Regulation of Gene Expression, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris Cedex 15, France1
Hong Kong University Pasteur Research Centre, Dexter HC Man Building, 8 Sassoon Road, Pokfulam, Hong Kong2
Genome and Informatics, Université de Versailles-Saint-Quentin, 45 Avenue des Etats Unis, 78035 Versailles Cedex, France3
Author for correspondence: Antoine Danchin. Tel: +852 2816 8402. Fax: +852 2168 4427. e-mail: adanchin{at}hkucc.hku.hk
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ABSTRACT |
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Keywords: secondary metabolism, Helicobacter pylori, Bacillus subtilis, Synechocystis PCC6803, discriminant amino acid residue
Abbreviations: FCA, Factorial Correspondence Analysis
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INTRODUCTION |
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From the chemical standpoint, the origin of life is often reduced to the question of incorporation of carbon into the simple building blocks needed for biopolymer synthesis. However, the universal requirement for nucleotides and amino acids demonstrates that nitrogen metabolism was extremely important in the first steps of life (Granick, 1957 ). It is likely that a nitrogen-fixing cycle predated life, placing nitrogen-rich molecules in the limelight (Danchin, 1989
), polyamines being crucial molecules (Cohen, 1998
). In this context, Ouzounis & Kyprides (1994)
constructed an interesting evolutionary tree of agmatinases, the polyamine-synthesizing enzymes, with emphasis on their universal presence. Since this seminal work, many new sequences have been obtained and annotated by their similarity with the known sequences. We therefore undertook a comparative analysis of the corresponding set of sequences. Genes that were deemed important were cloned and attempts were made to identify their functions. We reconsidered the phylogeny trees of arginases/agmatinases and constructed new ones for the enzymes involved in this first step in polyamine metabolism. To incorporate evolutionary constraints of different types, we first considered the usual types of phylogeny trees constructed based on the variation of the amino acid sequence in these proteins, without taking into account the presence of gaps in the sequences. Several discrepancies with respect to the expected position of some organisms in the trees were found. In a second approach, we reconstructed trees based only on the presence and evolution of gap-containing regions in the sequences (Baldauf & Palmer, 1993
; Gupta, 1998a
; see Fitch & Yasunobu, 1975
, for appropriate caveats), because gaps would be much less sensitive to genetic drift or amino acid metabolism. The crucial enzyme activities that presumably evolved from ancestral ureohydrolases were validated by cloning, expressing and measuring activity of the corresponding enzymes. The emerging picture is consistent with a bacterial origin of hydrolases (ureohydrolases and related activities), which later evolved to those of the Archaea and the Eukarya.
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METHODS |
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The reference Escherichia coli strain for the study of polyamine biosynthesis was obtained from the E. coli Genetic Stock Center, through the kind help of Dr Mary Berlyn. Strain MA255 was used: K12 thr-1 leuB6 fhuA2? lacY1 glnV44(AS)? gal-6 - relA1? can-1 speB2 speC3 rpsL133(strR) xylA7 mtlA2 thi-1. For cloning experiments strain XL-1 Blue was used (K12 supE44 hsdR17 recA1 endA1 gyrA46 thi relA1
lac F'[proAB+ lacIq lacZ
M15 Tn10(tetR)]; laboratory collection).
Synechocystis PCC6803, Helicobacter pylori 26695 and Neisseria gonorrheae MS11-E DNAs were kind gifts from Dr N. Tandeau de Marsac (Physiologie Microbienne, Institut Pasteur, Paris), Dr H. De Reuse (Pathogénie Bactérienne des Muqueuses, Institut Pasteur, Paris) and Dr M.-K. Taha (Unité des Neisseria, Institut Pasteur, Paris), respectively.
Sequence alignment and construction of trees.
