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Variability of the transporter gene complement in ammonia-oxidizing archaea Pierre Offre1, Melina Kerou1, Anja Spang2, and Christa Schleper1 1

University of Vienna, Department of Ecogenomics and Systems Biology, Archaea Biology and Ecogenomics Division, Althanstrasse 14, A-1090 Wien, Austria 2 Uppsala University, Department of Cell and Molecular Biology, Science for Life Laboratory, Box 596, SE-75123, Uppsala, Sweden

Ammonia-oxidizing archaea (AOA) are a widespread and abundant component of microbial communities in many different ecosystems. The extent of physiological differences between individual AOA is, however, unknown. Here, we compare the transporter gene complements of six AOA, from four different environments and two major clades, to assess their potential for substrate uptake and efflux. Each of the corresponding AOA genomes encode a unique set of transporters and although the composition of AOA transporter complements follows a phylogenetic pattern, few transporter families are conserved in all investigated genomes. A comparison of ammonia transporters encoded by archaeal and bacterial ammonia oxidizers highlights the variance among AOA lineages as well as their distinction from the ammonia-oxidizing bacteria, and suggests differential ecological adaptations. Ammonia-oxidizing archaea: a widespread and diverse group of organisms Ammonia-oxidizing archaea (AOA) were discovered approximately a decade ago [1,2] and are now a major research focus in environmental microbiology. These organisms belong to a recently described phylum, the Thaumarchaeota [3,4], and can be found in almost every aquatic and terrestrial habitat, including extreme environments such as hot springs and Antarctic waters [5–7]. Owing to their high abundance in the oceans and soils, AOA might be among the most abundant groups of organisms in the biosphere [8–10]. AOA are able to grow autotrophically by oxidizing ammonia (NH3) to nitrite (NO2) as their main source of electrons and energy (see [6] and references therein). Together with their long-known bacterial counterparts, the aerobic autotrophic ammoniaoxidizing bacteria (AOB) [11], AOA might play a significant role in the microbial oxidation of ammonia. Ammonia oxidation represents the first and rate-limiting step of the nitrification process, which is an essential component of the nitrogen cycle [12], and recent studies indicate that AOA Corresponding authors: Offre, P. ([email protected]); Schleper, C. ([email protected]). Keywords: Thaumarchaeota; ecological niche; genomics; transporters; ammonium. 0966-842X/ ß 2014 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.tim.2014.07.007

might also play significant roles in the production of two greenhouse gases, nitrous oxide [13–15] and methane [16]. Cultivated AOA belong to four lineages [group 1.1a, group 1.1a-associated, group 1.1b, and thermophilic AOA (ThAOA)], which form a well-supported monophyletic clade in many 16S rRNA gene phylogenies (e.g., [17]). Representatives of group 1.1a and 1.1b account for most AOA-related 16S rRNA gene sequences obtained in molecular surveys and are particularly prominent in marine waters and soils, respectively [6,7,18]. Both groups are also represented in other environments and include many sub-clades that can share as little as 92% 16S rRNA gene sequence identity, which reflects their considerable phylogenetic breadth. Cultivation and environmental studies have shown that AOA are functionally heterogeneous [17,19,20], as indicated by their different ammonia concentration and pH preferences [17,19,21–23], their variable ability to grow on urea as an ammonia source [22,24], and their variable requirements for small amounts of organic compounds to sustain growth [22]. The number and extent of physiological differences among individual AOA have, however, not been systematically investigated and the different environmental resources supporting their growth remain largely unknown. The set of transport systems encoded in an organism’s genome define, to a large extent, its potential to take up environmental resources (e.g., nutrients, water, and ions), resist toxic substances, and sense intercellular signaling compounds. Therefore, the transporter gene complement of an organism also contains information on this organism’s ecological preferences. Accordingly, comparative analyses of archaeal and bacterial genomes have indicated that transporter-coding genes are over-represented among recently acquired (horizontally transferred) genes [25–27], which enable microorganisms to sustain activity in their local environment. To explore the metabolic versatility and interspecies variations within AOA, in this study, we compare the transporter gene complements from six AOA strains and discuss both common and unique characteristics of these organisms (lists of the fully annotated transporter genes are available at the Archaea Biology and Ecogenomics Division, University of Vienna website: http://genetics-ecology.univie.ac.at/aoa_transport_systems.html). The analyzed AOA genomes include members of group 1.1a and Trends in Microbiology xx (2014) 1–11

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Table 1. Number of transport systems and transporter families in selected AOA genomes Organism

Nitrosopumilus maritimus SCM1

Nitrosoarchaeum limnia SFB1

Nitrosoarchaeum koreensis MY1

Cenarchaeum symbiosum A

Nitrososphaera gargensis

Affiliation

Group 1.1a (Alpha Iclade) a Marine water aquarium sediment

Group 1.1a (Alpha IIclade) a Brackish water sediment

Group 1.1a (Alpha IIclade) a Rhizosphere soil of Caragana sinica

Group 1.1a (Beta-clade) a

Group 1.1b

Tissues of the sponge Axinella mexicana

+ n.d. + n.d. 1.65 (1)

+ n.d. + n.d. 1.77 (76)

+ n.d. + n.d. 1.61 (1)

n.d. n.d. n.d. n.d. 2.05 (1)

Microbial mat (Garga spring outflow pond) + + + n.d. 2.83 (1)

+ + + +c 2.53 (1)

[45]

[43]

This study

Habitat

Ammonia sources b Carbon sources b

Ammonia Urea Bicarbonate Organic acids

Genome size in Mb (Nb. contigs) [87] [88] [44] Refs Number of transport systems and relative abundance of transporter classes e, f, g 60 50 51 Total 11 21.6% 9 15.0% 8 16.0% Channels/pores 21 41.2% 32 53.3% 25 50.0% Carrier-type transporters 10 19.6% 9 15.0% 8 16.0% Primary active transporters 9 17.6% 10 16.7% 9 18.0% Uncharacterized transporters Number and distribution of transporter families within the main classes of transporters g 35 42 36 Total 17.1% 5 11.9% 5 13.9% 6 Channels/pores 15 42.9% 21 50.0% 17 47.2% Carrier-type transporters 8 22.9% 8 19.0% 7 19.4% Primary active transporters 17.1% 8 19.0% 7 19.4% 6 Uncharacterized transporters

Nitrososphaera viennensis EN76 Group 1.1b

Garden soil

d

33 7 10 9 7

21.2% 30.3% 27.3% 21.2%

83 15 42 14 12

18.1% 50.6% 16.9% 14.5%

78 11 36 19 12

14.1% 46.2% 24.4% 15.4%

27 5 8 9 5

18.5% 29.6% 33.3% 18.5%

47 8 22 9 8

17.0% 46.8% 19.1% 17.0%

43 h 6 20 10 7

14.0% 46.5% 23.3% 16.3%

a

Clade designations are according to [89].

