1 Evidence for different contributions of archaea and bacteria to the ammonia-oxidizing 1 potential

35 Pages·2010·0.54 MB·English

AEM Accepts, published online ahead of print on 1 October 2010
Appl. Environ. Microbiol. doi:10.1128/AEM.01324-10
Copyright © 2010, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.
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1 Evidence for different contributions of archaea and bacteria to the ammonia-oxidizing
2 potential of diverse Oregon soils
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4 Running title: Contributions of archaea and bacteria to soil ammonia oxidation
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6 Anne E. Taylor1,2, Lydia H. Zeglin1, Sandra Dooley3, David D. Myrold1, Peter J. Bottomley1,4 w
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8 1Department of Crop and Soil Science, 3017 ALS, Oregon State University, Corvallis, OR 97331 d

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9 3Ecology and Environmental Biology, University of California, Irvine - Irvine, CA 92697 m

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10 4Department of Microbiology, 220 Nash Hall, Oregon State University, Corvallis, OR 97331 tt
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12 23017 ALS, Oregon State University, Corvallis, OR 97331 .a
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13 541-737-4136 .
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14 Fax [phone] /

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15 [email protected]
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16 Abstract
17 A method was developed to determine the contributions of ammonia-oxidizing archaea (AOA)
18 and bacteria (AOB) to the nitrification potential (NP) of soils taken from forest, pasture, cropped,
19 and fallowed (19 y) lands. Soil slurries were exposed to acetylene to irreversibly inactivate
20 ammonia monooxygenase, and upon removal of acetylene, recovery of the nitrification potential
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21 (RNP) was followed in the presence and absence of bacterial or eukaryotic protein synthesis w
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22 inhibitors. For unknown reasons, and despite measureable NPs, RNP did not occur consistently lo
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23 in forest soil samples; however, pasture, cropped, and fallowed soil RNPs commenced after lags d

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24 that ranged from 12 to 30 h post acetylene removal. Cropped soil RNP was completely prevented m

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25 by the bacterial protein synthesis inhibitor, kanamycin (800 µg/ml), whereas a combination of tt
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26 kanamycin plus gentamycin (800 µg/ml each) only partially prevented RNP (60%) of fallowed a
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27 soils. Pasture soil RNP was completely insensitive to either kanamycin, gentamycin or a .
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28 combination of the two. Unlike cropped soil, pasture and fallowed soil RNP occurred at both 30 .
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29 and 40oC and without supplemental NH + (≤10 µM NH + in solution), plus pasture soil RNP /
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30 demonstrated ~50% insensitivity to 100 µM allyl thiourea (ATU). In addition, fallowed and A
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31 pasture soil RNPs were insensitive to fungal inhibitors, nystatin and azoxystrobin. This il
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32 combination of properties suggests that neither fungi nor AOB contributed to pasture soil RNP, 0
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33 and that AOA were responsible for RNP of the pasture soils. Both AOA and AOB may b
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34 contribute to RNP in fallowed soil, while RNP in cropped soils was dominated by AOB. u
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35 Introduction
36 Until 2005 the majority of ammonia (NH ) oxidation in natural environments was
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37 thought to be carried out by a phylogenetically distinct group of NH -oxidizing bacteria (AOB).
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38 This nitrification paradigm was challenged by reports that thaumarchaeota carry the genes that
39 encode the enzyme ammonia monooxygenase (AMO) (55), the isolation of ‘Candidatus
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40 Nitrosopumilus maritimus’ strain SCM1, an obligate autotrophic thaumarchaeal NH3-oxidizer w
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41 (35), the detection of archaeal amoA gene transcripts in soil (54, 55), and the realization that lo
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42 NH3-oxidizing archaea (AOA) were widely distributed (19), and often more numerous than AOB d

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43 in soil (36), marine (40), and hot spring ecosystems (52, 57). Others have correlated either AOA m

