Polyphosphate (polyP), a linear polymer of hundreds of orthophosphate residues, exists in all tested cells in nature, from pathogenic bacteria to mammals. In bacteria, polyP has a crucial role in stress responses and stationary-phase survival. Polyphosphate kinase (PPK) is the principal enzyme that catalyses the synthesis of polyP in bacteria. It has been shown that PPK is required for bacterial motility, biofilm formation and the production of virulence factors. PPK inhibitors may thus provide a unique therapeutic opportunity against antibiotic-resistant pathogens. Here, we report crystal structures of full-length Escherichia coli PPK and its complex with AMPPNP (b-cimidoadenosine 5-phosphate). PPK forms an interlocked dimer, with each 80 kDa monomer containing four structural domains. The PPK active site is located in a tunnel, which contains a unique ATP-binding pocket and may accommodate the translocation of synthesized polyP. The PPK structure has laid the foundation for understanding the initiation of polyP synthesis by PPK.

Pseudomonas aeruginosa, of medical, environmental, and industrial importance, depends on inorganic polyphosphate (poly P) for a wide range of functions, especially survival. Mutants of PAO1 lacking poly P kinase 1, PPK1, the enzyme responsible for most poly P synthesis in Escherichia coli and other bacteria, are defective in motility, quorum sensing, biofilm formation, and virulence. We describe here multiple defects in the ppk1 mutant PAOM5, including a striking compaction of the nucleoid, distortion of the cell envelope, lack of planktonic motility and exopolymer production, and susceptibility to the ␤-lactam antibiotic carbenicillin as well as desiccation. We propose that P. aeruginosa with reduced poly P levels undergoes ultrastructural changes that contribute to profound deficiencies in cellular functions.carbenicillin ͉ exopolymer ͉ motility ͉ desiccation ͉ nucleoid P olyphosphate (poly P), a linear chain of phosphate residues linked by phosphoanhydride bonds, is present in all cells and was likely present throughout evolution (1). Poly P is synthesized in prokaryotic cells from ATP by poly P kinases (PPKs) for which two families, PPK1 and PPK2, have been identified. PPK1 is responsible principally for the synthesis of poly P in Escherichia coli, Salmonella enterica serovar Typhimurium, Shigella flexneri, Vibrio cholerae, Helicobacter pylori, Bacillus cereus, Myxococcus xanthus, and, as described here, P. aeruginosa. Homologous PPK1 amino acid sequences are in the databases of Ͼ40 organisms, including many bacterial pathogens (2). Knockout mutants of ppk1 in several pathogens demonstrate phenotypes including responses to stresses, motility, and virulence (3-7) as well as developmental defects in other species (8, 9). The only apparent eukaryotic homolog is in the social slime mold Dictyostelium discoideum (2), the mutant of which exhibits growth and developmental phenotypes (10).We describe here the construction and biochemical characterization of a P. aeruginosa ppk1 knockout mutant, PAOM5, for which the motility, biofilm formation, burned-mouse, and ocular virulence phenotypes were reported in refs. 3, 6, and 11 and which, unlike the WT, is susceptible to predation by D. discoideum (10). Electron microscopy studies reveal significant differences in ultrastructure between the WT and mutant, including compaction of the nucleoid, withdrawal of the cytoplasm from the inner membrane, abnormal envelope structure, as well as a failure to produce exopolymer. Video microscopy studies show that the mutant is almost immotile in liquid medium, despite having about the same number of polar flagella. We also demonstrate that the ppk1 mutant exhibits much reduced viability after exposure to a ␤-lactam antibiotic, carbenicillin, or the hyperosmolarity of desiccation. Results PPK1 Activity and poly P Levels Are Reduced but Not Abolished in the ppk1Mutant. P. aeruginosa PAO1 (WT) grown in rich (LB) medium exhibits high levels of PPK1 activity during midexponential phase growth, almost wholly associated with the membrane f...

