* Department of Biology and Center for Genetics and Molecular Medicine, University of Louisville
Department of Microbiology, University of Manitoba, Winnipeg, Canada
Correspondence: E-mail: martin.klotz{at}louisville.edu.
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Abstract |
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Key Words: catalase catalase-peroxidase nonheme catalase lateral gene transfer
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Introduction |
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During a long coevolution of pathogens and their hosts, the pathogens amassed an amazing complement of antioxidative functions to maintain their potential for infection. Whereas superoxide (degraded by superoxide dismutase to hydrogen peroxide) and hydroxyl radical are highly reactive and have thus a limited radius of action, hydrogen peroxide can easily penetrate cellular structures (cell walls, plasma membranes, etc.) and form adducts with sugars and amino acids that are transported by numerous dedicated permeases (Schubert and Wilmer 1991). Hence, hydrogen peroxide can inflict damage anywhere in a cell or tissue through oxidation of proteins (sulfhydryl groups) and single strand breakage in nucleic acids (Richter and Loewen 1982; Imlay, Chin, and Linn 1988). It is not surprising to find a very high level of functional redundancy among enzymes involved in the detoxification of hydrogen peroxide.
Enzymes that reduce hydrogen peroxide or use it as a reductant are generally termed hydroperoxidases (HP). Catalatic hydroperoxidases (CHPs) primarily dismutate hydrogen peroxide to water and dioxygen by two-electron transfer redox reactions, and there is considerable diversity among the enzymes that exhibit catalatic activity. Generally, these proteins can be placed into four main groups: (1) the "classic" heme-containing monofunctional catalases for which hydrogen peroxide is both electron donor and acceptor, (2) the heme-containing bifunctional CPXs in which the catalatic activity is much higher than the peroxidatic activity, (3) the nonheme-containing catalases, and (4) a miscellaneous group containing proteins with minor catalatic but no peroxidatic activities (Jones and Wilson 1978). CHPs have a variety of subunit sizes, a number of quaternary structures, at least two different heme prosthetic groups, and the reductant for bifunctional CPXs can vary. This article set out to investigate the evolutionary history of the first three categories, that is, enzymes with a considerable catalatic activity.
Monofunctional Catalases
Monofunctional catalases (hydrogen peroxide oxidoreductase E.C. 1.11.1.6) degrade hydrogen peroxide to oxygen and water (2H2O2 2H2O + O2). They are present in both empires of life and are the most extensively studied, beginning with the first report of a biochemical characterization of the enzyme in 1900 (Loew 1900). The enzyme's subsequent history has been well documented (Loewen 1997; Zamocky and Koller 1999). Investigations on catalases from a variety of sources, including tobacco, yeast, blood, and bovine liver (BLC), resulted in catalase being one of the first enzymes to be crystallized in 1937 (Sumner and Dounce 1937), and the enzyme has continued to be an object of interest and of intense study ever since. Monofunctional catalases use hydrogen peroxide alternately as oxidizing and reducing species. The oxidation involves a two-electron oxidation of heme iron, which in turn is reduced in a two-electron transfer from a second molecule of hydrogen peroxide to regenerate the resting enzyme. Catalases can also undergo two sequential one-electron reductions involving organic reductants, similar to peroxidases; however, the peroxidatic reaction is weak and usually restricted to small organic substrates because of limited accessibility to the active site. Biochemically, it is possible to divide the monofunctional catalases into two subgroups based on subunit size. One group contains small-subunit enzymes of 55 to 69 kd, and a second group contains large-subunit enzymes of 75 to 84 kd. All small-subunit enzymes so far characterized have heme b (or a degradation product of heme b) associated, and some have NADPH bound (Kirkman and Gaetani 1984; Almarsson et al. 1993; Hillar et al. 1994). With the exception of some mutant variants, all large-subunit enzymes so far characterized have heme d associated and none have been found with NADPH bound. The monofunctional catalases characterized in greatest detail, either by crystal structure analysis or electrospray mass spectrometry, have all been found to be homotetramers, although dimeric, hexameric, and even heterotrimeric enzymes have been reported (Nicholls, Fita, and Loewen 2001). There are currently over 250 known sequences of monofunctional catalases. Whereas the evolutionary relationships of subsets of enzymes have been described previously (Mayfield and Duvall 1996; Klotz, Klassen, and Loewen 1997; Scandalios, Guan, and Polidoros 1997; Frugoli et al. 1998; Kim, Sha, and Mayfield 2000; Loewen, Klotz, and Hassett 2000), we have set out to investigate the phylogeny of the entire monofunctional catalase gene family based on this prior literature and a comprehensive analysis of all presently available catalase gene and protein sequences, a number that has more than tripled since our last review (Klotz, Klassen, and Loewen 1997).
