Ammonia-oxidizing bacteria and ammonia-oxidizing archaea in soils of the Mount Everest on ...

32 Pages·2009·0.33 MB·English

1 Altitude ammonia-oxidizing bacteria and archaea in soils of Mount
2 Everest
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4 Li-Mei Zhang, 1† Mu Wang, 2† James I. Prosser, 3 Yuan-Ming Zheng,1 Ji-Zheng He1*
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6 1State Key Laboratory of Urban and Regional Ecology, Research Centre for
7 Eco-environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China
8 2 Agricultural and Animal Husbandry College of Tibet, Linzhi, Tibet 860000, China
9 3 Institute of Biological and Environmental Sciences, University of Aberdeen,
10 Cruickshank Building, St. Machar Drive, Aberdeen AB24 3UU, UK
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12 † These authors contributed equally to this work.
13 *For correspondence. E-mail: [email protected]; Telephone: [phone]; Fax:
14 ([phone].
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21 This is an Accepted Article that has been peer-reviewed and approved for publication in the FEMS
22 Microbiology Ecology, but has yet to undergo copy-editing and proof correction. Please cite this
23 article as an “Accepted Article”; doi: 10.1111/j.1574-6941.2009.00775.x
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27 Summary
28 To determine the abundance and distribution of bacterial and archaeal ammonia
29 oxidizers in alpine and permafrost soils, 12 soils at altitudes of 4,000 to 6,550 m above
30 sea level (masl) were collected from the northern slope of the Mount Everest (Tibetan
31 Plateau) where the permanent snow line is at 5,800 – 6,000 masl. Communities were
32 characterized by real-time PCR and clone sequencing by targeting on amoA genes,
33 putatively encode ammonia monooxygenase subunit A. Archaeal amoA abundance
34 was greater than bacterial amoA abundance in lower altitude soils (≤5,400 masl) but
35 this situation was reversed in higher altitude soils (≥5,700 masl). Both archaeal and
36 bacterial amoA abundance decreased abruptly in higher altitude soils. Communities
37 shifted from a Nitrosospira amoA cluster 3a-dominated AOB community in lower
38 altitude soils to communities dominated by a newly designated Nitrosospira ME and
39 cluster 2-related groups and Nitrosomonas cluster 6 in higher altitude soils. All
40 archaeal amoA sequences fell within soil and sediment clusters, and the proportions of
41 the major archaeal amoA clusters changed between the lower altitude and the higher
42 altitude soils. These findings imply that the shift in the relative abundance and
43 community structure of archaeal and bacterial ammonia oxidizers may result from
44 selection of organisms adapted to altitude-dependent environmental factors in elevated
45 soils.
46 Keywords: Alpine soil, Ammonia oxidizer, amoA gene, Community shift, Relative
47 abundance, Tibetan Plateau
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49 Until recently, chemolithoautotrophic ammonia-oxidizing bacteria (AOB) within
50 the Proteobacteria were thought to be the major microorganisms performing ammonia
51 oxidation, the first and rate-limiting step of nitrification. Metagenomic studies first
52 suggested that ammonia oxidation was potentially driven by members within the
53 domain Archaea that contained putative ammonia monooxygenase subunits (amoA,
54 amoB and amoC) (Venter et al., 2004; Treusch et al., 2005). The first successful
55 isolation of a mesophilic crenarchaeon, Nitrosopumilus maritimus, growing
56 autotrophically with ammonia as the sole energy source, confirmed the association of
57 these archaeal ammonia monooxygenase genes with ammonia oxidation (Könneke et
58 al., 2005).Given the functional significance and conserved phylogeny, the amoA gene
59 was used as a molecular marker for both AOB and these putative ammonia-oxidizing
60 archaea (AOA). Subsequent studies have demonstrated that archaeal amoA genes are
61 ubiquitous in marine environments (Francis et al., 2005; Wuchter et al., 2006),
62 wastewater bioreactors (Park et al., 2006), terrestrial hot springs (Weidler et al., 2007;
63 Reigstad et al., 2008) and soils (He et al., 2007; Nicol et al., 2008; Shen et al., 2008).
64 Active expression of archaeal amoA gene has also been detected in natural
65 environments (Lam et al., 2007; Tourna et al., 2008) and enrichment cultures
66 (Hatzenpichler et al., 2008).
