TCA Cycle Replenishing Pathways in Photosynthetic Purple Non-Sulfur Bacteria Growing with Acetate

Purple non-sulfur bacteria (PNSB) are anoxygenic photosynthetic bacteria harnessing simple organic acids as electron donors. PNSB produce a-aminolevulinic acid, polyhydroxyalcanoates, bacteriochlorophylls a and b, ubiquinones, and other valuable compounds. They are highly promising producers of molecular hydrogen. PNSB can be cultivated in organic waste waters, such as wastes after fermentation. In most cases, wastes mainly contain acetic acid. Therefore, understanding the anaplerotic pathways in PNSB is crucial for their potential application as producers of biofuels. The present review addresses the recent data on presence and diversity of anaplerotic pathways in PNSB and describes different classifications of these pathways.

These pathways take part in catabolism and anabolism. Sugars are oxidized via a glycolytic pathway (and similar reactions) with pyruvate (PA) production. The reversible pyruvate-oxidoreductase oxidizes PA into acetyl-CoA and CO 2 . The TCA cycle then oxidizes acetyl-CoA, some organic acids, and acetyl-CoA-metabolized substrates into CO 2 . The reducing equivalents derived from the degradation of carbohydrates and TCA cycle substrates initiate the ATP synthesis via aerobic and anaerobic respiration. This is the catabolic function of carbon metabolism pathways.
The intermediates of these crucial metabolic pathways include 12 major precursor metabolites that are used to synthesize 70-100 cell building blocks (Figure 1). The most important ones are L-amino acids, nucleotides, activated sugars, and fatty acids. Thus, the biosynthesis stems from the degradation reactions.
In purple bacteria, TCA cycle is active during the phototrophic anaerobic growth and dark aerobic respiration. In case of anaerobic phototrophic growth, when the bulk of ATP is a product of the light-dependent electron transport, the TCA cycle performs cellular biosynthesis and participates in the constructive metabolism (by converting one group of central building blocks into another for further biosynthesis, Figure 1). In addition, the carbon dioxide released during the oxidation of substrates in the cycle can act as C1units for synthesizing certain groups of compounds. For example, the photoheterotrophic growth of Rps. palustris using acetate transforms 22% of acetate into CO 2 , 67% of which is then fixed into the cellular material via ribulose bisphosphate carboxylase/oxygenase (Rubisco) [3]. then fixed into the cellular material via ribulose bisphosphate carboxylase/oxygenase (Rubisco) [3].
TCA cycle activity is varied depending on the carbon substrate type of the medium [4] and on the physiological state of the culture. It effectively acts as a roundabout on a busy highway, redistributing molecules in a similar way to cars to avoid traffic congestion or disruption [5]. Carboxylases are involved in this mutual conversion (reconstruction), e.g., amino/fatty acids being reconstructed into the carbohydrate precursors by the TCA cycle reactions [1]. Cells can assimilate acetate as a sole organic substrate in the case of anaplerotic pathways replenishing the oxaloacetate (OAA) pool. The PNSB produce polyhydroxyalcanoates, a-aminolevuline acid, bacteriochlorophylls a and b and molecular hydrogen. During the anoxygenic photosynthesis, PNSB use organic acids as electron donors. Taking into account the fact that many organic waste TCA cycle activity is varied depending on the carbon substrate type of the medium [4] and on the physiological state of the culture. It effectively acts as a roundabout on a busy highway, redistributing molecules in a similar way to cars to avoid traffic congestion or disruption [5]. Carboxylases are involved in this mutual conversion (reconstruction), e.g., amino/fatty acids being reconstructed into the carbohydrate precursors by the TCA cycle reactions [1]. Cells can assimilate acetate as a sole organic substrate in the case of anaplerotic pathways replenishing the oxaloacetate (OAA) pool.
The PNSB produce polyhydroxyalcanoates, a-aminolevuline acid, bacteriochlorophylls a and b and molecular hydrogen. During the anoxygenic photosynthesis, PNSB use organic acids as electron donors. Taking into account the fact that many organic waste waters contain acetate as the key organic acid, scrutinizing anaplerotic pathways in PNSB is essential.
Here, we overview the latest data on the presence and diversity of anaplerotic pathways in PNSB, and describe different classifications of these pathways.

