Phylogeny and evolution of the ribulose 1, 5-bisphosphate ...

ISSN 0026-8933, Molecular Biology, 2009, Vol. 43, No. 5, pp. 713–728. © Pleiades Publishing, Inc., 2009. Original Russian Text © T.P. Tourova, E.M. Spiridonova, 2009, published in Molekulyarnaya Biologiya, 2009, Vol. 43, No. 5, pp. 772–788.

UDC 585.852

Phylogeny and Evolution of the Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase Genes in Prokaryotes T. P. Tourova and E. M. Spiridonova Winogradskii Institute of Microbiology, Russian Academy of Sciences, Moscow, 117811 Russia; e-mail: [email protected] Received December 1, 2008 Accepted for publication February 4, 2009

Abstract—The review considers the phylogeny and evolution of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO), which is the key enzyme of the autotrophic Calvin–Benson cycle and the most abundant protein on Earth. RuBisCO occurs in several structural and functional forms, including fully functional forms I, II, and III, which catalyze carboxylation/oxygenation of ribulose 1,5-bisphosphate, and RuBisCO-like form IV, which lacks carboxylating activity. The genomic localization, operon structure, and copy number of the RuBisCO genes vary among different autotrophic organisms. The RuBisCO gene phylogeny substantially differs from the phylogeny of other conserved genes, including the 16S rRNA gene. The difference is due to duplication/deletion and horizontal gene transfer events that were common in the evolution of autotrophic organisms. DOI: 10.1134/S0026893309050033 Key words: ribulose 1,5-bisphosphate carboxylase/oxygenase, RuBisCO, bacteria, archaea, phylogeny, evolution

INTRODUCTION The primary production of organic carbon in the biosphere is based on ëé2 assimilation by autotrophic organisms. Groups of autotrophs developed various mechanisms of ëé2 fixation in the course of evolution: the ribulose bisphosphate (Calvin–Benson) cycle, reduction tricarboxylic acid cycle, 3-hydroxypropionic acid cycle, reduction dicarboxylic acid cycle, and the reduction acetyl-CoA pathway [1]. Recent studies have revealed a new mechanism in certain archaea [2]. The Calvin–Benson cycle is the most common mechanism of ëé2 assimilation. Although oxygenic phototrophs (higher plants and algae) make a major contribution to the global ëé2 fixation, studies of ëé2 fixation in oxygenic (cyanobacteria) and anoxygenic (photo- and chemoautotrophic bacteria) prokaryotes are of special interest for reconstructing the phylogeny of autotrophic metabolism and autotrophic organisms. Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO, EC 4.1.1.39) is the key enzyme of autotrophic ëé2 fixation via the Calvin–Benson cycle. While the structural and biochemical properties of the enzyme have been studied in detail by classical biochemical methods, scarce data are available for its origin and evolution. It is only known that autotrophic organisms that assimilate ëé2 via the Calvin–Benson cycle belong to different and rather distant evolution-

ary branches. In addition, the structure and organization of RuBisCO substantially varies among organisms, and four RuBisCO forms are currently known. Hence, it is still an open question as to whether the RuBisCO origin is monophyletic or polyphyletic. It proved efficient to study the origin and evolution of RuBisCO with the use of molecular phylogenetic methods, by comparing the nucleotide sequences of the RuBisCO genes for various groups of prokaryotes. A comparison of phylogenetic trees reconstructed via a conventional analysis of 16S rRNA genes and a nucleotide sequence analysis of the RuBisCO genes (and/or deduced amino acid sequences) and, in some cases, other structural genes may reveal some specifics of the evolution and function of RuBisCO genes in various prokaryotes. In addition, such data may clarify questionable taxonomic positions or evolutionary issues for individual groups of autotrophic prokaryotes. STRUCTURE AND BIOCHEMICAL PROPERTIES OF RUBISCO As a key enzyme of the Calvin–Benson cycle [3], RuBisCO catalyzes the addition of a carbon atom from atmospheric CO2 to ribulose 1,5-bisphosphate, a five-carbon sugar, to yield an unstable six-carbon intermediate, which immediately decays into two molecules of 3-phosphoglycerate. The product is then reduced to triose phosphates, and the CO2 acceptor is

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TOUROVA AND SPIRIDONOVA xylulose 5-phosphate ribose 5-phosphate

erythrose 4-phosphate

ribulose 5-phosphate

sedoheptulose fructose 6-phosphate 7-phosphate fructose bisphosphate

ATP

sedoheptulose 1,7-bisphosphate dioxyacetone phosphate

glyceraldehyde 3-phosphate

ribulose 1.5-bicphosphate

RuBisCO

CO2 H2O

3-phosphoglycerate

NAD(P)H

ATP

Fig. 1. Ribulose bisphosphate cycle of CO2 assimilation [1].

regenerated via subsequent molecular transformations. The fixation of three ëé2 molecules on three ribulose 1,5-bisphosphate molecules yields one molecule of phosphoglycerate, which is utilized in organic syntheses of the cell (Fig. 1) [1]. Apart from carboxylation, RuBisCO is capable to catalyze the oxygenase reaction in the presence of oxygen, cleaving ribulose bisphosphate into phosphoglycolic and phosphoglyceric acids [4]. In fact, the carboxylation reaction provides a basis for all metabolic pathways in living organisms on Earth. The oxygenation reaction arose as oxygen was released into the atmosphere by oxygenic phototrophs. RuBisCO is the most widespread protein [5] and is best understood among all 11 enzymes involved in the Calvin–Benson cycle. Exhaustive data on the structure and activity of RuBisCO are available [6]. Two main forms of RuBisCO are known today: forms I and II. The forms are involved in the Calvin– Benson cycle and differ in structure and activity. In addition, form III was found in archaea, but its functional significance is still unclear, since the Calvin– Benson cycle is unknown for archaea. Form IV is recognized, which combines the so-called RuBisCO-like proteins, which are incapable of catalyzing the ribulose 1,5-bisphosphate-dependent reaction of ëé2 fixation and are presumably involved in serum metabolism in response to oxidative stress [7]. RuBisCO Form I Form I is the most widespread and consists of eight large (L, Mr ~ 55 kDa) and eight small (S, Mr ~ 15 kDa) subunits. The molecular mass of the native enzyme (hexadecamer L8S8) is approximately 550 kDa [8].

