Bioinformatic analysis of an unusual gene-enzyme relationship in the arginine biosynthetic pathway among marine gamma proteobacteria: implications concerning the formation of N-acetylated intermediates in prokaryotes
© Xu et al; licensee BioMed Central Ltd. 2006
Received: 19 July 2005
Accepted: 12 January 2006
Published: 12 January 2006
The N-acetylation of L-glutamate is regarded as a universal metabolic strategy to commit glutamate towards arginine biosynthesis. Until recently, this reaction was thought to be catalyzed by either of two enzymes: (i) the classical N-acetylglutamate synthase (NAGS, gene argA) first characterized in Escherichia coli and Pseudomonas aeruginosa several decades ago and also present in vertebrates, or (ii) the bifunctional version of ornithine acetyltransferase (OAT, gene argJ) present in Bacteria, Archaea and many Eukaryotes. This paper focuses on a new and surprising aspect of glutamate acetylation. We recently showed that in Moritella abyssi and M. profunda, two marine gamma proteobacteria, the gene for the last enzyme in arginine biosynthesis (argH) is fused to a short sequence that corresponds to the C-terminal, N-acetyltransferase-encoding domain of NAGS and is able to complement an argA mutant of E. coli. Very recently, other authors identified in Mycobacterium tuberculosis an independent gene corresponding to this short C-terminal domain and coding for a new type of NAGS. We have investigated the two prokaryotic Domains for patterns of gene-enzyme relationships in the first committed step of arginine biosynthesis.
The argH-A fusion, designated argH(A), and discovered in Moritella was found to be present in (and confined to) marine gamma proteobacteria of the Alteromonas- and Vibrio- like group. Most of them have a classical NAGS with the exception of Idiomarina loihiensis and Pseudoalteromonas haloplanktis which nevertheless can grow in the absence of arginine and therefore appear to rely on the arg(A) sequence for arginine biosynthesis. Screening prokaryotic genomes for virtual argH-X 'fusions' where X stands for a homologue of arg(A), we retrieved a large number of Bacteria and several Archaea, all of them devoid of a classical NAGS. In the case of Thermus thermophilus and Deinococcus radiodurans, the arg(A)-like sequence clusters with argH in an operon-like fashion. In this group of sequences, we find the short novel NAGS of the type identified in M. tuberculosis. Among these organisms, at least Thermus, Mycobacterium and Streptomyces species appear to rely on this short NAGS version for arginine biosynthesis.
The gene-enzyme relationship for the first committed step of arginine biosynthesis should now be considered in a new perspective. In addition to bifunctional OAT, nature appears to implement at least three alternatives for the acetylation of glutamate. It is possible to propose evolutionary relationships between them starting from the same ancestral N-acetyltransferase domain. In M. tuberculosis and many other bacteria, this domain evolved as an independent enzyme, whereas it fused either with a carbamate kinase fold to give the classical NAGS (as in E. coli) or with argH as in marine gamma proteobacteria. Moreover, there is an urgent need to clarify the current nomenclature since the same gene name argA has been used to designate structurally different entities. Clarifying the confusion would help to prevent erroneous genomic annotation.
Results and Discussion
Occurrence of the argH(A) gene
While studying arginine biosynthetic genes in two vibrio-like strains (later characterized as novel psychropiezophilic Moritella species M. abyssi and M. profunda ) we found most arg genes clustered into a divergent operon-like structure composed of two wings: a leftward one comprising the sole argE gene and a rightward one argCBFGH(A) where argH is extended by a ± 170-long codon stretch, in translational continuity. This extension was shown to complement an E. coli auxotroph deficient in NAGS , demonstrating that it encodes an ArgA-like activity (EC 18.104.22.168).
(i) argH(A) appears to be restricted to this particular group of marine Bacteria. It is noticeable that V. cholerae, which is not a marine organism, does not have argH(A) whereas the three marine Vibrio species do: V. fischeri, V. vulnificus and V. parahaemolyticus. This pattern suggests that the presence of argH(A) is the result of orthologous transfer in diverging lines of descent sharing a common habitat (the sea), perhaps accompanied by some lateral transfer among them as discussed below. Fig 3 suggests that the H(A) fusion occurred in an ancestor common to clades 1 and 2, but this can not be ascertained without an extensive search among the numerous members of this group, in particular among different Idiomarina and Pseudoalteromonas species, as well as in the genera branching early on this tree.
