REVIEW ARTICLE published: 21 February 2012 doi: 10.3389/fmicb.2012.00061 Trace requirements for microbial involved in the production and consumption of methane and nitrous oxide

Jennifer B. Glass* and Victoria J. Orphan

Division of Geological and Planetary Sciences, California Institute of Technology, Pasadena, CA, USA

Edited by: Fluxes of greenhouse gases to the atmosphere are heavily influenced by microbiological Martha Gledhill, University of activity. Microbial enzymes involved in the production and consumption of greenhouse Southampton, UK gases often contain metal cofactors. While extensive research has examined the influ- Reviewed by: Aubrey L. Zerkle, Newcastle ence of Fe bioavailability on microbial CO2 cycling, fewer studies have explored metal University, UK requirements for microbial production and consumption of the second- and third-most Lisa Y. Stein, University of Alberta, abundant greenhouse gases, methane (CH4), and nitrous oxide (N2O). Here we review Canada the current state of biochemical, physiological, and environmental research on transition *Correspondence: metal requirements for microbial CH4 and N2O cycling. Methanogenic archaea require large Jennifer B. Glass, Division of Geological and Planetary Sciences, amounts of Fe, Ni, and Co (and some Mo/W and Zn). Low bioavailability of Fe, Ni, and Co California Institute of Technology, limits in pure and mixed cultures and environmental studies. Anaerobic 1200 E. California Blvd., MC 170-25, methane oxidation by anaerobic methanotrophic archaea (ANME) likely occurs via reverse Pasadena, CA 91125, USA. methanogenesis since ANME possess most of the enzymes in the methanogenic path- e-mail: [email protected] way. Aerobic CH4 oxidation uses Cu or Fe for the first step depending on Cu availability, and additional Fe, Cu, and Mo for later steps. N2O production via classical anaerobic denitrifica- tion is primarily Fe-based, whereas aerobic pathways (nitrifier denitrification and archaeal ammonia oxidation) require Cu in addition to, or possibly in place of, Fe. Genes encoding the Cu-containing N2O reductase, the only known capable of microbial N2O con- version to N2, have only been found in classical denitrifiers. Accumulation of N2O due to low Cu has been observed in pure cultures and a lake ecosystem, but not in marine sys- tems. Future research is needed on metalloenzymes involved in the production of N2Oby enrichment cultures of ammonia oxidizing archaea, biological mechanisms for scavenging scarce , and possible links between metal bioavailability and greenhouse gas fluxes in anaerobic environments where metals may be limiting due to sulfide-metal scavenging.

Keywords: methane, nitrous oxide, microbes, metals, enzymes

INTRODUCTION built wastewater and sewage treatment plants where With increasing concern about the future impacts of global cli- proliferate. Humans have also extensively applied inorganic N mate change, a detailed understanding of the sources and sinks as to agricultural soils, leading to increased microbial of greenhouse gases is essential to mitigating their environmental N2O emissions in soils, wetlands, and coastal hypoxic zones impact. Although CO2 is the most abundant greenhouse gas,many (Schlesinger, 2009). Understanding the controls and regulation of other climatically important gases exist (Montzka et al., 2011). microbial greenhouse gas emissions is therefore fundamental to The second- and third-most abundant naturally produced green- quantifying and managing both natural and anthropogenic fluxes house gases are methane (currently ∼1.8 ppm; Heimann, 2011) of these gases. and nitrous oxide (currently ∼322 ppb; Montzka et al., 2011) and This review will focus on two potent greenhouse gases, CH4 are ∼25× and 300× more efficient at absorbing infrared radiation and N2O, both of which are produced as natural by-products of than CO2, respectively. Furthermore, CH4 is oxidized to CO2 in microbial energy-generating metabolisms. CH4 is the final prod- the atmosphere, contributing to rising CO2 levels. N2Ohasavery uct of the anaerobic degradation of organic matter, whereas N2O long residence time in the atmosphere (120 years) and reacts with formation results from incomplete conversion of nitrate or nitrite atomic O to form nitric oxide (NO), which is involved in ozone to N2. At the heart of the pathways that generate these gases are destruction (Montzka et al., 2011). enzymes that catalyze reactions. Many of these enzymes Both humans and microbes play important, and often inter- contain transition metals as cofactors for electron transport or woven, roles in the production of CH4 and N2O. Humans as catalytic centers at active sites. Important transition metals in have bred and expanded the habitats of ruminants that contain the pathway of CH4 production (methanogenesis) and anaerobic methanogenic archaea in their guts, cultivated rice paddies, and methane oxidation include Fe, Ni, Co, Mo/W, and Zn, whereas

www.frontiersin.org February 2012 | Volume 3 | Article 61 | 1 Glass and Orphan Trace metals in microbial enzymes

aerobic (and intra-aerobic) methanotrophy and N2O production In the first section we review the metal inventory of metal- require Fe-, Cu-, and Mo-containing proteins. Only one protein, loenzymes in each of the three pathways. This is not intended the Cu-rich nitrous oxidase reductase, is known to reduce N2Oto as a definitive review as many of these enzymes are still under- N2. Physiological studies of pure cultures have shown that opti- studied and more complete reviews of methanogenic enzymes mal metal concentrations for microbial metabolism are orders of are available (Thauer, 1998; Thauer et al., 2008, 2010). Instead, magnitude higher than in situ concentrations in most aquatic envi- our goal is to estimate the transition metal stoichiometry of the ronments. These findings lead to the question: are some microbes three methanogenic pathways, illustrated in Figure 1. In the next perennially metal-limited in nature? If so, does metal availability section we discuss physiological studies of the metal requirements exert influence on the flux of greenhouse gases? If not, what mech- for methanogenesis and relate these findings back to the metal anisms do microbes use in natural environments to acquire trace content of each pathway. Pure culture, anaerobic digester, and metals? environmental studies are reviewed. Lastly, a summary of pre- Previous environmental studies of metal requirements for vious studies examining metal requirements for methanogenesis microbes have largely focused on those microbes directly involved is provided, and topics warranting further research are discussed. in CO2 cycling, principally phytoplankton that consume CO2 dur- This general format is also used in the sections on methanotrophy ing photosynthesis. Driving these studies was the “Fe hypothesis” (see Methanotrophy) and nitrous oxide cycling (see Nitrous Oxide by John Martin positing that CO2-consuming marine phyto- Production and Consumption). plankton could be fertilized by Fe (Martin and Fitzwater, 1988). Far less attention has been aimed at metal requirements for METALLOENZYMES IN METHANOGENESIS microbes involved in the cycling of non-CO2 greenhouse gases, Methanogenesis is one of the most metal-rich enzymatic pathways although many of these organisms also live in ecosystems with in biology (Zerkle et al., 2005). Depending on the pathway, exact very low metal bioavailability. A notable exception is the enor- metal requirements may differ, but the general trends remain the mous wealth of literature generated by the wastewater scientific same: Fe is the most abundant metal, followed by Ni and Co, and community about metal (particularly Fe, Ni, and Co) controls on smaller amounts of Mo (and/or W) and Zn. Fe is primarily present methanogenesis in anaerobic digesters (see Demirel and Scherer, as Fe–S clusters used for electron transport and/or catalysis. Ni is 2011). either bound to Fe–S clusters or in the center of a porphyrin unique The purpose of this article is to review the current state of to methanogens, F430. Co(balt) is present in cobamides literature on the metalloenzymes and trace metal physiology of involved in methyl group transfer. Zn occurs as a single structural organisms involved in CH4 and N2O processing. In each section, atom in several enzymes. Mo or W is bound to a pterin cofactor we discuss environmental studies if they exist and compare metal to form “” or “tungstopterin,” which catalyze two- concentrations for optimal growth of pure cultures to measured electron redox reactions. Other alkali metals and metalloids, such values of trace metals in natural environments. In the final sections as Na and Se, are essential for methanogenesis, but the discussion of the article, we compare the metal requirements of microbes here is limited to the transition metals. So far, no Cu-dependent involved in the CH4 and N2O cycles and discuss future research methanogenesis enzymes have been identified, which is striking directions. Readers are referred to previous reviews (Rogers and given the huge importance of Cu in aerobic methanotrophy (see Whitman, 1991; Conrad, 1996) for other aspects of microbial the Section “Aerobic Methanotrophy”). It is plausible that scarce controls on greenhouse gas cycling. Cu in anaerobic early-earth marine ecosystems selected for use of other metals (i.e., Fe, Ni, and Co) in primitive methanogens METHANOGENESIS whereas Cu was more bioavailable to aerobic methanotrophs after Methanogenesis is the microbial process whereby CO2, acetate, or the rise of atmospheric oxygen (Dupont et al., 2006, 2010; David methyl-compounds are converted to methane in order to generate and Alm, 2010). ATP through the build-up of a sodium ion or proton gradient. All Most orders – Class I (Methanobacteriales, methanogens are in the Euryarchaeota phylum (Whitman et al., Methanococcales, and Methanopyrales) and Class II (Metha- 2006) and account for 75–80% of the annual global production of nomicrobiales) – obtain energy by reducing CO2 to CH4 using CH4 (IPCC, 2007). Approximately two-thirds of biologically pro- electrons from H2 or in some cases formate (Anderson et al., duced CH4 is generated by aceticlastic methanogens that oxidize 2009). These methanogens do not contain cytochrome proteins, the carbonyl group of acetate to CO2 and reduce the methyl group in which Fe is bound to . Only one order of methanogens, the to CH4 (Rogers and Whitman, 1991; Ferry, 2010b). The remain- Methanosarcinales (Class III), contain cytochromes and are capa- ing one-third of biogenic methane is produced by reduction of ble of metabolizing a wider range of substrates for methanogen- CO2 with electrons from H2 or formate and the conversion of esis, including methanol, methylamines, methyl sulfides, acetate, methyl groups from compounds such as methanol, methylamines, and/or CO2 with H2. Members of the Methanosarcinales play a and dimethylsulfide. Therefore, methanogenesis is divided into key ecological role because the aceticlastic pathway is estimated to three pathways: hydrogenotrophic (i.e., CO2 reduction), aceticlas- account for two-thirds of biologically sourced methane (Rogers tic (acetate disproportionation), and methylotrophic (C1 utiliza- and Whitman, 1991). tion). The three pathways differ in the enzymes used to generate The Fe requirement for methanogenesis is vast: almost every methyl-tetrahydro(methano/sarcina)pterin (CH3-H4(M/S)PT), metalloenzyme involved in the pathway contains multiple Fe2S2, the intermediate common to all pathways, but converge in the Fe3S4,orFe4S4 clusters. The first enzyme in the CO2 reduction last two steps used to generate CH4 (Ferry, 2010b). pathway, formylmethanofuran dehydrogenase (abbreviated Fmd

Frontiers in Microbiology | Microbiological Chemistry February 2012 | Volume 3 | Article 61 | 2 Glass and Orphan Trace metals in microbial enzymes

FIGURE 1 | Metal content of metalloenzymes in the three pathways energy-converting hydrogenase; Fd, ferredoxin; Fmd/Fwd, Mo/W

of methanogenesis: H2/CO2 (black arrows), aceticlastic (green formylmethanofuran dehydrogenase; Frh, F420-reducing hydrogenase; Hdr, arrows), and methylotrophic (blue arrows). Each circle represents one heterodisulfide reductase; Hmd, Ni-free Fe hydrogenase; Mcr, methyl metal atom. Parentheses show varying metal content of a given enzyme. reductase; Mta, methanol-coenzyme M methyltransferase;

Question marks mean that enzyme may not be present in all Mtr, CH3-H4M(S)PT-coenzyme M methyltransferase; Vh(o/t)/Mvh, Ni–Fe

methanogens (6×[Fe4S4] and 10×[Fe4S4]polyferredoxins are encoded in hydrogenase. For simplicity, only metalloenzymes are labeled. See the

the eha operon and a 14×[Fe4S4]polyferredoxin is encoded in the ehb Section “Metalloenzymes in Methanogenesis” for more details, operon). Abbreviations: CA, carbonic anhydrase; Cdh, carbon monoxide abbreviations for intermediates and enzyme substitutions that occur in dehydrogenase/acetyl-CoA synthase; Ech/Eha/Ehb/Mbh, specific conditions and species.

for the Mo form and Fwd for theW form) can bind up to nine Fe4S4 to eight additional clusters in polyferredoxin (Vorholt et al., 1996; clusters: one cluster in the Mo/W-pterin binding subunit and up Figure 1). It is likely that Fwd can bind additional Fe4S4 clusters

www.frontiersin.org February 2012 | Volume 3 | Article 61 | 3 Glass and Orphan Trace metals in microbial enzymes

(Ferry, 1999). Ferredoxins that transfer electrons from H2 to other Methanophenazine is in turn reduced by the Ni–Fe hydrogenase methanogenesis enzymes require additional Fe in the form of two Vht/Vho, which contains one Ni–Fe center, two b-type , one Fe4S4 clusters (Daas et al., 1994). Fe3S4 cluster, and two Fe4S4 clusters (Deppenmeier et al., 1992; All hydrogenases involved in methanogenesis are Ni–Fe Thauer et al., 2010). In the marine strain Methanosarcina acetivo- enzymes that oxidize H2 and reduce ferredoxin, coenzyme F420, rans, Vht/Vho is replaced by Rnf, which contains six Fe4S4 clusters and other electron carriers (Thauer et al., 2010). The four different and no Ni, and uses reduced ferredoxin instead of H2 (Li et al., types of Ni–Fe hydrogenases involved in methanogenesis all con- 2006; Ferry, 2010a). tain abundant Fe. The energy-converting membrane-associated Methyl coenzyme M reductase (Mcr), common to all hydrogenase [abbreviated Ech, Eha, Ehb, or Mbh depending on methanogenic pathways, catalyzes the final step in methanogen- the class of methanogen (Anderson et al., 2009)] contains three esis: the reduction of CH3-S-CoM to CH4 and the formation of Fe4S4 clusters and a Ni–Fe active site (Thauer et al., 2010), and can CoM–CoB heterodisulfide with electrons derived from HS-CoB. contain additional polyferredoxin subunits with 6, 10, or 14 Fe4S4 The crystal structure of Mcr has been solved and contains two clusters (Tersteegen and Hedderich, 1999; Figure 1). coenzyme F430 Ni tetrapyrroles (Ermler et al., 1997). Previous Another Ni–Fe hydrogenase is used to reduce coenzyme F420. studies have shown that the F430 content of cells is dependent on Coenzyme F420, a flavin derivative that plays a critical role in two cellular Ni content (Diekert et al., 1980, 1981; Lin et al., 1989). intermediate electron-transfer steps in methanogenesis, is reduced Methanogens actively transport Ni and Co using ATP-dependent with H2 by the cytoplasmic hydrogenase Frh (Alex et al., 1990). uptake systems in order to fulfill enzymatic requirements (Jarrell The Frh enzyme complex contains a Ni–Fe active site and four and Sprott, 1982; Baudet et al., 1988; Rodionov et al., 2006; Zhang Fe4S4 clusters (Figure 1), and forms large aggregates, multiplying et al., 2009). its metal requirements approximately eightfold (Fox et al., 1987). When grown on acetate, methanogens use two metalloenzymes Under conditions of Ni limitation, some methanogens without for the conversion of the methyl group from acetate to CH3- cytochromes substitute Frh for a Ni-free Fe hydrogenase (Hmd) H4SPT,which differ from those used in CO2 reduction and methy- to decrease Ni requirements (Afting et al., 1998). The Ni-free Fe lotrophic pathways. The most metal-rich aceticlastic enzyme is hydrogenase has a significantly lower Fe requirement, using only CO dehydrogenase/acetyl-CoA synthase (Cdh), which cleaves the two Fe atoms per complex (Zirngibl et al., 1992; Shima et al., methyl group off of acetyl-CoA and transfers it to CH3-H4SPT. 2008; Thauer et al., 2010). When formate is used as an electron The Cdh complex contains one Fe4S4 cluster bridged to an Ni–Ni source instead of H2, Frh is replaced by formate dehydrogenase site (Funk et al., 2004), four Fe4S4 clusters and a NiFe4S4 cluster (Fdh), which contains one Mo/W-pterin cofactor, two Zn atoms (Gong et al., 2008), and reduces a 2×[Fe4S4]ferredoxin (Terlesky and between 21 and 24 Fe atoms (Schauer and Ferry, 1986). and Ferry, 1988; Ferry, 2010b). The CO2 by-product of Cdh is Co(balt)-containing methyltransferases are essential for converted to bicarbonate by an Fe-containing carbonic anhydrase methanogenesis. All methanogens utilize the energy-conserving (CA) instead of the Zn-form, which is commonly in CAs from CH3-H4M(S)PT-coenzyme M methyltransferase (Mtr) to trans- other species (Macauley et al., 2009). Overall, the metal content of fer the methyl group from CH3-H4M(S)PT to HS-CoM. The Mtr enzymes involved in aceticlastic methanogenesis is similar to the enzyme complex contains two cobamide cofactors (with one Co H2/CO2 pathway (Figure 1). The Fe and Mo/W requirements are each) and eight Fe atoms (Gartner et al., 1993). A wide range likely lower in the aceticlastic pathway because the Fe-rich poly- of other Co(balt)-containing methyltransferases are required for ferredoxins involved in the CO2 reduction route are not present, methyl coenzyme M formation in methylotrophic methanogens, nor is Fmd/Fwd. some of which contain additional metals (Thauer, 1998). For instance, the methanol-coenzyme M methyltransferase enzyme PHYSIOLOGICAL STUDIES OF TRACE METAL REQUIREMENTS FOR (Mta) expressed by methanol-utilizing methanogens contains one METHANOGENESIS Zn in addition to one cobalamin Co (Hagemeier et al., 2006). Previous physiological studies of metals as micronutrients for Heterodisulfide reductase (Hdr) is an Fe–S and Zn-containing methanogenesis have been compromised by significant metal enzyme that catalyzes the reduction of heterodisulfide (CoM- contamination in growth vessels and sampling equipment. The S-S-CoB) to form HS-CoM and HS-CoB in the second-to-last requirements for gas-tight material to maintain anaerobic con- step in methanogenesis. In methanogens without cytochromes, ditions has favored the use of glass culturing bottles with butyl Hdr has three subunits (HdrABC) whereas methanogens with rubber stoppers and stainless steel needles for sampling, both of cytochromes have a two-subunit protein (HdrDE; Hedderich et al., which are notoriously dirty with respect to metals. In fact, Ni 2005; Thauer et al., 2008). HdrABC contains seven Fe4S4 clus- requirements for methanogens were overlooked in early studies tersandonestructuralZnatom(Hamann et al., 2007) and due to the high Ni content (∼10%) of stainless steel syringe nee- forms a tight complex with the Ni–Fe hydrogenase Mvh, which dles. Dissolution of Ni in stainless steel needles by H2S in the contains one Ni–Fe active site, one Fe2S2 cluster, two Fe4S4 clus- media supplied ample Ni for growth of methanogens on media ters, one Fe3S4 cluster, and polyferredoxin with 12 Fe4S4 clusters not supplemented with Ni salts (S. Zinder, personal comm, 2011; (Reeve et al., 1989; Figure 1). In Methanosarcinales, HdrDE is Diekert et al., 1981; Whitman et al., 1982). The need for anaerobic reduced by methanophenazine, a lipid-soluble electron, and pro- conditions excludes the use of acid-washed plastics, which have ton carrier with very low redox potential. HdrDE contains three extremely low metal contamination and are commonly used for Fe4S4 clusters, one cofactor, and one structural Zn atom trace metal limitation experiments with aerobes, but are perme- (Heiden et al., 1994; Künkel et al., 1997; Simianu et al., 1998). able to O2 diffusion. To address these concerns, researchers have

