REVIEW ARTICLE published: 01 August 2011 doi: 10.3389/fmicb.2011.00165 Carbon metabolic pathways in phototrophic and their broader evolutionary implications

Kuo-HsiangTang1,2*†,Yinjie J.Tang3 and Robert Eugene Blankenship1,2*

1 Department of Biology, Washington University in St. Louis, St. Louis, MO, USA 2 Department of Chemistry, Washington University in St. Louis, St. Louis, MO, USA 3 Department of Energy, Environment, and Chemical Engineering, Washington University in St. Louis, St. Louis, MO, USA

Edited by: Photosynthesis is the biological process that converts solar energy to biomass, bio- Thomas E. Hanson, University of products, and biofuel. It is the only major natural solar energy storage mechanism on Delaware, USA Earth. To satisfy the increased demand for sustainable energy sources and identify the Reviewed by: Thomas E. Hanson, University of mechanism of photosynthetic carbon assimilation, which is one of the bottlenecks in pho- Delaware, USA tosynthesis, it is essential to understand the process of solar energy storage and associated Ulrike Kappler, University of carbon metabolism in photosynthetic organisms. Researchers have employed physiological Queensland, Australia studies, microbiological chemistry, enzyme assays, genome sequencing, transcriptomics, *Correspondence: and 13C-based metabolomics/fluxomics to investigate central carbon metabolism and Kuo-Hsiang Tang, One Brookings Drive, Campus Box 1137, St. Louis, enzymes that operate in . In this report, we review diverse CO2 assimilation MO, USA. pathways, acetate assimilation, carbohydrate catabolism, the tricarboxylic acid cycle and e-mail: [email protected]; some key, and/or unconventional enzymes in central carbon metabolism of phototrophic Robert Eugene Blankenship, One microorganisms. We also discuss the reducing equivalent flow during photoautotrophic Brookings Drive, Campus Box 1137, St. Louis, MO, USA. and photoheterotrophic growth, evolutionary links in the central carbon metabolic network, e-mail: [email protected] and correlations between photosynthetic and non-photosynthetic organisms. Considering †Current Address the metabolic versatility in these fascinating and diverse photosynthetic bacteria, many Kuo-Hsiang Tang, Carlson School of essential questions in their central carbon metabolism still remain to be addressed. Chemistry and Biochemistry, and 13 Department of Biology, Clark Keywords: acetate assimilation, autotrophic and anaplerotic CO2 assimilation, biomass and biofuel, C-based University, 950 Main Street, metabolomics, citrate metabolism, photosynthesis, unconventional pathways and enzymes Worcester, MA 01610, USA.

INTRODUCTION plastoquinone oxidoreductase) through the cytochrome b6f com- Phototrophic bacteria use light as the energy source to produce plex to NADP+ in Photosystem I (plastocyanin: ferredoxin oxi- phosphate bond energy (ATP) and reductants [e.g., NAD(P)H and doreductase; Blankenship, 2002). This non-cyclic electron trans- reduced ferredoxin] through photosynthetic electron transport. port is represented in the Z-scheme of photosynthesis. A cyclic Oxygenic phototrophic bacteria, known as , share electron transport pathway also operates in oxygenic phototrophs similar electron transport pathways with other oxygenic pho- during phototrophic growth and synthesizes ATP instead of totrophs (e.g., plants and algae). Photosynthetic electron trans- NADPH (Iwai et al., 2010). Photosynthetic electron transport port in cyanobacteria proceeds from oxidation of H2Oin pathways are different in anoxygenic (non- evolving) pho- the oxygen evolving complex of Photosystem II (or H2O: totrophs, all of which contain either a type II (quinone type) or a type I (Fe-S type) reaction center (RC). Five out of six phyla of photosynthetic bacteria are anoxygenic phototrophs: proteobac- teria [anaerobic anoxygenic phototrophic Proteobacteria (AnAPs) Abbreviations: Abbreviations of phototrophic bacteria. Three-letter abbrevia- tion for the generic name of phototrophic bacteria follows the information and aerobic anoxygenic phototrophic Proteobacteria (AAPs)], listed on LPSN (List of Prokaryotic names with Standing in Nomenclature) (GSBs), filamentous anoxygenic phototrophs (http://www.bacterio.cict.fr/index.html). (FAPs; or green non-sulfur/gliding bacteria), heliobacteria, and Other abbreviations. AAPs, aerobic anoxygenic phototrophic Proteobacteria; ACL, the recently discovered chloroacidobacteria (Blankenship, 2002; ATP citrate lyase; AnAPs, anaerobic anoxygenic phototrophic Proteobacteria; CCL, Bryant and Frigaard, 2006; Bryant et al., 2007). AAPs, AnAPs, citryl-CoA lyase; CCS, citryl-CoA synthetase; (Re)/(Si)-CS, (Re)/(Si)-citrate syn- thase; ED, Entner–Doudoroff; EMP, Emden–Meyerhof–Parnas; FAPs, filamen- and FAPs contain a type II RC, and GSBs, heliobacteria and tous anoxygenic phototrophs; FBP, fructose 1,6-bisphosphate; Fd, ferredoxin; chloroacidobacteriahaveatypeIRC.BacteriawithatypeIIRC GAP, d-glyceraldehyde-3-phosphate; Glc, glucose; GSBs, green sulfur bacteria; operate cyclic electron transport to generate ATP and reverse elec- 3HOP, 3-hydroxypropionate; KDPG, 2-keto-3-deoxy-6-phosphogluconate; α-KG, tron transport to produce NAD(P)H. GSBs operate both cyclic α-ketoglutarate; OAA, oxaloacetate; OPP pathway, oxidative pentose phosphate pathway; OTCA/RTCA cycle, oxidative/reductive tricarboxylic acid cycle; PGM, and non-cyclic electron transport to make NAD(P)H and ATP, phosphoglycerate mutase; RC, reaction center; R5P, ribose-5-phosphate; RuBisCO, and heliobacteria have been suggested to employ cyclic electron ribulose 1,5-bisphosphate carboxylase/oxygenase. transport (Kramer et al., 1997).

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The energy and reducing equivalents generated from light- induced electron transport drive diverse carbon metabolic path- ways for producing cellular material, bioactive products and biofuel. Additionally, numerous photosynthetic bacteria play essential roles in global carbon, nitrogen, and sulfur cycles, as many of them are known to assimilate nitrogen and/or oxi- dize sulfide in conjunction with their central carbon and energy metabolisms. Inorganic and organic carbon sources are used for building cellular building blocks of photoautotrophic and photoheterotrophic bacteria, respectively. Cyanobacteria, AnAPs, FAPs, and GSBs can grow autotrophically via a variety of autotrophic CO2 assimilation pathways, while AAPs and heliobac- teria are obligate . AAPs have been discov- ered in some ocean surface waters (Kolber et al., 2001) and certain (extreme) ecosystems (Yurkov and Csotonyi, 2009), and may play important roles in global carbon regulation (Kol- ber et al., 2001). Heliobacteria are the most recently discov- ered anaerobic anoxygenic phototrophs. Compared to other phototrophic bacteria, heliobacteria have the simplest photo- system (Heinnickel and Golbeck, 2007; Sattley and Blanken- ship, 2010) and some unique features: they are the only Gram- positive phototrophic bacteria in the bacterial phylum Firmi- cutes (and form heat resistant ), and are the only obligate anaerobic photoheterotrophs. All known heliobacteria are active nitrogen fixers and hydrogen producers (Madigan, 2006). The evolution of eukaryotes has been suggested to have occurred through endosymbiosis (Margulis, 1968; Doolittle, 1998). It has been proposed that the ancestral eukaryotes were anaerobes, and that aerobic α-Proteobacteria, which include FIGURE1|Schematic representation of central carbon metabolism in many AAPs, were the origins of mitochondria of eukaryotes and phototrophic bacteria. The metabolic pathways of autotrophic CO2 fixation (red lines), anaplerotic CO2 assimilation (green lines), carbohydrate cyanobacteria were the forerunners of the chloroplast of higher metabolism (blue lines), acetate assimilation (brown lines), and the TCA plant cells (Margulis, 1968; Hohmann-Marriott and Blankenship, cycle (black lines) in phototrophic bacteria are shown. Some metabolic 2011). pathways are employed only in phototrophic bacteria, and others are While photosynthetic bacteria have simplified, and some- distributed in phototrophic and non-phototrophic microbes. times unique, photosystems compared to higher plants and green algae (Blankenship, 2002), their carbon metabolism pathways are rather complex (Figure 1). Previous studies on central car- RESULTS AND DISCUSSIONS bon metabolism of photosynthetic organisms have suggested OVERVIEW: THE LIGHT AND DARK REACTIONS IN PHOTOTROPHIC that some phototrophic and non-phototrophic organisms use BACTERIA some distinct carbon assimilation and carbon metabolic path- No two phyla of phototrophic bacteria have the same metabolic ways (Hugler et al., 2011). In this report, we review the stud- pathways, while some ecological and metabolic features of pho- ies of key central carbon metabolic pathways, including CO2 totrophic bacteria are correlated with the type of RC employed. and acetate assimilations, carbohydrate catabolism and the tri- Only the five most characterized phyla of phototrophic bacteria are carboxylic acid (TCA) cycle, and also highlight several essen- discussed here. Bacteria with a type II (quinone type) RC (e.g.,Pro- tial and/or unconventional enzymes contributing to the critical teobacteria and FAPs) are facultative phototrophs (can grow with metabolic pathways in phototrophic bacteria. Some related meta- or without light) and are metabolically versatile (can grow aerobi- bolic pathways and enzymes distributed in non-phototrophic cally and anaerobically). Since various types of quinone molecules organisms are also discussed. General approaches to investigate in the type II RC can freely transport electrons in the membrane, the carbon metabolism of phototrophs are presented in Appen- severaltypeIIRCbacteriahaveanactiveTCAcycleandcanper- dix and Table A1 for readers who are interested in further form respiration. Some ancient aerobic α-Proteobacteria may have information. Overall, experimental evidence indicates that pho- been the ancestors of the mitochondria of modern eukaryotes totrophic bacteria containing the same type of RC often have (Margulis, 1968; Doolittle, 1998). distinct central carbon metabolism pathways, suggesting horizon- Bacteria with only a type I (Fe-S type) RC (e.g., GSBs and tal/lateral gene transfers and late adaptation during the evolution heliobacteria) are strict anaerobes and metabolic specialists. GSBs of photosynthesis. direct these reducing equivalents through light-induced electron

