In many cases, the insects carrying bacteriocyte endosymbionts have specialized to a diet devoid of or scarce in certain nutrients; examples include aphids feeding on plant sap and tsetse flies feeding on mammalian blood (6
). While plant sap is very poor in certain nitrogen compounds, in particular in amino acids essential for the aphids (102
), the blood-sucking tsetse flies make do with meals lacking several essential vitamins (86
). Accordingly, the establishment of a symbiosis with bacteria may have enabled these insects to specialize to these food resources and thereby to occupy ecological niches which, without the assistance of the metabolic abilities of these bacteria, would have been impossible to colonize efficiently. In agreement with the specific nutrient composition of their diets, previous work has suggested that Buchnera aphidicola
may provide essential amino acids to the aphid hosts whereas Wigglesworthia glossinidia
was thought to synthesize vitamines of the B group for the tsetse flies. Several results of these early experiments have been confirmed and extended by modern genome technology (see below) (2
However, food specialization of the host insects is not obvious in all cases. For example, carpenter ants (Camponotus
spp.) generally feed on a complex diet composed of dead and alive insects, bird excrement, and sweet food wastes. Despite their complex diet, these animals are endowed with bacteriocytes carrying obligate intracellular bacteria of the genus “Candidatus
). On the other hand, a recent survey of ants living in tropical rain forest canopies has shown that at least in this geographical region, ants, including many Camponotus
species, can be considered to be “secondary herbivores” since they may feed mainly on plant or insect exudates and are not predators or scavengers (24
). In fact, there seems to be a general tendency in members of the genus Camponotus
to feed on honey dew derived from sap-sucking insects, at least in certain seasons. It is possible that the endosymbiosis developed in these ants at a time where the animals were feeding mainly on such a specialized diet. In this scenario, the endosymbiotic bacteria of many “modern” Camponotus
species with a less specialized diet may be an evolutionary relic of a former nutrient-based relationship. On the other hand, since little is known about the diet of many Camponotus
species in nature and of seasonal changes in the food sources during the year, it is conceivable that there are ephemeral periods during which certain nutrients such as honey dew may be predominant. For survival during such periods, the animals may need the bacteria to enrich the restricted diet; concomitantly, a strong selection may favor the retention of relevant amino acid and other biosynthetic pathways in the endosymbionts. In addition to assistance in nutrient provision, the bacteria may provide other benefits for the animals. Since ants are social insects which have developed complex interaction strategies with each other and require a high hygiene standard in their nest, it is possible that the endosymbiotic bacteria are essential not only for the individual animals but also for purposes relevant at the colony level; e.g., they may contribute to the chemical language of the animals by assistance in the biosynthesis of trace pheromones or they may be engaged in the biosynthesis of antimicrobial compounds, as recently shown for a symbiosis of an extracellular actinomycete with leaf cutter ants, which protects the fungus gardens of these ants from attack by a pathogenic fungus (22
Currently, the genome sequences of five bacteriocyte endosymbionts are available (2
). These include the genomes of three Buchnera
species resident in the aphids Acyrthosiphum pisum
, Baizongia pistacea
, and Schizaphis graminum
, the genome of Wigglesworthia glossinidia
resident in tsetse flies; and that of “Candidatus
Blochmannia floridanus,” the endosymbiont of the carpenter ant Camponotus floridanus.
The genome sizes of these organisms vary between 615 and 705 kbp. With the exception of functions involved in translation, ribosome structure, and biogenesis, genome reduction has concerned all other functional categories currently classified in the COG database (Clusters of Orthologous Groups of Proteins; http://www.ncbi.nlm.nih.gov/COG/
) by comparison to the free-living Enterobacteriaceae
such as Escherichia coli.
In the following, we focus mainly on aspects concerning the primary metabolism of these microorganisms.
Consequences of an Obligate Intracellular Life for the Central Intermediate Metabolism
Glycolysis and citric acid cycle.
Glycolysis, in which glucose is oxidized to pyruvate, is the major catabolic pathway of sugar utilization and is conserved in all kingdoms of life. This pathway is also present in Buchnera and “Candidatus Blochmannia.” Accordingly, both organisms have a sugar-phosphotransfer import system (PTS) which may enable an efficient uptake of glucose, mannose, or related sugars and their subsequent oxidation, indicating that they take up hexoses from their host cell as an important energy and carbon source. Consistent with the presence of a PTS, no hexokinases are present in these endosymbionts. Interestingly, the Wigglesworthia genome encodes all glycolytic proteins but lacks the gene to encode phosphofructokinase (PfkA), which is the key enzyme in glycolysis. However, Wigglesworthia has retained transketolase and transaldolase of the nonoxidative branch of the pentose phosphate pathway. Wigglesworthia should therefore be able to oxidize hexoses to pyruvate, although without an energy yield (see below). In agreement with the lack of the glycolytic pathway and the oxidative branch of the pentose phosphate pathway (see below), it does not encode any obvious sugar uptake system. It is therefore tempting to assume that Wigglesworthia does not oxidize hexoses for energy generation. Instead, and in contrast to Buchnera and “Candidatus Blochmannia,” the enzymes of the Embden-Meyerhoff-Parnass pathway seem to be used in gluconeogenesis rather than glycolysis, because fructose bisphosphatase (Fbp) is present, which is the key enzyme of gluconeogenesis (see below). For energy generation, Wigglesworthia may therefore mainly oxidize amino acids or other organic compounds derived from the host cell (Fig. ).
FIG. 1. Glycolysis, TCA cycle, and gluconeogenesis in the different endosymbiotic bacteria. In Buchnera and “Candidatus Blochmannia,” glucose is oxidized to acetyl-CoA, while in Wigglesworthia, the pathway works in the opposite, gluconeogenetic (more ...)