67 Archaeal amoA genes are more abundant than those of bacteria in various
68 environments. In marine environments, archaeal amoA gene abundance exceeded that
69 of bacteria by up to four orders of magnitude (Wuchter et al., 2006; Lam et al., 2007;
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70 Mincer et al., 2007). In a study of 12 pristine and agricultural soils crossing three
71 climate zones, archaeal amoA gene was up to 3000 times that of bacterial amoA and
72 correlated with Crenarchaeota-specific lipids (Leininger et al., 2006). Consistently
73 higher abundance of archaeal amoA genes has also been reported in Chinese acid and
74 alkaline soils with different fertilization regimes (He et al., 2007; Shen et al., 2008)
75 and in Scottish agricultural plots (Nicol et al., 2008). Numerical dominance of archaeal
76 over bacterial amoA genes in natural environments suggested its putative greater
77 ecological function in global nitrogen cycling, but the presence or high abundance of a
78 functional gene does not mean that the function is operating (Prosser and Nicol, 2008).
79 Moreover, some recent studies reported greater abundance of AOB amoA genes in
80 estuarine sites (Caffrey et al., 2007), with increasing salinity in coastal sediments
81 (Mosier & Francis 2008; Santoro et al., 2008) and in anoxic sediments (Jiang et al.,
82 2009). It is important to understand whether there are more habitats in which AOB
83 dominate, especially in soils, and the physical and chemical factors that determine the
84 relative abundance and diversity of these two distinct groups of ammonia oxidizers.
85 A number of studies have shown that the distribution, community composition and
86 abundance of AOB and AOA are influenced greatly by temperature (Avrahami et al.,
87 2003), pH (Nicol et al., 2008), simulated global changes (Horz et al., 2004), salinity
88 (Mosier & Francis 2008; Santoro et al., 2008) and fertilization regime (He et al., 2007;
89 Shen et al., 2008). Distinct changes in AOA community composition have also been
90 observed in a soil profile, in which AOB were not detected (Hansel et al., 2008), and a
91 marked decrease in the abundance of archaeal amoA gene was found with increasing
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92 depth, from subsurface waters to 4,000 m depth in the North Atlantic Ocean (Agogue
93 et al., 2008). However, AOB and AOA communities at high altitude soils have not
94 been investigated.
95 The Tibetan Plateau is the Earth’s largest (2 × 106 km2) and highest (mean altitude
96 4,500 m above sea level, masl) plateau. Tectonic uplifting of the Tibetan Plateau has
97 led to a unique system, with snow cover, high UV exposure and lower oxygen and
98 nutrient concentrations. As a result, microbial communities in topsoil layers encounter
99 extreme conditions that may lead to unique survival adaptations and differences in
100 community composition. The Tibetan Plateau is also one of the most special regions
101 sensitive to global climate change (Wu et al., 2007) and the global warming and
102 elevated CO concentration may have complex impact on below-ground
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103 microorganisms (Xu et al., 2009). In the present study, real-time PCR and clone-library
104 sequencing were used to characterize the distribution and community composition of
105 AOA and AOB in these alpine soils, where the altitude-dependent environmental
106 factors and changing global climate may select for particular populations of ammonia
107 oxidizers.
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109 Materials and methods
110 Sampling sites and soil collection
111 During the fourth expedition on Mt. Everest of the Chinese Academy of Sciences
112 from April to June 2005, 12 soil samples (M1–M12) were collected from the northern
113 slope of Mt. Everest and adjoining areas (Table 1). M1 to M3 are field or farmland
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114 soils in the Rikaze area, Tibet, at an altitude of 4,000 masl. M4 to M12 are bare soils
115 with no visible plant development (Fig. 1). The permanent snow line is at 5,800 –
116 6000 masl. For each site, three subsamples were removed from the soil surface (0 – 10
117 cm depth) and were mixed after removal of large stones and snow. Samples were
118 transported to a refrigerator with temperature at –10(cid:31) in two days and stored until
119 further analysis. Soils was passed through a 2.0 mm sieve before usage.