Frontiers in Microbiology | Microbiological Chemistry February 2012 | Volume 3 | Article 61 | 4 Glass and Orphan Trace metals in microbial enzymes

acid-washed glassware with sulfuric acid, treated butyl rubber concentrations of Fe, relatively high levels of Ni and Co, trace stoppers, and polypropylene pipet tips with NaHCO3/EDTA and Zn and Mo/W, and negligible amounts of Cu and Mn. This covered cannula needles with Teflon tubing (Whitman et al., 1982; prediction does not take into account complications if enzymes Sowers and Ferry, 1985). Such methods were used in some, though have differing K m values for their respective substrates, which not all, of the studies reviewed in the Section “Pure Cultures.” To could result in shifts in enzyme ratios, nor does it consider metal our knowledge, Teflon-lined glass bottles have not been used to requirements for metabolic pathways besides methanogenesis. minimize metal contamination in previous anaerobic studies, but Nevertheless, it roughly agrees with the cellular metal contents may be a good option for future research. of 11 previously analyzed methanogens, which contained 700– The experiments discussed in Section “Mixed Cultures and 2800 ppm Fe, 17–180 ppm Ni, 23–1810 ppm Zn, 10–120 ppm Co, Anaerobic Bioreactors” on mixed cultures and anaerobic biore- 10–70 ppm Mo, <10–160 ppm Cu, and 2–27 ppm Mn, in terms of actors were conducted, for the most part, not in defined media dryweight(Diekert et al., 1981; Whitman et al., 1982; Scherer but rather in sludge or wastewater treatment effluent. Studies of et al., 1983). These data suggest that the metal requirements metal requirements for pure cultures and anaerobic digesters typi- for methanogenesis have a strong influence on whole cell metal cally report only total added metal, although these reactors usually contents. have higher background metal contents than pure cultures. How- Early studies on Methanobacterium thermoautotrophicum ever, the ubiquitous presence of sulfide, carbonate, and phosphate (renamed Methanothermobacter thermautotrophicus)grownon in methanogenic cultures leads to metal precipitation, so dis- H2 and CO2 showed that low concentrations of Fe, Ni, Co, and Mo solved metal concentrations generally decline throughout exper- in growth media could limit growth (Taylor and Pirt, 1977; Schön- iments (Callander and Barford, 1983a,b). Indeed, precipitation– heit et al., 1979). For the formation of 1 g of biomass (dry weight), dissolution kinetics of metal sulfides may play a key role in 10 μmol of Fe, 150 nmol of Ni, 20 nmol of Co, and 20 nmol of bioavailability of metals in anaerobic digesters (Gonzalez-Gil et al., Mo were required, a stoichiometry of 500 Fe: 7.5 Ni: 1 Co: 1 Mo 1999; Jansen et al., 2007). However, increased rates of methano- (Schönheit et al., 1979). Optimal growth was observed at 300– genesis after addition of a particular metal are clear evidence 500 μMFe(Patel et al., 1978) and 1 μM Ni, while addition of Cu, of environmental (albeit human-contributed) metal limitation as Zn,and Mn to the media did not stimulate growth (Schönheit et al., compared to axenic cultures. 1979; Table 1). Interestingly, optimal growth of another H2/CO2- utilizing methanogen, Methanococcus voltae, occurred at much Pure cultures lower Fe and Ni concentrations (10 and 0.2 μM, respectively), Based on the metal content of the enzymes involved in methano- despite the more rigorous cleaning protocols used in this study genesis, one would predict that methanogens require very high (Whitman et al., 1982).

Table 1 | Optimal dissolved metal concentrations (calculated from total amounts of salt added) in defined growth media for pure methanogenic cultures grown on H2/CO2, formate, acetate, methanol, or trimethylamine.

Metal Substrate Concentration (μM) Species Reference

Fe H2/CO2 300–500 Methanospirillum hungatei, Patel et al. (1978) Methanobacterium bryantii strain MOH

H2/CO2 >15 Methanococcus voltae Whitman et al. (1982) Acetate 100 Methanothrix soehngenii VNBF1 Fathepure (1987) Methanol 50 Methanosarcina barkeri Lin et al. (1990) Trimethylamine 5 Methanococcoides methylutens Sowers and Ferry (1985) 2 Ni H2/CO2 1 Methanobacterium thermoautotrophicum Schönheit et al. (1979)

H2/CO2 0.2 Methanococcus voltae Whitman et al. (1982) Acetate 2 Methanothrix soehngenii VNBF1 Fathepure (1987) Methanol 0.1 Methanosarcina barkeri Scherer and Sahm (1981) Trimethylamine 0.25 Methanococcoides methylutens Sowers and Ferry (1985) Co Acetate 2 Methanothrix soehngenii VNBF1 Fathepure (1987) Methanol 1 Methanosarcina barkeri Scherer and Sahm (1981) Trimethylamine 0.1 Methanococcoides methylutens Sowers and Ferry (1985) Mo Acetate 2 Methanothrix soehngenii VNBF1 Fathepure (1987) + Methanol (with NH4 ) 0.5 Methanosarcina barkeri Scherer and Sahm (1981), Scherer (1988)

Methanol (N2-fixing) 5 Methanosarcina barkeri Scherer (1988)

WH2/CO2 1 Methanocorpusculum parvum Zellner and Winter (1987) Formate 0.5 Methanocorpusculum parvum Zellner and Winter (1987)

1Renamed Methanosaeta concilii; 2Renamed Methanothermobacter thermautotrophicus. Adapted from Takashima and Speece (1990).

www.frontiersin.org February 2012 | Volume 3 | Article 61 | 5 Glass and Orphan Trace metals in microbial enzymes

How do metal requirements change when methanogens are 5–10 mM FeCl2 added due to precipitation as ferrous carbonate), grown on substrates other than H2/CO2? Optimal growth of whereas background soluble Fe was ∼25 μM(Hoban and van Methanothrix soehngenii VNBF (renamed Methanosaeta concilii) den Berg, 1979). The authors suggested that mixed methanogenic grown on acetate was observed at 100 μM Fe, 2 μM Ni, 2 μM cultures might have higher Fe requirements than pure cultures. Co, and 2 μMMo(Fathepure, 1987; Table 1). Optimal growth of Addition of Ni and Co stimulated aceticlastic methanogenesis methanol-utilizing Methanosarcina barkeri was observed at lower and increased the thickness of biofilms in anaerobic food waste concentrations: 50 μM Fe, 0.1 μM Ni, 1 μM Co and 0.5 μMMo, digesters (Murray and van den Berg, 1981), municipal sludge and addition of Cu and Mn had no effect on growth (Scherer and digesters (Speece et al., 1983), and synthetic wastewaters (Kida Sahm, 1981; Lin et al., 1990). Optimal growth of trimethylamine- et al., 2001). Addition of Mo (50 nM) had a smaller effect (Murray grown Methanococcoides methylutens was lower still: 5 μMFe, and van den Berg, 1981). In another study, individual addition 0.25 μM Ni, and 0.1 μM Co, with no stimulation by addition of of Fe, Ni, Co, Mn, Zn, Mo, and Cu to a wastewater treatment Mo, Zn, Mn, W, and Cu (Sowers and Ferry, 1985). These studies sludge did not produce any significant increase in the conversion suggest that metal requirements are higher on acetate than methyl- of acetate to methane (Florencio et al., 1993). It is possible that compounds, although the opposite – particularly for Co – has been the sludge in the latter study contained sufficient metal carry-over found in mixed culture anaerobic digester studies (see the Section that these metals were not limiting for aceticlastic methanogen- “Mixed Cultures and Anaerobic Bioreactors”). esis. Metal speciation almost certainly influences bioavailability In some methanogens, W-containing formylmethanofuran in these bioreactors, where up to 95% of dissolved Ni and Co dehydrogenases (Fwd) and W-containing formate dehydrogenases is present in strongly bound forms (Jansen et al., 2005). It is cur- (W-Fdh) are expressed in place of Mo-containing forms of the rently unknown whether methanogens can directly take up soluble enzymes. W was first shown to stimulate growth of Methanococcus metal-sulfide complexes or metal-organic complexes (Jansen et al., vannielii on formate (Jones and Stadtman, 1977). A subsequent 2007). Additionally, temperature may exert influence on the trace study revealed that optimal growth on H2/CO2 required more W metal requirements; higher trace metal requirements have been (1 μM) than growth on formate (0.5 μM) and that more W was found for thermophilic than mesophilic mixed reactors, despite incorporated into cells during growth on H2/CO2 than on formate smaller biomass yield (Takashima et al., 2011). (Zellner and Winter, 1987), suggesting that Fwd has a higher W Limitation of methanogenesis by low Co(balt) bioavailability requirement than W-Fdh. has been identified in numerous studies of anaerobic mixed cul- In addition to metal cofactors in enzymes directly involved in tures grown on methanol.A study of methylotrophic methanogens methanogenesis, additional metals are required for other meta- in wastewater treatment sludge showed a pronounced increase bolic pathways in methanogens, such as nitrogen fixation. In in methanogenic activity when Co was added, and a significant a study of metal requirements for two strains of M. barkeri, decline when it was absent from the media (Florencio et al., 1993). Scherer (1988) found that optimal Mo media concentrations for Follow-up studies have confirmed the high Co(balt) requirements diazotrophic growth were 10× greater (5 μM Mo) than NH4- for methanol-grown anaerobic bioreactors (Zandvoort et al., based growth (0.5 μM Mo). One of the two strains (strain 227) 2006). The same trend was observed for Ni, although to a lesser could grow just as well with (V) instead of Mo in extent, but minimal effect was found for other metals (Fe, Zn, Mo, the media, suggesting that it contained a V-nitrogenase (Scherer, Cu, and Zn). Optimal Ni and Co concentrations for methano- 1988) as later confirmed by Chien et al. (2000). However, dif- genesis was ∼2 μM(Florencio et al., 1993, 1994; Zandvoort et al., fering results have been published regarding the extent to which 2002). The increased need for Co for methanol-based growth vs. V stimulates nitrogen fixation in this strain (Lobo and Zinder, other substrates is likely due to additional Co(balt)-containing 1988). Other methanogens, such as Methanococcus maripaludis, methyltransferases required for growth on methyl-compounds. lack the V-nitrogenase and have an absolute requirement for Mo This concurs with previous findings that the highest amount of for diazotrophy (Kessler et al., 1997). corrinoids are produced in methanogens grown on methanol com- pared to other substrates, with about 3× as much corrinoids in Mixed cultures and anaerobic bioreactors methanol-grown cell vs. acetate-grown cells (Krzycki and Zeikus, The potential of metals to increase the efficiency of methanogen- 1980) and that cobalamin production increases with media Co esis in industrial settings has led to a steady stream of studies on concentration up to ∼50 μM(Lin et al., 1989). metal requirements for optimal functioning of anaerobic waste (sewage, agricultural, and food-processing) treatment bioreactors Environmental studies since the late 1970s (Demirel and Scherer, 2011). The produc- Very few studies have investigated the effects of metal additions tion of methane from small organic molecules such as acetate and on natural methanogenic ecosystems. To our knowledge, the only methanol in waste treatment reactors is a beneficial by-product published experiment was performed in five North American because it can be captured and used as a renewable fuel source. peatlands (Basiliko and Yavitt, 2001). Ambient dissolved metal Several studies suggest that the addition of Fe, Ni, and Co concentrations in -poor peatlands were low (2–20 nM Ni, can increase the rate of aceticlastic methanogenesis in anaerobic 30 nM Co, and <2 μM Fe) due to the high metal-binding capac- digesters. An early study of aceticlastic methanogenesis in mixed ity of peat. In mineral-rich peatlands, concentrations were 40– cultures originally obtained from sewage sludge and maintained 60 nM Ni, 2–5 μM Co, and 20–30 μM Fe. The authors found that in a defined medium showed that the optimal soluble Fe concen- combined addition of Fe (12 μM), Ni (12 μM), and Co (6 μM) trations for methanogenesis were 0.2–2 mM (lower than the total to mineral-poor surface samples resulted in an enhancement of

Frontiers in Microbiology | Microbiological Chemistry February 2012 | Volume 3 | Article 61 | 6 Glass and Orphan Trace metals in microbial enzymes