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44 (7, 46, 58) or AOB amoA gene copy abundance (17, 32) with nitrification activity, and detected tt
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45 expression of AOA amoA mRNA (43, 60). To our knowledge there is no published work that a
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46 has directly distinguished NH -oxidizing activity by AOA from NH -oxidizing activity by AOB .
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47 in soils. .
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48 As yet, it is unknown what factors determine whether AOA or AOB nitrify in an o
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49 environment. We hypothesized that AOA and AOB might contribute differentially to p
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50 nitrification in diverse soils across a landscape with different physical and chemical ,

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51 characteristics, plant community compositions, and management practices. We know that 1
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52 nitrification by AOA and AOB is inhibited by acetylene (28, 30, 46); and we hypothesized that y

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53 antibiotics that prevent protein synthesis could be used to discriminate between AMO activity of e
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54 AOA and AOB during recovery of nitrification potential (RNP) after acetylene exposure that
55 specifically and irreversibly inactivates AMO (28, 30). Upon removal of acetylene, NH
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56 oxidation resumed in Nitrosomonas europaea after a 1 to 2 h delay, and was prevented by the
57 bacterial protein synthesis and transcriptional inhibitors, chloramphenicol and rifampicin (28).
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58 We hypothesized that our RNP assays should be of relatively short duration (<48 h) and be
59 independent of population growth occurring during the assay. In the presence of the sulfa drug
60 sulfadiazine (SDZ), which inhibits folic acid synthesis, AOB proliferation was completely
61 prevented during a 32 d microcosm study of soil amended with pig manure (50), whereas AOA
62 proliferation was marginally or unaffected. Although SDZ clearly prevented proliferation of
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63 AOB to a greater extent than AOA, it could not be discerned whether AOB, AOA, or both w
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64 contributed to the ammonia oxidation occurring throughout the 32d incubation. Because the a
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65 mechanism of protein synthesis in archaea is quite different from that in bacteria, and more d

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66 closely resembles the mechanism of eukaryotic protein synthesis, archaea are insensitive to many m

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67 of the traditional bacterial protein synthesis inhibitors (6, 16, 37). To our knowledge, there are tt
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68 no antibiotics or protein synthesis inhibitors specifically developed to inhibit archaea. Those that a
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69 have been successfully used with Haloferax mediterranei (31) and Sulfolobus solfataricus (2, 9) .a
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70 were identified initially as eukaryotic inhibitors and fortuitously inhibited these two archaea. It .
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71 remains to be determined how other archaea will respond to these inhibitors. /
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72 Although only a few strains of AOA have been cultured or enriched (15, 25, 35), a A
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73 number of potentially discriminating traits have been discovered among these il
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74 isolates/enrichments that lead us to believe that it might be possible to use these traits, in 0
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75 combination with acetylene inactivation and recovery of nitrification to discriminate between b
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76 AOA and AOB NH -oxidizing activity in soil samples. These traits include differences between u
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77 AOA and AOB to allyl thiourea (ATU) sensitivity (1, 20, 25, 27), a relatively narrow
78 temperature optimum (25 – 33oC) for all known cultures of AOB versus some AOA (3, 10, 33),
79 and a much lower apparent K for NH by ‘Candidatus Nitrosopumilus maritimus’ strain SCM1
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80 than AOB (39). To evaluate these ideas, we sampled a range of Oregon soils along a transect of
81 different land uses that included forest, pasture, cropped, and nearby fallowed sites.
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83 Materials and Methods
84 Chemicals. Allyl thiourea (ATU), N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid
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85 (TES) buffer, neomycin trisulfate salt, nystatin, dimethyl sulfoxide (DMSO, 99.5%), w
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86 Actinomycin D, and NH4Cl, were obtained from Sigma (St. Louis , MO). Acetylene was a
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87 obtained from Airgas (Radnor, PA), and cycloheximide (96.4%), kanamycin sulfate, and d

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88 gentamycin sulfate from EMD Biosciences, Inc. (La Jolla, CA), and emetine was purchased from m