Part 1: TCA Cycle Replenishing Pathways
For cells using acetate as a sole organic substrate, the TCA cycle should be sustainable. An anaplerotic pathway replenishing the oxaloacetic pool should function due to the outflow of the TCA cycle intermediates. Evidently, the existence of a single anaplerotic pathway is necessary and sufficient. However, there is evidence on several anaplerotic pathways functioning simultaneously in one bacterium [6,7]. Currently, several anaplerotic pathways replenishing the OAA pool are known: the glyoxylate pathway, the ethylmalonyl-CoA pathway, pyruvate synthase in combination with carboxylating enzymes [8], the citramalate cycle [9,10], and the methylaspartate cycle [11,12]. Carbon dioxide produced via the TCA cycle can be used not only in the pyruvate synthase reaction with the following PA carboxylation, but also in autotrophic CO 2 fixation [13]. Under the conditions of bacterial growth with acetate or butyrate used as the only organic substrate, the main function of CO 2 -fixing reactions is believed to be maintaining the redox balance of the cell [14,15].
The glyoxylate cycle is the most common pathway for replenishing the OAA pool in the TCA [25]. In a functional TCA cycle ( Figure 2, reactions 1 to 13), the presence of this anaplerotic pathway depends on two enzymes: isocitrate lyase, ICL (EC: 4.1.3.1), and malate synthase (EC: 2.3.3.9) (Figure 2, reactions 14 and 15). The full glyoxylate cycle is a combination of glyoxylate shunt and some TCA cycle reactions, and includes reactions 1-3, 11-13, and 14, 15. One full turn of the glyoxylate cycle produces a TCA cycle intermediate (succinate) and a precursor molecule (glyoxylate) following the malate synthesis from glyoxylate and acetyl-CoA (reactions 14, 15, Figure 2). Both of them maintain the concentration of oxaloacetate during TCA cycle intermediate outflow. Glyoxylate shunt is believed to be non-functional in anaerobic growth [26,27]. However, it is active in anaerobic bacteria, including phototrophs, provided that ferredoxin is present as a cofactor, and pyruvic acid (and α-ketoglutarate) can be produced by reductive carboxylation [28,29].
Several PNSB (Rhodobacter sphaeroides, renamed as Cereibacter sphaeroides [30]; Rsp. gelatinosus, renamed as Rubrivivax gelatinosus [31]; Rs. rubrum; Phaeospirillum fulvum, renamed as Magnetospirillum fulvum [30]) do not have ICL activity [6,7,32,33]. It should be noted that the domain Bacteria has been substantially re-classified (www.bacterio. net, accessed on 17 February 2020). As a result, many strains were renamed. In this review, we use the names after the authors of the publications with a new name in parentheses at the first mention. Some of them do not have malate synthase activity (Rubrivivax gelatinosus [33]).
The genomic analysis shows that the ethylmalonyl-CoA pathway is the main anaplerotic pathway for the genus Paracoccus, allowing the replenishment of the oxaloacetate pool [42]. Of note is that the key genes of two anaplerotic pathways (the glyoxylate cycle and the ethylmalonyl-CoA pathway) were found in P. denitrificans when the cells grew with acetate as the only organic substrate [42]. The quantities of these enzymes were different in different growth phases. However, neither the ICL gene deletion nor that of the crotonyl-CoA reductase/carboxylase gene were lethal for the acetate assimilation by this bacterium. Each of the anaplerotic pathways was found to give the organism a specific advantage. The mutants' analysis revealed that the ethylmalonyl-CoA pathway in P. denitrificans apparently improves the biomass yield, whilst the glyoxylate cycle enables a faster growth on a medium with acetate being the only organic substrate. Utilizing the succinate, both strains with deletions grew similarly to the wild type. However, the strain without ethylmalonyl-CoA pathway showed a longer lag phase.
The authors [42] compared the translational activity of ccr and icl genes (encoding crotonyl-CoA reductase/carboxylase and ICL, respectively) in P. denitrificans grown with succinate or acetate. In the bacterial cells grown with succinate, only the ccr preserved their quantity at the low basal level.
In Rba. capsulatus, which also has both the glyoxylate cycle and ethylmalonyl-CoA pathway genes, the ICL activity had patterns similar to the ccr in P. denitrificans when growing on acetate at an uncontrolled pH [40]. However, when the optimal pH level was maintained, the ICL activity in the acetate culture increased similarly to those of icl in P. denitrificans [37].
The recent data show that this particular cycle can be combined in the ICL + PNSB as in other bacteria with the glyoxylate cycle.
The methylaspartate cycle [11,52,53]. The reaction sequence of this metabolic pathway was first demonstrated for the haloarchaeon Haloarcula marismortui ( Figure 4). The methylaspartate cycle was elucidated combining proteomics and the enzyme activities measurements with the H. marismortui [11]. Using the gene deletion mutants of H. hispanica, enzyme assays and metabolite analysis, the authors bridged the knowledge gaps by an unambiguous identification of the genes encoding all characteristic enzymes of the cycle [54].
In the methylaspartate cycle, acetyl-CoA is transformed to glutamate via reactions of the TCA cycle and glutamate dehydrogenase. The rearrangement of glutamate into methylaspartate and its subsequent deamination leads to mesaconate (2-methylfumarate) production. Mesaconate is then activated to mesaconyl-CoA (2-methylfumaryl-CoA), which is hydrated to β-methylmalyl-CoA, and β-methylmalyl-CoA is finally cleaved to propionyl-CoA and glyoxylate. Propionyl-CoA carboxylation leads to the methylmalonyl-CoA and subsequently to succinyl-CoA production, thus completing the cycle, whereas the condensation of glyoxylate with another molecule of acetyl-CoA yields malate, the final product of the methylaspartate cycle [11]. The enzymes of this pathway are discussed below. In the methylaspartate cycle, acetyl-CoA is transformed to glutamate via reactions of the TCA cycle and glutamate dehydrogenase. The rearrangement of glutamate into methylaspartate and its subsequent deamination leads to mesaconate (2-methylfumarate) production. Mesaconate is then activated to mesaconyl-CoA (2-methylfumaryl-CoA), which is hydrated to β-methylmalyl-CoA, and β-methylmalyl-CoA is finally cleaved to propionyl-CoA and glyoxylate. Propionyl-CoA carboxylation leads to the methylmalonyl-CoA and subsequently to succinyl-CoA production, thus completing the cycle, whereas the condensation of glyoxylate with another molecule of acetyl-CoA yields malate, the final product of the methylaspartate cycle [11]. The enzymes of this pathway are discussed below.
A functioning methylaspartate cycle is expected to require a high intracellular glutamate concentration, because the affinity for substrate (Km) of methylaspartate ammonialyase for methylaspartate in H. marismortui was determined as 26 ± 5 mM, and the equilibrium of the preceding glutamate mutase reaction is not on the side of methylaspartate (Km = 0.093 at 30 °C) [55]. Indeed, the cytoplasmic glutamate concentration in the acetategrown H. marismortui cells was 35 ± 5 mM (compared with 6 ± 1 mM in acetate-grown, ICL + H. volcanii) [11].
It was shown that methyaspartate and glyoxylate cycles are evenly distributed in haloarchaea [54]. Interestingly, 83% of the species using the methylaspartate cycle also possess the genes for polyhydroxyalkanoate biosynthesis, whereas only 34% of the species with the glyoxylate cycle are capable of synthesizing this storage compound. The authors suggest that the methylaspartate cycle is shaped for the polyhydroxyalkanoate utilization during carbon starvation, whereas the glyoxylate cycle is probably adapted for growing on substrates metabolized via acetyl-CoA. Interestingly, some haloarchaea possess genes for both cycles in the genome [54]. This suggests the possibility that the pathways may be adapted to fulfill various functions or allow microorganisms to adapt to a fast-changing environment. A functioning methylaspartate cycle is expected to require a high intracellular glutamate concentration, because the affinity for substrate (Km) of methylaspartate ammonialyase for methylaspartate in H. marismortui was determined as 26 ± 5 mM, and the equilibrium of the preceding glutamate mutase reaction is not on the side of methylaspartate (Km = 0.093 at 30 • C) [55]. Indeed, the cytoplasmic glutamate concentration in the acetategrown H. marismortui cells was 35 ± 5 mM (compared with 6 ± 1 mM in acetate-grown, ICL + H. volcanii) [11].
It was shown that methyaspartate and glyoxylate cycles are evenly distributed in haloarchaea [54]. Interestingly, 83% of the species using the methylaspartate cycle also possess the genes for polyhydroxyalkanoate biosynthesis, whereas only 34% of the species with the glyoxylate cycle are capable of synthesizing this storage compound. The authors suggest that the methylaspartate cycle is shaped for the polyhydroxyalkanoate utilization during carbon starvation, whereas the glyoxylate cycle is probably adapted for growing on substrates metabolized via acetyl-CoA. Interestingly, some haloarchaea possess genes for both cycles in the genome [54]. This suggests the possibility that the pathways may be adapted to fulfill various functions or allow microorganisms to adapt to a fast-changing environment.
There has been no evidence in favor of the methylaspartate cycle functioning in PNSB. Analysis of genomes of Rba. capsulatus SB1003 and 9 strains of Rsp. palustris did not reveal any genes necessary for this pathway in bacteria [13,39].
The citramalate cycle ( Figure 5) was found in the purple non-sulfur bacterium Rsp. rubrum [9] and later proposed for Rba. sphaeroides 2R [56]. In this pathway, acetyl-CoA and PA are condensed into citramalate, which is converted to mesaconate. The latter is converted to mesaconyl-CoA, which is further converted to methylmalyl-CoA. Methylmalyl-CoA is cleaved into glyoxylate and propionyl-CoA. Glyoxylate can be involved in the TCA cycle through a malate synthase reaction or, alternatively, used for the biosynthetic needs of the cells grown with C4-substrates. In turn, propionyl-CoA is converted into PA during a series of reactions with the succinyl-CoA formation step, which completes the cycle.
Leroy [7] reported that in the Rs. rubrum S1H, citramalate formed from PA and acetyl-CoA may not be a metabolite of the citramalate cycle, but an intermediate compound of the branched chain amino acid biosynthesis/degradation pathway (isoleucine and valine, ILV synthesis pathway) or a novel alternative anaplerotic acetate assimilation pathway through PA and part of the ILV biosynthesis pathway [7,51,57]. These pathways are activated during the acetate assimilation. It should be noted that once grown with the substrates such as malate or fumarate, the citramalate-dependent route of isoleucine synthesis does not function in Rs. rubrum even in mutant strains with an inactivated CBB cycle [58]. In this case, the redox balance is maintained by the threonine-dependent synthesis pathway ILV. The authors detected the reversal of some TCA cycle enzymes, which carried the reductive flux from malate or fumarate to α-ketoglutarate. This pathway and the reductive synthesis of amino acids derived from α-ketoglutarate are likely to be important for the electron balance. This suggestion is supported by the fact that adding α-ketoglutarate-derived amino acids to the medium prevented the Rs. rubrum CBB cycle mutant growth when a terminal electron acceptor was absent [58]. The flux estimates also suggested that the CBB cycle mutant preferentially synthesized isoleucine using the reductive threonine-dependent pathway instead of a less-reductive citramalate-dependent pathway. Interestingly, unlike a similar mutant strain of Rps. palustris with an inactivated CBB cycle, mutant Rs. rubrum was not capable of growing with succinate. There has been no evidence in favor of the methylaspartate cycle functioning in PNSB. Analysis of genomes of Rba. capsulatus SB1003 and 9 strains of Rsp. palustris did not reveal any genes necessary for this pathway in bacteria [13,39].
The citramalate cycle ( Figure 5) was found in the purple non-sulfur bacterium Rsp. rubrum [9] and later proposed for Rba. sphaeroides 2R [56]. In this pathway, acetyl-CoA and PA are condensed into citramalate, which is converted to mesaconate. The latter is converted to mesaconyl-CoA, which is further converted to methylmalyl-CoA. Methylmalyl-CoA is cleaved into glyoxylate and propionyl-CoA. Glyoxylate can be involved in the TCA cycle through a malate synthase reaction or, alternatively, used for the biosynthetic needs of the cells grown with C4-substrates. In turn, propionyl-CoA is converted into PA during a series of reactions with the succinyl-CoA formation step, which completes the cycle. Leroy [7] reported that in the Rs. rubrum S1H, citramalate formed from PA and acetyl-CoA may not be a metabolite of the citramalate cycle, but an intermediate compound of the branched chain amino acid biosynthesis/degradation pathway (isoleucine and valine, ILV synthesis pathway) or a novel alternative anaplerotic acetate assimilation pathway through PA and part of the ILV biosynthesis pathway [7,51,57]. These pathways are activated during the acetate assimilation. It should be noted that once grown with the substrates such as malate or fumarate, the citramalate-dependent route of isoleucine synthesis does not function in Rs. rubrum even in mutant strains with an inactivated CBB cycle [58]. In this case, the redox balance is maintained by the threonine-dependent synthesis pathway ILV. The authors detected the reversal of some TCA cycle enzymes, which carried the reductive flux from malate or fumarate to α-ketoglutarate. This pathway and the reductive synthesis of amino acids derived from α-ketoglutarate are likely to be important for the electron balance. This suggestion is supported by the fact that adding α-ketoglutarate-derived amino acids to the medium prevented the Rs. rubrum CBB cycle mutant growth when a terminal electron acceptor was absent [58]. The flux estimates also Later, additional reactions of conversion of acetyl-CoA to propionyl-CoA, involving pyruvate synthase and part of the pathway of biosynthesis/degradation of IVL, were revealed [59]. The authors called this pathway methylbutanoyl-CoA. The ethylmalonyl-CoA pathway and methylbutanoyl-CoA pathways appear to be simultaneously used by Rs. rubrum.
