Hamilton, 1980; Wolfinbarger, 1980). While some amino acid transport systems are constitutive, the majority of the systems are under N regulation. Ammonium and high intracellular concentrations of amino acids repress amino acid transport systems (Table 2), whereas C, N, or S starvation activates the synthesis of amino acid transport systems (Oxender et al., 1980; Wolfinbarger, 1980). Like the amino acid transporters, microbial transport systems for peptides are energy dependent and are located in the cytoplasmatic membrane. The upper size limit of peptide transport systems across membranes seems to be about 600 Da, which corresponds to a penta- or hexapeptide (Payne and Smith, 1994). The uptake of peptides as opposed to corresponding mixtures of amino acids generally requires less energy and has been shown to be nutritionally superior (Matthews and Payne, 1980). Transporters for the uptake of amino sugars originating from chitin and peptidoglycan have been identified for different bacteria and fungal species. The transporters have been found to be induced by the presence of the substrate and repressed by glucose (Plumbridge, 1990; Brinkkötter et al., 2000; Ezquerro-Sáenz et al., 2006; Alvarez and Konopka, 2007; Vollmer et al., 2008). Given the widespread occurrence of the N regulation system, it seems likely that these uptake systems follow the same regulatory mechanisms as amino acid transporters. 5. Nitrogen uptake pathways in soil Our review of the enzyme systems involved in the utilization of N by microorganisms and their regulation shows that in soils, being an environment with many different N sources and often limited supply of N, the conditions favoring the direct as well as the MIT route may be met. Several studies determined the relative importance of the two pathways in soil. In a study with labeled leucine and glycine added to soil, Barraclough (1997) showed that the conventional MIT route was not operative and the results were consistent with the direct route. Drury et al. (1991) came to a similar conclusion, based on the observation that addition of 15NHþ 4 to soil in which net mineralization was occurring resulted in no incorporation of the added 15N into the microbial biomass. The authors of two other studies concluded that both pathways, the direct and MIT route, operated concurrently (Hadas et al., 1992; Luxhøi et al., 2006). Different approaches were used in these studies to determine the relative importance of the two routes. Hadas et al. (1992) used the NCSOIL model to simulate the CeN turnover and 15N distribution among soil pools in soils amended with NHþ 4 and alanine. Luxhøi et al. (2006) compared gross N mineralization rates with gross litter N decomposition rates in soil amended with litter using a NH4-pool dilution approach. They estimated that about 70% of the litter N was directly assimilated by soil microorganisms in organic form. These studies are in line with Geisseler et al. (2009), who added labeled amino acids to soil samples. They estimated the fraction of N taken up via the direct route in a microcosm study with amendment of wheat residues to be 55 and 62% for N-rich and N-poor residues,

respectively. The increased importance of the direct route in the soil amended with N-poor residues was likely due to the lower mineral N availability in this treatment (Geisseler et al., 2009). This finding is supported by the fact that the uptake of organic N molecules is subject to N regulation. The pathway of N acquisition, however, is not only dependent on N availability, but also on that of C and energy. In the presence of readily available C, organic N molecules are primarily used as N sources, while in the absence of other C sources, organic N molecules are used for both C and N favoring the direct route (Gibbs and Barraclough, 1998). This hypothesis is supported by studies reported earlier; the expression of the genes encoding for amino acid oxidases have been found to be induced by amino acids in the presence of a C source (Vallon et al., 1993; Davis et al., 2005), suggesting that under C limiting conditions, the direct uptake of the amino acids may be favored. 5.1. Conceptual model for the nitrogen uptake pathway How is the relative importance of the MIT and direct route affected by the composition of residues and soil properties? The literature indicates that N regulation is the dominant mechanism governing the enzyme systems involved in the utilization of N containing compounds. Therefore, when the availability of NHþ 4 is high, enzyme systems for the utilization of alternative N sources are repressed. Under these conditions, the direct route may be insignificant (Fig. 2). However, the synthesis of extracellular N mineralizing enzymes is also repressed by high NHþ 4 levels reducing the supply of NHþ 4 (Palenik and Morel, 1990; Vallon et al., 1993). But enzymes already excreted are no longer under the control of microorganisms and may continue to contribute to the availability þ of NHþ 4 . As long as their activity is high enough to maintain the NH4 concentration at an elevated level, enzymes systems for the uptake of other N sources are not de-repressed and the MIT route remains the dominant N uptake pathway. In contrast, when the NHþ 4 availability is low, enzyme systems for the uptake of organic N molecules are de-repressed and the direct route becomes dominant. In aerated soils, the NHþ 4 concentration is generally low due to plant uptake and nitrification, which favors the direct route. When the ratio between available C to N is high, N becomes limiting relative to C. Under these conditions, net N immobilization occurs, which results in a depletion of the mineral soil N pool. This in turn should result in the de-repression of enzyme systems used for the acquisition of alternative N sources. The direct route should