b

Positive signs ‘‘+’’: evidence for growth on the corresponding substrate. n.d.: not determined on the basis of growth experiments.

c

Pyruvate, oxaloacetate, 2-oxoglutarate and glyoxylate stimulate N. viennensis EN76 growth on ammonia and bicarbonate [22,42].

d

The genome of N. viennensis EN76 has been submitted to GenBank under Accession Number CP007536.

e

In the case of heteromultimeric transporters, only complete transport systems were counted and not every open reading frames encoding the protein subunits.

f

Fragmented open reading frames were considered as pseudogenes and transport systems encoded by fragmented open reading frames were therefore excluded from the counts.

g

Several proteins and protein complexes referenced in the transporter classification database (TCDB) [28] and present in all six AOA genomes were not taken in account in this analysis, i.e., proton-pumping complexes of the respiratory electron-transfer chain, the general and twin-arginine secretory pathways for protein export, a putative group-translocating polysaccharide exporter, transmembrane electron carriers and accessory factors involved in transport.

h

One transport system in N. viennensis EN76 could not be assigned to a transporter family (Figure 1) and was excluded from this count.

1.1b (Table 1). Two of the organisms investigated are pelagic marine (Nitrosopumilus maritimus SCM1) or brackish water (Nitrosoarchaeum limnia SFB1) organisms, one is a sponge symbiont (Cenarchaeum symbiosum A), two are from soil (Nitrosoarchaeum koreensis MY1 and Nitrososphaera viennensis EN76) and one is from a moderately thermophilic biofilm (Nitrososphaera gargensis). The transport systems of AOA Transporters are classified into four major classes according to the mechanism and energy source driving the transport process: (i) channels that facilitate the diffusion of substrates, usually by a passive mechanism; (ii) carriers that include both passive and active transporters, the latter using chemiosmotic energy; (iii) primary active transporters (PAT), which use primary energy sources (e.g., ATP) to transport solutes; and (iv) group translocators (e.g., phosphotransferase systems (PTS)), which chemically modify the substrate during the transport process [28]. Results from a genome study [29] suggest that channels, carriers, and PAT are well represented in the 2

Crenarchaeota and Euryarchaeota phyla, whereas group translocators and PTS in particular appear less common in archaea, which is in stark contrast to their wide occurrence in bacteria. Thus far, the genomes of haloarchaeal euryarchaea, including Haloferax volcanii, Haloarcula marismortui, Haloarcula hispanica, Halalkalicoccus jeotgali, and Haloterrigena turkmenica [30,31], are the sole archaeal genomes known to encode PTS. The composition, relatedness, and adaptive function of archaeal transporter complements remain however largely unknown, especially in recently discovered archaeal lineages. The genomes of AOA encode different numbers of transport systems All six of the examined AOA genomes encode channels, carriers, and PAT, whereas PTS are absent (Table 1). In addition, several partially characterized and putative transport systems, classified here as uncharacterized transporters, are also present. The relative abundance patterns of the four classes of transporters (channels, carriers, PAT, and uncharacterized transporters) are similar in the free-living

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Opinion AOA, in which carrier-type transporters represent the most abundant and diverse class of transporters (Table 1). By contrast, in the sponge symbiont Cenarchaeum symbiosum, the four transporter classes are more evenly represented. Individually, the AOA genomes encode a variable number of transporters, ranging from 33 to 83 (Table 1). Interestingly, the investigated members of group 1.1a possess fewer transport systems than the investigated Nitrososphaera species, which is consistent with the smaller size of their genomes (Table 1). This trend is, to some extent, reflected in the number of transporter families, which varies from 27 to 42 in the group 1.1a genomes and from 43 to 47 in the two group 1.1b members (Table 1). The genome of C. symbiosum stands out because of its intermediate genome length but simplest transporter complement, which is characterized by a reduced set of carrier-type transporters (Table 1). This suggests that C. symbiosum might take up fewer compounds than free-living AOA and is therefore specialized for a lower number of resources and/or inhabits a niche characterized by low physicochemical fluctuations. The transporter complement encoded by individual AOA genomes is highly variable The individual transporter complements of the six analyzed AOA account for a varying fraction (44–77%) of the total 61 transporter families that are represented in all of the six genomes (Figure 1). Only 18 transporter families are conserved in all six AOA (MIP, Amt, MscS, MIT, DHA1, ZIP, CPA2, TSUP, PepT, PhoT, MZT, TauT, DrugE1, MacB, Sweet, DedA, CopD, and CbtAB; Figure 1), emphasizing the large fraction (>70%) of the 61 transporter families that forms the variable component of the AOA transporter complements. Furthermore, the genomes of the analyzed free-living AOA share six additional transporter families (APC, CDF, DMT, RhtB, NicO, and CrcB; Figure 1), which are not represented in the reduced set of transport systems encoded in the C. symbiosum genome. Most transporter families are, in addition, variably represented in the individual AOA genomes by different number of representative transporters (Figure 1); for example, Nitrosopumilus maritimus encodes two DrugE1 family transporters, whereas Nitrososphaera viennensis encodes five, thus highlighting supplementary differences between the organisms investigated. The variable component of the six AOA transporter complements includes 43 transporter families, among which 23 are distributed in a group-specific pattern. That is, ten of these families are represented in group 1.1a genomes but absent from group 1.1b organisms and 13 of these families are present in group 1.1b AOA but absent from group 1.1a genomes. However, none of the families that are specific for group 1.1a is conserved in all four of the group 1.1a genomes and might not, therefore, represent group-specific features. By contrast, six out of the 13 group 1.1b-specific transporter families are represented in the two Nitrososphaera species genomes (UT, UMF10, NCS1, ACR3, 3.A.3.5 P-ATPase family, and ArsAB; Figure 1). However, generalizations for the whole diversity of organisms within group 1.1a and group 1.1b cannot be made on the basis of only a few genomes. Eleven families are found only once, and each of the analyzed AOA genomes encodes