All three endosymbionts encode the pyruvate dehydrogenase complex and are able to oxidize pyruvate to CO2
and acetyl coenzyme A (acetyl-CoA), although Wigglesworthia
appears to be the only endosymbiont which is able to synthesize CoA from panthotenate and therefore to generate acetyl-CoA without the assistance of the host cell. In contrast, Buchnera
Blochmannia” have to rely on their host cell for the supply of this essential coenzyme. No regular citric acid cycle is present in the endosymbionts. “Candidatus
Blochmannia” and Wigglesworthia
have lost the C2
-fixing steps of the citric acid cycle, while most energy-yielding reactions, i.e., those catalyzed by α-ketoglutarate dehydrogenase to fumarase, are present. Although both microorganisms have lost the malate dehydrogenase encoded by the mdh
gene, a dissimilatory malate:quinone oxidoreductase (Mqo) is present which may participate in the cycle by production of oxaloacetate and in energy generation by feeding electrons to the ubiquinone pool of the respiratory chain. This enzyme was shown to contribute to the citric acid cycle in E. coli
, although its role is not well understood since it cannot entirely substitute for malate dehydrogenase (128
). Thus, the citric acid cycle of “Candidatus
Blochmannia” and Wigglesworthia
starts with α-ketoglutarate and seems to end with oxaloacetate. Consistent with the presence of a glutamate transport system, GltP, a secondary carrier, or the GltJKL ATP-binding cassette (ABC) transporter, respectively (9
), it is possible that transamination of glutamate to aspartate, catalyzed by AspC and using oxaloacetate as a cosubstrate, takes place, thus closing the cycle. In Wigglesworthia
oxaloacetate can also be used for gluconeogenesis. Interestingly, in Buchnera
the complete citric acid cycle, except α-ketoglutarate dehydrogenase, is missing (Fig. ). α-Ketoglutarate dehydrogenase activity results in the production of succinyl-CoA, which is required for lysine biosynthesis. Since Buchnera
encodes neither an obvious α-ketoglutarate transporter nor a transaminase to generate α-ketoglutarate from gluta-
mate, the source of α-ketoglutarate is currently not known (see below) (Fig. ).
Acetyl-CoA produced by the endosymbiotic bacteria should therefore be used mainly for biosynthetic processes. In fact, “Candidatus Blochmannia” and Wigglesworthia can build up fatty acids from acetyl-CoA, whereas Buchnera lacks the relevant enzymes (see below). Buchnera and Wigglesworthia but not “Candidatus Blochmannia” have retained phosphotransacetylase (Pta) and acetate kinase (AckA) and may be able to generate ATP by the production of acetate from acetyl-CoA as an additional energy supply, which may compensate to some extent for the lack of glycolysis in Wigglesworthia and for the missing citric acid cycle in Buchnera (Fig. ).
All three endosymbionts are strictly aerobic bacteria. No genes involved in fermentative pathways could be found in either genome. As in E. coli
, the electron transport chain consists of a primary dehydrogenase and a terminal reductase, which are linked by ubiquinone (127
Blochmannia” and Buchnera
contain the nuo
operon, which codes for NADH dehydrogenase I (Ndh I). This enzyme couples substrate oxidation to proton translocation by acting as a proton pump. In contrast, Wigglesworthia
contains only the ndh
gene, which codes for NADH dehydrogenase II (Ndh II). This enzyme does not couple substrate oxidation to proton translocation. The electrons from both NADH dehydrogenases are transferred to ubiquinone, which finally donates them to cytochrome o
oxidase. Cytochrome o
oxidase again acts as a proton pump, which for Wigglesworthia
appears to be the only proton pump of the respiratory chain. All three species contain typical F0
-type ATP synthases. Figure summarizes the features of the respiratory chains of the three microorganisms as deduced from their genome sequences.
FIG. 2. Electron transport chains of the endosymbiotic bacteria. In Buchnera, the electron transport chain consists merely of NADH dehydrogenase I (alternative designation, NUO) and cytochrome o oxidase (CYO). As indicated in red, ubiquinone (UQ) cannot be synthesized (more ...)
Due to the lack of the tricarboxylic acid TCA cycle in Buchnera
and glycolysis in Wigglesworthia
, the energy yield differs strongly between the three endosymbionts, rendering “Candidatus
Blochmannia” the fittest and Buchnera
the least effective (Fig. ). However, it is not known if this reflects differences in the energy requirement or supply of these bacteria. Interestingly, although a manganese-containing superoxide dismutase (SodA) is present in all endosymbionts, other detoxifying systems, including catalase, are missing. If is therefore possible that the generation of toxic oxygen species and free radicals during respiration contributes to the higher mutation rate of these bacteria than of their free-living relatives (47
). Another surprising feature is the complete lack of ubiquinone biosynthetic genes in Buchnera
, which must obtain this essential electron carrier from its host organism (112
FIG. 3. Energy yield and proton translocation. Buchnera, “Candidatus Blochmannia,” and Wigglesworthia are shown in the colors indicated in the graphic. The right panel summarizes proton translocation in the respective organisms. In Buchnera, a (more ...) Pentose phosphate pathway.
A major purpose of the pentose phosphate pathway is the generation of NADPH, which serves as reducing agent in many endergonic biosynthetic pathways such as fatty acid and nucleotide biosynthesis. The pathway consists of two distinct branches. In the oxidative branch, glucose-6-phosphate is oxidized and decarboxylated to ribulose-5-phosphate and NADPH is generated. First, glucose-6-phosphate dehydrogenase generates 6-phosphogluconolactone, which is converted enzymatically by 6-phosphogluconolactonase to 6-phosphogluconate, although this reaction may also occur spontaneously. Then phosphogluconate dehydrogenase further oxidizes and decarboxylates its substrate to ribulose-5-phosphate, a central building block required, e.g., for nucleotide and cofactor biosynthesis. The nonoxidative branch of the pathway leads to the recovery of the starting substrate glucose-6-phosphate, by the concerted action of ribulose-5-phosphate epimerase and ribulose-5-phosphate isomerase as well as transketolase and transaldolase (Fig. ).
FIG. 4. Pentose phosphate pathway. In Wigglesworthia, the oxidative branch of the pentose phosphate pathway is missing and only the regenerative steps are present. Missing steps are shown in grey. The YbhE protein is thought to catalyze the conversion of d-glucono-1,5-lactone-6-phosphate (more ...)