120 Soil chemical analysis
121 Soil pH was determined with a soil to water ratio of 1:10. Soil organic matter was
122 determined by the K Cr O oxidation method and total nitrogen by the Kjeldahl
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123 method (Bremner 1996).
124 DNA extraction
125 DNA was extracted from 0.8 – 1.0 g (fresh weight) of soil using the FastDNA SPIN
126 Kit for Soil (Q·BIOgene, Inc., Carlsbad, CA) with a bead beating time of 20 s and a
127 speed setting of 5.5 m. Extracted DNA was checked on a 1% agarose gel and the
128 concentration was determined using a Nanodrop® ND-1000 UV-Vis
129 Spectrophotometer (NanoDrop Technologies, Wilmington, DE).
130 Quantification of amoA genes by real-time PCR
131 Primer pairs amoA1F/amoA2R (Rotthauwe et al., 1997) and
132 Arch-amoAF/Arch-amoAR (Francis et al., 2005) were used for real-time PCR
133 quantification of bacterial and archaeal amoA genes, respectively. Real-time PCR was
134 performed on an iCycler iQ 5 thermocycler (Bio-Rad). The 25 µl-reaction volume
135 contained 12.5 µl SYBR(R) Premix Ex TaqTM (TaKaRa Bio Inc., Shiga, Japan), 0.4 mg
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136 ml-1 bovine serum albumin (BSA), 400 nM of each AOB primer or 200 nM each of
137 AOA primer and 2 µl of 10-fold diluted or undiluted extracted DNA (1-10 ng) as
138 template. Three replicates were analyzed for each sample. Amplifications were carried
139 out as follows: 95°C for 1 min, followed by 40 cycles of 10 s at 95°C, 30 s at 55°C for
140 AOB or 53°C for AOA, 1 min at 72°C and plate read at 83°C. Melting curve analysis
141 was performed at the end of PCR runs to check the specificity of the products. PCR
142 products amplified from extracted DNA with the primers for real-time PCR assays
143 were gel-purified and ligated into the pGEM-T Easy Vector (Promega, Madison, USA),
144 and the resulting ligation products were transformed into E. coli JM109 competent
145 cells following the manufacturer’s instructions. After reamplification with the
146 vector-specific primers T7 and SP6, the positive clones were selected to extract
147 plasmid DNA with a MiniBEST Plasmid Purification Kit (TaKaRa) and used as amoA
148 gene standards. The plasmid DNA concentration was determined on a Nanodrop®
149 ND-1000 UV-Vis Spectrophotometer (NanoDrop Technologies, Wilmington, DE) and
150 amoA gene copy number was calculated directly from the concentration of extracted
151 plasmid DNA. Ten-fold serial dilutions of a known copy number of the plasmid DNA
152 were subjected to real-time PCR in triplicate to generate an external standard curve.
153 PCR efficiency and correlation coefficients for standard curves were 95.4% and r2 =
154 0.996 for AOB and 93.4% and r2 = 0.996 for AOA.
155 Cloning and sequence analysis of bacterial and archaeal amoA genes
156 PCR products of bacterial and archaeal amoA genes amplified with the primers for
157 the real-time PCR assays were purified and cloned, as described above, for each site,
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158 generating 12 clone libraries for bacterial and 12 for archaeal amoA genes. Clones
159 were screened for inserts of the expected size with the vector-specific primers T7 and
160 SP6. For restriction fragment length polymorphism (RFLP) assay, approximately 60
161 positive clones from each clone library were digested with restriction endonuclease
162 MboI (Takara Bio Inc., Shiga, Japan). Digested DNA fragments were separated by
163 electrophoresis on a 2% agarose gel and imaged using a GBOX-HR Gel
164 Documentation System (Syngene, UK) after ethidium bromide staining.
165 The RFLP patterns were grouped and 1 – 3 clones representing unique band types
166 were sequenced. Sequences were subjected to homology analysis using the software
167 DNAMAN version 6.0.3.48 (Lynnon Biosoft, USA). Sequences of chimeric origin
168 were checked by partial treeing analysis and compared with GenBank sequences using
169 BLASTn searches. For each clone library, for sequence types that exhibited more than
170 98% identity to each other, only one representative was used for construction of the
171 tree. The GenBank sequences most similar to clone sequences in this study and
172 reference sequences for defining clusters were included in phylogenetic tree
173 construction. Phylogenetic analysis based on nucleotide sequences was performed
174 using MEGA version 4.0 (Tamura et al., 2007) and a neighbor-joining tree was
175 constructed using Kimura 2-parameter distance with 1000 replicates to produce
176 Bootstrap values (1000 replicates).
177 The sequences determined in this study were deposited in the GenBank database
178 and assigned accession numbers from FJ853214 to FJ853366.