(ii) No correlation appears to exist between the presence of argH(A) and either psychrophily or piezophily. Indeed argH(A) was found among mesophiles (V. fischeri, V. vulnificus, V. parahaemolyticus), psychrophiles (Colwiella psychrerythraea, Pseudoalteromonas haloplanktis), psychro-piezophiles (P. profundum, M. abyssi, M. profunda) and meso-piezophiles (Idiomarina loihiensis). As the cardinal temperatures and hydrostatic pressures of these closely related organisms actually overlap, lateral transfer among them seems feasible even if the reality of the phenomenon is beyond experimental proof.
(iii) Sequences homologous to the full E. coli argA gene were found in several of the organisms harboring the H(A) fusion but not in I. loihiensis and P. haloplanktis (for M. abyssi and M. profunda, their genomes have not yet been sequenced). In I. loihiensis and P. haloplanktis, the (A) sequence therefore does not appear to be functionally redundant, which in turn suggests that these organisms, which can grow in absence of arginine [12, 13], depend exclusively on domain (A) for the first step of arginine biosynthesis.
Origin of argH(A)
The data suggest that argH(A) results from a fusion that occurred between argH and a gene coding for an acetyltransferase able to acetylate L-glutamate in the N- position. The fusion could have been selected for in an organism devoid of a canonical NAGS, such as I. loihiensis and P. haloplanktis, perhaps as the result of gene loss, or it may reflect a more primordial event. Interestingly, in I. loihiensis, the genes of the argCBFGH(A) cluster are tightly coupled, either overlapping by 3 nt (argC and B, argF and G), separated by 3 nt (argB and F) or by 4 (argG and H). This arrangement suggests that, at the time the fusion originated, the capacity to derepress the recruited acetyltransferase from the rightward promoter of the operon may have been essential and actually explains why this fusion took place. In keeping with this hypothesis, the genome of I. loihiensis and P. haloplanktis do contain a sequence homologous to the E. coli argR regulatory gene.
Occurrence of arg(A)-like sequences with putative function in other organisms
Furthermore, we fused these arg(A)-like sequences in silico with argH sequences from the same organism in order to build so-called argHX sequences. Since argH is in the the last step of the pathway, and the (A) determinant in the first one, we focused the search on organisms presumed to possess the whole pathway and, took into account any structural and/or functional significance of the association of the two determinants. All 26 species found using this approach form a homogeneous group presenting the following features: (i) they do not possess a multidomain homologue of a NAGS protein, (ii) they contain an ornithine acetyltransferase (ArgJ) and (iii) they lack an acetylornithinase gene (ArgE). Thus, these 26 species should use an alternative to NAGS in order to acetylate glutamate and are presumed to recycle the transfer of the acetyl group from acetylornithine to glutamate. In contrast, the species retrieved in the first step of the screening, before implementing the virtual fusion approach, form a wider and less homogeneous group (Fig. 4), where NAGS and/or ArgE can be found. This suggests that the virtual fusion approach identifies a functionally significant group.
i. The argH(A) fusions form a monophyletic group branching close to homologous sequences present in the genomes of both Thermus and Deinococcus, two phylogenetically related organisms. The two groups join at a node position where the bootstrap value is less than 60 %, but it is remarkable that the Thermus and Deinococcus sequences (annotated as homologues of argH and a putative acetyltransferase gene), are actually adjacent, suggesting that together they play the role of a functional analogue of argH(A). T. thermophilus argH (which overlaps argG by 10 nt at the proximal end) and the putative acetyltransferase gene are separated by only 2 nt, strongly suggesting that the Thermus arg(A)- like sequence is part of an argGH(A) operon in the arginine regulon of this organism . In D. radiodurans the situation is similar but more complex: no less than three putative acetyltransferase genes are adjacent to argG (the first one separated by only 2 nt) while argH is very closely linked to another three putative acetyltransferase genes; of these three it is the last one (Q9RWI5_DEIRA Hypothetical protein DR0683) that is retrieved by our homology search.
ii. The other part of the tree contains various prokaryotic species including mesophilic Archaea. Clustering with these Archaea, we note a clade of Actinobacteria including M. tuberculosis. This is highly significant since the M. tuberculosis sequence was shown tocode for an enzyme whose functional characterization was reported while this paper was being prepared for publication: it displays acetylglutamate synthetase activity  and is required for the growth of its host as shown by previous high-density mutagenesis .