Blochmannia” have a complete pentose phosphate pathway and also encode PTS sugar import systems. In contrast, and in agreement with the lack of any sugar uptake system, Wigglesworthia
lacks the oxidative branch of the pentose phosphate pathway which enables a direct oxidation of glucose-6-phosphate, although the nonoxidative branch of the pathway is maintained in this organism (Fig. ). Buchnera
Blochmannia” encode the genes for the first and third steps of the oxidative branch of the pathway, glucose-6-phosphate dehydrogenase and 6-phosphogluconatedecarboxylase, respectively. Interestingly, although the enzymatic activity of the phosphogluconolactonase (Pgl), the second enzyme of this pathway, which converts d
-6-phosphoglucono-δ-lactone to 6-phosphogluconate, has been described in E. coli
), no gene could be assigned to this enzyme activity. Until recently, the biological role of a 6-phosphogluconolactonase was unclear, because its substrate is very unstable and subject to rapid spontaneous hydrolysis. However, the delta form, 1-5, of the lactone is the only product of glucose-6-phosphate oxidation, which by intramolecular rearrangement subsequently leads to the gamma form, 1-4. Only the delta but not the gamma form hydrolyzes spontaneously, demonstrating that the gamma form is a “dead end.” Since only the delta form is a substrate for 6-phosphogluconolactonase, lactonase activity accelerates hydrolysis of the delta form, thus preventing its conversion to the useless gamma form (74
In a review, Cordwell (18
) proposed the investigation of genes of unknown function for Pgl activity, which are present in the genomic region of E. coli
between the modCEF
genes and the lambda attachment site, which according to classical mapping procedures should be the genome region carrying the pgl
gene. Interestingly, of the four genes with unassigned functions in this region, only the ybhE
gene is present in Buchnera
Blochmannia,” whereas it is absent from the Wigglesworthia
genome, which also lacks the other genes of the oxidative pentose phosphate pathway. It is therefore likely that the ybhE
gene encodes the missing Pgl enzyme of the oxidate pentose phosphate pathway. In fact, BLAST searches with YbhE reveal a weak similarity to a putative 6-phosphoglucolactonase from Bacillus cereus
, which was assigned this function on the basis of its sequence similarity to an enzyme (Pgl) from Pseudomonas aeruginosa
(data not shown). Reductive power required for anabolic processes in the form of NADPH can therefore be directly generated by Buchnera
Blochmannia” via the oxidative pentose phosphate pathway. All of these bacteria have retained a NAD kinase, and NADP can be generated by this enzyme. In addition, a few dehydrogenases which depend on NADP for their activity are present (Table ).
Dehydrogenases present in the endosymbiotic bacteria
Gluconeogenesis and LPS biosynthesis.
Retaining the gene for fructose bisphosphate phosphatase (fbp), only Wigglesworthia appears to be able to build up complex carbohydrates by gluconeogenesis. It is able to synthesize glucose from pyruvate, and it is the only endosymbiont encoding phosphoenolpyruvate (PEP) carboxylase, which allows gluconeogenesis starting from oxaloacetate (Fig. ). Oxaloacetate can be generated either from aspartate provided by the host cell through the action of aspartate transaminase AspC or by the malate-chinone oxidoreductase (Mqo) described above (Fig. ). Gluconeogenesis is not conserved in Buchnera and “Candidatus Blochmannia” due to the deletion of the fbp gene.
In line with the lack of gluconeogenesis, Buchnera
has lost nearly the entire genetic equipment required for lipopolysaccharide (LPS) biosynthesis, which in virtually all gram-negative bacteria is an essential structural feature of the outer membrane and determines many properties in their interaction with the environment. In contrast, “Candidatus
Blochmannia” and Wigglesworthia
, which are located in the cytoplasm, have retained several LPS biosynthetic functions. While Wigglesworthia
should be able to build up the sugar backbone of the LPS by gluconeogenesis, “Candidatus
Blochmannia” is endowed with a PTS and seems to rely on external sugar resources. The LPS of Enterobacteriaceae
typically consists of three parts: lipid A, the core oligosaccharide, and the O-specific polysaccharide. In E. coli
, lipid A biosynthesis starts with UDPGlcNac, which first undergoes a 3-O substitution and then an N substitution with β-hydroxymyristic acid. Subsequently, the diacyl derivative is dimerized and the UMP moiety is released. Next, the 1-phospho dimer is substituted by 2-keto-3-deoxy-mannooctonic acid (KDO) derived from CMP-KDO and the hydroxyl groups of β-hydroxymyristic acid are esterified with fatty acids and phosphorylated at C-4 (94
) (Fig. ).
FIG. 5. Lipid A biosynthesis. Biosynthesis pathways of lipid A in the different endosymbiotic bacteria are shown. Steps missing in the respective organisms are highlighted in grey; e.g., Buchnera is missing the entire pathway. In contrast, in “Candidatus (more ...)
Blochmannia,” the biosynthesis and membrane assembly of a basic LPS structure is possible. It is likely to be composed of a lipid A moiety linked to KDO (possibly KDO2
). In “Candidatus
Blochmannia,” the KDO2
moiety can be further modified by the addition of a fatty acid, probably a lauroyl residue (by analogy to E. coli
), to the distal glucosamine unit, because it encodes the LpxL (HtrB) acyltransferase. In E. coli
K-12, a second acyl residue consisting of a myristoyl residue is added by the LpxM acyltransferase, which is missing in “Candidatus
Blochmannia.” Interestingly, in Wigglesworthia
, both acyltransferases, LpxL and LpxM, are missing, although in general the presence of at least one of these acyl residues in the LPS is considered essential for the viability of the bacteria. However, there are also other examples in which the genome sequences do not reveal the presence of the respective acyltransferases, indicating that some uncharacterized enzymes may have related activities (96
In free-living enterobacteria, the lipid A moiety is further modified by the addition of heptoses. However, the heptose biosynthesis pathway is completely missing in Wigglesworthia
. In contrast, in “Candidatus
Blochmannia,” the heptosyl transferases WaaC (RfaC) and WaaF (RfaF) have been conserved and are involved in the modification of the LPS core with heptose. However, the heptose biosynthesis pathway which leads from sedoheptulose-7-phosphate to ADP-l
-mannoheptose is heavily impaired, since only the HldD (RfaD) and HldE (RfaE) proteins are retained whereas the isomerase GmhA is missing and GmhB (YaeD) is a pseudogene (Fig. ). In line with the degeneration of LPS biosynthetic enzymes, Buchnera
does not code for the outer membrane protein Imp (for “Increased Membrane Permeability”), which was recently shown to be implicated in the transport of LPS to the cell surface and which is highly conserved in most gram-negative bacteria (11
). In agreement with an apparently intact LPS core structure, the other two endosymbionts carry the imp
FIG. 6. Modification of LPS by heptoses. In the free-living Enterobacteriaceae, the LPS core is further modified with heptoses. All three endosymbionts apparently are unable to synthesize the respective heptoses. The biosynthesis and modification enzymes were (more ...)