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89 TCI America (Portland, OR). The fungicide Abound® (active ingredient azoxystrobin: methyl tt
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90 (E)-2-{2-[6-(cyanophenoxy) pyrimidin-4-yloxy]phenyl}-3-methoxyacrylate), was obtained from a
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91 Syngenta Crop Protection, Inc. (Greensboro, NC), and Szechrome NAS reagent from .a
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92 Polysciences Inc. (Warrington, PA). .
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93 Soils. After removal of litter or sod, mineral soil samples (0 to 10 cm) were collected from sites /
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94 along a west-east transect of different land uses approximately 6 miles (9.5 km) in length and A
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95 located 10 miles (16 km) north of Corvallis, Oregon (44.7° N 123.3° W). Soils were taken from il
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96 three sites under mature red alder stands (forest) in the uplands (~350 m elevation) of the 0
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97 MacDonald-Dunn Forest (Jory-Gelderman series, Paleo/haplo-humults, pH 6.0 to 6.3), from b
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98 three sites under permanent grass pastures situated on a toe-slope below the forest (~125 m u
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99 elevation) on the OSU Beef Farm (Dixonville-Gellatly-Witham complex, Argi/haploxerolls pH
100 5.7 to 6.4), and from three cropped (under wheat or barley at the time of sampling) and three
101 fallowed fields (unfertilized and uncultivated since 1990, and colonized by volunteer grasses and
102 forbs which are mowed twice yearly) at the OSU Hyslop Field Research Laboratory (Woodburn
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103 series, Argixerolls, pH 5.9 to 6.5) on the Willamette Valley floor (~85 m elevation). Four to five
104 soil samples were recovered from each of the three locations that were spaced between 100 m to
105 ~1000 m apart in each of the forest, pasture, cropped and fallowed areas. The samples at each
106 location were composited and thoroughly mixed in the field, and brought to the laboratory where
107 they were sieved (4.75 mm) and stored at 4oC. Samples of soil (5 – 10 g) were oven-dried to
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108 determine the water content. The levels of net readily mineralizable nitrogen (N Min) were w
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109 determined in soils adjusted to ~60% of water holding capacity, incubated at 30oC, and sampled loa
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110 at 7, 14, 21 and 30 days, and the accumulations of ammonium (NH +) plus nitrate (NO -) d
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111 measured. Physical and chemical properties of soils were determined by the standard procedures m