CH4 production, whereas no effect or inhibition was observed at and/or organism. Every laboratory likely had different levels of mineral-rich peatlands. The effect of individual metals was not trace metal contamination and metal-sulfide precipitation, and tested. No effect was observed for CO2 production in mineral- thus the given optimal metal concentrations are all estimates to poor peatlands, suggesting that heterotrophic microbes are not as varying degrees. Nonetheless, a few trends are clear: (1) Fe con- metal-limited as methanogens. Addition of the chelator citrate,but centrations required for optimal growth in media are much higher −5 −4 not EDTA, led to increased CH4 production in the peat with the than all other metals (in the 10 to 10 M range); (2) optimal lowest metal concentrations, suggesting either that methanogens Ni, Co, Mo, and W concentrations are in the 10−7 to 10−6 M can take up metal–citrate complexes or that they can compete with range, and higher Mo concentrations (10−5 M) are needed for citrate for metal-binding. diazotrophic growth; (3) addition of Cu and Mn is not needed Other anoxic ecosystems have similar trace metal concentra- for growth. Few studies have investigated Zn requirements for tions to peat bogs, suggesting that metal limitation of methano- methanogenesis, although Zn is present in at least two enzymes in genesis may be widespread in natural environments. The con- the pathway. centration range of Ni, Co, and Mo in five representative anoxic Studies of anaerobic digesters and peatlands suggest that Fe, waters was consistently below the optima for growth of pure cul- Ni, and Co – and possibly other metals – may limit methanogen- tures (Tables 1 and 2). Optimal methanogenic growth occurs at esis in the natural ecosystems. The high Co(balt) requirement for 0.2–2 μM Ni and in natural anoxic waters Ni ranges from 0.002 methylotrophic methanogenesis in anaerobic digesters is of par- to 0.07 μM. Optimal media concentrations of Co are 0.1–2 μM ticular interest. To our knowledge, only one study has looked into and in natural environments Co is generally <0.02 μM. Optimal the possibility of metal limitation of methanogenesis in a natural Mo concentrations are 0.5–5 μM; ambient Mo is 0.01–0.2 μMin setting (Basiliko and Yavitt, 2001). More environmental studies anoxic environments. Zn is also generally very low (<0.04 μM) are essential to better understand the nature of metal bioavailabil- in anoxic waters, and may contribute to trace metal limitation of ity and/or limitation in the ecosystems that contribute 75–80% methanogenesis, although pure culture studies are not available of the world’s annual CH4 sources, but preliminary comparison to determine optimal Zn concentrations. The opposite is true for of optimal concentrations for growth of pure cultures and in situ W: few data are available on in situ W concentrations, but in most levels shows that trace metal (particularly Ni and Co) limitation ecosystems W is below the growth optima of 0.5–1 μM. Optimal of methanogenesis may be widespread in anoxic settings. Fe concentrations are present in anoxic sediment pore waters (such as the Santa Monica Basin) where Fe reaches 200 μM, but other METHANOTROPHY anoxic waters have significantly lower Fe (Table 2). Therefore, it is Methanotrophy is the microbial process whereby CH4 is oxidized quite reasonable to expect that bioavailability of trace metals may as an energy and carbon source. Aerobic methanotrophy is esti- be important limiting growth factors in natural methanogenic mated to consume ∼35% (0.6 Gt) of the total global CH4 produced environments. per year, whereas anaerobic methanotrophy is estimated to con- sume ∼18% (0.3 Gt; Thauer, 2011). Aerobic methanotrophy is SUMMARY AND AREAS FOR FUTURE RESEARCH performed by certain bacteria that combine molecular oxygen Based on the metal content of enzymes in methanogenesis, one with CH4 to produce methanol (CH3OH). Methanol is then would predict (1) roughly equal Fe, Ni, and Zn requirements oxidized to formaldehyde (CH2O), which is incorporated into for CO2 reduction, aceticlastic, and methylotrophic pathways; organic compounds using either the serine or ribulose monophos- (2) higher Mo/W requirements for CO2 reduction and methy- phate pathway (Semrau et al., 2010). Sulfate-dependent anaerobic lotrophic pathways than aceticlastic methanogenesis; and (3) methane oxidation (AOM) occurs primarily in anoxic marine sed- higher Co(balt) requirement for the methylotrophic pathway than iments and is performed by archaea that have not been isolated CO2 reduction and aceticlastic pathways. Previous studies are in pure culture, but are most closely related to Methanosarcinales difficult to compare because few of them simultaneously vary and Methanomicrobiales. These archaea are believed to oxidize methanogenic pathways using the same experimental conditions methane anaerobically through a reverse methanogenesis pathway

Table 2 | Compiled dissolved trace metal concentrations in representative anoxic waters.

Sample site Fe Ni Co Cu Zn Mo Reference

Peat bog waters 0.4–40 0.002–0.07 0.002–4 0.02–2 Basiliko and Yavitt (2001), Bragazza (2006) Santa Monica basin sediment pore 100–200 0.01 0.01–0.02 <0.005 0.2 Shaw et al. (1990) waters (>5 cm depth) Black Sea sulfidic water column 0.01–0.3 0.01 <0.004 <0.002 <0.004 <0.01 Haraldsson and Westerlund (1988), (>200 m) Emerson and Huested (1991) Framvaren Fjord water column (>20 m) 0.03–2 <0.07 0.001–0.01 <0.001 0.002–0.01 <0.04 Haraldsson and Westerlund (1988), Emerson and Huested (1991) Baltic Sea water column (>150 m) 0.1–2 0.01 <0.002 <0.007 0.01–0.04 Kremling (1983)

All concentrations are in units of micromolar (μM).

www.frontiersin.org February 2012 | Volume 3 | Article 61 | 7 Glass and Orphan Trace metals in microbial enzymes

(Scheller et al., 2010; Thauer, 2011). Thus, phylogenetically unre- between the pMMO-B and pMMO-C subunits (Lieberman and lated aerobic and anaerobic methanotrophs accomplish the same Rosenzweig, 2005; Hakemian et al., 2008). Since pMMO is a het- task using very different enzymes (Chistoserdova et al., 2005) and erotrimer, it therefore contains nine Cu atoms (and possibly three are therefore predicted to have different metal requirements. Cu Zn atoms; Figure 2). Recent Mössbauer evidence suggests that Fe requirements for aerobic methanotrophy have been the subject of could also be present in pMMO, as supported by previous protein a great deal of research, whereas metal requirements for AOM are preparations that contained Fe (Martinho et al., 2007; Semrau just beginning to be explored, but are expected to be similar to et al., 2010). The electron donor to pMMO is a cytochrome bc1 metal requirements for methanogenesis. complex, containing three heme groups and one Fe2S2 cluster (Zahn and DiSpirito, 1996). AEROBIC METHANOTROPHY Several strains of aerobic methanotrophs are also capable of Aerobic methanotrophs (abbreviated “MOB” for “methane- expressing a cytoplasmic soluble MMO (sMMO), which con- oxidizing bacteria”) are commonly found at oxic/anoxic interfaces tains a dimeric hydroxylase with two Fe ions in a di-Fe active of environments where CH4 is produced. Methanotroph genera, site cluster and an additional Fe2S2 cluster in a reductase sub- beginning with “Methylo-,” are found in the Gammaproteobacte- unit (Lipscomb, 1994; Merkx et al., 2001). Expression of partic- ria and Alphaproteobacteria (Hanson and Hanson, 1996; Semrau ulate vs. soluble MMO is regulated by Cu availability (Murrell et al., 2010). Recently, MOB have also been discovered in the Ver- et al., 2000). Work with pure cultures has shown that a “Cu rucomicrobia (Op den Camp et al., 2009). Furthermore, the newly switch” occurs at 0.85–1 μmol/g dry weight: at lower Cu:biomass discovered freshwater phylum NC10 bacterium Methylomirabilis ratios, sMMO is expressed, and at higher Cu:biomass ratios, oxyfera performs intra-aerobic methane oxidation using pMMO pMMO is expressed (Stanley et al., 1983; Hanson and Hanson, and internally produced O2 from NO (Ettwig et al., 2010; Wu et al., 1996; Nielsen et al., 1997). In contrast to pMMO, the elec- 2011). In addition to being important in greenhouse gas process- tron donor to sMMO is NADH (Lipscomb, 1994; Merkx et al., ing, some MOB play important roles in pollutant degradation and 2001). bioremediation (Semrau, 2011). After CH4 is oxidized to CH3OH by either pMMO or sMMO, the periplasmic -dependent enzyme Metalloenzymes in aerobic methanotrophy methanol dehydrogenase (abbreviated MDH or Mxa) oxidizes Classical aerobic methanotrophy. The metal requirements for CH3OH to formaldehyde (CHOH) using two cytochrome c pro- MOB are largely regulated by (Cu) availability. When Cu teins (cL and cH)aselectronacceptors(Hanson and Hanson, is abundant, MOB express a membrane-bound Cu (and possibly 1996). These cytochromes are then oxidized by cytochrome aa3, Fe and Zn)-containing enzyme (particulate methane monooxy- containing two hemes and two Cu atoms (Figure 2). MDH/Mxa genase, pMMO) that catalyzes the first step of CH4 oxidation. does not contain any transition metal cofactors, but does pos- When Cu is lacking, some MOB can synthesize a soluble pro- sess 2 mol of Ca per mole of tetramer. Approximately 50% of the tein for methane oxidation that contains Fe instead of Cu (soluble CHOH produced by MDH/Mxa is assimilated into biomass, and methane monooxygenase,sMMO). Subsequent enzymatic steps in the other half is further oxidized to formate (CHOOH) and then aerobic methanotrophy rely primarily on Fe, with lesser amounts to CO2 to generate reducing equivalents (Hanson and Hanson, of Ca, Mo, and possibly Zn (Figure 2). Below is a general overview 1996; Trotsenko and Murrell, 2008; Chistoserdova, 2011). This of the metal content of enzymes in methanotrophy. For a more step is mediated by either a membrane-bound cytochrome-linked extensive discussion of the of aerobic methanotro- formaldehyde dehydrogenase (DL-FalDH) when cells are grown phy, the reader is referred to other reviews (Hanson and Hanson, under high Cu conditions or a soluble NAD(P)+-linked enzyme 1996; Hakemian and Rosenzweig, 2007; Trotsenko and Murrell, (N-FalDH) when cells are grown under low Cu conditions (Zahn 2008; Semrau et al., 2010; Chistoserdova, 2011). et al., 2001). Neither protein contains metal cofactors, but DL- The first step of aerobic methanotrophy, the oxidation of CH4 FalDH does require cytochrome bc1 as an electron acceptor (Zahn to CH3OH, is catalyzed by methane monooxygenase (MMO). All et al., 2001). The final step in aerobic methane oxidation is formate + aerobic methanotrophs (with the exception of facultative methan- oxidation to CO2, catalyzed by NAD –formate dehydrogenase otrophic species in the genus Methylocella) are capable of express- (Fdh). The only characterized Fdh protein in MOB (from M. ing pMMO (Hanson and Hanson, 1996). The metal content of trichosporium OB3b) contains four FexSx clusters and one molyb- pMMO has been a matter of debate for many years, namely dopterincofactor(Jollie and Lipscomb, 1991; Figure 2). Multiple regarding whether the active site coordinates Fe or Cu for func- Fdh enzymes are present in M. capsulatus Bath genome (Ward tion (Bollinger, 2010). Crystal structures and experiments have et al., 2004) and it is possible that one or more of them contains provided strong evidence that the active site is a two Cu cen- W in place of Mo, similar to Fdh proteins from methylotrophs ter in the pMMO-B subunit (Lieberman and Rosenzweig, 2005; (Laukel et al., 2003). Balasubramanian and Rosenzweig, 2007; Hakemian et al., 2008; Balasubramanian et al., 2010). An additional mono-nuclear Cu Intra-aerobic methane oxidation coupled to denitrification. atom is present in one of crystal structures (from Methylococcus The genome of the denitrifying bacterium M. oxyfera, coupled capsulatus Bath) but not in Methylosinus trichosporium OB3b and to proteomic, transcriptomic, and stable isotope labeling exper- is not required for CH4 oxidation (Balasubramanian et al., 2010). iments, have revealed that this organism also contains pMMO Crystal structures predict coordination of another metal ion (Zn in but uses a different pathway for methane oxidation than other M. capsulatus Bath and Cu in M. trichosporium OB3b), positioned MOB. Instead of obtaining O2 from the environment, M. oxyfera

Frontiers in Microbiology | Microbiological Chemistry February 2012 | Volume 3 | Article 61 | 8 Glass and Orphan Trace metals in microbial enzymes

FIGURE 2 | Metal content of the enzymes involved in aerobic Fe–nitrite reductase; NOD, NO dismutase (putative enzyme). Putative methanotrophy at high or low Cu, and intra-aerobic methane intra-aerobic methane oxidation coupled with denitrification pathway is oxidation coupled with denitrification. Each circle represents one adapted from Wu et al. (2011). For simplicity, only metalloenzymes are metal atom. Abbreviations: Cyt, cytochrome; DL-FalDH, dye-linked labeled. Question marks mean that the metal content of the enzyme formaldehyde dehydrogenase; FDH, formate dehydrogenase; MDH, is not well known, and for the case of NOD, that the enzyme is methanol dehydrogenase; pMMO, Cu-particulate methane unknown. See the Section “Metalloenzymes in Aerobic monooxygenase; sMMO, soluble methane monooxygenase; NirS, Methanotrophy” for more details.

generates its own supply of O2 internally from NO. Therefore, Aerobic Methanotrophy”; Ettwig et al., 2010; Wu et al., 2011). this metabolism has been called intra-aerobic methane oxida- Instead of using FalDH for oxidation of CH2O to CHOOH, tion coupled to denitrification (Ettwig et al., 2010; Wu et al., M. oxyfera contains several genes encoding enzymes responsi- 2011). In the proposed pathway, an Fe-based cytochrome cd1 ble for (H4MPT)-dependent C1 transfer nitrite reductase [cd1NIR or NirS; see the Section “Nitrate/Nitrite reactions (Ettwig et al., 2010; Wu et al., 2011). The enzymes ReductiontoN2O/N2 Pathway (Denitrification)”], likely con- involved in these reactions are not known to contain metals taining four Fe atoms (Fülöp et al., 1995), is used to reduce (Vorholt, 2002). In the last step, CHOOH is oxidized to CO2 − NO2 to NO (Ettwig et al., 2010; Wu et al., 2011). In con- by formate dehydrogenase [Fdh; see the Section “Nitrate/Nitrite trast to classical denitrification, where NO is reduced to N2O ReductiontoN2O/N2 Pathway (Denitrification)”]. Carbon assim- and then N2, M. oxyfera disproportionates two molecules of ilation in M. oxyfera likely proceeds via CO2 fixation with the NO to O2 and N2 (Figure 2). The NO dismutase enzyme Calvin–Benson–Bassham cycle instead of the serine and ribu- that performs this reaction is currently unknown. The O2 gen- lose monophosphate (RuMP) pathways used by other MOB erated by this uncharacterized NO dismutase is then used to (Ettwig et al., 2010; Wu et al., 2011). This pathway remains to oxidize CH4 to CH3OH using pMMO, followed by oxidation be confirmed biochemically, but likely requires abundant Cu for of CH3OH to CH2O by MDH/Mxa (see the Section “Classical pMMO.

www.frontiersin.org February 2012 | Volume 3 | Article 61 | 9 Glass and Orphan Trace metals in microbial enzymes

Physiological studies of metal requirements for aerobic Low Cu minerals were dissolved more quickly by methanobactin methanotrophy than high Cu minerals (Kulczycki et al., 2007). Similarly, increas- Pure cultures. Studies of the physiological importance of metals ing concentrations of surface sites on minerals lead to decreased for MOB have focused largely on Cu. Addition of Cu to the growth Cu bioavailability to M. trichosporium OB3b in common soil min- media in strains encoding both the particulate and the soluble erals (Morton et al., 2000). Therefore, particulate Cu content and forms of MMO fundamentally changes methanotroph physiology sediment or soil mineral may display a more robust by triggering the“Cu switch,”turning on the expression of pMMO correlation with methanotrophic activity than dissolved Cu levels (Stanley et al., 1983; Choi et al., 2003). Further increases in Cu in aquatic systems or pore waters (Knapp et al., 2007; Fru, 2011), concentration can lead to up to 55-fold higher pMMO expres- although this remains to be tested in natural systems. sion, with optimal pMMO activity occurring at 60 μMCuforM. capsulatus Bath (Choi et al., 2003; Semrau et al., 2010). Cellu- ANAEROBIC METHANOTROPHY lar Cu contents in laboratory cultures of aerobic methanotrophs The process of anaerobic methane oxidation (AOM) has been amended with high Cu in the growth media can be extremely high, known for over 30 years based on geochemical data (Martens and up to ∼250 nmol Cu/mg protein (Choi et al., 2003). Berner, 1977), but the identity of the microorganisms carrying In order to maintain Cu homeostasis and protect against out this metabolism remained elusive until the early 2000s due the potentially toxic effects of high levels of Cu, some aerobic to the extreme difficulty of culturing AOM microbes. Combined methanotrophs express the Cu-chelating “chalkophore” molecule molecular and stable isotope studies revealed that archaeal groups methanobactin (Kim et al., 2004; Balasubramanian and Rosen- (anaerobic methanotrophic archaea, abbreviated “ANME”) were zweig, 2008). Methanobactin is a 1217-Da molecule that binds responsible for AOM activity in CH4 seep sediments through one atom of Cu(I) with high affinity (Kim et al., 2004; Choi syntrophic associations with sulfate-reducing bacteria (Hinrichs et al., 2006) and accumulates in the growth media when Cu is et al., 1999; Boetius et al., 2000; Orphan et al., 2001, 2002). <0.7 μM(DiSpirito et al., 1998). Cu-loaded methanobactin is Described ANME lineages currently belong to three distinct phy- actively transported into methanotrophic cells when Cu concen- logenetic groups, with members of the ANME-1 forming a novel trations rise between 0.7 and 1 μM(Semrau et al., 2010; Balasub- clade between the Methanomicrobiales and Methanosarcinales ramanian et al., 2011). Furthermore, it has recently been shown orders, and groups ANME-2 and ANME-3 belonging to the that M. capsulatus Bath contains numerous proteins on its surface Methanosarcinales. Together, these methanotrophic lineages are that respond to changing Cu concentrations in growth medium, believed to be responsible for the oxidation of ∼80% of the including abundant Fe-containing cytochrome c peroxidases and methane naturally seeping upward from marine sediments, or multi--type cytochromes (Karlsen et al., 2011). It is per- ∼7–25% of total global methane (Reeburgh, 2007). Although haps interesting to note that abundant extracellular is evident ANME have not been cultured, metagenomic, and environmen- on other methanotrophs in the genera Crenothrix and Clonothrix tal proteomic studies have provided insight into their metabolic (Kolk, 1938; Vigliotta et al., 2007), although separate pathways and strategies for AOM (Hallam et al., 2003, 2004; Krüger et al., 2003; functional roles may be at play in these cases. Meyerdiercks et al., 2005; Pernthaler et al., 2008).