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at least one of these families (FNT, SP, ACS, UMF12, DAACS, ArsB, NAAT, CUT2, HAAT, 3.A.3.7 P-ATPase family, and 3.A.3.25 P-ATPase family; Figure 1). This analysis indicates that the transporter complements encoded in the six analyzed AOA genomes are clearly distinct from each other (Figure 1) and suggests that even closely related AOA (as indicated by 16S rRNA gene identity), such as Nitrosoarchaeum limnia/Nitrosoarchaeum koreensis or Nitrososphaera gargensis/N. viennensis, possess many different functional traits. This trend has been observed in several instances in well-studied microbial populations [27]. The composition of AOA transporter gene complements may be primarily determined by the phylogenetic position of the host organism The relatedness of the six AOA transporter complements was investigated using a hierarchical cluster analysis. The clustering of AOA transporter complements (Figure 1) matches the 16S rRNA gene-based phylogeny of the respective organisms, suggesting that the overall composition of these AOA transporter complements could reflect the evolutionary relatedness of the corresponding microbial strains and, very tentatively, that AOA clades define functionally cohesive groups of microorganisms. Closely related AOA strains might therefore have similar habitat preferences, although this seems to contradict the habitat of origin of at least two of the investigated AOA (Table 1): N. limnia and N. koreensis share 99.1% 16S rRNA gene identity but originate from brackish water sediments and rhizosphere soil, respectively. This discrepancy could indicate that N. limnia and/or N. koreensis have been enriched from environments that are not representing their general habitat preference, but it could also suggest that similar, although not necessarily identical, microhabitats are present in macroscopically different environments. However, some of the differences between N. limnia and N. koreensis transporter complements might reflect environmental selection, such as the presence of putative bicarbonate transporters (SBT family; Figure 1) in N. limnia, and their absence in N. koreensis: SBT transporters provide the ability to take up inorganic carbon as bicarbonate when the local concentration of CO2 is low (CO2 passively diffuses across biological membranes) but are unnecessary in environments with higher CO2 partial pressure, such as in rhizosphere soil. Assessing the links between AOA genomic diversity, habitat preference, molecular adaptations, and biogeography will ultimately rely on technological and methodological developments that will enable large-scale AOA population genomics studies, high-throughput characterization of AOA physiology, gene knockouts, and characterization of their microhabitat in situ. Solute transport capabilities of individual AOA differ as suggested by the composition of their transporter gene complement The functional characteristics of the transport systems encoded in the analyzed AOA genomes (Table 1) will be briefly outlined in this section. For additional information on the transporter families discussed in this article, we refer the reader to the corresponding entries in the 3

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0.65

∗

∗

0.80

is

1 1

1 1

1 1 1

1

1

1

1

2

2

2

1

1

1

1

1

1 1 4 1

1 3 1 2

iosu ymb

ienn ens N. v

1 1

arge nsis N. g

m C. s

1 1

mnia

N. k ore ens is

PFAM

N. m ari m

Transporter family

N. li

us

1.00

Channels/Pores 1.A.1. The voltage-gated Ion channel (VIC) superfamily 1.A.8. The major intrinsic protein (MIP) family 1.A.11. The ammonia channel transporter (Amt) family

Type 1 Type 2 Type 1 Type 2 Type 1 Type 2

PF00520 PF00230 PF00909

1

1

1.A.13. The epithelial chloride channel (E-ClC) family 1.A.16. The formate-nitrite transporter (FNT) family 1.A.22. The large conductance mechanosensive Ion channel (MscL) family 1.A.23. The small conductance mechanosensive Ion channel (MscS) family 1.A.28. The urea transporter (UT) family

PF00092 PF01226 PF01741 PF00924 PF03253

1

3

1 2

1 2

1

1.A.35. The CorA metal Ion transporter (MIT) family

PF01544

2

2

1

1

2

2

2 1 1

3 1 1

2

4 1

Electrochemical potenal-driven transporters 2.A.1.1. The sugar porter (SP) family

1

SSF103473

2.A.1.14. The anion:caon symporter (ACS) family

2 3 1

2.A.1.59. unidenfied major facilitator-10 (UMF10) family 2.A.1.63. The unidenfied major Facilitator-12 (UMF12) family

1

1

2 1

1 1 1

1

2.A.1. unclassified 2.A.3. The amino acid-polyamine-organocaon (APC) superfamily

Type 1 Type 2

PF13520

1

2.A.4. The caon diffusion facilitator (CDF) family

PF01545

3

2.A.7. The drug/Metabolite transporter (DMT) superfamily

PF02535 PF00892

2 2 1

Type 1 Type 2

2.A.21. The solute:sodium symporter (SSS) family, type 1

Type 1 Type 2

2.A.23. The dicarboxylate/Amino acid:caon (Na+ or H+) symporter (DAACS) family 2.A.36. The monovalent caon:proton anporter-1 (CPA1) family 2.A.37. The monovalent caon:proton anporter-2 (CPA2) family

2.A.39. The nucleobase:caon symporter-1 (NCS1) family 2.A.45. The arsenite-anmonite (ArsB) Efflux family 2.A.59. The arsenical resistance-3 (ACR3) family

PF01699 PF01384

2.A.20. The inorganic phosphate transporter (Pit) family

Type 1 Type 2

†

3

1

2

2

1 2 1

1 1

1

1

4

†

1 1

PF00474

†

1 2 1 1

1 1 1

1

7

5

1 1

1

1 1 2

1 3

5

1 2

PF02386

1

3

2

1

PF02133

1

PF03600 PF01758

1

PF13440

1

1

2.A.76. The resistance to homoserine/Threonine (RhtB) family

1 1

1 2 1

1

1

2

1

1 1

1 1

1 2

1

1

1

2

2.A.89. The vacuolar iron transporter (VIT) family

PF01914 PF01925

2.A.108. The iron/Lead transporter (ILT) family 2.A.109. The tellurium Ion tesistance (TerC) family

PF03239 PF03741

2.A.113. The nickel/cobalt transporter (NicO) family

PF03824

1

2

2.A.66. The muldrug/Oligosaccharidyl-lipid/Polysaccharide (MOP) flippase S/family

2.A.95. The 6TMS neutral amino acid transporter (NAAT) family 2.A.102. The putave 4-toluene sulfonate uptake permease (TSUP) family

2 1 1

1

PF00375 PF00999 PF00999

PF01810 PF05982 PF01988

4

†

1

1 1

1

1

1

1

1 2

2 1 1

†

1

†

Primary acve transporters 3.A.1.2. The carbohydrate uptake transporter-2 (CUT2) family

PF02653

3.A.1.4. The hydrophobic amino acid uptake transporter (HAAT) family 3.A.1.5. The pepde/Opine/Nickel uptake transporter (PepT) family

PF02653 PF00528

1

1

1

1 1

1

1

3.A.1.7. The phosphate uptake transporter (PhoT) family

PF00528

1

1

1

1

1

1

3.A.1.9. The phosphonate uptake transporter (PhnT) family

PF00528

1

3.A.1.15. The manganese/Zinc/Iron chelate uptake transporter (MZT) family 3.A.1.17. The taurine uptake transporter (TauT) family

