Prokaryotic DNA segregation by an actin-like filament

The mechanisms responsible for prokaryotic DNA segregation are largely unknown. The partitioning locus (par) encoded by the Escherichia coli plasmid R1 actively segregates its replicon to daughter cells. We show here that the ParM ATPase encoded by par forms dynamic actin-like filaments with properties expected for a force-generating protein. Filament formation depended on the other components encoded by par, ParR and the centromere-like parC region to which ParR binds. Mutants defective in ParM ATPase exhibited hyperfilamentation and did not support plasmid partitioning. ParM polymerization was ATP dependent, and depolymerization of ParM filaments required nucleotide hydrolysis. Our in vivo and in vitro results indicate that ParM polymerization generates the force required for directional movement of plasmids to opposite cell poles and that the ParR-parC complex functions as a nucleation point for ParM polymerization. Hence, we provide evidence for a simple prokaryotic analogue of the eukaryotic mitotic spindle apparatus.     

Direct nucleation and bundling of actin by the SipC protein of invasive Salmonella.

Salmonella causes severe gastroenteritis in humans, entering non-phagocytic cells to initiate intracellular replication. Bacterial engulfment occurs by macropinocytosis, which is dependent upon nucleation of host cell actin polymerization and condensation ('bundling') of actin filaments into cables. This is stimulated by contact-induced delivery of an array of bacterial effector proteins, including the four Sips (Salmonella invasion proteins). Here we show in vitro that SipC bundles actin filaments independently of host cell components, a previously unknown pathogen activity. Bundling is directed by the SipC N-terminal domain, while additionally the C-terminal domain nucleates actin polymerization, an activity so far known only in eukaryotic proteins. The ability of SipC to cause actin condensation and cytoskeletal rearrangements was confirmed in vivo by microinjection into cultured cells, although as SipC associates with lipid bilayers it is possible that these activities are normally directed from the host cell membrane. The data suggest a novel mechanism by which a pathogen directly modulates the cytoskeletal architecture of mammalian target cells.     

High-density functional display of proteins on bacteriophage lambda

We designed a bacteriophage lambda system to display peptides and proteins fused at the C terminus of the head protein gpD of phage lambda. DNA encoding the foreign peptide/protein was first inserted at the 3' end of a DNA segment encoding gpD under the control of the lac promoter in a plasmid vector (donor plasmid), which also carried lox P(wt) and lox P(511) recombination sequences. Cre-expressing cells were transformed with this plasmid and subsequently infected with a recipient lambda phage that carried a stuffer DNA segment flanked by lox P(wt) and lox P(511) sites. Recombination occurred in vivo at the lox sites and Amp(r) cointegrates were formed. The cointegrates produced recombinant phage that displayed foreign protein fused at the C terminus of gpD. The system was optimised by cloning DNA encoding different length fragments of HIV-1 p24 (amino acid residues 1-72, 1-156 and 1-231) and the display was compared with that obtained with M13 phage. The display on lambda phage was at least 100-fold higher than on M13 phage for all the fragments with no degradation of displayed products. The high-density display on lambda phage was superior to that on M13 phage and resulted in selective enrichment of epitope-bearing clones from gene-fragment libraries. Single-chain antibodies were displayed in functional form on phage lambda, strongly suggesting that correct disulphide bond formation takes place during display.This lambda phage display system, which avoids direct cloning into lambda DNA and in vitro packaging, achieved cloning efficiencies comparable to those obtained with any plasmid system. The high-density display of foreign proteins on bacteriophage lambda should be extremely useful in studying low-affinity protein-protein interactions more efficiently compared to the M13 phage-based system.     

Implications of treadmilling for the stability and polarity of actin and tubulin polymers in vivo

In this report, we examine how the cell can selectively stabilize anchored filaments and suppress spontaneous filament assembly. Because microtubules and actin filaments have an organized distribution in cells, the cell must have a mechanism for suppressing spontaneous and random polymerization. Though the mechanism for suppressing spontaneous polymerization is unknown, an unusual property of these filaments has been demonstrated recently, i.e., under steady-stae conditions, in vitro actin filaments and microtubules can exhibit a flux of subunits through the polymers called "treadmilling." In vivo, however, most, if not all, of these polymers are attached at one end to specific structures and treadmilling should not occur. The function of treadmilling in vivo is, therefore, unclear at present. However, as shown here, the same physicochemical property of coupling assembly to ATP or GTP hydrolysis that leads to treadmilling in vitro can act to selectively stabilize anchored polymers in vivo. I show here that the theory of treadmilling implies that the concentration of subunits necessary for assembly of the nonanchored polymer will in general be higher than the concentration necessary for the assembly of polymers anchored with a specific polarity. This disparity in the monomer concentrations required for assembly can lead to a selective stabilization of anchored polymers and complete suppression of spontaneous polymerization at apparent equilibrium in vivo. It is possible, therefore, that the phenomenon of treadmilling is an in vitro manifestation of a mechanism designed to use ATP or GTP hydrolysis to control the spatial organization of filaments in the cell.     