The citramalate pathway has no potential for functioning in the studied strains Rps. palustris [13]. The possibilities of the citramalate cycle functioning in Rba. capsulatus have not been clearly defined, since the data on the presence of enzymes catalyzing 2methylfumarate formation from (S)-citramalate and (S)-citramalyl-CoA conversion into (S)citramalate are not available [39]. Additional research is necessary to clarify the distribution of this cycle in purple bacteria.
The reversible pyruvate:ferredoxin oxidoreductase (or pyruvate synthase). In strict anaerobes, acetyl-CoA is generally transformed into PA by pyruvate:ferredoxin oxidoreductase, the same enzyme that functions in the reversed TCA cycle (rTCA cycle), the DC/HB cycle, and the reductive acetyl-CoA pathway [60]. With no active glyoxylate cycle reported, green sulfur bacteria and heliobacteria use pyruvate synthase for acetate assimilation [8]. Pyruvate synthase synthesizes PA from CO 2 and acetyl-CoA produced through acetate uptake and the rTCA cycle during mixotrophic growth of green sulfur bacteria [20,61,62]. This PA formation pathway in combination with carboxylating enzymes can also function in facultative anaerobes growing in the absence of oxygen [8] since the enzyme is sensitive to oxygen. It has been shown that pyruvate:oxidoreductase (NifJ) can be used in Rs. rubrum S1H for direct absorption of acetyl-CoA through PA [7].
Since this enzyme is sensitive to oxygen, the acetate assimilation via the above route is not possible under the aerobic conditions; thus, alternative pathways exist in (micro)aerobic organisms. Furdui and Ragsdale [63] measured a significant carboxylating activity under physiological concentrations and showed that the reaction rate depends on the concentration of acetyl-CoA and PA only.
It has been shown that PA could be synthesized from acetate in nine examined strains of Rps. palustris via the reaction of pyruvate:ferredoxin oxidoreductase. Meanwhile, this pathway is transcriptionally active in acetate cultures of Rps. palustris CGA009 and some acetate cultures of Rba. capsulatus [13]. Whereas PA synthesis from acetyl-CoA and formate is possible in Rps. palustris strains BisB18 and BisA53, the genes coding proteins for these reactions were not expressed in the Rba. capsulatus cultures grown with acetate.
The CBB cycle. In this cycle, carbon dioxide is fixed to form C6-compounds. It can serve as a substrate for carboxylases; 3-PGA formed in this metabolic pathway under the glycolytic pathway enzymes can be further converted into PEP and PA. They can be subsequently carboxylated to malate or oxaloacetate the replenishing oxaloacetate pool of TCA cycle. The Rubisco, phosphoribulokinase and sedoheptulose bisphosphatase [64] are unique, i.e., functioning only in this cycle.
The key enzyme that fixes CO 2 in the CBB cycle is Rubisco. Currently, different forms of Rubisco have been found in microorganisms. Four main forms of Rubisco are distinguished [65]. Rubiscos of forms I, II, and III exhibit carboxylase and oxygenase activity, but for potentially different physiological purposes. Form III is distinguished into a separate category, since it is found only in archaea, having a unique ancient evolutionary history. Proteins of form IV include Rubisco-like proteins (Rubisco-like protein, RLP), catalyzing reactions that are important for sulfur metabolism. In many organisms, the function of RLP is unknown.
It should be noted that part of genes annotated as Rubisco subunits in the recently sequenced Rps. palustris strains display high protein sequence identity with the Rubiscolike proteins rlp1 and rlp2 [13]. Rba. sphaeroides has two forms of Rubisco. Form I consists of eight large and eight small subunits that encode rbcL and rbcS genes, respectively. Form II is a hexamer that consists only of large subunits encoded by either the rbcL gene or the rbcR gene [66]. Rba. capsulatus [67] and Rps. palustris [68] also have two forms of Rubisco. They are encoded by the genes cbbLS (form I Rubisco) and cbbM (form II Rubisco). Unlike these bacteria, Rsp. rubrum has only form II Rubisco, which consists of two subunits encoded by the cbbM gene [66,69]. Surprisingly, this protein was able to function not only as Rubisco, but also as an enolase in the methionine utilization pathway (a methionine salvage pathway, MSP) anaerobic conditions [70,71].
Mutant strains of Rba. sphaeroides that synthesize either form I or form II Rubisco grew in the photoautotrophic conditions, although slower than the wild type [73]. The phototrophic growth RubisCO gene-deletion strains of the Rs. rubrum and Rba. sphaeroides was observed when CO 2 was the only carbon source using inorganic electron donors less reduced than molecular hydrogen, i.e., thiosulfate or sulfide [92,93]. The authors suggested that there are two independent CO 2 fixation pathways that support photolithoautotrophic growth in purple non-sulfur photosynthetic bacteria.
Rubisco can catalyze a reaction of ribulose-1,5-bisphosphate and molecular oxygen (O 2 ) instead of carbon dioxide (CO 2 ) [94]. This explains the release of glycolate into the medium in the presence of molecular oxygen [94]. In cyanobacteria and algae, glycolate is either released into the medium or oxidized by the enzyme glycolate oxidase to glyoxylate, which can be converted to glycerate through the serine pathway [94]. In the glycolate pathway, one molecule of PGA is formed from two molecules of glycolate, while a molecule of CO 2 is released. Thus, during the functioning of Rubisco as oxygenase, oxygen uptake and carbon dioxide release occur. The combination of the light-dependent absorption of oxygen and the release of CO 2 is commonly called photorespiration [64]. However, in most purple bacteria, oxygen has an inhibitory effect on both Rubisco activity and its synthesis [66].
The synthesis and activity of Rubisco depends on the concentration of CO 2 in the medium: the amount of enzyme in cells increases with an increase in CO 2 [95]. The level of the CBB cycle enzymes activity in PNSB is different under different growth conditions and decreases in the presence of organic substrates [96,97]. The activity of this pathway is reduced in cells after the transition from the phototrophic growth using succinate to medium with acetate [7]. This may be due to the outflow of CO 2 to the synthesis of other significant intermediates from the acetyl-CoA (for example, carboxylation of acetyl-CoA with reversible pyruvate: oxidoreductase, as well as to the steps of carboxylation in the ethylmalonyl-CoA pathway). In some cases, PNSB fix significant amounts of CO 2 in the CBB cycle regardless of the organic compounds. For example, 22% of acetate carbon is converted to carbon dioxide and 67% of this carbon dioxide is fixed in the cell material through Rubisco during photoheterotrophic growth of the Rps. palustris using acetate medium [3]. Rubisco activity of Rps. palustris was repressed by chemoheterotrophic growth but was not decreased during photoheterotrophic growth [43]. It has also been demonstrated that the enzyme level of Form I increased significantly during photoautotrophic growth or during growth on a very reduced substrate such as butyrate in both Rba. sphaeroides [98] and Rba. capsulatus [99]. The CBB cycle is necessary during photoheterotrophic growth with succinate, malate, fumarate or acetate to maintain a pool of oxidized electron carriers. Rs. rubrum, Rs. fulvum, renamed as Magnetospirillum fulvum [30,100,101] and Rps. palustris CBB cycle phosphoribulokinase mutants that cannot produce ribulose-1,5-bisphosphate demonstrate an inability to grow photoheterotrophically on succinate unless an electron acceptor is provided or H 2 production is permitted [100]. CBB cycle mutants of Rs. rubrum, but not of Rps. palustris, grew photoheterotrophically on malate [100] or fumarate [55] without electron acceptors or H 2 production.
Wang D. and coauthors suggested an alternative model wherein disrupting Rubisco activity prevents photoheterotrophic growth due to the accumulation of toxic ribulose-1,5bisphosphate [91]. It was later demonstrated that the CBB cycle is still needed to oxidize electron carriers even in the absence of toxic ribulose-1,5-bisphosphate [100]. Considering available data, Rubisco regulation in PNSB is connected with CO 2 fixation, or with the maintenance of the redox balance in cells but not with TCA cycle activity. The reductive TCA cycle. Some rTCA cycle reactions can be considered as anaplerotic pathways replenishing TCA intermediates. They start from the reductive carboxylation of acetyl-CoA to PA catalyzed by ferredoxin-dependent pyruvate synthase. PA is converted to oxaloacetate [20] or PEP and further to oxaloacetate [102]. In PNSB with a complete set of TCA cycle enzymes, the presence of active pyruvate synthase (or pyruvate synthase and PEP synthase) in combination with carboxylating enzymes is sufficient to replenish the pool of its intermediates during the photoassimilation of acetate. So, the activity of pyruvate synthase (and PA or PEP carboxylatyng enzyme) as a part of rTCA could be considered as anaplerotic pathways replenishing the oxaloacetate pool. The same can be said about another pathway of autotrophic fixation of carbon dioxide-the dicarboxylate/hydroxybutyrate cycle [22] and the reductive glycine pathway [24].
The 3-hydroxypropionate bi-cycle ( Figure 6) is found in the green non-sulfur bacterium Chloroflexus aurantiacus [16,17]. It may serve as an anaplerotic pathway, replenishing the oxaloacetate pool. This autotrophic pathway of carbon dioxide fixation consists of two cycles [18] with common reactions (reactions 1-3, Figure 6). In the first cycle, (S)-malyl-CoA is formed from acetyl-CoA (reactions 1-10, Figure 6). Then, (S)-malyl-CoA is cleaved (reaction 11a) into acetyl-CoA (it is returned to the cycle) and glyoxylate. The latter is further converted in the second cycle reactions of the 3-hydroxypropionate pathway to form (3S)-citramalyl-CoA (reactions 11b-14, Figure 6). The synthesized (3S)-citramalyl-CoA is finally cleaved to PA and acetyl-CoA. As a result of the complete turnover of 3-hydroxypropionate bi-cycle PA molecule is formed from three bicarbonate molecules. Reactions 1-6 of 3-hydroxypropionate bi-cycle ( Figure 6, or in more detail reactions 1-9 in Figure 7) could replenish the TCA cycle. The step of converting propionyl-CoA to succinyl-CoA includes additional reactions discussed in Part 2.
The 3-hydroxypropionate bi-cycle is a way to coassimilate organic substrates such as glycolate, acetate, propionate, 3-hydroxypropionate, lactate, butyrate, or succinate [6]. The rate of CO 2 assimilation by carboxylation of organic substrates might even be higher than the autotrophic carbon assimilation rate. The 3-hydroxypropionate bi-cycle makes a balanced redox state of the cell possible, since CO 2 fixation consumes electrons. C. aurantiacus uses the 3-hydroxypropionate bi-cycle, together with the glyoxylate cycle, to channel organic substrates into the central carbon metabolism both in autotrophic and heterotrophic growth conditions (with and without oxygen). Only a fraction of acetate during photoheterotrophic growth on medium with acetate and bicarbonate was oxidized to CO 2 , probably in the course of the assimilation process.
The 3-hydroxypropionate bi-cycle of the Chloroflexi phylum evolved late in the Earth's history as a result of a series of horizontal gene transfer events. This explains the lack of geological evidence for this pathway based on the carbon isotope record [103]. Data on the activity of this cycle in PNSB are not available. This cycle does not take part in OAA pool replenishment in Rba. capsulatus [39]. Genes of virtually all enzymes participating in this pathway are present in the genomes of some strains Rps. palustris [13]. However, additional research to confirm the presence of this metabolic pathway in Rps. palustris is necessary.
The 3-hydroxypropionate/4-hydroxybutyrate cycle was found in Metallosphaera sedula [21]. This metabolic pathway ( Figure 7) begins with the carboxylation reaction of acetyl-CoA to malonyl-CoA. Some intermediates and carboxylation reactions of this pathway coincide with the reactions of the 3-hydroxypropionate cycle.
One complete turn of this cycle under the autotrophic growth leads to the formation of two acetyl-CoA molecules. One molecule is recycled in the biosynthesis of the cell material. In the presence of exogenous acetate, the functioning of enzymes of the 3hydroxypropionate bi-cycle ( Figure 7; reactions 1-9) is sufficient to replenish the pool of TCA cycle intermediates. Data on the activity of this full cycle in PNSB are not available.

