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The discovery of an extraordinarily high level of mobile elements in the genome of Wolbachia, a widespread arthropod and nematode endosymbiont, suggests that this bacterium could be an excellent model for assessing the evolution and function of mobile DNA in specialized bacteria. In this paper, we discuss how studies on the temperate bacteriophage WO of Wolbachia have revealed unexpected levels of genomic flux and are challenging previously held views about the clonality of obligate intracellular bacteria. We also discuss the roles this phage might play in the Wolbachia-arthropod symbiosis and infer how this research can be translated to combating human diseases vectored by arthropods. We expect that this temperate phage will be a preeminent model system to understand phage genetics, evolution and ecology in obligate intracellular bacteria. In this sense, phage WO might be likened to phage lambda of the endosymbiont world.
Copyright (c) 2010 Elsevier Ltd. All rights reserved.
Bacteriophage lambda protein phosphatase (lambdaPP) is a member of a large family of metal-containing phosphoesterases, including purple acid phosphatase, protein serine/threonine phosphatases, 5'-nucleotidase, and DNA repair enzymes such as Mre11. lambdaPP can be activated several-fold by various divalent metal ions, with Mn(2+) and Ni(2+) providing the most significant activation. Despite the extensive characterization of purified lambdaPP in vitro, little is known about the identity and stoichiometry of metal ions used by lambdaPP in vivo. In this report, we describe the use of metal analysis, activity measurements, and whole cell EPR spectroscopy to investigate in vivo metal binding and activation of lambdaPP. Escherichia coli cells overexpressing lambdaPP show a 22.5-fold increase in intracellular Mn concentration and less dramatic changes in the intracellular concentration of other biologically relevant metal ions compared to control cells that do not express lambdaPP. Phosphatase activity assessed using para-nitrophenylphosphate as substrate is increased 850-fold in cells overexpressing lambdaPP, indicating the presence of metal-activated enzyme in cell lysate. EPR spectra of intact cells overexpressing lambdaPP exhibit resonances previously attributed to mononuclear Mn(2+) and dinuclear [(Mn(2+))(2)] species bound to lambdaPP. Spin quantitation of EPR spectra of intact E. coli cells overexpressing lambdaPP indicates the presence of approximately 40 microM mononuclear Mn(2+)-lambdaPP and 60 microM [(Mn(2+))(2)]-lambdaPP. The data suggest that overexpression of lambdaPP results in a mixture of apo-, mononuclear-Mn(2+), and dinuclear-[(Mn(2+))(2)] metalloisoforms and that Mn(2+) is a physiologically relevant activating metal ion in E. coli.
Bacteriophage lambda protein phosphatase (lambdaPP) with Mn(2+) as the activating metal cofactor was studied using phosphatase inhibition kinetics and electron paramagnetic resonance (EPR) spectroscopy. Orthophosphate and the oxoanion analogues orthovanadate, tungstate, molybdate, arsenate, and sulfate were shown to inhibit the phosphomonoesterase activity of lambdaPP, albeit with inhibition constants (K(i)) that range over 5 orders of magnitude. In addition, small organic anions were tested as inhibitors. Phosphonoacetohydroxamic acid (PhAH) was found to be a strong competitive inhibitor (K(i) = 5.1 +/- 1.6 microM) whereas phosphonoacetic acid (K(i) = 380 +/- 45 microM) and acetohydroxamic acid (K(i) > 75 mM) modestly inhibited lambdaPP. Low-temperature EPR spectra of Mn(2+)-reconstituted lambdaPP in the presence of oxoanions and PhAH demonstrate that inhibitor binding decreases the spin-coupling constant, J, compared to the native enzyme. This suggests a change in the bridging interaction between Mn(2+) ions of the dimer due to protonation or replacement of a bridging ligand. Inhibitor binding also induces several spectral shifts. Hyperfine splitting characteristic of a spin-coupled (Mn(2+))(2) dimer is most prominent upon the addition of orthovanadate (K(i) = 0.70 +/- 0.20 microM) and PhAH, indicating that these inhibitors tightly interact with the (Mn(2+))(2) form of lambdaPP. These EPR and inhibition kinetic results are discussed in the context of establishing a common mechanism for the hydrolysis of phosphate esters by lambdaPP and other serine/threonine protein phosphatases.
