Nitrous oxide reduction by an obligate aerobic bacterium Gemmatimonas aurantiaca strain T-27

47 Pages·2017·1.18 MB·English

AEM Accepted Manuscript Posted Online 7 April 2017
Appl. Environ. Microbiol. doi:10.1128/AEM.00502-17
Copyright © 2017 American Society for Microbiology. All Rights Reserved.
1 Nitrous oxide reduction by an obligate aerobic bacterium Gemmatimonas
2 aurantiaca strain T-27
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4 Doyoung Park1, Hayeon Kim1 and Sukhwan Yoon1 o
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6 1Department of Civil and Environmental Engineering, Korea Advanced Institute of d
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7 Science and Technology, Daejeon, 350-701, Korea
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9 Running Title: Nitrous oxide reduction by Gemmatimonas aurantiaca .
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11 Corresponding author o
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12 Sukhwan Yoon, [email protected] v
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20 Abstract
21 N O-reducing organisms with nitrous oxide reductases (NosZ) are known as the only
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22 biological sink of N O in the environment. Among the most abundant nosZ genes
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23 found in the environment are nosZ genes affiliated to the understudied o
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24 Gemmatimonadetes phylum. In this study, a unique regulatory mechanism of N O lo
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25 reduction in Gemmatimonas aurantiaca strain T-27, an isolate affiliated to
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26 Gemmatimonadetes phylum, was examined. Strain T-27 was incubated with N2O h
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27 and/or O2 as the electron acceptor. Significant N2O reduction was observed only when //a
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28 O2 was initially present. When batch cultures of strain T-27 were amended with O2 .a
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29 and N2O, N2O reduction commenced after O2 was depleted. In a long-term incubation .o
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30 with addition of N O upon depletion, N O reduction rate decreased over time and o
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31 came to an eventual stop. Spiking of the culture with O2 resulted in the resuscitation v
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32 of N2O reduction activity, supporting the hypothesis that N2O reduction by strain T- e
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33 27 required the transient presence of O2. The highest level of nosZ transcription (8.97 ,
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34 nosZ transcripts/recA transcript) was observed immediately after O depletion and 8
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35 transcription decreased ~25-fold within 85 hours, supporting the observed phenotype. g
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36 The observed difference between responses of strain T-27 cultures amended with and
37 without N O to O starvation suggested that N O helped sustain viability of strain T-
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38 27 during temporary anoxia, although N O reduction was not coupled to growth. The
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39 findings in this study suggest that obligate aerobic microorganisms with nosZ genes
40 may utilize N O as a temporary surrogate for O to survive periodic anoxia.
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42 Importance o
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43 Emission of N O, a potent greenhouse gas and ozone depletion agent, from soil lo
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44 environment is largely determined by microbial sources and sinks. N2O reduction by f
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45 organisms with N2O reductases (NosZ) is the only known biological sink of N2O at h
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46 environmentally relevant concentrations (up to ~1000 ppmv). Although a large //
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47 fraction of nosZ genes recovered from soil is affiliated to nosZ found in the genomes .
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48 of the obligate aerobic phylum Gemmatimonadetes, N2O reduction has not yet been .o
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49 confirmed in any of these organisms. This study demonstrates N O reduction by an o
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50 obligate aerobic bacterium Gemmatimonas aurantiaca strain T-27 and suggests a v
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51 novel regulation mechanism for N2O reduction identified in this organism, which may e
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52 also be applicable to other obligate aerobic organisms possessing nosZ genes. We ,

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53 expect that these findings will significantly advance understanding of N O dynamics 8
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54 in environments with frequent transitions between oxic and anoxic conditions. g
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56 Introduction
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57 The recent meteorological data from the first half of the year 2016 witnessed the
58 highest worldwide average temperature ever recorded for the same period of the year,
59 supporting the concern of the scientific world on on-going climate change (GISS
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60 Surface Temperature Analysis; http://www.ncdc.noaa.gov/sotc/global/201606). The o
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61 increase in greenhouse gas emission due to various anthropogenic activities has been lo
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62 regarded as the major culprit of the general upward trend in the global temperature
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63 and climate anomalies occurring with alarming frequency across the globe (1).
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64 Nitrous oxide (N2O) is a greenhouse gas with a global warming potential ~300 times //a
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65 that of CO2 and is the third most important contributor to global warming (6.2%) after .a
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66 CO2 (78%) and CH4 (16%) (1-4). Further, abolition of chlorofluorocarbon has left .o
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67 N O as the largest contributor to destruction of the ozone layer in the stratosphere (5, o
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68 6). Therefore, control of N2O emissions is indispensable in the efforts to curb global v
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69 warming and climate change. e
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70 ,