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112 of the Central Analytical Services Laboratory, Department of Crop and Soil Science, Oregon tt
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113 State University. a
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114 Nitrification Potential (NP). NP assays are used primarily to characterize the potential activity .a
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115 of the nitrifying population under conditions of non-limiting NH3, O2, and neutral pH (51). Soils .o
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116 were removed from 4oC storage and pre-incubated field moist at room temperature (22 ± 2oC) /
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117 for two days prior to experimental use. The fallowed and cropped soils from Hyslop Farm had A
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118 low water content (≤ 11% wt/wt), therefore deionized water was added (1 ml/15 g soil) before il
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119 pre-incubation to achieve a water content which approximates field capacity (18% wt/wt). For 0
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120 NP assays, 5 g portions of moist soil were added to 50 ml aliquots of 30 mM TES, buffered to b
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121 pH 7.2 with 10 M KOH, in 150 ml bottles with black phenolic caps fitted with gray butyl u
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122 stoppers. 30 mM TES buffer was used because its pKa is pH 7.4, it did not extract humic
123 compounds from the soil, and its buffering capacity was sufficient to maintain the soil slurries at
124 pH 6.8 to 7.0 for the duration of the assays. Preliminary experiments showed that 1 mM NH +
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125 was a saturating, non-inhibiting concentration, and all NP assays included 1 mM supplemental
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126 NH + (as NH Cl) unless otherwise stated. Soil slurries were shaken at 200 rpm in either a
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127 Brunswick orbital constant temperature shaker maintained at either 30 or 40oC, or on a bench top
128 orbital shaker at room temperature (22 ± 2oC). At time intervals, samples (1.8 ml) of soil slurry
129 were removed to microcentrifuge tubes, centrifuged for 3 min at 13.4 x103 g, and nitrite (NO -)
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130 and NO - concentrations were determined immediately on the supernatants, or stored at -20oC
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131 until they could be processed. NO2- and NO3- concentrations were calculated using the oven- w
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132 dried weight of the soil used in the assay. Background NO2- and NO3- concentrations were loa
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133 measured after soil slurries were shaken for 15 min. NP rates were determined as NO - plus d
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134 NO - accumulations over 24h. Although background NO - was undetected in any of the soils, m
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135 NO2- accumulated to a varying fraction of the total NO2- plus NO3- in actively nitrifying soil ttp
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136 slurries. NO - was determined as described elsewhere (22). NO - concentrations were a
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137 determined by using either Szechrome reagent as described by the manufacturer, or with an .a
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138 Astoria Pacific (Clackamas, OR) flow solution auto analyzer. It was determined that both ATU .
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139 and cycloheximide interfered with the Szechrome assay, and in those cases NO3- was always o/
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140 determined by autoanalyzer. In a number of experiments NH + was also determined using the A
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141 flow solution auto analyzer. In some cases, high concentrations of background soil NO - were il
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142 removed prior to NP assays as follows: 5 g portions of moist soils were dispensed into 40 ml 0
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143 centrifuge tubes with ~ 30 ml of dH O, vortexed briefly, centrifuged at 7.5 x103 g for 15 min, b
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144 and the supernatant carefully decanted. Soil pellets were resuspended in NP assay buffer and u
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145 transferred to assay bottles. Preliminary experiments showed that pretreating the soils in this
146 manner lead to minimal loss of NP.
147 The effects of bacterial and eukaryotic protein synthesis inhibitors, ATU, and fungicides
148 on nitrification were determined by comparing nitrification in the presence and absence of the
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149 individual compounds. During preliminary experiments with the relatively water insoluble
150 antibiotic, actinomycin D, we found that nitrification in soil slurries was inhibited by extremely
151 small volumes of DMSO and other solvents such as methanol, thereby preventing use of
152 actinomycin D concentrations > 200 µg/ml. Due to their insolubility in aqueous solutions, we
153 did not pursue use of antibiotics such as rifampicin or chloramphenicol or fungicides such as
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154 triazoles. The water soluble bacterial protein synthesis inhibitors neomycin, kanamycin and w
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155 gentamycin were utilized. Kanamycin and gentamycin have been proven effective in preventing a
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156 growth of the AOB Nitrosomonas europaea (13, 21, 56). The effect of the water soluble d

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157 eukaryotic protein synthesis inhibitors emetine and cycloheximide were evaluated. A stock of m

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158 the nitrification inhibitor ATU was mixed immediately before use. The water soluble fungicides tt
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159 nystatin (1.2 ml/50 ml, 12,000 units) or azoxystrobin (20 µl/50 ml, 13 mg active ingredient) were a
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160 used as recommended by the manufacturers. .a
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161 Recovery of Nitrification Potential (RNP). For RNP assays, 5 g portions of moist soil were .
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162 dispensed into 50 ml aliquots of 30 mM TES buffer, pH 7.2, in 150 ml bottles with black /
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163 phenolic caps fitted with gray butyl stoppers. Acetylene was added (0.025% vol/vol, or 0.025 A
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164 kPa) to the headspace for 6 h. From preliminary experiments it was determined that a 6 h il
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165 acetylene exposure was sufficient to inactivate all NH -oxidation in the soils used in the study. 0
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166 Acetylene inhibition and RNP steps were carried out at 30oC with 1 mM supplemental NH +, b
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167 unless otherwise indicated. These conditions in the absence of inhibitors were considered to be u
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168 the standard RNP, designated as RNPo. NP controls comprised soil suspensions to which t
169 acetylene was not added. An acetylene containing control was also included to evaluate the
170 possibility of heterotrophic nitrification. Removal of acetylene was achieved by placing the soil
171 slurries under a vacuum (-100 kPa) and degassed. Analysis of headspace samples by gas
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172 chromatography with flame-ionization detection (FID) as described previously (59), determined
173 that degassing for 5 min under vacuum was adequate to remove all traces of acetylene from soil
174 slurries.
175 After degassing, all bottles were incubated with caps loosened to permit aeration.
176 Although RNP varied with soils from different land use regimens, replicate soils from the same
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177 land use regimen responded similarly. In the case of pasture and cropped soils, the length of lag w
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178 time was evaluated by determining NO2- plus NO3- accumulation at 6 h intervals for 24 to 30 h, loa
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179 followed by sampling at less frequent intervals for a total of 48 h. Fallowed soil had a lower NP, d

