bc1 Complexes of anammox bacteria
In the proposed model for the anammox energy metabolism, the bc1 complex has a central role (Fig. 3). As it is crucial for many other organisms, mitochondria and chloroplasts (cyt b6f), the complex has been well investigated over the years, and the crystal structures were resolved for several species (see for reviews: Crofts, 2004; Osyczka et al., 2005; Crofts et al., 2006; Cooley, 2010; Mulkidjanian, 2010). Anammox bacteria have invented some interesting variations on the common theme. These are encoded in the K. stuttgartiensis genome by three gene clusters: kuste3096-3097, kustd1480-1485 and kuste4570-4574 (Fig. 6a). mRNA deep sequencing and proteomic analyses revealed that all three complexes are expressed at the transcriptional and protein levels, albeit in different amounts, kuste4570-4574 being the major species (Strous et al., 2006; de Almeida et al., 2011; Kartal et al., 2011a,b).
Figure 6. Gene cluster organization of the three bc1 complexes in the Kuenenia stuttgartiensis genome (a) and the proposed functional organization of their gene products (b). (a) Lengths of the gene products and the position of structural motifs are drawn to scale (aa: amino acids). (b) Putative heme b- and quinone (Q)-binding sites were derived from sequence comparison with respect to the canonical bacterial bc1 complex (left-hand figure), which is represented as its monomeric three-subunit complex for simplicity (see also text). Numbers refer to the kust gene numbers as in (a). Structural motifs are specified in the Figure. 2Fe2S, Rieske 2Fe-2S iron-sulfur cluster; cleavage site, N-terminal cleavage site; tat signal, twin-arginine translocation signal; TMH, transmembrane-spanning helix. The catalytic heme c is as specified in Fig. 4.
Download figure to PowerPoint
The canonical bc1 complex is a dimer of three core components: the Rieske 2Fe-2S iron-sulfur protein, a monoheme cyt c, and membrane-bound quinone-binding cytochrome b6 (Crofts, 2004; Osyczka et al., 2005; Cooley, 2010) (Fig. 6b). Cyt c and the Rieske iron-sulfur protein reside at the (periplasmic) p-side. After translation, the latter protein is transported by the twin-arginine (tat) translocon. Cyt b6 traverses the membrane eight times (TMH-A–H), at which four highly conserved histidines at the entrances and exits of TMH-B and TMH-D coordinate two heme b molecules, facing the cytoplasmic (bH) and periplasmic (bL) sides, respectively (Fig. 6b). The architecture resembles FdnI (cyt b556) described above with the major difference that cyt b6 can bind two quinones: one near the cytoplasm (Qi) and the other positioned at the periplasm (Qo). Binding of Qi is achieved by amino acids at the TMH-A membrane entrance and the loop in between TMH-D and TMH-E (Fig. 6b). Qo is sandwiched by amino acid stretches at the end of TMH-C and TMH-E. The catalytic function of the bc1 complex is coupling the oxidation of the two-electron carrier quinol with the reduction of two cyt c-type cytochromes. Hereby, two protons from QoH2 are released at the p-side. By an ingenious mechanism (‘Q-cycle’), proposed by Peter Mitchell (1975a,b), an oxidized Qi gets reduced and takes two protons from the cytoplasm, altogether giving an apparent net proton translocation stoichiometry of 4H+/2e. The mechanism, also known as ‘oxidant-induced reduction’ or ‘electron bifurcation’, exploits the large difference in midpoint redox potentials of the quinone-semiquinone (i.e. the one-electron reduced species) and the semiquinone-quinol couples.