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71 Both natural and anthropogenic sources of N O have predominantly biological origin, 8
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72 with biological transformation of N-fertilizer applied to agricultural soils being the g
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73 single largest source (1, 7, 8). Nitrification produces N O as a byproduct of ammonia
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74 oxidation, and denitrification emits N O as a stable intermediate or an end-product
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75 (8-11). Other relatively minor sources of N O include a dissimilatory reduction to
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76 ammonium (DNRA) and chemodenitrification (8, 12-14). In contrary to the diverse
77 pathways leading to the production of N O, the sole biological sink process of N O in
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78 the environment is its reduction by the organisms expressing nitrous oxide reductases
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79 (NosZ) (8, 15-17). N2O reduction was originally regarded merely as a part of the o
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80 denitrification cascade. N O reduction as an independent respiratory reaction had not lo
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81 garnered deserved interest until recent discoveries unveiled the unexpectedly broad
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82 diversity of nosZ (15, 18). The novel clade of nosZ (clade II nosZ) genes is often
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83 discovered in organisms lacking the genes encoding for the key denitrification //
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84 enzymes, namely nirK or nirS, indicating that these organisms utilize N2O reduction .a
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85 as an independent respiratory reaction from denitrification (19). Thus, these non- .o
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86 denitrifying N O reducers function as de facto sinks of N O, as confirmed by o
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87 physiological observations from experiments with isolates possessing NosZ (20, 21). v
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88 Indeed, a recent study on the kinetics of N2O reductions revealed that the clade II e
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89 nosZ-encoding organisms have significantly higher affinity to N2O than the clade I ,
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90 nosZ-encoding organisms, supporting the hypotheses that the organisms with clade II 8

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91 nosZ may contribute to the mitigation of N O emission from non-point sources (17). g
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93 Metagenomic analyses of environmental DNA have revealed that clade II nosZ are in
94 fact abundant in diverse environments ranging from tropical forest and hot desert to
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95 Arctic tundra and polar deserts (18, 22). Among the most abundant phylogenetic group
96 of nosZ in the environment are clade II nosZ genes affiliated to the
97 Gemmatimonadetes phylum (16, 18). Gemmatimonadetes have been identified as one
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98 of the most abundant phyla of bacteria in the soil environments and often constitutes o
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99 >2% of the total bacterial population (23, 24); however, only three representative lo
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100 strains of this phylum (Gemmatimonas aurantiaca strain T-27, Gemmatirosa
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101 kalamazoonesis strain KBS708, and Gemmatimonas phototropica strain AP64) have
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102 been isolated to date, and the physiological characteristics of these organisms are //
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103 virtually unknown (25-28). Although clade II nosZ genes were found in the genomes .
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104 of G. aurantiaca strain T-27 and G. kalamazoonesis strain KBS708, these strains were .o
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105 both characterized as obligate aerobes and respiration on any other electron acceptors, o
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106 e.g., N2O, has yet to be explored (25-27). Therefore, questions remain unanswered v
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107 regarding the functionality of this Gemmatimonadetes NosZ and its potential e
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108 physiological role as a respiratory enzyme. As this particular group of nosZ constituted ,

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109 up to 33% of the entire nosZ gene pools identified in soil metagenomes, understanding 8

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110 the N O reduction phenotype of Gemmatimonadetes phylum is crucial for predicting g
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111 N O sink capabilities of subsurface soil environments. In this study, N O reduction by
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112 G. aurantiaca strain T-27 was observed both in absence and presence of oxygen. The
113 inability of this organism to consume N O in the complete absence of oxygen and the
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114 unexpected transcription pattern of nosZ, i.e., up-regulation in presence of oxygen and
115 down-regulation in absence of oxygen, suggest a novel regulatory mechanism for N O
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116 respiration by obligate aerobic microorganisms.
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118 Materials and Methods
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119 Bacterial culture and growth conditions. Gemmatimonas aurantiaca strain T-27
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120 was acquired from Japan Collection of Microorganisms (accession number:
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121 JCM11422). The culture medium used in this study was developed from NM-1 //
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122 medium, a semi-defined medium previously developed for isolation and culturing of .
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123 G. aurantiaca strain T-27 (26). The medium contained, per liter, 0.5 g glucose, 0.5 g .o
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124 polypepton, 0.5 g monosodium glutamate, 0.5 g yeast extract, 0.44 g K HPO , 0.1 g o
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125 (NH4)2SO4 and 0.1 g MgSO4. As nitrous oxide reductase is a copper-dependent v
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126 enzyme and lack of copper may hinder the activity of the enzyme (29), CuCl2 was e
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127 added to a concentration of 5 μM. pH was adjusted to 7.0 with 5 N NaOH solution. ,

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128 For preparation of anoxic or partial oxic cultures, 100 mL aliquots of the medium 8