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180 showed a longer lag (~24 to 30 h), and recovered more slowly. The RNPs were compared with m

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181 the initial NP controls by assessing the amount of NO2- plus NO3- that accumulated in the 24 h ttp
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182 post lag period relative to the accumulation occurring between 0 to 24 h in the NP. From a
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183 preliminary experiments we established there were no detrimental effects of degassing the NP .a
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184 controls after 6 h of incubation on the subsequent rate. Aqueous solutions of antibiotics, .
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185 nitrification inhibitors, or fungicides were added after acetylene was evacuated. Preliminary /
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186 experiments were carried out with a range of concentrations of the antibiotics to determine their A
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187 minimum effective concentrations. il
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188 Extraction of nucleic acids and qPCR. DNA was extracted from 0.25 to 0.3 g dry soil using a 0
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189 MoBio PowerSoil (Carlsbad, CA) extraction kit, and DNA quantified using a NanoDrop ND- b
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190 1000 UV-Vis Spectrophotometer (ThermoScientific, Rockwood, TN). Q-PCR of the archaeal u
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191 and bacterial amoA genes was performed using the Brilliant II SYBR Green Master Mix
192 (Stratagene, La Jolla, CA) and an ABI 7500 Real Time PCR System (Foster City, CA). Each 25
193 µL reaction volume included 10 ng template DNA. Primers and thermalcycler protocols for both
194 bacterial (amoA_1R and amoA_2F primers) and archaeal (Arch-amoAF and Arch-amoAR
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195 primers) amoA genes are described elsewhere (41, 48). Standard curves were constructed with
196 2.0 x 101 to 2.0 x 10-4 ng Nitrosomonas europaea genomic DNA (bacterial amoA,
197 efficiency=91-94%, R2=0.996) or 1.25 x 101 to 1.25 x 10-3 ng of a TOPO plasmid containing the
198 ‘Candidatus Nitrosopumilus maritimus’ strain SCM1 amoBAC gene insert (archaeal amoA,
199 efficiency=95-100%, R2=0.994-0.995). Each reaction was run in triplicate. Copy numbers were
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200 standardized by the mass of DNA and g dry soil analyzed for each sample and log10 transformed w
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201 before further analysis. a
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202 Statistics. We used one-way ANOVA with Bonferonni post-hoc analysis using SPSS 11 for d

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203 Mac to determine significance of temperature effects within each incubation set. A single-tailed m

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204 Student’s T-test assuming unequal variance was used to evaluate the effect of supplemental tt
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205 NH + on NP, and to determine the effect of inhibitors and temperature on RNP. a
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206 .a
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207 Results .
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208 Soil Properties /

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209 Due to their close geographic proximity, the soils taken along the landscape transect A
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210 experience approximately the same precipitation and temperature regimen. Archived soil il
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211 temperature data from the cropped and fallowed sites shows annual maxima and minima of 0
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212 37.2±1.5 and 2.9±0.9oC, respectively (http://cropandsoil.oregonstate.edu/weather/archive). A b
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213 suite of chemical and physical properties were determined for the soils (Table 1). While there u
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214 were only small differences in pH among the sites, other soil characteristics varied to a greater t
215 extent. Notably, high extractable P concentrations in cropped and fallowed soils reflect the
216 legacy of P fertilization to promote crop yield, and the high levels of Mg, Na, Ca and clay in the
217 pasture soils are likely due to landscape position (toe slope). The cropped soils had the least
218 amount of total C; the adjacent fallowed soils had been out of production for 19 y and had C