Heme-Containing Catalase-Peroxidases
Catalase-peroxidases exhibit a predominant catalatic activity but biochemically differ from the monofunctional catalases in exhibiting a significant peroxidatic activity (Sun et al. 1994; Thomas, Morris, and Hager 1970). Of the more than 60 currently available CPX sequences, most have been identified in prokaryotes (including Negibacteria, Posibacteria, and Archaebacteria), but there is recent evidence for their presence in fungi. CPXs have sequence similarity to class I peroxidases in plants (e.g., plastid ascorbate peroxidase) and fungi (e.g., cytochrome C peroxidase); however, there is no resemblance in sequence to the monofunctional catalases. CPXs are active as either dimers or tetramers, and the large size of the subunits composed of two domains with similar sequences has lead to the hypothesis that the CPXs may have arisen through a gene duplication and fusion (Welinder 1991, 1992). The N-terminal domain has retained activity along with greater sequence similarity to other CPXs. The C-terminal domain has evolved with greater sequence deviation, does not bind heme, and is presumably inactive. The evolutionary relationships of CPXs have been described briefly in the past, including a critical discussion of hypothetical lateral transfer between Archaebacteria and Eubacteria (Klotz, Klassen, and Loewen 1997; Faguy and Doolittle 2000; Loewen, Klotz, and Hassett 2000; Zamocky et al. 2001; Cavalier-Smith 2002a).
Nonheme Catalases
A very different catalatic mechanism has evolved in the nonheme catalases in which the reaction center is a manganese complex. The nonheme catalases make up a small group of enzymes, so far only of prokaryotic origin, with only three examples purified and characterized, but a larger number sequenced. A manganese-rich reaction center was identified as the active site in each of the three characterized enzymes (Kono and Fridovich 1983; Allgood and Perry 1986; Whittaker et al. 1999), and this has been confirmed to be a bridged binuclear manganese cluster by crystal structure analysis (Waldo, Fronko, and Penner-Hahn 1991). The mechanism of catalytic action in these enzymes is currently under investigation.
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Material and Methods |
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The refined three alignments were also used for inference of phylogeny. Phylogenetic relationships were investigated by using a distance algorithm and character-based tree-searching methods with maximum-parsimony (MP) or maximum-likelihood (ML) object functions. Distance neighbor-joining trees were constructed by using the BioNJ function in PAUP* (v. 4.10b [Swofford 1999]). The obtained trees were used as guide trees and for comparison with phylograms obtained with MP and ML methods. MP (50% majority consensus; random taxon-addition order replicates with Tree-Bisection-Reconnection branch-swapping, Mulpars and steepest decent functions in effect) trees were built independently from all three alignments by using the PAUP* program. The quality of the branching patterns was assessed by bootstrap resampling of the data sets using 100 replications. Because inclusion or exclusion of a few characters can highly affect bootstrap proportions of MP trees derived from limited data sets, we also conducted ML inferences for the CPX and nonheme catalase gene families. The sequence alignments of both gene families were each subjected to a Bayesian (ML) inference of phylogeny by using the program MrBayes v. 2.01 (written by Huelsenbeck and Ronquist; http://morphbank.ebc.uu.se/mrbayes/). The nonheme catalase and CPX protein sequence alignments were subjected to Metropolis-Coupled Monte Carlo Markov Chain sampling of 30,000 and 50,000 generations, respectively. Four equally heated Markov chains were utilized to build a sufficient number of reliable trees after the likelihoods of the trees have converged on a stable value and to allow successful swapping between chains. The searches were conducted assuming equal rates across sites and using the JTT empirical amino acid substitution model (Jones, Taylor, and Thornton 1992). In a postrun analysis, MrBayes summarized the results concerning tree topology and branch lengths. By ignoring the trees generated before the search converged on stable likelihood values (removed as "burn-in"), a 50% majority rule consensus phylogram was constructed that displayed the mean branch lengths and posterior probability values of the observed clades. These probability values were comparable to the bootstrap proportions calculated for the branches in the MP consensus trees. Additionally, programs contained in the PHYLIP package (Felsenstein 1994) were used as before (Klotz, Klassen, and Loewen 1997; Loewen, Klotz, and Hassett 2000) for additional analysis of the much larger data set of the monofunctional heme-catalase sequences.