An essential question is whether any other of these 26 prokaryotes harboring an arg(A)- like sequence actually depend on it for acetylation of glutamate in vivo. We know (see above) that the complete sequence of their genome lacks a classical NAGS. The presumption would be even stronger if these species possessed a monofunctional OAT, and were thus unable to acetylate glutamate with acetyl-CoA [5, 16, 17]. Currently, T. thermophilus and Streptomyces coelicolor fulfill this second criterium [ibid, ]. The (A)-like sequence of T. thermophilus moreover appears co-regulated with the argGH cluster. It is not known whether M. tuberculosis OAT is monofunctional, but the fact that the arg(A)- like sequence of this organism was shown to be essential by transposon-mediated inactivation  actually suggests that it is. Since it is not yet possible to predict in silico whether a particular OAT is bifunctional , biochemical evidence is needed to decide which of the other microorganisms actually depend on their arg(A)- like sequence for arginine biosynthesis.
Comparative analysis of putative glutamate N-acetyltransferases
The discovery of a novel type of biosynthetic arg locus, coding for a classical argininosuccinase ArgH fused with a putative N-acetyltransferase able to complement an argA deficiency was extended by genomic analysis to a group of phylogenetically and ecologically related marine gamma proteobacteria. The case of I. loihiensis  and P. haloplanktis  is particularly significant from the functional point of view since the cognate genomes do not appear to contain a genuine argA sequence and the organisms are nevertheless arginine-independent, indicating that they depend on Arg(A) for arginine biosynthesis. Note there is widespread occurrence of sequences homologous to arg(A) in organisms lacking a classical NAGS (Fig. 5), including instances (Thermus and Deinococcus) where the sequence is adjacent-to, and coexpressed with argH. Moreover T. thermophilus and S. coelicolor which do not possess an OAT able to acetylate glutamate with acetylCoA, most probably depend on their arg(A) homologue for arginine biosynthesis.
The gene-enzyme relationship for the first committed step of arginine biosynthesis must now be considered in a new perspective. Several alternatives can be recognized:
i. The classical NAGS originally found in E. coli and Pseudomonas has two domains: an N-terminal one, with a carbamate kinase fold, displays extensive similarity with acetylglutamate kinase (NAGK), while the C-terminal one contains an N-acetyltransferase fold. This classical NAGS may occur in organisms with an acetylornithinase (ArgE) or an ornithine acetyltransferase (ArgJ), two situations epitomized by E. coli and P. aeruginosa. In P. aeruginosa, where ArgJ only recycles the acetylgroup from acetylornithine, NAGS fulfils an anaplerotic, but essential function, priming arginine biosynthesis with the acetyl group from acetyl-CoA [1, 2].
ii. In B. stearothermophilus and T. neapolitana the ArgJ (OAT) protein is bifunctional: not only does it recycle the acetyl group, but it also catalyzes the first step (EC 22.214.171.124). Early data concerning expression of B. subtilis genes in E. coli , reinterpreted after sequencing of the cognate DNA show that this bacterium also has a bifunctional OAT. In principle, such organisms do not need a NAGS, an assumption corroborated by the actual lack of a NAGS gene in the genomes of their close relatives, B. subtilis and T. maritima. It is worth noting that bifunctional OAT does not show recognizable similarity with NAGS despite the fact that they catalyze the same reaction [5, 19].
Full names of in alphabetical order species used in phylogenetic studies
Complete species namea
iv. The discovery of the in vivo active Moritella arg(A) sequence fused to the argH gene, and the detection of several homologous argH(A) sequences among marine Proteobacteria including species devoid of NAGS (I. loihiensis, P. haloplanktis) indicates that reaction EC 126.96.36.199 can be catalyzed by a short version of NAGS corresponding to the C-terminal domain of the bimodular NAGS. Furthermore, a number of previously uncharacterized acetyltransferases from different prokaryotes are homologous to this shorter version and the recent biochemical characterization of one of them in M. tuberculosis strongly suggests that many organisms rely on this monodomain form of NAGS to synthesize acetylglutamate.
Due to a lack of biochemical evidence for OAT, we do not know which of the two isoforms, mono or bifunctional, is the more widespread and possibly the primordial one. One possibility is that an arginine pathway using a bifunctional OAT and devoid of NAGS is the most ancestral version of the biosynthesis and that the various forms of NAGS we have been discussing appeared under selection after a mutation transformed a bifunctional OAT into a monofunctional enzyme. The acetylornithinase found in enteric and vibrio- like bacteria may have emerged after loss of such an OAT (7).