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transport to the reductive (reverse) TCA (RTCA) cycle, which cycle for carbon fixation (Kusian and Bowien, 1997; Sorokin et al., is basically the reversal of the oxidative (forward) TCA (OTCA) 2007). Moreover, a complete Calvin–Benson cycle has been found cycle, for fixing CO2 and producing biomass (Evans et al., 1966a). in the genomes of several heterotrophic bacteria (McKinlay and Heliobacteria have an incomplete RTCA cycle, and can utilize a Harwood, 2010), and the roles of key gene products in these limited set of carbon sources. Reduced ferredoxins that are elec- bacteria remain to be investigated. In addition to the Calvin– tron donors to pyruvate synthase and α-ketoglutarate synthase in Benson cycle, five other autotrophic carbon fixation pathways the RTCA cycle are oxygen-sensitive, which may correlate with have also been identified (Ensign, 2006; Thauer, 2007; Raven, GSBs and heliobacteria being strict anaerobes. 2009; Berg et al., 2010; Hugler and Sievert, 2011), including Most cyanobacteria utilize the Calvin–Benson cycle for the RTCA cycle (the Arnon–Buchanan–Evans cycle; Figure 2B), autotrophic CO2 assimilation and can grow photoautotrophically, the 3-hydroxypropionate (3HOP) bi-cycle (Figure 2C), the 3- photomixotrophically, and/or photoheterotrophically. Fermenta- hydroxypropionate/4-hydroxybutyrate (3HOP/4HOB) cycle, the tive growth of cyanobacteria with carbohydrates has also been dicarboxylate/4HOB cycle, and the reductive acetyl-CoA pathway reported (Stal and Moezelaar, 1997). One group of nitrogen- (the Wood–Ljungdahl pathway). The 3HOP/4HOB cycle (Berg fixing marine cyanobacteria, which do not have Photosystem II, et al., 2007), which is similar to the 3HOP bi-cycle and uses the autotrophic carbon assimilation pathways and the TCA cycle, are same carbon assimilation enzymes (e.g., acetyl-CoA carboxylase suggested to be obligate photoheterotrophs (Zehr et al.,2008; Tripp and propionyl-CoA carboxylase), the dicarboxylate/4HOB cycle et al., 2010). Anoxygenic phototrophic bacteria are either pho- (Huber et al.,2008),and the reductive acetyl-CoA pathway (Ljung- toautotrophs or photoheterotrophs. Anoxygenic photoautotrophs dahl,1986;Wood,1991)havenotyetbeenreportedinphototrophs operate at least three different autotrophic CO2 fixation path- and are not discussed further here. ways, including the Calvin–Benson cycle. As molecular oxygen Among these autotrophic carbon fixation pathways, cyanobac- is a substrate for RuBisCO (ribulose 1,5-bisphosphate carboxy- teria and AnAPs use the Calvin–Benson cycle (Tabita, 1995; lase/oxygenase) that competes with CO2 in the Calvin–Benson Blankenship,2002). GSBs use the RTCA cycle (Evans et al.,1966a,b; cycle, giving rise to photorespiration. Oxygenic phototrophs con- Buchanan and Arnon, 1990) and the FAP bacterium Chloroflexus tain CO2 concentrating mechanisms and carboxysomes to ele- aurantiacus utilizes the 3HOP bi-cycle (Holo, 1989; Strauss and vate CO2 concentration at the RuBisCO active site (Kaplan and Fuchs, 1993; Herter et al., 2002; Zarzycki et al., 2009). Several Reinhold, 1999). Note that some chemolithoautotrophs, such as Oscillochloridaceae strains in FAPs have been reported to use the Thiomicrospira crunogena (Dobrinski et al., 2005) and Thiobacil- Calvin–Benson cycle, instead of the 3HOP bi-cycle, and those lus neopolitanus (Holthuijzen et al., 1986), contain carboxysomes strains also have a branched TCA cycle (Berg et al., 2005; Table 1). and appear to employ a carbon concentrating mechanism, so The RTCA cycle is also distributed in many non-phototrophic that these adaptations are not exclusively by phototrophic organ- bacteria (Hugler and Sievert, 2011), including Hydrogenobacter isms. No such mechanisms have been identified in anoxygenic thermophilus (Shiba et al., 1985) and some members of the ε- phototrophs. In response to the intrinsic problem of RuBisCO Proteobacteria (Hugler et al., 2005, 2007; Takai et al., 2005). While and carbon fixation via the Calvin–Benson cycle, most anoxy- biochemical analyses indicate that certain enzymes in the RTCA genic bacteria may have either retreated to low oxygen envi- cycle are oxygen-sensitive, several autotrophic microbes (such as ronments and/or adopted different CO2 assimilation pathways H. thermophilus and Aquifex aeolicus) that employ the RTCA cycle (autotrophic or anaplerotic) and various carboxylases for car- are not strict anaerobes and can grow well aerobically (Kawasumi bon metabolism. Thus, various autotrophic CO2 fixation cycles, et al., 1984; Pandelia et al., 2011). The Calvin–Benson cycle and the as well as some unconventional carbon metabolic pathways, oper- 3HOP bi-cycle can operate in both aerobic and anaerobic condi- ating in anoxygenic phototrophs are possibly correlated with their tions, although oxygen is the substrate for RuBisCO that competes special ecological niches, such as high sulfide and low oxygen with CO2 in the Calvin–Benson cycle. environments. No autotrophic carbon assimilation pathways have been reported in AAPs (Yurkov and Beatty, 1998; Fuchs et al., 2007; CENTRAL CARBON METABOLISM PATHWAYS Swingley et al., 2007), heliobacteria (Madigan, 2006), and the Figure 1 presents a general scheme of central carbon metabo- Roseiflexus in FAPs (Hanada et al., 2002). The genus Rosei- lism pathways, including CO2 and acetate assimilation pathways, flexus in FAPs has been suggested to be photoheterotrophic and carbohydrate metabolism and the TCA cycle. Detailed informa- not photoautotrophic (Hanada et al.,2002),although genes encod- tion for each metabolic pathway in five phyla of phototrophic ing the 3HOP bi-cycle have been found in the genome (Klatt bacteria [e.g., the Proteobacteria (AnAPs and AAPs), GSBs, FAPs, et al., 2007). Further, preliminary studies indicate that the most cyanobacteria, and heliobacteria] is elaborated below. recently identified photosynthetic bacterium Candidatus Chlo- racidobacterium (Cab.) thermophilum (Bryant et al., 2007), which Autotrophic CO2 fixation pathways is a member of the sixth photosynthetic bacteria phylum the Aci- One of the landmarks of photosynthesis is assimilating CO2 dobacteria, is perhaps also a , due to the lack of autotrophically into cellular material with light, and the most evidence supporting autotrophic carbon assimilation and growth. well-known CO2 assimilation pathway is the reductive pentose Many metabolites, intermediates or products produced by phosphate (PP) pathway (the Calvin–Benson cycle; Figure 2A; these autotrophic carbon fixation pathways are essential for Calvin and Benson, 1948; Bassham et al., 1950; Calvin, 1989). building cellular material. The Calvin–Benson cycle synthesizes 3- Note that some chemolithoautotrophs also use the Calvin–Benson phosphoglycerate (3PG) and sugar phosphates (including triose-,

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FIGURE 2 | Autotrophic CO2 fixation pathways in phototrophic organisms. The CO2 assimilation steps in the Calvin–Benson cycle (A), reductive TCA (RTCA)

cycle (B) and 3-hydroxypropionate bi-cycle (C) are shown in bold and colored red. Reactions catalyzed by PEP carboxylase (i.e., the enzyme for anaplerotic CO2 assimilation) and pyruvate synthase are included in the RTCA cycle (B). Participations or consumption of ATP and reducing equivalents are shown. tetrose-, pentose-, hexose-, and heptose phosphates), and all Acetate assimilation of the other autotrophic carbon fixation pathways produce or Many AAPs, AnAPs, FAPs, GSBs, and heliobacteria have been regenerate acetyl-CoA, and several of them also generate pyru- reported to grow heterotrophically or mixotrophically (in the vate, phosphoenolpyruvate (PEP), oxaloacetate (OAA), glyoxy- presence of CO2) on acetate with three acetate assimilation late, succinyl-CoA, and/or α-ketoglutarate (Table 2). All of these mechanisms: the (oxidative) glyoxylate cycle (Figure 3A), pyru- metabolites are essential for the central carbon metabolism of vate synthase (Figure 3B), and the ethylmalonyl-CoA path- microorganisms. way (Figure 3C). The (oxidative) glyoxylate cycle and the

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Table 1 | The carbon assimilation pathways reported in phototrophic bacteria.