Now, what is different about bc1 complexes in anammox bacteria? Nothing special regarding the Rieske iron-sulfur proteins in the three bc1 complexes; according to alignments, these are conserved with regard to other known species. The N-terminal signal sequences (tat signals) indicate that the encoded proteins are exported (Fig. 6). By use of the tat signal, fully assembled proteins – usually equipped with iron-sulfur clusters – are carried across the membrane (Coulthurst & Palmer, 2008; Yuan et al., 2010; Robinson et al., 2011). Kuste3097 appears to be a fusion protein between cyt b6 and a diheme cyt c at the C-terminal part. The N-terminal amino acid sequence is fully conserved with respect to known cyt b6 proteins. Apart from their Rieske factors, the arrangements of kustd1480-1485 and kuste4570-4574 are more complex (Fig. 6b). Rather than mono- or diheme cyt c, the kustd1480-1485 complex harbors an octaheme c-type protein (kustd1485). The presence of an N-terminal cleavage site suggests that it is exported after translation. In the kuste4570-4574 complex, even two multiheme proteins are present: a hexaheme c-type protein (kuste4573) and the HAO-like octaheme protein kuste4574 referred to previously. Their N-terminal leaders are indicative of protein export. Remarkably, in both complexes kustd1480-85 and kuste4569-74, cyt b6 is split into two genes with their division at similar positions. Kustd1481 and kuste4571 contain four TMHs each and both show a high degree of sequence identity (69%) to each other and to TMHs A-D in common cyt b6. The conserved regions include histidines involved in the coordination of the two b-type hemes and the first halves of the amino acid stretches related to Qo and Qi binding. Kustd1484 and kustd4572 are 47% identical, and both have five TMHs that are homologous to the C-terminal part of cyt b6. Sequence identities include the second halves of Qo-binding and – to a lesser extent – the Qi-binding motifs. It remains to be established whether the complexes will bind one or two quinones. The presence of genes coding for FAD-containing NAD(P) oxidoreductase in both complexes is surprising (Fig. 6a and b). Both gene products lack N-terminal cleavage sites indicating their residence in the cytoplasm. The question then is what these particular bc1 complexes are doing. Thinking of the bifurcation principle, which also applies to flavines and their semiquinones (Buckel & Thauer, 2012), it is possible to speculate that they couple the oxidation of (mena)quinol to the reduction of an electron acceptor of higher redox potential and one of low redox potential: NAD(P) ( = −0.32 V). This would solve the serious problem of NAD(P)H synthesis in an elegant way. In the most abundant complex, which harbors HAO-like kuste4574, the high-redox-potential electron acceptor might be nitrite with NO as the reduced product ( = +0.38 V). Again, this is what the reading of the genome and comparative literature analyses suggest. Ultimate proof will come from the isolation and characterization of these complexes.
The anammoxosome and energy metabolism
The anammoxosome is hypothesized to constitute the power station of the anammox cell. As already pointed out, there is compelling evidence for such a role: Major part of cytochrome c proteins is present in close proximity to the inner rim of the anammoxosome membrane, the HAO-like kustc1061 and HZS are specifically present inside the organelle, and most of ATPase-1 can be detected at its membrane (see above and Neumann et al., 2011). Still, conclusive evidence will only come from the isolation of the anammoxosomes and the demonstration that the conversion of ammonium and nitrite in these organelles results in the generation of pmf. This will be a formidable task, requiring dedicated methods to peel off the cell wall and outer membrane layers one by one.
If the anammoxosome is the power plant, the straightforward question is: For what purpose do these microorganisms place their bioenergetic machinery inside this organelle? With the current state of our knowledge, an answer can only be speculative. Denitrifying bacteria reduce nitrate to N2 via nitrite, NO, and N2O. Except for nitrate reduction by the nitrate reductase (Nar) system, all reactions reside at the periplasm, which represents the p-side of the chemiosmotic system (Richardson, 2000; Simon, 2002; Simon et al., 2008; Kraft et al., 2011). Nitrite, NO, and N2O reduction by themselves do not contribute to pmf generation, except when electron transfer in these reactions proceeds via the bc1 complex. Nevertheless, the high catalytic activities of these reductases enable the organisms to metabolize at high rate, which results in rapid growth. In contrast, anammox bacteria have to deal with the very sluggish HZS, and they have to express this enzyme at high levels to achieve appreciable metabolic activity. The periplasmic space presumably would not be sufficient to harbor the required amount of enzyme. Next, when localized at the periplasm, the enzymes are exposed to a large surface where intermediates can diffuse out. Indeed, it is well known that denitrifying bacteria release significant amounts of NO and N2O, especially during metabolic shifts as a result of environmental changes (pH, aerobic–anaerobic transitions) (Betlach & Tiedje, 1981; Baumann et al., 1996, 1997; Otte et al., 1996; Saleh-Lakha et al., 2009). These microorganisms may cope with these losses because they metabolize very rapidly and NO and N2O conversions contribute relatively little to energy conservation. However, for anammox bacteria, such a loss of intermediates would be detrimental. The ‘simple’ solution is the containment of the catabolism within a special organelle. Curvature of the membrane system provides extra space for respiratory enzymes, whereas NO and hydrazine that escape from the anammoxosome can partly diffuse back into the organelle. Membranes are the barriers for the passage of charged compounds even as small as protons, but with a flaw. Protons passively diffuse through the membrane at a certain rate, independent of the metabolic activity of the cell, thus dissipating the pmf. In mitochondria that operate at a high rate, leakage accounts for an estimated 10% energy loss (Haines, 2001). Again, this would also be detrimental for the slowly metabolizing anammox bacteria. Obviously, densely packed ladderanes might raise a better barrier to proton, NO, or hydrazine leakage than common lipids. Nevertheless, the finding that those intermediates can be detected outside the cell indicates that ladderanes are not perfect. Moreover, cell aggregation could also be beneficial, allowing anammox bacteria to share residual losses with their companions in the biofilm.