Mgm101 is a Rad52-related protein required for mitochondrial DNA recombination

Homologous recombination is a conserved molecular process that has primarily evolved for the repair of double-stranded DNA breaks and stalled replication forks. However, the recombination machinery in mitochondria is poorly understood. Here, we show that the yeast mitochondrial nucleoid protein, Mgm101, is related to the Rad52-type recombination proteins that are widespread in organisms from bacteriophage to humans. Mgm101 is required for repeat-mediated recombination and suppression of mtDNA fragmentation in vivo. It preferentially binds to single-stranded DNA and catalyzes the annealing of ssDNA precomplexed with the mitochondrial ssDNA-binding protein, Rim1. Transmission electron microscopy showed that Mgm101 forms large oligomeric rings of ～14-fold symmetry and highly compressed helical filaments. Specific mutations affecting ring formation reduce protein stability in vitro. The data suggest that the ring structure may provide a scaffold for stabilization of Mgm101 by preventing the aggregation of the otherwise unstable monomeric conformation. Upon binding to ssDNA, Mgm101 is remobilized from the rings to form distinct nucleoprotein filaments. These studies reveal a recombination protein of likely bacteriophage origin in mitochondria and support the notion that recombination is indispensable for mtDNA integrity.     

Maturation of bacteriophage T4 lagging strand fragments depends on interaction of T4 RNase H with T4 32 protein rather than the T4 gene 45 clamp

In the bacteriophage T4 DNA replication system, T4 RNase H removes the RNA primers and some adjacent DNA before the lagging strand fragments are ligated. This 5'-nuclease has strong structural and functional similarity to the FEN1 nuclease family. We have shown previously that T4 32 protein binds DNA behind the nuclease and increases its processivity. Here we show that T4 RNase H with a C-terminal deletion (residues 278-305) retains its exonuclease activity but is no longer affected by 32 protein. T4 gene 45 replication clamp stimulates T4 RNase H on nicked or gapped substrates, where it can be loaded behind the nuclease, but does not increase its processivity. An N-terminal deletion (residues 2-10) of a conserved clamp interaction motif eliminates stimulation by the clamp. In the crystal structure of T4 RNase H, the binding sites for the clamp at the N terminus and for 32 protein at the C terminus are located close together, away from the catalytic site of the enzyme. By using mutant T4 RNase H with deletions in the binding site for either the clamp or 32 protein, we show that it is the interaction of T4 RNase H with 32 protein, rather than the clamp, that most affects the maturation of lagging strand fragments in the T4 replication system in vitro and T4 phage production in vivo.     

C-terminal hydrophobic interactions play a critical role in oligomeric assembly of the P22 tailspike trimer

The tailspike protein from the bacteriophage P22 is a well characterized model system for folding and assembly of multimeric proteins. Folding intermediates from both the in vivo and in vitro pathways have been identified, and both the initial folding steps and the protrimer-to-trimer transition have been well studied. In contrast, there has been little experimental evidence to describe the assembly of the protrimer. Previous results indicated that the C terminus plays a critical role in the overall stability of the P22 tailspike protein. Here, we present evidence that the C terminus is also the critical assembly point for trimer assembly. Three truncations of the full-length tailspike protein, TSPΔN, TSPΔC, and TSPΔNC, were generated and tested for their ability to form mixed trimer species. TSPΔN forms mixed trimers with full-length P22 tailspike, but TSPΔC and TSPΔNC are incapable of forming similar mixed trimer species. In addition, mutations in the hydrophobic core of the C terminus were unable to form trimer in vivo. Finally, the hydrophobic-binding dye ANS inhibits the formation of trimer by inhibiting progression through the folding pathway. Taken together, these results suggest that hydrophobic interactions between C-terminal regions of P22 tailspike monomers play a critical role in the assembly of the P22 tailspike trimer.     