AnAPs AAPs FAPs GSBs Heliobacteria Cyanobacteria

AUTOTROPHIC CO FIXATION PATHWAYS AND ANAPLEROTIC CO ASSIMILATIONS 2 √√2 √ The Calvin–Benson cycle √√ The reductive TCA cycle (incomplete) √ The 3-hydroxypropionate bi-cycle √√√√√ √ Anaplerotic CO2 assimilations ACETATE ASSIMILATION PATHWAYS √√ √ The (oxidative) glyoxylate cycle √√ √ Pyruvate synthase √√ The ethylmalonyl-CoA pathway

AnAPs, anaerobic anoxygenic phototrophic Proteobacteria; AAPs, aerobic anoxygenic phototrophic Proteobacteria; FAPs, filamentous anoxygenic phototrophs; GSBs, green sulfur bacteria.

Table 2 | Metabolites generated from CO2 and acetate assimilation pathways for producing cellular material in phototrophic organisms.

Pathways 3PG, triose Acetyl-CoA Pyruvate PEP OAA malate Glyoxylate Succinyl-CoA/ α-KG and sugar succinate phosphate

AUTOTROPHIC CO FIXATION PATHWAYS AND ANAPLEROTIC CO ASSIMILATIONS 2 √ 2 The Calvin–Benson cycle √√√√√ √ √ The reductive TCA cycle √√ √√ The 3-hydroxypropionate bi-cycle √√√√ Anaplerotic CO2 assimilations ACETATE ASSIMILATION PATHWAYS √√ √ √ The (oxidative) glyoxylate cycle √√ Pyruvate synthase √√√√√ The ethylmalonyl-CoA pathway

3PG, 3-phosphoglycerate; PEP,phosphoenolpyruvate; OAA, oxaloacetate; and α-KG, α-ketoglutarate. ethylmalonyl-CoA pathway can replenish the metabolites in the whereas other AnAPs apparently lack essential genes/enzymes in TCA cycle. Pyruvate synthase and the ethylmalonyl-CoA pathway the pathway (e.g., Rubrivivax gelatinosus, Rba. Sphaeroides, and −) assimilate both acetate and inorganic carbon (CO2/HCO3 . Dis- Rhodospirillum rubrum; Albers and Gottschalk, 1976; Lim et al., tributions of the acetate assimilation pathways in phototrophic 2009). Genes in the glyoxylate cycle have also been identified in bacteria are listed in Table 1. the genome of several FAPs (Cfl. aurantiacus, Cfl. aggregans, Chlo- roflexus sp. Y-400-fl, Roseiflexus castenholzii and Roseiflexus sp. RS-1; Tang et al., 2011). GSBs,AAPs, and heliobacteria do not have The (oxidative) glyoxylate cycle. The (oxidative) glyoxylate cycle, an active glyoxylate cycle, and use alternative acetate assimilation discovered more than 50 years ago by Kornberg and Krebs (1957), pathways (discussed below). produces four-carbon units (malate, OAA, and succinate) by assembling two acetyl-CoA molecules (Figure 3A). It is essen- tial not only to convert acetate, lipids and some amino acids to Pyruvate synthase. With no active glyoxylate cycle reported, carbohydrates, but also to other central biosynthetic precursors in GSBs and heliobacteria use pyruvate synthase (or pyruvate: ferre- the TCA cycle (anaplerotic function). The glyoxylate cycle has also doxin oxidoreductase) for acetate assimilation. Pyruvate synthase been found in plants, some (non-phototrophic) bacteria, archaea, converts acetyl-CoA to pyruvate and can assimilate both acetate protists, fungi, and nematodes. The glyoxylate cycle is active and (i.e., acetyl-CoA) and CO2 (Figure 3B). The catalysis requires has been suggested to channel excess carbon flow away from the reduced ferredoxin and thus only anaerobic anoxygenic bacteria branched TCA cycle during mixotrophic and heterotrophic, but operate pyruvate synthase. Pyruvate synthase synthesizes pyruvate not autotrophic, growth of the cyanobacterium Synechocystis sp. from acetyl-CoA produced through acetate uptake and the RTCA PCC 6803 (Yang et al., 2002; Shastri and Morgan, 2005). cycle during mixotrophic growth of GSBs (Evans et al.,1966a; Feng Some AnAPs either operate an active glyoxylate cycle (e.g., Rps. et al., 2010b; Tang and Blankenship, 2010). CO2 is required for the palustris; McKinlay and Harwood, 2011) or have genes in the path- growth of heliobacteria in the medium containing acetate as the way identified (e.g., Rhodospirillum centenum, Rps. palustris and only organic carbon source (Madigan, 2006; Tang et al., 2010b), Rba. capsulatus; Oda et al.,2008; Lu et al.,2010; Strnad et al.,2010), and the supplemented CO2 is required not only for anaplerotic

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FIGURE 3 | Acetate assimilation pathways. Three acetate assimilation oxaloacetate. The CO2 assimilation steps in the pathway/cycle are shown in pathways, the (oxidative) glyoxylate cycle (A), the reaction catalyzed by bold and colored red. Different ethylmalonyl-CoA pathways have been pyruvate synthase (B) and the ethylmalonyl-CoA pathway (C), are shown. All reported in Methylobacterium extorquens AM1 [(C), left] and of the acetate assimilation pathways may directly or indirectly synthesize sphaeroides [(C), right].

CO2 assimilation, but also the generation of pyruvate catalyzed by (chemotrophic) methylotrophs (e.g.,Methylobacterium extorquens pyruvate synthase (Pickett et al., 1994; Tang et al., 2010a,b). AM1; Korotkova et al., 2002; Peyraud et al., 2009; Smejkalova et al., 2010; Figure 3C, left; net reaction: acetyl-CoA + 2CO2 → 2 + The ethylmalonyl-CoA pathway. The ethylmalonyl-CoA glyoxylate + CoA + H ), and has also been proposed in some pathway is responsible for producing glyoxylate in some actinobacteria (Erb et al., 2009). The produced glyoxylate can be

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further converted to PEP via the serine pathway, which assimilates suggest that the Calvin–Benson cycle is not only assimilating CO2 one carbon unit through N 5, N 10-methylene-tetrahydrofolate and producing biomass during photoautotrophic growth, but is (Erb et al., 2007). The ethylmalonyl-CoA pathway has also been also accepting reducing equivalents during photoheterotrophic identified in the AnAP bacterium (Alber growth (e.g., when the Calvin–Benson cycle becomes expend- et al., 2006; Erb et al., 2007) and is proposed in several AnAPs able; McKinlay and Harwood, 2010). Furthermore, the choice (Albers and Gottschalk, 1976; Ivanovsky et al., 1997; Alber et al., of the pathway for assimilating organic compounds during pho- 2006; Erb et al., 2009). In contrast to the ethylmalonyl-CoA path- toheterotrophic growth is also important (Laguna et al., 2011). wayreportedinM. extorquens AM1, Rba. sphaeroides condenses During photoheterotrophic growth, Rps. palustris uses reducing glyoxylate with acetyl-CoA to produce malyl-CoA,which is further equivalents to produce hydrogen rather than assimilate carbon hydrolyzed to malate and CoA (Figure 3C, right). (McKinlay and Harwood, 2011) and Rba. sphaeroides directs Acetate can support the phototrophic and chemotrophic (in most of the reducing equivalents to assimilate acetate using darkness) growth of several AAPs (Shiba and Simidu, 1982; the ethylmalonyl-CoA pathway and therefore hydrogen produc- Shiba and Harashima, 1986; Shiba, 1991; Yurkov et al., 1996; tion decreases (Laguna et al., 2011). These studies are consis- Koblizek et al., 2003; Biebl et al., 2005, 2006). The mechanism tent with observations that RuBisCO-knockout Rba. sphaeroides of acetate assimilation remains to be understood in these organ- can grow photolithoautotrophically using thiosulfate or sul- isms because some AAPs do not have an active glyoxylate cycle fide, which is not as a potent reductant as H2, as the elec- and no AAPs have pyruvate synthase identified. Thus, acetate- tron source and thus consuming the excess reducing equiva- grown AAPs require alternative pathways to produce OAA, which lents produced by photosynthesis is less essential (Wang et al., is required for producing biomass and building blocks of cells. 1993), although the pathway for assimilating CO2 in these exper- Note that the ethylmalonyl-CoA pathway has been recently sug- iments is not clear. Alternative pathways for removing excess gested to be employed by AAPs (Alber et al., 2006). It is of interest reducing equivalents are utilized by certain AAPs during photo- to verify if the proposed ethylmalonyl-CoA pathway is active in heterotrophic growth; for example, nitrate reduction to ammo- AAPs, and whether it plays an important role in CO2 assimilation nium by Roseobacter denitrificans (Shiba, 1991; Tang et al., by AAPs. 2009a). Several heterocystous and non-heterocystous cyanobacteria are Reducing equivalent flow in photoautotrophs and photoheterotrophs known to be active nitrogen fixers and hydrogen producers (Sum- Photosynthesis generates reducing equivalents through light- mers et al., 1995; Chen et al., 1998; Bandyopadhyay et al., 2010; Li induced electron transport. In photoautotrophic bacteria, chan- et al., 2010). The non-heterocystous cyanobacterium Cyanothece neling reducing equivalents to autotrophic CO2 fixation pathways sp. ATCC 51142, which can produce hydrogen aerobically (Bandy- is essential when phototrophs cannot respire. For example, GSBs opadhyay et al., 2010), guides reducing equivalents to reduce are obligate photoautotrophs and cannot grow heterotrophically nitrate to ammonium during photoheterotrophic growth with and in darkness. The reducing equivalents generated from photo- glycerol (Feng et al., 2010a). synthetic electron transport in the type I (Fe-S type) RC of GSBs can be utilized for CO2 assimilation (via the RTCA cycle) and Anaplerotic CO2 assimilations other types of cellular metabolism during phototrophic growth. Anaplerotic reactions replenish the intermediates in a metabolic A similar scenario can be found in chloroplasts in photosynthetic pathway. Anaplerotic CO2 assimilations are those that replenish eukaryotes, NADPH produced from non-cyclic electron transport OAA or malate for replenishing the TCA cycle, and are important is employed for assimilating CO2 via the Calvin–Benson cycle. to synthesize cellular building blocks. Anaplerotic CO2 assimila- Alternatively, heliobacteria, the other anaerobic anoxygenic bac- tions cannot serve as an autotrophic CO2 fixation pathway because teria with a type I RC, do not have an autotrophic CO2 fixation they require organic substrates that are not subsequently pro- mechanism. The reducing equivalents generated from their pho- duced from the incorporation of CO2. Four enzymes catalyzing tosynthetic electron transport are utilized to reduce organic and anaplerotic CO2 assimilation have been reported: pyruvate car- inorganic molecules in cells, and all known heliobacterial species boxylase,phosphoenolpyruvate (PEP) carboxylase,PEP carboxyk- are active nitrogen fixers and hydrogen producers (Madigan, inase,and malic enzyme (Figure 4). Anaplerotic CO2 assimilations 2006). have been reported in all types of phototrophic bacteria. Among In contrast to GSBs and heliobacteria, AnAPs can grow these, AnAPs, AAPs have an active complete OTCA cycle and can both photoautotrophically and photoheterotrophically. Unlike the regenerate OAA, and can also replenish OAA via anaplerotic CO2 wild-type, RuBisCO-, and phosphoribulokinase (PRK)-knockout assimilations (Yurkov and Beatty, 1998; Furch et al., 2009; Tang Rba. sphaeroides mutants require dimethyl sulfoxide as an alter- et al., 2009a) and the glyoxylate cycle and/or the ethylmalonyl- native external electron recipient to accept (excess) reducing CoA pathway (Albers and Gottschalk, 1976; Alber et al., 2006; Erb equivalents produced during photoheterotrophic growth (Hallen- et al.,2007) to produce OAA. Alternatively,anaplerotic CO2 assim- beck et al., 1990a,b; Falcone and Tabita, 1991). Also, RuBisCO- ilations are essential for the growth of heliobacteria, cyanobacteria, knockout Rsp. rubrum and Rba. sphaeroides can grow photo- and GSBs, all of which can only use anaplerotic CO2 assimilations heterotrophically under nitrogen fixation conditions (Joshi and to replenish OAA. Tabita, 1996) and produce abundant H2 presumably from uti- lizing excess reducing equivalents. Recent metabolic flux analy- Heliobacteria. The activity of PEP carboxykinase has been ses on the AnAP bacterium Rhodopseudomonas palustris also reported in heliobacteria (Pickett et al.,1994; Tanget al.,2010b). In