Fig. 2. Conceptual model of the factors affecting the relative importance of the MIT and direct route of N uptake by soil microorganisms.


therefore be favored over the MIT route, as long as N is limiting relative to C (Fig. 2). When C becomes limiting relative to N, N containing organic molecules may be used as C sources. As long as the NHþ 4 concentration in soil solution is low, the direct route remains the dominant N uptake pathway. However, when microorganisms use large amounts of N containing molecules as C sources, excess N is released as NH3, which results in a shift from the direct to the MIT route. Therefore, the relative importance of the two pathways depends on the relative availability of C and N as well as on the concentration of NHþ 4 in soil solution. Carbon and N availability most likely have the strongest effect on the N uptake route between a C to N ratio of the available substrates of 20 and 40. While a C to N ratio greater than about 40 results in net N immobilization, a C to N ratio below 20 normally leads to net N mineralization (Stevenson, 1986; Whitmore, 1996; Cabrera et al., 2005). However, other properties of residues, such as lignin and tannin content, may affect N mineralization rates from litter (Fox et al., 1990; Valenzuela-Solano and Crohn, 2006). When most of the available C is in the form of molecules that also contain N, such as amino acids and amino sugars, the direct route may even be important when NHþ 4 is available at high concentrations because microorganisms have to meet their C demand by taking up organic N molecules. In contrast, when carbohydrates or other readily available C sources are available, the C of organic N molecules is not needed and NHþ 4 is again preferred as N source over organic N molecules, favoring their extracellular deamination. This may explain why extracellular amino acid oxidase has been found to be induced by amino acids in the presence of a C source (Vallon et al., 1993; Davis et al., 2005). In plant residues, a large proportion of the C is in the form of carbohydrates, which reduces the need to take up N containing molecules as C sources. The MIT route may therefore be dominant when plant residues are the main source of organic material, as long as the C to N ratio of the residues is not too high to result in a depletion of the NHþ 4 pool. In contrast, a large proportion of the C in microbial residues is in the form of amino sugars and amino acids, which also contain N. Therefore, when a large proportion of the available organic material is of microbial origin, the direct route may be more important. However, the narrow C to N ratio of the organic molecules will result in the release of NH3. If alternative C sources become available, the microorganisms may switch to the MIT route. Therefore, we hypothesize that the relative importance of the direct and MIT route during the decomposition of residues is determined by three factors: (i) the form of N available, (ii) the source of C, and (iii) the availability of N relative to C (Fig. 2). In our conceptual model, the relative amount of N acquired by the MIT and the direct route is not static, but dynamically changes over time in response to changing availabilities of different C and N sources. Carbon and N availability in turn is affected by environmental factors such as temperature, soil moisture or aeration. In addition, the heterogeneity of soil makes it likely that both pathways may be dominant at the same time in different microsites.

6. Conclusions Nitrogen turnover in soil, including N mineralization and N uptake by soil microorganisms have been investigated in a large number of studies. The enzyme systems responsible for these processes and their regulation, however, have found less attention. Our review of the enzyme systems involved in the utilization of N by microorganisms and their regulation shows that in soils, being an environment with many different N sources and often limited supply of N, the conditions favoring the direct as well as the MIT route may be met.

