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BMC Evol BiolBMC Evolutionary Biology1471-2148BioMed Central London 1471-2148-4-471555506310.1186/1471-2148-4-47Research ArticleWidespread presence of "bacterial-like" PPP phosphatases in eukaryotes Andreeva Alexandra V 12aandreeva@brookes.ac.ukKutuzov Mikhail A 12m.kutuzov@usa.net1 Research School of Biological and Molecular Sciences, Oxford Brookes University, Headington, Oxford OX3 OBP, UK2 Present address: University of Illinois, College of Medicine, Department of Pharmacology, 835 S. Wolcott Ave, Chicago, IL 60612, USA2004 19 11 2004 4 47 47 25 8 2004 19 11 2004 Copyright © 2004 Andreeva and Kutuzov; licensee BioMed Central Ltd.2004Andreeva and Kutuzov; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Background
In eukaryotes, PPP (protein phosphatase P) family is one of the two known protein phosphatase families specific for Ser and Thr. The role of PPP phosphatases in multiple signaling pathways in eukaryotic cell has been extensively studied. Unlike eukaryotic PPP phosphatases, bacterial members of the family have broad substrate specificity or may even be Tyr-specific. Moreover, one group of bacterial PPPs are diadenosine tetraphosphatases, indicating that bacterial PPP phosphatases may not necessarily function as protein phosphatases.

Results
We describe the presence in eukaryotes of three groups of expressed genes encoding "non-conventional" phosphatases of the PPP family. These enzymes are more closely related to bacterial PPP phosphatases than to the known eukaryotic members of the family. One group, found exclusively in land plants, is most closely related to PPP phosphatases from some α-Proteobacteria, including Rhizobiales, Rhodobacterales and Rhodospirillaceae. This group is therefore termed Rhizobiales / Rhodobacterales / Rhodospirillaceae-like phosphatases, or Rhilphs. Phosphatases of the other group are found in Viridiplantae, Rhodophyta, Trypanosomatidae, Plasmodium and some fungi. They are structurally related to phosphatases from psychrophilic bacteria Shewanella and Colwellia, and are termed Shewanella-like phosphatases, or Shelphs. Phosphatases of the third group are distantly related to ApaH, bacterial diadenosine tetraphosphatases, and are termed ApaH-like phosphatases, or Alphs. Patchy distribution of Alphs in animals, plants, fungi, diatoms and kinetoplasts suggests that these phosphatases were present in the common ancestor of eukaryotes but were independently lost in many lineages. Rhilphs, Shelphs and Alphs form PPP clades, as divergent from "conventional" eukaryotic PPP phosphatases as they are from each other and from major bacterial clades. In addition, comparison of primary structures revealed a previously unrecognised (I/L/V)D(S/T)G motif, conserved in all bacterial and "bacterial-like" eukaryotic PPPs, but not in "conventional" eukaryotic and archaeal PPPs.

Conclusions
Our findings demonstrate that many eukaryotes possess diverse "bacterial-like" PPP phosphatases, the enzymatic characteristics, physiological roles and precise evolutionary history of which have yet to be determined.
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Background
Reversible phosphorylation of proteins is a ubiquitous mechanism, indispensable for regulation of virtually any cellular function. Therefore, protein kinases and phosphatases are of paramount importance for normal functioning of all metabolic and signalling pathways. In eukaryotes, PPP family is one of the two known protein phosphatase families specific for Ser and Thr [1-4]. Unlike eukaryotic (and archaeal [5]) PPP phosphatases, bacterial members of the family have broad substrate specificity [6] or may even be Tyr-specific [7-9]. Moreover, one group of bacterial PPPs are diadenosine tetraphosphatases [10,11]. Unlike eukaryotes, in prokaryotes PPP phosphatases appear to be facultative, since entirely sequenced genomes of some bacteria and archaea do not encode them [5,12]. Nevertheless, when present, they appear to play essential roles [13-15].

Three motifs (GDXHG, GDXXDRG and GNH(E/D)), highly conserved in the N-terminal subdomains of the catalytic domains of all PPP phosphatases [10,11], contain most residues which coordinate metal ions in the active centre [16] and are considered as the signature of the PPP family.

In a previous work [17] we identified an unusual cDNA fragment from a moss Physcomitrella patens, showing no similarities to the known PPP phosphatases beyond the presence of the GDXHG and GDXXDRG motifs. Detection of homologous cDNA sequences from Arabidopsis and rice suggested the presence of an unknown PPP group in plants, distinct from "conventional" eukaryotic PPP phosphatases [17].

We have now taken advantage of a much greater representation (as compared to 1999) of sequence databases for various species to further explore this initial observation. We present the evidence for the existence in eukaryotes of three "non-conventional" branches of the PPP family. We also identify a previously unrecognised conserved motif in the PPP catalytic domain, which can be used as a signature of "bacterial"-type PPP phosphatases.

Results
"Rhizobiales / Rhodobacterales / Rhodospirillaceae – like" PPP phosphatases in plants
Two Arabidopsis sequences, At3g09960 and At3g09970, were retrieved using the P. patens fragment [17] as a query in TBlastN searches. They share 85% identity with each other at the protein level (see Figure 1). Both genes are transcribed, since full-length cDNAs have been detected in a large-scale transcription study [18]. They are arranged on chromosome 3 in tandem, suggesting their origin by a recent duplication. A number of ESTs from other plant species (but none from non-plant eukaryotes) were also detected by TBlastN searches, which in most cases provide no evidence for the existence of more than one isoform.

Figure 1 Comparison of the primary structures of plant Rhilphs, related α-proteobacterial phosphatases and human PP1α as a prototype of "conventional" eukaryotic PPP phosphatases. Amino acid residues conserved in at least all but one Rhilphs and α-proteobacterial phosphatases are shown in white and shaded in black. Residues conserved in at least two thirds of the sequences are shown in white and shaded in dark grey. Residues conserved in at least half of the sequences are shown in black and shaded in light grey. Following substitutions were considered as conserved residues: Ile/Leu, Phe/Tyr, Asp/Glu, Asn/Gln, Arg/Lys and Ser/Thr. Catalytic site residues that interact with metal ions are indicated by asterisks according to [20]. SAPNY motif in PP1, conserved in most eukaryotic PPP phosphatases, is double underlined. Solanum tuberosum sequence is translation of the EST entries BQ516856, BQ516857 and BI435517. Physcomitrella patens sequence is translation of the EST entry BQ039171. Other accession numbers are indicated in Table 1.