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Table 3 | The carbohydrate catabolic pathways reported in phototrophic bacteria.

The EMP The ED The oxidative The non-oxidative pathway pathway pentose pentose phosphate phosphate pathway pathwaya √√ √ AnAPs – √ √ AAPs – – √ √√ FAPs – √ GSBs – – – √ √ Heliobacteria –– √ √√ Cyanobacteria –

–: Not shown in the experimental evidences or/and not present in the genomes. aThe non-oxidative pentose phosphate pathway is unequal to the reductive pen- tose phosphate pathway (or the Calvin–Benson cycle), as illustrated in Figures 2

FIGURE 4 | Anaplerotic CO2 assimilation reactions. The CO2-anaplerotic and 5. reactions catalyzed by pyruvate carboxylase, PEP carboxylase, PEP carboxykinase, and malic enzyme are shown. Participations or consumption that the autotrophic carbon fixation pathways support photoau- of ATP and reducing equivalents are presented. Abbreviation: PEP, phosphoenolpyruvate; OAA, oxaloacetate. totrophic growth because they require pre-existing/endogenous organic compounds that are not produced by the pathway, anaplerotic CO2 assimilations are nonetheless crucial for the cell contrast to AnAPs, AAPs, and FAPs, heliobacteria have an incom- growth of phototrophic organisms. plete OTCA cycle (Tang et al., 2010a), which cannot regenerate OAA, and do not have the glyoxylate cycle and ethylmalonyl-CoA Carbohydrate metabolism pathway (Table 1). As a result, the anaplerotic CO2 assimilation is Most chemoheterotrophic organisms use carbohydrates as carbon the only approach to synthesize OAA and is essential for the growth sources to build up cellular material and provide reductants. GSBs of heliobacteria, consistent with high flux in the anaplerotic CO2 are the only group of phototrophic bacteria that do not appar- assimilation in heliobacteria (Tang et al., 2010a). ently grow on carbohydrates as sole carbon sources (Table 3). Three carbohydrate catabolic pathways in photosynthetic organ- Cyanobacteria and GSBs. Active anaplerotic CO2 assimilations isms are known: the Emden–Meyerhof–Parnas (EMP) pathway have been identified in GSBs (Evans et al., 1966a; Sirevag, 1995; (glycolysis), Entner–Doudoroff (ED) pathway, and PP pathway Feng et al., 2010b; Tang and Blankenship, 2010) and cyanobacteria (also called the phosphogluconate pathway), which includes the (Neuera and Bothe,1983; Owttrim and Colman,1988; Zhang et al., oxidative PP (OPP) and non-OPP pathways (Figure 5A). The 2004; Stockel et al., 2011). Cyanobacteria have a branched TCA EMP pathway and the ED pathway convert one molecule of glu- cycle (enzymes that catalyze the interconversion of α-ketoglutarate cose into two molecules of pyruvate, although different courses and succinate are absent) and cannot regenerate OAA (Stanier and are employed by these two pathways. Regulation of these two Cohen-Bazire, 1977). An active glyoxylate cycle in Synechocystis sp. pathways provides flexibility for energy generation and bypasses PCC 6803 has been reported (Yanget al.,2002; Shastri and Morgan, bottlenecks for glucose oxidation (Selig et al., 1997). For instance, 2005), although key genes in the glyoxylate cycle are absent in the glucose oxidation through the ED pathway can avoid the phos- genomes of most cyanobacteria. GSBs require OAA to initiate the phofructokinase reaction [fructose 6-phosphate (F6P) → fructose RTCA cycle and do not have the glyoxylate cycle and ethylmalonyl- 2,6-bisphosphate (FBP)], which is one of the irreversible and rate- CoA pathway. Although GSBs can produce OAA via ATP citrate determining steps in the EMP pathway (Nelson and Cox, 2008). lyase (ACL) in the RTCA cycle (see Enzymes that dissimilate citrate), Only the OPP pathway, not the non-OPP pathway, can produce OAA needs to be replenished by anaplerotic CO2 assimilations if reducing equivalents. Note that the non-OPP pathway is not the large amounts of α-ketoglutarate and other metabolites are pulled same as the reductive PP pathway (or the Calvin–Benson cycle). out of the cycle for nitrogen assimilation and other cellular metab- R5P produced via the PP pathway is a precursor for the biosyn- olism. As a result, cyanobacteria (if the glyoxylate cycle is absent) thesis of nucleic acids, ATP, histidine, and coenzymes. Net energy and GSBs can only replenish OAA through anaplerotic CO2 assim- output and reductant production for the three glucose catabolic ilations. It has been shown that malic enzyme is essential for pathways are: 2 ATP and 2 NADH via the EMP pathway, 2 NADPH optimal photoautotrophic growth of cyanobacteria (Bricker et al., via the (oxidative) PP pathway, and 1 NADPH (the reaction that 2004). generates the first NADPH in the OPP pathway is part of the ED Altogether, active anaplerotic reactions are essential not only pathway), 1 ATP and 1 NADH via the ED pathway. Moreover, ATP for photoheterotrophs (heliobacteria), but are also for the pho- can be also generated in the oxidative pentose phosphate path- toautotrophs (cyanobacteria and GSBs). Even though anaplerotic way. depending on the flux mode considered (Kruger and von CO2 assimilations cannot support phototrophic growth in the way Schaewen, 2003).

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FIGURE 5 | Carbohydrate catabolism. The “classical” EMP,ED, and PP EMP/gluconeogenic pathway and KDPG aldolase in the ED pathway are pathways, which have been found in phototrophic bacteria, are shown in (A), shown in (B). Abbreviation: EMP,Emden–Meyerhof–Parnas; ED, and aldol condensation reactions catalyzed by FBP aldolase in the Entner–Doudoroff; KDPG, 2-keto-3-dehydro-6-phosphogluconate.