Herpes simplex virus 1 contains three origins of replication; two copies of oriS and one of a similar sequence, oriL. Here, the combined action of multiple factors known or thought to influence the opening of oriS are examined. These include the viral originbinding protein, UL9, and single-strand binding protein ICP8, host cell topoisomerase I, and superhelicity of the DNA template. By using electron microscopy, it was observed that when ICP8 and UL9 proteins were added together to oriS-containing supertwisted DNA, a discrete preunwinding complex was formed at oriS on 40% of the molecules, which was shown by double immunolabeling electron microscopy to contain both proteins. This complex was relatively stable to extreme dilution. Addition of ATP led to the efficient unwinding of Ϸ50% of the DNA templates. Unwinding proceeded until the acquisition of a high level of positive supertwists in the remaining duplex DNA inhibited further unwinding. Addition of topoisomerase I allowed further unwinding, opening >1 kb of DNA around oriS.T he initiation of DNA replication for most genomes begins with the recognition of the origin by specific origin-binding proteins. Binding frequently is accompanied by a structural alteration in the DNA that promotes unwinding and entry of the proteins required to initiate DNA synthesis (1, 2). Some origin-binding proteins also exhibit helicase activity, which facilitates origin unwinding, and several form hexamers or double hexamers on the DNA, a structure typical of many helicases; examples include simian virus 40 T antigen (3-5) and the E1 protein of the papillomaviruses (6, 7). Herpes simplex virus 1 (HSV-1) UL9 protein provides origin recognition function but binds as a double dimer to the HSV-1 origins rather than as a hexamer (8-10).HSV-1 provides an excellent system for study of these early replication steps in a mammalian system. The HSV-1 genome encodes seven proteins required for origin-dependent DNA replication consisting of a DNA polymerase and its accessory protein, a heterotrimeric helicase-primase, a single-stranded (ss) DNAbinding protein, ICP8, and the origin-binding protein, UL9 protein (11-16). HSV-1 contains three functional origins of DNA replication. One, oriL, is present in the long unique segment of the genome, whereas the other highly homologous origin, oriS, is present twice in the repeat region flanking the short unique segment (17)(18)(19)(20). The minimal functional oriS sequence (79 bp) consists of a 45-bp inverted repeat containing a central A͞T-rich element flanked on each side by two high-affinity UL9 proteinbinding sites designated box I and box II. A third weaker UL9 protein-binding site, box III, is located adjacent to box I.ICP8 is the major ssDNA-binding protein coded by the HSV-1 genome. ICP8 stimulates the helicase activity of UL9 protein (21, 22) and binds to its C-terminal domain (23). The ICP8-UL9 protein interaction is stable in the presence of double-stranded DNA but not ssDNA likely due to the strong affinity of ICP8 for ssDNA (9,24).UL9 prot...

The herpes simplex virus type 1 (HSV-1) genome contains three origins of replication: ori L and two copies of ori S . These origins contain specific sequences, box I and box II, linked by an AT-rich segment, that are recognized by an HSV-1-encoded origin binding protein (UL9 protein) which also possesses DNA helicase activity. Despite its intrinsic helicase activity, the UL9 protein is unable to unwind ori S or the box I element of ori S , either in the presence or absence of the HSV-1-encoded single-strand DNA binding protein, ICP8. However, a complex of the UL9 protein and ICP8 can unwind box I if it contains a 3 single-stranded tail at least 18 nt in length positioned downstream of box I. These findings suggest a model for the initiation of HSV-1 DNA replication in which a complex consisting of the UL9 protein bound to box I, and ICP8 bound to single-stranded DNA generated at the A؉T rich linker, perhaps as a consequence of transcription, unwinds an HSV-1 origin of replication to provide access to the replication machinery with the consequent initiation of viral DNA replication. This mode of unwinding is distinct from that observed for other animal viruses-e.g., simian virus 40 or bovine papilloma virus-in which the initiator protein, T antigen, or E1 protein alone, unwinds elements of the origin sequence, and the single-strand DNA binding protein serves only to keep the separated strands apart.Herpes simplex virus type 1 (HSV-1) encodes a 94-kDa origin binding protein, the product of the UL9 gene (1, 2). Studies of HSV-1 DNA replication in vivo have shown the origin binding protein (UL9 protein, also known as OBP) to be essential for viral DNA replication (3, 4). In addition to the UL9 protein, six other viral gene products are required (for a review, see ref. 5). These include a DNA polymerase with its associated processivity enhancing factor, a heterotrimeric helicaseprimase or primosome, and a single-strand DNA binding protein (ICP8).The HSV-1 genome contains three highly homologous origins of replication, ori L and two copies of ori S (5). The UL9 protein that exists in solution as a homodimer (6, 7), specifically and cooperatively binds the two inverted pentanucleotide repeats in boxes I and II of ori S which are separated from each other by an AϩT-rich sequence of 18 nt (8, 9). Ori L contains a second copy of box I in place of box II (see Fig. 1 A). A similar arrangement of binding sites has been observed in the origins of several other herpes viruses including herpes simplex virus type 2 (10), varicella zoster virus (11), equine herpesvirus 1 (12), and human herpes virus type 6 (13). Amino acid sequence comparisons between the HSV-1 UL9 protein and herpes simplex virus type 2, varicella zoster virus, and human herpes virus type 6 homologs show the helicase and DNA binding domains to be highly conserved (14,15).In addition to its origin binding activity, the UL9 protein exhibits DNA-dependent ATPase and 3Ј-5Ј helicase activities (6,7,16,17). Although the existence of these activities suggests t...