Substrate uptake and substrate trafficking
Anammox bacteria have to acquire their substrates, ammonium and nitrite in particular, from environments where the concentrations are generally low. CO2/bicarbonate as the sole carbon source is sufficiently present in most anaerobic systems, but it still has to be taken up. Nitrate is subjected to export or import depending on its role in the metabolism. The anammox cell is layered by three membrane systems. Consequently, substrates will pass two or three membrane layers to be used in anabolic (cytoplasm) or catabolic (anammoxosome) reactions, respectively. Considering that transcription and translation occur in the cytoplasm (‘riboplasm’), inherent problems are the proper sorting toward the anammoxosome or the other membrane systems, and the correct topological orientation of membrane-bound transport systems. Anammox bacteria possess the common bacterial set of protein export systems: the sec-translocon for the translocation of proteins that typically have a cleavable N-terminal TMH and the tat-translocon (Medema et al., 2010). Cleavage of the N-terminal leader is performed by type I signal peptidase that serves both the sec and tat translocons (Auclair et al., 2012). The encoding gene and all other components of the sec and tat systems are found as single copies in the K. stuttgartiensis genome. Next, genes encoding type II and type IV signal peptidases are detectable in the genome, also as single copies. Their presence suggests additional transport systems for subsets of proteins including those that are located extracellularly (Paetzel et al., 2002). The peptidases recognize specific amino acids in the N- or sometimes C-terminal region for cleavage. Despite detailed analyses, Medema et al. (2010) were unable to detect any features that could be related to targeting toward the anammoxosome or cytoplasmic membranes.
In the outer membrane of Gram-negative bacteria, numerous proteins function as porins and transporters (see for a recent review: Fairman et al., 2011). These outer membrane proteins (OMPs) are structured by 8-24 β-barrel strands forming a channel through which components can pass the membrane. Passage can be aspecific for a range of compounds, but many OMPs act as very specific molecular sieves. The specific ones are equipped with an ingenious ratchet mechanism, preventing substrate backflow out of the cell. In this way, compounds can be accumulated. Substrate-specific sieves can be expressed in high copy numbers. The analysis of the K. stuttgartiensis genome by the HHomp toolkit (http://toolkit.tuebingen.mpg.de/hhomp) reveals the presence of at least 25 different OMP-like proteins in the organism, belonging to different families, but most of these are still annotated as ‘unknown’ or ‘hypothetical’ (Speth et al., 2012b). Such an annotation also concerns kuste1878, which is in fact one of the most abundant proteins encountered during protein fractionation (N. M. de Almeida and W. J. Maalcke, unpublished result). Hence, the outer membrane of K. stuttgartiensis seems to be gated. This would leave anammox substrates to cross one or two more membrane barriers.
From bioenergetic and topological points of view, substrate trafficking in anammox bacteria represents an interesting case. Experimentally, these matters are still terra incognita, and the following discussion is solely based on genome analysis. Assuming that the outermost membrane is not fully closed, the compartment surrounding the cytoplasm/riboplasm represents a periplasmic space (p-side), while the cytoplasm itself is the negative (n-) side, which is alkaline in common bacteria. The anammoxosome constitutes a second p-side, presumably of acidic pH (Van der Star et al., 2010). Consequently, negatively charged molecules to be directed to the anammoxosome first have to be taken up against the pmf and subsequently benefit from it during export from the cytoplasm. The opposite holds for positively charged compounds. The net result should be an increase in concentration to serve the need of metabolic enzymes. As outlined next, anammox bacteria employ general sets of channel proteins. Thus, a similar protein should support both import (into the cytoplasm) and export (into the anammoxosome) of its substrate. Anammox bacteria rely on members of the major facilitator superfamily (MFS) for the transport of their key substrates (Fig. 8a). Strictly speaking, these are not transporters that derive energy from ATP hydrolysis or the pmf to drive processes, but they facilitate the channeling through a membrane (see for a review: Law et al., 2008). Importantly, MFS proteins work bidirectionally: They mediate both substrate import and substrate export. The resolution of the crystal structures of a number of key members of MFS proteins allowed a detailed insight into the molecular mechanism of substrate translocation. As a common principle, the membrane-spanning helices surround a pore with a narrow slit permitting passage of only the dedicated substrates. These substrates are scavenged in a vestibule at the entrance side. The channels occur in open or closed conformations to control transport.