Anaerobic anoxygenic phototrophic Proteobacteria and aerobic among photosynthetic bacteria. SomeAnAPs are known to employ anoxygenic phototrophic Proteobacteria. Anaerobic anoxygenic either the EMP or ED pathway, depending on growth condi- phototrophic Proteobacteria are the most metabolically versatile tions and nutrients, for carbohydrate catabolism, whereas the

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OPP pathway has not been reported to be active in AnAPs. Vrije et al., 2007). An active ED pathway has not been reported in Employing activity assays, sugar consumption, analysis of labeled any heliobacteria, consistent with the fact that very few Gram-(+) amino acids using 14C-labeled sugar in wild-type and Glc-6- bacteria employ the ED pathway. phosphate dehydrogenase-deficient mutant, previous studies indi- cate that Rba. capsulatus and Rba. sphaeroides utilize the EMP Filamentous anoxygenic phototrophs. Most of the reports of (or the ED) pathway to oxidize fructose during phototrophic (or carbohydrate metabolism of FAPs have focused on Cfl. auran- chemotrophic) growth, and operate the ED pathway to dissimi- tiacus, which is the most investigated FAP bacterium (or green late glucose during both phototrophic and chemotrophic (dark) non-sulfur/gliding bacterium). Cfl. aurantiacus has been reported growth (Conrad and Schlegel, 1977, 1978; Fuhrer et al., 2005). to grow well on glucose and a number of other sugars during Consistent with the experimental data, all of the genes in the ED aerobic respiration (Madigan et al., 1974), and it uses the EMP pathway have been identified in the genome of Rba. capsulatus and pathway for carbohydrate catabolism (Krasilnikova et al., 1986). Rba. sphaeroides. Other AnAPs are missing either the gene encod- An active ED pathway has not been identified in FAPs. Gene anno- ing 6-phosphogluconate dehydratase (such as Bradyrhizobium sp. tation in Cfl. aurantiacus indicates that the PP pathway is complete BTAi1) or all of the genes (i.e., Rsp. centenum, Rsp. Rubrum, and (Tang et al., 2011), consistent with the activities reported for Glc- all of the strains of Rps. palustris) in the ED pathway. 6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate Genes in the ED pathway are present in the genomes of AAPs dehydrogenase (6PGDH), two essential enzymes in the OPP (Tang et al., 2009a), and an active ED pathway has been sug- pathway (Krasilnikova et al., 1986). Note that the fbaA gene gested in several AAPs (Yurkov and Beatty,1998). Moreover, recent encoding FBP aldolase in the EMP/gluconeogenic pathway is 13C-based metabolomics/fluxomics studies, transcriptomics, and missing in the genome of Chloroflexi species (e.g., Cfl. auran- activity assays indicate that two members of the AAPs, R. denitri- tiacus, Chloroflexus sp. Y-400-fl, and Cfl. aggregans; Tang et al., ficans OCh114 and Dinoroseobacter shibae DFL12, exclusively or 2011). If Cfl. aurantiacus were unable to synthesize FBP aldolase, mainly use the ED pathway to oxidize glucose. Both AAPs have an an active OPP pathway would be employed for the intercon- inactive EMP pathway (Furch et al., 2009; Tang et al., 2009a), even version of d-Glc-6-phosphate and GAP, so sugars can be con- though all of the genes in the EMP pathway have been identified verted to pyruvate and other energy-rich species, and vice versa. in the D. shibae genome (Furch et al., 2009). Like AnAPs, an active However, higher activities of phosphofructokinase and fructose OPP pathway has also not been reported in AAPs (Yurkov and 1,6-bisphosphate (FBP) aldolase have been found in Cfl. auran- Beatty, 1998). tiacus grown with glucose than with acetate (Krasilnikova and All of the marine Roseobacter species that have been sequenced Kondrateva, 1987; Kondratieva et al., 1992; Hanada and Pierson, have genes in the ED pathway in their genome (Moran et al., 2006). Thus, Cfl. aurantiacus and other Chloroflexi species may 2007; Brinkhoff et al., 2008). Many marine Roseobacter species are employ a novel FBP aldolase. Note that Roseiflexi species (Rosei- non-phototrophs, some of which have been shown to operate an flexus sp. RS-1 and Roseiflexus castenholzii), which are closely active ED pathway (Yurkov and Beatty, 1998; Furch et al., 2009). related to Chloroflexi species, have a putative a bifunctional FBP Thus, both phototrophic and non-phototrophic Roseobacters uti- aldolase/phosphatase gene identified (Say and Fuchs, 2010). Genes lize the ED pathway. Note that many strains of AAPs and AnAPs encoding various types of aldolase are present in the Cfl. auranti- that use the ED pathway do not have the pgd gene (encoding acus genome (Tang et al., 2011). Further work is needed to clarify 6-phosphogluconate dehydrogenase) in the OPP pathway (Lim this picture. et al., 2009; Strnad et al., 2010) and/or the pfk gene (encoding 6- phosphofructokinase) in the EMP pathway (Swingley et al., 2007; Cyanobacteria. Cyanobacteria can use the OPP pathway and the Tang et al., 2009a). The selective evolutionary advantages of losing EMP pathway for carbohydrate dissimilation (Smith, 1982). Some the pfk gene and/or the pgd gene, and having an inactive EMP cyanobacteria, such as Synechocystis sp. PCC 6803, can assimi- pathway or/and the OPP pathway (Furch et al., 2009; Tang et al., late glucose during mixotrophic growth (Astier et al., 1984), and 2009a), remain unclear. other species can grow on fructose (Wolk, 1973). Further, genes in the EMP pathway and the PP pathway have been identified in Heliobacteria. d-Fructose and d-glucose can support pho- the genome of cyanobacteria. Fermentative growth of cyanobac- totrophic growth of Heliobacterium gestii, one of the heliobacteria teria with carbohydrates has been reported (Stal and Moezelaar, discovered in tropical paddy (Madigan,2006). Hbt. modestical- 1997), and the OPP pathway has been recognized to be the main dum is closely related to Hbt. gestii in phylogeny (Bryantseva et al., route for oxidizing glucose (Carr, 1973; Smith, 1982) and pro- 1999). All of the genes in the EMP pathway are present in the Hbt. viding the reducing equivalents to nitrogenase in diazotrophic modesticaldum genome. Consistent with genomic annotation and cyanobacteria (Summers et al., 1995; Bergman et al., 1997). The phylogenetic analyses, recent studies demonstrate that d-glucose, importance of G6PDH and 6PGDH in the OPP pathway for the d-fructose, and d-ribose can support the phototrophic growth of growth of cyanobacteria has been demonstrated (Scanlan et al., Hbt. modesticaldum with a trace amount of yeast extract supplied 1995; Hagen and Meeks, 2001; Knowles and Plaxton, 2003), and (Tang et al., 2010b). Because Hbt. modesticaldum has a complete the reaction of G6PDH in cyanobacteria is also known to be EMP pathway, but does not have key genes in the OPP and ED regulated by thioredoxin (Cossar et al., 1984; Lindahl and Flo- pathways (Sattley et al., 2008), it is likely that Hbt. modesticaldum rencio, 2003). In addition, the EMP pathway in cyanobacteria is employs the EMP pathway for carbohydrate catabolism and hydro- also suggested to be active. Other than glucose, some cyanobac- gen production as suggested in certain thermophilic microbes (de teria, such as Cyanothece, can grow on glycerol (Feng et al.,

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2010a). Glycerol is a precursor for biosynthesis of triacylglyc- phosphorylated and non-phosphorylated substrates, i.e., KDPG, erols, and can be also converted to GAP in the EMP/gluconeogenic 2-keto-3-deoxy-gluconate (KDG), GAP, and d-glyceraldehyde, pathway. pyruvate and its analogs, and a wide range of aldehyde com- ponents (Griffiths et al., 2002; Ahmed et al., 2005). Further, Aldolases employed in the EMP and ED pathways. the enzyme from the aerobic thermophilic archaeon Sulfolobus Figure 5B shows the reactions catalyzed by fructose 1,6- solfataricus, and perhaps Thermoplasma acidophilum, and other bisphosphate (FBP) aldolase (EC 4.1.2.13) and 2-keto-3-deoxy-6- thermoacidophilic Archaea, also has high relative activity with 2- phosphogluconate (KDPG) aldolase (EC 4.1.2.14). The reactions keto-3-deoxygalactonate (KDGal; Lamble et al., 2003). Directed catalyzed by both aldolases are reversible. FBP aldolase, catalyz- evolution and site-directed mutagenesis have also been employed ing the interconversion of FBP and d-glyceraldehyde-3-phosphate to further broaden the substrate specificities of the class I KDPG (GAP)/dihydroxyacetone phosphate (DHAP), is widely distrib- aldolase (Franke et al., 2004; Cheriyan et al., 2007). uted in phototrophs due to being employed by both the EMP pathway and gluconeogenesis. The tricarboxylic acid cycle Forward, reverse, and branched TCA cycles. Many metabolites FBP aldolase in the TCA cycle are the precursors of amino acids and biomass. Different classes of FBP aldolases have been identified in archaea, At least three forms of the TCA cycle have been identified in pho- bacteria, and eukaryotes (Say and Fuchs, 2010), and some FBP totrophic bacteria: the forward TCA cycle (or the OTCA cycle), aldolases are also responsible for additional biological functions the reverse TCA cycle (or the RTCA cycle), and the branched (or (Grochowski and White, 2008). All of the phototrophic bacte- incomplete) TCA cycle. The OTCA cycle or the Krebs cycle, dis- ria, except FAPs, have the class II FBP aldolase (encoded by the covered by Sir Hans Krebs more than seven decades ago (Krebs fbaA gene), which has been mainly found in bacteria and fungi. and Johnson, 1937), produces reducing equivalents and ATP, and The fbaB gene encoding the class I FBP aldolase, mainly found is one of the most important, if not the most essential, central in eukaryotes, can also be identified in the genomes of AnAPs, carbon and energy metabolic pathway in phototrophs and non- AAPs, cyanobacteria, and heliobacteria. Further, a bifunctional phototrophs. AnAPs (Imhoff et al., 2005; Madigan and Jung, 2009) FBP aldolase/phosphatase enzyme, which has no sequence homol- and AAPs (Yurkov and Beatty, 1998; Furch et al., 2009; Tang et al., ogy with the classical FBP aldolases, was recently identified in 2009a) employ an active OTCA cycle, like most non-phototrophic some archaea. The bifunctional enzyme has been suggested to be organisms. Cfl. aurantiacus operates the OTCA cycle under either an ancestral enzyme in gluconeogenesis (Say and Fuchs, 2010). aerobic (in darkness) or anaerobic (in the light) growth conditions Genes encoding the bifunctional FBP aldolase/phosphatase have (Sirevag and Castenholtz, 1979), whereas the family Oscillochlo- also been found in the genomes of phototrophs Roseiflexi species ridaceae in FAPs have a partial RTCA cycle (Berg et al., 2005). (Say and Fuchs, 2010). GSBs channel the reducing equivalents generated via photosyn- thetic electron transport to the RTCA cycle, which functions as KDPG aldolase an autotrophic CO2 assimilation pathway for producing cellular KDPG aldolase, which catalyzes the interconversion of KDPG material (Evans et al., 1966a; Sirevag, 1995). GSBs also operate and GAP/pyruvate (Figure 5B), is one of the two enzymes spe- the incomplete OTCA cycle (from citrate to α-ketoglutarate) dur- cific for the ED pathway, and the other specific enzyme is 6- ing mixotrophic growth with acetate (Feng et al., 2010b; Tang phosphogluconate dehydratase. While KDPG aldolase is required and Blankenship, 2010). Cyanobacteria have a branched TCA in the ED pathway,KDPG aldolase is not specific to the ED pathway cycle and do not have enzymes catalyzing interconversion of α- and is also responsible for Arg, Pro, glyoxylate, and dicarboxylase ketoglutarate and succinate (Stanier and Cohen-Bazire,1977). The metabolism (Conway,1992). KDPG aldolase has been identified in carbon flux via the branched TCA cycle is very low during pho- AnAPs (Conrad and Schlegel,1977,1978; Conway,1992) andAAPs toautotrophic growth of cyanobacteria. In this case, cyanobacteria (Yurkov et al., 1991; Furch et al., 2009; Tang et al., 2009a) that use use the branched TCA cycle for synthesizing biomass rather than the ED pathway for carbohydrate oxidation. The ED pathway has producing ATP and reducing equivalents (Shastri and Morgan, not been reported in cyanobacteria, while the eda gene encoding 2005). KDPG aldolase has been found in the genome of many cyanobac- Compared to the energy-producing OTCA cycle,metabolic flux terial strains.Although no detailed studies of KDPG aldolases from in the RTCA cycle is energetically unfavorable. While several reac- phototrophs have been reported, sequence alignments suggest that tions in the TCA cycle are reversible, the reductive carboxylation all of the phototrophic bacterial KDPG aldolases are the class I of succinyl-CoA to α-ketoglutarate (catalyzed by α-ketoglutarate KDPG aldolases,which form an internal active site lysine-substrate synthase) and of acetyl-CoA to pyruvate (catalyzed by pyruvate aldimine for catalysis (Fullerton et al., 2006). synthase) in the RTCA cycle are not thermodynamically favorable. The structure-function relationships and reaction mech- The standard Gibbs free energy change of the decarboxylation of anism of the class I KDPG aldolase in non-phototrophs pyruvate in pyruvate synthase is estimated to be −4.6 kcal/mol have been investigated intensively (Allard et al., 2001; Ahmed (Thauer et al., 1977). Thus, GSBs and other organisms using the et al., 2005; Fullerton et al., 2006). These studies indi- RTCA cycle recruit strong reductants, such as reduced ferredoxin, cate that the class I KDPG aldolase in the phosphorylative, to operate the carboxylation reactions catalyzed by pyruvate syn- semi-phosphorylative and non-phosphorylative ED pathways thase and α-ketoglutarate synthase and to proceed the RTCA cycle has promiscuous substrate specificities, and can interact with efficiently.