Sequence alignments, contig alignments for intron analysis (Sequencher 4.1, Gene-Codes, Madison, Wis.), phylogenetic inferences (PAUP*, MrBayes, PHYLIP 3.5), tree constructions (TreeViewPPC, v. 1.6.5, Roderick Page, personal communication), and graphics were accomplished by using Apple Macintosh G3 computers. The SEQBOOT, PROTPARS, PROTDIST, CONSENSE, NEIGHBOR, FITCH, and TREE programs of the PHYLIP package were also run on a SGI Impact R10000 workstation. Alignments generated from full-length and core sequences resulted in similar or identical phylogenetic trees.
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Results and Discussion |
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The phylogenetic trees constructed by distance neighbor-joining (Phylip 3.5) or MP (PAUP*) inferences yielded nearly identical trees, showing that the monofunctional catalase gene family falls into three member clades (fig. 1). While clade 1 and 3 contain small-subunit catalase sequences, clade 2 contains exclusively large-subunit catalase sequences. The observed branching pattern implies an evolution of the three sequence lineages after a minimum of two gene duplications (Klotz, Klassen, and Loewen 1997; Loewen, Klotz, and Hassett 2000). Catalases are found in both empires of life, the Prokaryota and Eukaryota (Mayr 1998; Cavalier-Smith 2002b), but their abundance and diversity in some taxa is limited. In the Eukaryota, catalases are present in all major taxa, the Protista, Animalia, Fungi, and Planta, but overall, catalases do not group according to ssu-rRNAbased species-phylogenetic relationships (Olsen, Woese, and Overbeek 1994). For example, all three catalase clades contain bacterial enzymes, and analysis of the individual clades shows that each clade has a base of bacterial sequences that branches off into a bacterial branch and an eukaryotic branch (fig. 1).
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All algal/plant catalases reside in clade 1 together with a subset of small-subunit catalases from Posibacteria and Negibacteria. Multiple catalase genes in the plant genome have likely arisen by an initial duplication of a clade 1type catalase gene. Plant genomes harbor small catalase gene subfamilies of up to four member genes, and their phylogenetic relationships (Frugoli et al. 1996; Guan and Scandalios 1996) and gene structures (Frugoli et al. 1996) have been analyzed recently. The expression products of these genes can assemble to various different tetrameric isozymes, and the regulation of this allows the synthesis of organ-specific catalases (Frugoli et al. 1998). Clade 1type catalases have not yet been found in animals or fungi. Fungi generally have multiple catalase genes per genome. Some fungi express catalases that group in a single clade (either clade 2 or clade 3), and others show distribution of their catalases into both clade 2 and clade 3. Clade 2 catalases are also found in bacteria, whereas clade 3 catalases are found in all phylogenetic groups except for plants. Eukaryotic clade 3type catalases are usually peroxisomal.
To refine the relative groupings of eukaryotic catalases, the number and locations of introns in each gene was analyzed as previously described (Johnson et al. 2002), and the results were used to construct a phylogenetic tree inferring maximum parsimony (fig. 2). Whereas a grouping of plant (clade 1) and animal catalases (clade 3) in the intron tree implies the existence of single-rooted intron lineages, multiple independent intron lineages are evident for protozoan (clade 3) and fungal (clades 2 and 3) catalase genes. Prokaryotic catalase genes naturally lack introns.