In summary, Buchnera
cannot synthesize a typical outer cell membrane whereas Wigglesworthia
Blochmannia” still are able to build up a core LPS structure. Why did these bacteria experience such a reduction in their LPS structure? The lipid A moiety of the LPS molecule is an extremely potent toxin and, when released from the bacteria, can cause harm to a host animal. Interestingly, E. coli
acyltransferase mutants are viable, but pathogenic strains lacking the LpxM acyltransferase are attenuated in their virulence properties (115
). Therefore, it is tempting to speculate that the stable symbiotic integration of endotoxin-carrying bacteria into a eukaryotic host has required the detoxification of this potentially dangerous compound, which, in the case of Buchnera
, has even led to its complete removal from the bacteria. Whether the observed differences in the degree of degeneration of the LPS structure among the various endosymbiotic bacteria is due to their different locations within the eukaryotic host cell or to significant differences in the age of the respective symbioses is not known (see below). It is interesting that another intracellular bacterium of many arthropods, Wolbachia pipientis
, belonging to the alpha-group of the Proteobacteria
, has also lost its capacitiy to synthesize LPS (139
Consistent with the reduction in the potential of these bacteria to synthesize LPS, there is also a significant slimming of the murein biosynthetic pathways; however, this appears to be quite variable in these bacteria. “Candidatus
Blochmannia” and Wigglesworthia
are able to synthesize the amino sugars N
-glucosamine and N
-acetylmuramic acid, whereas Buchnera
strains have lost part of this pathway (Fig. ). However, since N
-glucosamine-1-phosphate is also produced by the host animals, the bacteria may be able to import this compound to produce the aminosugars required for murein biosynthesis. Table lists proteins and enzymes involved in peptidoglycan biosynthesis present in the endosymbionts. Based on the enzyme equipment, it is likely that all endosymbionts can synthesize a peptidoglycan structure, although the conservation of various biosynthetic enzymes, such as transpeptidases and transglycosylases, and of shape-determining scaffold proteins is quite variable among them. Moreover, Buchnera
appears to be much more impaired in its murein biosynthesis capacity, since, for example, RodA and the Mre and Mrd proteins, which are involved in the determination of bacterial shape, are missing. E. coli rodA
mutants form round, osmotically stable cells. MreB is part of an intracellular spiral scaffold, which assembles on the cytoplasmic face of the inner membrane. MreB mutants form spheroids or misshapen rods. In agreement with these findings, Buchnera
but not the other endosymbionts has lost its rod-like shape and the cells are round (142
FIG. 7. Synthesis of amino sugars. “Candidatus Blochmannia” and Wigglesworthia are able to build up UDP-N-acetylmuramate from fructose-6-phosphate. In Buchnera, the transition of d-glucosamine-6-phosphate to UDP-N-acetylglucosamine seems to be (more ...)
Proteins involved in cell wall biogenesis and cell division
It is interesting that, with the exception of Wigglesworthia
, the endosymbionts lack alanine racemase. These bacteria either use the l
variants of the amino acids for murein biosynthesis or are supplied with d
-alanine by their host; d
-alanine may be derived from the gut microflora or directly from the host. In fact, d
-amino acids have been detected in peptides of various cells from animals such as amphibians, snails, crustaceans, and spiders, which are able to generate the d
isomer from l
-amino acids by a posttranslational reaction (57
). With regard to amino acid biosynthesis, it is also interesting that Wigglesworthia
, which has lost most of these pathways, is still able to synthesize diaminopimelic acid (DAP), which is an intermediate of the lysine biosynthesis pathway, although lysine itself cannot be synthesized by Wigglesworthia.
DAP, however, is an important compound in cell wall biosynthesis because it is required for the cross-linking of the peptidoglycan chains.
The prediction that these endosymbiotic bacteria are still able to synthesize a murein layer is further supported by the fact that all of them encode lipoproteins which covalently link the peptidoglycan layer with the outer membrane. In addition, lipoprotein signal peptidases and parts of the LolABCDE lipoprotein release system are present, which in E. coli
is essential for survival (71
). Moreover, sequence similarities between the proteins of the LolCDE ABC transporter of E. coli
and the hypothetical YcfUVW proteins of Yersinia pestis
were recently noted (91
). Since several of the Ycf proteins are also present in the endosymbionts, they may have taken over the function of the missing Lol proteins in the release and placement of lipoproteins. It remains curious, however, that the otherwise essential lipoprotein-specific periplasmic chaperon LolA is missing entirely from all three sequenced Buchnera
strains. Possibly a gene of unknown function is substituting for LolA in Buchnera.
In contrast to free-living Enterobacteriaceae
, the endosymbionts lack all enzymes and transport systems required for the recycling of periplasmic peptidoglycan fragments which are generated during normal growth of bac-teria.
Fatty acid metabolism.
In E. coli
, fatty acid biosynthesis is carried out by a type II fatty acid synthase, a multienzyme complex encoded by the accABCD
). Acetate residues in their activated forms as acetyl-CoA and malonyl-CoA (generated from acetyl-CoA by acetyl-CoA carboxylase AccA) are linked to the enzyme complex as thioesters. Acetate is bound to the so-called condensing enzyme and malonate is linked to the acyl carrier protein (ACP). Next, by the activity of FabD, malonate is converted to acetoacetate via elimination of CO2
and condensation with acetate. Acetoacetate remains linked to ACP as a thioester. In the following steps, a NADPH-dependent reduction mediated by FabG, a dehydration step catalyzed by FabA and another reduction step performed by FabI follow, resulting in the production of a saturated fatty acid after several bouts of this reaction cycle.
In “Candidatus Blochmannia” the entire pathway is present, whereas in Wigglesworthia 3-oxoacyl-ACP synthase III, FabH, which catalyzes the first condensation step of acetyl-CoA with ACP, is missing. However, it is likely that this enzyme can be substituted by FabB, the 3-oxoacyl-ACP synthase I, enabling Wigglesworthia to perform a complete fatty acid biosynthesis. The situation in Buchnera is more complex, and strain-specific differences are found, although all sequenced Buchnera strains are probably no longer capable of fatty acid biosynthesis. All strains lack acetyl-CoA carboxylase, AccA, and FabH. In Buchnera strains APS but not SG or BP, FabD, which catalyzes the condensation of malonate with ACP, is also missing. Finally, FabA, catalyzing the dehydration of the growing fatty acid chain, is absent from all Buchnera strains, although both reductases and the acyl carrier protein are still present. In conclusion, in line with the fact that “Candidatus Blochmannia” and Wigglesworthia are both able to synthesize complex lipids such as phospholipids or LPS, they are also able to build up fatty acids from acetyl-CoA. In fact, they have retained virtually the same biosynthetic capability as E. coli K-12 and can synthesize saturated and unsaturated fatty acids. In contrast, Buchnera is severely impaired in fatty acid biosynthesis. Since Buchnera very probably has to import phospholipids from the host organism (see below) and does not require fatty acids for LPS biosynthesis, it may not need its own fatty acid biosynthesis machinery and the respective pathways may be in the process of degeneration. Interestingly, all three endosymbionts are unable to oxidize fatty acids for energy generation, since the enzymes required for β-oxidiation are missing entirely.