The large subunits are catalytic; the role of the small subunits is unclear. The small subunits lack catalytic activity, but the enzyme activity substantially increases in their presence, possibly because the hexadecameric structure is stabilized and conformational shifts arise in the active center of the enzyme [9, 10]. The nucleotide and amino acid sequences of the small subunits greatly vary among different organisms, while the primary structure of the large subunits is conserved to a greater extent [11]. RuBisCO form I is major in phototrophic organisms (both pro- and eukaryotes) and aerobic chemilithoautotrophs [12]. RuBisCO form I is divided into two types, greenlike and red-like, which differ in the primary structure of the large subunits. Amino acid sequence homology of the large subunits is 69–92% within each type and is only 53–60% between the two types [13]. The green-like type occurs in two variants, IA and IB [11]. Variant IB is found in chloroplasts of higher plants, plastids of green algae, and cyanobacteria and is phylogenetically related to variant IA, which is found in α-, β-, and γ-proteobacteria [12]. The enzymes of the cyanobacteria Prochlorococcus marinus and Synechococcus sp. WH7803 are phylogenetically closer to proteobacterial variant IA than to cyanobacterial variant IB [14, 15]. The red-like type of form I is also divided into two relatively close variants, IC and ID, which occur, respectively, in α- and β-proteobacteria and many nongreen algae [11]. Since the oxygenation reaction may cause a 50% loss of the fixed carbon [16], thus dramatically suppressing the ëé2-depending growth of the organism, the most important kinetic parameter of RuBisCO is the specificity factor τ, which is a ratio of carboxylase to oxygenase activity at any ëé2 and O2 concentraMOLECULAR BIOLOGY

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PHYLOGENY AND EVOLUTION OF PROKARYOTIC RuBisCO

tions [τ = (vc[O2])/(vo[CO2])] and reflects the capability of the enzyme to select the reaction substrate [17]. It is unknown how the enzymes prefer one substrate over another one. The τ factor is usually higher than 80 for form I of higher plants, 25–75 for form I of bacteria, and lower than 20 for RuBisCO form II [17, 18]. A high τ factor means that RuBisCO is capable of selectively fixing ëé2 regardless of the presence of oxygen and, therefore, is better adapted to lower ëé2 concentrations and/or aerobic conditions. RuBisCO Form II RuBisCO form II consists of only large subunits (Ln), whose number varies from two to eight depending on the organism. The catalytic subunits of forms I and II differ both biochemically and immunologically, and the homology between their amino acid sequences is only 25–30% [8]. In spite of the low overall homology, the amino acid sequences share highly conserved regions, which are related to the active center. Conservation of important active-center amino acid residues determines the uniformity of active centers and the three-dimensional structures in all carboxylases [19]. RuBisCO type II was first isolated from Rhodospirillum rubrum [20] and, more recently, from other nonsulfur purple bacteria, Rhodobacter sphaeroides [21] and Rhodobacter capsulatus [22]. It was long believed that form II is restricted to these bacteria. However, RuBisCO was later found in other bacteria, including the Vestimentifera symbiont Riftia pachyptila [23], Hydrogenovibrio marinus [24], Thiomonas intermedia [25], Thiobacillus denitrificans [26], Halothiobacillus neapolitanus [27], and Magnetospirillum magnetotacticum [28], as well as in eukaryotic dinoflagellates [29]. In addition, genes presumably coding for RuBisCO type II were found in the completely sequenced genomes of various bacteria, but experimental evidence for their functional activity is still lacking. The amino acid sequences of the form II large subunits are highly homologous in phototrophic (Rh. sphaeroides and Rh. rubrum) and chemotrophic (H. marinus and T. denitrificans) prokaryotes and eukaryotic dinoflagellates. While four variants are recognized for form I by amino acid sequence homology, form II is a uniform group of enzymes with an evolutionarily conserved sequence [30]. There is a hypothesis that RuBisCO form II is the most ancient and, possibly, ancestral for the other forms, since it has an extremely low specificity factor τ and, consequently, is best adapted to higher ëé2 concentrations [31]. It is possible that RuBisCO form II was the first to arise in an anaerobic environment, while form I evolved as the Cé2 concentration grew lower and é2 appeared in the atmosphere [32, 33]. MOLECULAR BIOLOGY

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Archaeal RuBisCO (Form III) In archaea, RuBisCO activity was initially observed in extracts of the extreme halophile Haloferax medditerranei [33] and, more recently, in the hyperthermophiles Pyrodictium abyssi and P. occultum [34]. In addition, genome sequencing revealed putative RuBisCO genes in the hyperthermophilic archaea Methanococcus jannaschii, Archaeoglobus fulgidus, and Thermococcus kodakaraensis [35, 36]. Since both methane-producing and sulfate-reducing archaea utilize pathways other than the Calvin–Benson cycle (the reduction acetyl-CoA pathway, the tricarboxylic acid cycle, etc.) to fix ëé2, it is quite surprising that their genomes have sequences potentially coding for the large subunit of RuBisCO [36]. Expression of the putative archaeal RuBisCO genes in Escherichia coli and subsequent purification and examination of the recombinant proteins showed that the genes code for functional proteins that possess RuBisCO activity [35, 36]. Moreover, it was demonstrated that the TÒ. kodakaraensis KOD1 gene coding for RuBisCO is indeed transcribed and translated and that its protein product Tk-RuBisCO does occur in native cells [35]. Further studies revealed that the product of M. jannaschii, A. fulgidus, Methanosarcina acetivorans, and Ms. earkeri rbcL is a catalytically active enzyme that catalyzes the ribulose 1,5-bisphosphate-dependent reaction of ëé2 fixation in cell extracts [37]. The Ms. acetivorans RuBisCO gene proved to function in Rh. capsulatus and Rh. sphaeroides mutant strains defective in the RuBisCO genes under both photoheterotrophic and photoautotrophic conditions [37]. Thus, archaeal RuBisCO is capable of CO2 fixation via the Calvin–Benson cycle. A phylogenetic analysis of the amino acid sequences of all known RuBisCO forms showed that the archaeal proteins cluster in a group that substantially differs from RuBisCO forms I and II. The archaeal RuBisCO sequences have motifs characteristics of the RuBisCO large subunits. The amino acid sequence homology between form III and forms I and II is approximately 50%. All active-center amino acid residues that are directly involved in catalysis are conserved in archaeal carboxylases identified as RuBisCO form III [36, 38]. Archaeal RuBisCO consists only of large subunits. Small subunits were not found in the enzyme, nor were their coding sequences detected in archaeal genomic DNA [36, 38]. A high sequence homology (>70%) is observed for sequences belonging to the same form in forms I and II. In contrast, archaeal RuBisCO greatly vary in amino acid sequence and three-dimensional structure [39]. The homology of RbcL-1, the product of one of the two RuBisCO genes found in A. fulgidus, to the M.