Environmental studies. Environmental studies on Cu availability Metalloenzymes in anaerobic methanotrophy to MOB are lacking, which is striking considering that Cu has been Evidence is accumulating that ANME anaerobically oxidize known to be involved in methanotrophy for many years. Knapp methane by running the methanogenesis pathway in reverse et al. (2007) suggest that the reason that correlations between envi- (Scheller et al., 2010), as was predicted based on the close phyloge- ronmental dissolved Cu (which in oxic settings is <1 μM; Table 3) netic relationship of ANME to methanogens in the orders Metha- and methanotrophic activity have not been noted is because some nomicrobiales and Methanosarcinales (Knittel and Boetius, 2009). MOB can access mineral- and organic-bound Cu using small Cu- The discovery of gene homologs to mcrA in ANME provided the binding peptides, most notably methanobactin. In the absence first clues that these microbes were using reverse methanogene- of dissolved Cu, M. trichosporium OB3b acquired sufficient Cu sis to oxidize CH4 (Hallam et al., 2003). High concentrations of from Cu-containing minerals to express pMMO, but only when the Ni-containing and a slightly modified variant of methanobactin was present (Knapp et al., 2007). CH4 oxidation F430 (Mayr et al., 2008) were found in microbial mats performing rates were highest at ∼100 ppm mineral Cu (Kulczycki et al.,2011). AOM in the Black Sea (Krüger et al., 2003), adding further support

Table 3 | Compiled dissolved trace metal concentrations in representative oxic waters.

Sample site Fe Ni Co Cu Zn Mo Reference

Seawater <0.001 0.002–0.01 <0.001 <0.001–0.005 <0.001–0.009 0.1 Bruland (1980), Collier (1985), Johnson et al. (1997) Soil pore water 1–20 0.1–0.5 0.1–2 Kinniburgh and Miles (1983) Oxic lake water 0.01–10 <0.001–0.03 0.01–0.8 0.02–0.6 0.004–0.3 Wetzel (2001)

All concentrations are in units of micromolar (μM).

Frontiers in Microbiology | Microbiological Chemistry February 2012 | Volume 3 | Article 61 | 10 Glass and Orphan Trace metals in microbial enzymes

for the model. Immunolocalization and biochemical studies have occur at levels lower than their growth optima (Table 2), assuming provided clear evidence that Mcr catalyzes the first step of AOM in that their metal requirements are similar to those of methanogens. ANME (Heller et al., 2008; Scheller et al., 2010) and metagenomic For a full review of the biochemistry of AOM, readers are referred studies of ANME communities identified the presence of genes to Thauer and Shima (2008). encoding almost all of the other methanogenic enzymes with the exception of the Ni–Fe hydrogenases Eha/Ehb and two of the three SUMMARY subunits of the Ni–Fe hydrogenase Mvh (Hallam et al., 2004; Mey- Extensive research has been focused on the importance of Cu erdiercks et al., 2005; Thauer, 2011). Therefore, it is reasonable to for particulate methane monooxygenase, resulting in a detailed predict that metal requirements for the enzymes involved in AOM understanding of physiological and biochemical aspects of Cu are similar to those used for methanogenesis, involving high lev- in pure cultures of MOB. However, few studies have attempted els of Fe, Ni, Co, and additional Mo/W and Zn (see the Section to relate Cu bioavailability, particularly in mineral form, to rates “Metalloenzymes in Methanogenesis”), but possibly with slightly of aerobic CH4 oxidation in the environment. Furthermore, few lower Fe and Ni requirements due to the absence of two of the studies have investigated additional metal requirements, particu- Ni–Fe hydrogenases involved in methanogenesis. The finding that larly Fe, but also small amounts of Mo and Zn, for aerobic and ANME can also fix N2 (Dekas et al., 2009) suggests that they may intra-aerobic methanotrophy, although clearly Fe in particular have additional Fe and Mo/V requirements.ANME occur in anoxic is important for these metabolisms (Figure 2) and cell surface and sulfidic environments where Ni, Co, Mo, and possibly Fe likely Cu acquisition mechanisms (Karlsen et al., 2011). In contrast to

FIGURE 3 | Metal content of three pathways of N2O production: NXOR, nitroxyl oxidoreductase (putative enzyme). Putative archaeal classical denitrification, bacterial nitrifier denitrification, and archaeal ammonia oxidation pathway is adapted from Walker et al. (2010).For ammonia oxidation. Each circle represents one metal atom. Parentheses simplicity, only metalloenzymes are labeled. Question marks depict show varying metal content of a given enzyme. Abbreviations: AMO, unknown or unconfirmed enzymes. Enzymes colored a solid purple ammonia monooxygenase; HAO, hydroxylamine oxidoreductase; Nar, indicate that an unknown amount of Cu is present. See the Section dissimilatory nitrate reductase; NirS, Fe–nitrite reductase; NirS, Cu–nitrite “Metalloenzymes Involved in the Production and Consumption of Nitrous reductase; cNOR, nitric oxide reductase; Nos, nitrous oxide reductase; Oxide” for more details.

www.frontiersin.org February 2012 | Volume 3 | Article 61 | 11 Glass and Orphan Trace metals in microbial enzymes

aerobic methanotrophy, essentially nothing is known about the although such alternative nitrate reductases have so far only been metal requirements for AOM besides the need for large amounts found in metal-reducing bacteria (Antipov et al., 1998, 2005). of Ni in cofactor F430 (Krüger et al., 2003). Presumably, metal The next step in denitrification, nitrite reduction to nitric oxide requirements for reverse methanogenesis by ANME are similar to (NO), is catalyzed by either a cytochrome cd1 (four Fe)-containing those of the canonical seven-step methanogenic pathway, but this nitrite reductase (abbreviated cd1NIR or NirS; Fülöp et al., 1995) hypothesis remains to be tested. or a Cu-containing nitrite reductase (abbreviated CuNIR or NirK) that possesses either 6 or 24 Cu atoms depending on its subunit content (Godden et al., 1991; Nojiri et al., 2007; Figure 3). NITROUS OXIDE PRODUCTION AND CONSUMPTION NO reduction to N O occurs almost instantaneously after A diverse range of organisms using several different pathways 2 its formation due to the of NO. In denitrifying bacte- are known to produce N O. Whereas CH production is only 2 4 ria, NO reductases (NORs) are members of the heme/copper known to occur within the archaea, N Oproductionismore 2 cytochrome oxidase enzyme superfamily that substitute Fe in place widespread. Pathways for biological N O production include dis- 2 of Cu at their active site (Hendriks et al., 2000). Other enzymes similatory nitrate/nitrite reduction (e.g., denitrification), nitrifier in this family cannot efficiently reduce NO, suggesting that the denitrification (Wrage et al., 2001), hydroxylamine oxidation, and replacement of Cu with Fe is important for the enzyme mecha- NO detoxification (also known as the “nitrosative stress” path- x nism. The most common type of NOR in denitrifying bacteria way; Stein, 2011). N O is also produced by MOB (Sutka et al., 2 is NorBC (or cNOR), a cytochrome bc complex that contains 2006; Campbell et al., 2011) and enrichment cultures of ammonia Fe in the form of two hemes and one non-heme Fe and uses oxidizing archaea (AOA; Jung et al.,2011; Santoro et al.,2011). Bac- cytochrome c as an electron donor (Hendriks et al., 2000; Zumft, terial denitrification and nitrifier denitrification together account 2005; Figure 3). The nitrosative stress pathway uses a different NO for ∼70% of the global N O flux (IPCC, 2007), although recent 2 reductase called qNOR, which contains only the NorB subunit data suggest that AOA may also be an important source of N O 2 and accepts electrons from quinols (Hendriks et al., 2000; Zumft, in marine and terrestrial systems (Jung et al., 2011; Santoro et al., 2005). Two additional Fe-containing NOR enzymes, the flavohe- 2011). For an in-depth review of the methods used to identify moglobin Hmp and the flavorubredoxin NorVW, are important N O-producing bacteria by their key enzymes, readers are referred 2 for relieving nitrosative stress under anoxic conditions by NO to Stein (2011). reductiontoN O(Poole and Hughes, 2000; Gardner et al., 2003). Metalloenzymes involved in N O production primarily con- 2 2 Fungal NORs are members of the cytochrome P-460 family and tain Fe and/or Cu, with additional Mo needed for the dis- also contain only Fe (Hendriks et al., 2000). similatory nitrate reduction pathway to N O and/or N and 2 2 After its production, N O can either escape to the atmosphere possibly additional Zn in nitrifier denitrification. A schematic 2 or be further reduced to N by nitrous oxide reductase (Nos). of the metal content of proteins involved in N O production 2 2 Nos is a Cu-rich enzyme, binding 12 atoms of Cu per homodimer and consumption is shown in Figure 3. Below we review bio- (Brown et al., 2000; Figure 3). Low pH and/or trace amounts of O chemical and physiological studies of the metal requirements 2 tend to increase the ratio of N O:N released because Nos is inhib- for N O production through the standard and nitrifier den- 2 2 2 ited at low pH and is more sensitive to O than other reductases itrification pathways, and touch on new discoveries of met- 2 in denitrification (Knowles, 1982). alloenzymes putatively involved in archaeal ammonia oxida- tion. Metalloenzymes involved in the nitrosative stress path- Bacterial ammonia oxidation to N O (nitrifier denitrification and way are mentioned briefly, but are outside the scope of this 2 hydroxylamine oxidation) review. Autotrophic ammonia oxidizing Bacteria (AOB; Shaw et al., 2006) and MOB (Yoshinari, 1985; Ren et al., 2000) are capable of aero- METALLOENZYMES INVOLVED IN THE PRODUCTION AND bic production of N2O during the process of ammonia oxidation → → − → −) CONSUMPTION OF NITROUS OXIDE (NH3 NH2OH NO2 NO3 .N2Oisproducedfrom Nitrate/nitrite reduction to N2O/N2 pathway (denitrification) two offshoots of the nitrification pathway: nitrifier denitrification Nitrous oxide is a gaseous intermediate in the process of deni- (Ritchie and Nicholas, 1972; Poth and Focht, 1985; Wrage et al., trification, the anaerobic microbial respiratory process whereby 2001; Shaw et al., 2006) and hydroxylamine oxidation (Cantera − − → − → → − NO3 isreducedtoN2 in the sequence: NO3 NO2 NO and Stein, 2007b; Stein, 2011). In nitrifier denitrification, NO2 is N2O → N2. The process is leaky and therefore N2O is almost reduced to NO, and then N2OorN2, instead of being oxidized − always found in trace abundances in environments where den- to NO . In hydroxylamine oxidation, NH2OH is oxidized to NO, − 3 itrification occurs (Rogers and Whitman, 1991). Since NO is which is then reduced to N2O. Little is known about the controls − 3 typically present at higher abundances than NO2 in natural envi- on N2O:N2 ratios for both of these processes. ronments, denitrification typically starts with dissimilatory nitrate In the first step of nitrifier denitrification and hydroxy- reduction. The most well-studied dissimilatory nitrate reductase lamine oxidation, ammonia (NH3) is oxidized to hydroxylamine is the membrane-bound NarGHI complex, which contains two b- (NH2OH), catalyzed by the enzyme ammonia monooxygenase type cytochromes (each binding one Fe atom), one Fe3S4 cluster, (AMO). Although the crystal structure of AMO has not been four Fe4S4 clusters, and a molybdopterin (Mo) active site (Bertero solved, AMO shares high similarity to pMMO of aerobic methan- et al., 2003; Jormakka et al., 2004; Figure 3). It is possible for dis- otrophs (Holmes et al., 1995; Arp and Stein, 2003 and references similatory nitrate reduction to occur with Fe or V in place of Mo, therein), suggesting that it also contains three Cu atoms per

Frontiers in Microbiology | Microbiological Chemistry February 2012 | Volume 3 | Article 61 | 12 Glass and Orphan Trace metals in microbial enzymes