Among prokaryotes, related sequences were detected in some α-Proteobacteria, the closest matches being with Rhizobiales, Rhodobacterales and Rhodospirillaceae. Therefore, we designate this group as Rhizobiales / Rhodobacterales / Rhodospirillaceae-like phosphatases, or Rhilphs (Figure 1; see also Table 1 and Figure 4), and the two Arabidopsis genes products At3g09960 and At3g09970 as "Rhizobiales-like" phosphatases 1 (RLP1) and 2 (RLP2), respectively.

Table 1 Species, accession numbers (UniProt, EMBL, NCBI or TIGR Gene Index) and common names (where available) of PPP phosphatase sequences shown in Figure 4. For A. thaliana sequences, gene numbers are also indicated. Sequence No. 67 is available from Chlamydomonas reinhardtii draft genome [65].

No.	Accession	Species name	
Rhilphs	
1	Q9SR61; At3g09960	Arabidopsis thaliana	
2	Q9SR62; At3g09970	Arabidopsis thaliana	
3	BE034080	Mesembryanthemum crystallinum	
4	BQ995369	Lactuca sativa	
5	AW034786	Lycopersicon esculentum	
6	BU875146	Populus balsamifera	
7	BG457803	Medicago truncatula	
8	AV425727	Lotus japonicus	
9	BM731295	Glycine max	
10	BF261816	Hordeum vulgare	
11	BQ788728	Triticum aestivum	
12	AL731641	Oryza sativa	
13	CF670562	Pinus taeda	
14	BQ039171	Physcomitrella patens	
Group I (α-Proteobacteria)	
15	Q987U4	Mesorhizobium loti	
16	Q8UA33	Agrobacterium tumefaciens	
17	ZP_00054691	Magnetospirillum magnetotacticum	
18	ZP_00015226	Rhodospirillum rubrum	
19	ZP_00014771	Rhodospirillum rubrum	
20	CAE28794	Rhodopseudomonas palustris	
21	Q9ABQ8	Caulobacter crescentus	
22	ZP_00051041	Magnetospirillum magnetotacticum	
23	ZP_00093979	Novosphingobium aromaticivorans	
24	Q92V37	Sinorhizobium meliloti	
Group VII (heterogeneous)	
25	Q8YZT4	Anabaena sp.	
26	Q9WZK1	Thermotoga maritima	
27	O34205	Fervidobacterium islandicum	
28	NZ_AABE01000101	Cytophaga hutchinsonii	
Group III (γ-Proteobacteria and bacteriophage λ)	
29	P03772	Bacteriophage λ	
30	P55798	E. coli PrpA	
31	Q8VPE2	Salmonella typhimurium PrpA	
32	Q8Z487	Salmonella enterica	
33	P55799	E. coli PrpB	
Group IV (Firmicutes)	
34	Q81YR3	Bacillus anthracis	
35	Q9FB69	Lactococcus lactis	
36	Q97FF3	Clostridium acetobutylicum	
Alphs	
37	BM291808	Amblyomma variegatum	
38	TC9835	Ciona intestinalis	
39	BU652795	Chlamydomonas reinhardtii	
40	AC091781	Trypanosoma brucei	
41	AC084046	Trypanosoma brucei	
42	AL499620	Leishmania major	
43	BQ143558	Metarhizium anisopliae	
44	AC127427	Magnaporthe grisea	
45	AA966318	Aspergillus nidulans	
46	P40152	Saccharomyces cerevisiae	
ApaH	
47	Q8Y1K9	Ralstonia solanacearum ApaH	
48	Q9JVF4	Neisseria meningitidis ApaH	
49	P05637	Escherichia coli ApaH	
Group VI (heterogeneous)	
50	O31614	Bacillus subtilis	
51	O69213	Anabaena sp. PrpA	
52	Q93JF4	Streptomyces coelicolor	
53	Q9RS78	Deinococcus radiodurans	
Group II (Cyanobacteria)	
54	O54390	Microcystis aeruginosa PP1-Cyano 1	
55	P74150	Synechocystis sp.	
56	Q8YP31	Anabaena sp.	
57	ZP_00072257	Trichodesmium erythraeum	
58	Q8DGA2	Thermosynechococcus elongatus	
Shelphs	
59	AC119500*	Leishmania major	
60	Q8EBN0	Shewanella oneidensis	
61	Q9S427	Shewanella sp.	
62	TIGR_167879 Contig1731	Colwellia psychrerythraea	
63	CF394707	Pinus taeda	
64	TC31593	Solanum tuberosum	
65	Q944L7; At1g18480	Arabidopsis thaliana	
66	BF645180	Medicago truncatula	
67	Scaffold_45	Chlamydomonas reinhardtii	
68	Q9LMJ5; At1g07010	Arabidopsis thaliana	
69	AW266595	Mesembryanthemum crystallinum	
70	BG644111	Lycopersicon esculentum	
71	BG450922	Medicago truncatula	
72	BI787505	Glycine max	
73	Q8L676	Oryza sativa	
74	TC21958	Hordeum vulgare	
75	AC007863	Trypanosoma brucei	
76	AL499621	Leishmania major	
77	TIGR_246197 Contig433	Myxococcus xanthus	
78	EAK84303	Ustilago maydis	
79	O74480	Schizosaccharomyces pombe	
80	EAK87480	Cryptosporidium parvum	
81	Q7RIH8	Plasmodium yoelii	
82	Q8IKE5	Plasmodium falciparum	
83	Q8I5Y5	Plasmodium falciparum	
84	Q7RR22	Plasmodium yoelii	
Group V (heterogeneous)	
85	O87639	Streptomyces coelicolor	
86	Q9RVT7	Deinococcus radiodurans	
Archaea	
87	O28453	Archaeoglobus fulgidus PPA	
88	Q8ZW26	Pyrobaculum aerophilum	
89	O34200	Methanosarcina thermophila PP1-arch2	
90	Y12396	Pyrodictium abyssi	
"Conventional" eukaryotic PPP	
91	Q9U493	Plasmodium falciparum PPJ	
92	Q8I728	Trypanosoma cruzi PPEF	
93	BH900132	Ostreococcus tauri	
94	O14829	Homo sapiens PPEF1 (PP7)	
95	Q8IDE7	Plasmodium falciparum PP5	
96	P53041	Homo sapiens PP5	
97	P53043	Saccharomyces cerevisiae PPT	
98	P32838	Saccharomyces cerevisiae PPG	
99	P05323	Homo sapiens PP2A	
100	O00743	Homo sapiens PP6	
101	Q08209	Homo sapiens Calcineurin (PP2B)	
102	P08129	Homo sapiens PP1	
103	P32945	Saccharomyces cerevisiae PPQ	
104	O49346	Arabidopsis thaliana PP7	
* This phosphatase is unlikely to be catalytically active due to replacements of essential residues in the active centre.