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Compared to other phototrophic bacteria, the carbon flow of have also been suggested (Qian et al., 2008). Also, bacteria with heliobacteria has not been understood until recently. The enzyme the activity of (Re)-CS reported often employ citramalate syn- activity of ATP citrate lyase, the key enzyme in the RTCA cycle, and thase for isoleucine synthesis (Feng et al., 2009; Tang et al., 2009b, (Si)-citrate synthase, the common enzyme for synthesizing citrate 2010a). Together, (Re)-CS, homocitrate synthase (Figure 6A), 2- to initiate the OTCA cycle (see below), have not been detected isopropylmalate synthase, and citramalate synthase (Figure A1 in in heliobacteria (Pickett et al., 1994; Tang et al., 2010a). Genes Appendix) catalyze Claisen condensation type reactions with sim- encoding ATP citrate lyase and (Si)-citrate synthase have not been ilar α-ketoacid substrates, and may have evolved from the same found in heliobacteria, whereas all other genes in the RTCA cycle ancestor (Figure 7). have been found in Hbt. modesticaldum genome (Sattley et al., Sequence comparisons between (Re)-CS and (Si)-CS also sug- 2008). Feeding the cultures with [2-13C]acetate and probing the gest that these two types of CS are phylogenetically distinct (Li 13C-labeling patterns of Glu, Kelly, and coworkers suggested that et al., 2007). As indicated by other enzyme pairs that catalyze the Heliobacterium strain HY-3 uses the OTCA cycle to synthesize α- formation of stereroisomers (Lamzin et al., 1995), dissimilar active ketoglutarate (Pickett et al., 1994). However, the authors cannot site structures between (Re)-CS and (Si)-CS are expected to gen- explain these observations with the lack of the (Si)-citrate syn- erate different citrate isomers. Moreover, unlike the potent inhi- thase. Our recent studies indicate that Hbt. modesticaldum uses bition of aconitase by (−)-erythro-2-F-citrate, previous studies (Re)-citrate synthase to initiate the incomplete OTCA cycle (from have suggested that (+)-erythro-2-F-citrate, synthesized from 2- citrate to α-ketoglutarate) to synthesize Glu, and that carbon flux fluoroacetate, and OAA by (Re)-CS, is not an inhibitor of aconitase is mostly carried out through the incomplete OTCA cycle (Tang (Lauble et al., 1996; Tang et al., 2010a). et al., 2010b). While Hbt. modesticaldum does not have the gene encoding (Re)-CS, two genes encoding putative homocitrate synthases have Enzymes operating in citrate metabolism been identified in the Hbt. modesticaldum genome (Sattley et al., Enzymes that synthesize citrate 2008). Homocitrate has only one known task in cells: serving as Citrate synthase is required for producing citrate through assimi- the precursor for lysine biosynthesis via the α-aminoadipate path- lating acetyl-CoA and OAA to initiate the OTCA cycle that gener- way. However, except for genes encoding putative homocitrate ates reducing equivalents. The citrate synthase reported in current synthase, other genes in the α-aminoadipate pathway (Figure A2 textbooks and most of the literature is (Si)-citrate synthase [(Si)- in Appendix) have not been identified in the Hbt. modesticaldum CS; EC 2.3.3.1]. The majority of organisms, including four types genome, so the function of the putative homocitrate synthases of photosynthetic microbes: AAPs, AnAPs, FAPs, and cyanobacte- remains to be verified. Whether one of the putative homoci- ria, use (Si)-CS to synthesize citrate from acetyl-CoA and OAA for trate synthases or another gene product functions as (Re)-CS in initiating the OTCA cycle. The gltA gene encoding (Si)-CS has also heliobacteria should be investigated. been identified in the genome of GSBs (Eisen et al., 2002; Daven- port et al., 2010). Alternatively, the activity of (Si)-CS has not been Enzymes that dissimilate citrate detected in heliobacteria (Pickett et al., 1994; Tang et al., 2010b), Cleavage of citrate through the RTCA cycle produces acetyl- and the gltA gene is absent in the Hbt. modesticaldum genome CoA and OAA for building cellular material. Citrate cleavage in (Sattley et al.,2008). However,when performing the enzyme assays biological organisms can be accomplished by three enzymes: cit- under anaerobic conditions,product turnover has been detected in rate lyase (EC 4.1.3.6), ATP citrate lyase (ACL; EC 4.1.3.8), or cell-free extracts of Hbt. modesticaldum with the addition of acetyl- citryl-CoA synthetase (CCS; or so called citryl-CoA ligase) fol- CoA, OAA, and Mn2+ metal ions (Tang et al., 2010a). Further, the lowed by citry-CoA lyase (CCL; Aoshima et al., 2004b; Hugler fifth carbon position of Glu has been found to be 13C-labeled using et al., 2007). The reaction of citrate lyase, which cleaves cit- [1-13C]pyruvate, also suggesting that Hbt. modesticaldum synthe- rate into acetate and oxaloacetate (OAA), is distinct from ACL sizes citrate via (Re)-citrate synthase [(Re)-CS; EC 2.3.3.3; Tang and CCS/CCL, which catalyze ATP-dependent cleavage of cit- et al., 2010a]. If citrate is synthesized via (Si)-CS, the first carbon rate to OAA and acetyl-CoA (Figure 6B). The phototrophic position of Glu is expected to be labeled using [1-13C]pyruvate. GSBs and some anaerobic bacteria use ACL to dissimilate cit- Differences in the labeling patterns of Glu result from whether rate produced via the RTCA cycle into acetyl-CoA and OAA (Si)-CS or (Re)-CS attaches the acetyl group at the pro-S or pro- (Evans et al., 1966a; Wahlund and Tabita, 1997; Kanao et al., R arm of OAA, respectively. The final product of (Re)-CS versus 2001; Kim and Tabita, 2006; Hugler et al., 2007; Voordeckers (Si)-CS is the same, since citrate is a symmetric molecule, while the et al., 2008). The reaction catalyzed by the Cba. tepidum ACL intermediate, (3R)-citryl-CoA versus (3S)-citryl-CoA, is different in vivo has been suggested to be reversible (Tang and Blanken- (Figure 6A). ship, 2010), and ACL is also responsible for the citrate synthe- Compared to (Si)-CS, (Re)-CS is an unconventional enzyme sis for acetate oxidation in the OTCA cycle in some anaerobic for citrate biosynthesis, and is phylogenetically related to homoci- chemotrophic bacteria (Thauer et al., 1989). Moreover, ACL is trate synthase (EC 2.3.3.14) and 2-isopropylmalate synthase (Li also responsible for producing cytoplasmic acetyl-CoA for many et al., 2007). Homocitrate synthase catalyzes the formation of biological processes in eukaryotic cells, such as histone acety- (3R)-homocitrate via the Claisen condensation of acetyl-CoA lation, lipid production, a signal for activating glucose catabo- and α-ketoglutarate (Tucci and Ceci, 1972; Wulandari et al., lism and apoptosis (Rathmell and Newgard, 2009; Wellen et al., 2002; Figure 6A). Similar sequences and active site structures 2009; Chu et al., 2010; Morrish et al., 2010; Wang et al., 2010; between 2-isopropylmalate synthase and homocitrate synthase Zhao et al., 2010).