The herpes simplex type 1 (HSV-1) origin binding protein, the UL9 protein, exists in solution as a homodimer of 94-kDa monomers. It binds to Box I, the high affinity element of the HSV-1 origin, Ori s , as a dimer. The UL9 protein also binds the HSV-1 single strand DNA-binding protein, ICP8. Photocross-linking studies have shown that although the UL9 protein binds Box I as a dimer, only one of the two monomers contacts Box I. It is this form of the UL9 homodimer that upon interaction with ICP8, promotes the unwinding of Box I coupled to the hydrolysis of ATP to ADP and P i . Photocross-linking studies have also shown that the amount of UL9 protein that interacts with Box I is reduced by its interaction with ICP8.Antibody directed against the C-terminal ten amino acids of the UL9 protein inhibits its Box I unwinding activity, consistent with the requirement for interaction of the C terminus of the UL9 protein with ICP8. Inhibition by the antibody is enhanced when the UL9 protein is first bound to Box I, suggesting that the C terminus of the UL9 protein undergoes a conformational change upon binding Box I.

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The herpes simplex virus 1 (HSV-1) genome encodes seven polypeptides that are required for its replication. These include a heterodimeric DNA polymerase, a singlestrand-DNA-binding protein, a heterotrimeric helicase/primase, and a protein (UL9 protein) that binds specifically to an HSV-1 origin of replication (oris). We demonstrate here that UL9 protein interacts specifically with the 180-kDa catalytic subunit of the cellular DNA polymerase a-primase. This interaction can be detected by immunoprecipitation with antibodies directed against either of these proteins, by gel mobility shift of an oris-UL9 protein complex, and by stimulation of DNA polymerase activity by the UL9 protein. These findings suggest that enzymes required for cellular DNA replication also participate in HSV-1 DNA replication.In vivo studies of the replication of the linear 152-kb herpes simplex virus 1 (HSV-1) genome have demonstrated that it circularizes shortly after infection and then enters a rolling circle mode of DNA replication (1). However, the existence of three origins of replication, OriL and the diploid oris, as well as an HSV-1-encoded protein (UL9 protein) that binds specifically to these origins (for review, see ref.2), suggests that origin-dependent, theta-type DNA replication, analogous to that observed for the simian virus 40 (SV40) minichromosome, may also occur. A recent analysis in vivo of the replication of plasmids containing an HSV-1 origin (oris) in HSV-1-infected cells by two-dimensional gel analysis (3) has identified "bubble arcs" indicative of theta-type DNA replication (K. Kelly and I.R.L., unpublished data).The HSV-1 genome encodes seven polypeptides that are essential for its replication (2, 4). These include, in addition to the UL9 protein, a heterodimeric, highly processive DNA polymerase, a single-strand-DNA-binding protein (ICP8), and a heterotrimeric helicase/primase. We have recently found that a complex consisting of the HSV-1 DNA polymerase, helicase/primase, and ICP8, isolated from Sf21 insect cells multiply infected with baculoviruses recombinant for the genes encoding these enzymes, can promote the rolling circle replication of a 3-kb plasmid that is independent of oris and the UL9 protein (5). This reaction may reflect the rolling circle phase of viral DNA replication.The UL9 protein, which exists in solution as a homodimer of 83-kDa subunits (6, 7), binds to specific sequences within onts (sites I, II, and III) (8-11). The two high-affinity sites (sites I and II) are separated by an 18-nt A+T-rich spacer. The UL9 protein also has DNA-dependent ATPase and DNA helicase activities (6,7,12,13 Studies of SV40 DNA replication in vitro have shown that the SV40 T antigen interacts specifically with the cellular DNA polymerase a-primase (Pol a) to form a complex that initiates DNA replication at the SV40 origin (16-19). We have therefore inquired whether the HSV-1-encoded UL9 protein forms an analogous complex with Pol a. We demonstrate here that the UL9 protein interacts specifically with the cata...