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jannaschii and Tc. kodakaraensis is so low (41.0 and 50.1%, respectively) that RbcL-1 was assigned to form IV (see below). The highest sequence homology (81.9%) is observed between A. fulgidus RbcL-2 and the Tc. kodakaraensis enzyme [35]. The most comprehensive data are available for RuBisCO of M. jannaschii and Tc. kodakaraensis. The M. jannaschii enzyme is a homodimer (L2, år ~105 kDa) and is extremely sensitive to oxygen, losing most of its activity upon exposure in air; the effect is reversible. The protein is thermostable and can be exposed at 85°ë for up to 60 min without any loss of activity. In spite of its inhibition by oxygen, this unusual enzyme has low oxygenase activity. Its specificity factor τ is 0.5, the lowest known for RuBisCO [36]. RuBisCO of Th. kodakaraensis is a homodecamer ((L2)5, år ~ 450 kDa) and consists of five dimeric subunits, which form a pentagonal ring structure. Analysis of the three-dimensional structure of the enzyme revealed unique ionic interactions between the surfaces of neighboring dimers (L2/L2 interactions), which are involved in maintaining the pentagonal decameric structure. The unique quaternary structure improves the thermostability of the enzyme and increases its denaturation temperature from 90°C (the denaturation temperature of the L2 dimer) to 113°ë. Since TÒ. kodakaraensis grows at a temperature ranging from 65 to 100°ë, the decameric structure is probably essential for RuBisCO stabilization in the cell [38]. In contrast to M. jannaschii RuBisCO, the TÒ. kodakaraensis enzyme is not inhibited by oxygen and has the highest known factor τ, 310 at 90°ë [38]. The difference in biochemical properties between the M. jannaschii and TÒ. kodakaraensis enzymes possibly reflects the relatively low homology (61.3%) of their amino acid sequences. In addition, the difference may be explained by the fact that the properties of M. jannaschii RuBisCO were assayed at room temperature, while a temperature of 65°ë is optimal for the enzyme [36, 38]. Putative RuBisCO genes were found in the genomes of other archaea: Pyrococcus furiosus, P. abyssi, Pc. horikoshii, and Ms. mazei. Although the three-dimensional structure was not studied for their protein products, sequence comparisons do not support the idea that decamerization is a common feature of archaeal RuBisCOs. Their high thermostability is possibly due to stronger and/or more numerous atom– atom interactions within dimers [38]. Likewise, Ar. fulgidus and Ms. acetivorans RuBisCOs are homodimers [37]. The functional role of archaeal RuBisCO is unknown. The M. jannaschii and A. fulgidus genomes harbor putative genes for enzymes involved in the Calvin–Benson cycle, such as ribose 5-phosphate

isomerase, phosphoglycerate kinase, glyceraldehyde 3-phosphate dehydrogenase, and triose phosphate isomerase. However, a gene potentially coding for phosphoribulokinase, which produces a substrate of RuBisCO, was not found in any archaeal genome. Active phosphoribulokinase was also not detected in archaea in enzymatic studies [37]. Recent experiments [39] showed that M. jannaschii, as well as other methane-producing archaea, utilize an alternative pathway to synthesize ribulose 1,5-bisphosphate, whose key precursor is 5-phospho-D-ribose 1-pyrophosphate (PRPP). The new pathway points to a unique evolutionary relationship between the Calvin– Benson cycle and purine metabolism, since PRPP is a central metabolite that is common for these two pathways. A phylogenetic analysis of various form III RuBisCOs showed that, diverse as they are, these archaeal enzymes followed a separate evolutionary path and represent a separate RuBisCO form [35]. Since RuBisCOs are found in archaea, which are the most ancient microorganisms, it is likely that RuBisCO is one of the most ancient enzymes that appeared at the pre-oxygen stage of the evolution of the biosphere. RuBisCO-like Proteins (Form IV) Genome sequencing in the green sulfur bacterium Chlorobaculum tepidum revealed a gene for a protein that is structurally similar to RuBisCO but is incapable of catalyzing the ribulose 1,5-bisphosphate dependent reaction of ëé2 fixation. The protein was termed a RuBisCO-like protein (RLP) and assigned to form IV [7]. To date, RLPs have been found in many microorganisms, including C. thiosulfatophilum, Rhodopseudomonas palustris (RLP1 and RLP2), Allochromatium vinosum, Mesorhizobium loti, Burkholderia cepacia, Bordetella bronchiseptica, A. fulgidus (RbcL-1), Bacillus subtilis (YkrW), and B. anthracis. RLPs are highly diverse in structure and form at least one subgroup, which includes RLPs of the green sulfur bacteria C. tepidum and Chlorobium limicola, the purple nonsulfur bacterium Rps. palustris, and the purple sulfur bacterium Al. vinosum. While amino acid sequence homology in this subgroup of RuBisCO form IV is approximately 60%, other RLPs have only 25–30% homology. In addition, the phylogenetic relationships among certain RLPs are no closer than between form IV and the other RuBisCO forms. This sequence diversity most likely reflects the fact that phylogenetically different RLPs perform physiologically different functions [41]. A common feature of RLPs is that they all have amino acid substitutions in conserved active-center motifs that determine carboxylase activity. Of the 19 conserved amino acid residues of the active center, MOLECULAR BIOLOGY

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substitutions affect ten residues in Chlorobaculum, eight in B. subtilis, and four in A. fulgidus. All of the known RLPs lack carboxylase activity [7]. Like enzymes of the other three forms, C. tepidum RLP is a homodimer with the active center formed between the two subunits. Since the shape and chemical properties of the active center are changed by amino acid substitutions, RLP cannot bind ribulose 1,5-bisphosphate, but is probably capable of binding its structural analogs. It is thought that C. tepidum RLP catalyzes reactions other than carboxylation, possibly, enolization. In spite of the sequence diversity of the four RuBisCO forms, their dimers as functional units have a conserved structure [42]. The physiological role remains unclear for the majority of RLPs. Experiments with deletion mutants showed that C. tepidum RLP is not absolutely essential for cell growth. The rates of autotrophic growth and autotrophic ëé2 assimilation decreased in the mutant compared with the wild-type strain by factors of 3–4 and 2.9, respectively, and defects were observed in serum metabolism (the accumulation of elementary sulfur globules was fourfold higher) and the content of photosynthetic pigments (the BChlc content was 20% lower than in the wild-type strain). Elementary serum is a product of H2S oxidation during phototrophic groups and is usually oxidized to sulfate in later growth phases, when the H2S and thiosulfate pools are exhausted. The observed decrease of photoautotrophic growth and ëé2 fixation rates in the mutant with a disrupted RLP gene probably results from distorted oxidation of elementary serum and thiosulfate, which serve as electron donors for ëé2 fixation upon H2S depletion [7]. In addition, the C. tepidum mutant accumulates many oxidative stress proteins [7]. It is thought that C. tepidum RLP plays an important role in sulfur metabolism and the response to oxidative stress [7]. Chlorobaculum tepidum, Ch. limicola, Rps. palustris, and Al. vinosum are phototrophic sulfur-oxidizing bacteria. Their RLPs form a separate cluster in RuBisCO form IV. Since, C. tepidum RLP probably affects elementary sulfur and thiosulfate oxidation, a similar function may be assumed for RLPs of the other organisms of the cluster [41]. Studies showed that B. subtilis RLP, which is encoded by ykrW, plays a main role in the accessory pathway of methionine synthesis via a recyclization of methylthioadenosine, which is a byproduct of polyamine biosynthesis [43, 44]. Hence, it is possible that phylogenetically distant C. tedium and B. subtilis RLPs play different physiological roles in bacterial cells in accord with the above hypothesis [41]. The gene coding for RuBisCO form II in the phototrophic bacterium R. rubrum complements the ykrW defect in the B. subtilis mutant strain, restoring the MOLECULAR BIOLOGY