179 Statistical analyses
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180 amoA gene abundance data were log-transformed to provide variance homogeneity.
181 Statistical analyses were performed using SPSS version 13.0. Paired-samples t-test
182 were used to compare archaeal and bacterial amoA gene copy numbers and
183 independent-sample t-tests were used to compare archaeal or bacterial amoA gene
184 copy numbers at lower altitude and higher altitudes. Bivariate correlations were carried
185 out to link different parameters. P < 0.05 was considered to be significant.
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187 Results
188 Soil properties
189 Three soils (M1 – M3) at altitude of 4000 masl were sampled from field or
190 farmland with pH values of 8.6 to 8.7. Organic matter in these three soils varied
191 between 8.4 and 12.6 g kg-1 soil and total nitrogen between 0.51 and 0.66 g kg-1 soil
192 (Table 1). Soils (M4 – M12) at altitudes above 5,000 masl were less well developed
193 with high content of gravel and pH was in the range 9.0 – 9.1. Organic matter content
194 in these soils ranged from 2.9 to 6.3 g kg-1 soil, and total nitrogen from 0.06 to 0.21 g
195 kg-1 soil. These values are much lower than in M1 – M3 soils and showed no
196 significantly negative correlation with altitude (Table 2).
197 AOA and AOB abundance
198 The abundance of archaeal and bacterial amoA genes decreased abruptly in soils
199 above 5,400 masl and significant difference in archaeal and bacterial amoA
200 abundances were observed between lower altitude (M1 – M5, ≤5,400 masl) and higher
201 altitude (M6 – M12, ≥5,700 masl) soils (df = 10, P < 0.05; Fig. 2A). Differences were
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202 also seen in the relative abundances of archaeal and bacterial amoA genes. Archaeal
203 amoA abundance in lower altitude soils (5.17 × 107 – 3.79 × 108 g-1 soil) was
204 significantly higher than bacterial amoA abundance (6.97 × 105 – 1.59 × 107g-1 soil) (df
205 = 4, P < 0.001; Fig. 2A). In contrast, at high altitude, archaeal amoA abundance (4.63 ×
206 103 – 2.72 × 104 g-1 soil) was less than that for bacteria (5.41 × 104 – 3.1 × 106 g-1 soil)
207 (df = 6, P < 0.001, Fig. 2A). As a consequence, the mean Log ratio of
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208 archaeal:bacterial amoA decreased from 1.45 in low altitude soils to – 1.34 in high
209 altitude soils (Fig. 2B), with significantly negative and positive correlations,
210 respectively, with altitude and archaeal amoA abundance (Table 2). There is a
211 significantly negative correlation between the abundance of AOA and altitude, and a
212 non-significant negative correlation between the abundance of AOB and altitude
213 (Table 2). The abundance of AOA was positively correlated with the abundance of
214 AOB, and significantly positive correlations were also observed between both AOA
215 and AOB abundance and organic matter and total nitrogen (Table 2).
216 Community composition of AOA and AOB
217 Phylogenetic trees of the bacterial and archaeal amoA gene sequences and related
218 NCBI sequences are shown in Fig. 3 and Fig. 4. All AOB sequences fell within
219 Nitrosospira amoA clusters 3a.1, 3a.2, ME, cluster 2-related and Nitrosomonas amoA
220 clusters 6, 7 (Avrahami et al., 2002; Avrhami & Conrad 2003). Sequences in cluster 3a
221 fell within two sub-clusters 3a.1 and 3a.2 with bootstrap value at 90, and 2 distinct
222 clades were designated as cluster ME and cluster 2-related. Cluster ME was related to
223 but outside of cluster 1 and 4 groups with a bootstrap value of 90 and cluster 2-related
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