Desmin filaments are stably associated with the outer nuclear surface in chick myoblasts

Eukaryotic cells have highly organized, interconnected intracellular compartments. The nuclear surface and cytoplasmic cytoskeletal filaments represent compartments involved in such an association. Intermediate filaments are the major cytoskeletal elements in this association. Desmin is a muscle-specific structural protein and one of the earliest known muscle-specific genes to be expressed during cardiac and skeletal muscle development. Desmin filaments have been shown to be associated with the nuclear surface in the myogenic cell line C2C12. Previous studies have revealed that mice lacking desmin develop imperfect muscle, exhibiting the loss of nuclear shape and positioning. In the present work, we have analyzed the association between desmin filaments and the outer nuclear surface in nuclei isolated from pectoral skeletal muscle of chick embryos and in primary chick myogenic cell cultures by using immunofluorescence microscopy, negative staining, immunogold, and transmission electron microscopy. We show that desmin filaments remain firmly attached to the outer nuclear surface after the isolation of nuclei. Furthermore, positive localization of desmin persists after gentle washing of the nuclei with high ionic strength solutions. These data suggest that desmin intermediate filaments are stably and firmly connected to the outer nuclear surface in skeletal muscles cells in vivo and in vitro.     

Dynamics of the T4 bacteriophage DNA packasome motor: endonuclease VII resolvase release of arrested Y-DNA substrates

Conserved bacteriophage ATP-based DNA translocation motors consist of a multimeric packaging terminase docked onto a unique procapsid vertex containing a portal ring. DNA is translocated into the empty procapsid through the portal ring channel to high density. In vivo the T4 phage packaging motor deals with Y- or X-structures in the replicative concatemer substrate by employing a portal-bound Holliday junction resolvase that trims and releases these DNA roadblocks to packaging. Here using dye-labeled packaging anchored 3.7-kb Y-DNAs or linear DNAs, we demonstrate FRET between the dye-labeled substrates and GFP portal-containing procapsids and between GFP portal and single dye-labeled terminases. We show using FRET-fluorescence correlation spectroscopy that purified T4 gp49 endonuclease VII resolvase can release DNA compression in vitro in prohead portal packaging motor anchored and arrested Y-DNA substrates. In addition, using active terminases labeled at the N- and C-terminal ends with a single dye molecule, we show by FRET distance of the N-terminal GFP-labeled portal protein containing prohead at 6.9 nm from the N terminus and at 5.7 nm from the C terminus of the terminase. Packaging with a C-terminal fluorescent terminase on a GFP portal prohead, FRET shows a reduction in distance to the GFP portal of 0.6 nm in the arrested Y-DNA as compared with linear DNA; the reduction is reversed by resolvase treatment. Conformational changes in both the motor proteins and the DNA substrate itself that are associated with the power stroke of the motor are consistent with a proposed linear motor employing a terminal-to-portal DNA grip-and-release mechanism.     

The helicase domain of phage P4 alpha protein overlaps the specific DNA binding domain.

Replication initiation depends on origin recognition, helicase, and primase activities. In phage P4, a second DNA region, the cis replication region (crr), is also required for replication initiation. The multifunctional alpha protein of phage P4, which is essential for DNA replication, combines the three aforementioned activities on a single polypeptide chain. Protein domains responsible for the activities were identified by mutagenesis. We show that mutations of residues G506 and K507 are defective in vivo in phage propagation and in unwinding of a forked helicase substrate. This finding indicates that the proposed P loop is essential for helicase activity. Truncations of gene product alpha (gp alpha) demonstrated that 142 residues of the C terminus are sufficient for specifically binding ori and crr DNA. The minimal binding domain retains gp alpha's ability to induce loop formation between ori and crr. In vitro and in vivo analysis of short C-terminal truncations indicate that the C terminus is needed for helicase activity as well as for specific DNA binding.     