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recyclization of toxic compounds to methionine. Thus, there is a functional link between the B. subtilis RLP gene involved in the accessory methionine biosynthesis pathway and the R. rubrum RuBisCO gene; the two genes possibly originate from a common ancestor [44]. A hypothesis was advanced that RLPs are evolutionary ancestors of RuBisCOs currently occurring in plants, bacteria, and archaea [7]. Gupta et al. [45, 46] proposed a linear phylogeny of bacteria on the basis of an analysis of conserved inserts and deletions in highly conserved catalytic domains of various proteins. Gram-positive bacteria (including B. subtilis), whose DNAs have a low GC content, were assumed to directly descend from ancient bacteria. Moreover, such Gram-positive bacteria are thought to appear earlier than archaea, Gram-negative bacteria, cyanobacteria, and phototrophic bacteria. Synonymous codon usage and the GC content of the B. subtilis RLP gene are typical of the B. subtilis genome, indicating that the RLP gene was not acquired from another organism via horizontal transfer [47]. These findings made it possible to assume that all other RuBisCO forms originate from a common ancestral RLP (which then evolved into B. subtilis RLP) or even a particular RLP involved in the accessory methionine biosynthesis pathway and that B. subtilis RLP represent an ancestral protein that was preserved up to date and gave origin to all other RuBisCO forms [48]. At the same time, it cannot be excluded that RLPs are intermediates in the evolution from the true RuBisCO function to alternative activities [7]. ORGANIZATION AND LOCALIZATION OF RUBISCO GENES The genes coding for the large and small subunits of RuBisCO form I may form an operon with the genes coding for other enzymes involved in the Calvin–Benson cycle or certain proteins that modify the activity of these enzymes or regulate transcription of their genes. In this case, each gene of the operon has a prefix cbb, meaning that the gene is a structural or regulatory gene of the Calvin–Benson–Bassham cycle [49]. Such organization of the RuBisCO form I genes (cbbLS) was observed in proteobacteria. Cyanobacterial and plant RuBisCO operons do not contain other structural genes of the Calvin–Benson cycle and consist exclusively of the large and small subunit genes. In this case, the genes are usually designated as rbcL and rbcS for the large and small subunits, respectively. The genes coding for RuBisCO type II are always organized in a cbb operon and are designated as cbbM [6]. The localization and number of functional RuBisCO genes may substantially vary among different and even closely related (at the level of species)

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microorganisms. Many chemoautotrophic bacteria display both chromosomal and plasmid localizations of cbbLS, and the genes occur in more than one functional copy in some cases. For instance, autotrophic CO2 fixation via the Calvin–Benson cycle in the facultative autotroph Cupriavidus necator H16 is genetically determined by two cbbLS operons, of which one is in the chromosome and the other is in the megaplasmid pHG1. Duplication of the cbb genes in chromosomes and megaplasmids was observed for several other strains of the species. At the same time, the heavy metal-resistant Cv. metallidurans strain CH34 lacks pHG-like plasmids and has only chromosomal cbb genes [50, 51]. A second cbbLS gene set occurs in pGH21-a in Alcaligenes hydrogenophilus M50 [52]. The genes coding for the large subunit of RuBisCO form I (cbbL) were found in certain aerobic carboxydobacteria. Like in Cv. necator H16, these genes are duplicated in Oligotropha carboxydovorans OM5: one copy is in the chromosome, and the other is in pHCG3 [52]. The nitrifying bacterium Nitrobacter hamburgensis X14 similarly has cbbLS in both the chromosome and pPB13 [53]. The genes of the cbb operon are exclusively in pHG22-a in the aquatic bacterium Acidovorax facilis K, while the strain J of the same species has only a chromosomal copy of the cbb operon [52]. An exclusively plasmid localization of cbbLS is characteristic of Rhodococcus opacus, the only Grampositive hydrogen bacterium examined in this respect. Its strains MR11 and MR22 have the genes in the linear plasmids pHG201 and pGH205, respectively [54]. An exclusively chromosomal localization of the cbb genes is observed for the symbiotic nitrogen-fixing bacterium Bradyrhizobium japonicum, which is capable of chemoautotrophic growth as a free-living organism [55]. A plasmid localization of cbbLS was not detected in phototrophic bacteria [52]. Several copies of functional RuBisCO genes may occur in a chromosome of one organism. For instance, two cbbLS copies are found in the chromosome in many strains of the obligate chemolithoautotroph Acidithiobacillus ferrooxidans. However, there is only one cbbLS gene set in the Ac. ferrooxidans strain ATCC 19859 [56]. In phototrophic bacteria, two sets of the cbb genes for form I (cbbAB and cbbLS) were found only in the purple sulfur bacterium Al. vinosum [57] and, in our recent study, in closely related Al. minutissimum [58]. Although both of the copies are functional, there is a certain difference between them. RuBisCO encoded by the genes designated as rbcAB dominates, while the rbcLS genes are expressed to an extremely low level. The enzymes encoded by the two gene sets are both functionally active, but they differ in constants and CO2 specificity [57]. At the same time, we observed a substantial sequence similarity between

Allochromatium rbcL and the only (and, consequently, preserving a high physiological activity) cbbL copy of purple sulfur bacteria of the genus Thiocapsa. This finding made it possible to assume that these genes both originate from a common ancestor, a corresponding gene of purple sulfur bacteria of the family Chromatiaceae. None of the above bacteria possessing a double set of the cbbLS genes for RuBisCO form I combines both green-like and red-like types of the enzyme. The two gene sets both code for form I of the green-like type in Ac. ferrooxidans, Al. vinosum, and Al. minutissimum and of the red-like type in Cv. necator. The purple nonsulfur bacterium Rh. azotoformans is the only known organism whose genome has the cbbLS operons for both types of form I [59]. There is no data on duplication of the genes for RuBisCO form II (cbbM) in the genome of one organism or their plasmid localization, but many autotrophic bacteria, including certain purple nonsulfur bacteria and thiobacilli, have both RuBisCO forms I and II. The purple nonsulfur bacterium Rh. sphaeroides was the first organism whose genome was found to code for both forms of the enzyme [21]. The bacterium has two chromosomes, each carrying a cluster of the cbb genes with a different organization. Chromosome I contains the cbbI operon, whose cbbLS genes code for RuBisCO form I of the red-like type. The cbbII operon is in chromosome II and includes cbbM, which codes for RuBisCO form II. Each of the operons has additional genes for other enzymes involved in the Calvin–Benson cycle, and some of these genes are duplicated [60]. The only chromosome of Rh. capsulatus, another purple nonsulfur bacterium, contains two spatially separate cbb operons. A smaller operon includes cbbLS, coding for RuBisCO form I of the green-like type [61]. In contrast to the Rh. sphaeroides cbbI operon, the Rh. capsulatus cbbLS genes are not associated with structural genes for other enzymes of the Calvin–Benson cycle [62]. A larger operon is highly similar to Rh. sphaeroides cbbII, including cbbM and genes for other enzymes of the Calvin–Benson cycle. An analysis of the cbbL and cbbM mutants of Rh. sphaeroides and Rh. capsulatus showed that disruption of the gene for one RuBisCO form increases transcription of the other operon and production of its protein product so that the total RuBisCO concentration does not significantly differ from that in the wild-type strain. Thus, a loss of one RuBisCO form is compensated for by the other form [60, 63]. RuBisCO form I is necessary for cell growth at a low CO2 level (which agrees with the hypothesis that form I has higher affinity for CO2 as compared with form II), and an increase in CO2 strongly inhibits RuBisCO form I [64]. MOLECULAR BIOLOGY