Figure 2 Comparison of the primary structures of "Shewanella-like" phosphatases (Shelphs) and human PP1α as a prototype of eukaryotic PPP phosphatases. Designations for conserved amino acid residues are as in Figure 1. For Oryza sativa, dashed underlined C-terminal sequence has been corrected by comparison with ESTs. Accession numbers: Plasmodium falciparum 1, Q8I5Y5; 2, Q8IKE5; Trypanosoma brucei 1, AC007863; 2, AC084046.12. Chlamydomonas reinhardtii sequence is translation of the EST entries BG855683 and BI995255. Other accession numbers are indicated in Table 1.

Figure 3 Characteristic modifications (shaded in black) in the conserved PPP signature motif GDXXDRG in bacterial diadenosine tetraphosphatases and eukaryotic Alphs. Eukaryotic species are shown in bold. Plus signs indicate that gene expression is confirmed by the presence of ESTs.

Figure 4 Neighbor-Net analysis of the conserved N-terminal subdomains (starting 5 amino acid residues before conserved GDXHG and ending 25 residues after GNH(E/D) of 104 bacterial, archaeal and eukaryotic PPP phosphatases. Bootstrap values exceeding 50% (out of 1000 resamplings) were obtained in a separate neihbour-joining analysis and are shown in brackets. Species and accession numbers are listed in Table 1. Note that groups designated as I, IV and VII did not receive significant bootstrap support; corresponding sequences are grouped together for convenience of their representation in Table 1. This image (and bootstrap values for the alternative splits) can be viewed at a higher resolution as the Additional File 1.

Figure 5 Distinct conserved motifs in the C-termini of bacterial and "bacterial-like" PPP phosphatases from eukaryotes as opposed to archaeal and eukaryotic PPP phosphatases. A His residue directly binding a metal ion in the catalytic centre (marked with asterisk), and the elements of secondary structure are shown for bacteriophage λ phosphatase and for human PP1 according to ref. [20] and [19], respectively. The (I/L/V)D(S/T)G motif, highly conserved in bacterial and "bacterial-like" phosphatases, is highlighted. An expanded version of this alignment can be viewed as the Additional File 2.

Structural features of Rhilphs
All residues that are expected to bind metal ions in the catalytic centre are conserved in Rhilphs (Figure 1). Rhilphs do not have N- or C-terminal extensions beyond their catalytic domains, which in many PPP phosphatases have regulatory function and / or interact with regulatory proteins / subunits. Instead, they have characteristic inserts between the conserved motifs GNH(E/D) and HAG (corresponding to HGG in "conventional" eukaryotic PPPs, see Figure 1). Notably, much shorter inserts are found at a similar position in α-proteobacterial phosphatases (group I in Figure 1). Inserts in both plant Rhilphs and α-proteobacterial phosphatases contain a conserved motif LXXAXPXXH (Figure 1). Similarly to bacteriophage λphosphatase (λPP [19]), Rhilphs lack a region corresponding to β8, β9 and α9 of eukaryotic PPPs [20-22]. Like bacterial PPP phosphatases, Rhilphs do not have a SAPNY motif, conserved in the β12-β13 loop of eukaryotic PPPs. Analysis of Rhilph sequences did not reveal targeting or signal peptides.

While this work was in progress, a phosphatase encoded by an Arabidopsis gene At1g07010 was reported in an independent study [4].

"Shewanella-like" PPP phosphatases in plants, red algae, fungi and unicellular parasites
We undertook further TBlastN searches using full-length Arabidopsis RLP2 as a query to see whether Arabidopsis genome encodes additional "bacterial-like" PPPs. These searches identified two more genes for putative PPP phosphatases, only distantly related to Rhilphs and to any other members of the family (Figure 2).

One of these genes is At1g070101. At least three different predicted products of this gene could be found in protein databases. On the basis of comparison with EST sequences, we consider as the correct structure that of Q8RY10 with Asp and Gly at positions 109 and 208, respectively (see Figure 2). The other detected gene, At1g18480, is also represented in the databases by three distinct deduced proteins. Comparison with A. thaliana ESTs confirms Q944L7 as the correct structure.

Genomic and EST database searches provided ample evidence for the presence of related phosphatases in a number of green plants, including multiple angiosperm species, pine and a unicellular green alga Chlamydomonas reinhardtii (Figure 2; see also Table 1 and Figure 4). Related sequences were also identified in some fungi (several basidiomycetes and an ascomycete Schizosaccharomyces pombe, but not other ascomycetes), in Apicomplexa, Trypanosomatidae, and in a red alga Porphyra yezoensis (for available sequence from the latter species, see Figure 5).

The most closely related prokaryotic phosphatases were detected in Myxococcus xanthus (δ-Proteobacteria) and psychrophilic bacteria Alteromonadales (γ-Proteobacteria): uncharacterised phosphatases from Shewanella oneidensis and Colwellia psychrerythraea and a Tyr-specific phosphatase PPI from Shewanella sp. [8]. Therefore, we designate this phosphatase group as "Shewanella-like" phosphatases, or Shelphs, and the products of the two prototype Arabidopsis genes At1g07010 and At1g18480 as "Shewanella-like" phosphatases 1 (SLP1) and 2 (SLP2), respectively.