monomer and possibly additional Zn and Fe (see the Section PHYSIOLOGICAL STUDIES “Metalloenzymes in Aerobic Methanotrophy”; Figure 3). A Cu As reviewed in the Section “Metalloenzymes Involved in the Pro- requirement for AMO is supported by experiments showing that duction and Consumption of Nitrous Oxide,”the metal content of Cu addition activates AMO (Ensign et al., 1993). enzymes involved in N2O production and consumption is com- The next step of nitrifier denitrification, NH2OH oxidation posed primarily of Fe and Cu (Figure 3). The influence of Cu on − to NO2 , is catalyzed by the enzyme hydroxylamine oxidoreduc- N2O processing by denitrifying bacteria has been the subject of tase (HAO), which contains 24 Fe atoms in c-type cytochromes a number of pure culture and environmental investigations. The (Igarashi et al., 1997; Figure 3). Orthologs of the copper- influence of Fe availability on growth of AOB and denitrifiers is containing NirK enzymes are typically used for the reduction less studied, and very little is known about the importance of Fe − of NO2 to NO (Casciotti and Ward, 2001; Cantera and Stein, for N2O processing in these microbes. Research is also lacking on 2007a,b). NO, a toxic intermediate, is then reduced to N2O the influence of Fe and Cu availability on N2O processing during by an unknown enzyme, likely either NorBC (Beaumont et al., nitrifier denitrification and in AOA enrichment cultures. These 2004; Casciotti and Ward, 2005), the tetraheme cytochrome c554 pathways are of particular interest due to the likelihood of high (Upadhyay et al., 2006), or NorS (Stein, 2011). In hydroxy- Cu requirements for their first step (AMO), whereas N2O can be lamine oxidation, NH2OH is oxidized by HAO to NO instead produced (but not consumed) without Cu by denitrifiers. − of NO2 (Figure 3).ReductionofNOtoN2O is then catalyzed by cytochrome c554 or NorS as with nitrifier denitrification (Stein, Pure cultures 2011). The importance of Cu for the consumption of N2O by denitrify- Although N2 has been shown to be produced by the nitri- ing bacteria has been clearly demonstrated by laboratory studies fier denitrification pathway (Poth, 1986), nos genes have not (Iwasaki and Terai, 1982; Matsubara et al., 1982; Granger and been found in the genomes of bacterial nitrifiers (Stein et al., Ward, 2003; Twining et al., 2007). These studies showed that 2007) so it is unclear how N2 is formed during nitrifier N2O accumulates when denitrifying bacteria are grown without denitrification. added Cu, whereas complete denitrification to N2 occurs when Cu is present above a certain threshold. In early studies with Archaeal ammonia oxidation Pseudomonas perfectomarinus and two Alcaligenes strains, back- Archaeal nitrification was first reported in 2005 (Könneke et al., ground Cu contamination in growth medium was not quantified 2005) and archaeal enrichment cultures have recently been shown and high levels of Cu were added in the replete condition (1–6 μM; to produce N2O(Jung et al.,2011; Santoro et al.,2011). For a review Iwasaki and Terai, 1982; Matsubara et al., 1982). In follow-up of AOA, see Hatzenpichler (in preparation). The genomes of AOA studies with Paracoccus denitrificans and Pseudomonas denitrifi- Cenarchaeum symbiosum and Nitrosopumilus maritimus suggest cans, trace metal-clean growth protocols were utilized and total that AOA have higher Cu requirements than AOB (Hallam et al., dissolved Cu background contamination was measured to be 0.3– 2006; Walker et al., 2010). C. symbiosum and N. maritimus contain 0.5 nM (Granger and Ward, 2003; Twining et al., 2007). At less amo genes with conserved motifs that bind the metal centers in than 1 nM total dissolved Cu, N2O accumulated; above 1 nM, it M. capsulatus Bath, suggesting that archaeal AMO also contains was consumed (Granger and Ward, 2003; Twining et al., 2007). abundant Cu (Hallam et al., 2006; Walker et al., 2010). Both AOA Enzyme activity of nitrous oxide reductase (Nos) was below the genomes lack genes to encode a bacterial-type HAO complex. AOA detection limit in denitrifiers grown at 0.3 nM total dissolved Cu, may use a Cu-based electron-transfer system instead of the Fe- suggesting that the reason for build-up of N2O was Cu limitation based system used by AOB, as implied by the abundance of genes of Nos (Granger and Ward, 2003). Supplementation of 10 nM Cu encoding copper-containing proteins, including numerous multi- to Cu-limited cultures caused increased growth rate and complete copper oxidases and small Cu-binding plastocyanin-like domains consumption of N2O(Granger and Ward, 2003). These labora- (Hallam et al.,2006;Walker et al.,2010). The N. maritimus genome tory studies led to the hypothesis that low Cu concentrations in contains nirK genes (Walker et al., 2010) and AOA nirK homologs natural aquatic ecosystems may promote N2O accumulation (see have been found to be widespread in environmental samples (Bar- the Section “Environmental Studies”). tossek et al., 2010). Walker et al. (2010) propose a model for the Metal requirements for AOB have not been studied as exten- AOA nitrification pathway whereby the products of AMO (either sively as those for denitrifiers, but preliminary studies support a NH2OH, or alternatively the highly reactive intermediate nitroxyl, high importance of Fe for nitrification. An early study of nitrify- HNO) are oxidized to nitrous acid (HNO2) by a Cu-containing ing bacterial cultures showed that addition of Fe alone produced − protein, either a Cu-HAO or a multi-copper oxidase acting as a a 56% increase in NO2 production whereas addition of Cu alone nitroxyl oxidoreductase (NXOR; Figure 3). Following these reac- produced a 17% increase (Lees and Meiklejohn, 1948). The few − tions, NO2 is reduced to NO by NirK, with plastocyanin acting as subsequent studies of metal requirements for AOB have focused an electron donor. Future studies are needed to test whether pure on Fe requirements (Meiklejohn, 1953; Wei et al., 2006). Both cultures of AOA can produce N2O or whether an N intermediate AMO and HAO activity were reduced in Nitrosomonas europaea such as NO is produced by AOA and reduced to N2Obybacterial cultures grown at 0.2 vs. 10 μMFe(Wei et al., 2006). Lowered partners. If AOA can indeed produce N2O as supported by iso- HAO activity is expected due to the high heme content of HAO in topic studies (Santoro et al., 2011), future biochemical studies are AOB cells (Whittaker et al., 2000). AMO activity dependence on required to determine what pathway is used, and whether they can Fe suggests that Fe is a component of AMO or that Fe is present consume it. in the electron donor to AMO. The Fe content of AMO is difficult

www.frontiersin.org February 2012 | Volume 3 | Article 61 | 13 Glass and Orphan Trace metals in microbial enzymes

to assess due to the lack of in vitro protein preparations. It is also present in methanogenic enzymes, but not in enzymes involved possible that AOB scavenge/co-opt Fe-loaded siderophores pro- in aerobic methanotrophy and N2O cycling. In contrast, Cu is duced by other bacteria; if supplied with exogenous siderophores, not used in methanogenesis, but is very important for aerobic N. europaea expresses genes for siderophore transporters and fer- methanotrophy and N2O cycling. Small amounts of Mo and Zn ric transporters when Fe is limiting (Wei et al., 2006; Stein et al., are common in most pathways. If ongoing biochemical studies 2007). Lastly, the fact that AOB cells are bright red in color further confirm that anaerobic methanotrophs use the reverse methano- supports the major requirement for Fe in their metabolism. genesis pathway to oxidize methane, they likely require the same suite of metals as methanogens: Fe, Co, Ni, Zn, and Mo/W. Environmental studies How do metal requirements for CH4 and N2O cycling compare Several recent field studies have suggested that Cu limitation with other ecologically important microbes? The metal stoichiom- prohibits N2O consumption. Cu addition stimulated N2Ocon- etry of 15 marine eukaryotic phytoplankton showed an average sumption in Linsley Pond, a temperate lake ecosystem with 3 nM trace metal stoichiometry of Fe1Mn0.53Zn0.08Cu0.05Co0.03Mo0.005 Cu (Twining et al., 2007). Similarly, the ratio of N2O/N2 decreased (Ho et al., 2003; Barton et al., 2007). High Mn requirements as water-extractable Cu concentration increased in freshwater sed- are likely reflective of the oxygen-generating Mn-containing pho- iments (Jacinthe and Tedesco, 2009). While scarce Cu can limit tosystem II complex. The elemental stoichiometry of CH4 and −1 denitrification, excess Cu (above 100 mg kg sediment) in pol- N2O processing microbes is not nearly as well-constrained as luted ecosystems can inhibit the pathway (Sakadevan et al., 1999; that of eukaryotic phytoplankton, but preliminary studies of Magalhães et al., 2011). On the other hand, denitrification in three methanogens suggest that, after Fe, Ni is an extremely impor- major marine oxygen minimum zones does not appear to be Cu- tant metal for methanogenesis and anaerobic methanotrophy. limited: addition of Cu had minimal effect on denitrification rates Very little is known about the metal stoichiometry of aerobic in seawater incubations with background Cu concentrations of and intra-aerobic methanotrophs and N2O-producing microbes, 1–3 nM (Ward et al., 2008). Therefore, it is likely that marine den- although it is predicted that their Cu content is much higher than itrifiers have evolved mechanisms to satisfy their Cu requirements both methanogens and phytoplankton, especially in the case of by taking up Cu that is strongly bound to natural ligands, which archaeal ammonia oxidizers. Metallomics – the comprehensive can bind 99% of the total Cu in temperate lakes (Twining et al., analysis of the entirety of metal-containing species within a cell 2007) and in the ocean (Moffett and Dupont, 2007). (Szpunar, 2005) – may reveal still more unexpected metals or nat- Metal bioavailability varies considerably between freshwater ural products excreted by microbes to alter metal bioavailability. and seawater oxic environments. In general, dissolved Fe and Cu For example, a recent metallomics study of the hyperthermophilic are present at much lower concentrations in seawater than in lakes sulfur-reducing Archeon, Pyrococcus furiosus, revealed 158 unas- and soil pore waters (Table 3). Therefore, N2O production by signed peaks, 75 of which contained metals that marine ammonia oxidizers may be Fe or Cu-limited and/or they the organism was not known to assimilate, such as Pb and U may have evolved strategies to scavenge these metals. (Cvetkovic et al., 2010). Furthermore, it is likely that the metal requirements for each of the pathways reviewed here are amplified SUMMARY by additional metalloenzymes involved in complex metal cofactor In contrast to CH4,N2O can be produced by diverse microorgan- biosynthesis, such is the case for hydrogenases and nitrogenases isms via numerous pathways containing varying amounts of Fe (Rubio and Ludden, 2008; Shepard et al., 2011). and Cu, small amounts of Mo and possibly Zn. To date, only one More studies are needed to address gaps in our knowledge of enzyme has been found to convert N2OtoN2: the Cu-containing microbial trace metal physiology. Ideally these studies will make Nos enzyme found in denitrifiers. The absence of nos genes from use of well-defined media and trace metal-clean conditions, such the genomes of AOA and AOB (Hallam et al., 2006; Stein et al., as Teflon-lined glass bottles for anaerobic studies. Understudied 2007; Walker et al., 2010) suggests either that N2O is their final topics include Zn requirements for methanogenesis, trace metal gaseous product or that they have alternative nitrous oxide reduc- content of ANME enzymes (other than the well-studied Mcr), tases. The ability of some AOB to produce N2 (Poth, 1986) lends the influence of Cu on N2O production by archaeal enrichment support to the presence of alternative reductases. A search for cultures, the biochemistry of Cu-enzymes in archaeal ammo- such alternative enzymes deserves attention, as ammonia oxidiz- nia oxidizers, and strategies for metal-binding ligand production ers are important players in the global N2O budget. Research on (i.e., methanobactin) in greenhouse gas cycling microbes. The the biochemistry of N2O production in AOA enrichment cultures search for alternative N2O-consuming enzymes in ammonia oxi- is essential to understand the exciting findings of recent studies dizers deserves particular attention, as suggested by the ability of suggesting that large amounts of Cu may be involved in AOA some bacterial nitrifiers lacking the Cu-containing nitrous oxide metabolism. reductase enzyme to convert N2OtoN2. In order to better connect the findings from biochemical and COMPARISONS, CONCLUSION, AND FUTURE RESEARCH physiological studies of pure cultures with the larger picture of Trace metals present in microbial enzymes involved in CH4 and global greenhouse gas cycling and environmental metal bioavail- N2O cycling include Fe, Co, Ni, Cu, Zn, Mo, and W. While Fe- ability, more in situ studies are necessary. Preliminary studies containing enzymes are present in almost all pathways we reviewed suggest that rates of methanogenesis in peatlands may be con- (with the possible exception of archaeal ammonia oxidation), trolled by Fe, Ni, and Co availability, since addition of all three other metals differ by metabolism. For instance, Ni, Co, and W are metals stimulated methanogenesis in mineral-poor peats. It is

Frontiers in Microbiology | Microbiological Chemistry February 2012 | Volume 3 | Article 61 | 14 Glass and Orphan Trace metals in microbial enzymes

currently unknown whether one metal was the primary limit- Knoll, 2002; Scott et al., 2008; Dupont et al., 2010). Several such ing growth element or whether multiple metals were co-limiting scenarios have been proposed for metal limitation of microbial (Saito et al., 2008). If co-limitation was occurring, the addition of greenhouse gas cycling: a decline in seawater Ni due to cool- all three elements would result in higher rates of methanogenesis ing of the Earth’s mantle in the late Archean may have limited than any metal added solely. On the other hand, other systems and methanogenesis and contributed to the Great Oxidation Event pathways may be dominantly regulated by the bioavailability of 2.4 billion years ago (Konhauser et al., 2009) while low Cu due only one scarce metal; this seems to be the case for Co(balt) lim- to sulfide scavenging in the Proterozoic ocean could have led to itation of methylotrophic methanogenesis in anaerobic digesters, global warming through the build-up of N2O(Buick, 2007). The Cu limitation of N2O consumption by freshwater denitrifying bac- question of how metal bioavailability influences greenhouse gas teria and possibly Cu limitation of ammonia oxidizing archaea as cycling in ancient and modern ecosystems is just beginning to be suggested by numerous genes encoding Cu-containing proteins in investigated. AOA genomes. Lastly, relationships between metal bioavailability and fluxes of ACKNOWLEDGMENTS greenhouse gases remain largely unexplored, particularly in anaer- The authors acknowledge funding from the National Aeronautics obic ecosystems. High sulfide environments likely pose particular and Space Administration (NASA) Astrobiology Postdoctoral Fel- challenges for microbial metal acquisition due to abiotic sulfide lowship (to Jennifer B. Glass), the NASA Astrobiology Institute scavenging and precipitation of metal sulfides. Of the bioessen- (NNA04CC06A), and the U.S. Department of Energy’s Office of tial metals involved in greenhouse gas cycling, Mo, Fe, and Cu Biological and Environmental Research Early Career Award (to are most susceptible to sulfide scavenging, followed by Co, Ni, Victoria J. Orphan). Thanks to Roland Hatzenpichler, Patricia and Zn (Morse and Luther, 1999). Microbial metal limitation Tavormina, Shawn McGlynn, Anne Dekas, Joshua Steele, Hiroyuki due to sulfide drawdown may have driven major evolutionary Imachi, Lisa Stein and Aubrey Zerkle for helpful comments on episodes and geochemical transitions in Earth history (Anbar and drafts of this manuscript.

REFERENCES Zvyagilskaya, R. A., and L’vov, Bartossek, R., Nicol, G. W., Lanzen, A., Bragazza, L. (2006). Heavy metals Afting, C., Hochheimer, A., and Thauer, N. P. (2005). Characterization of Klenk, H. P.,and Schleper, C. (2010). in bog waters: an alternative way R. (1998). Function of H2-forming molybdenum-free nitrate reductase Homologues of nitrite reductases in to assess atmospheric precipitation methylenetetrahydromethanopterin from haloalkalophilic bacterium ammonia-oxidizing archaea: diver- quality? Glob. Planet. Change 53, dehydrogenase from Methanobac- Halomonas sp strain AGJ 1-3. Bio- sity and genomic context. Environ. 290–298. terium thermoautotrophicum in chemistry 70, 799–803. Microbiol. 12, 1075–1088. Brown, K., Tegoni, M., Prudencio, M., coenzyme F420 reduction with H2. Arp, D. J., and Stein, L. Y. (2003). Basiliko, N., and Yavitt, J. B. (2001). Pereira, A. S., Besson, S., Moura, J. J., Arch. Microbiol. 169, 206–210. Metabolism of inorganic N com- Influence of Ni, Co, Fe, and Na Moura, I., and Cambillau, C. (2000). Alex, L. A., Reeve, J. N., Orme- pounds by ammonia-oxidizing bac- additions on methane production A novel type of catalytic copper clus- Johnson, W. H., and Walsh, C. T. teria. Crit. Rev. Biochem. Mol. Biol. in Sphagnum-dominated Northern ter in nitrous oxide reductase. Nat. (1990). Cloning, sequence deter- 38, 471–495. American peatlands. Biogeochem- Struct. Biol. 7, 191–195. mination, and expression of the Balasubramanian, R., Kenney, G. E., and istry 52, 133–153. Bruland, K. W. (1980). Oceanographic genes encoding the subunits of Rosenzweig,A. C. (2011). Dual path- Baudet, C., Sprott, G. D., and Patel, G. distributions of cadmium, , the nickel-containing 8-hydroxy-5- ways for copper uptake by methan- B. (1988). Adsorption and uptake of nickel, and copper in the North deazaflavin reducing hydrogenase otrophic bacteria. J. Biol. Chem. 286, nickel in Methanothrix concilii. Arch. Pacific. Earth Planet. Sci. Lett. 47, from Methanobacterium thermoau- 37313–37319. Microbiol. 150, 338–342. 176–198. totrophicum delta H. Biochemistry Balasubramanian, R., and Rosen- Beaumont, H. J. E., Van Schooten, Buick, R. (2007). Did the Protero- 29, 7237–7244. zweig, A. C. (2007). Structural and B., Lens, S. I., Westerhoff, H. zoic Canfield Ocean cause a laugh- Anbar, A. D., and Knoll, A. H. (2002). mechanistic insights into methane V., and Van Spanning, R. J. ing gas greenhouse? Geobiology 5, Proterozoic ocean chemistry and oxidation by particulate methane M. (2004). Nitrosomonas europaea 97–100. evolution: a bioinorganic bridge? monooxygenase. Acc. Chem. Res. 40, expresses a nitric oxide reductase Callander, I., and Barford, J. (1983a). Science 297, 1137–1142. 573–580. during nitrification. J. Bacteriol. 186, Precipitation, chelation, and the Anderson, I., Ulrich, L. E., Lupa, B., Balasubramanian, R., and Rosen- 4417–4421. availability of metals as nutri- Susanti, D., Porat, I., Hooper, S. zweig, A. C. (2008). Copper Bertero, M. G., Rothery, R. A., Palak, M., ents in anaerobic digestion. I. D., Lykidis, A., Sieprawska-Lupa, methanobactin: a molecule whose Hou, C., Lim, D., Blasco, F., Weiner, Methodology. Biotechnol. Bioeng. 25, M., Dharmarajan, L., Goltsman, E., time has come. Curr. Opin. Chem. J. H., and Strynadka, N. C. J. (2003). 1947–1957. Lapidus, A., Saunders, E., Han, C., Biol. 12, 245–249. Insights into the respiratory electron Callander, I., and Barford, J. (1983b). Land, M., Lucas, S., Mukhopadhyay, Balasubramanian, R., Smith, S. M., transfer pathway from the structure Precipitation, chelation, and the B., Whitman, W. B., Woese, C., Bris- Rawat, S., Yatsunyk, L. A., Stemm- of nitrate reductase A. Nat. Struct. availability of metals as nutri- tow, J., and Kyrpides, N. (2009). ler, T. L., and Rosenzweig, A. C. Biol. 10, 681–687. ents in anaerobic digestion. II. Genomic characterization of Metha- (2010). Oxidation of methane by a Boetius, A., Ravenschlag, K., Schu- Applications. Biotechnol. Bioeng. 25, nomicrobiales reveals three classes of biological dicopper centre. Nature bert, C. J., Rickert, D., Widdel, F., 1959–1972. methanogens. PLoS ONE 4, e5797. 465, 115–119. Gieseke, A., Amann, R., Jørgensen, Campbell, M. A., Nyerges, G., doi:10.1371/journal.pone.0005797 Barton, L. L., Goulhen, F., Bruschi, M., B. B., Witte, U., and Pfannkuche, O. Kozlowski, J. A., Poret-Peterson, Antipov, A. N., Lyalikova, N. N., Khi- Woodards, N. A., Plunkett, R. M., (2000). A marine microbial consor- A. T., Stein, L. Y., and Klotz, M. jniak, T. V., and L’vov, N. P. (1998). and Rietmeijer, F. J. M. (2007). The tium apparently mediating anaero- G. (2011). Model of the mol- Molybdenum-free nitrate reductases bacterial metallome: composition bic oxidation of methane. Nature ecular basis for hydroxylamine from vanadate-reducing bacteria. and stability with specific reference 407, 623–626. oxidation and nitrous oxide FEBS Lett. 441, 257–260. to the anaerobic bacterium Desul- Bollinger, J. M. (2010). Biochemistry: production in methanotrophic Antipov, A. N., Morozkina, E. V., fovibrio desulfuricans. Biometals 20, getting the metal right. Nature 465, bacteria. FEMS Microbiol. Lett. 322, Sorokin, D. Y., Golubeva, L. I., 291–302. 40–41. 82–89.