The 50 ArgI and SpeB amino acid sequences and the 22 spermidine (spermine) synthase sequences currently available were aligned with the programs DIALIGN version 2 (Morgenstern et al., 1998 ), as well as CLUSTALW (Higgins et al., 1996
), using default parameters (similarity matrix BLOSUM, 30; gap open penalty, 10; gap extension penalty, 0·1). A second putative ArgI sequence (encoded by the rocF gene) from H. pylori was extracted from the Astra server for comparison to the one present in our alignments. Because the differences were very small and did not change the positions of gaps, this sequence was not incorporated further in our analysis. The resulting multiple alignment was then checked for the conservation of important residues (Perozich et al., 1998
) and manually edited. Regions where the two alignment programs gave widely different solutions were removed (see Fig. 1
). Phylogenetic analyses were performed using the PHYLIP 3.57c suite of programs (Felsenstein, 1993
). The pairwise distance matrices were calculated by PROTDIST with the Dayhoff option to estimate the expected amino acid replacements per position. The neighbour-joining (NJ) trees were obtained with NEIGHBOR. The most parsimonious trees were determined with PROTPARS. In each case, we performed 1000 bootstrap resamplings with SEQBOOT. The consensus trees were calculated by CONSENSE and drawn with the program TREEVIEW (Page, 1996
).
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Cloning procedures and biochemical assays.
Cloning was performed by PCR amplification of the DNA regions of the putative argI or speB genes under study and followed by subsequent ligation of PCR products into plasmid pTrc99A (Pharmacia Biotech).
To clone the putative argI gene from Synechocystis PCC6803, a DNA fragment beginning at the translational start point and ending 5 bp after the stop codon of argI was amplified by PCR, using primers introducing a BspHI cloning site at the 5' end and a BglII cloning site at the 3' end of the fragment. The PCR product was ligated and inserted into the NcoI and BamHI sites of pTrc99A, creating pDIA5600. To clone the putative speB gene from Synechocystis PCC6803, a DNA fragment beginning at the translational start point and ending 52 bp after the stop codon of speB was amplified by PCR, using primers introducing an AflIII cloning site at the 5' end and a BamHI cloning site at the 3' end of the fragment. The PCR product was ligated as described above, creating pDIA5601. To clone the rocF gene (encoding ArgI) from H. pylori 26695, a DNA fragment beginning at the translational start point and ending at the stop codon of rocF was amplified by PCR using primers introducing an NcoI cloning site at the 5' end and a BamHI cloning site at the 3' end of the fragment. The PCR product was ligated as described above, creating pDIA5602. To clone the putative argI gene from N. gonorrheae MS11-E, a DNA fragment beginning at the translational start point and ending 4 bp after the stop codon of argI was amplified by PCR using primers introducing a BspHI cloning site at the 5' end and a BamHI cloning site at the 3' end of the fragment. The PCR product was ligated as described above, creating pDIA5603.
The plasmids containing these putative argI or speB genes were expressed in an E. coli mutant unable to synthesize polyamines. To assay agmatinase/arginase activities, strain MA255 was used.
For determination of enzymic activities, the bacteria from 200 ml LB overnight cultures were centrifuged, washed with PBS and centrifuged again; the pellets were then weighed. Extracts were prepared by grinding the bacterial paste for several minutes in a mortar with alumina (equal to twice the pellet weight). The mixtures were resuspended in 50 mM Tris/HCl, pH 7·6, containing 1 mM EDTA and 1 mM DTT and centrifuged at 10000 r.p.m. for 30 min in the cold. Supernatants were used for determination of enzyme activities. Urea was produced as described by Hirshfield et al. (1970) using either agmatine or arginine as substrates (10 mM each). The assay was slightly modified by raising the pH of the Tris/HCl buffer from 7·5 to 9·0 as described by Yamamoto et al. (1988)
. Urea measurement was performed using the Blood urea nitrogen assay kit (Sigma).