Part 2: Integration of TCA Cycle Replenishing Pathways. Separation of Them on the Basis of Main Metabolites
Biochemical research has recently shown a surprising diversity in the central carbon metabolism. Amounting evidence indicates that anaplerotic pathways function in complex. This provides bacteria with additional advantages, such as an increased growth rate or higher efficiency of substrate utilization. For example, purple non-sulfur bacterium Rba. capsulatus does not have ICL activity during the shift from lactate to acetate medium [37]. Most probably, the growth is supported by the function of the ethylmalonyl-CoA pathway [39,41]. Once induced ICL in addition to the ethylmalonyl-CoA pathway boosts the growth rate of bacterium [37]. ICL − purple non sulfur bacterium in addition to the citramalate cycle [9] or alternative isoleucine-valine pathway [7] have an ethylmalonyl-CoA pathway and pyruvate:oxidoreductase which can be used for PA synthesis from acetyl CoA [7]. The presence of several anaplerotic pathways gives higher flexibility in TCA intermediates replenishment and redox balance regulation in Rs. rubrum.

Four Groups of Pathways
The data on the currently known enzymes show that anaplerotic pathways of replenishing the OAA pool have both common and variable chains of reactions [13,39]. The TCA cycle intermediates are formed through the stage of one of the four main metabolites being synthesized: glyoxylate, propionyl-CoA, and PA/PEP ( Figure 8). Further conversion of these metabolites into TCA cycle components ( Figure 9) is also versatile [39,40]. Based on this fact, a new classification of anaplerotic pathways was suggested [13]. Four groups of possible reactions giving essential intermediates were proposed: Group (I) includes pathways of glyoxylate formation. It consists of part of glyoxylate cycle reactions, photorespiration and glyoxylate formation pathway from glycine formed at one stage of the reductive glycine pathway.
Group (II) includes pathways of propionyl-CoA and glyoxylate simultaneous formation: namely, part of methylaspartate cycle reactions, part of citramalate cycle reactions and the set of reactions of ethylmalonyl-CoA pathway.
Group (IV) includes pathways of PA/PEP formation. This group consists of subgroup A (consisting of two pathways of PA formation from exogenous acetate and CO 2 ), subgroup B (including pathways of PA/PEP formation from the stored carbohydrates (glycogen)), and subgroup C (consisting of PEP/PA formation using CO 2 ).
Subgroup A combines two pathways: the first pathway is PA formation through pyruvate oxidoreductase; the second pathway is PA formation involving reversible formate dehydrogenase and reversible formate-C-acetyltransferase.
Subgroup C includes the PA/PEP formation pathway (through serine) from glycine synthesized in the reductive glycine pathway and PEP/PA formation from the CBB cycle intermediates.
Pathways of further conversion of synthesized glyoxylate, propionyl-CoA, PA/PEP into TCA cycle components ( Figure 9) also appear to be branched [13,39] The bacterium with all pathways represented in Figures 8 and 9 is unlikely to exist. However, this combination of all known anaplerotic pathways in general could give a key for a quick and simple search for the anaplerotic pathway(s) in a new bacterium of interest. Additionally, it could provide insight into the active anaplerotic pathway in new bacterium on the basis of metabolomic analysis.
Subgroup C includes the PA/PEP formation pathway (through serine) from glycine synthesized in the reductive glycine pathway and PEP/PA formation from the CBB cycle intermediates.
Pathways of further conversion of synthesized glyoxylate, propionyl-CoA, PA/PEP into TCA cycle components (Figure 9) also appear to be branched [13,39] 37-38, 39 (or 40a, 41-43, or 43-44)) and to β-D-Glucose-6-phosphate (reactions 37, 38, 40a, 41-42 (or 43)). Subgroup C includes the PA/PEP formation pathway from CO2: the PA/PEP formation pathway (through serine) from glycine synthesized in the reductive glycine pathway (reaction 75,79-84); and PEP/PA formation from the CBB cycle intermediates (reactions 21-32, 51, 50, 49). The scheme is based on the KEGG Pathway database and pertinent literature analyzed. The functional numbers of enzymes and the description of each function according to KEGG Orthology are presented in the tables under the same number as the catalyzed reaction illustrated in this figure. The bacterium with all pathways represented in Figures 8 and 9 is unlikely to exist. However, this combination of all known anaplerotic pathways in general could give a key for a quick and simple search for the anaplerotic pathway(s) in a new bacterium of interest. Additionally, it could provide insight into the active anaplerotic pathway in new bacterium on the basis of metabolomic analysis.