Structural features of Shelphs
Like in Rhilphs, all residues that are expected to bind metal ions in the catalytic centre are conserved in Shelphs (see Figure 1). Another feature common with Rhilphs is the presence of inserts (as compared to "conventional" eukaryotic PPPs) between the GNHE and H(A/G)G motifs (Figure 1), which are especially long in plant Shelphs. However, these inserts share no sequence similarity between Rhilphs and Shelphs and probably appeared in the two phosphatase groups independently. Like in Rhilphs, a region corresponding to β8, β9 and α9 of eukaryotic PPPs is absent in Shelphs, and the primary structure of the region corresponding to the β12-β13 loop is similar to that of typical bacterial PPPs. A.thaliana SLP1 and corresponding Shelph isoform from rice have chloroplast targeting sequence, which could not be detected in A.thaliana SLP2 and corresponding isoform from Medicago truncatula.

Eukaryotic PPPs distantly related to bacterial diadenosine tetraphosphatases
Identification of Rhilphs and Shelphs prompted us to perform extensive searches of eukaryotic sequence databases. These searches revealed the existence of other "bacterial-like" PPP phosphatases throughout eukaryotes. Sequences only distantly related to Rhilphs, Shelphs or any other PPP phosphatases were detected in several fungi (including a putative S. cerevisiae phosphatase reported previously [23]), in Trypanosomatidae, a tick Amblyomma, an ascidian Ciona, Chlamydomonas, pine and diatoms Fragilariopsis cylindrus and Phaeodactylum tricornutum. Blast searches using these sequences revealed that all of them share higher similarity to bacterial diadenosine tetraphosphatases (ApaH) than to other PPP groups. Therefore, we tentatively designate them as ApaH-like phosphatases, or Alphs (Figures 3 and 4; Table 1; partial sequences available for pine and diatoms are shown in Figure 5).

Alphs share a distinctive common structural feature. In the GDXXDRG motif, absolutely conserved in other PPPs, the second Asp (which stabilises the protonation of a His that directly participates in catalysis [20]) is replaced by a neutral amino acid, and the Arg residue (which coordinates phosphate [24]) is replaced, with one exception, by Lys (Figure 3). The former of these replacements is also found in ApaH, while the latter is unique to Alphs. While higher overall sequence similarity and a common alteration in the GDXXDRG motif are compatible with closer relatedness of Alphs to bacterial diadenosine tetraphosphatases, phylogenetic analysis using full length sequences failed to produce a robust tree due to high sequence diversity (not shown).

Relationship of novel eukaryotic PPP groups to known PPP phosphatases
In order to better understand the relationship of "bacterial-like" PPP phosphatases in eukaryotes to each other and to bacterial PPPs, we attempted to extend our previous phylogenetic analysis of eukaryotic PPP phosphatases [25] by including PPP sequences from a number of bacteria and archaea. Primary structures of bacterial PPP phosphatases are extremely diverse and, outside the relatively conserved N-terminal subdomain of about 100 amino acids containing the GDXHG, GDXXDRG and GNH(E/D) motifs, they share only a few conserved residues. Moreover, many of the sequences have long insertions at different positions. This leads to the failure to produce informative alignments of full-length catalytic domains. Therefore, we aligned more conserved N-terminal subdomains only, an approach applied previously by Kennelly [6] to a much smaller set of PPP sequences available at that time. Phylogenetic reconstruction was attempted with either neighbor-joining (as implemented in PHYLIP [26] or SplitsTree [27]) or maximum likelihood analysis using quartet puzzling (TreePuzzle [28]); in the latter case a smaller dataset consisting of with consisting of 32 representative sequences was analyzed due to the inability of the algorithm to handle large datasets. Due to the relatively short length of the sequences and their high diversity, some of the major clades did not receive significant bootstrap support and were different depending on the method used, although most major clades, including Rhilphs and Shelphs, were recovered by both methods. Alphs tended to be grouped together by neighbor-joining but were split into smaller clades when maximum likelihood analysis was used. However, we still tentatively consider Alphs as a single group due to the characteristic replacements in their catalytic centre.

To circumvent the ambiguity of the results, we used Neighbor-Net [29], a neighbor-joining based method that constructs phylogenetic networks rather than trees and thus represents conflicting signals and visualises feasible trees in a single plot (Figure 4; for a high-resolution image, see Additional file 1). The Neighbor-Net analysis accurately identified the major clades such as eukaryotic and archaeal phosphatases, as well as their closer relationship to each other than to bacterial PPPs [6]. Separation of "conventional" eukaryotic PPPs into two branches, suggested previously from the analysis of the full-length catalytic domains [25], was also recovered. As it was suggested by initial sequence similarity searches, Rhilphs, Shelphs and Alphs represent distinct major clades of the PPP family, as divergent from "conventional" eukaryotic and archaeal PPP phosphatases as they are from major bacterial clades (Figure 4; Additional file 1).

Common structural elements in all "bacterial-like" PPP phosphatases from eukaryotes and bacterial phosphatases
C-terminal regions of the catalytic domain of all "conventional" eukaryotic PPP phosphatases share a highly conserved (with minor variations) SAPNY motif, located in the β12-β13 loop. This loop and the Tyr residue of the SAPNY motif in particular are implicated in interaction with regulators and inhibitors [21,30-32]. β strands (β9 and β10) corresponding to β12 and β13 are conserved in λPP [19]. However, the sequence on the C-terminal side of β9 is dissimilar to SAPNY in λPP and in bacterial PPPs. A conservative replacement of the first Ser of SAPNY by Thr is found in many bacterial sequences (this Thr is however only moderately conserved and is replaced by Glu or Gln in all Rhilphs and by Val or Phe in most Shelphs). The two adjacent positions are occupied by highly conserved Asp and Gly residues, respectively, thus defining a previously unrecognised motif (I/L/V)D(S/T)G. This motif is present in all examined bacterial PPPs, as well as in all "bacterial-like" phosphatases from eukaryotes described above (Figure 5; see also Additional file 2 for a more complete alignment).

In addition, we note the presence of another characteristic feature of bacterial and "bacterial-like" PPPs: the His residue (H248 in PP1; H186 in λPP) coordinating one of the metal ions in the catalytic centre is preceded by an absolutely conserved Gly; this residue is conserved in some archaeal but not in "conventional" eukaryotic PPPs (Figure 5; Additional file 2).

Discussion
In this report, we have documented the presence in different eukaryotic lineages of the genes that encode PPP phosphatases resembling those of bacterial origin, rather than "conventional" eukaryotic members of the family. Catalytic domains of these "bacterial-like" phosphatases are characterised by relatively conserved structure of the N-terminal subdomains, but very diverse organisation of the C-terminal subdomains, where conserved motifs and residues forming the active centre are separated by sequences of various length that share little or no similarity between different clades (Figure 6). In most cases, corresponding EST sequences could be detected, which confirms that these genes are expressed.