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FIGURE 6 | Enzymes involved in citrate metabolism and central carbon metabolism. Claisen condensation catalyzed by (Re)-citrate synthase, (Si)-citrate synthase, and homocitrate synthase (A) and retro-aldol reactions catalyzed by ATP citrate lyase (ACL) and citryl-CoA synthetase (CCS)/citryl-CoA lyase (CCL) (B) are shown.

Different isoforms of ACLs have been identified. While a two-subunit ACL (non-mACL) suggest the one-subunit ACL is metazoan ACL (mACL) consists of a single polypeptide (Sun a gene fusion product of the two-subunit ACL. Also, a sec- et al., 2010), ACL from other organisms, including other ond type of ACL (the type II ACL) has also been reported eukaryotic cells (fungi, yeast, and plants) and microorganisms, recently in the magnetotactic α-Proteobacterium Magnetococ- including GSBs (Wahlund and Tabita, 1997; Kim and Tabita, cus sp. MC-1, which uses the RTCA cycle for autotrophic CO2 2006), has two subunits (encoded by aclA and aclB). Sequence fixation (Williams et al., 2006), and has also been proposed comparisons between the one-subunit ACL (mACL) and the in several proteobacterial genomes (Hugler and Sievert, 2011).

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FIGURE7|Proposed evolutionary perspectives of some central carbon another common ancestor. The oxidative TCA cycle was thought to have metabolic pathways. Previous studies suggested that 2-isopropylmalate evolved from the primitive reductive TCA cycle, whereas the reductive TCA synthase, homocitrate synthase, citramalate synthase, and (Re)-citrate cycle operated in the GSBs has been proposed to be an adaptive form that synthase may have evolved from a common ancestor, and that (Si)-citrate may have evolved from the OTCA cycle. The proposed evolutionary links are synthase, ATP citrate lyase and citryl-CoA lyase may have evolved from shown in dashed lines.

The function of the type II ACL has not been biochemically evolved from CCL [or (Si)-CS), CCS and succinyl-CoA synthetase confirmed. (Aoshima et al., 2004b]. Note that some species in the Aquificae While eukaryotes and most operate ACL, certain phylum use CCL/CCS and others operate ACL for citrate cleav- non-photosynthetic bacteria use CCL and CCS,instead of ACL,for age (Hugler et al., 2007), suggesting that ACL and CCL/CCS may citrate cleavage (Figure 6B). An obligately chemolithoautotrophic share some evolutionary history. ACL, (Si)-CS, and CCL may have bacterium H. thermophilus (Aoshima et al., 2004a,b) and some evolved from a common ancestor (Figure 7). members of the family Aquificaceae (Hugler et al., 2007)havebeen The OTCA cycle has been suggested to have evolved from the reported recently to use CCS and CCL to cleave citrate. Further, RTCA cycle (Wachtershauser, 1990). GSBs, which use ACL for the iron-oxidizing bacterium Leptospirillum ferriphilum has genes citrate cleavage, may operate the RTCA cycle through an adap- encoding CCL and CCS, but not ACL, identified in the genome tation and horizontal gene transfer in response to environmental (Levican et al., 2008). changes. Note that genes encoding (Si)-CS (gltA) and ACL (aclBA) are present in the genomes of GSBs (Eisen et al., 2002; Davenport Citrate metabolism and the TCA cycle: evolution or adaptation? et al.,2010). The sequence information suggests thatACL may have Citrate metabolism operates in every living cell. Citrate biosyn- evolvedfrom(Si)-CS and succinyl-CoA synthetase (Aoshima et al., thesis is required for initiating the OTCA cycle, and oxidation 2004b). In addition to the RTCA cycle, GSBs also operate a partial of citrate through the OTCA cycle creates reducing equivalents OTCA cycle during mixotrophic growth (Tang and Blankenship, and energy. ATP-dependent citrate cleavage produces acetyl-CoA 2010). Phylogenetic analyses with 16S rRNA genes of photosyn- and OAA to build cellular material. (Re)-CS and (Si)-CS cat- theticbacteria(Woese, 1987; Pace, 1997) suggest that GSBs evolved alyze citrate biosynthesis and start the OTCA cycle, and ACL and later than FAPs, which have an active OTCA cycle. Taken together, CCS/CCL operate ATP-dependent citrate cleavage via the RTCA the RTCA cycle in GSBs may have evolved from the OTCA cycle. cycle. Sequence alignments suggest that (Si)-CS and CCL are phy- The proposed evolutionary lineages of citrate metabolism and the logenetically relevant, and that ACL is likely a gene fusion product TCA cycle in the central carbon metabolic network are illustrated of (Si)-CS (or CCL) and succinyl-CoA synthetase. Sequences of in Figure 7. CCS and succinyl-CoA synthetase are also similar (Aoshima et al., 2004a). Additionally, slight CCL activity has been reported in (Si)- EVOLUTIONARY PERSPECTIVES OF CENTRAL CARBON METABOLISM CS (Srere, 1963) and low (Si)-CS activity has also been found Understanding the evolution of photosynthesis is an important in the H. thermophilus CCL (Aoshima et al., 2004b). Based on objective in a deeper understanding of the origin and develop- the sequence homology of enzymes involved in citrate metabo- ment of photosynthetic diversity. Lateral and horizontal transfers lism, (Si)-CS may have evolved from CCL and ACL may have of photosynthetic genes among phototrophic bacteria have been

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proposed during the evolution of photosynthesis (Nagashima Clostridium), use iPGM, whereas most of the Gram-(−)bacteria et al., 1997; Raymond et al., 2002; Raymond and Blankenship, employ dPGM. Moreover, although both GSBs and heliobacte- 2004), although it has not been generally accepted which bac- ria have pyruvate synthase/α-ketoglutarate synthase instead of teria were donors and which bacteria were recipients during pyruvate dehydrogenase/α-ketoglutarate dehydrogenase,their car- gene transfers. The photosystems of photosynthetic bacteria have bon flow patterns in the TCA cycle are entirely different (Feng been well studied. Proteobacteria (AnAPs and AAPs) and FAPs et al., 2010b; Tang and Blankenship, 2010; Tang et al., 2010a). have a type II (quinone type) RC, and GSBs, chloroacidobacte- Together, gene/enzyme pairs in citrate biosynthesis and sugar ria and heliobacteria have a type I (Fe-S type) RC (Blankenship, metabolism, and carbon flow in the TCA cycle of heliobacte- 2002). Phototrophic bacteria containing the same type (or dif- ria versus GSBs clearly indicate that their carbon metabolism ferent types) of RCs have been known to use distinct (or similar) correlates with the 16S rRNA-based phylogeny of the group light-harvesting antenna complexes. For example,chlorosomes are (), rather than with the presence of the photosyn- present in Chloroflexi species (FAPs), GSBs, and chloroacidobac- thetic RC. According to many similarities in carbon metabolism teria but absent in Proteobacteria, the Roseiflexi species (FAPs), between heliobacteria and non-phototrophic Firmicutes, either and heliobacteria. Also, GSB, chloroacidobacteria and heliobacte- genes encoding carbon metabolism in non-phototrophic Firmi- ria have a type I RC, while heliobacteria have no light-harvesting cutes may have been transferred from or to heliobacteria, or genes complexes other than antenna pigments that are part of the RC encoding photosystems in heliobacteria may have been trans- core complex. This is distinct from GSB and chloroacidobacteria, ferred from other photosynthetic bacteria. The latter hypothesis is both of which contain chlorosomes and the Fenna–Matthews– consistent with the fact that heliobacteria have the simplest photo- Olson (FMO) complex. So there seems to be little correlation system among phototrophic bacteria (Madigan, 2006; Heinnickel between the RC types and the light-harvesting antenna com- and Golbeck, 2007; Sattley et al., 2008; Sattley and Blankenship, plexes in anoxygenic phototrophic bacteria. These features suggest 2010). horizontal gene transfers of RC or light-harvesting complexes among phototrophic bacteria. Moreover, some features in the CONCLUSION carbon metabolism of phototrophic bacteria suggested below All life on Earth requires carbon sources and energy. Phototrophic also suggest horizontal gene transfers between phototrophic and bacteria use light as the energy source for autotrophic,mixotrophic non-phototrophic bacteria. and also heterotrophic growth. The carbon metabolism in photo- synthetic bacteria is not only essential for constructing cellular Central carbon metabolism of heliobacteria versus material, but also providing reducing equivalents for photosyn- non-phototrophic Firmicutes thetic electron transport during photoheterotrophic growth. The Among GSBs, chloroacidobacteria, and heliobacteria, only GSBs central carbon metabolism of photosynthetic bacteria has received are known to be photoautotrophs. Heliobacteria, the only more attention recently, mainly due to their metabolic versatil- Gram-(+) phototrophic bacteria, use (Re)-CS to produce cit- ity and/or uniqueness as well as the information acquired from rate possibly via the (3R)-citryl-CoA intermediate, and GSBs systematic analyses. This review illustrates that photosynthetic contain the gene encoding (Si)-CS and use ACL and/or (Si)- bacteria employ many unconventional central carbon metabolic CS to synthesize (Si)-CS. Previous studies indicate that several pathways and novel enzymes in response to their ecological niches, species exclusively use (Re)-CS to synthesize citrate whereas certain carbon assimilation pathways remain to be under- (Li et al., 2007). Additionally, two types of phosphoglycerate stood. Further, both evolutionary origin and late adaptation have mutase (PGM) have been reported to catalyze the interconversion been suggested for several of the central metabolic enzymes and of 2-phosphoglycerate and 3-phosphoglycerate in carbohydrate pathways. Together, the rich knowledge accumulated for central metabolism: the cofactor-dependent PGM (dPGM) and cofactor- carbon metabolism, along with the information obtained from independent PGM (iPGM). It has been suggested that dPGM may the genes of 16S rRNA, various enzymes and photosynthetic com- be evolutionarily related to a family of acid phosphatases and ponents, is expected to shed more light on the origin and evolution to fructose 2,6-bisphosphatase (Schneider et al., 1993), and that of photosynthesis. iPGM has been derived from the family of alkaline phosphatase and sulfatases (Galperin et al., 1998). Heliobacteria have the gpmI ACKNOWLEDGMENTS gene (encoding iPGM; Sattley et al., 2008) and GSBs have the This work was supported by the Exobiology Program of NASA gpmA gene (encoding dPGM). Likewise, members of the Gram- Grant NNX08AP62G (Robert Eugene Blankenship) and National (+) bacteria, including spore-forming Firmicutes (Bacillus and Science Foundation Career Grant MCB0954016 (Yinjie J. Tang).