Enzymes Participating in Group I Reactions
Some glyoxylate cycle reactions. This pathway includes some of the TCA cycle reactions ( Figure 8, reactions 1-3, 11, 12, 13), the key enzyme of the glyoxylate cycle (ICL, EC: 4.1.3.1, Figure 8, reaction 14) and enzymes converting glyoxylate into malate (two pathways of glyoxylate conversion into TCA cycle intermediates are known), which are described below (Figure 9, reactions 1 or 2-3). Enzymes of TCA cycle and ICL are  Figure 8, reaction 14) and enzymes converting glyoxylate into malate (two pathways of glyoxylate conversion into TCA cycle intermediates are known), which are described below (Figure 9, reactions 1 or 2-3). Enzymes of TCA cycle and ICL are presented in Table 1. As a matter of fact, the same reaction can be performed by different enzymes (Table 1). Table 1. Pathways of glyoxylate formation including some glyoxylate cycle reactions (reactions 1-3, 14, 11-13) and TCA cycle enzymes (Figure 8, reactions 1-13). Figure 8 Function Photorespiration. This reaction sequence is shown in Figure 8 ( Tables 2 and 3, respectively) is either released into the medium or enters one of four known phosphoglycolate salvage pathways [105]: the glycolate pathway (the canonical plant-like C2 cycle), where one PGA molecule is formed from two glycolate molecules, releasing one CO 2 molecule [106]; the bacterial-like glycerate pathway; the malate cycle; and the oxalate decarboxylation pathway. Since the term photorespiration is ill-suited to describe the recycling of 2 phosphoglycolate in lightindependent autotrophs, it was suggested to use a more general term "Rubisco-related 2-phosphoglycolate salvage", or, for short, "phosphoglycolate salvage" [105,[107][108][109]. The glycolate pathway is confirmed for PNSB [66,67,94].