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RESULTS |
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Phylogeny trees
This alignment allowed us to construct phylogenies with two commonly employed approaches for phylogenetic reconstructions, i.e. distance and parsimony (maximum likelihood was also used, but did not give significantly different results; data not shown). Fig. 2 displays the trees obtained by using these methods. The trees are, on the whole, rather similar. They are firmly split into two main parts, one corresponding to a majority of identified or putative arginases (ARGI) and the other to agmatinases (SPEB). However, the bootstrap scores within the SPEB cluster are generally poor, which means that a detailed view of the evolution of agmatinases and, in some parts, of arginases cannot be obtained from this study. An interesting observation, however, is that the sequence of SpeB from Bacillus subtilis (Sekowska et al., 1998
) groups in both trees with the corresponding enzymes from the Archaea. Most of the sequences from the Eukarya, on the other hand, are supposed to be of the ArgI type (no agmatinases have been identified among the vertebrates) and group far from the archaeal ones. The sequences from Schizosaccharomyces pombe are also grouped in both trees with the sequences from Gram-negative bacteria (presumably agmatinases; see below). Enzyme activities corresponding to other functions that are related to, but distinct from urea hydrolysis (e.g. HutG encodes formiminoglutamase and PahA encodes proclavaminate amidino hydrolase, which synthesizes a precursor of clavulanic acid, etc.) are generally scattered in the tree.
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As shown in Table 1, the H. pylori enzyme is an arginase. In contrast, the gene product labelled ArgI in Synechocystis PCC6803 (Cyanobase; http://www.kazusa.or.jp/cyano/cyano.orig.html) is an agmatinase. The gene product labelled SpeB in this organism might have been a second agmatinase. However, we failed to detect this activity or to identify this gene product as an arginase under conditions where the other gene products produced urea from either arginine or agmatine. It is possible that this is an enzyme used in the degradation of histidine similar to the hutG gene product of B. subtilis. More likely, it is involved in secondary metabolism comparable to the pahA gene product, which is implicated in synthesis of clavulanic acid in Streptococcus clavuligerus (see below). The N. gonorrheae enzyme labelled ArgI is clearly identified as an agmatinase, not an arginase.
Discrimination between arginases and agmatinases
Arginases and agmatinases release urea and ornithine or putrescine, respectively, from substrates that differ from each other only by the presence of a carboxylate group (in arginine). Because arginases and agmatinases can be divided into two well-defined categories, we investigated whether they could be separated according to a consensus sequence. The manganese-binding sites, as shown above, make an identical consensus in both enzymes. This could be expected since these enzymes display the same catalytic activity. Ten invariant conserved residues are found in the Prosite motif (http://www.expasy.ch/cgi-bin/nicedoc.pl?PDOC00135), modified below, using the data from our study. Among these, six bind to the manganese-ion cofactors (indicated by #):
Pattern 1 [ILV]-X-[FILMV]-G-G-[ED]-H#-X-[ILMV]-[ASTV]-X-[AGP]-X(3)-[AGST];
Pattern 2 [ILMV](2)-X-[FILMVY]-D#-[AS]-H#-X-D#;
Pattern 3 [FHY]-[ILV]-[ST]-[FILMVY]-D#-[ILMV]-D#-X(3)-[APQ]-X(3)-P-[AGS]-X(7)-G.
A fourth conserved pattern also seems to be important:
Pattern 4 [AGSV]-[ACFILMV]-[DE]-[FILMTV]-[AIMTV]-E-[FILMV]-[AGHNS]-[GPS].
If only the arginase side of the tree is retained, one uncovers several further constraints that specify the arginase family. These include restriction to [ILMV] at the third residue in pattern 1 (no F), a conserved W (Y in the widely deviant H. pylori sequence; see Discussion) at position 3 in pattern 2 and restriction to H at position 1 in pattern 3 (neither F nor Y). In contrast, several regions generally differ between the ArgI and SpeB families precisely where the patterns are restricted in arginases. In addition, the polypeptide sequence intervening between patterns 2 and 3 is significantly shorter (20 or so residues less) in agmatinases compared to arginases. In agmatinases, position 3 of pattern 2 is more variable than in arginases, H, Q or N being present instead of W (Y). Likewise, the H at position 1 of pattern 3 of arginases is replaced by an aromatic residue in agmatinases.
Once these general features are identified, one may also remark that several sequences appear to differ significantly from the others. This is particularly true in the case of the H. pylori arginase. Some sequences also appear to be much longer than the mean. These include certain sequences of Schizosaccharomyces pombe, the sequence of Caenorhabditis elegans and the sequence of Synechocystis PCC6803 (labelled SPEB).