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Fairly soon after the emergence of clade 2type catalases, a gene duplication event accompanied by losses of sequence at both the 5' and the 3' ends resulted in the ancestral gene encoding small-subunit catalases. Such a "sequence loss upon duplication" scenario is supported by recent experimental data in that an N-terminally and C-terminally digested clade 2type catalase was found to retain catalase activity (Loewen, in preparation). The first small-subunit catalase gene was likely a clade 1type gene, as it is present in the Eobacteria in which a clade 3type gene has not yet been found (fig. 3). Furthermore, the orientation of heme in clade 2type and clade 1type catalases is identical but differs by 180° in clade 3type catalases (Carpena et al. 2002). In addition, the evolutionary distance between clades 2 and 1 on the phylogenetic tree is always shorter than the distance between clade 3 and either one of clade 1 or clade 2 (fig. 1), independent of the method of inference. The tree in figure 1 also tells us that the clade 1type catalase gene has further evolved paraphyletically and orthologously in the evolutionarily younger lineages of the Negibacteria and Posibacteria. Interestingly, like in the case of the clade 2type catalases, the clade 1type catalase genes of the Posibacteria are more similar in sequence to the eobacterial gene found in Deinococcus than the negibacterial clade 1type genes (figs. 1 and 3).
Clade 3type catalase genes are absent from modern representatives of the phylogenetically old taxonomic groups and abundant only in Posibacteria, Proteobacteria, and Eukaryota (fig. 3 and table 1 ). If clade 3type catalase genes were as old as the other two members of the gene family, then the gene must have been lost massively in all these divergently evolving taxa after the emergence of the Posibacteria and Proteobacteria. Although the scantiness of molecular traces (pseudogenes) in bacterial genomes after gene loss can be explained theoretically (Lawrence, Hendrix, and Casjens 2001), a selective massive simultaneous gene loss in some but not in other taxa is very unlikely. We reason instead, that the clade 3type, small-subunit catalase gene has likely developed last by a second gene duplication event in either the Posibacteria or Proteobacteria and was subsequently horizontally acquired by the other taxon and a few species in other bacterial taxa (see below). We propose that the clade 3type catalase gene has evolved in the Posibacteria. This is concluded from the branching pattern in figure 1 because posibacterial sequences group near the root of the clade, forming two subclades, one being exclusively Actinobacteria and the other being a mix of actinobacterial and endobacterial sequences. The latter subclade contains predominantly proteobacterial catalase genes, which subgroup into
- proteobacterial and ß/
-proteobacterial lineages. Hence we propose that (a) posibacterial clade 3type catalase gene(s) were (was) laterally acquired by Proteobacteria. This laterally acquired clade 3type catalase gene was retained in addition to other types of catalase genes in environmental genera such as Pseudomonas, Ralstonia, and Rhizobium, whereas animal hostassociated genera such as Neisseria, Brucella, and Bordetella have kept this gene at the expense of all other catalase gene types. Interestingly, the animal hosts also harbor clade 3type catalases, suggesting that the similar physiological selection pressures resulted in the selection of the same enzyme in both parasite and host. The proteobacterial subclusters in clade 3 (fig. 1) are "spiked" with taxonomic "exceptions": a sphingobacterial gene (Bacteroides), a planktobacterial gene (Pirellula), a spirochaeteal gene (Leptospira), a cyanobacterial gene (Nostoc), and an archaebacterial gene (Methanosarcina mazei). The Bacteroides and Methanosarcina and Nostoc and Pirullela catalases group closely together on the phylogenetic tree (fig. 1), although the organisms are rather distantly related (fig. 3). A clade 3type catalase gene was isolated just recently from the spirochaete Leptospira interrogans (GenBank accession number AE011360). In addition to its close grouping with clade 3type catalases from pseudomonads and Wolbachia on the phylogenetic tree (fig. 1), the Leptospira catalase gene is also in a tandem arrangement with ankyrin as found for several pseudomonad catalases (Ma et al. 1999). Thus, these clade 3type catalase genes likely arrived in their respective host cells by lateral transfer, as they share habitats with many of the prospective gene-donating eubacterial cousins. Extant representatives of the Spirochaetae, Planctobacteria, Sphingobacteria, and Cyanobacteria otherwise lack clade 3type catalase genes, and the other detected clade 3type catalase genes in the Euryarcheota are too distant from the Methanosarcina mazei gene on the tree to propose a direct common ancestor for all three archaebacterial genes (fig. 1). Hence, clade 3type catalase genes have likely been horizontally transferred in several events between Eubacteria and at least once between Eubacteria and the euryarchaebacterium Methanosarcina mazei. The latter organism seems to have acquired both of its catalase genes horizontally from different donors: the clade 2type catalase gene from bacilli and the clade 1type catalase gene from a proteobacterial donor (fig. 1). Horizontal gene transfer has been addressed recently as a widespread and evolutionary important event among bacteria (e.g., Doolittle 1999; Ochman, Lawrence, and Groisman 2000; Nesbø et al. 2001; Gogarten, Doolittle, and Lawrence 2002).