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Rhodobacter sphaeroides and Rh. capsulatus were assigned to one genus on the basis of classical morphological and chemotaxonomic traits [65]. The two bacteria are closely related on evidence of a phylogenetic analysis of their 16S rRNAs [66]. The RuBisCO form II amino acid sequences of Rh. sphaeroides and Rh. capsulatus have more than 94% homology, which confirms their close phylogenetic relatedness. However, form I RuBisCOs of these bacteria belong to different types (green-like and red-like) and substantially differ in amino acid sequence (58% homology) [67]. Substantial differences were observed for the structure and regulation of the cbb operons coding for RuBisCO form I [60, 62], suggesting different mechanisms for the evolution of RuBisCO form I in these phototrophic bacteria.

bacteria start producing CbbLS-2, which has the highest factor τ (33.1). All three carboxylases are expressed at an extremely low CO2 concentration (0.03%). It seems that CbbLS-1 is produced when the level of CO2 fixation by CbbLS-2 and/or CbbM is insufficient for cell growth. Thus, H. marinus adapts to various CO2 concentrations by utilizing different RuBisCO forms [69]. More recent studies revealed three RuBisCO gene sets in several sulfur-oxidizing oblicate chemolithoautotrophes of the genus Thiomicrospira, which is phylogenetically related to H. marinus [70]. The presence of three RuBisCO gene sets (two cbbLS sets and one cbbM gene) in the complete genome sequence was demonstrated for several Ac. ferrooxidans strains.

The sulfur-oxidizing bacterium T. denitrificans was the first nonphotosynthetic organism whose genome was found to contain genes for RuBisCO forms I and II. This bacterium is an obligate autotroph that is capable of CO2 fixation via the Calvin–Benson cycle in both aerobic and anaerobic conditions. Both RuBisCO forms are expressed in T. denitrificans in anaerobic conditions. An increase in CO2 dramatically inhibits RuBisCO form I, as in Rh. sphaeroides and Rh. capsulatus [26, 30].

PHYLOGENY AND EVOLUTION OF RUBISCO

The genome of the obligatory aerobe Halothiobacillus neapolitanus similarly preserved functional cbbM, and the microorganism synthesizes both RuBisCO forms at a higher CO2 concentration (5%). The level of form II expression is extremely lower than that of form I expression even in these conditions. It is possible that form II dominates when the oxygen concentration decreases to a minimum essential for cell growth. When bacteria grow in air, the production of form II decreases and that of form I considerably increases. RuBisCO form I, which is more tolerant of oxygen, becomes the only form involved in CO2 assimilation in this case. Disruption of cbbL increases the cbbM expression, but the mutant strain is incapable of growth at the atmospheric CO2 concentration [27]. Finally, the genomes of certain microorganisms have three sets of RuBisCO genes: two cbbLS sets and one cbbM gene. Three RuBisCO gene sets were first found in the hydrogen-oxidizing bacterium H. marinus [24, 68]. The three RuBisCO genes vary in expression depending on the CO2 concentration, and the expression profile agrees with the specificity factor τ for each carboxylase. When the CO2 concentration is 15%, bacteria synthesize only RuBisCO form II (CbbM), which has the lowest τ (14.8). When CO2 concentration decreases to 2%, bacteria start producing CbbLS-1, whose specificity factor is intermediate (26.6). CbbM expression is also observed in this case. When CO2 concentration decreases to 0.15%, CbbLS-1 becomes undetectable, the CbbM level decreases, and MOLECULAR BIOLOGY

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Generally, the results of phylogenetic analyses of RuBisCO sequences do not always coincide with the data obtained by comparing other macromolecules, in particular, the 16S rRNA [12]. Specifically, the accepted classification of the genes coding for RuBisCO form I into the green- and red-like types disagrees with the current views of the origin of plastids and bacterial evolution. According to the results obtained by comparing the 16S and 23S rRNA genes and supported by sequence analyses of several conserved structural genes (tufA for the elongation factor EF-Tu, atpB for H+-ATPase subunit β, rpoC1 for RNA polymerase subunit β', etc.), all plastids are of a cyanobacterial origin and possibly have a common ancestor [71–75]. However, plastids fail to form a monophyletic groups on phylogenetic trees based on the RuBisCO gene sequences. The plastid RuBisCO genes of green algae, plants, and glaucophytes belong to the IB cluster of the green-like type of form I, while the plastid RuBisCO genes of cryptomonads and red and brown algae form the ID cluster of the red-like type [13]. The phylogenetic relationships within the IB and ID clusters generally agree with the taxonomic positions of the corresponding organisms [76]. Compared with plastid trees, phylogenetic trees constructed for bacteria on the basis of the 16S and 23S rRNA gene or cbbL/rbcL differ in topology to a far greater extent. According to the ribosomal gene analysis, α-, β-, and γ-proteobacteria form monophyletic groups within the same phylogenetic division Proteobacteria. The phylogenetic tree based on the cbbL/rbcL sequences has a far more intricate topology. For instance, the α-proteobacterium N. vulgaris appears in the cluster of green-like sequences, while Rh. sphaeroides, Xanthobacter, a Mn-oxidizing bacterium, and an α-proteobacterium appear in the cluster of red-like sequences. In addition, the β-proteobacterium Cv. necator is in the cluster of red-like

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sequences, while T. denitrificans is in the green-like sequence cluster. Some aspects of the RuBisCO phylogeny within the green- and red-like sequence clusters also disagree with the phylogeny of the 16S rRNA genes. For instance, RuBisCO sequences of α and β-proteobacteria are intermixed within each type at variance with the monophyletic origin assumed for the two groups of bacteria on the basis of the 16S rRNA analysis. The most striking difference between the two phylogenetic trees concerns the position of the cyanobacteria Prochlorococcus marinus and Synechococcus sp. WH7803, which cluster with γ-proteobacteria in the RuBisCO tree, in contrast to other cyanobacteria [12]. The discrepancies between the phylogenetic data based on RuBisCO genes sequences and other marker genes are hardly attributable to methodical errors. The majority, if not all, of the differences observed for the cbbL/rbcL phylogeny reflect the difference between phylogenies of genes and organisms. The differences in the cbbL/rbcL phylogeny are most likely explained by horizontal transfer and duplication of genes and a differential loss of one of the gene copies during evolution [13]. In the context of the horizontal transfer hypothesis, four independent horizontal transfer events are assumed to explain the division of plastids and proteobacteria into the green- and red-like groups (greenlike/red-like division of form I). First, the RuBisCO operon of the red-like type was transferred from an αproteobacterium to a common ancestor of red and brown plastids (nongreen eukaryotic algae). The donor cannot be identified unequivocally, since α and β-proteobacterial red-like sequences are intermixed in the RuBisCO tree and data are available for only a few bacterial genes coding for red-like RuBisCO. At least three horizontal transfer events presumably occur during the subsequent distribution of green- and red-like genes among proteobacteria. A relatively simple scenario suggests that a cyanobacterial green-like rbcL sequence was transferred to an ancestor of γ-proteobacteria in their early evolution (after the divergence of β-proteobacteria from γ-proteobacteria). More recently, this green-like γ-proteobacterial sequence was transferred to the ancestors of the α-proteobacteirum N. vulgaris and β-proteobacterium T. denitrificans [13]. Several additional horizontal transfer events are assumed to explain the discrepancies observed for the cbbL/rbcL phylogeny and unrelated to the greenlike/red-like division of form I. For example, plastids of certain peridinin-containing dinoflagellates (such as Gonyaulax) differ from plastids of other algal groups in utilizing RuBisCO type II encoded by the nuclear genome. Since dinoflagellate chloroplasts are of a cyanobacterial origin, it is clear that Gonyaulax