Figure 6 Schematic diagram depicting organisation of the catalytic domains of the phosphatase groups discussed in this study. N-terminal subdomains (used in the alignment for the Neighbor-Net analysis, Figure 4) and C-terminal subdomains are shown in red and yellow, respectively. Positions of the conserved motifs in PPλ and the residues forming the active centre (underlined; shown according to ref. [19]) are shown. Positions of the LXXAXPXXH motif in plant Rhilphs and related phosphatases from Rhizobiales are indicated (green boxes). For more detailed information on the position of inserts in Rhilphs and Shelphs relative to the elements of the secondary structure, see Figures 1 and 2, respectively.

The most conspicuous presence of "bacterial-like" PPPs has been detected in plants. Plants possess phosphatases from all three novel groups described in this work. Rhilphs are most closely related to PPP phosphatases from a number of α-proteobacteria, including purple photosynthetic bacteria and Rhizobiales. The absence of related sequences in eukaryotes other than land plants suggests that Rhilphs may have been acquired after plants started colonising land. Although bacterial lineage that could be the source for plant Rhilphs could not be unambiguously identified by phylogenetic reconstruction, Rhizobiales (or purple photosynthetic bacteria from which Rhizobiales are thought to have originated [33,34]) appear to be likely candidates. Indeed, Rhizobiales have transmissible chromosomal elements and some, like Agrobacterium, are able to integrate their plasmid genes into plant genome [35] or even transform animal cells [36], a situation that would be ideally suited for a horizontal gene transfer to occur. Interestingly, the presence of genes of rhizobial origin has been detected in plant parasitic nematodes [37]. Possible origin of plant Rhilphs from α-proteobacterial phosphatases is also supported by the presence in the enzymes of both groups of characteristic inserts in similar positions, which share some sequence similarity (see Results).

Phosphatases of another group, designated as Shelphs, are found in green plants, in a red alga, in Apicomplexa, Trypanosomatidae, as well as in some fungi. The similarity between proteins from Apicomplexa and Trypanosomatidae and those from plants is well documented. Trypanosomatidae are related to photosynthetic euglenoids and are thought to have lost plastids secondarily [38]. Apicomplexan parasites have a relict plastid, originated from the engulfment of a red alga [39]. Thus, the presence of phosphatases shared by plants, red algae, Apicomplexa and Trypanosomatidae is not surprising and probably reflects the presence of Shelphs in a common ancestor of photosynthetic eukaryotes. The presence of chloroplast targeting sequence in SLP1 suggests a possible origin of Shelphs from a bacterial precursor of the chloroplast (however it should be noted that Shelphs are absent from cyanobacteria); alternatively, this sequence may have appeared secondarily. Protein Ser/Thr phosphorylation / dephosphorylation is essential for regulation of photosynthesis, and unidentified okadaic acid-insensitive protein phosphatases in chloroplasts have been reported [40,41]. SLP1 appears to be a good candidate for such a phosphatase.

The origin of fungal Shelphs is unclear. Curiously, they are found in basidiomycetes and in an ascomycete S. pombe, but not in a number of other ascomycetes, whose genomes have been completed. Current data do not permit to discriminate between (i) the presence of Shelphs in a common ancestor of eukaryotes and their loss in such lineages as animals and many fungi, and (ii) independent acquisition of Shelphs from bacteria by an ancestor of photosynthetic eukaryotes and by fungi. Further sequencing of eukaryotic genomes may shed light on the evolutionary history of this PPP group.

The third group of "bacterial-like" phosphatases detected in eukaryotes, designated here as Alphs, appears to be distantly related to bacterial diadenosine tetraphosphatases ApaH. Patchy distribution in several eukaryotic kingdoms suggests that Alphs were probably present in the common ancestor of eukaryotes, but were independently lost in many lineages, including insects, vertebrates and flowering plants. A characteristic modification of the conserved GDXXDRG motif shared only with ApaH further supports a suggestion that Alphs may represent a divergent branch of diadenosine tetraphosphatases, rather than protein phosphatases. However, relatedness of eukaryotic Alphs to bacterial diadenosine tetraphosphatases remains hypothetical, since Alph sequences are too divergent from ApaH, as well as from each other, to permit a reliable phylogenetic reconstruction.

Diadenosine oligophosphates are considered as emerging signalling molecules in both intra- and intercellular signalling in eukaryotes [42,43]. In particular, human diadenosine oligophosphate hydrolase FHIT has been identified as a tumor suppressor [44]. It seems plausible that appearance of eukaryotic diadenosine oligophosphate hydrolases (structurally unrelated to the PPP phosphatases) may have made bacterial-type diadenosine tetraphosphatases redundant, leading to their loss in many eukaryotic lineages. It would be interesting to test experimentally whether Alphs are indeed diadenosine oligophosphatases.

More generally, an important implication of our findings is that many eukaryotes possess PPP phosphatases with yet undetermined substrate specificity. Eukaryotic PPP phosphatases are generally considered as Ser/Thr specific in vivo, although they may be able to dephosphorylate phosphoTyr-containing substrates in vitro (e.g. [45,46]). This is probably true for archaeal PPPs as well [5]. However, Ser/Thr specificity is not a feature of bacterial PPP phosphatases [7-9,13,47-49]. Thus, it would not be possible to predict substrate specificity of uncharacterised "bacterial-like" PPP phosphatases without experimental evidence. In particular, since Shewanella PPI is Tyr-specific [8], it would be interesting to determine substrate specificity of eukaryotic Shelphs. It is also worth noting that interest in tyrosine phosphorylation in plants has recently been stimulated by identification of plant Tyr phosphatase genes and by the finding that Tyr phosphorylation is involved in the regulation of stomatal movement (reviewed by Luan [50]).