Reaction Number in
In the phosphoglycolate salvage pathway reactions, glyoxylate is formed from glycolate [40] with the enzymes shown in Table 2. Several different enzymes can catalyze the formation of glyoxylate from glycolate (reactions 17, 18-19 or 20, Figure 8,  Figure 8).

Enzymes Participating in Group II Reactions
This set of reactions lead to the formation of 2-methylfumaryl-CoA, which is converted into glyoxylate and propionyl-CoA via two reactions common for these pathways ( Figure 8 and Table 5 (Figure 8, reaction 68). The subsequent transformation of synthesized propionyl-CoA and glyoxylate into the TCA cycle intermediates could occur in several reactions described below. It should be noted that even in the absence of active pathways of propionyl-CoA conversion into the TCA cycle intermediates, ethylmalonyl-CoA pathway could have an impact on TCA pool replenishment via glyoxylate emerging simultaneously with propionyl-CoA. In this case, propionyl-CoA could be used for biosynthetic needs, since it is the main participant in odd-chain fatty acid formation during growth on acetate.   Table 5 presents information on enzymes capable of catalyzing these reactions.

Reaction Number in
Citramalate cycle reactions. This reaction sequence is shown in Figure 8 (reactions 52-56, Table 6; 67-68, Table 5). One of two molecules (propionyl-CoA or glyoxylate) should turn into PA to replenish the first substrate of the cycle (Figure 9). This cycle is not present in the KEGG PATHWAY database in explicit form, but the sequence is shown on a separate chemical reaction level [9,10].
Propionyl-CoA and glyoxylate are formed from 2-methylfumaryl-CoA in reactions common for the ethylmalonyl-CoA pathway and the methylaspartate cycle ( Figure 9, Table 5, reactions 67, 68). The conversion reactions of propionyl-CoA and glyoxylate to the intermediates of the TCA cycle or PA are discussed in the corresponding sections below. The PA consumed in the first reaction of the citramalate cycle is replenished by decarboxylation of oxaloacetate or malate (reaction 52a, 52b or 52c, Table 6).

Reaction Number
in Figure 8 Function

Enzymes Participating in Group IV Reactions
Group IV includes PA/PEP formation pathways and subdivided by three subgroups by substrates for PA/PEP synthesize.
Subgroup A consists of two pathways of PA formation from exogenous acetate and CO 2 (Figure 8, reactions 75-76 or 77).