www.frontiersin.org February 2012 | Volume 3 | Article 61 | 15 Glass and Orphan Trace metals in microbial enzymes

Cantera, J. J. L., and Stein, L. Y. (2007a). J., Trauger, S. A., Kalisiak, E., Apon, vanadium in seawater. Mar. Chem. water and synthesis of nitric oxide by Molecular diversity of nitrite reduc- J. V., Siuzdak, G., Yannone, S. M., 34, 177–196. cytochrome cd1. Cell 81, 369–377. tase genes (nirK) in nitrifying bacte- Tainer, J. A., and Adams, M. W. W. Ensign, S. A., Hyman, M. R., and Arp, Funk, T., Gu, W., Friedrich, S., Wang, ria. Environ. Microbiol. 9, 765–776. (2010). Microbial metalloproteomes D. J. (1993). In vitro activation H., Gencic, S., Grahame, D. A., Cantera, J. J. L., and Stein, L. Y. are largely uncharacterized. Nature of ammonia monooxygenase from and Cramer, S. P. (2004). Chem- (2007b). Role of nitrite reductase in 466, 779–782. Nitrosomonas europaea by copper. J. ically distinct Ni sites in the A- the ammonia-oxidizing pathway of Daas, P. J. H., Hagen, W. R., Keltjens, J. Bacteriol. 175, 1971–1980. cluster in subunit of the acetyl- Nitrosomonas europaea. Arch. Micro- T., and Vogels, G. D. (1994). Char- Ermler, U., Grabarse, W., Shima, S., CoA decarbonylase/synthase com- biol. 188, 349–354. acterization and determination of Goubeaud, M., and Thauer, R. K. plex from Methanosarcina ther- Casciotti, K. L., and Ward, B. B. (2001). the redox properties of the 2[4Fe- (1997). Crystal structure of methyl- mophila: Ni L-edge absorption and Dissimilatory nitrite reductase 4S] ferredoxin from Methanosarcina coenzyme M reductase: the key X-ray magnetic circular dichroism genes from autotrophic ammonia- barkeri strain MS. FEBS Lett. 356, enzyme of biological methane for- analyses. J. Am. Chem. Soc. 126, oxidizing bacteria. Appl. Environ. 342–344. mation. Science 278, 1457–1462. 88–95. Microbiol. 67, 2213–2221. David,L. A.,and Alm,E. J. (2010). Rapid Ettwig, K. F.,Butler, M. K., Le Paslier, D., Gardner, A. M., Gessner, C. R., and Casciotti, K. L., and Ward, B. B. (2005). evolutionary innovation during an Pelletier, E., Mangenot, S., Kuypers, Gardner, P. R. (2003). Regulation of Phylogenetic analysis of nitric oxide Archaean genetic expansion. Nature M. M. M., Schreiber, F.,Dutilh, B. E., the nitric oxide reduction operon reductase gene homologues from 469, 93–96. Zedelius, J., De Beer, D., Gloerich, J., (norRVW) in Escherichia coli. J. Biol. aerobic ammonia oxidizing bacteria. Dekas, A., Poretsky, R. S., and Orphan, Wessels, H. J., van Alen, T., Luesken, Chem. 278, 10081–10086. FEMS Microbiol. Ecol. 52, 197–205. V. J. (2009). Deep-sea archaea fix F., Wu, M. L., van de Pas-Schoonen, Gartner, P., Ecker, A., Fischer, R., Chien, Y. T., Auerbuch, V., Brabban, and share nitrogen in methane- K. T., Op den Camp, H. J., Janssen- Linder, D., Fuchs, G., and Thauer, A. D., and Zinder, S. H. (2000). consuming microbial consortia. Sci- Megens, E. M., Francoijs, K. J., Stun- R. K. (1993). Purification and Analysis of genes encoding an alter- ence 326, 422–426. nenberg, H., Weissenbach, J., Jetten, properties of N5 methyltetrahy- native nitrogenase in the archaeon Demirel, B., and Scherer, P. (2011). M. S., and Strous M. (2010). Nitrite- dromethanopterin: coenzyme M Methanosarcina barkeri 227. J. Bac- Trace element requirements of agri- driven anaerobic methane oxidation methyltransferase from Metha- teriol. 182, 3247–3253. cultural biogas digesters during bio- by oxygenic bacteria. Nature 464, nobacterium thermoautotrophicum. Chistoserdova, L. (2011). Modularity logical conversion of renewable bio- 543–548. Eur. J. Biochem. 213, 537–545. of methylotrophy, revisited. Environ. mass to methane. Biomass Bioenergy Fathepure, B. Z. (1987). Factors Godden, J. W., Turley, S., Teller, D. C., Microbiol. 13, 2603–2622. 35, 992–998. affecting the methanogenic activ- Adman, E. T., Liu, M. Y., Payne, Chistoserdova, L., Vorholt, J. A., and Deppenmeier, U., Blaut, M., Schmidt, ity of Methanothrix soehngenii W. J., and Legall, J. (1991). The 2.3 Lidstrom, M. E. (2005). A genomic B., and Gottschalk, G. (1992). VNBF. Appl. Environ. Microbiol. 53, angstrom X-ray structure of nitrite view of methane oxidation by aero- Purification and properties of a F420- 2978–2982. reductase from Achromobacter cyclo- bic bacteria and anaerobic archaea. nonreactive, membrane-bound Ferry, J. G. (1999). Enzymology clastes. Science 253, 438–442. Genome Biol. 6, 208–213. hydrogenase from Methanosarcina of one carbon metabolism in Gong, W., Hao, B., Wei, Z., Fer- Choi, D. W., Corbin, J., Do, Y. S., strain Gö1. Arch. Microbiol. 157, methanogenic pathways. FEMS guson, D. J., Tallant, T., Krzycki, Semrau, J. D., Antholine, W. E., 505–511. Microbiol. Rev. 23, 13–38. J. A., and Chan, M. K. (2008). Hargrove, M. S., Pohl, N. L., Boyd, E. Diekert, G., Konheiser, U., Piechulla, K., Ferry, J. G. (2010a). The chemical biol- Structureofthe22Ni-dependent S., Geesey, G., Hartsel, S. C., Shafe, and Thauer, R. K. (1981). Nickel ogy of methanogenesis. Planet. Space CO dehydrogenase component of P. H., McEllistrem, M. T., Kisting, requirement and factor F430 con- Sci. 58, 1775–1783. the Methanosarcina barkeri acetyl- C. J., Campbell, D., Rao, V., de la tent of methanogenic bacteria. J. Ferry, J. G. (2010b). How to make a liv- CoA decarbonylase/synthase com- Mora, A. M., and Dispirito, A. A. Bacteriol. 148, 459–464. ing by exhaling methane. Annu. Rev. plex. Proc. Natl. Acad. Sci. U.S.A. 105, (2006). Spectral, kinetic, and ther- Diekert, G., Weber, B., and Thauer, R. Microbiol. 64, 453–473. 9558–9563. modynamic properties of Cu(I) and K. (1980). Nickel dependence of Florencio, L., Field, J. A., and Lettinga, Gonzalez-Gil, G., Kleerebezem, R., and Cu(II) binding by methanobactin factor F430 content in Methanobac- G. (1994). Importance of Lettinga, G. (1999). Effects of nickel from Methylosinus trichospo- terium thermoautotrophicum. Arch. for individual trophic groups in an and cobalt on kinetics of methanol rium OB3b. Biochemistry 45, Microbiol. 127, 273–277. anaerobic methanol-degrading con- conversion by methanogenic sludge 1442–1453. DiSpirito, A. A., Zahn, J. A., Gra- sortium. Appl. Environ. Microbiol. as assessed by on-line CH4 moni- Choi, D.-W., Kunz, R. C., Boyd, E. S., ham, D. W., Kim, H. J., Larive, C. 60, 227–234. toring. Appl. Environ. Microbiol. 65, Semrau, J. D., Antholine, W. E., Han, K., Derrick, T. S., Cox, C. D., and Florencio, L., Jenicek, P., Field, J. A., 1789–1793. J.-I., Zahn, J. A., Boyd, J. M., De La Taylor, A. (1998). Copper-binding and Lettinga, G. (1993). Effect of Granger, J., and Ward, B. B. (2003). Mora, A. M., and DiSpirito, A. A. compounds from Methylosinus tri- cobalt on the anaerobic degradation Accumulation of nitrogen oxides in (2003). The membrane-associated chosporium OB3b. J. Bacteriol. 180, of methanol. J. Ferment. Bioeng. 75, copper-limited cultures of denitrify- methane monooxygenase (pMMO) 3606–3613. 368–374. ing bacteria. Limnol. Oceanogr. 48, and pMMO-NADH: quinone oxi- Dupont, C. L., Butcher, A., Ruben, R. E., Fox, J. A., Livingston, D. J., Orme- 313–318. doreductase complex from Methylo- Bourne, P. E., and Caetano-Anollés, Johnson, W. H., and Walsh, C. Hagemeier, C. H., Kruer, M., Thauer, coccus capsulatus Bath. J. Bacteriol. G. (2010). History of biological T. (1987). 8-Hydroxy-5-deazaflavin- R. K., Warkentin, E., and Ermler, U. 185, 5755–5764. metal utilization inferred through reducing hydrogenase from Metha- (2006). Insight into the mechanism Collier, R. W. (1985). Molybdenum in phylogenomic analysis of protein nobacterium thermoautotrophicum: of biological methanol activation the northeast Pacific Ocean. Limnol. structures. Proc. Natl. Acad. Sci. 1. Purification and characterization. based on the crystal structure of the Oceanogr. 30, 1351–1354. U.S.A. 107, 10567–10572. Biochemistry 26, 4219–4227. methanol-cobalamin methyltrans- Conrad, R. (1996). Soil microorganisms Dupont, C. L., Yang, S., Palenik, B., Fru, E. C. (2011). Copper biogeo- ferase complex. Proc. Natl. Acad. Sci. as controllers of atmospheric trace and Bourne, P. E. (2006). Modern chemistry: a cornerstone in aero- U.S.A. 103, 18917–18922. gases (H2,CO,CH4, OCS, N2O, and proteomes contain putative imprints bic methanotrophic bacterial ecol- Hakemian, A. S., Kondapalli, K. C., NO). Microbiol. Mol. Biol. Rev. 60, of ancient shifts in trace metal ogy and activity? Geomicrobiol. J. 28, Telser, J., Hoffman, B. M., Stemm- 609–640. geochemistry. Proc. Natl. Acad. Sci. 601–614. ler, T. L., and Rosenzweig, A. C. Cvetkovic,A.,Menon,A. L.,Thorgersen, U.S.A. 103, 17822. Fülöp, V., Moir, J. W. B., Ferguson, S. J., (2008). The metal centers of partic- M. P., Scott, J. W., Poole Ii, F. L., Jen- Emerson, S. R., and Huested, S. S. and Hajdu, J. (1995). The anatomy ulate methane monooxygenase from ney, F. E. Jr., Lancaster, W. A., Praiss- (1991). Ocean anoxia and the con- of a bifunctional enzyme: structural Methylosinus trichosporium OB3b. man, J. L., Shanmukh, S., Vaccaro, B. centrations of molybdenum and basis for reduction of oxygen to Biochemistry 47, 6793–6801.

Frontiers in Microbiology | Microbiological Chemistry February 2012 | Volume 3 | Article 61 | 16 Glass and Orphan Trace metals in microbial enzymes