The other three documented occurrences of a clade 3type catalase in Euryarcheota (Methanobacteriales and Methanosarcinales [figs. 1 and 3]) represent likely vertical holophyletic molecular evolution from a mycobacterial/ streptomycetal clade 3type catalase gene in the transitional state of exoskeleton revision (Cavalier-Smith 2002a, see fig. 3 and below). A careful reexamination of the sequencing data by the Methanosarcina acetivorans genome project staff (chad{at}genome.wi.mit.edu) confirmed a frame-shift mutation immediately downstream of the catalytic region. The sequence corrected by us for this mutation and included in the analysis (fig. 1) represents therefore the hypothetical expression product of a pseudogene. We hypothesize that similar loss of function mutations occurred also in other methanogens whose molecular traces have been erased (Lawrence, Hendrix, and Casjens 2001). All other crenarchaeal and euryarchaeal descendents of the "neoexoskeletal ancestor" likely have lost catalase genes among many other genes early in the process of adaptation to extreme environments (acidic, high temperature, etc.) and ongoing genome economization (Cavalier-Smith 2002a and references therein).
Eukaryotic Monofunctional Catalases
There are only a few reports on LGT from bacteria into the Eukaryota. Some genes were suspected of having been transferred as a result of acquisition of endosymbionts by the eukaryotic cell, and a few others have been reported between bacteria and protozoa (Andersson and Roger 2002; Nixon et al. 2002 and references therein). Eukaryotic cells and Archaebacteria have likely acquired catalase genes from eubacteria both vertically and laterally after the three types of catalase genes evolved as outlined above, and we are going to discuss in the following how the Eukaryota have acquired them. It will be helpful in this discussion to also consider the physiological roles of individual catalases in their host organisms and to ask whether they are housekeeping enzymes or induced or secreted enzymes that confer advantageous properties to their hosting cell. Furthermore, our analysis of intron residence (number and location) in eukaryotic catalase genes, summarized in figure 2, provides further information about the directedness of catalase gene evolution andif lateral transfer is suspectedhow many times such transfer might have had taken place. Although the phylogenetic tree derived from alignments of catalase protein sequences reveals at least one lateral transfer event of a clade 3type catalase gene from eubacteria to methanogens, the neoexoskeletal ancestors of the Archae-bacteria and Eukaryota seem to have acquired their housekeeping catalase gene from descendents of the Actinobacteria. This is in line with the numerous recently reviewed similarities between and lines of descent of many structures and functions in the Actinobacteria and Archaebacteria (Cavalier-Smith 2002a) and lower Eukaryota such as the Protozoa (Cavalier-Smith 2002b).
It is almost certain that acquisition of the type 3 catalase gene by eukaryotic cells from bacteria succeeded both catalase gene duplication events. If this were not the case, residence of all three types of catalases or the molecular remnants of their encoding genes should be expected in most eukaryotic taxa. Instead, catalase-positive Protista, Animalia, and Fungi contain a clade 3type catalase as a housekeeping enzyme, whereas modern algae and plants lack type 3 catalases. It is well documented (Cavalier-Smith 2002b and references therein) that plants evolved from ancestral protista other than the ancestors of the fungi and animals. Therefore, absence of clade 3type catalases in plants can be well explained by a single gene loss event early in the alga/plant evolutionary lineage. Modern plants and algae contain exclusively small clade 1type catalase gene families (Frugoli et al. 1996). These likely did not originate from the endosymbiontic -bacterium ancestral to the mitochondria or the oxygenic photosynthetic endosymbiotic cyanobacterium ancestral to chloroplasts because
-Proteobacteria and Cyanobacteria did not holophyletically acquire clade 1type catalase genes (table 1
and fig. 3). Instead and because clade 1type catalase genes are not abundant among the Actinobacteria (table 1
), the protistan cell(s) ancestral to the alga/plant lineage likely acquired their clade 1type catalase gene directly via LTG from an endobacterial/ eobacterial or proteobacterial donor whose recent descendants carry clade 1type genes (figs. 1 and 3). The single lineage of intron residence in plant catalase genes (fig. 2) supports the singularity of the gene loss (clade 3type gene) and acquisition (clade1type gene) events in the alga/plant ancestral protistan cell.