Enzymes Participating in Group IV Reactions
Group IV includes PA/PEP formation pathways and subdivided by three subgroups by substrates for PA/PEP synthesize.
Subgroup A consists of two pathways of PA formation from exogenous acetate and CO2 (Figure 8, reactions 75-76 or 77).
The formate hydrogenlyase (FHL; formate dehydrogenase) enzyme complex is the key element of fermentative H2 production by E. coli [122], which catalyzes the disproportionation of formate to hydrogen and carbon dioxide: HCO − 2 + H + ↔ CO2 + H2. The 'forward' reaction (CO2 and H2 production from formate) under physiological fermentative conditions is observed [123,124]. The expression of active formate hydrogenlyase is repressed at a low formate concentration accompanied by a relatively high pH. However, theoretical arguments and experimental data indicate that this enzyme exists under certain conditions [125,126]. An evolutionary progenitor of formate hydrogenlyase on the early Earth could be responsible for a hydrogen-dependent CO2 fixation [127].
Pyruvate formate-lyase (PFL) is an oxygen-sensitive enzyme widespread in nature. This enzyme typically produces an additional ATP molecule (3 ATP molecules instead of two) during glucose fermentation [121]. Despite the reverse reaction of this enzyme being demonstrated in vivo, until recently its physiological function has been unknown. Zelcbuch et al. [128] demonstrated PFL-dependent co-assimilation of acetate and formate by the E. coli mutant strains unable to assimilate acetate through a glyoxylate shunt. In these experiments, the entire cellular pool of PA is derived from acetate and formate supporting the activity of PFL in H2 uptake reaction.
The oxygen-tolerant and NAD + -dependent formate dehydrogenase from Rba. capsulatus catalyzes the reduction of CO2 to formate [129]. The biochemical properties of this enzyme from the Rba. capsulatus has attracted a lot of attention [130,131].
The 'forward' reaction (CO 2 and H 2 production from formate) under physiological fermentative conditions is observed [123,124]. The expression of active formate hydrogenlyase is repressed at a low formate concentration accompanied by a relatively high pH. However, theoretical arguments and experimental data indicate that this enzyme exists under certain conditions [125,126]. An evolutionary progenitor of formate hydrogenlyase on the early Earth could be responsible for a hydrogen-dependent CO 2 fixation [127].
Pyruvate formate-lyase (PFL) is an oxygen-sensitive enzyme widespread in nature. This enzyme typically produces an additional ATP molecule (3 ATP molecules instead of two) during glucose fermentation [121]. Despite the reverse reaction of this enzyme being demonstrated in vivo, until recently its physiological function has been unknown. Zelcbuch et al. [128] demonstrated PFL-dependent co-assimilation of acetate and formate by the E. coli mutant strains unable to assimilate acetate through a glyoxylate shunt. In these experiments, the entire cellular pool of PA is derived from acetate and formate supporting the activity of PFL in H 2 uptake reaction.
The oxygen-tolerant and NAD + -dependent formate dehydrogenase from Rba. capsulatus catalyzes the reduction of CO 2 to formate [129]. The biochemical properties of this enzyme from the Rba. capsulatus has attracted a lot of attention [130,131].
Subgroup B includes pathways of PA/PEP formation from the stored carbohydrates (glycogen). The use of storage compounds is possible in periods of shift from the growth using TCA cycle intermediates to acetate as the sole organic substrate (several hours). The duration of the transition period is determined by the amount of storage compounds and the formation of the enzymes that are to be synthesized in sufficient quantity to adapt to new growth conditions. For example, the lag phase characteristic of the growth of Rs. rubrum with acetate with low bicarbonate concentration is also characterized by the mobilization of internal glycogen by the cell acting as a source of PA/PEP. Therefore, it may substitute the ethylmalonyl-CoA pathway as an anaplerotic pathway supporting the viability of bacteria [57]. Many bacteria use the stored carbohydrates for PEP/PA formation in the Entner-Doudoroff pathway (Figure 8 reactions 45-48, Table 3) along with the reactions of glycogen decomposition to β-D-glucose-6-phosphate (reactions 37, 38, 40a, 41-42 (or 43), Table 3) and in the Embden-Meyerhof-Parnas pathway (reactions 40, 39, 27/78, 26, 25, 24, 23, 51, 50, 49; Table 3) combined with reactions of glycogen decomposition to β-D-fructose-6-phosphate (reactions 37-38, 39 (or 40a, 41-43, or 43-44), Table 3).
Subgroup C includes the PA/PEP formation reaction using CO 2 . This consists of PEP/PA formation from the CBB cycle intermediates and the PA/PEP formation pathway (through serine) from glycine synthesized in reductive glycine pathway.
PEP/PA formation from the CBB cycle intermediates. This cycle includes reactions 21-32 shown in Figure 8 and corresponding enzymes ( Table 3). The main product of CBB in heterotrophic growth conditions is 3-PGA [132]. For consequent conversion of 3-PGA into 2-PGA and then into PEP and PA, Embden-Meyerhof-Parnas pathway enzymes could be activated, namely 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (EC: During the photoheterotrophic growth using acetate, lactate, malate, and succinate, PNSB produce carbon dioxide, since the carbon atom in the biomass is more reduced than in these compounds [133]. However, this does not mean that purple bacteria do not require exogenous carbon dioxide or carbonate ions. This follows from the fact that at elevated pH and a high content of Ca and Mg ions in the medium, the produced mineral carbon can participate in reactions of precipitation. It should be noted that exogenous carbon dioxide is required at high concentrations of organic acids in the growth medium (apparently it is not connected with the redox balancing of cells) or under bacterial photoheterotrophic growth conditions at high light intensities (discussed in Part 3).
In some cases, especially when the acetate cultures grow at the maximum growth rate, the role of the cycle might be shifted to the anaplerotic pathway for the replenishment of TCA cycle intermediates. This suggestion needs further experimental verification.
The PA/PEP formation pathway (through serine) from glycine synthesized in reductive glycine pathway (reaction 84, 88-93 Table 4, Figure 8). An alternative hypothetical variant of the reductive glycine pathway relies on the synthesis of PA from serine [24]. In this pathway, instead of the deamination step, serine can be synthesized from glycine and 5,10-methylen-THF (product of previous glycine formation). After that, serine can be converted into PA. This sequence of reactions in combination with pyruvate carboxylase can also replenish the pool of TCA cycle intermediates.
Glycerate Pathway for PEP Formation from Glyoxylate. The pathways of glyoxylate conversion through the reaction of the glycerate pathway were determined [138,139]. The reactions of this pathway are shown in Figure 9 (reactions 32-35, Table 8, simultaneously with pathways of PA/PEP conversion into TCA cycle intermediates (Table 9)). Two molecules of glyoxylate by glyoxylate carboligase condense into tartronate semialdehyde with CO 2 released (reaction 32). Tartronate semialdehyde reductase catalyzes the formation of D-glycerate from tartronate semialdehyde (reaction 33). D-glycerate is converted to 2-phospho-D-glycerate under the action of the glycerate kinase (reaction 34 PA formation step is converted to TCA cycle intermediates, Figure 9; more details in the section on the carboxylation of PA/PEP are given below). Table 8. Glyoxylate to malate converting enzymes (reactions 1 or reaction 2-3); PEP formation from glyoxylate in the glycerate pathway (reactions 32-35); OAA formation from glyoxylate in the β-hydroxyaspartate cycle (reaction [28][29][30][31]28). Figure 9 Function    , 18b)). Figure 9 Function Number, Enzyme Name and Number (KEGG Orthology Database)

Reaction Number in
19a, 19 and (Figure 9) 52a, 52c ( Figure 8 β-hydroxyaspartate Cycle for OAA Formation from Glyoxylate. The β-hydroxyaspartate cycle was discovered in the middle of the last century [140], and yet was only described in detail in 2019 [141]. In this cycle, one molecule of oxaloacetate is formed from two molecules of glyoxylate ( Figure 9 of the reaction 28-31, 28; Table 8). The enzyme aspartateglyoxylate aminotransferase catalyzes one reaction, which is both the first and the last reaction in the cycle (Figure 9, reaction 28): glycine (involved in subsequent reactions of the cycle) and oxaloacetate (the final product of β-hydroxyaspartate cycle) are formed from glyoxylate and aspartate. Glycine and the second molecule of glyoxylate under the action of β-hydroxyaspartate aldolase are converted to (2R, 3S)-β-hydroxyaspartate (Figure 9, reaction 29). From the latter, under the action of β-hydroxyaspartate dehydratase, iminosuccinate and H 2 O are formed (reaction 30). Iminosuccinate reductase catalyzes the formation of an aspartate molecule from iminosuccinate (reaction 31), which enters the first cycle reaction (reaction 28). One of its products is an oxaloacetate molecule synthesized from two glyoxylate molecules; in addition, glycine is formed as described above (one of the substrates of the second reaction of the cycle). The β-hydroxyaspartate cycle is present in the genomes of some Proteobacteria that also encode the CBB cycle [141], but the presence in PNSB needs additional investigation.

Pathways of Propionyl-CoA Conversion into Succinyl-CoA or PA
There are three pathways of propionyl-CoA conversion into TCA cycle intermediates replenishing OAA pool known up to date. The methylmalonyl-CoA pathway leads to the formation of succinyl-CoA from propionyl-CoA [142]). In other pathways (methylcitrate pathway and oxidative pathway via lactate), propionyl-CoA is converted into PA, which could yield malate or OAA in several reaction sequences, as described in the corresponding sections.