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Zhao, S., Xu, W., Jiang, W., Yu, W., Lin, Conflict of Interest Statement: The Citation: Tang K-H, Tang YJ and Copyright © 2011 Tang, Tang and Y., Zhang, T., Yao, J., Zhou, L., Zeng, authors declare that the research was Blankenship RE (2011) Carbon meta- Blankenship. This is an open-access arti- Y., Li, H., Li, Y., Shi, J., An, W., Han- conducted in the absence of any bolic pathways in phototrophic bacteria cle subject to a non-exclusive license cock, S. M., He, F., Qin, L., Chin, J., commercial or financial relationships and their broader evolutionary impli- between the authors and Frontiers Media Yang, P., Chen, X., Lei, Q., Xiong, that could be construed as a potential cations. Front. Microbio. 2:165. doi: SA, which permits use, distribution and Y., and Guan, K. L. (2010). Regula- conflict of interest. 10.3389/fmicb.2011.00165 reproduction in other forums, provided tion of cellular metabolism by pro- This article was submitted to Frontiers in the original authors and source are cred- tein lysine acetylation. Science 327, Received: 06 April 2011; accepted: 18 July Microbial Physiology and Metabolism, a ited and other Frontiers conditions are 1000–1004. 2011; published online: 01 August 2011. specialty of Frontiers in Microbiology. complied with.

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APPENDIX pyruvate, is the first and the only specific enzyme employed in APPROACHES FOR INVESTIGATING CARBON METABOLISM OF the citramalate pathway, which directly converts pyruvate to 2- PHOTOTROPHS ketobutyrate for isoleucine biosynthesis (Figure A2 in Appendix). Prior to the availability of genomic information, the carbon All of the other enzymes in the citramalate pathway are also metabolism of phototrophic organisms was investigated by activ- engaged in leucine biosynthesis (Xu et al., 2004). While citramalate ity assays,together with stable isotope-labeling (Strauss et al.,1992; synthase can only be identified in organisms that operate the citra- Pickett et al., 1994) or radioactive isotopic-labeling experiments malate pathway (Howell et al., 1999), 2-isopropylmalate synthase (Bassham et al., 1950; Evans et al., 1966b; Conrad and Schlegel, (EC 2.3.3.13), catalyzing the formation of (2S)-2-isopropylmalate 1977). Recently, functional characterization of phototrophic via the Claisen condensation of 2-oxoisovalerate and acetyl-CoA, organisms has been accelerated because: (1) The instruments and is universally required for leucine biosynthesis. 2-oxoisovalerate data analyses of GC-/LC-MS (gas/liquid chromatography-mass is also the precursor of valine. High sequence homology (∼50% spectrometry) and NMR (nuclear magnetic resonance) have been identity and ∼70% similarity) between citramalate synthase and significantly improved; (2) high-throughput sequencing methods 2-isopropylmalate synthase has been reported (Howell et al., 1999; have determined complete genomes of photosynthetic organisms Xu et al., 2004; Risso et al., 2008), in agreement with similar and many of their genomes have been fully annotated; and (3) substrates for these two enzymes (Figure A1 in Appendix). more systematic approaches (i.e., “omics”), such as transcrip- tomics, proteomics, metabolomics, and fluxomics (Sauer, 2006; Peyraud et al., 2009; Singh et al., 2009; Stitt et al., 2010;Tripp et al., 2010), have been developed. Among these metabolomics approaches, 13C-based metabolic analysis and 13C-flux analysis have been recognized as powerful tools to probe active carbon metabolic pathways in vivo via feeding cells with a 13C-labeled carbon source and tracing 13C-labeling patterns in biomass and metabolites. Table A1 in Appendix illustrates several carbon meta- bolic pathways in phototrophic bacteria probed by 13C-labeled protein-based amino acids (Pickett et al., 1994; Furch et al., 2009; Peyraud et al., 2009; Tang et al., 2009a, 2010a; Feng et al., 2010a,b; Wu et al., 2010). In cooperation with in vivo physiological studies (or so called“physiomics”; Sanford et al., 2002),activity assays,and in vitro biochemical characterizations (Alber et al.,2006; Berg et al., 2007; Zarzycki et al., 2009), more knowledge and new insights into carbon metabolism of photosynthetic and non-photosynthetic microbes have been revealed.

CITRAMALATE SYNTHASE AND 2-ISOPROPYLMALATE SYNTHASE FIGURE A1 | Reactions catalyzed by citramalate synthase and Citramalate synthase (EC 2.3.1.182), catalyzing the formation of 2-isopropylmalate synthase. (2R)-citramalate via the Claisen condensation of acetyl-CoA and

www.frontiersin.org August 2011 | Volume 2 | Article 165 | 21 Tang et al. Central carbon metabolism and evolution in photosynthesis , Tang Tang , , Feng et al. Tang et al. Feng et al. – studies on , , , Tang et al. (2010a) Tang et al. (2010a) , , Pickett et al. (1994) Pickett et al. (1994) , , . a,b,c Feng et al. (2010b) Feng et al. (2010a) Peyraud et al. (2009) non-phototrophs (2010b) Feng et al. (2010a) Feng et al. (2010a) Feng et al. (2010a) (2010b) Furch et al.(2009a) (2009) et al. (2009a) Feng et al. (2010a) et al. (2009a) Wu et al. (2010) Tang et al. (2010a) ) Rba. sphaeroides C-labeled carbon sources 13 GSBs, heliobacteria (incomplete) FAPs and possibly AAPs cyanobacteria Cyanobacteria Phototrophic bacteria References FAPs, cyanobacteria Heliobacteria AnAPs, cyanobacteria, some AnAPs (e.g., AAPs, GSBs, heliobacteria, AAP,AnAPs 60% C]Glc) or > -KG 13 α C]Glc) labeling -KG because -KG labeled )-citrate synthase) 13 α -KG and OAA α α Si b,c in the OPP pathway leads -KG than pyruvate due to α 2 ways. C-labeling in Glc released as CO OAA/ assimilating non-labeled inorganic carbon and R5P due to assimilating non-labeled inorganic carbon and metabolites in the pathway and Ile because both amino acids are synthesized from acetyl-CoA and pyruvate OAA versus in labeled carbons are not scrambled in to non-labeled pyruvate Features 13 13 (versus C1 position of labeled with ( in GAP than in pyruvate higher (with [6- His. → a Glu, and R5P His, Ser Significantly low labeling in GAP Not applicable Analysis of the intermediates Ala, Asp, Glu Notably lower labeling in Asp Enriched labeling in OAA All of the photosynthetic bacteria Asp, Glu Different labeling patterns in → -KG -ketoglutarate; OAA, oxaloacetate; R5P,ribose-5-phosphate. α α Ala, C]- C]- -KG, α 13 13 C]- C]acetate Leu, Ile Identical labeling patterns in Leu →→ 13 13 and [2- C]Glc Ser, Ala Notably lower (with [1- 2 13 C]- pyruvate Asp, pyruvate 13 → C]pyruvate or [2- C]pyruvate or [1- C]Glc or [6- C]pyruvate GluC]Glc C5 position of C]acetyl-CoA and Ala 13 13 13 13 13 13 C-carbon sources Biomarkers C-bicarbonate or non-labeled C-bicarbonate [1- [1- 13 13 13 glycerol pyruvate inorganic carbon with [1- and [3- glycerol [2- Ser. OAA → ways C-isotopomer labeling patterns of protein-based amino acids in central metabolic pathways of phototrophic bacteria using 13 Amino acids: GAP → -anaplerotic path 2 C-labeling patterns of protein-based amino acids serve as biomarkers for probing metabolic path 13 )-citrate synthase (via the Abbreviations: GAP,d-glyceraldehyde-3-phosphate; Glc, glucose, Precursors Re The Pathways and enzymes a b c pathway OTCA cycle) Citrate synthesized by ( isoleucine biosynthesis The branched TCA cycle [3- The oxidative PP pathway [1- The Entner–Doudoroff (ED) The ethylmalonyl-CoA pathway [1- The reductive TCA cycle Non-labeled inorganic carbon The Calvin–Benson cycle Non-labeled CO The CO The citramalate pathway in TableA1 |The

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FIGURE A2 | Amino acids biosynthesis in phototrophic and The pathways for isoleucine and lysine biosynthesis are shown in blue and non-phototrophic bacteria. Biosynthesis of amino acids directly and red, respectively. Phototrophic bacteria have not yet been known to indirectly employing pyruvate and oxaloacetate (OAA) as precursors is shown. synthesize lysine via the α-aminoadipate pathway.

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