Hakemian, A. S., and Rosenzweig, Saraste, M. (2000). Nitric oxide dissolved iron concentrations in Knittel, K., and Boetius, A. (2009). A. C. (2007). The biochemistry reductases in bacteria. Biochim. Bio- the world ocean? Mar. Chem. 57, Anaerobic oxidation of methane: of methane oxidation. Annu. Rev. phys. Acta 1459, 266–273. 137–161. progress with an unknown process. Biochem. 76, 223–241. Hinrichs, K.-U., Hayes, J. M., Sylva, Jollie, D., and Lipscomb, J. D. (1991). Annu.Rev.Microbiol.63, 311–334. Hallam, S. J., Girguis, P. R., Pre- S. P., Brewer, P. G., and DeLong, Formate dehydrogenase from Knowles, R. (1982). Denitrification. ston, C. M., Richardson, P. M., E. F. (1999). Methane-consuming Methylosinus trichosporium OB3b. Microbiol. Mol. Biol. Rev. 46, 43–70. and Delong, E. F. (2003). Identifica- archaebacteria in marine sediments. Purification and spectroscopic Kolk, L. A. (1938). A comparison tion of methyl coenzyme M reduc- Nature 398, 802–805. characterization of the cofactors. J. of the filamentous iron organ- tase A (mcrA) genes associated with Ho, T. Y., Quigg, A., Finkel, Z. V., Mil- Biol. Chem. 266, 21853–21863. isms, Clonothrix fusca Roze and methane-oxidizing archaea. Appl. ligan, A. J., Wyman, K., Falkowski, Jones, J., and Stadtman, T. C. (1977). Crenothrix polyspora Cohn. Am. J. Environ. Microbiol. 69, 5483–5491. P. G., and Morel, F. M. M. (2003). Methanococcus vannielii: culture and Bot. 25, 11–17. Hallam, S. J., Mincer, T. J., Schleper, The elemental composition of some effects of selenium and tungsten on Konhauser, K. O., Pecoits, E., Lalonde, S. C., Preston, C. M., Roberts, K., marine phytoplankton. J. Phycol. 39, growth. J. Bacteriol. 130, 1404–1406. V., Papineau, D., Nisbet, E. G., Bar- Richardson, P. M., and DeLong, E. F. 1145–1159. Jormakka, M., Richardson, D., Byrne, ley, M. E., Arndt, N. T., Zahnle, K., (2006). Pathways of carbon assimi- Hoban, D., and van den Berg, L. B., and Iwata, S. (2004). Architecture and Kamber, B. S. (2009). Oceanic lation and ammonia oxidation sug- (1979). Effect of iron on conver- of NarGH reveals a structural clas- nickel depletion and a methanogen gested by environmental genomic sion of acetic acid to methane dur- sification of Mo-bisMGD enzymes. famine before the Great Oxidation analyses of marine Crenarchaeota. ing methanogenic fermentations. J. Structure 12, 95–104. Event. Nature 458, 750–753. PLoS Biol. 4, e95. doi:10.1371/jour- Appl. Microbiol. 47, 153–159. Jung, M.-Y., Park, S.-J., Min, D., Kim, Könneke, M., Bernhard, A. E., De La nal.pbio.0040095 Holmes, A. J., Costello, A., Lid- J.-S., Rijpstra, W. I. C., Sinninghe Torre, J. R., Walker, C. B., Waterbury, Hallam, S. J., Putman, N., Preston, strom, M. E., and Murrell, J. Damsté, J. S., Kim, G.-J., Mad- J. B., and Stahl, D. A. (2005). Iso- C. M., Detter, J. C., Rokhsar, D., C. (1995). Evidence that partici- sen, E. L., and Rhee, S.-K. (2011). lation of an autotrophic ammonia- Richardson, P. M., and DeLong, E. pate methane monooxygenase and Enrichment and characterization of oxidizing marine archaeon. Nature F. (2004). Reverse methanogenesis: ammonia monooxygenase may be an autotrophic ammonia-oxidizing 437, 543–546. testing the hypothesis with envi- evolutionarily related. FEMS Micro- archaeon of mesophilic crenarchaeal Kremling, K. (1983). The behavior of ronmental genomics. Science 305, biol. Lett. 132, 203–208. group I. 1a from an agricultural Zn, Cd, Cu, Ni, Co, Fe, and Mn in 1457–1462. Igarashi, N., Moriyama, H., Fujiwara, T., soil. Appl. Environ. Microbiol. 77, anoxic Baltic waters. Mar. Chem. 13, Hamann, N., Mander, G. J., Shokes, J. Fukumori,Y.,and Tanaka,N. (1997). 8635–8647. 87–108. E., Scott, R. A., Bennati, M., and The 2.8 A structure of hydroxy- Karlsen, O. A., Larsen, O., and Jensen, Krüger, M., Meyerdierks, A., Glöckner, Hedderich, R. (2007). A cysteine- lamine oxidoreductase from a nitri- H. B. (2011). The copper responding F. O., Amann, R., Widdel, F., Kube, rich CCG domain contains a novel fying chemoautotrophic bacterium, surfaceome of Methylococccus cap- M., Reinhardt, R., Kahnt, R., Böcher, [4Fe-4S] cluster binding motif as Nitrosomonas europaea. Nat. Struc. sulatus Bath. FEMS Microbiol. Lett. R., Thauer, R. K., and Shima, S. deduced from studies with subunit Biol. 4, 276–284. 323, 97–104. (2003). A conspicuous nickel pro- B of heterodisulfide reductase from IPCC. (2007). The Physical Science Basis: Kessler, P. S., Mclarnan, J., and Leigh, tein in microbial mats that oxidize Methanothermobacter marburgensis. Contribution of Working Group I to J. A. (1997). Nitrogenase phylogeny methane anaerobically. Nature 426, Biochemistry 46, 12875–12885. the Fourth Assessment Report of the and the molybdenum dependence 878–881. Hanson, R. S., and Hanson, T. E. (1996). Intergovernmental Panel on Climate of nitrogen fixation in Methanococ- Krzycki, J., and Zeikus, J. (1980). Methanotrophic bacteria. Microbiol. Change. New York: Cambridge Uni- cus maripaludis. J. Bacteriol. 179, Quantification of corrinoids in Rev. 60, 439–471. versity Press. 541–543. methanogenic bacteria. Curr. Micro- Haraldsson, C., and Westerlund, S. Iwasaki, H., and Terai, H. (1982). N2 Kida, K., Shigematsu, T., Kijima, J., biol. 3, 243–245. (1988). Trace metals in the water and N2O produced during growth Numaguchi, M., Mochinaga, Y., Kulczycki, E., Fowle, D. A., Kenward, columns of the Black Sea and of denitrifying bacteria in copper- Abe, N., and Morimura, S. (2001). P. A., Leslie, K., Graham, D. W., + + Framvaren Fjord. Mar. Chem. 23, depleted and -supplemented media. Influence of Ni2 and Co2 on and Roberts, J. A. (2011). Stim- 417–424. J. Gen. Appl. Microbiol. 28, 189–193. methanogenic activity and the ulation of methanotroph activity Hedderich, R., Hamann, N., and Jacinthe, P.A., and Tedesco, L. P. (2009). amounts of coenzymes involved in by Cu-substituted borosilicate glass. Bennati, M. (2005). Heterodisul- Impact of elevated copper on the methanogenesis. J. Biosci. Bioeng. 91, Geomicrobiol. J. 28, 1–10. fide reductase from methanogenic rate and gaseous products of deni- 590–595. Kulczycki, E., Fowle, D. A., Knapp, C., archaea: a new catalytic role for an trification in freshwater sediments. Kim, H. J., Graham, D. W., DiSpir- Graham, D. W., and Roberts, J. A. iron-sulfur cluster. Biol. Chem. 386, J. Environ. Qual. 38, 1183–1192. ito, A. A., Alterman, M. A., (2007). Methanobactin-promoted 961–970. Jansen, S., Gonzalez-Gil, G., and van Galeva, N., Larive, C. K., Asun- dissolution of Cu-substituted boro- Heiden,S.,Hedderich,R.,Setzke,E.,and Leeuwen,H. P.(2007). The impact of skis, D., and Sherwood, P. M. silicate glass. Geobiology 5, 251–263. Thauer, R. K. (1994). Purification of Co and Ni speciation on methano- A. (2004). Methanobactin, a Künkel, A., Vaupel, M., Heim, S., a two subunit cytochrome b contain- genesis in sulfidic media – biouptake copper-acquisition compound from Thauer, R. K., and Hedderich, ing heterodisulfide reductase from versus metal dissolution. Enzyme methane-oxidizing bacteria. Science R. (1997). Heterodisulfide reduc- methanol grown Methanosarcina Microb. Technol. 40, 823–830. 305, 1612–1615. tase from methanol grown cells barkeri. Eur. J. Biochem. 221, Jansen, S., Steffen, F., Threels, W. F., and Kinniburgh, D. G., and Miles, D. L. of Methanosarcina barkeri is not a 855–861. van Leeuwen, H. P. (2005). Specia- (1983). Extraction and chemical flavoenzyme. Eur. J. Biochem. 244, Heimann, M. (2011). Atmospheric sci- tion of Co (II) and Ni (II) in anaer- analysis of interstitial water from 226–234. ence: enigma of the recent methane obic bioreactors measured by com- soils and rocks. Environ. Sci. Technol. Laukel, M., Chistoserdova, L., Lidstrom, budget. Nature 476, 157–158. petitive ligand exchange-adsorptive 17, 362–368. M. E., and Vorholt, J. A. (2003). The Heller, C., Hoppert, M., and Reitner, J. stripping voltammetry. Environ. Sci. Knapp, C. W., Fowle, D. A., Kulczy- tungsten-containing formate dehy- (2008). Immunological localization Technol. 39, 9493–9499. cki, E., Roberts, J. A., and Graham, drogenase from Methylobacterium of coenzyme M reductase in anaer- Jarrell, K., and Sprott, G. (1982). D. W. (2007). Methane monooxy- extorquens AM1: purification and obic methane-oxidizing archaea of Nickel transport in Methanobac- genase gene expression mediated properties. Eur. J. Biochem. 270, ANME 1 and ANME 2 type. Geomi- terium bryantii. J. Bacteriol. 151, by methanobactin in the pres- 325–333. crobiol. J. 25, 149–156. 1195–1203. ence of mineral copper sources. Lees, H., and Meiklejohn, J. (1948). Hendriks, J., Oubrie, A., Castresana, J., Johnson, K. S., Gordon, R. M., and Proc. Natl. Acad. Sci. U.S.A. 104, Trace elements and nitrification. Urbani, A., Gemeinhardt, S., and Coale, K. H. (1997). What controls 12040–12045. Nature 161, 398–399.

www.frontiersin.org February 2012 | Volume 3 | Article 61 | 17 Glass and Orphan Trace metals in microbial enzymes

Li, Q., Li, L., Rejtar, T., Lessner, D. the products of nitrite respiration of a hexameric copper-containing nitric oxide by three methan- J., Karger, B. L., and Ferry, J. G. in Pseudomonas perfectomarinus. J. nitrite reductase. Proc. Natl. Acad. otrophic bacteria. Appl. Environ. (2006). Electron transport in the Bacteriol. 149, 816–823. Sci. U.S.A. 104, 4315–4320. Microbiol. 66, 3891–3897. pathway of acetate conversion to Mayr, S., Latkoczy, C., Krüger, M., Gün- Op den Camp, H. J. M., Islam, T., Ritchie, G. A., and Nicholas, D. J. (1972). methane in the marine archaeon ther, D., Shima, S., Thauer, R. K., Stott, M. B., Harhangi, H. R., Hynes, Identification of the sources of Methanosarcina acetivorans. J. Bac- Widdel, F., and Jaun, B. (2008). A., Schouten, S., Jetten, M. S. M., nitrous oxide produced by oxidative teriol. 188, 702–710. Structure of an F430 variant from Birkeland, N.-K., Pol, A., and Dun- and reductive processes in Nitro- Lieberman, R. L., and Rosenzweig, archaea associated with anaerobic field, P. F. (2009). Environmental, somonas europaea. Biochem. J. 126, A. C. (2005). Crystal structure of oxidation of methane. J. Am. Chem. genomic and taxonomic perspec- 1181–1191. a membrane-bound metalloenzyme Soc. 130, 10758–10767. tives on methanotrophicVerrucomi- Rodionov, D. A., Hebbeln, P., Gelfand, that catalyses the biological oxi- Meiklejohn,J. (1953). Iron and the nitri- crobia. Environ. Microbiol. Rep. 1, M. S., and Eitinger, T. (2006). Com- dation of methane. Nature 434, fying bacteria. J. Gen. Microbiol. 8, 293–306. parative and functional genomic 177–182. 58–65. Orphan, V. J., House, C. H., Hin- analysis of prokaryotic nickel and Lin, D., Kakizono, T., Nishio, N., Merkx, M., Kopp, D. A., Sazinsky, M. H., richs, K.-U., McKeegan, K. D., and cobalt uptake transporters: evidence and Nagai, S. (1990). Enhanced Blazyk, J. L., Müller, J., and Lippard, DeLong, E. F. (2001). Methane- for a novel group of ATP-binding cytochrome formation and stimu- S. J. (2001). Dioxygen activation and consuming archaea revealed by cassette transporters. J. Bacteriol. lated methanogenesis rate by the methane hydroxylation by soluble directly coupled isotopic and phy- 188, 317–327. increased ferrous concentrations methane monooxygenase: a tale of logenetic analysis. Science 293, Rogers, J. E., and Whitman, W. B. in Methanosarcina barkeri culture. two and three proteins. Angew. 484–487. (1991). Microbial Production and FEMS Microbiol. Lett. 68, 89–92. Chem. Int. Ed. Engl. 40, 2782–2807. Orphan, V. J., House, C. H., Hin- Consumption of Greenhouse Gases: Lin, D. G., Nishio, N., Mazumder, T. Meyerdiercks, A., Kube, M., Lombardot, richs, K.-U., McKeegan, K. D., and Methane, Nitrogen Oxides, and K., and Nagai, S. (1989). Influ- T., Knittel, K., Bauer, M., Glöckner, DeLong, E. F. (2002). Multiple Halomethanes. Washington: Ameri- + + + ence of Co2 ,Ni2 and Fe2 F. O., Reinhardt, R., and Amann, R. archaeal groups mediate methane can Society for Microbiology. on the production of tetrapyrroles (2005). Insights into the genomes oxidation in anoxic cold seep sedi- Rubio, L. M., and Ludden, P. W. by Methanosarcina barkeri. Appl. of archaea mediating the anaero- ments. Proc. Natl. Acad. Sci. U.S.A. (2008). Biosynthesis of the iron- Microbiol. Biotechnol. 30, 196–200. bic oxidation of methane. Environ. 99, 7663–7668. molybdenum cofactor of nitroge- Lipscomb, J. D. (1994). Biochemistry Microbiol. 7, 1937–1951. Patel, G., Khan, A., and Roth, L. (1978). nase. Annu.Rev.Microbiol.62, of the soluble methane monooxy- Moffett, J. W., and Dupont, C. (2007). Optimum levels of sulphate and iron 93–111. genase. Annu.Rev.Microbiol.48, Cu complexation by organic ligands for the cultivation of pure cultures Saito, M. A., Goepfert, T. J., and Ritt, 371–399. in the sub-arctic NW Pacific and of methanogens in synthetic media. J. T. (2008). Some thoughts on Lobo, A. L., and Zinder, S. H. Bering Sea. Deep Sea Res. Part I J. Appl. Microbiol. 45, 347–356. the concept of colimitation: three (1988). Diazotrophy and nitroge- Oceanogr. Res. Pap. 54, 586–595. Pernthaler, A., Dekas, A. E., Brown, C. definitions and the importance of nase activity in the archaebacterium Montzka, S., Dlugokencky, E., and But- T., Goffredi, S. K., Embaye, T., and bioavailability. Limnol. Oceanogr. 53, Methanosarcina barkeri 227. Appl. ler, J. (2011). Non-CO2 greenhouse Orphan, V. J. (2008). Diverse syn- 276–290. Environ. Microbiol. 54, 1656–1661. gases and climate change. Nature trophic partnerships from deep-sea Sakadevan, K., Zheng, H., and Bavor, Macauley, S. R., Zimmerman, S. A., 476, 43–50. methane vents revealed by direct H. J. (1999). Impact of heavy met- Apolinario, E. E., Evilia, C., Hou, Morse, J. W., and Luther, G. W. (1999). cell capture and metagenomics. als on denitrification in surface wet- Y.-M., Ferry, J. G., and Sowers, K. Chemical influences on trace metal- Proc. Natl. Acad. Sci. U.S.A. 105, land sediments receiving wastewater. R. (2009). The archetype gamma- sulfide interactions in anoxic sed- 7052–7057. Water Sci. Technol. 40, 349–355. class carbonic anhydrase (Cam) con- iments. Geochim. Cosmochim. Acta Poole, R. K., and Hughes, M. N. (2000). Santoro, A. E., Buchwald, C., McIl- tains iron when synthesized in vivo. 63, 3373–3378. New functions for the ancient glo- vin, M. R., and Casciotti, K. L. Biochemistry 48, 817–819. Morton, J. D., Hayes, K. F., and Sem- bin family: bacterial responses to (2011). Isotopic signature of N2O Magalhães, C. M., Machado, A., Matos, rau, J. D. (2000). Bioavailability of nitric oxide and nitrosative stress. produced by marine ammonia- P., and Bordalo, A. A. (2011). Impact chelated and soil-adsorbed copper to Mol. Microbiol. 36, 775–783. oxidizing archaea. Science 333, of copper on the diversity, abun- Methylosinus trichosporium OB3b. Poth, M. (1986). Dinitrogen produc- 1282–1285. dance and transcription of nitrite Environ. Sci. Technol. 34, 4917–4922. tion from nitrite by a Nitrosomonas Schauer, N. L., and Ferry, J. G. (1986). and nitrous oxide reductase genes in Murray, W. D., and van den Berg, isolate. Appl. Environ. Microbiol. 52, Composition of the coenzyme F420- an urban European estuary. FEMS L. (1981). Effects of nickel, cobalt, 957–959. dependent formate dehydrogenase Microbiol. Ecol. 77, 274–284. and molybdenum on performance Poth, M., and Focht, D. D. (1985). from Methanobacterium formicicum. 15 Martens, C. S., and Berner, R. A. (1977). of methanogenic fixed-film reac- N kinetic analysis of N2Opro- J. Bacteriol. 165, 405–411. Interstitial water chemistry of anoxic tors. Appl. Environ. Microbiol. 42, duction by Nitrosomonas europaea: Scheller, S., Goenrich, M., Boecher, R., Long Island Sound sediments. 1. 502–505. an examination of nitrifier denitrifi- Thauer, R. K., and Jaun, B. (2010). Dissolved gases. Limnol. Oceanogr. Murrell, J. C., McDonald, I. R., and cation. Appl. Environ. Microbiol. 49, The key nickel enzyme of methano- 22, 10–25. Gilbert, B. (2000). Regulation of 1134–1141. genesis catalyses the anaerobic oxi- Martin, J. H., and Fitzwater, S. E. (1988). expression of methane monooxyge- Reeburgh, W. S. (2007). Oceanic dation of methane. Nature 465, Iron deficiency limits phytoplank- nases by copper ions. Trends Micro- methane biogeochemistry. Chem. 606–608. ton growth in the north-east Pacific biol. 8, 221–225. Rev. 107, 486–513. Scherer, P. (1988). Vanadium and subarctic. Nature 331, 341–343. Nielsen, A. K., Gerdes, K., and Mur- Reeve, J. N., Beckler, G. S., Cram, molybdenum requirement for the Martinho, M., Choi, D. W., DiSpirito, rell, J. C. (1997). Copper-dependent D. S., Hamilton, P. T., Brown, J. fixation of molecular nitrogen by A. A., Antholine, W. E., Semrau, J. reciprocal transcriptional regulation W., Krzycki, J. A., Kolodziej, A. F., two Methanosarcina strains. Arch. D., and Münck, E. (2007). Möss- of methane monooxygenase genes Alex, L., Orme-Johnson, W. H., and Microbiol. 151, 44–48. bauer studies of the membrane- in Methylococcus capsulatus and Walsh, C. T. (1989). A hydrogenase- Scherer, P., Lippert, H., and Wolff, G. associated methane monooxygenase Methylosinus trichosporium. Mol. linked gene in Methanobacterium (1983). Composition of the major from Methylococcus capsulatus Bath: Microbiol. 25, 399–409. thermoautotrophicum strain delta H elements and trace elements of 10 evidence for a diiron center. J. Am. Nojiri, M., Xie, Y., Inoue, T., Mat- encodes a polyferredoxin. Proc. Natl. methanogenic bacteria determined Chem. Soc. 129, 15783–15785. sumura, H., Kataoka, K., Deligeer, Acad. Sci. U.S.A. 86, 3031–3035. by inductively coupled plasma emis- Matsubara, T., Frunzke, K., and Zumft, Yamaguchi, K., Kai, Y., and Suzuki, Ren, T., Roy, R., and Knowles, R. (2000). sion spectrometry. Biol. Trace Elem. W. (1982). Modulation by copper of S. (2007). Structure and function Production and consumption of Res. 5, 149–163.