Figure 8. Genes coding for different members of the major facilitator superfamily (MFS) in the Kuenenia stuttgartiensis genome (a) and the putative localization of their gene products in the anammox cell (b). In (a) the parts of the gene products showing high sequence identity with known MFS members, including the positions of transmembrane-spanning helices (vertical black lines), are highlighted in blue. N- or C-terminal segments localized in the cytoplasm or at its opposite side are colored orange and light blue, respectively. Lengths of the polypeptides are drawn to scale (aa: amino acids). Expression values are expressed as n-fold coverage of Solexa deep RNA sequencing of the K. stuttgartiensis transcriptome (Strous et al., 2006; Kartal et al., 2011b). Codes in (b) refer to the kust codes specified in (a). PII: PII protein involved in the regulation of ammonium metabolism, hisK: His Kinase, histidine kinase domain.
Download figure to PowerPoint
Inspection of the K. stuttgartiensis genome reveals that the organism uses members of the AmtB/Rh family for ammonium uptake. In fact, five distinct genes coding for such proteins were annotated, and these are all expressed (Fig. 8a) (Strous et al., 2006; Kartal et al., 2011b). Kustc1009, kustc1012, and kustc1015 are located in the same gene cluster, the latter two of which are preceded by PII proteins (kustc1010 and kustc1014). The cytoplasmic PII protein is the master controller of ammonium metabolism, a covalent linkage with UMP directing its activity (Arcondéguy et al., 2001; Leigh & Dodsworth, 2007; Forchhammer, 2008). Among others, the proteins in the non-UMP-bound state are able to dock to the AmtB exit, in this way plugging the transport channel (Andrade et al., 2005). The cytoplasmic C-terminal amino acids of AmtB are essential in this interaction. The homology to known AmtBs and the presence of PII proteins in the kustc1009-1015 gene cluster suggest that they are involved in ammonium transport, kustc1012, and kustc1015 in direct connection with their cognate PII proteins. Atomic structures of AmtB proteins from three different microbial species have been resolved (Khademi et al., 2004; Zheng et al., 2004; Andrade et al., 2005). All three share identical homotrimeric architecture of protomers, each having 11 TMHs. The comparison of the kustc1009, kustc1012, and kustc1015 amino acid sequences with those of structurally well-studied AmtBs demonstrates the conservation of essential structural and functional features. However, anammox proteins contain an additional N-terminal TMH, which might be cleaved during maturation. A second difference is that kustc1009 contains a much longer C-terminal amino acid stretch than the others (Fig. 8a), which possibly prevents PII binding. (one may note that kustc1009 lacks a cognate PII). By analogy with known systems, one might infer that kustc1012 and kustc1015 mediate ammonium import from the periplasm into the cytoplasm (Fig. 8b). This would leave kustc1009 as the ammonium channel into the anammoxosome. The K. stuttgartiensis genome encodes a fourth AmtB homologue, kuste3690. Its characteristic feature is a histidine kinase domain near the C-terminus that is predicted to be localized in the cytoplasm. The presence of the kinase domain suggests a signaling function for kuste3690. The fifth candidate, kustc0381, belongs to the Rh subfamily and shares all essential sequence motifs with the Nitrosomonas europaea Rh protein, which was identified as a CO2 transporter (Li et al., 2007). Like other MFS proteins, translocation seems to be regulated by open–closed state conformers. The molecular trigger behind its opening or closing is not understood, but Li et al. (2007) suggested that a currently unknown (metabolic) protein could play a role in its control. Intriguingly, kustc0381 has an N-terminal extension of ~230 amino acids. This extension follows a noncleavable TMH right after the translation start, and the TMHMM program (http://www.cbs.dtu.dk/services/TMHMM-2.0/) predicts its localization outside the cytoplasm. The polypeptide could have a role in substrate (CO2) accumulation, channel closing, or both. However, the lack of homology to a known protein(s) leaves its function elusive. Considering that CO2 fixation takes place in the cytoplasm (see next section), kustc0381 should be bound to the cytoplasmic membrane (Fig. 8b).