RuBisCO does not originate from plastids because RuBisCO form II is not found in cyanobacteria. It is possible that the Gonyaulax RuBisCO form II gene initially occurred in the mitochondrial genome and then was transferred into the nucleus, while the original plastid gene for RuBisCO form I was lost [29]. Thus, the evolution of Gonyaulax RuBisCO provides an example of RuBisCO gene transfer and replacement. Another possible horizontal transfer event involved the rbcL genes that are found in the cyanobacteria Pr. marinus and Synechococcus sp. WH7803 and substantially differ from the RuBisCO genes of other cyanobacteria. The genes appear in the cluster IA, which consists totally of proteobacterial greenlike sequences. Apart from their rbcL sequences, no data relate these cyanobacteria to proteobacteria. Hence, Pr. marinus and Synechococcus sp. WH7803 acquired their RuBisCO genes via lateral transfer from the green-like proteobacterial group [12, 13]. Finally, since α and β-proteobacterial sequences do not form the expected monophyletic groups within the red-like cluster of RuBisCO form I, lateral gene transfer is assumed to occur within the cluster [12, 13]. However, the available data are too scarce to clarify the evolution of the RuBisCO genes in this group. On the other hand, a gene duplication hypothesis was advanced as an alternative of the horizontal gene transfer hypothesis to explain the division of plastids and proteobacteria into the green- and red-like groups (green-like/red-like division of form I). This hypothesis suggests that a single duplication event affected the RuBisCO operon before the divergence of cyanobacteria and proteobacteria. Subsequent evolution involved multiple independent events of gene loss in each of the lineages. This assumption fully explains the green-like/red-like division of form I, and the distribution of taxa between the two types simply reflects which one of the copies was preserved. However, the gene duplication hypothesis suggests that the two types of form I coexisted in some lineages for a long time (approximately 1–2 Ma, at least until plastids appeared and the green- and red-like lineages diverged). If so, some cyanobacteria and protebacteria would still preserve the RuBisCO genes of both types. However, not a single cyanobacterium is now known to have a multiple gene set coding for RuBisCOs of both types [13]. The only case where genes for both types of RuBisCO form I occur in one organism, the purple nonsulfur bacterium Rh. azotoformans, most likely results from lateral transfer rather than gene duplication [59]. A phylogenetic analysis of cbbL was performed and its evolution was studied in the genus Rhodobacter [59]. The results showed that Rh. sphaeroides and Rh. azotoformans cluster together within the red-like type of form I, suggesting a comMOLECULAR BIOLOGY

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Green-like Red-like

?

Rh. sphaeroides

Red-like

Rh. azotoformans

Red-like Green-like

Rh. blasticus

Green-like

Rh. capsulatus

Green-like

Rh. veldkampii

Green-like

Green-like

Fig. 2. Hypothetical evolutionary history of cbbL in the genus Rhodobacter [59].

mon origin of the red-like cbbL for the two species. The green-like cbbL genes of Rh. veldkampii, Rh. blasticus, Rh. capsulatus, and Rh. azotoformans similarly cluster together in the group of green-like sequences and, probably, originate from a common ancestor. Since Rh. veldkampii occupies the most basal position in the Rhodobacter cluster in the phylogenetic tree based on the 16S rRNA sequences and has only green-like form I, it was assumed that the common ancestor of the genus Rhodobacter also had only green-like form I. If so, the ancestor of Rh. sphaeroides and Rh. azotoformans received the red-like gene via horizontal transfer, and the ancestor of Rh. sphaeroides subsequently lost the green-like gene (Fig. 2) [59]. If duplication was responsible for the presence of two cbbL genes of different types in the Rh. azotofomans genome, such a duplication had to occur in a common ancestor of the genus Rhodobacter. This hypothesis suggests that cbbL genes of both types coexisted for a long time and that multiple independent losses of either green- or red-like genes occurred in various evolutionary lineages, which seems unlikely. Thus, lateral gene transfer is the most plausible cause of the coexistence of the green- and red-like types of form I in Rh. azotoformans. In turn, if the ancestor of all species of the genus Rhodobacter acquired the green-like form I gene via horizontal transfer in addition to the ancestral gene for RuBisCO form II, the above hypothesis does not contradict the earlier hypothesis that form I appeared in the ancestor of Rh. capsulatus as a result of horizontal gene transfer [67]. MOLECULAR BIOLOGY

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Another evidence of horizontal gene transfer is the discrepancy between the ribosomal and RuBisCO phylogenies of alkalophilic bacteria of the genus Thioalkalivibrio [77, 78]. According to an analysis of the 16S rRNA gene sequences, strains of this genus form a separate cluster within the family Ectothiorhodospiraceae [79, 80] (Fig. 3a). All autotrophic bacteria of the family have only one RuBisCO gene, which codes for green-like form I. However, the phylogenetic tree based on its analysis considerably differs in topology from the tree based on the ribosomal sequences, especially in the region of two species of the genus, Tv. nitratireducens and Tv. paradoxus. The species form one phylogenetic group within the genus on the tree based on the 16S rRNA sequences. A common origin of the two species is confirmed by their cell morphology (both species have large immobile coccoid cells with large sulfur inclusions), which is unusual for the genus Thioalkalivibrio [80]. The cbbL genes of the group (Fig. 3b) are also of a common origin and are highly divergent from the genes of the other species of the genus. Moreover, the two species reliably cluster together with the purple sulfur bacteria Thiocapsa roseopersicina and Al. vinosum, which belong to Chromatiaceae, another family of γ-proteobacteria. This strongly suggests a common origin for the cbbLS genes of these microorganisms differing in phylogenetic position and physiological and biochemical properties. Horizontal gene transfer is again one of the possible evolutionary mechanisms in this case. Such transfer possibly occurred in a common ancestor of all bacteria of the cluster and involved a gene block rather than a single gene, judging from certain common morphological and physiological characteristics.