Alignment of gaps in the sequences
Until now, the phylogeny trees constructed were based only on the differences between amino acids at equivalent positions. However, changes yielding gaps in the alignment also correspond to one or more mutational events. In general, gaps specify the insertion or deletion of loops in the three-dimensional structure of the protein (Briozzo et al., 1998 ). For this reason, their length is usually variable, whereas the place where they occur is fixed. This is due to structural constraints in the architecture of the protein active site(s). It is therefore interesting to consider gaps as the hallmark of some mutational events, noting that conservation of both the presence and the length of the insertions/deletions may indicate some kinship between the corresponding proteins. We therefore constructed a matrix based on the lengths of insertions occurring between well-conserved regions (see Fig. 1
). As expected, the insertions/deletions often occur at places where the multiple alignment is less reliable. However, since they are anchored by highly conserved residues, their length is unequivocal. In addition, protein length was also included in the matrix because the length of the protein introduces a constraint in evolution of the same structural nature as the introduction of gaps. This matrix was used to calculate the co-ordinates of the sequences using FCA. The latter enabled us to obtain pairwise distances that were subsequently used as input in the program NEIGHBOR.
The resulting distance tree is displayed in Fig. 3. One of its prominent features is that arginases and agmatinases are clearly separated. It should be noted that this separation does not merely come from the fact that the lengths of the proteins were used in the pairwise distance calculation, even if the agmatinases tend to be shorter than the arginases. For example, the protein labelled SPEB_SYNEC is one of the longest in the set (390 aa) and is nevertheless clearly not a member of the arginase family. In addition, it is probable that gap regions III and VI (Fig. 1
) are the most discriminant. Interestingly, some of the sequences that were located in a continuum near the junction of the arginase and agmatinase trees when using evolution of the sequences without consideration of gaps, moved to less ambiguous positions. The ambiguous situation of the H. pylori enzyme has been solved by demonstrating experimentally that it is an arginase. Remarkably, the enzymes that differ both from arginases and from agmatinases also group together in this tree. The protein labelled SPEB from Synechocystis is located next to the agmatinase cluster in a group comprising poorly defined activities (a protein labelled ARGI from a Streptomyces sp. and the SPEB protein from Rhodobacter capsulatus). Our experiments suggest that it might not be an agmatinase but an enzyme related to secondary metabolism activities linked to polyamines (Cohen, 1998
). Furthermore, the plant enzymes may be a sister group of their cyanobacterial counterpart, labelled ARGI, but that we identified as an agmatinase. Finally, we observed that the B. subtilis agmatinase clustered with the archaeal enzymes, as in the previous phylogenetic reconstructions, while the enzymes from E. coli and N. gonorrheae are now correctly grouped together (the N. gonorrheae ARGI enzyme being in fact an agmatinase).
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DISCUSSION |
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The H. pylori sequence is of particular interest because, although from a Gram-negative organism, it does not group with E. coli and N. gonorrheae. Furthermore, the SSEH motif differs significantly from the consensus GGD(or E)H motif present in all other arginases. Using a second type of H. pylori ARGI, it is evident that SSEH is specific to these bacteria and does not result from a sequencing error (data not shown). Indeed, the lifestyle of H. pylori is very unusual. Possibly, this is reflected in the style of its genome, constraints on the availability of metabolites affecting the composition of its proteins (Danchin et al., 2000 ). Alternatively, catalytic mechanisms might be affected in a more extreme environment. Variation in the nature of the preferred amino acids (or codons) would account for the rather odd place of the sequence in the non-gapped trees. The sequence from Bacillus halodurans, which is also present in bacteria living in unusual environments, is also similarly biased, a GGDC sequence replacing the (almost) universal GGD(or E)H sequence (and it groups with H. pylori in the gap tree). Several methods have been devised to deal with composition biases in alignments, for example by taking into account biases in the G+C content of genomes (Tourasse & Gouy, 1997
; Foster & Hickey, 1999
; Wilquet & Van de Casteele, 1999
) or by taking care of long-branch attraction artefacts in the statistical methods used (Lyons-Weiler & Hoelzer, 1997
). But, as seen here, this is probably of limited interest when divergence is very ancient. As a consequence, we emphasize the utility of considering gaps when constructing trees, especially for proteins that have been evolving for a very long time (Gupta, 1998b
). Gaps are less sensitive to the nature of the genetic code or amino acids, but only to the relationship between the three-dimensional structure of a protein and its function. Besides allowing better prediction of protein function, our alignment identified residues of major importance for the two binding sites of the manganese co-factor as well as residues and regions separating arginases from agmatinases.