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The pathway of N uptake not only affects the competition between plants and microorganisms, but also the interpretation of gross N mineralization rates as measured by isotope dilution methods. If the MIT route is dominant, all the N passes through the NHþ 4 pool. Therefore, gross N mineralization represents the total amount of bioavailable N. If in contrast the direct route is dominant, gross N mineralization determined by isotope pool dilution only measures the surplus N released from cells. Based on the literature, the factors most likely affecting the relative importance of the two pathways during the decomposition of residues are the form of N available, the source of C, and the availability of N relative to C. However, most of our knowledge about the enzyme systems involved in the microbial N utilization comes from studies conducted with cultured microorganisms. As the species used as well as the experimental conditions may not be representative for soil, it is important to determine the relative importance of the direct and MIT route as well as the factors affecting the enzyme systems required for these two pathways in soil directly. This knowledge should result in a better understanding of the soil N cycle in general and the competition between soil microorganisms and plants for N in particular. Acknowledgements We would like to thank two anonymous reviewers for their valuable comments on the manuscript. References Abdel-Fattah, G.M., Mohamedin, A.H., 2000. Interactions between a vesiculararbuscular mycorrhizal fungus (Glomus intraradices) and Streptomyces coelicolor and their effects on sorghum plants grown in soil amended with chitin of brawn scales. Biology and Fertility of Soils 32, 401e409. Acosta-Martínez, V., Harmel, R.D., 2006. Soil microbial communities and enzyme activities under various poultry litter application rates. Journal of Environmental Quality 35, 1309e1318. Adams, D.J., 2004. Fungal cell wall chitinases and glucanases. Microbiology 150, 2029e2035. Adesina, M.F., Lembke, A., Costa, R., Speksnijder, A., Smalla, K., 2007. Screening of bacterial isolates from various European soils for in vitro antagonistic activity towards Rhizoctonia solani and Fusarium oxysporum: site-dependent composition and diversity revealed. Soil Biology & Biochemistry 39, 2818e2828. Ahearn, D.G., Meyers, S.P., Nichols, R.A., 1968. Extracellular proteinases of yeasts and yeastlike fungi. Applied Microbiology 16, 1370e1374. Allison, C., Macfarlane, G.T., 1992. Physiological and nutritional determinants of protease secretion by Clostridium sporogenes: characterization of six extracellular proteases. Applied Microbiology and Biotechnology 37, 152e156. Allison, S.D., Vitousek, P.M., 2005. Responses of extracellular enzymes to simple and complex nutrient inputs. Soil Biology & Biochemistry 37, 937e944. Alvarez, F.J., Konopka, J.B., 2007. Identification of an N-acetylglucosamine transporter that mediates hyphal induction in Candida albicans. Molecular Biology of the Cell 18, 965e975. Andrade, S.L.A., Einsle, O., 2007. The Amt/Mep/Rh family of ammonium transport proteins (Review). Molecular Membrane Biology 24, 357e365. Anraku, Y., 1980. Transport and utilization of amino acids by bacteria. In: Payne, J.W. (Ed.), Microorganisms and Nitrogen Sources. Wiley, Chichester, pp. 9e33. Asmar, F., Eiland, F., Nielsen, N.E., 1992. Interrelationship between extracellular enzyme activity, ATP content, total counts of bacteria and CO2 evolution. Biology and Fertility of Soils 14, 288e292. Baars, J.J., Op den Camp, H.J.M., van der Drift, C., Van Griensven, L.J.L.D., Vogels, G.D., 1995. Regulation of nitrogen-metabolizing enzymes in the commercial mushroom Agaricus bisporus. Current Microbiology 31, 345e350. Badalucco, L., Kuikman, P.J., Nannipieri, P., 1996. Protease and deaminase activities in wheat rhizosphere and their relation to bacterial and protozoan populations. Biology and Fertility of Soils 23, 99e104. Barak, P., Molina, J.A.E., Hadas, A., Clapp, C.E., 1990. Mineralization of amino acids and evidence of direct assimilation of organic nitrogen. Soil Science Society of America Journal 54, 769e774. Barraclough, D., 1997. The direct or MIT route for nitrogen immobilization: a 15N mirror image study with leucine and glycine. Soil Biology & Biochemistry 29, 101e108. Bhattacharya, D., Nagpure, A., Gupta, R.K., 2007. Bacterial chitinases: properties and potential. Critical Reviews in Biotechnology 27, 21e28. Bidochka, M.J., Khachatourians, G.G., 1988. Regulation of extracellular protease in the entomopathogenic fungus Beauveria bassiana. Experimental Mycology 12, 161e168.

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