The cytoplasmic membrane of E. coli
consists of several phospholipids, mainly phosphatidylethanolamine, which makes up 70 to 80% of all phospholipids, and phosphatidylglycerol. A minor but important component is cardiolipin (21
). The building blocks required for glycerolipid biosynthesis are acyl-CoA and glycerone phosphate, which is dehydrogenated to glycerol phosphate. In two consecutive steps acyl-CoA is transferred to glycerol phosphate by two different acyltransferases to yield 1,2-diacylglycerol-3-phosphate, which is subsequently activated by CTP to CDP-diacylglycerol, the major intermediate of glycerolipid metabolism. CDP-diacylglycerol can be metabolized to phosphatidyl-l
-serine and decarboxylated to phosphatidylethanolamine. Phosphatidylglycerol is synthesized from glycerol-3-phosphate and CDP-diacylglycerol, which react to give phosphatidylglycerol phosphate, which is converted to phosphatidylglycerol. Cardiolipin is made from CDP-diacylglycerol and phosphatidylglycerol by cardiolipin synthase (21
) (Fig. ).
FIG. 8. Phospholipid synthesis. In Buchnera the complete biosynthetic pathway except cardiolipin synthase is missing. If cardiolipin synthase is still active, the respective precursors, CDP-diacylglycerol and phosphatidylglycerol, have to be provided by the host. (more ...)
Wigglesworthia is equipped with the full set of genes necessary for the biosynthesis of all three glycerolipids, while in “Candidatus Blochmannia” only the plsB gene, encoding the first acyltransferase is missing. Thus, both organisms are very probably able to synthesize their own membrane phospholipids. The situation is strikingly different in Buchnera. In line with the degeneration of its potential involvement in cell wall and outer membrane biosynthesis, Buchnera has lost the ability to produce phospholipids and must import either the phospholipids themselves or their biosynthetic enzymes from the host cell (Fig. ). Therefore, although electron micrographs still show the presence of a gram-negative double membrane in Buchnera, the cell wall and outer membrane may structurally be very impaired by comparison with the apparently intact outer membrane of the cytosolic endosymbionts.
has retained only one enzyme involved in phospholipid biosynthesis, cardiolipin synthase, which is also present in the other endosymbionts. In E. coli
, anionic phospholipids, in particular cardiolipin, have several important functions and are involved in protein secretion (75
), recruitment of the replication initiatior protein DnaA to the membrane (46
), and provision of diacylglycerol moieties to outer membrane lipoproteins (118
). In eukaryotic organisms, cardiolipin is found in the inner mitochondrial membrane, where it is essential for mitochondrial function (72
). The conservation of cardiolipin synthase even in Buchnera
, which has lost all the other phospholipid biosynthetic functions, is intriguing and may suggest that this enzyme is very important for the symbiotic organisms, although its precursors must be imported from the host. It is also possible that the bacteria provide the host cell with cardiolipin to enhance the function of the mitochondria, which are probably required by the endosymbionts to satisfy their energy demands. Activation of mitochondrial activity by an endosymbiotic bacterium, Sitophilus oryzae
principal endosymbiont of weevils (S. oryzae
), which is phylogenetically closely related to the endosymbionts discussed in this review has recently been described, although it was suggested that this may be achieved by the supply of vitamins such as pantothenic acid and riboflavin to the host cell (41
). Finally, cardiolipin is known to function as a proton reservoir in particular for bacteria living in basic habitats and may therefore have a quite general importance for proton pumping in biological membranes (52
). Such adaptations may be critical, since in general the pH in the cytosol provides reducing conditions in a near-neutral environment (pH 6.8 to 7.1) whereas the extracellular environment in general has a near-neutral, slightly alcaline environment (pH 7.4).
The biosynthesis of purines proceeds in a series of 10 reactions by stepwise addition of functional groups to 5-phosphoribosyl-1-diphosphate, the activated form of ribose-5-phosphate (Fig. ). 5′-Phosphoribosyl-5-amino-4-imidazole carboxamide (AICAR) and IMP are important intermediates of the purine biosynthesis pathway. Pyrimidine biosynthesis is less complex and proceeds in three steps, with orotate formed from aspartate and carbamoyl phosphate. Orotate is then linked to 5-phosphoribosyl-1-diphosphate and decarboxylated to UMP (Fig. ).
FIG. 9. Purine biosynthesis. In Wigglesworthia, with the exception of the phosphoribosylglycinamide formyltransferase PurN, the complete purine biosynthetic pathway is present. In Buchnera and “Candidatus Blochmannia,” the first steps, leading (more ...)
FIG. 10. Pyrimidine biosynthesis. Buchnera is able to synthesize UMP, but most successive steps are missing, while “Candidatus Blochmannia” seems to need UMP as the starting material but, with the exception of trymidylate synthase (ThyA), can catalyze (more ...)
Purine and pyrimidine nucleotides are very abundant in the host cell, with ATP being one of the most abundant compounds in the cytosol. Nevertheless, regarding nucleotide biosynthesis, the endosymbionts have retained much of their autonomy. In fact, the purine and pyrimidine biosynthesis pathways of Wigglesworthia are virtually identical to those of E. coli K-12, although in the purine pathway the purN gene encoding the phosphoribosylglycinamide formyltransferase responsible for formylation of 5′-phosphoribosylglycinamide (GAR) cannot be detected (Fig. ). However, since all other enzymes appear to be well conserved, it is likely that this reaction is carried out by an alternative enzyme, leading to a complete purine biosynthesis pathway in this microorganism. Also, Buchnera and Blochmannia are expected to be able to produce purines, although in both bacteria the pathway transforming PRPP via GAR and 5′-phosphoribosylformylglycinamidine (FGAM) to the purine biosynthesis intermediate AICAR was lost. However, in contrast to Wigglesworthia, in Buchnera and “Candidatus Blochmannia” the histidine biosynthesis pathway has been conserved (see below) and so these organisms are able to synthesize AICAR starting from PRPP via phosphoribulosyl-formimino-AICAR phosphate which is then cleaved to AICAR as an intermediate spinoff product and to imidazole-glycerol-3-phosphate, which is then further processed to generate histidine. If this kind of combination of biosynthetic pathway turns out to be operating in vivo, which in free-living bacteria such as E. coli are strictly separate and have their individual specific regulatory systems, this would imply that during the evolution of these endosymbiotic bacteria the regulation of such pathways may have been changed or even abolished due to a constant environment. In fact, it no longer makes sense to regulate ATP phosphoribosyltransferase of the histidine pathway by end product regulation if the same enzyme is also required for purine biosynthesis.
Not all enzymes encoded by E. coli
required for interconversion of the nucleotides between their mono-, di-, and triphosphorylated and deoxy forms are present in the endosymbionts. However, it is likely that they can synthesize the whole set of nucleotides, since they have the capacity to synthesize all basic purine nucleotides such as IMP, AMP, and XMP (Fig. ). To compensate for this insufficiency, there are several possibilities: (i) broader specificities of the enzymes involved, which allow further parts of the nucleotide metabolism to occur, as exemplified in several other organisms with a reduced genome, e.g., Mollicutes
); (ii) activation of salvage pathways which allow sufficient compensation in a nutrient-rich environment (possibly provided by the host organism) (110
); and (iii) direct transport of nucleotides into the cytosol, similarly to several parasites. However, as mentioned above, on the basis of similarity to currently known nucleotide transporters, there are no indications in favor of this option.
