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Bacteria play host to a wide range of protein phosphorylation-dephosphorylation systems (Fig. 1). As little as five years ago the known systems were thought to be late-emerging and absolutely prokaryote specific. Today we know that most protein kinases and protein phosphatases are descended from a set of common, and possibly quite ancient, prototypes. Prokaryote- and eukaryote-specific protein kinases and protein phosphatases are rare and represent exceptions, not the rule as previously thought. Commonality suggests that a dynamic and versatile regulatory mechanism was first adapted to the modulation of protein function as early if not earlier than more "basic" mechanisms such as allosterism, etc. The existence of common molecular themes confirms that the microbial world offers a unique, largely untapped library and a powerful set of tools for the understanding of a regulatory mechanism which is crucial to all organisms, tools whose diversity and experimental malleability will provide new avenues for exploring and understanding key modes of cellular regulation.

Protein phosphorylation/dephosphorylation has long been considered a recent addition to Nature's regulatory arsenal. Early studies indicated that this molecular regulatory mechanism existed only in higher eukaryotes, suggesting that protein phosphorylation/dephosphorylation had emerged to meet the particular signal-transduction requirements of multicellular organisms. Although it has since become apparent that simple eukaryotes and even bacteria are sites of protein phosphorylation/dephosphorylation, the perception widely persists that this molecular regulatory mechanism emerged late in evolution, i.e. after the divergence of the contemporary phylogenetic domains. Only highly developed cells, it was reasoned, could afford the high 'overhead' costs inherent in the acquisition of dedicated protein kinases and protein phosphatases. The advent of genome sequencing has provided an opportunity to exploit Nature's phylogenetic diversity as a vehicle for critically examining this hypothesis. In tracing the origins and evolution of protein phosphorylation/dephosphorylation, the members of the Archaea, the so-called 'third domain of life', will play a critical role. Whereas several studies have demonstrated that archaeal proteins are subject to modification by covalent phosphorylation, relatively little is known concerning the identities of the proteins affected, the impact on their functional properties, or the enzymes that catalyse these events. However, examination of several archaeal genomes has revealed the widespread presence of several ostensibly 'eukaryotic' and 'bacterial' protein kinase and protein phosphatase paradigms. Similar findings of 'phylogenetic trespass' in members of the Eucarya (eukaryotes) and the Bacteria suggest that this versatile molecular regulatory mechanism emerged at an unexpectedly early point in development of 'life as we know it'.

Inspection of the genomes for the bacteria Bacillus subtilis 168, Borrelia burgdorferi B31, Escherichia coli K-12, Haemophilus influenzae KW20, Helicobacter pylori 26695, Mycoplasma genitalium G-37, and Synechocystis sp PCC 6803 and for the archaeons Archaeoglobus fulgidus VC-16 DSM4304, Methanobacterium thermoautotrophicum delta H, and Methanococcus jannaschii DSM2661 revealed that each contains at least one ORF whose predicted product displays sequence features characteristic of eukaryote-like protein-serine/threonine/tyrosine kinases and protein-serine/threonine/tyrosine phosphatases. Orthologs for all four major protein phosphatase families (PPP, PPM, conventional PTP, and low molecular weight PTP) were present in the bacteria surveyed, but not all strains contained all types. The three archaeons surveyed lacked recognizable homologs of the PPM family of eukaryotic protein-serine/threonine phosphatases; and only two prokaryotes were found to contain ORFs for potential phosphatases from all four major families. Intriguingly, our searches revealed a potential ancestral link between the catalytic subunits of microbial arsenate reductases and the protein-tyrosine phosphatases; they share similar ligands (arsenate versus phosphate) and features of their catalytic mechanism (formation of arseno-versus phospho-cysteinyl intermediates). It appears that all prokaryotic organisms, at one time, contained the genetic information necessary to construct protein phosphorylation-dephosphorylation networks that target serine, threonine, and/or tyrosine residues on proteins. However, the potential for functional redundancy among the four protein phosphatase families has led many prokaryotic organisms to discard one, two, or three of the four.

For many years, the regulation of protein structure and function by phosphorylation and dephosphorylation was considered a relatively recent invention that arose independently in each phylogenetic domain. Over time, however, incidents of apparent domain trespass involving the presence of 'eukaryotic' protein kinases or protein phosphatases in prokaryotic organisms were reported with increasing frequency. Today, genomics has provided the means to examine the phylogenetic distribution of 'eukaryotic' protein kinases and protein phosphatases in a comprehensive and systematic manner. The results of these genome searches challenge previous conceptions concerning the origins and evolution of this versatile regulatory mechanism.