Three motifs, GDXHG, GDXXDRG and GNH(E/D) form the diagnostic signature of all PPP phosphatases [10,11]. We detected a (I/L/V)D(S/T)G motif, which appears to be a characteristic signature of "bacterial"-type PPPs. The existence of such a motif is striking per se, taking into account extreme structural diversity of bacterial PPP phosphatases. It indicates that (I/L/V)D(S/T)G was probably present as the fourth "universal" signature motif in the common ancestor of PPP phosphatases, and was lost in the common lineage of archaeal and "conventional" eukaryotic PPPs. An alternative possibility could be that the (I/L/V)D(S/T)G motif was acquired by a bacterium and propagated by lateral gene transfer, replacing the ancestral SAPNY-related motif. However this scenario seems less likely, since the (I/L/V)D(S/T)G motif is present, with minor variations, in virtually all bacterial phosphatases, despite their great diversity, and is replaced by SAPNY-related sequences only in archaeal and "conventional" eukaryotic PPPs.

The Asp residue in the 2nd position of (I/L/V)D(S/T)G is highly conserved and can only be replaced by Glu, indicating that the negative charge is essential. The presence of a highly conserved Gly in the 4th position indicates that flexibility of the polypeptide chain is likely to be important. The crystal structure of bacteriophage λ phosphatase (PPλ; [19]) shows that the Asp residue of (I/L/V)D(S/T)G (Asp202) is just downstream of the β9 strand, which corresponds to the β12 strand in mammalian PP1. In PPλ, Asp202 is hydrogen bonded to a water molecule coordinated to one of the metal ions in the catalytic centre, which probably accounts for its conservation. In eukaryotic or archaeal PPPs, corresponding position is occupied by neutral residues (see Figure 5).

It would be tempting to speculate that this difference in the region just downstream of the β9 (β12) may be responsible for a feature that is common to all "bacterial"-type but not to eukaryotic / archaeal PPPs. One such feature is the Ser/Thr specificity of the latter group. The Tyr residue of the SAPNY motif has been suggested to provide a bulky phenol ring in the β12-β13 loop, sufficient to sterically block access of phosphoTyr-containing substrates to the active site [32]. However this is unlikely to be the sole determinant of Ser/Thr specificity, since residues containing bulky aromatic rings (Tyr, Phe or Trp) are found in the same or adjacent positions in many bacterial phosphatases (Figure 5). Since the (I/L/V)D(S/T)G motif, absent in eukaryotic and archaeal PPPs, is involved in organisation of the catalytic centre [19], it is possible that this difference in the catalytic centre organisation may be one of the determinants of broad substrate specificity vs. Ser/Thr specificity.

Conclusions
So far, eukaryotic PPP phosphatases were considered as a well-defined monophyletic group of enzymes, specifically dephosphorylating phosphoSer and phosphoThr, while a much more structurally and enzymatically diverse PPP phosphatases were known to be present in prokaryotes. Our findings demonstrate that, in addition to "conventional" eukaryotic PPP Ser/Thr-specific protein phosphatases, many eukaryotes possess very diverse "bacterial-like" PPP phosphatases. Enzymatic characteristics, physiological roles and evolutionary history of these phosphatases have yet to be revealed.

Methods
Detection of PPP phosphatase-coding sequences
Sequence similarity searches were conducted using BlastP or TBlastN [51] at NCBI [52] in the following databases: "non-redundant" (NR), "expressed sequence tags" (EST), "genomic sequence survey" (GSS) and "high-throughput genomic sequences" (HTGS). Additional Blast searches of the following databases were performed: finished and unfinished genomes of eukaryotes at the NCBI [53]; fungal genomes at the Broad Institute [54]; plant genomes at The Arabidopsis Information Resource (TAIR) [55]; Gene Index databases of tentative consensus sequences (EST assemblies) at The Institute for Genomic Research (TIGR) [56]; Chlamydomonas reinhardtii draft genome [57]. In all cases, reciprocal searches were used, i.e. hits retrieved by Blast searches were in their turn used as queries in the following Blast searches. Accuracy of gene prediction was examined by comparison of the retrieved sequences with translations of corresponding EST entries. In the absence of available ESTs, closely related sequences from other species were used. Taxonomy of the species from which the phosphatase sequences is given according to the NCBI taxonomy web site [58].

Phylogenetic analysis
Multiple alignments were generated using CLUSTAL W [59] at Kyoto University Bioinformatics Centre [60] and edited manually. During manual editing, particular attention was paid to correct alignment of the PPP family signature motifs and other conserved residues known to constitute the catalytic site of PPP phosphatases. Phylogenetic tree construction by the neighbor-joining method [61] and bootstrap analysis were performed using the PHYLIP package, version 3.573 [26]. Maximum likelihood analysis was performed using TreePuzzle [28]. Possible alternative neighbor-joining based phylogenies were visualised using Neighbor-Net [29] as implemented in SplitsTree, version 4.β10 [62]

Analysis of the primary structure
The presence of signal peptides and targeting sequences was analyzed using TargetP [63] at the the Centre for Biological Sequence Analysis, Technical University of Denmark [64].

Abbreviations
EST, expressed sequence tag, Alph, ApaH – like phosphatase; PPP, protein phosphatases of the P family; Rhilph (RLP), Rhizobiales / Rhodobacterales / Rhodospirillaceae – like phosphatase; Shelph (SLP), Shewanella – like phosphatase.

Authors' contributions
Both authors contributed equally to this work.

Supplementary Material
Additional File 1
Neighbor-Net analysis of the conserved N-terminal subdomains (starting 5 amino acid residues before conserved GDXHG and ending 25 residues after GNH(E/D) of 104 bacterial, archaeal and eukaryotic PPP phosphatases. This version of Figure 4 is the original SplitsTree file that can be viewed using SplitsTree, freely available for download (see Methods). Bootstrap values (out of 100 resamplings) are shown and can be highlighted by selecting corresponding alternative splits. The file also contains the alignment used for the analysis (Input).

Click here for file

 Additional File 2
Distinct conserved motifs in the C-termini of bacterial and "bacterial-like" PPP phosphatases from eukaryotes as opposed to archaeal and eukaryotic PPP phosphatases. This is an expanded version of Figure 5.