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Horizontal gene transfer was also assumed for the evolution of the cbbL genes in sulfur-oxidizing bacteria of the genus Thiobacillus, since several representatives of the genus form separate branches clustering with various species of β and γ-proteobacteria on the RuBisCO tree [81]. At the same time, the fact that several bacteria have more than one RuBisCO gene provides the best evidence for duplication of RuBisCO genes in the cases unrelated to the green-like/red-like division of form I. For instance, high nucleotide sequence homology between the chromosomal and plasmid cbbLS genes in Cv. necator H16 made it possible to assume that the two gene clusters most likely originate via duplication and intragenomic rearrangements and that this event is relatively recent [50, 51]. A similar explanation was assumed on the same grounds for the presence of near identical chromosomal cbbLS sets in Ac. ferrooxidans Fe1 [82]. A similar assumption is possible for the fact that RuBisCO forms I and II coexist in several proteobacteria. This fact possibly reflects a primary duplication that occurred before the diversification of forms I and II. Since RuBisCO form II has a simple homodimeric structure (Lx) and low substrate specificity, it is thought to be an ancestral form of the enzyme. If so, the original RuBisCO form would be widespread, while form II (found only in proteobacteria and several dinoflagellates) is less common than form I. Hence, it cannot be excluded that form II appeared during the evolution of proteobacteria via duplication of a form I gene and a subsequent loss of small subunit-coding nucleotide sequences and an increase in the evolutionary rate of large subunit-coding sequences [13]. At the same time, the mechanism generating multiple gene copies may differ even between strains of the same species. The two cbbL genes of Ac. ferrooxidans ATCC 23270 not only have low nucleotide sequence homology (approximately 75%), but they also substantially differ in other characteristics, such as GC content (55.4 mol % in cbbL-1 and 58.9 mol % in cbbL-2) and synonymous codon usage. Hence, lateral gene transfer was assumed as the most plausible mechanism that generated two sets of genes coding for RuBisCO form I in Ac. ferrooxidans ATCC 23270 [83]. It should be noted that the hypotheses of horizontal gene transfer and gene duplication are not mutually exclusive. For instance, both of the mechanisms are probably responsible for the presence of a triple set of RuBisCO genes (two cbbLS sets and one cbbM gene) in the genome of the hydrogen-oxidizing bacterium H. marinus [24, 68]. Nucleotide sequence homology between cbbL-1 and cbbL-2 is 76.5%. The two genes display near identical codon usage and have much the same GC content (45.4 mol % in cbbL-1 and

46.4 mol % in cbbL-2), which is comparable with the GC content in H. marinus total DNA (44.1 mol %). Taken together, these findings made it possible to assume a common origin for the two cbbLS gene sets; i.e., gene duplication in an ancestor of H. marinus is more likely than lateral transfer of one of the gene sets. It cannot be excluded, however, that genes were transferred from an organism similar in codon usage and GC content or that the transfer was so ancient that the new gene had enough time to adapt to the genome structure of the recipient cell [68]. However, further studies of the three RuBisCO gene sets in H. marinus showed that, in spite of the high nucleotide sequence homology, the cbbLS-1 operon with its accompanying genes strikingly differs in structure from the cbbLS-2 operon and is highly similar in organization of the cbbM operon, which codes for another RuBisCO form [69]. The earlier hypothesis of simple duplication of the cbbLS genes in an ancestor of H. marinus [68] fails to explain this finding. The organization of the operons coding for RuBisCO and related enzymes probably results from a set of duplication, lateral transfer, and genome reorganization events that took place during evolution. The following explanation can be assumed for the presence of three RuBisCO genes and their organization. An H. marinus ancestor, which had the cbbM operon, acquired the cbbLS-2 genes via lateral transfer. Duplication of the cbbLS-2 genes and their subsequent reorganization with genes of the cbbM operon yielded the cbbLS-1 operon [69]. Additional data that refine the evolutionary scenario assumed for the RuBisCO genes were obtained in studies of alkalophilic sulfur-oxidizing bacteria of the genera Thiomicrospira and Thioalkalimicrobium, which form a common Thiomicrospira phylogenetic group along with H. marinus [70]. We found that Tms. kuenenii and Tms. crunogena similarly have three RuBisCO gene sets each, while Tms. pelophila, which is phylogenetically separate from this group of species, has two gene sets corresponding to H. marinus cbbL-2 and cbbM. Thioalkalimicrobium species have only one cbbL gene, which corresponds to H. marinus cbbL-2. Codon usage analysis did not relate the Thiomicrospira group to other autotrophic sulfuroxidizing bacteria whose sequences might result from horizontal gene transfer. Moreover, the analysis of codon usage in cbbL identified the cyanobacterium Anabaena sp. PCC 7120 as the species most closely related to the Thiomicrospira group (Fig. 4). The above findings, together with the data on H. marinus, made it possible to assume a scenario of RuBisCO gene evolution for the total Thiomicrospira group of genera (Fig. 5). The similarity of cbbL-2 and cbbL-1 in nucleotide sequence, GC content, and codon usage supports gene duplication in the genome of an ancestral form of the Tms. crunogena cluster, MOLECULAR BIOLOGY

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100

Allochromatium vinosum

Thialkalispira microaerophla

Eclothiorhodospira shaposhnikovii Alkalilimmnikola ehrlichii

Thialkalivibrio denitrificans

Thialkalivibrio thiocyanodenitrificans

Thialkalivibrio nitratireducens

Thialkalivibrio paradoxus

Thialkalivibrio thiocyanoxidans

Nitrobacter winogradskyi

Rhodobacter capsulatus

Hydrogenovibrio marinus

Halothiobacillus neapolitanus

Svnechcoccus sp. PCC 6301

Prochlorothrix hollandica

Prochlorococcus marinus

Anabaeba sp. PCC 7120

a

2

1

Cyanobacteria

Hydrogenophaga pseudoflava

Wautersia metallidurans

Tiomonas intermedia

Thiobacillus denitrificans

Hydrogenophilus thermoluteolus

Acidithiobacillus ferroxidons

Synechococcus sp. PCC

85

98

Thialkalivibrio versutus

Thialkalivibrio nitratis

Methylococcus capsulatus

100

100

82 100

83

97

100

100

Thialkalivibrio jannaschii

b

3

γ

P r o t e o b a c t e r i a

82 100

100 80

0.05

b

Thialkalivibrio nitratis

Prochlorococcus sp. GP2 Synechcoccus sp. WH7803

Nitrobacter winogradskyi Nitrobacter vulgaris Tioalkalispira microaerophila



Hydrogenovibrio marinus cbbL-2 Anabaeba sp. PCC 7120 Synechococcus sp. PCC

2

Cyanobacteria

Thialkalivibrio nitratireducens Thialkalivibrio paradoxus

Cyanobacteria

PB-a

Eclothiorhodospira shaposhnikovii Thialkalivibrio denitrificans Allochromatium vinosum cbbL-1 Tiomonas intermedia PB-b

1

Rhodobacter capsulatus Methylococcus capsulatus Alkalilimmnikola ehrlichii

Thialkalivibrio thiocyanoxidans Thialkalivibrio jannaschii Halothiobacillus neapolitanus

100

PB-b

PB-a

Hydrogenovibrio marinus cbbL-1 Hydrogenophilus thermoluteolus PB-b

Acidithiobacillus ferroxidons Thioalkalivibrio versutus

100

Thiobacillus denitrificans PB-b Acidithiobacillus ferroxidons Hydrogenophaga pseudoflava Wautersia metallidurans Thialkalivibrio thiocyanodenitrificans

Allochromatium vinosum cbbL-2 Thiocapsa roseopersicino

90

100

100

84

86

PB-γ

Fig. 3. Phylogenetic tree of alkalophilic autotrophic bacteria of the genus Thioalkalivibrio as constructed on the basis of (a) the nucleotide sequences of the 16S rRNA gene or (b) the amino acid sequences encoded by cbbL. Bar, evolutionary distance corresponding to five nucleotide or amino acid substitutions per 100 bp. Bootstrap supports are indicated (values of more than 80 are considered to be significant). Organisms examined in our studies [78–80] are in bold.