PF00950 PF00528

2 1

1 1

1 1

1 1

2 2

2 2

3.A.1.105. The drug exporter-1 (DrugE1) family 3.A.1.122. The macrolide exporter (MacB) family

PF01061 PF02687

2 1

2 1

2 1

1 1

3 2

5 2

3.A.3. The P-type ATPase (P-ATPase) superfamily (3.A.3.5. family) 3.A.3. The P-type ATPase (P-ATPase) superfamily (3.A.3.7. family)

PF00122 PF00122

1

2 1

3.A.3. The P-type ATPase (P-ATPase) superfamily (3.A.3.25. family) 3.A.4. The arsenite-anmonite (ArsAB) efflux family

PF00122 PF03600

1

2

1

1

1

PF03030

1

1

1

1

PF03471 PF03083

2 1

1 1

1 1

1 1

1

Incompletely characterized transport systems 9.A.40. The HlyC/CorC (HCC) family of putave transporters 9.A.58. The sweet; PQ-loop; Saliva; MtN3 (sweet) family

9.B.27. The DedA or YdjX-Z (DedA) family 9.B.45. The Arg/Asp/Asp (RDD) family 9.B.62. The copper resistance (CopD) family 9.B.69. The putave cobalt transporter (CbtAB) family 9.B.71. The camphor resistance (CrcB) family

PF04193 PF02308 PF09335

1 2

1 1 3

2

1 2

2

2

1 2

PF06271 PF04234

1

1 1

1 1

1

2 1

2 2

PF05425

1

1

1

1

1

1

PF09490 PF02537

1 1

1 1

2 1

1

1 1

1 1

TRENDS in Microbiology

Figure 1. Numbers and types of transport systems encoded in the genomes of six ammonia-oxidizing archaea (AOA). Transporter families were classified according to the transporter classification database (TCDB) [28] and the corresponding protein families are indicated by matching PFAM [90] or SUPERFAMILY [91] domains. Subtypes in (Figure legend continued on the bottom of the next page.)

4

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Opinion Transporter Classification Database (TCDB; http:// www.tcdb.org/) and references therein [28]. Identification of the substrates of AOA transport systems will ultimately rely on experimental work, but information extrapolated from functionally characterized proteins related to AOA transporters can be used to predict which type(s) of solute may be transported. Half of the 18 transporter families conserved in all AOA genomes analyzed in this study play a role in the transport of inorganic solutes and include ammonia, phosphate, and metal ion uptake transporters (Figure 1). Families present in all six AOA also include drug exporter families (DHA1, DrugE1, and MacB; Figure 1), which may be involved in the efflux of toxic compounds, secondary metabolites, or signaling molecules. Transporter families that are thought to be primarily involved in the uptake of organic molecules (e.g., the MIP, PepT, Sweet, TauT, and TSUP families [28]) are also represented in all six genomes (Figure 1). However, the substrates of the corresponding transporters remain undetermined and proteins within those families may conduct various additional metabolites, protein cofactors, and, in some cases, even inorganic substances (e.g., [32–34]). Members of the MIP family, for example, can function as both uptake and efflux proteins and may transport glycerol, dihydroxyacetone, and/or urea but this is not a universal feature of MIP proteins, which can also transport water, ammonia and/or various inorganic ions (see the respective entry in the TCDB and references therein; http://www.tcdb.org/search/result.php?tc=1.A.8). By contrast, characterized Sweet family proteins (Sweet; Figure 1) function as sugar, amino acid, and organic acid transporters [35–37] and might represent genuine organic substrate transporters in AOA. The uptake and assimilation of organic molecules was inferred in environmental studies (e.g., [20,38–41]), and pyruvate and other 2-oxoacids have been demonstrated to be essential for growth in N. viennensis laboratory cultures [22,42]. These organic acids may fuel the tricarboxylic acid cycle under primarily autotrophic growth conditions in at least some AOA but the biological and ecological significance of these growth modes is still unknown. The variable component of AOA transporter complements includes transport systems for organic as well as inorganic substances (Figure 1). Interestingly, several of the corresponding transporter families (UT, SP, MHS, ACS, APC, DMT, SSS, DAACS, NCS1, NAAT, CUT2, HAAT, and PhnT) have been reported to play a role in the uptake of various classes of organic compounds (e.g., carbohydrates, amino acids, organic acids, (poly)amines, urea, vitamins, nucleobases, nucleosides, and organophosphorus compounds), suggesting that individual AOA could take up different subsets of organic molecules and ions. Substrate uptake experiments with cultivated AOA strains and biochemical characterization of AOA transport

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systems will be necessary to determine whether the varying complements of transporters represent genuine functional diversity or instead functional redundancy. Current evidence suggests that at least part of the variations in the composition of AOA transporter complements represent genuine functional diversity. For example, putative urea uptake transporters (UT, SSS type 1) are only found in C. symbiosum, N. gargensis, and N. viennensis, and growth on urea was verified experimentally in the latter two organisms [22,43]. Putative organophosphorus transporters (PhnT) are only found in N. maritimus and C. symbiosum [44,45], whereas putative bicarbonate transporters (SBT) are found in N. maritimus and N. limnia and a putative cyanate transporter (FNT) in N. gargensis [43]. The transport systems belonging to the VIC, MIP, Amt, APC, CaCA, SSS, NCS1, Sweet, and CopD transporter families are classified into two different subtypes within each of these families based on sequence alignments (type1 and type 2; Figure 1). These sequence variants may have different functions and represent an additional source of variation between individual AOA. This will be discussed in more detail in the following section for proteins belonging to the ammonia channel transporter (Amt) family. Ammonia transporters in AOA and AOB The overall function of ammonia transporters is to increase the transmembrane flux of ammonia when passive diffusion is limited by low permeability of the cytoplasmic membrane, low extracellular pH, or low ammonia concentration [46]. The relative increase in ammonia diffusion provided by a dedicated transporter is a function of the transporter conductance and density of transport units in the membrane. Ammonia transporters encoded in sequenced genomes of AOA and AOB belong to the Amt family (Figures 1 and 2). This family is widely distributed across the three domains of life [47] and has been intensively studied over the past 20 years [48,49]. The transport mechanism of Amt family proteins remains, however, unclear, even for the best-characterized of these transporters, which is the AmtB protein of Escherichia coli (e.g., [50,51]). Several studies have indicated that some aspects of the transport process (e.g., nature of the transported substrates (ammonia versus ammonium), electrogenicity, and energetics of the transport) may vary between different representatives of the family (e.g., [52–54]). Thus far, the role of facilitated ammonia uptake in the metabolism of AOA and AOB remains unclear [55]. Published models of AOA and AOB metabolism favor the hypothesis of a periplasmic location for the ammonia oxidation process [44,55,56], suggesting that facilitated ammonia uptake would contribute solely to nitrogen assimilation. However, several physiological and biochemical investigations of AOB have yielded results consistent with