Click here for file
==== Refs
Cohen PTW  Novel protein serine/threonine phosphatases: Variety is the spice of life Trends Biochem Sci 1997 22 245 251 9255065 10.1016/S0968-0004(97)01060-8 
Andreeva AV Kutuzov MA  RdgC/PP5-related phosphatases: novel components in signal transduction Cell Signal 1999 11 555 562 10433516 10.1016/S0898-6568(99)00032-7 
Ariño J  Novel protein phosphatases in yeast Eur J Biochem 2002 269 1072 1077 11856338 10.1046/j.0014-2956.2002.02753.x 
Kerk D Bulgrien J Smith DW Barsam B Veretnik S Gribskov M  The complement of protein phosphatase catalytic subunits encoded in the genome of Arabidopsis Plant Physiol 2002 129 908 925 12068129 10.1104/pp.004002 
Kennelly PJ  Archaeal protein kinases and protein phosphatases: insights from genomics and biochemistry Biochem J 2003 370 373 389 12444920 10.1042/BJ20021547 
Kennelly PJ  Protein phosphatases – a phylogenetic perspective Chem Rev 2001 101 2291 2312 11749374 10.1021/cr0002543 
Tsuruta H Aizono Y  Enzymatical properties of psychrophilic phosphatase I J Biochem 1999 125 690 695 10101281 
Tsuruta H Aizono Y  Cloning of phosphatase I gene from a psychrophile, Shewanella sp, and some properties of the recombinant enzyme J Biochem 2000 127 143 149 10731677 
Iwanicki A Herman-Antosiewicz A Pierechod M Seror SJ Obuchowski M  PrpE, a PPP protein phosphatase from Bacillus subtilis with unusual substrate specificity Biochem J 2002 366 929 936 12059787 
Koonin EV  Bacterial and bacteriophage protein phosphatases Mol Microbiol 1993 8 785 786 8392657 
Barton GJ Cohen PTW Barford D  Conservation analysis and structure prediction of the protein serine/threonine phosphatases. Sequence similarity with diadenosine tetraphosphatase from Escherichia coli suggests homology to the protein phosphatases Eur J Biochem 1994 220 225 237 8119291 
Kennelly PJ  Protein kinases and protein phosphatases in prokaryotes: a genomic perspective FEMS Microbiol Lett 2002 206 1 8 11786249 10.1016/S0378-1097(01)00479-7 
Missiakas D Raina S  Signal transduction pathways in response to protein misfolding in the extracytoplasmic compartments of E.coli : role of two new phosphoprotein phosphatases PrpA and PrpB EMBO J 1997 16 1670 1685 9130712 10.1093/emboj/16.7.1670 
Umeyama T Naruoka A Horinouchi S  Genetic and biochemical characterization of a protein phosphatase with dual substrate specificity in Streptomyces coelicolor A3(2) Gene 2000 258 55 62 11111042 10.1016/S0378-1119(00)00450-9 
Shi L  Manganese-dependent protein O-phosphatases in prokaryotes and their biological functions Front Biosci 2004 9 1382 1397 14977554 
Barford D  Molecular mechanisms of the protein serine/threonine phosphatases Trends Biochem Sci 1996 21 407 412 8987393 10.1016/S0968-0004(96)10060-8 
Andreeva AV Kutuzov MA  Physcomitrella patens gene/cDNA fragments related to genes encoding protein Ser/Thr phosphatases J Plant Physiol 1999 155 153 158 
Haas BJ Volfovsky N Town CD Troukhan M Alexandrov N Feldmann KA Flavell RB White O Salzberg SL  Full-length messenger RNA sequences greatly improve genome annotation Genome Biol 2002 3 research0029. 12093376 10.1186/gb-2002-3-6-research0029 
Voegtli WC White DJ Reiter NJ Rusnak F Rosenzweig AC  Structure of the bacteriophage λ Ser/Thr protein phosphatase with sulfate ion bound in two coordination modes Biochemistry 2000 39 15365 15374 11112522 10.1021/bi0021030 
Egloff MP Cohen PTW Reinemer P Barford D  Crystal structure of the catalytic subunit of human protein phosphatase 1 and its complex with tungstate J Mol Biol 1995 254 942 959 7500362 10.1006/jmbi.1995.0667 
Goldberg J Huang HB Kwon YG Greengard P Nairn AC Kuriyan J  Three-dimensional structure of the catalytic subunit of protein serine/threonine phosphatase-1 Nature 1995 376 745 753 7651533 10.1038/376745a0 
Kissinger CR Parge HE Knighton DR Lewis CT Pelletier LA Tempczyk A Kalish VJ Tucker KD Showalter RE Moomaw EW Gastinel LN Habuka N Chen X Maldonado F Barker JE Bacquet R Villafranca JE  Crystal structures of human calcineurin and the human FKBP12-FK506-calcineurin complex Nature 1995 378 641 644 8524402 10.1038/378641a0 
Coster F van Dyck L Jonniaux J-L Purnelle B Goffeau A  The sequence of a 13.5 kb DNA segment from the left arm of yeast chromosome XIV reveals MER1; RAP1; a new putative member of the DNA replication complex and a new putative serine/threonine phosphatase gene Yeast 1995 11 85 91 7762305 
Zhang J Zhang Z Brew K Lee EYC  Mutational analysis of the catalytic subunit of muscle protein phosphatase-1 Biochemistry 1996 35 6276 6282 8639569 10.1021/bi952954l 
Andreeva AV Kutuzov MA  PPP family of protein Ser/Thr phosphatases: Two distinct branches? Mol Biol Evol 2001 18 448 452 11230548 
Felsenstein J  PHYLIP (phylogeny inference package) Version 35731999 Distributed by the author, Department of Genetics, University of Washington, Seattle 
Huson DH Bryant D  Estimating phylogenetic trees and networks using SplitsTree4, in preparation 
Schmidt HA Strimmer K Vingron M von Haeseler A  TREE-PUZZLE: maximum likelihood phylogenetic analysis using quartets and parallel computing Bioinformatics 2002 18 502 504 11934758 10.1093/bioinformatics/18.3.502 
Bryant D Moulton V  Neighbor-Net: An agglomerative method for the construction of phylogenetic networks Mol Biol Evol 2004 21 255 65 14660700 10.1093/molbev/msh018 