Bacteriophage lambda phosphoprotein phosphatase (lambdaPP) has structural similarity to the mammalian Ser/Thr phosphoprotein phosphatases (PPPs) including the immunosuppressant drug target calcineurin. PPPs possess a conserved active site containing a dinuclear metal cluster, with metal ligands provided by a phosphoesterase motif plus two additional histidine residues at the C-terminus. Multiple sequence alignment of lambdaPP with 28 eubacterial and archeal phosphoesterases identified active site residues from the phosphoesterase motif and in many cases 2 additional C-terminal His metal ligands. Most highly similar to lambdaPP are E. coli PrpA and PrpB. Using the crystal structure of lambdaPP [Voegtli, W. C., et al. (2000) Biochemistry 39, 15365-15374] as a structural and active site model for PPPs and related bacterial phosphoesterases, we have studied mutant forms of lambdaPP reconstituted with Mn(2+) by electron paramagnetic resonance (EPR) spectroscopy, Mn(2+) binding analysis, and phosphatase kinetics. Analysis of Mn(2+)-bound active site mutant lambdaPP proteins shows that H22N, N75H, and H186N mutations decrease phosphatase activity but still allow mononuclear Mn(2+) and [(Mn(2+))(2)] binding. The high affinity Mn(2+) binding site is shown to consist of M2 site ligands H186 and Asn75, but not H22 from the M1 site which is ascribed as the lower affinity site.
The protein phosphatase encoded by bacteriophage lambda (lambda PP) belongs to a family of Ser/Thr phosphatases (Ser/Thr PPases) that includes the eukaryotic protein phosphatases 1 (PP1), 2A (PP2A), and 2B (calcineurin). These Ser/Thr PPases and the related purple acid phosphatases (PAPs) contain a conserved phosphoesterase sequence motif that binds a dinuclear metal center. The mechanisms of phosphoester hydrolysis by these enzymes are beginning to be unraveled. To utilize lambda PP more effectively as a model for probing the catalytic mechanism of the Ser/Thr PPases, we have determined its crystal structure to 2.15 A resolution. The overall fold resembles that of PP1 and calcineurin, including a conserved beta alpha beta alpha beta structure that comprises the phosphoesterase motif. Substrates and inhibitors probably bind in a narrow surface groove that houses the active site dinuclear Mn(II) center. The arrangement of metal ligands is similar to that in PP1, calcineurin, and PAP, and a bound sulfate ion is present in two novel coordination modes. In two of the three molecules in the crystallographic asymmetric unit, sulfate is coordinated to Mn2 in a monodentate, terminal fashion, and the two Mn(II) ions are bridged by a solvent molecule. Two additional solvent molecules are coordinated to Mn1. In the third molecule, the sulfate ion is triply coordinated to the metal center with one oxygen coordinated to both Mn(II) ions, one oxygen coordinated to Mn1, and one oxygen coordinated to Mn2. The sulfate in this coordination mode displaces the bridging ligand and one of the terminal solvent ligands. In both sulfate coordination modes, the sulfate ion is stabilized by hydrogen bonding interactions with conserved arginine residues, Arg 53 and Arg 162. The two different active site structures provide models for intermediates in phosphoester hydrolysis and suggest specific mechanistic roles for conserved residues.
Monoclonal antibody 51F recognizes determinants in the helix 4 region of the native form of the N-terminal domain of lambda repressor. A cassette mutagenesis method was used to introduce changes within this region, and antibody-reactive candidates were isolated and sequenced. The resulting data allow the identification of repressor side chains that are critical determinants of antibody binding. Four of these side chains are on the surface of the N-terminal domain and probably contact the antibody directly. These contact positions were then mutagenized individually, and the antibody binding phenotypes of a large number of singly mutant repressors were determined. Taken together, the mutational data allow a functional map of the recognition surface to be constructed and the physical nature of some of the specific interactions that stabilize the antibody-antigen complex to be surmised.
Monoclonal antibodies reactive with distinct regions of the N-terminal domain of the lambda repressor protein have been isolated. By comparing the affinities of these antibodies for mutant repressors with increased and decreased thermal stabilities, each of the antibodies can be shown to bind to epitopes accessible in the native conformation of the N-terminal domain. Experiments probing antibody binding to protein fragments, mutant variants, and peptides have also been used to define likely regions of contact between the antibodies and the N-terminal domain.