Transport of nitrite is facilitated by the FocA/NirC proteins, which are also members of the MFS. FocA proteins mediate formate import and export, while NirC proteins catalyze reversible nitrite transfer across the membrane at high rates (Moir & Wood, 2001; Falke et al., 2010). Unlike NirC, FocA proteins are well defined by crystal structures from three bacterial species (Wang et al., 2009; Waight et al., 2010; Lü et al., 2011). At the moment, a third branch (FNT3) has been added to the family represented by the hydrosulfide ion channel (HSC), the crystal structure of which was recently resolved from Clostridium difficile (Czyzewski & Wang, 2012). In this organism, HSC efficiently expels HS− from the cell. Like other anion channels, the protein is not very specific and it is capable of formate and nitrite translocation as well. FocA and HSC share many structural properties. Both form symmetric pentamers with each protomer comprising six TMHs. In addition, both proteins have many conserved amino acids in common that are related to substrate accumulation and transport. As many of these features are shared with NirCs, on the basis of amino acid sequences alone, it is rather difficult to predict what the physiological function of a particular protein is. However, their localization in the genome may give a hint: The channel proteins tend to be present in close proximity to genes coding for enzymes that deal with the metabolism of the molecules they translocate.
The genome of K. stuttgartiensis has five genes that code for FocA/NirC-like proteins, four of these occurring in tandem (kusta0004 and kusta0009; kustd1721 and kustd1720). Kusta0004 shares the highest sequence identity (67%) with kustd1721, while kusta0009 is most related to kustd1720 (66% sequence identity). A particular property of kusta0009, and to a lesser extent of kustd1720, is the presence of a relatively long N-terminal sequence predicted to be in the cytoplasm. In known FocA proteins, this part of the protein was demonstrated to undergo a pH-dependent structural change, thereby opening or closing the transport channel (Lü et al., 2011). Multiple sequence alignment places these four anammox proteins inbetween FocA and NirC proteins. The kustd1720-1721 tandem is localized immediately upstream of nitrate:nitrite oxidoreductase reductase (Nxr) locus (kustd1713-1699; see below), indicating that all four could have a role in nitrite transport with one partner localized on the anammoxosome membrane and the counterpart on the cytoplasmic membrane (Fig. 8b). The fifth gene coding for a FocA/NirC protein (kuste4324) is an orphan. Kuste4324 shows higher sequence identity to FocA than to NirC proteins. If localized on the cytoplasmic membrane, kuste4324 could be a good candidate to serve in formate uptake. Re-evaluation of the K. stuttgartiensis genome resulted in the identification of one more member of the FocA/NirC/FNT3 family, kuste3055, which is annotated as a conserved hypothetical protein. This protein is 38% identical to HSC, and all structural and functional amino acids are conserved. Remarkably, kuste3055 is by far the most highly expressed transporter (Fig. 8a). Considering (1) the aspecific substrate use of HSC, (2) its proficient properties in substrate export from the cytoplasm, and (3) the absence of a clear role for hydrogen sulfide in anammox metabolism, kuste3055 could be an additional shuttle to supply the anammoxosome with nitrite.
Nitrate plays a dual role in anammox metabolism. It is the product of nitrite oxidation, generating reducing equivalents for CO2 fixation, and it is the terminal electron acceptor for organic electron donor oxidation. Both nitrite oxidation and nitrate reduction are catalyzed by Nxr, which is most likely localized in the anammoxosome (see below). Such localization would need at least two transporters to take nitrate across the anammoxosome and cytoplasmic membranes. Indeed, genes coding for two of these are found in the genome of K. stuttgartiensis (kuste2308 and kuste2335) (Fig. 8a). The gene products of these are 66% identical to each other and belong to the NarK MFS of nitrate channel proteins. The family splits into two branches: NarK1 members act as high-affinity nitrate/H+ symporters, and NarK2 proteins are low-affinity nitrate/nitrite antiporters (Moir & Wood, 2001; Goddard et al., 2008). Multiple sequence alignments assign both kuste2308 and kuste2335 to the NirK1 subfamily. Unfortunately, the amino acid sequences do not give an indication about their specific localization, and the assignment made in Fig. 8b is provisional.
This ends our discussion on the wide-open status of our knowledge on the transfer of key substrates; the summarizing scheme presented in Fig. 8b is tentative at best. The ultimate picture on transport systems will be much more complicated due to the need of common inorganic compounds (phosphate, calcium, magnesium, etc.), the role of trace metals (Fe, Ni, Cu, Mn, Mo) in cellular metabolism, and the fact that the K. stuttgartiensis genome possesses a variety of putative uptake systems for organic compounds, including ABC transporters. Besides these, the genome of S. profunda contains many genes involved in oligopeptide transport systems, which suggests these bacteria are capable of oxidizing decaying organic matter (Van de Vossenberg et al., 2012). Again, the presence of transporters for organic compounds in the genomes of anammox bacteria indicates that these microorganisms are not just chemolithoautotrophic specialists, but are metabolically much more versatile.