Although not substantiated experimentally, some of the sequences which differ from arginases may have other important activities (Cohen, 1998 ). Secondary metabolism activities or degradation of histidine are placed on the agmatinase side in the trees. Interestingly, in the highly conserved region, [FYMLIV]-[MLIV]-[WHYNQV]-[FMLIV]-D-[AS]-H, the alanine or serine residue before the final histidine is replaced by an arginine in the Synechocystis SPEB sequence (clustered in the secondary metabolism sequences). This alteration replaces a small non-polar or polar amino acid with a very large basic one. We did not identify the corresponding enzyme as an arginase or an agmatinase. This prompted us to check whether this was due to a sequencing error: arginine and alanine are coded by CGN or GCN codons and it is well known that C and G residues often migrate at the same rate on gels, resulting in frequent GC swaps in the final sequence. After PCR amplification of the corresponding region from the chromosome, we found that the sequence is exact (data not shown). We therefore propose that this residue is involved in defining a new specificity for the enzyme, probably for some reaction of secondary metabolism (cyanobacteria are known to produce secondary metabolites that may derive from molecules related to polyamines; Cohen, 1998
). This observation illustrates the identification of an amino acid residue that may play a discriminant role in catalysis and could be a target for oriented in vitro evolution of enzyme activities.
The question of the origin and fate of arginine early in evolution is important because it is related both to the fixation of nitrogen and to the universality of the genetic code. Remarkably, the Archaea are only present in the agmatinase tree. Also, the pathways leading to putrescine (the primary polyamine, Fig. 4) involve ornithine, an amino acid not incorporated into proteins. The cell concentration of ornithine (an analogue of lysine) must therefore be finely controlled to avoid misincorporation into proteins. As a consequence, the arginase pathway is likely to have evolved later than that involving arginine decarboxylation and agmatinase. In this respect, one should note that the consensus sequence corresponding to pattern 2 is highly variable in the non-arginase sequences while it is extremely conserved in arginases, suggesting indeed that these sequences are of more recent descent. In particular, the W residue in WXDAHXD could be chosen as a discriminant residue to identify arginases (Fig. 1
). One can therefore confidently assume that agmatinases predated arginases. The latter would have appeared in the Bacteria by recruitment of a wide specificity agmatinase and then transferred to eukaryotes (perhaps through transfer from mitochondria).
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Remarkably, the only Gram-negative arginase is that from H. pylori (substantiated experimentally in this study). The Bacteria are split into several groups in the gap tree. In the case of agmatinases, those of B. subtilis and the Archaea go together. A group comprising the Synechocystis PCC6803 agmatinase (wrongly annotated as an arginase) together with enzymes involved in secondary metabolism, and in broader specificity hydrolases, is also prominent in the gap tree. If one reasons in terms of acquisitive evolution (Thompson & Krawiec, 1983 ), assuming that an ancient activity with broad specificity has been progressively specified during evolution (Jensen, 1976
; Danchin, 1989
; Roy, 1999
), then the origin of the ureohydrolase tree would be near the PahA HutG group, leading to agmatinases and enzymes involved in secondary metabolism and, subsequently, to arginases. To substantiate the grouping of B. subtilis and archaeal polyamine enzymes, we performed a phylogenetic reconstruction of spermidine synthases. These enzymes are less universally distributed than ureohydrolases. They exert their function downstream from the synthesis of putrescine, the precursor of polyamines. Fig. 5
displays the most parsimonious tree obtained in this case. Again, B. subtilis groups with its archaeal counterparts, while the Eukarya form another well separated group. In addition, we find not only a group made of spermine synthases (SPSY and SPEE), which use spermine as the substrate instead of spermidine, but also a group of related methyltransferases, which use S-adenosylmethionine rather than decarboxylated S-adenosylmethionine as a substrate (PUTR).
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ACKNOWLEDGEMENTS |
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Received 25 January 2000;
revised 1 May 2000;
accepted 5 May 2000.