Interestingly, in contrast to the other endosymbionts, “Candidatus
Blochmannia” shows a complete degeneration of its pyrimidine biosynthesis pathway. This implies that the ant endosymbiont requires the import of pyrimidines from its host organism. In fact, the nucleoside permease NupC is present in “Candidatus
Blochmannia.” This permease may satisfy the nucleoside demands of “Candidatus
Blochmannia,” since in E. coli
the homolog NupC has specificity toward pyrimidine nucleosides and their deoxy derivatives (Fig. ) (20
). Nucleoside transporters are apparently missing from Buchnera
Another interesting feature of pyrimidine biosynthesis is that Buchnera
is able to produce only the basic pyrimidine nucleotide UMP, but several subsequent steps generating cytidine and thymidine nucleotides and their deoxy variants are missing. Again, based on the mechanisms described above for the purine nucleotides, it is assumed that Buchnera
should be able to synthesize the respective compounds.
FIG. 11. Overview of the transport capacities in the different endosymbionts. In most cases, only the general transport capabilities of the endosymbiotic bacteria are shown. The dedicated transport systems may differ among the various bacteria. The Buchnera genome (more ...) Sulfur metabolism.
Sulfate must be reduced to sulfide in order to become incorporated into biomolecules. Insects generally are not able to reduce oxidized sulfur compounds and must rely on their diet to provide them. B. aphidicola APS and “Candidatus B. floridanus” are able to reduce sulfate via the APS-PAPS pathway. In this pathway, sulfate is first reduced to sulfite. For this purpose, ATP sulfurylase forms an energy-rich anhydride, adenosine-5′-phosphosulfate (APS), followed by the formation of 3′-phosphoadenosine-5′-phosphosulfate (PAPS), catalyzed by APS kinase. PAPS is then reduced to sulfite by PAPS reductase. Next, sulfite is reduced to H2S by sulfite reductase under consumtion of NADPH. H2S is immediately fixed to O-acetylserine, resulting in cysteine.
B. floridanus” not only is able to reduce sulfate via the APS-PAPS pathway but also has retained a sulfate-specific ABC transport system (CysAUW) (Fig. ), which very probably enables these bacteria to metabolize even trace amounts of sulfate (114
APS is also capable of sulfate reduction, but no known sulfate carrier was identified in its genome, indicating that sulfate is taken up by an unknown transport system. W. glossinidia
, B. aphidicola
SGR, and B. aphidicola
BP are not able to reduce sulfate via the APS-PAPS pathway, which indicates that their diet contains sufficient amounts of sulfur compounds to sustain their life or that sufficient amounts of reduced sulfur are provided by the gut flora.
Transport systems. (i) Small-molecule transport systems.
The majority of transport systems present in the endosymbiotic bacteria is constituted by secondary carriers, in which transport activity is driven by an ion gradient across the membrane (48
). Most remaining transport systems are ABC-type carriers, consisting of a membrane-spanning permease and an ATP-binding subunit which energizes transport by ATP hydrolysis (Fig. ) (108
). Interestingly, periplasmic substrate-binding proteins, which usually are an integral part of such transport systems, are missing in most ABC-type carriers of the endosymbiotic bacteria. Very few permeases catalyzing transport by a concentration gradient are found.
Only a single transport system is shared by all three endosymbionts: the secondary carrier for inorganic phosphate PitA (Fig. ) (40
). All three microorganisms also encode multidrug efflux systems with a broad substrate specificity. While “Candidatus
Blochmannia” and Wigglesworthia
contain EmrE, a secondary carrier which makes the bacteria resistant to a wide variety of toxic cationic hydrophobic compounds such as ethidium bromide, methyl viologen, and tetracycline, as well as intercalating dyes (67
share the Mdl multidrug efflux system, which is an ABC-type carrier (3
). Only Buchnera
contains the aquaglyceroporin GlpF involved in glycerol and water transport, which may also accept small uncharged organic molecules such as urea, glycine, and glycerolaldehyde as substrates (50
). In accordance with the advanced degeneration of its amino acid biosynthetic capability, Wigglesworthia
has retained several transport systems for amino acids, which are absent from “Candidatus
Blochmannia” and Buchnera
, e.g., BrnQ, a secondary carrier for branched amino acids, and SdaC, a secondary carrier for serine and threonine (111
Blochmannia” and Wigglesworthia
but not Buchnera
encode putative Na/H antiporters. In Wigglesworthia
the NhaA Na/H antiporter is present (136
), while “Candidatus
Blochmannia” harbors a homolog of the yjcE
gene, to which the function of a Na/H antiporter was assigned by similarity. In Buchnera
, a sodium-dependent NADH dehydrogenase (Rfn) is present, which couples NADH oxidation to the export of sodium (39
), indicating that sodium export is important for these bacteria, which may imply a detoxification function of these systems.
Surprisingly, only in Wigglesworthia
are two different potassium transporters found, the secondary carrier Kup and the ATP-dependent Trk system composed of several subunits. TrkA is a peripheral membrane protein bound to the cytoplasmic side of the membrane and is essential for transport activity. TrkH and TrkG are the K+
-translocating subunits, and TrkE seems to be involved in energy transfer (106
). Interestingly, in Wigglesworthia
only TrkA and TrkH are conserved, posing the question whether this system is functional. Potassium plays a central role in turgor maintenance in the free-living relatives of the endosymbiotic bacteria. However, since both transport systems present in Wigglesworthia
are characterized by a low affinity for potassium and since the high-affinity Kdp transport system is not present, a function of these transport systems in turgor maintenance is uncertain.
Manganese is essential for the activity of enzymes such as oxalate oxidase and glutamine synthetase. Manganese-containing superoxide dismutase is the principal antioxidant enzyme of mitochondria. A number of manganese-activated enzymes play important roles in the metabolism of carbohydrates and amino acids (64
). Manganese-containing enzymes such as pyruvate carboxylase and PEP carboxykinase play important roles in gluconeogenesis, and arginases required for the urea cycle also contain manganese. Despite the obvious importance of manganese for all living cells, only “Candidatus
Blochmannia” encodes a dedicated manganese carrier, MntH (68
). Additionally, the bf140 and bf141 gene products may constitute an ABC transport system which has significant similarity to other manganese carriers, so that there may be two manganese transport systems in “Candidatus
Blochmannia.” Since manganese is required for many enzyme activities, it is expected that the other endosymbionts import manganese via other systems that have not yet been identified. Both “Candidatus
Blochmannia” and Wigglesworthia
contain CorA, a permease specific for magnesium and cobalt (53
). In addition, “Candidatus
Blochmannia” encodes a putative cobalt efflux carrier, CorC, which also has a distinct similarity to hemolysin-related proteins (90
). Finally, an ABC carrier for zinc, the ZnuAB system (89
), is present in Buchnera.