PA/PEP Carboxylating Enzymes (Anaplerotic Carboxylases).
Synthesis of the TCA cycle intermediates from PA or PEP requires their carboxylation, which could lead to (S)-malate or oxaloacetate formation ( Figure 9, reactions 19, 19a, 20-22;  The favorable direction of the malate dehydrogenase (oxaloacetate decarboxylating) reaction is decarboxylation. However, some malic enzymes demonstrated a pH-dependence of this favorable reaction direction (to PA or malate formation) [146]. In addition to this, malic enzyme was shown to be a key enzyme in replenishing TCA cycle intermediates in Synechocystis sp. PCC 6803, because it catalyzes PA carboxylation [147] [13]. However, in Rba. capsulatus, it is encoded by two genes.
Among the enzymes catalyzing the reversible PEP carboxylation to OAA (Figure 9, reaction 20), one PEP carboxykinase (EC: 4.1.1.49) gene is present in all studied Rps. palustris strains and Rba. capsulatus SB1003 [13]. Studies have described the contribution of this enzyme to PA and acetate metabolism [35,132]. This enzyme was shown to be able to catalyze the reverse reaction, i.e., C3-carboxylation, for example, in Rl. eutropha [148,149].
Transformation of (R)-malate to (S)-malate could be provided by enzyme having racemase activity (Figure 9, reaction 26; Table 9). The possibility of this reaction was demonstrated experimentally in Rba. capsulatus cultures [151]. Unfortunately, information on the coding gene (genes), properties and structure of this enzyme is not available.
Cis-Aconitate Formation from PA and Acetyl-CoA. A reaction sequence related to the itaconate metabolism ( Figure 9, reactions 15-17, 18 (or 18a, 18b); Table 9) could hypothetically take part in TCA cycle intermediate replenishment on the cis-aconitate level [40]. This path is considered to be the main means of assimilating itaconate in bacteria [152]. The itaconic metabolism acid has been elucidated in the ascomycetous fungus Aspergillus terreus [153], in the basidiomycetous fungus Ustilago maydis [154] via trans-aconitate, and in human macrophages via cis-aconitate. All these reactions, except for cis-aconitate decarboxylation to itaconate (reaction 18 or 18b), are considered to be reversible nowadays. As mentioned above, some carboxylyases (decarboxylases) direct the reaction depending on the environmental conditions (substrate/product concentrations, pH, etc.) [146,147]. The reversion of reaction 18 (or 18a) could lead to the formation of aconitate from acetate and PA. There are no sufficient data on the possibility of converting itaconate in reactions 18a and 18b accounted for scarce information on itaconate-converting enzymes. As of today, only a few enzymes from quite a low number of organisms have been characterized [155]. However, there are a few experimental studies into enzymatic activities of itaconate converting reactions.

Part 3: Alternative Functions of Anaplerotic Pathways
Evidently, the replenishment of the TCA cycle intermediates is vital for any microorganism, and purple bacteria are no exception. Nevertheless, the carbon metabolism in purple bacteria, as in other prokaryotes, is highly complex and flexible. That is why the anaplerotic pathways, in addition to TCA intermediate pool replenishment, could play different alternative roles. Most of them are also vital for the bacterium's survival, and improve its ability to withstand stress or compete with other species in the environment.
For example, an elevated concentration of acetate prevents the growth of E. coli and other bacteria [156]. There are several reports on the inhibiting effect of elevated acetate concentration for purple bacteria such as Rba. capsulatus [38,157] and Rs. rubrum [57]. This property of acetate is widely used in the food industry, which applies acetic acid as a preservative. Several explanations have been put forward to account for the reasons why a high acetate concentration inhibits microorganism growth. Among them are difficulty of the membrane's potential maintenance [158], increased intracellular osmotic pressure [159,160], and a disruption of carbon flows in central metabolism, which itself inhibits growth [161][162][163]. None of these hypotheses have been proven. The disruption of acetyl-phosphate showed some growth-inhibiting effects (~20%), and uncoupling the inhibiting effect plays only a limited role [156]. In all cases, anaplerotic pathways actively contribute to acetate consumption/degradation, with a decreased inhibitory effect of high acetate concentrations on bacterial growth [1,57].
Sustainable functioning of CO 2 -consuming anaplerotic pathways under a high acetate concentration can be supported by adding bicarbonate or carbon dioxide as an additional electron acceptor [3,15,164]). For example, the increase in acetate concentration from 1 to 100 mM increases the lag period and reduces the growth rate of Rba. capsulatus strain St. Luis [38]. Another strain of this bacterium could not grow at a high concentration of acetate without bicarbonate addition [165]. The CBB cycle and other pathways of carbon dioxide assimilation in this case work as electron sink spending excess of electrons but not as anaplerotic pathway [15,51]. Another way of maintaining the redox balance for purple bacteria grown with acetate is not connected with anaplerotic pathways but with nitrogen fixation. In this process, the nitrogenase system acts as an electron sink using electrons for nitrogen fixation and hydrogen production. The regulatory system in organisms regulates both CBB and nitrogen fixation processes [83]. This system is different in various types of purple bacteria (see above, PART 1, The CBB cycle), but the main goal of them is redox balance maintenance inside cells.
Excessive illumination is a stress for purple bacteria grown with acetate. An increase in the light intensity results in an excess of the proton motive force. This brings about an excessive NADH synthesis via a reverted electron flow by NADH dehydrogenase [166,167]. A decrease in illumination or additional bicarbonate restored the growth of bacteria [57].
Anaplerotic pathways acceleration could eliminate strong light growth inhibition. For example, Rs. rubrum S1H after a prolonged cultivation with acetate produced an "acetatecompetent" strain [51]. It appears that this strain contained several additional copies of ethylmalonyl-CoA pathway genes due to a reversible gene duplication and amplification. This particular strain did not have any lag-phase at the growth onset with acetate and tolerated strong light.
Changes in the synthesis of storage compounds are yet another mechanism to adapt to the stress of elevated light and acetate assimilation [168,169]. Purple bacteria are capable of accumulating polyhydroxyalkanoates (PHA) [170,171]. PHA is the major storage compound in bacteria grown on acetate. A protein profile with analysis of the PHA content in Rs. rubrum wild type and "acetate-competent" strains showed that the "acetate-competent" strain could resist strong light much better than a wild strain [57]. During the switch to strong light, the "acetate-competent" strain was demonstrated to grow better and did not accumulate the elevated quantity of PHA, in contrast to the wild strain. Thus, the wild strain involved PHA synthesis in light-stress adaptation, whereas "acetate-competent strain" did not due to the higher activity of PHA synthesis.
Thus, the genetically determined anaplerotic pathways may take part in various critical processes underlying the adaptation of purple bacteria to environmental stresses.

Conclusions
The data reviewed in this paper reveal that our understanding of anaplerotic pathways replenishing the TCA cycle intermediates in microorganisms and, particularly, in purple bacteria has expanded significantly in the last decade. This success results from advances in genomic sequencing and proteomic, transcriptomic, and metabolomic research. We have already known that the simultaneous functioning of more than one pathway is not an exception but rather a rule for numerous microorganisms [172]. The integrated network of all known anaplerotic pathways in one scheme has appeared recently [13,39]. This shows that central carbon metabolism is very flexible. Simultaneously, this network shows that all known anaplerotic pathways share many common reaction chains. This integrated scheme might efficiently outline active anaplerotic pathways in a newly discovered strain. Nowadays, the quickest way to elucidate it is the genome sequencing followed by the analysis of genetic potential for different anaplerotic pathways. After this initial step, the exact transcriptomic experiments with subsequent proteomic analysis provide clues for the final metabolomics study.
Our overview of possible anaplerotic pathways show that many reaction chains/cycles able to replenish TCA cycle intermediates have some additional functions. Furthermore, some chains were described previously as having another function (for example, CBB cycle). Thus, central carbon metabolism is a sophisticated system supporting the lives of microbials life, and our classification of different chains for anaplerotic function, inorganic carbon acquisition, synthesis of different blocks for overall metabolism or energetic reactions is just our simplification of their nature.