81

100

100

92

92

0.05

a

PHYLOGENY AND EVOLUTION OF PROKARYOTIC RuBisCO 723

TOUROVA AND SPIRIDONOVA Frequency of codons ending with G or C

724

ÒbbL 1.3

Pro

1.1 0.9 0.7 0.5 0.3 0.1

–0.1

Afel-1

Tcrl-2 Tkul-2 Tpel-2 Hmal-2 Tac Hmal-1 Tkul-1 Tcrl-1 Taa Tas Ana

–0.3 –1.0

–0.8

Tsae Syne Htne

Syn

Esp Rhca Avil-2 Tvni Hhps Afel-2 Tvde TminTvpa ThpaTvnit Tvthi Pho Synec Avil-1 Hpps Tvja Tbde Tvve Tvth

–0.6 –0.4 –0.2 0 0.2 0.4 Frequency of codons ending with C or U versus A or G

0.6

Fig. 4. Analysis of synonymous codon usage in the cbbL genes of representatives of the Thiomicrospira group: () Thiomicrospira”. cbbL-2 and () cbbL-1 found in organisms of the group; () cbbL found in other organisms. Designations: Taa is Tm. aerophilum, Tas is Tm. sibiricum, Tac is Tm. cyclicum, TcrI-2 is Tms. crunogena cbbL-2, TkuI-2 is Tms. kuenenii cbbL-2, TpeI-2 is Tms. pelophila cbbL-2, HmaI-2 is H. marinus cbbL-2, TcrI-1 is Tms. crunogena cbbL-1, TkuI-1 is Tms. kuenenii cbbL-1, HmaI-1 is H. marinus cbbL-1, AfeI-1 is Acidithiobacillus ferrooxidans cbbL-1, AfeI-2 is A. ferrooxidans cbbL-2, Tbde is Thiobacillus denitrificans, Htne is Halothiobacillus neapolitanus, Tmin is Thiomonas intermedia, Tsae is Thioalkalispira microaerophila, AviI-1 is Allochromatium vinosum cbbA, AviI-2 is Alc. vinosum cbbL, Tvni is Thioalkalivibrio nitratireducens, Tvpa is Tv. paradoxus, Tvth is Tv. thiocyanoxidans, Tvve is Tv. versutus, Tvja is Tv. jannaschii, Tvnit is Tv. nitratis, Tvde is Tv. denitrificans, Tvthi is Tv. thiocyanodenitrificans, Esp is Ectothiorhodospira shaposhnikovii, Hhps is Hydrogenophaga pseudoflava, Ana is Anabaena sp. PCC 7120, Pho is Prochlorothrix hollandica, Pro is Prochlorococcus marinus, Syn is Synechococcus sp. PCC 7002, Syne is Synechococcus sp. PCC 6301, Synec is Synechococcus sp. WH7803, Thpa is Thioclava pacifica, Rhca is Rhodobacter capsulatus, and Hpps is Hydrogenophilus thermoluteolus.

including Tms. crunogena, Tms. kuenenii, and H. marinus. Based on codon usage, it was cyanobacteria that most probably donated cbbL-2 to the ancestor of the Trhimicrospira group. Two scenarios are possible for the subsequent evolution of the Tms. pelophila cluster: either cbbL-2 was not duplicated in the genome of an ancestor of the cluster, or its duplication did occur, but cbbL-1 was lost during further evolution of the group. Species of the genus Thioalkalimicrobium additionally lost cbbM. Selective loss of cbb genes from a multiple set might result from evolutionary selection of the optimal RuBisCO form. Since RuBisCO form I is capable of selective CO2 assimilation in the presence of oxygen and is thereby better fit to modern atmospheric conditions, additional cbbM or cbbL genes are not essential. Hence, these genes are likely to be inactivated and lost depending on the living conditions of their host organisms. If so, cbbL-1 and cbbM loss in Thioalkalimicrobium species (obligate alkalophiles isolated from bicarbonate lakes) and cbbL-1 loss in Tms. pelophila (an alkali-tolerant bacterium) were associated with adaptation to their ecological niches, namely, to low CO2 concentrations at pH > 8.

However, the presence of several RuBisCO forms with different catalytic properties provides certain ecological advantages and flexibility in some ecological niches. This is supported by the fact that Tms. crunogena, which has three RuBisCO gene sets, has the highest growth rate among mesophilic chemolithotrophs. Thus, both evolutionary mechanisms should be considered to provide the most plausible explanation of the RuBisCO phylogeny in many cases. A discrepancy due to lateral gene transfer and/or duplication or selective loss of genes is also observed between the phylogenetic data based on the 16S rRNA sequences and other molecular markers, including glyceraldehyde phosphate dehydrogenase [84], Hsp60 [85], ATPase [86], etc. The cbbL/rbcL gene is widespread and sufficiently conserved, thus providing a convenient model for studying this phenomenon. A better understanding of RuBisCO evolution can be expected to facilitate the interpretation of similar situations for other genes [13]. CONCLUSIONS Although ribosomal phylogenetics became one of the most common methods to study the relationships MOLECULAR BIOLOGY

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Ancestor of the Thiomicrospira group

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Cyanobacterium

cbbM

Lateral transfer of the cbbLS-2 operon

cbbLS-2 duplication and reorganization with genes of the cbbM cluster

cbbLS-2 duplication did not take place

cbbM cbbLS-2 cbbLS-1

cbbM cbbLS-2

Loss of the cbbM operon

Loss of the cbbLS-1 operon cbbLS-2 Thiomicrospira crunogena Hydrogenovibrio marinus

Thiomicrospira pelophila Thiomicrospira kuenenii Thioalkalimicrobium sp.

Thiomicrospira crunogena cluster

Thiomicrospira pelophila cluster

Fig. 5. Hypothetical evolution of cbbL in members of the Thiomicrospira group.

in prokaryotes, it is still unclear whether the evolution of a ribosomal gene (or any other single gene) reflects the evolution of the entire genome. To answer this question, it is probably necessary to compare ribosomal phylogenetic reconstructions with reconstructions based on complete genome sequences. As long as such data are unavailable, any reconstruction is approximate and lacks sufficient detail. As a palliative, ribosomal trees are now compared with other monogenic trees based on the genes that are functionally unrelated to ribosomal genes. Since the main objective of molecular phylogenetics is to reconstruct the phylogeny of organisms rather than macromolecules, the best approximation to the actual phylogeny can be expected from a comparative analysis of individual dendrograms via a phylogeny mixing approach, which makes it possible to reveal the main trends in the evolution of living organisms. The following rules are accepted in such studies. Evolutionary inferences from trees obtained by different methods should coincide to be accepted as reflecting the actual events that took place in evolution. Coincident topologies of evolutionary reconstructions suggest the most probable order of evolutionary events. It should be noted, however, that discrepancies between dendrograms may point to certain specifics of the evolutionary process in closely related organisms. In other words, the above rules are not always absolute because differences in evolution rate may arise as a MOLECULAR BIOLOGY

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