The deduced protein product of open reading frame slr0946 from Synechocystis sp. strain PCC 6803, SynArsC, contains the conserved sequence features of the enzyme superfamily that includes the low-molecularweight protein-tyrosine phosphatases and the Staphylococcus aureus pI258 ArsC arsenate reductase. The recombinant protein product of slr0946, rSynArsC, exhibited vigorous arsenate reductase activity (V max ‫؍‬ 3.1 mol/min ⅐ mg), as well as weak phosphatase activity toward p-nitrophenyl phosphate (V max ‫؍‬ 0.08 mol/ min ⅐ mg) indicative of its phosphohydrolytic ancestry. pI258 ArsC from S. aureus is the prototype of one of three distinct families of detoxifying arsenate reductases. The prototypes of the others are Acr2p from Saccharomyces cerevisiae and R773 ArsC from Escherichia coli. All three have converged upon catalytic mechanisms involving an arsenocysteine intermediate. While SynArsC is homologous to pI258 ArsC, its catalytic mechanism exhibited a unique combination of features. rSynArsC employed glutathione and glutaredoxin as the source of reducing equivalents, like Acr2p and R773 ArsC, rather than thioredoxin, as does the S. aureus enzyme. As postulated for Acr2p and R773 ArsC, rSynArsC formed a covalent complex with glutathione in an arsenate-dependent manner. rSynArsC contains three essential cysteine residues like pI258 ArsC, whereas the yeast and E. coli enzymes require only one cysteine for catalysis. As in the S. aureus enzyme, these "extra" cysteines apparently shuttle a disulfide bond to the enzyme's surface to render it accessible for reduction. SynArsC and pI258 ArsC thus appear to represent alternative branches in the evolution of their shared phosphohydrolytic ancestor into an agent of arsenic detoxification.Arsenate and related compounds (e.g., arsenite, antomite) are naturally occurring, broadly acting toxins frequently encountered at biologically deleterious concentrations in the environment (reviewed in reference 44). The strong chemical parallels between phosphorous and arsenic, which reside in the same column of the periodic table, limit the ability of phosphate transport systems to discriminate between this vital nutrient and arsenate (45), thus exacerbating the latter's toxic potential. Microorganisms combat the collateral importation of arsenate by a two-step mechanism in which this compound is first reduced to arsenite. Although arsenite is a more potent toxicant than arsenate, the former can be selectively banished from the cell's interior through the intervention of a dedicated, inducible transporter.While the basic strategy for conferring arsenical resistance is broadly conserved among microorganisms, the arsenate reductases responsible for catalyzing the conversion of arsenate to arsenite are not. To date, three "detoxifying" arsenate reductases have been identified and characterized in molecular detail: Acr2p from the microbial eukaryote Saccharomyces cerevisiae (2), ArsC encoded by plasmid R773 from the gramnegative bacterium Escherichia coli (R773 ArsC) (7), and ArsC encod...

The structural gene for a putative PPP family proteinserine/threonine phosphatase from the microcystin-producing cyanobacterium Microcystis aeruginosa PCC 7820, pp1-cyano1, was cloned. The sequence of the predicted gene product, PP1-cyano1, was 98% identical to that of the predicted product of an open reading frame, pp1-cyano2, from a cyanobacterium that does not produce microcystins, M. aeruginosa UTEX 2063. By contrast, PP1-cyano1 displayed less than 20% identity with other PPP family protein phosphatases from eukaryotic, archaeal, or other bacterial organisms. PP1-cyano1 and PP1-cyano2 were expressed in Escherichia coli and purified to homogeneity. Both enzymes exhibited divalent metal dependent phosphohydrolase activity in vitro toward phosphoserine-and phosphotyrosine-containing proteins and 3-phosphohistidine-and phospholysine-containing amino acid homopolymers. This multifunctional potential also was apparent in samples of PP1-cyano1 and PP1-cyano2 isolated from M. aeruginosa. Catalytic activity was insensitive to okadaic acid or the cyanobacterially produced cyclic heptapeptide, microcystin-LR, both potent inhibitors of mammalian PP1 and PP2A. PP1-cyano1 and PP1-cyano2 displayed diadenosine tetraphosphatase activity in vitro. Diadenosine tetraphosphatases share conserved sequence features with PPP family protein phosphatases. The diadenosine tetraphosphatase activity of PP1-cyano1 and PP1-cyano2 confirms that these enzymes share a common catalytic mechanism.Strains of the cyanobacterium Microcystis aeruginosa sp. synthesize the cyclic heptapeptide microcystin-LR, a potent toxin toward humans and other animals (1, 2). In eukaryotes, the immediate targets of microcystin-LR are the structurally homologous catalytic subunits of the protein-serine/threonine phosphatases PP2A and PP1 1 (3), to which it binds with nanomolar or near nanomolar affinity, respectively (4). Sensitivity to these and other toxins, such as okadaic acid, is so highly conserved among eukaryotic PP1 and PP2A that it serves as a criterion for the identification of these enzymes in cell extracts (5). Moderate sensitivity to microcystin-LR extends to homologs recently identified in members of the Archaea such as Methanosarcina thermophila TM-1 (6, 7) and Pyrodictium abyssi TAG11 (8).Recently, the presence of open reading frames potentially encoding PP1/2A-like, also known as the PPP family (9), protein phosphatases has been reported in two strains of M. aeruginosa, the microcystin-producing strain M. aeruginosa PCC 7820 and the non-producing strain M. aeruginosa UTEX 2063 (10). These open reading frames have been designated pp1-cyano1 and pp1-cyano2, respectively. Given the observation that microcystins accumulate within the interior of toxin-producing cyanobacteria (11), and the near absolute conservation of sensitivity to these compounds by members of the PP1/2A superfamily (5), how do microcystin-producing cyanobacteria protect themselves from the action of endogenous toxins against their presumptive PP1/ 2A-like protein phosphatases? It ...

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