Connor JH Kleeman T Barik S Honkanen RE Shenolikar S  Importance of the β12-β13 loop in protein phosphatase-1 catalytic subunit for inhibition by toxins and mammalian protein inhibitors J Biol Chem 1999 274 22366 22372 10428807 10.1074/jbc.274.32.22366 
Lee EY Zhang L Zhao S Wei Q Zhang J Qi ZQ Belmonte ER  Phosphorylase phosphatase: new horizons for an old enzyme Front Biosci 1999 4 D270 D285 10077543 
Watanabe T Huang HB Horiuchi A da Cruze Silva EF Hsieh-Wilson L Allen PB Shenolikar S Greengard P Nairn AC  Protein phosphatase 1 regulation by inhibitors and targeting subunits Proc Natl Acad Sci USA 2001 98 3080 3085 11248035 10.1073/pnas.051003898 
Sprent JI  Graham PH, Sadowsky MJ, Vance CP  Evolution and diversity in the legume-rhizobium symbiosis: Chaos theory? In Symbiotic Nitrogen Fixation 1994 Dordrecht: Kluwer Academic Publishers 1 10 
Fleischman D Kramer D  Photosynthetic rhizobia Biochim Biophys Acta  1998 1364 17 36 9554937 10.1016/S0005-2728(98)00011-5 
Escobar MA Dandekar AM  Agrobacterium tumefaciens as an agent of disease Trends Plant Sci 2003 8 380 386 12927971 10.1016/S1360-1385(03)00162-6 
Kunik T Tzfira T Kapulnik Y Gafni Y Dingwall C Citovsky V  Genetic transformation of HeLa cells by Agrobacterium Proc Natl Acad Sci USA 2001 98 1871 1876 11172043 10.1073/pnas.041327598 
Scholl EH Thorne JL McCarter JP Bird DM  Horizontally transferred genes in plant-parasitic nematodes: a high-throughput genomic approach Genome Biol 2003 4 R39 12801413 10.1186/gb-2003-4-6-r39 
Hannaert V Saavedra E Duffieux F Szikora JP Rigden DJ Michels PA Opperdoes FR  Plant-like traits associated with metabolism of Trypanosoma parasites Proc Natl Acad Sci USA 2003 100 1067 1071 12552132 10.1073/pnas.0335769100 
Foth BJ McFadden GI  The apicoplast: a plastid in Plasmodium falciparum and other Apicomplexan parasites Int Rev Cytol 2003 224 57 110 12722949 
Hast T Follmann H  Identification of two thylakoid-associated phosphatases with protein phosphatase activity in chloroplasts of the soybean (Glycine max) J Photochem Photobiol B -Biology 1996 36 313 319 10.1016/S1011-1344(96)07383-6 
Hammer MF Markwell J Sarath G  Purification of a protein phosphatase from chloroplast stroma capable of dephosphorylating the light-harvesting complex-II Plant Physiol 1997 113 227 233 9064687 10.1104/pp.113.1.227 
Kisselev LL Justesen J Wolfson AD Frolova LY  Diadenosine oligophosphates (Ap(n)A), a novel class of signalling molecules? FEBS Lett 1998 427 157 163 9607303 10.1016/S0014-5793(98)00420-7 
Vartanian AA Suzuki H Poletaev AI  The involvement of diadenosine 5 ',5 "'-P-1,P-4-tetraphosphate in cell cycle arrest and regulation of apoptosis Biochem Pharmacol 2003 65 227 235 12504798 10.1016/S0006-2952(02)01481-8 
Pekarsky Y Zanesi N Palamarchuk A Huebner K Croce CM  FHIT: from gene discovery to cancer treatment and prevention Lancet Oncology 2002 3 748 754 12473516 10.1016/S1470-2045(02)00931-2 
Chan CP Gallis B Blumenthal DK Pallen CJ Wang JH Krebs EG  Characterization of the phosphotyrosyl protein phosphatase activity of calmodulin-dependent protein phosphatase J Biol Chem 1986 261 9890 9895 2426255 
MacKintosh C Garton AJ McDonnell A Barford D Cohen PTW Tonks NK Cohen P  Further evidence that inhibitor-2 acts like a chaperone to fold PP1 into its native conformation FEBS Lett 1996 397 235 238 8955354 10.1016/S0014-5793(96)01175-1 
Zhuo S Clemens JC Hakes DJ Barford D Dixon JE  Expression, purification, crystallization, and biochemical characterization of a recombinant protein phosphatase J Biol Chem 1993 268 17754 17761 8394350 
Shi L Carmichael WW Kennelly PJ  Cyanobacterial PPP family protein phosphatases possess multifunctional capabilities and are resistant to microcystin-LR J Biol Chem 1999 274 10039 10046 10187782 10.1074/jbc.274.15.10039 
Shi L Kehres DG Maguire ME  The PPP-family protein phosphatases PrpA and PrpB of Salmonella enterica serovar Typhimurium possess distinct biochemical properties J Bacteriol 2001 183 7053 7057 11717262 10.1128/JB.183.24.7053-7057.2001 
Luan S  Protein phosphatases in plants Annu Rev Plant Biol 2003 54 63 92 14502985 10.1146/annurev.arplant.54.031902.134743 
Altschul SF Madden TL Schaffer AA Zhang J Zhang Z Miller W Lipman DJ  Gapped BLAST and PSI-BLAST: a new generation of protein database search programs Nucleic Acids Res 1997 25 3389 3402 9254694 10.1093/nar/25.17.3389 
NCBI Blast search 
NCBI BLAST with eukaryotic genomes 
Fungal Genomes Blast search at the Broad Institute 
The Arabidopsis Information Resource (TAIR) Blast search 
Gene Index databases of tentative consensus sequences at the Institute for Genomic Research (TIGR) 
Chlamydomonas reinhardtii draft genome BLAST search 
NCBI Taxonomy Homepage 
Higgins DG Thompson JD Gibson TJ  Using CLUSTAL for multiple sequence alignments Methods Enzymol 1996 266 383 402 8743695 
CLUSTAL W  Multiple Sequence Alignment at Kyoto University Bioinformatics Centre 
Saitou N Nei M  The neighbor-joining method: a new method for reconstructing phylogenetic trees Mol Biol Evol 1987 4 406 425 3447015 
SplitsTree  Analyzing and Visualizing Evolutionary Data 
Emanuelsson O Nielsen H Brunak S von Heijne G  Predicting subcellular localization of proteins based on their N-terminal amino acid sequence J Mol Biol 2000 300 1005 1016 10891285 10.1006/jmbi.2000.3903 
TargetP Server at the Centre for Biological Sequence Analysis, Technical University of Denmark 
Chlamydomonas reinhardtii draft genome, scaffold 45