some families were classified on the basis of distinct local alignments between translated AOA gene sequences and reference proteins in the TCDB. Crosses (y) indicate open reading frames that do not significantly hit a protein family domain but were assigned to a transporter family based on significant psi-blast hits (e-values < 5e-15 in the first three iterations). In the case of heteromultimeric transporters, only the PFAM domain corresponding to the integral membrane protein was indicated. Similarity between individual AOA transporter complements was assessed using a cluster analysis, which is depicted by the ultrametric tree and similarity bar above the data matrix. Distance was calculated according to the Bray–Curtis dissimilarity Index, agglomerative hierarchical clustering performed with the complete linkage method, and significance of linkages determined by a non-parametric bootstrap analysis. Asterisks indicate significant linkages at 95% confidence level and values in the data matrix are color-coded according to statistically supported linkages.

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100

Eukaryoc cluster (Viridiplantae)

50 Archaeal cluster (Sulfolobales and Picrophilus torridus DSM 9790) Vulcanisaeta distributa DSM 14429 Metallosphaera cuprina Ar-4 Methanobacteriales cluster

100 99 100

54

Group 1.1a AOA cluster 1

99 100 100

69

AOA Amt-1

Group 1.1b AOA cluster 1

Euryarchaeal cluster

97

Chloroflexi cluster

MEP Bacterial cluster (Firmicutes and Microcyss aeruginosa NIES-843)

Bacterial cluster (Planctomycetes, Nitrospira, NC10 and Deltaproteobacteria) 100 93

100

100 99 100

82

74

100

Eukaryoc cluster (Fungi)

100 Methanosarcinales cluster Geobacter sp. Cluster 100 Euryarchaeal cluster 100 Group 1.1a AOA cluster 2 91 100 Group 1.1b AOA cluster 2 100 Eukaryoc cluster (Euglenozoa, Heterolobsea and Amoebozoa)

AOA Amt-2

Bacterial cluster (Alphaproteobacteria and Methylococcus capsulatus str. Bath) Methylococcus capsulatus str. Bath

Cluster comprised of members of the Chloroflexi, Archaea, Planctomycetes, Cyanobacteria, Firmicutes and NC10

56

Grade-MEP

100 97

Haloarchaeal cluster Methylococcus capsulatus str. Bath 100 Bacterial cluster (Betaproteobacteria and Nitrospira)

Rhesus-type Amt of AOB

0.5

TRENDS in Microbiology

Figure 2. Maximum likelihood phylogenetic tree of ammonia channel transporter (Amt) family proteins. The alignment was based on 342 aligned amino acid positions and the phylogeny was calculated with RaxML [92] using the LG substitution matrix and the GAMMA model of rate heterogeneity. Numbers on branches represent rapid bootstrap inference values (only support values > 50% are shown) and the scale bar refers to 50% estimated sequence divergence. The tree was rooted with Rhesus-type Amt proteins from beta-proteobacterial ammonia-oxidizing bacteria. Monophyletic groups were collapsed to ease the readability of the tree (alignment and sequence accession numbers are available upon request). Bacterial clusters are shown in blue, archaeal clusters in green, eukaryotic clusters in violet, and mixed clusters are indicated by a mixture of the respective colors. Abbreviations: AOA, ammonia-oxidizing archaea; AOB, ammonia-oxidizing bacteria; MEP, methyl-ammonia permeases.

intracellular ammonia oxidation [57–59], which could be fuelled by an ammonia transport process. AOA genomes encode two types of ammonia transporters Ammonia transporters of AOA belong to a large group of proteins referred to as methyl-ammonia permeases (MEP) (Figure 2), which form a monophyletic group within the Amt family and include most bacterial and archaeal Amt proteins identified thus far [47]. MEP-type proteins do not always conduct monomethylamine [60] but all characterized members of this group consistently transport ammonia (e.g., [54,61–63]). The overall architecture and fold of Amt proteins is conserved in AOA MEP-type transporters, as indicated by sequence alignment (Figure 3) and comparative protein structure modeling. Furthermore, residues that have been found to determine important structural and mechanistic features of E. coli AmtB [64,65] and Archaeoglobus fulgidus Amt-1 MEP-type proteins [66], for example, the ammonium-binding site, the phenylalanine gate, and twin-histidine residues, are conserved in the Amt proteins of AOA (Figure 3). This suggests that AOA ammonia transporters recruit ammonium in a manner that is similar to E. coli AmtB and A. fulgidus Amt1, but the chemical substance passing through MEP-type proteins has not been clearly identified and could be ammonia, ammonium, or ammonia and H+ co-transport 6

[67,68]. A recent study [69] showed that A. fulgidus Amt-1 and Amt-3 are electrogenic ammonium transporters rather than ammonia channels, which is in favor of the ammonium or ammonia and H+ co-transport scenarios. Amt transporters encoded in AOA genomes define two separate and well-supported lineages within the MEP clade, and sequences within each of the two lineages, referred to as Amt-1 and Amt-2, cluster following the taxonomy of the host organisms (Figure 2). Amino acid sequences of Amt-1 and Amt-2 proteins share only 38–46% identity and Amt-1 proteins are 59–113 amino acids longer than those belonging to the Amt-2 lineage (Table 2). Additional amino acid residues in Amt-1 proteins are localized on extracellular protein segments and form an extended N terminus and two extended loops (Figure 3). Similar to E. coli AmtB and A. fulgidus Amt-2 [66,70], Amt-1 proteins of AOA contain a cleavable signal peptide sequence (Table 2) targeting those proteins to the general secretory pathway for translocation into the cytoplasmic membrane. Similar to A. fulgidus Amt-1 and Amt-3 transporters [66], Amt-2 proteins of AOA lack a characteristic signal peptide (Table 2), suggesting that these proteins are inserted into the cell membrane via a non-classical protein secretion pathway [71]. Sequence and structural dissimilarities between Amt-1 and Amt-2 AOA transporters thus indicate that these proteins might have different functions. This hypothesis is further supported by the consistently higher

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Amt2

Amt1

Opinion

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NVIE_002420 1 Ngar_c14320 1 NVIE_022110 1 Ngar_c33590 1 Nmar_1698 1 Nlim_1421 1 MY1_1789 1 CENSYa_1453 1 CENSYa_0526 1 NVIE_023840 1 Ngar_c30510 1 Nmar_0588 1 MY1_1932 1 Nlim_1564 1 AMTA_AFUL 1 AMTB_ECOLI -22