Type VI secretion and bacteriophage tail tubes share a common assembly pathway

The Type VI secretion system (T6SS) is a widespread macromolecular structure that delivers protein effectors to both eukaryotic and prokaryotic recipient cells. The current model describes the T6SS as an inverted phage tail composed of a sheath‐like structure wrapped around a tube assembled by stacked Hcp hexamers. Although recent progress has been made to understand T6SS sheath assembly and dynamics, there is no evidence that Hcp forms tubes in vivo. Here we show that Hcp interacts with TssB, a component of the T6SS sheath. Using a cysteine substitution approach, we demonstrate that Hcp hexamers assemble tubes in an ordered manner with a head‐to‐tail stacking that are used as a scaffold for polymerization of the TssB/C sheath‐like structure. Finally, we show that VgrG but not TssB/C controls the proper assembly of the Hcp tubular structure. These results highlight the conservation in the assembly mechanisms between the T6SS and the bacteriophage tail tube/sheath.     

Actin dynamics is controlled by a casein kinase II and phosphatase 2C interplay on Toxoplasma gondii Toxofilin

Actin polymerization in Apicomplexa protozoa is central to parasite motility and host cell invasion. Toxofilin has been characterized as a protein that sequesters actin monomers and caps actin filaments in Toxoplasma gondii. Herein, we show that Toxofilin properties in vivo as in vitro depend on its phosphorylation. We identify a novel parasitic type 2C phosphatase that binds the Toxofilin/G-actin complex and a casein kinase II-like activity in the cytosol, both of which modulate the phosphorylation status of Toxofilin serine53. The interplay of these two molecules controls Toxofilin binding of G-actin as well as actin dynamics in vivo. Such functional interactions should play a major role in actin sequestration, a central feature of actin dynamics in Apicomplexa that underlies the spectacular speed and nature of parasite gliding motility.     

Higher-order structure of long repeat chromatin.

The higher-order structure of chromatin isolated from sea urchin sperm, which has a long nucleosomal DNA repeat length (approximately 240 bp), has been studied by electron microscopy and X-ray diffraction. Electron micrographs show that this chromatin forms 300 A filaments which are indistinguishable from those of chicken erythrocytes (approximately 212 bp repeat); X-ray diffraction patterns from partially oriented samples show that the edge-to-edge packing of nucleosomes in the direction of the 300 A filament axis, and the radial disposition of nucleosomes around it, are both similar to those of the chicken erythrocyte 300 A filament, which is described by the solenoid model. The invariance of the structure with increased linker DNA length is inconsistent with many other models proposed for the 300 A filament and, furthermore, means that the linker DNA must be bent. The low-angle X-ray scattering in the 300-400 A region both in vitro and in vivo differs from that of chicken erythrocyte chromatin. The nature of the difference suggests that 300 A filaments in sea urchin sperm in vivo are packed so tightly together that electron-density contrast between individual filaments is lost; this is consistent with electron micrographs of the chromatin in vitro.     

Bovine whey proteins inhibit the interaction of Staphylococcus aureus and bacteriophage K

AIMS: To understand the potential use of bacteriophage K to treat bovine Staphylococcus aureus mastitis, we studied the role of whey proteins in the inhibition of the phage-pathogen interaction in vitro. METHODS AND RESULTS: The interaction of bacteriophage K and S. aureus strain Newbould 305 was studied in raw bovine whey and serum. Incubation of S. aureus with phage in whey showed that the bacteria are more resistant to phage lysis when grown in whey and also bovine serum. Whey collected from 23 animals showed a wide variation in the level of phage-binding inhibition. The role of the protein component of milk whey in this inhibition was established; treatment of the whey by heat, proteases and ultrafiltration removed the inhibitory activity. Brief exposure of S. aureus cells to whey, followed by resuspension in broth, also reduced phage binding. Microscopy showed the adhesion of extracellular material to the S. aureus cell surface following exposure to whey. Chromatographic fractionation of the whey demonstrated that the inhibitory proteins were present in the high molecular weight fraction. CONCLUSIONS: The adsorption of whey proteins to the S. aureus cell surface appeared to inhibit phage attachment and thereby hindered lysis. The inhibitory whey proteins are of high molecular weight in their native form and may sterically block phage attachment sites on the cell surface. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings have implications for any future use of phage therapy in the treatment of mastitis, and other diseases, caused by S. aureus. This pathogen is predicted to be much more resistant to phage treatment in vivo than would be expected from in vitro broth culture experiments.     