Only very few carriers of unknown function are present in the endosymbiotic genomes.
In agreement with the minimal genomes present in these bacteria, only minor transport capacity was retained in their genomes. This is somewhat surprising, since one would expect massive metabolite fluxes between the symbionts and their host cells and therefore a large number of transport systems, as is the case, for example, in parasitic and symbiotic bacteria such as Chlamydia
, respectively (51
). Interestingly, Wolbachia pipientis
, a frequent obligate intracellular parasitic companion of many arthropods belonging to the alpha- Proteobacteria
, also has a reduced genome of 1.27 Mb and encodes only a very limited number of transport systems (139
). The small number of transport systems might therefore allow conclusions about the importance of the transported substrate for the metabolism of the bacteria: the PTS systems of Buchnera
Blochmannia,” which both are able to oxidize glucose by glycolyis, the glutamate transporters of “Candidatus
Blochmannia” and Wigglesworthia
, which both seem to feed glutamate into their truncated TCA cycle, and the transport systems for sulfate and pyrimidine nucleotides in “Candidatus
Blochmannia,” which have already been described in various sections of this review. It is also worth mentioning that Buchnera
encodes a much smaller number of transport systems than the other two species (Fig. ), which may either be due to the longer evolutionary history of this symbiosis or be due to its close association with the host-derived vesicle membrane.
(ii) Transport of macromolecules.
The Sec protein export system enables protein translocation across the inner membrane into the periplasmic space or the integration of proteins in the cytoplasmic membrane. It consists of SecB, a chaperone which guides the proteins to the exit place, SecA, a peripheral membrane protein with ATPase activity, the SecYEG translocase complex, and the SecDF accessory proteins. The SecDF and SecG proteins are not essential for protein export (27
). In the endosymbiotic genomes, the Sec system is conserved to different degrees (Fig. ). In Buchnera
, all components except the nonessential secDF
genes are present, strongly arguing for a functional Sec protein export system in this bacterium. In “Candidatus
Blochmannia,” most sec
genes are conserved except for secG
, which is nonessential, and secB
, which may be functionally replaced by a different chaperone. Thus, there also seems to be a functional Sec protein export system in “Candidatus
Blochmannia.” Similarly, in Wigglesworthia
, the secA
, and secG
genes are conserved, indicating a functional Sec protein export system.
Blochmannia” and Wigglesworthia
, the Tol-Pal system, consisting of the tolQRAB
, and ybgF
genes, is present (Fig. ). This system forms a protein complex which spans the periplasm and has components in the inner and outer membrane. Tol-Pal systems confer outer membrane stability and are also involved in the translocation of group A colicins and other macromolecules across the cell envelope (63
). Recently it was observed that tol-oprL
mutants of Pseudomonas putida
are impaired in growth with glycerol, fructose, and arginine as a result of a reduced transport capacity of the respective carbon source (65
). From these findings, it was concluded that Tol-Pal systems are also required for the proper functioning of certain transport systems. Buchnera
does not encode Tol-Pal-related functions. Since Buchnera
has a strongly reduced cell wall and is tightly surrounded by a host cell-derived membrane, this different environment may have made the Tol-Pal system dispensable for Buchnera
, whereas the cytosolic “Candidatus
Blochmannia” and Wigglesworthia
still require this system to stabilize their membranes and cell wall.
Despite their spatially restricted habitat within host cells, Buchnera
contain an almost complete flagellar machinery. It is possible that during certain developmental stages of the host the bacteria are motile, leave the bacteriocytes, and move to different tissues, such as the ovaries of their host. On the other hand, it may well be that these proteins serve as type IV secretion systems and are involved in the exchange of proteins or other macromolecules with the host cell. Protein secretion via a related system has recently been described for Yersinia enterocolitica
Specific Metabolic Adaptations of Bacteriocyte Endosymbionts of Different Insects
Biosynthesis of essential amino acids by the aphid endosymbiont Buchnera.
As already mentioned for the endosymbiosis of Buchnera
with aphids, previous work had suggested that these bacteria supply nutrients such as essential amino acids to their host insects. This is necessary because these insects thrive on a very unbalanced diet that has an excess of carbohydrates relative to nitrogen compounds including certain amino acids (25
). Insects apparently require 10 essential amino acids which are particularly rare in plant sap. Although difficult to interpret due to side effects and negative effects on other resident bacteria in the animals, previous attempts to cure the insects of their primary endosymbionts (resulting in aposymbiotic animals), for example by adding antibiotics to the diet, indicated the absolute requirement of these bacteria for the development and survival of the aphids (137
). Various metabolic studies which compared symbiotic aphids with aposymbiotic animals indicated that at least tryptophan, valine, leucine, and phenylalanine are provided by the symbiotic bacteria (26
). The genome sequences of these organisms now show that they have in fact retained the biosynthesis pathways for essential amino acids required to enrich the respective diet of their host animals (112
). As shown in Table , with the exception of methionine, B. aphidicola
APS has retained the biosynthetic pathways of all amino acids essential to insects such as arginine, valine, leucine, isoleucine, lysine, threonine, histidine, phenylalanine, and tryptophan. Instead, only very few nonessential amino acids can be synthesized by the bacteria. Depending on the host organism, the degeneration of the amino acid biosynthetic potential of Buchnera
species may still be an ongoing process, since in B. aphidicola
BP and SG even the cysteine biosynthesis pathway was lost. Buchnera
strains have also lost the ability to synthesize the precursors of some essential amino acids. For example, glutamate and aspartate have to be imported from the host prior to biosynthesis of the respective essential amino acids, although no respective transport system has been identified so far in Buchnera.
This is a striking example of the mutual interdependence of the metabolic activities of the host and its endosymbiont (112
). Interestingly, glutamate is the major nitrogen component of phloem sap (102
), in agreement with the lack of the respective biosynthetic capability in Buchnera.
This conservation of amino acid biosynthetic genes by the various Buchnera
strains is in marked contrast to what is observed in several parasitic bacteria including Chlamydia trachomatis
, Borrelia burgdorferi
, Mycoplasma genitalium
, and Rickettsia prowazekii
, which have lost their capacity to synthesize amino acids (4
). Although earlier work indicated that ammonium ions may be utilized by Buchnera
as a nitrogen source (25
), the genome sequences show that Buchnera
lacks crucial enzymes required for assimilation of nitrogen such as the glutamine synthetase and a glutamate synthase, indicating a strict dependency of these bacteria on amino acid-derived nitrogen.