YAPDD-------------PAVPYHCW-QTNEDGTF-VI--QTGEDGSQNLVPCEINTG..64..DAGGYGFIGTLDWAGLQNVLHDVP------------SSQY---------------AIT YTPDD-------------PAAVYHLW-ETDEEGNY-VL----DEEGN--LIPAAIDTG..64..DATGHGFIGTLDWAGLQNVLHDAP------------SSQY---------------AVT YAPDD-------------PAVPYHCW-QTNEDGTFKIV--VDNSTGAQSLVPCEINHG..64..DAGGYGFIGTLDWAGLQNVLHDVP------------SDAYGGIT-----------GFT YTAND-------------PAVPYHCFIDQNGDGAITAADEVEDEEGNMALVPCEVSPG..64..DATGQGFIGDFSWVGLQNVLHDVP------------SNAYYGIN-------SDGGRNA QSVEDGMDGYVKGTSGIYTGNPNECW----------------FDDGEGGMLPCMIDTG..63..DEGANMFMGSLDYAGFNMVSAWAPIGELGPC-HDTWSAAYQMNEFKEGEYCSQSWPGT QSVNDGMDGYVKGTSGIYTGNPNECW----------------YDDGEGNMLPCKVDTG..63..DSDANMFMGSLQYVGFNDVSHFAPLGEPGPC-ADTWSAAYQMNAMVEGDVCGQGWPNT QNVNDGMDGYVAGTSGIYTGNPNECW----------------YDDGEGNMLPCKIDTG..63..DSDANMFMGSLQYAGFNEVSHYAPLGEAGPC-ADTWSAAYQMNAMVEGDVCSQGWPGT QSVEDGMDGYVLGNSGIYTGNPNECW----------------VDNGDGTFTACYIDTG..63..DNDANMFMGALDYAGFNQVSHYAPLGAPTECDGDIWSNAYQMQQMKPDVACSDSWPGT QTMEDDADGY--DNRGLYTGNPNECW----------------VDDGEGEFTACYIDSG..63..DSDWNMFAGSLDYAGFNQVSYFAPLGSPIEC-QDTASNEYLMQEIKSGVACSDTWPGT M----------------------------------------------------PIDTG..63..SQN--GWIGGLDWLFFNNVPFNDSV----------------------------DYAPT M----------------------------------------------------PIDTG..63..SQG--GFIGGLDWLFFNNVPFNDSL----------------------------DYAPT M----------------------------------------------------VLDSG..63..SEH--GLIGDMEWVFLKGVPSDDSL----------------------------PFAPT M----------------------------------------------------PIDTG..63..DESG-GFIGNMDWVFLKGVPWDEAL----------------------------DYAPT V----------------------------------------------------PIDSG..63..DESG-GLIGNMDWVFLKGVPWDEAL----------------------------DYAPT ------------------------------------------------------MSDG..63..DIS--GIIGGLNYALLSGVKGED--------------------------------------------------------------MKIATIKTGLASLAMLPGLVMAAPAVADKA..64..---GNNFFGNINWLMLKNIELTAVM-------------------------------GS

Amt2

Amt1

Loop 1 NVIE_002420 Ngar_c14320 NVIE_022110 Ngar_c33590 Nmar_1698 Nlim_1421 MY1_1789 CENSYa_1453 CENSYa_0526 NVIE_023840 Ngar_c30510 Nmar_0588 MY1_1932 Nlim_1564 AMTA_AFUL AMTB_ECOLI

174 170 179 186 202 201 201 202 199 135 135 135 136 136 123 156

Loop 2

IWATFVYDFAAHWTWQIAVPDNYGMNPGYCGFGW-----------TGCLGSLDFAGGTVIHITSGWSGLVIALMLGRRLGYGKVPMEPHNISLVVLGAALLWVGWFGFNAGSAG IWATFVYDFAAHWTWQIAAPDNYGRNPDYCGFGW-----------TGCLGSLDFAGGTVIHITSGWSGLVIALMLGRRLGYGKMPMEPHNVSLVVLGAALLWVGWFGFNAGSAG IWATFVYDFAAHWTWEITAPDNYGRNPGYCNFGW-----------GGCLGALDFAGGTVIHITSGWSGLVIALMLGRRLGYGKVPMEPHNISLVVLGAALLWVGWFGFNAGSAA IWATFVYDFAAHWTWQITATDNYGRTPGYCGFGW-----------GGCLGALDFAGGTVIHITSGWSGLVIALMLGRRLGYGKMPMEPHNVSLVVLGAALLWFGWFGFNAGSAA LWGTFVYDPIAHWVW---------------GGGFIGGGGLDLDPDLSPTFALDFAGGTVVHISSGFAALAGALVLGRRLGYGKVPMEPHNIPMVVLGASILWFGWFGFNAGSEV LWATFVYDPVAHWVW---------------GGGYIGGGAIDLNPDLSPSFALDFAGGTVVHITSGFSALAGALILGRRLGYGKVPMEPHNIPMVVLGAGILWFGWFGFNAGSEV LWATFVYDPVAHWVW---------------GGGYIGGGALDLNPDLSPSFALDFAGGTVVHITSGFSALAGALILGRRLGYGKVPMEPHNIPMVVLGAGILWFGWFGFNAGSEV LWGTFVYDPVAHWVW---------------GGGYIGGGTLDLDPDLSPTFALDFAGGTVVHITSGFSALAAALILGRRLGYGKVPMEPHNVPMVVLGASILWFGWFGFNAGSEV LWATFVYDPVAHWIW---------------GGGFVSAGSLDIDPDLSPSFALDFAGGIVVHVTSGFSALAAALVLGRRIGYGKVPMDPHNVPMMVLGVTILWFGWTGFNAGSEV AWSIFIYYPLAHWIW---------------GRGW-----------LADLGVFDFAGGIVIHTSAGMASLAAALILGRRKNFGPDIMVPHNIPLAVIGAALLWIGWFGFNAGSAL AWSILVYYPLAHWIW---------------GRGW-----------LADMGVFDFAGGIVIHTSAGLGSLAAALVLGRRKNFGPDIMVPHNIPLAVIGATLLWIGWFGFNAGSVL AWSMLIYYPLVHWVW---------------GGGW-----------LAQLGVVDFAGGIVIHTSVGMAALAAAIVLGKRRNYGPAIMIPHSIPLAVLGSSLLWLGWFGFNAGSAL SWSILIYYPLVHWVW---------------GGGW-----------LAELGVVDFAGGIVIHTSVGMGALAAAIVLGRRRFFGPAIEIPHSIPLAVVGSSLLWLGWFGFNAGSAL SWSILIYYPLVHWVW---------------GGGW-----------LAELGVVDFAGGIVIHTSVGMGALAAAIVLGRRRFFGPAIEIPHSIPLAVTGSALLWLGWFGFNAGSAL LWLTFVYAPFAHWLW---------------GGGW-----------LAKLGALDFAGGMVVHISSGFAALAVAMTIGKRAGFEEYSIEPHSIPLTLIGAALLWFGWFGFNGGSAL VWLTLSYIPIAHMVW---------------GGGL-----------LASHGALDFAGG GG GTV TVVHINA VVHINAAIAGLVGA AIAG GLVGA LVGA AYL YLIGKRVGFGKEAFKPH Y KPHNLPM NLPMVFT MVFTG GTA G TAILYIGW ILYIGW WFGFN NAG AGSAG GSAG