A minimized M13 coat protein defines the requirements for assembly into the bacteriophage particle

The M13 filamentous bacteriophage coat is a symmetric array of several thousand alpha-helical major coat proteins (P8) that surround the DNA core. P8 molecules initially reside in the host membrane and subsequently transition into their role as coat proteins during the phage assembly process. A comprehensive mutational analysis of the 50-residue P8 sequence revealed that only a small subset of the side-chains were necessary for efficient incorporation into a wild-type (wt) coat. In the three-dimensional structure of P8, these side-chains cluster into three functional epitopes: a hydrophobic epitope located near the N terminus and two epitopes (one hydrophobic and the other basic) located near the C terminus on opposite faces of the helix. The results support a model for assembly in which the incorporation of P8 is mediated by intermolecular interactions involving these functional epitopes. In this model, the N-terminal hydrophobic epitope docks with P8 molecules already assembled into the phage particle in the periplasm, and the basic epitope interacts with the acidic DNA backbone in the cytoplasm. These interactions could facilitate the transition of P8 from the membrane into the assembling phage, and the incorporation of a single P8 would be completed by the docking of additional P8 molecules with the second hydrophobic epitope at the C terminus. We constructed a minimized P8 that contained only nine non-Ala side-chains yet retained all three functional epitopes. The minimized P8 assembled into the wt coat almost as efficiently as wt P8, thus defining the minimum requirements for protein incorporation into the filamentous phage coat. The results suggest possible mechanisms of natural viral evolution and establish guidelines for the artificial evolution of improved coat proteins for phage display technology.     

Segregation and activation of myosin IIB creates a rear in migrating cells

We have found that MLC-dependent activation of myosin IIB in migrating cells is required to form an extended rear, which coincides with increased directional migration. Activated myosin IIB localizes prominently at the cell rear and produces large, stable actin filament bundles and adhesions, which locally inhibit protrusion and define the morphology of the tail. Myosin IIA forms de novo filaments away from the myosin IIB–enriched center and back to form regions that support protrusion. The positioning and dynamics of myosin IIA and IIB depend on the self-assembly regions in their coiled-coil C terminus. COS7 and B16 melanoma cells lack myosin IIA and IIB, respectively; and show isoform-specific front-back polarity in migrating cells. These studies demonstrate the role of MLC activation and myosin isoforms in creating a cell rear, the segregation of isoforms during filament assembly and their differential effects on adhesion and protrusion, and a key role for the noncontractile region of the isoforms in determining their localization and function.     

Mechanism of coliphage M13 contraction: intermediate structures trapped at low temperatures.

The filamentous coliphage M13 can be transformed into a spherical particle (termed spheroid) by exposure to an interface of water and slightly polar but hydrophobic solvent such as chloroform-water at 24 degrees C. We report here that exposure of M13 filaments to a chloroform-water interface at 2 degrees C trapped the phage particles in forms morphologically intermediate to filaments and spheroids. These structures were rods 250 nm long and 15 nm wide, and each had a closed, slightly pointed end, an open flaired end, and a hollow central channel. The final contraction of these intermediates (termed I-forms) into spheroids was dependent upon both temperature and the presence of the solvent-water interface but was apparently independent of both the minor phage coat proteins and the virion DNA. Although stable in an aqueous environment, I-forms, in contrast to filaments, were readily disrupted by detergents, suggesting that the phage structure had been altered to a form more easily solubilized by membrane lipids. These solvent-induced changes might be related to the initial steps of phage penetration in vivo.     

Tau polymerization: role of the amino terminus

The abnormal polymerization of the tau molecule into insoluble filaments is a seminal event in the neurodegenerative process underlying Alzheimer's disease. Previous experimentation has shown that the microtubule-binding repeat region of the molecule is vital for its ability to polymerize in vitro into filaments similar to those found in Alzheimer's disease. However, it is becoming clear that regions outside the microtubule-binding repeat, such as exons 2 and 3 and the carboxy-terminal tail, can greatly influence its polymerization. Since it has been previously postulated that the amino terminus of tau could be involved in generating pathological conformations in the disease state, its role in the polymerization process was investigated. This report demonstrates that the removal of the amino terminus greatly inhibits the polymerization of the tau molecule, reducing both the rate and extent of polymerization. These results support the hypothesis that the ability of tau to form specific conformations involving the amino terminus is an early event in the formation of tau polymers in the disease state. Furthermore, the mutation of arginine 5 to leucine ((R)5(L)), mimicking an amino-terminal tau mutation found in a single case of FTDP-17, enhances the polymerization of the tau molecule. Therefore, the amino terminus of the tau molecule, while largely overlooked in studies of its polymerization, is a significant contributor to the polymerization process.