Amino acid and cofactor biosynthetic capability of the endosymbiotic bacteriaa
Several plasmids which harbor amino acid biosynthetic genes have been identified in Buchnera
). There are plasmids encoding a putative anthranilate synthase (TrpEG), which catalyzes the first step of tryptophan biosynthesis. Other plasmids contain genes required for leucine biosynthesis (leuA
, and leuD
). Plasmid-mediated amplification of biosynthetic genes appears to be a more common phenomenon and has also been reported for other bacteria such as cyanobacteria, where genes required for cysteine biosynthesis can be plasmid located (85
), or for Vibrio anguillarum
and its histamine biosynthesis genes (5
). Gene amplification by transfer to plasmids with a high copy number may be a means of adaptation to specific new environments which may require high-level expression of certain gene products (100
). Surprisingly, some plasmids of Buchnera
that carry trpEG
contain mutations which should lead to a silencing of the expression level of anthranilate synthase, e.g., by mutations in the presumptive promoter regions or by pseudogene formation (7
). The biological significance of gene amplification and silencing for Buchnera
and the evolutionary forces leading to these events are not clear yet. As expected from their important function for host nutrition, it should be anticipated that the amino acid biosynthetic genes are subject to a strong host-level selection for their functionality; however, for the endosymbionts of several aphids of the genus Diuraphis
, it seems that pseudogenes are an ancient and universal feature of these bacteria and have arisen independently in several Diuraphis
). The assumption that amplification and subsequent silencing of amino acid biosynthesis genes is merely a direct reaction to the availability of the essential amino acid in the plant sap is therefore probably too simplistic, although it may explain this phenomenon to some extent. However, silencing or reduction activities may help in controlling the growth of Buchnera
or might be involved in reaching an adequate metabolic flux balance between the different pathways of the host and Buchnera.
In fact, recent data indicate that the genome of Buchnera
is highly polyploid and may be present at more than 100 copies per cell. Therefore, it is also possible that the bacteria lacking virtually all regulatory factors involved in the regulation of amino acid biosynthesis regulate the expression of certain amino acid pathways via the copy number of the respective plasmids (54
Cofactor biosynthesis by the tsetse fly endosymbiont Wigglesworthia as a possible key for its symbiotic function.
The gene content related to specific functions of the symbiosis is quite different in Wigglesworthia
, the endosymbiont of tsetse flies. Wigglesworthia
has retained many biosynthetic pathways required for cofactor and vitamine biosynthesis. There are about 62 genes involved in the biosynthesis of cofactors, prosthetic groups, and carriers. According to the genome sequence, Wigglesworthia
is able to synthesize pantothenate, biotin, thiazole, thiamine, flavin adenine dinucleotide, lipoic acid, pyridoxine, protoheme, nicotinamide, and folate (2
). The conservation of these biosynthetic pathways fits well with the fact that mammalian blood is quite poor in certain cofactors and vitamins, in particular vitamins of the B complex. The genome sequence nicely confirms previous experiments which already indicated that Wigglesworthia
might be implicated in providing the flies, in particular, with vitamins of the B complex (86
). In contrast to Buchnera, Wigglesworthia
has lost most of the amino acid biosynthetic pathways. It encodes factors engaged in a few steps involved in the biosynthesis of the nonessential amino acids glycine, glutamate, glutamine, aspartate, and DAP. Accordingly, although Wigglesworthia
encodes only very few transport systems, several of them apparently are involved in amino acid import (see above).
Metabolic interactions in the ant-“Candidatus Blochmannia” symbiosis.
Although ants of the genus Camponotus in general are omnivorous animals, they show a preference for honey dew and other sweet secretions from plants and animals, as well as for urea from animal exudates. The genome sequence of “Candidatus Blochmannia” indicates that this symbiosis also has a nutritional basis, with the bacteria having retained almost all biosynthetic pathways for amino acids which are essential for the host, with only the arginine biosynthetic pathway missing. The biosynthetic capability for nonessential amino acids, on the other hand, is largely reduced, and the most remarkable feature is the presence of tyrosine synthesis (Table ). Holometabolous insects need large amounts of aromatic amino acids such as tyrosine for the sclerotization and melanization of their cuticle during ecdysis, and it is likely that the bacteria contribute significantly to satisfy this demand. Since the conservation of entire biosynthetic pathways, despite the extreme genome reduction in these bacteria, may be indicative of an important role of the respective pathway for the symbiosis, it is tempting to speculate that bacterial tyrosine biosynthesis may have a prominent function for the ants. In accordance with the preference of the host for a diet rich in urea (N. Blüthgen, personal communication), a complete urease gene cluster is present in the bacterial genome. Urease hydrolyzes urea to produce CO2 and ammonia, the latter of which can be fed into amino acid metabolism by the activity of glutamine synthetase, which is also encoded by “Candidatus Blochmannia.” Another striking feature of “Candidatus Blochmannia” is the lack of arginine synthesis, although all other essential amino acids can be synthesized. This indicates that arginine is not limiting in this system and is degraded rather than synthesized. Arginine is an amino acid which is particularly rich in nitrogen and could serve as a nitrogen storage compound. It can be cleaved into ornithine and urea by arginases of the animal host or by a bacterial protein (Bf1253) of the arginase family. Thus, arginine could serve as a nitrogen store to keep amino acid synthesis running in times of high metabolic activity but no food uptake, e.g., during pupation.
Only two enzymes of the arginine synthesis pathway, carbamoyl-phosphate synthase (CarAB) and ornithin carbamoyltransferase (ArgI), are retained in “Candidatus Blochmannia,” enabling the bacteria to synthesize citrulline from ornithine. This includes the possibility that the endosymbionts take part in a urea cycle similar to that known of mammals, where the corresponding part of the urea cycle is localized in the mitochondria. However, this urea cycle would short-circuit the arginine-urea pathway suggested above. Therefore, if both reaction pathways are relevant to this symbiosis, they are likely to be operating during different stages of the life of the animal.
“Candidatus Blochmannia” has retained the glycolytic pathway and is able to synthesize acetyl-CoA from the oxidation of pyruvate. However, unlike the other endosymbionts, it is not able to synthesize acetate from acetyl-CoA and thus gain ATP. The only way to dispose of acetyl-CoA and recover Co A is to feed acetyl-CoA into fatty acid synthesis. Thus, “Candidatus Blochmannia” may supply its host not only with essential amino acids but also with fatty acids.