M4

Loop 3

M6

M5

TRENDS in Microbiology

Figure 3. Segments of the amino acid sequence alignment of ammonia-oxidizing archaea (AOA) ammonia channel transporter (Amt) family proteins. Aligned sequences were obtained from the genomes of Nitrososphaera viennensis (NVIE), Nitrososphaera gargensis (Ngar), Nitrosopumilus maritimus (Nmar), Nitrosoarchaeum koreensis (MY1), Nitrosoarchaeum limnia (Nlim), and Cenarchaeum symbiosum (CENSYa). Protein sequences from Archaeoglobus fulgidus (Amt-1_AFUL) and Escherichia coli (AmtB_ECOLI) Amt proteins are used as references. The alignment was generated with MAFFT [93]. AOA sequences are grouped into two sequence clusters, Amt-1 and Amt-2, which correspond to the respective clusters in Figure 2. The presented regions correspond to the first three extracellular loops (Loops 1–3) and transmembrane domains 4 to 6 (M4–M6) as deduced from the E. coli AmtB structure [64,65]. Numbering starts from the first residue of the mature protein. Residues corresponding to the highly conserved transmembrane domains 1 and 2, which separate loop 1 from loop 2, are masked and the number of these residues is shown in parentheses. The first of the two conserved histidines lining the transporter pore is highlighted in green (H168), and the three residues that constitute the proposed ammonium-binding site are indicated in orange (S219, W148, D160). Other residues conserved in all sequences are highlighted in black, and similar residues in gray. Residues with negatively charged side groups (D and E) in loops 1–3 are indicated in red. Conserved cysteine pairs, which could form disulfide bonds, potentially stabilizing the extracellular loops, are highlighted in yellow.

levels of Amt-1 transcripts over Amt-2 transcripts in N. maritimus cells grown in batch cultures [72]. Molecular dynamic simulations of ammonium recruitment events by E. coli AmtB have indicated that the affinity of MEP-type transporters might be determined, at least in part, by the presence of negative charges in the extracellular vestibule of these proteins [73]. This is consistent with the recent report of a decrease in A. fulgidus

Amt-1 affinity at low (< 5.5) pH [69]. Interestingly, both the extended N terminus and extracellular loops of AOA Amt-1 proteins contain several amino acids with negatively charged side groups (Figure 3), which contribute to an excess of negative charges in the extracellular vestibule, as suggested by protein structure modeling, and might increase the substrate affinity of Amt-1 transporters compared with Amt-2 proteins. This finding is consistent with

Table 2. Selected characteristics of ammonia transporters encoded in the genomes of six AOA Cluster Locus Tag a Length Extended N terminus Extended C terminus Signal peptide d Adjacent glnK gene

Amt-1 Nmar 1698 521 +b + -

Nlim 1421 520 +b + -

MY1 1789 520 +b + -

CENSYa 0526 516 +b + -

CENSYa 1453 521 +b + -

Ngar c14320 496 +b + -

Ngar c33590 519 +b + -

Nvie 002420 502 +b + -

Nvie 022110 502 +b + -

Amt-2 Nmar 0588 435 +c -e +

Nlim 1564 435 +c -e +

MY1 1932 437 +c -e +

Ngar c30510 408 -e -

Nvie 023840 408 -e -

a

Locus tags correspond to the following organisms: Nmar, Nitrosopumilus maritimus SCM1; Nlim, Nitrosoarchaeum limnia SFB1; MY1, Nitrosoarchaeum koreensis MY1; CENSYa, Cenarchaeum symbiosum A; Ngar, Nitrososphaera gargensis Ga9.2; Nvie, Nitrososphaera viennensis EN76.

b

The extended N terminus of Amt-1 transporters includes 60 amino acid residues.

c

The extended C terminus of Amt-2 transporters includes 25 amino acid residues.

d

The SignalP 4.1 server [94], available at the Center for Biological Sequence Analysis website (http://www.cbs.dtu.dk/services/SignalP/), was used to predict the presence and cleavage site of signal peptides.

e

Amt-2 transporters are predicted to be secreted via a non-classical protein secretion pathway using the SecretomeP 2.0 Server [71], which is available at the Center for Biological Sequence Analysis website (http://www.cbs.dtu.dk/services/SecretomeP/).