The alternatives emphasized in the present survey do not appear to exhaust the variety of solutions implemented in nature for the N-acetylation of glutamate. Archaea such as M. jannaschii have a monofunctional OAT  but no homologues of either mono- or two-domain ArgA could be revealed by our investigations. It is therefore possible that in such organisms the reaction EC 188.8.131.52 is carried out by yet another protein. In this respect, organisms such as Xanthomonas appear rather puzzling. In the entirely sequenced genomes of the three available species, a gene encoding a short protein similar to an acetyltransferase has been annotated argA. However, although this gene is clearly inside the arg cluster (between argC and argB) it does not appear as homologous to any of the known mono- or two-domain ArgA proteins and it is absent from the closely related Xylella species. It might therefore represent a new acetylglutamate synthetase or been incorrectly annotated.
In conclusion, our concept of the gene-enzyme relationships in arginine biosynthesis is undergoing a drastic revision among prokaryotes. Far from being universal, the patterns of acetylation of the intermediates may differ in phyla and even within the same phylum. The basic acetylation strategy that segregates arginine and proline precursors in different pathways is not brought into question, but the identity and the origin of the enzymes responsible for glutamate acetylation appears to betray extensive "natural tinkering" . Further phylogenetic analysis and the structural characterization of the cognate proteins will hopefully shed some light on the evolution of this crucial metabolic step.
Identifying all sequences homologous to the Arg(A) domain
In a first step, the M. abyssi sequence  was used as a query to collect the ArgH(A) homologous sequences from the last version (September 2005) of Uniprot (SwissProt and TREMBL) using the Blast facilities of the ExPaSy server  and the following criteria : ranking among the best E-values and aligning along the full length (around 620 aa) of the query. Note that several of the found homologues have been incorrectly annotated as "bifunctional protein ArgH" (V. parahaemolyticus), "argininosuccinate lyase" (I. loihiensis, V. vulnificus YJ016), "amino-acid acetyltransferase" (V. fischeri), N-acetylglutamate synthase (V. vulnificus CMCP6). These ArgH(A) sequences were immediately followed in the Blast outfile by the whole set of ArgH proteins (around 450 residues long). Preliminary sequence data was obtained from  in the case of Aeromonas hydrophila.
In a second step, the sequence of the domain (A) of the ArgH(A) protein of M. abyssi (KAVGTFAVTEKHNQVTGCASIYVYDTGLAELRSLGIEPGYQGGGQGKAVVEYMLRKAEQMAIQKVFVLTRVPEFFMKLGFRSTSKSMLPEKVLKDCDMCPRQHACDEVALEFKLNVVGQTINLKAEKLAS) was further used do detect (A) homologues. We first identified a list of short (150–180 residues) proteins generally annotated as putative acetyltransferases and, in a second, more distant wave, the acetyltransferase domain of the canonical ArgA (NAGS) such as that of E. coli.
Reconstructing phylogenetic trees
All the (A) homologous sequences were multiply aligned using ClustalX . The arg(A)-like sequences were virtually fused in silico with argH sequences from the same organism in order to build so-called argHX sequences. The ArgH(A) and ArgHX sequences were further mutiply aligned. Both automatic alignments were manually improved using the BioEdit software , saved in PHYLIP format and further used to reconstruct phylogenetic trees applying a two-step maximum likelihood approach as follows. After computing a BIONJ  distance tree using the Dayhoff model of evolution , program Phyml  was employed to refine this initial distance tree and to optimize its topology using a discrete-gamma model to accommodate rate variation among sites. The shape parameter alpha of the gamma distribution was estimated as described in  and found to be 3.74, the proportion of invariant sites being 0.065. The confidence limits for each node were further estimated using the non-parametric bootstrap approach of the Phyml program with 100 computed data sets.
We are very much indebted to Claudine Médigue and Antoine Danchin for kindly sending us before publication crucial information obtained from their annotation of the genome of Pseudoalteromonas haloplanktis that has been sequenced by Genoscope . We also thank our colleague Troy Philipps, at the University of Southern California, for critically reading the manuscript.
Aeromonas hydrophila preliminary sequence data was obtained from The Institute for Genomic Research .
BL wishes to thank the CNRS for support through the UMR 8621.
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