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129 were distributed into 160 mL serum bottles (Wheaton, Millville, NJ) and flushed with g
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130 >99.999% N for ~20 minutes to remove O . This degassing step was omitted for oxic
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131 precultures prepared with 50 mL medium in 160 mL serum bottles. The serum bottles
132 were then sealed with butyl rubber stoppers (Geo-Microbial Technologies, Ochelata,
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133 OK). After autoclaving, 10 % of headspace (6 mL of 60 mL headspaces) was
134 aseptically replaced with air to prepare partial oxic cultures (~2.1% O concentration
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135 in the headspace). No air was added to anoxic controls. 0.5 mL of 200X Wolin’s
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136 vitamin solution (30) was added and 2.0 mL of G. aurantiaca strain T-27 preculture o
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137 was inoculated. Three milliliters of >99.999% N O (Deokyang Co., Ulsan, Korea) lo
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138 was aseptically added using a disposable syringe connected to 0.2-µm syringe filters
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139 (Advantec INC, Tokyo, Japan) after the same volume of headspace gas was removed.
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140 All culture vessels were incubated at 30°C with shaking at 140 rpm. Glucose was //
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141 added in excess (0.5 g/L), as a theoretical stoichiometric calculation estimated that .
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142 only 15.0% (42.5 μmoles) of added glucose would be consumed to reduce 125.8 .o
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143 μmoles O and 258.9 μmoles N O (the maximum amounts of electron acceptors used o
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144 in this research), even if glucose is used as the sole electron donor. Glucose v
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145 concentrations were measured after reactions were complete to confirm that the e
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146 amount of electron donor added was not a limiting factor for growth of G. aurantiaca ,

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147 strain T-27. 8

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148 u
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149 Analytical procedure. The amounts of N O in the serum bottles were quantified
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150 using an HP6890 Series gas chromatograph equipped with an HP-PLOT/Q column
151 and an electron capture detector (Agilent, Santa Clara, CA). Injector, oven, and
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152 detector temperatures were set to 200, 85, and 250 °C, respectively (17). For each
153 measurement, 200 µL of headspace gas was taken using a 1700-series gas-tight
154 syringe (Hamilton Company, Reno, NV) and 100 µL of the withdrawn sample was
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155 manually injected into the gas chromatograph. The syringe was flushed at least o
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156 three times with pressurized N gas to remove O before use, and 200 µL N was lo
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157 added upon each sampling event to prevent a pressure drop in the culture bottles.
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158 The change in the amounts of N2O due to gas sampling was accounted for in the h
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159 subsequent calculations (31). Oxygen concentrations were monitored with fiber- //
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160 optic oxygen sensor spots and FireStingO2 oxygen meter (Pyroscience, Aachen, .
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161 Germany). The total amounts of N2O and O2 in the reaction vessels were calculated .o
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162 from the headspace concentrations as described previously (17). The dimensionless o
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163 Henry’s constants (moles in the headspace / moles in the aqueous phase) of N2O v
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164 and O2 at 30 °C were calculated to be 1.92 and 33.3, respectively (32). These e
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165 dimensionless Henry’s constants were used to calculate the aqueous concentrations ,

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166 of N O and O . The amounts of the gases in the headspace and the aqueous phase 8
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167 were summed up for total amounts of N O and O in the vessels. Glucose g
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168 concentration was measured colorimetrically using Glucose (HK) Assay Kit
169 (Sigma-Aldrich, St. Louis, MO).
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171 Monitoring of N O consumption. A series of incubation experiments was
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172 performed to investigate consumption of N O by G. aurantiaca strain T-27. Oxic
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173 precultures for the experiments were prepared from glycerol stocks of strain T-27.
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174 For anoxic control experiments, 50 mL of the oxic precultures were harvested at the o
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175 late exponential phase (OD ~ 0.043) and centrifuged at 4,800 x g for 10 minutes lo
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176 at 4°C. In an anaerobic chamber (Coy Laboratory Products Inc., Grass Lake, MI)
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177 filled with 95% N2 and 5% H2, the pellets were equilibrated for one hour and h
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178 resuspended into 2 mL medium taken from the anoxic culture bottles prepared as //
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179 described above. The concentrated precultures were reinjected into the anoxic .
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180 serum bottle and N2O concentration was monitored for 312 hours. After .o
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181 confirming the absence of N O consumption activity in the anoxic cultures, the o
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182 N2O consumption experiments were performed in partial oxic culture bottles v
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183 prepared with low concentration (2.1%) of O2 in the headspace. One milliliter e
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184 frozen stock of strain T-27 was grown in 50 mL of oxic medium in a 160 mL serum ,

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185 bottle until the late exponential phase. Each partial oxic culture bottle (with 100 mL 8

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186 medium) was inoculated with 2 mL of these late-exponential-phase cultures. O g
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187 concentrations were monitored until the concentrations dropped below the
188 detection limit of the oxygen meter and N O concentrations were measured with 5-
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189 26.5 hour intervals until no remaining N O was detected. The killed-cell negative
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