Protist, animal, and fungal lineages of clade 3type catalase genes delineate from separate bacterial branches that contain actinobacterial sequences (fig. 1). Hence, it is plausible to propose that protozoa, animals, and fungi acquired their catalase genes as a result of holophyletic molecular evolution from their Actinobacteria-derived ancestors (Cavalier-Smith 2002b). However, it is also clear from figure 1 that clade 3type catalase genes in the protists and animals and in the fungi have likely originated from different protozoan ancestors. Although there is currently no catalase sequence from a modern protozoon available that groups with the fungal clade 3type catalases, the sequence of catalase A from Dictyostelium subgroups with archaebacterial catalases, and the sequence of catalase A from Toxoplasma subgroups with the animal catalases. The clade 3type catalases from Dictyostelium, Toxoplasma, and the animals represent different lineages of gene development in that they constitute different lineages of intron acquisition (fig. 2). Clade 3type catalases from the fungi represent multiple lineages of eukaryotic gene evolution in that they have acquired from one to as many as seven introns, or they lack introns all together (fig. 2).
In contrast to most of the housekeeping clade 3type catalases, many of the clade 2type catalases in fungi are secreted and/or inducible by environmental cues (Johnson et al. 2002; Kawasaki et al. 1997). These properties suggest that clade 2type catalases have been acquired as supplements to constitutive housekeeping enzymes and play a role in the virulence of their recipients (Kawasaki et al. 1997). A recent characterization of the catalase complement of the fungus Histoplasma capsulatum led Johnson et al. (2002) to propose that the known large-subunit, clade 2type catalase genes in fungi were acquired from bacteria by at least two independent LGT events. The protein sequences of the two clade 2 catalase lineages were most similar to those of large-subunit catalases from Posibacteria such as Bacillus, Mycobacterium, and Streptomyces (fig. 1), which are abundant in aerobic soil habitats that overlap with those of the fungi. Although the description of Histoplasma catalase evolution is one of the first reports of direct LGT from bacteria into fungi, evidence for lateral transfer of genes from bacteria to fungi has been documented in laboratory experiments (Sprague 1991; Hayman and Bolen 1993). In this context, it is helpful to mention that there is also evidence for recent LGT of CPX genes from bacteria into fungi (see below).
The data of figure 2 support our hypothesis that the clade 1type, clade 2type, and clade 3type catalases evolved in the Eukaryota by intron acquisition. The number of introns in catalase genes correlates with general intron-richness of their host genome; thus the absence of introns in the Hemiascomycotina is not surprising. The small number of intron loss versus gain events (only three of the plant, two of the fungal, and two of the animal catalase introns were lost) supports the proposed polarity of the process: invasion of introns into intronless genes. This is in contrast to the model of intron loss (Frugoli 1998) based on the analysis of selected plant catalase sequences.
Taken together, we conclude that monofunctional catalase genes are of bacterial origin and that the clade 3type genes have holophyletically evolved in the Archaebacteria, Protista, Animalia, and Fungi. Catalase genes have also been laterally acquired by Archaebacteria (clade 3), plants (clade 1), and fungi (clade 2) more than once (direct LGT and/or phagocytosis), and they have further evolved in eukaryotes orthologously and paralogously by intron acquisition into formerly intron-free genes. In addition to analyses of evolutionary distance and intron residence, emerging structural/crystallographic information on catalases from all three clades (Carpena et al. 2002) supports a model of divergent evolution rather than convergent evolution of the monofunctional catalase gene family.