7

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Opinion transcription studies in N. maritimus [72], in which Amt-2 mRNA was more prominently detected during growth at 500 mM ammonium whereas the level of Amt-1 transcripts changed only moderately from 500 mM to 10 nM ammonium but decreased by two orders of magnitude within 1 day in the absence of substrate. This led the authors to also hypothesize that in N. maritimus, the Amt-2 protein had a lower substrate affinity than Amt-1 [72]. In this context, the absence of a gene encoding an Amt-2 protein in the C. symbiosum genome (Figure 1 and Table 2) suggests that this organism may never be exposed to ‘high’ ammonia concentrations or instead that ammonia uptake via Amt-1 facilitated diffusion and/or passive diffusion is sufficient to meet the ammonia demand of C. symbiosum at higher ammonia concentrations. Alternatively, C. symbiosum might primarily rely on the uptake of urea as a possible nitrogen/ammonia source via its SSS family urea transporter (Figure 1). Indeed, the broad diversity of bacterial urease genes and transcripts detected in tissues of the marine sponge Xestospongia testudinaria suggests that urea might represent an important source of nitrogen/ ammonia for at least some sponge-inhabiting microorganisms [74]. Amt-1 proteins of group 1.1a AOA share only 39–65% sequence identity with those of group 1.1b, and the extended N terminus and extracellular loops reveal group-specific sequence patterns (Figure 3). However, the functional significance of these differences, if any, remains unclear. Interestingly, the genomes of C. symbiosum, N. gargensis and N. viennensis each encode two Amt-1 transporters, whereas the genomes of N. maritimus, N. limnia, and N. koreensis encode only one (Figure 1 and Table 2). Amt-2 transporters of the free-living group 1.1a AOA contain an extended C terminus (Table 2). Additionally, the corresponding genes are located adjacent to two glnKlike genes (Table 2) that encode PII signal transduction proteins [75], which are typically encoded in the vicinity of amt genes. Some of these proteins interact with the C terminus of various bacterial and archaeal MEP-type transporters, regulating the influx of ammonium and the expression of genes involved in cellular nitrogen metabolism [48,75]. This suggests that Amt-2 proteins, in at least some group 1.1a AOA, could function as ammonia sensors [48] in addition to their function as transporters of ammonia. Ammonia transporters of AOA and AOB are functionally different Amt family proteins encoded in sequenced AOB genomes belong to the Rhesus clade (Figure 2) and are only distantly related to the MEP-type proteins of AOA, sharing only 24– 31% sequence identity. Although transporters of the Rhesus clade are present in only few bacterial groups [76], they are encoded in most characterized AOB genomes. Nitrosomonas eutropha C91 and Nitrosococcus oceani ATCC 19707 are notable exceptions as they do not encode known ammonia transporters [77,78], suggesting that these organisms solely rely on passive ammonia diffusion or use as yet unknown transporters. Structural investigations of MEP- and Rhesus-type proteins indicate that the overall fold of these proteins 8

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is very similar but members of both clades are characterized by distinct features [79,80]. For example, the ammonium-binding site characterizing MEP-type proteins is absent in the extracellular vestibule of Rhesus-type proteins, suggesting that Rhesus-type transporters recruit ammonia rather than ammonium (see also above) or have an alternative, as yet unrecognized, ammonium-binding site. Accordingly, genetic complementation experiments indicate that recruitment and transport of ammonia/ammonium by Nitrosomonas europaea Rh50 and E. coli AmtB proteins are not identical [81]. Furthermore, N. europaea Rh50 [58,81] and other Rhesus-type proteins [82] are low affinity (millimolar range) high capacity transporters, whereas investigated bacterial MEP clade transporters have micromolar affinities and low capacity [54,61]. The different characteristics of AOA and AOB ammonia transporters might therefore contribute to the different substrate thresholds and ammonia uptake halfsaturation constants (Km) of characterized AOA and AOB strains, which, in turn, determine their adaptation to ‘low’ and ‘high’ ammonia concentrations, respectively [19,21]. We predict that functional and structural investigations of Amt family proteins will reveal additional differences between AOA and AOB, reflecting their respective lifestrategy and shedding light on their ecological niche. For example, ammonia transporter genes of N. maritimus and N. europaea are differentially expressed in response to ammonia starvation [72,83]. Although the levels of both Amt-1 and Amt-2 transcripts in N. maritimus decrease markedly, albeit at different rates, upon short-term ammonia starvation [72], N. europaea Rh50 transcripts increased twofold when cells were starved for 16 hours [83], clearly indicating that AOA and AOB cope with substrate shortage by using different molecular mechanisms. Investigation of the radically different envelopes and cytoplasmic membranes of AOA and AOB [42,84,85] may also bring insight into the function of the corresponding ammonia transporters and clarify their role in the adaptation of cultivated AOA and AOB to different ammonia concentrations. Concluding remarks and perspectives Although all cultivated members of AOA seem to share similar overall metabolism and are characterized by a conserved set of information processing genes [4], functional differences among AOA emerge from physiological [19] and comparative genome studies [43]. The nature and diversity of ecological resources supporting their growth and activity remain, however, mostly unknown (Box 1). Our investigation of the transporter gene complements of six different AOA shows that each strain possesses a specific set of transport systems that varies, even among closely related organisms, in the type and number of transporters present, which suggests that there is potential functional heterogeneity within this microbial group. This also suggests that individual AOA have varying abilities for the uptake of different classes of compounds, including organic molecules (e.g., urea, organic acids, and organophosphorus), likely reflecting differences in their local microenvironment

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Opinion Box 1. Outstanding questions  How does the composition of individual transporter complements (and genomes) vary across the entire phylogenetic range of AOA?  Do individual transporter complements of AOA reflect the chemical composition of their respective microhabitat?  Can the transporter complement of AOA be used to predict their ecological preferences?  Do differences between AOA transporter complements reflect functional variability or redundancy?  What compounds are transported by the putative organic substrate transporters and what is the relevance of the corresponding transport processes to the ecological success of AOA?  What is the metabolic function of ammonia uptake in AOA and AOB (e.g., nitrogen assimilation and fueling of the ammonia oxidation process) and what are the cellular functions (i.e., uptake and/or sensing) of the different types of ammonia transporters in AOA and AOB?  How does genetic variability of ammonia transporters encoded in different AOA and AOB genomes translate into functional differences (e.g., different affinities, conductance, or capacity to sense ammonia)?

and emphasizing a general lack of knowledge of their individual ecological niche. The overall composition of the six analyzed transporter gene complements could reflect the evolutionary relatedness of the investigated strains, suggesting that corresponding AOA clades form functionally cohesive groups of microorganisms. The pattern suggested by our analysis, however, needs to be confirmed by future comparative genome analyses, including a larger sample of AOA genomes representing a broader range of clades. Follow-up analysis of the frequency of transporter genes in various AOA populations, the detection of gene loss and acquisition (e.g., horizontal gene transfer), and the characterization of AOA microhabitats will provide new insight into the links between AOA genomic diversity and ecological preferences and will clarify the molecular events leading to the adaptation of AOA to different environments [43,86]. Investigation of individual transporter families will most likely reveal additional levels of functional variability between different AOA, as indicated by our examination of their ammonia transporters. Analyzed AOA genomes encode two types of ammonia transporters that are likely to differ in their affinity for ammonium and cellular function. Sequence and structural variations in the ammonia transporters of different AOA, as well as differences in the number and type of ammonia transporters and associated regulatory proteins, could support the variable ammonia concentration preference and tolerance in these organisms [21,22]. Furthermore, the inferred high-affinity of AOA Amt-1 transporters could represent a crucial determinant of the adaptation of AOA to very low ammonia concentrations. We anticipate that experimental work addressing the function of AOA transporters and transport processes will increase our knowledge on the ecology and molecular cell biology of this important group of microorganisms. Acknowledgements We would like to thank Ricardo Alves and Jim Prosser for comments and suggestions on the manuscript. This work was supported by grant P25369 from the Austrian Science Fund and by research grant 09-EuroEEFG-FP034 from the European Science Foundation.

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