Frontiers in Microbiology | Microbiological Chemistry February 2012 | Volume 3 | Article 61 | 18 Glass and Orphan Trace metals in microbial enzymes

Scherer, P., and Sahm, H. (1981). Effect of anaerobic digestion.Water Res. 17, Thauer, R. K. (1998). Biochemistry of J., Hügler, M., Karr, E. A., Kön- of trace elements and on 677–683. methanogenesis: a tribute to Mar- neke, M., Shin, M., Lawton, T. the growth of Methanosarcina Stanley, S. H., Prior, S. D., Leak, D., jory Stephenson. Microbiology 144, J., Lowe, T., Martens-Habbena, W., barkeri. Acta Biotechnol. 1, and Dalton, H. (1983). Copper 2377–2406. Sayavedra-Soto, L. A., Lang, D., Siev- 57–65. stress underlies the fundamen- Thauer, R. K. (2011). Anaerobic oxi- ert, S. M., Rosenzweig, A. C., Man- Schlesinger, W. H. (2009). On the fate of tal change in intracellular loca- dation of methane with sulfate: on ning, G., and Stahl, D. A. (2010). anthropogenic nitrogen. Proc. Natl. tion of methane mono-oxygenase the reversibility of the reactions Nitrosopumilus maritimus genome Acad. Sci. U.S.A. 106, 203–208. in methane-oxidizing organisms: that are catalyzed by enzymes also reveals unique mechanisms for nitri- Schönheit, P.,Moll, J., and Thauer, R. K. studies in batch and continu- involved in methanogenesis from fication and autotrophy in glob- (1979). Nickel, cobalt, and molyb- ous cultures. Biotechnol. Lett. 5, CO2. Curr. Opin. Microbiol. 14, ally distributed marine crenarchaea. denum requirement for growth 487–492. 292–299. Proc. Natl. Acad. Sci. U.S.A. 107, of Methanobacterium thermoau- Stein, L. Y. (2011). Surveying N2O- Thauer, R. K., Kaster, A.-K., Goenrich, 8818–8823. totrophicum. Arch. Microbiol. 123, producing pathways in bacteria. M., Schick, M., Hiromoto, T., and Ward, B. B., Tuit, C. B., Jayakumar, A., 105–107. Meth. Enzymol. 486, 131–152. Shima, S. (2010). Hydrogenases Rich, J. J., Moffett, J., and Naqvi, S. Scott, C., Lyons, T. W., Bekker, A., Shen, Stein, L. Y., Arp, D. J., Berube, P. M., from methanogenic archaea, W. A. (2008). Organic carbon, and Y., Poulton, S. W., Chu, X., and Chain, P. S., Hauser, L., Jetten, M. nickel, a novel cofactor, and H2 not copper, controls denitrification Anbar,A. D. (2008). Tracing the step- S., Klotz, M. G., Larimer, F. W., Nor- storage. Annu. Rev. Biochem. 79, in oxygen minimum zones of the wise oxygenation of the Proterozoic ton, J. M., Op den Camp, H. J. M., 507–536. ocean. Deep Sea Res. Part I Oceanogr. ocean. Nature 452, 456–459. Shin, M., and Wei, X. (2007). Whole Thauer, R. K., Kaster, A.-K., See- Res. Pap. 55, 1672–1683. Semrau, J. D. (2011). Bioreme- genome analysis of the ammonia dorf, H., Buckel, W., and Hed- Ward, N., Larsen, Ø., Sakwa, J., Bruseth, diation via methanotrophy: oxidizing bacterium, Nitrosomonas derich, R. (2008). Methanogenic L., Khouri, H., Durkin, A. S., Dim- overview of recent findings eutropha C91: implications for niche archaea: ecologically relevant differ- itrov, G., Jiang, L., Scanlan, D., Kang, and suggestions for future adaptation. Environ. Microbiol. 9, ences in energy conservation. Nat. K. H., Lewis, M., Nelson, K. E., research. Front. Microbiol. 2:209. 2993–3007. Rev. Microbiol. 6, 579–591. Methé, B., Wu, M., Heidelberg, J. doi:10.3389/fmicb.2011.00209 Sutka, R. L., Ostrom, N. E., Ostrom, Thauer, R. K., and Shima, S. (2008). F., Paulsen, I. T., Fouts, D., Ravel, Semrau, J. D., DiSpirito, A. A., and P. H., Breznak, J. A., Gandhi, H., Methane as fuel for anaerobic J., Tettelin, H., Ren, Q., Read, T., Yoon, S. (2010). Methanotrophs and Pitt, A. J., and Li, F. (2006). Dis- microorganisms. Ann. N. Y. Acad. DeBoy, R. T., Seshadri, R., Salzberg, copper. FEMS Microbiol. Rev. 34, tinguishing nitrous oxide produc- Sci. 1125, 158–170. S. L., Jensen, H. B., Birkeland, N. 496–531. tion from nitrification and denitri- Trotsenko, Y. A., and Murrell, J. C. K., Nelson, W. C., Dodson, R. J., Shaw, L. J., Nicol, G. W., Smith, Z., fication on the basis of isotopomer (2008). Metabolic aspects of aerobic Grindhaug, S. H., Holt, I., Eid- Fear, J., Prosser, J. I., and Baggs, abundances. Appl. Environ. Micro- obligate methanotrophy. Adv. Appl. hammer, I., Jonasen, I., Vanaken, E. M. (2006). Nitrosospira spp. can biol. 72, 638–644. Microbiol. 63, 183–229. S., Utterback, T., Feldblyum, T. V., produce nitrous oxide via a nitri- Szpunar, J. (2005). Advances in Twining, B. S., Mylon, S. E., and Benoit, Fraser, C. M., Lillehaug, J. R., and fier denitrification pathway. Environ. analytical methodology for bio- G. (2007). Potential role of copper Eisen, J. A. (2004). Genomic insights Microbiol. 8, 214–222. inorganic speciation analysis: availability in nitrous oxide accumu- into methanotrophy: the complete Shaw, T. J., Gieskes, J. M., and Jahnke, R. metallomics, metalloproteomics lation in a temperate lake. Limnol. genome sequence of Methylococcus A. (1990). Early diagenesis in differ- and heteroatom- tagged proteomics Oceanogr. 52, 1354–1366. capsulatus (Bath). PLoS Biol. 2, e303. ing depositional environments: the and metabolomics. Analyst 130, Upadhyay, A. K., Hooper, A. B., doi:10.1371/journal.pbio.0020303 response of transition metals in pore 442–465. and Hendrich, M. P. (2006). NO Wei, X., Vajrala, N., Hauser, L., water. Geochim. Cosmochim. Acta 54, Takashima, M., Shimada, K., and reductase activity of the tetraheme Sayavedra-Soto, L. A., and Arp, D. 1233–1246. Speece, R. E. (2011). Minimum cytochrome c554 of Nitrosomonas J. (2006). Iron and phys- Shepard, E. M., Boyd, E. S., Broder- requirements for trace metals europaea. J. Am. Chem. Soc. 128, iological responses to iron stress in ick, J. B., and Peters, J. W. (2011). (iron, nickel, cobalt, and zinc) 4330–4337. Nitrosomonas europaea. Arch. Micro- Biosynthesis of complex iron-sulfur in thermophilic and mesophilic Vigliotta, G., Nutricati, E., Carata, biol. 186, 107–118. enzymes. Curr. Opin. Chem. Biol. 15, methane fermentation from glu- E., Tredici, S. M., De Stefano, M., Wetzel, R. G. (2001). Limnology: Lake 319–327. cose. Water Environ. Res. 83, Pontieri, P., Massardo, D. R., Prati, and River Ecosystems, 3rd Edn. San Shima, S., Pilak, O., Vogt, S., Schick, 339–346. M. V., De Bellis, L., and Alifano, P. Diego: Academic Press. M.,Stagni,M. S.,Meyer-Klaucke,W., Takashima,M.,and Speece,R. E. (1990). (2007). Clonothrix fusca Roze 1896, Whitman, W. B., Ankwanda, E., and Warkentin, E., Thauer, R. K., and Mineral requirements for methane a filamentous, sheathed, methan- Wolfe, R. S. (1982). Nutrition and Ermler, U. (2008). The crystal struc- fermentation. Crit. Rev. Environ. otrophic gamma-proteobacterium. carbon metabolism of Methanococ- ture of [Fe]-hydrogenase reveals the Control 19, 465–479. Appl. Environ. Microbiol. 73, cus voltae. J. Bacteriol. 149, 852–863. geometry of the active site. Science Taylor, G. T., and Pirt, S. J. (1977). 3556–3565. Whitman, W. B., Bowen, T. L., 321, 572–575. Nutrition and factors limiting the Vorholt, J. A. (2002). Cofactor-dep- and Boone, D. R. (2006). The Simianu, M., Murakami, E., Brewer, growth of a methanogenic bacte- endent pathways of formaldehyde methanogenic bacteria. Prokaryotes J. M., and Ragsdale, S. W. (1998). rium (Methanobacterium thermoau- oxidation in methylotrophic 3, 165–207. Purification and properties of the totrophicum). Arch. Microbiol. 113, bacteria. Arch. Microbiol. 178, Whittaker, M., Bergmann, D., Arciero, heme-and iron-sulfur-containing 17–22. 239–249. D., and Hooper, A. B. (2000). Elec- heterodisulfide reductase from Terlesky, K., and Ferry, J. (1988). Vorholt, J. A., Vaupel, M., and Thauer, tron transfer during the oxidation of Methanosarcina thermophila. Purification and characterization of R. K. (1996). A polyferredoxin ammonia by the chemolithotrophic Biochemistry 37, 10027–10039. a ferredoxin from acetate-grown with eight [4Fe-4S] clusters as a bacterium Nitrosomonas europaea. Sowers, K. R., and Ferry, J. G. Methanosarcina thermophila. J. Biol. subunit of molybdenum formyl- Biochim. Biophys. Acta 1459, (1985). Trace metal and Chem. 263, 4080–4082. dehydrogenase from 346–355. requirements of Methanococcoides Tersteegen, A., and Hedderich, R. Methanosarcina barkeri. Eur. J. Wrage, N., Velthof, G. L., van Beu- methylutens grown with trimethy- (1999). Methanobacterium thermo- Biochem. 236, 309–317. sichem, M. L., and Oenema, O. lamine. Arch. Microbiol. 142, autotrophicum encodes two mul- Walker, C., De La Torre, J., Klotz, (2001). Role of nitrifier denitrifi- 148–151. tisubunit membrane-bound [NiFe] M., Urakawa, H., Pinel, N., Arp, cation in the production of nit- Speece, R. E., Parkin, G. F., and Gal- hydrogenases. Eur. J. Biochem. 264, D., Brochier-Armanet, C., Chain, rous oxide. Soil Biol. Biochem. 33, lagher, D. (1983). Nickel stimulation 930–943. P., Chan, P., Gollabgir, A., Hemp, 1723–1732.

www.frontiersin.org February 2012 | Volume 3 | Article 61 | 19 Glass and Orphan Trace metals in microbial enzymes

Wu, M. L., Ettwig, K. F., Jetten, Zandvoort, M. H., Osuna, M. B., Geerts, Zhang, Y., Rodionov, D. A., Gelfand, that could be construed as a potential M. S., Strous, M., Keltjens, J. R., Lettinga, G., and Lens, P. N. M. S., and Gladyshev, V. N. (2009). conflict of interest. T., and van Niftrik, L. (2011). (2002). Effect of nickel depriva- Comparative genomic analyses of A new intra-aerobic metabolism tion on methanol degradation in nickel, cobalt and vitamin B12 Received: 05 December 2011; paper in the nitrite-dependent anaerobic a methanogenic granular sludge utilization. BMC Genomics 10, 1–26. pending published: 21 December 2011; methane-oxidizing bacterium Can- bioreactor. J. Ind. Microbiol. Biotech- doi:10.1186/1471-2164-10-78 accepted: 05 February 2012; published didatus ‘Methylomirabilis oxyfera.’ nol. 29, 268–274. Zirngibl, C., van Dongen, W., Schwörer, online: 21 February 2012. Biochem. Soc. Trans. 39, 243–248. Zandvoort, M. H., van Hullebusch, B., Bünau, R., Richter, M., Klein, Citation: Glass JB and Orphan VJ (2012) Yoshinari, T. (1985). Nitrite and nitrous E. D., Gieteling, J., and Lens, A., and Thauer, R. K. (1992). H2- Trace metal requirements for micro- oxide production by Methylosinus P. N. L. (2006). Granular sludge forming methylenetetrahydromet- bial enzymes involved in the produc- trichosporium. Can. J. Microbiol. 31, in full-scale anaerobic bioreactors: hanopterin dehydrogenase, a novel tion and consumption of methane and 139–144. trace element content and deficien- type of hydrogenase without iron- nitrous oxide. Front. Microbio. 3:61. doi: Zahn, J. A., Bergmann, D. J., Boyd, cies. Enzyme Microb. Technol. 39, sulfur clusters in methanogenic arc- 10.3389/fmicb.2012.00061 J. M., Kunz, R. C., and DiSpirito, 337–346. haea. Eur. J. Biochem. 208, 511–520. This article was submitted to Frontiers in A. A. (2001). Membrane-associated Zellner, G., and Winter, J. (1987). Zumft, W. G. (2005). Nitric oxide Microbiological Chemistry, a specialty of quinoprotein formaldehyde dehy- Growth promoting effect of tung- reductases of prokaryotes with Frontiers in Microbiology. drogenase from Methylococcus cap- sten on methanogens and incorpo- emphasis on the respiratory, Copyright © 2012 Glass and Orphan. sulatus Bath. J. Bacteriol. 183, ration of tungsten-185 into cells. heme–copper oxidase type. J. Inorg. This is an open-access article distributed 6832–6840. FEMS Microbiol. Ecol. 40, 81–87. Biochem. 99, 194–215. under the terms of the Creative Commons Zahn, J. A., and DiSpirito, A. A. (1996). Zerkle,A. L., House, C. H., and Brantley, Attribution Non Commercial License, Membrane-associated methane S. L. (2005). Biogeochemical signa- Conflict of Interest Statement: The which permits non-commercial use, dis- monooxygenase from Methylococcus tures through time as inferred from authors declare that the research was tribution, and reproduction in other capsulatus (Bath). J. Bacteriol. 178, whole microbial genomes. Am.J.Sci. conducted in the absence of any forums, provided the original authors and 1018–1029. 305, 467–502. commercial or financial relationships source are credited.

Frontiers in Microbiology | Microbiological Chemistry February 2012 | Volume 3 | Article 61 | 20