The Evolution of Bifunctional Catalase-Peroxidase
Bifunctional CPXs are encoded by a second family of catalase genes that are sequence-unrelated to the monofunctional catalases discussed above. In a recent article, Faguy and Doolittle (2000) aligned 19 CPX protein sequences (nine proteobacterial, five posibacterial, three archaebacterial, and two cyanobacterial sequences) and concluded from the derived trees that CPX genes had been laterally transferred from Archaebacteria into pathogenic Proteobacteria. Two years later and discounting a handful of short sequences that are likely the result of incomplete gene duplication events, we count a total of 58 usable sequences: 30 proteobacterial, 14 posibacterial, five archaebacterial, three cyanobacterial, and one planctobacterial sequences. Also five eukaryotic CPX gene sequences from pezizomycotinal fungi are reported. The tree resulting from alignment of these 58 sequences (fig. 4) is much less robust than previous trees that were based on a much smaller set of selected sequences (Klotz, Klassen, and Lowen 1997; Faguy and Doolittle 2000; Loewen, Klotz, and Hassett 2000). The majority-consensus tree obtained from Bayesian inference of CPX phylogeny shown in figure 4 allows for several different interpretations of CPX gene evolution; however, it is evident that several LGT processes contributed to CPX gene distribution. An interpretation of the phylogenetic tree (fig. 4) in conjunction with figure 3 suggests that the evolution of the CPX gene family has likely occurred much later than that of the heme-containing monofunctional catalases discussed above. The groupings of the CPX sequences from Desulfitobacterium (Posibacteria) and Geobacter (Negibacteria); Shewanella, Legionella, and Vibrio (Proteobacteria) and Cyanobacteria and Bacillus (Posibacteria); diverse Proteobacteria and Pirellula (Planctobacteria) and Archaebacteria; and Proteobacteria and Archaebacteria underline the frequency of lateral exchange of the CPX genes among prokaryotes. The claim by Faguy and Doolittle (2000) of directed LGT of CPX genes from Archaebacteria to pathogenic Eubacteria is incongruent with phylogenetic trees obtained with the larger data set (fig. 4), as the proposed "recipient group" contains nonpathogenic bacteria in the genera Nitrosomonas, Shewanella, and Pirellula. The CPX proteins found in fungi are functional (Johnson et al. 2002; Kawasaki and Aguirre 2001) and their sequences group closely on the phylogenetic tree as a subcluster of a larger cluster of proteobacterial sequences. Thus, it seems feasible to propose a single recent lateral acquisition event of a proteobacterial CPX gene by an ancestor of the Pezizzomycotina.
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The Evolution of Nonheme Catalase
To summarize and discuss the evolution of the nonheme (Mn) catalase gene family, 29 of the 32 available sequences were aligned and used for the construction of phylogenetic trees. The depicted majority-rule consensus tree obtained by Bayesian inference of nonheme catalase phylogeny (fig. 5) indicated that nonheme catalase genes cluster in two major groups that have likely arisen by gene duplication. Nonheme catalases have apparently evolved later than monofunctional catalases but preceded the emergence if bifunctional CPX. Consideration of figures 3 and 5 led us to propose that the ancestral nonheme catalase gene evolved and was maintained in the common negibacterial ancestor of Cyanobacteria and Posibacteria but lost in the common ancestor of Spirochaetae, Sphingobacteria, Planctobacteria, and Proteobacteria. Present residence of Mn-catalase genes in the Planctobacterium Pirellula sp. and several ß-Proteobacteria and -Proteobacteria is highly likely due to LGT from their consortial associates such as the nostocales or bacilli because the recipients contain only one or the other member gene of the family (fig. 5). Mn-catalasepositive Posibacteria and Cyanobacteria often contain representative genes from both members of the family. Whereas some of the Mn-catalasepositive organisms are opportunistic pathogens, Mn-catalase genes have only been found in fairly widely distributed and abundant species (fig. 5 and table 1
).
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Concluding Remarks |
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Our discussion of the composite tree in figure 3 leads to the conclusion that monofunctional heme-containing catalase and Mn-catalase evolved likely in the ancestors of modern Eobacteria and Negibacteria, and they were followed by the emergence of bifunctional CPX in the Proteobacteria. Compared with the monofunctional heme-containing catalase and Mn-catalase families, distribution of CPX by LGT was significantly higher. The recent finding of an Mn-catalase with uncharacteristically high catalatic activity in an aerobic hyperthermophilic Chrenarcheabacterium is surprising; however, the phylogenetic analysis revealed that this gene arrived in this organism by LGT.
We propose that catalatic hydroperoxidases have evolved in the Eukaryota through vertical holophyletic evolution and several independent lateral transfer events of genes from eubacterial donors into eukaryotic recipients. The evolution of catalase genes in the Eukaryota is also characterized by genome-specific acquisition of introns into formerly intronless prokaryotic genes.
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Acknowledgements |
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Footnotes |
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Anthony Dean, Associate Editor
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Literature Cited |
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