The Crystal Structure of Bacteriophage HK97 gp6: Defining a Large Family of Head-Tail Connector Proteins

The final step in the morphogenesis of long-tailed double-stranded DNA bacteriophages is the joining of the DNA-filled head to the tail. The connector is a specialized structure of the head that serves as the interface for tail attachment and the point of egress for DNA from the head during infection. Here, we report the determination of a 2.1 Å crystal structure of gp6 of bacteriophage HK97. Through structural comparisons, functional studies, and bioinformatic analysis, gp6 has been determined to be a component of the connector of phage HK97 that is evolutionarily related to gp15, a well-characterized connector component of bacteriophage SPP1. Whereas the structure of gp15 was solved in a monomeric form, gp6 crystallized as an oligomeric ring with the dimensions expected for a connector protein. Although this ring is composed of 13 subunits, which does not match the symmetry of the connector within the phage, sequence conservation and modeling of this structure into the cryo-electron microscopy density of the SPP1 connector indicate that this oligomeric structure represents the arrangement of gp6 subunits within the mature phage particle. Through sequence searches and genomic position analysis, we determined that gp6 is a member of a large family of connector proteins that are present in long-tailed phages. We have also identified gp7 of HK97 as a homologue of gp16 of phage SPP1, which is the second component of the connector of this phage. These proteins are members of another large protein family involved in connector assembly.     

噬菌体gp17基因2种重组产物的抗菌效应比较

通过重组技术获得大肠埃希菌噬菌体内溶素纯化蛋白和表面展示噬菌体,并观察产物的生物效应。将肠侵袭性大肠埃希菌EIEC 8401噬菌体LSB-1内溶素基因gp17构建到质粒pET300中,并在大肠埃希菌BL21中诱导表达,通过Ni柱纯化系统纯化产物;利用噬菌体展示技术构建T7-LSB-gp17重组噬菌体,通过双层琼脂法纯化噬菌体,并观察2种产物的抗菌效应。2 139 bp的gp17基因通过重组技术表达出78.3 ku的可溶性蛋白,纯化后浓度为2.38 mg/mL,其对EIEC8401有良好的抑菌活性,但对其他试验菌无抗性;通过噬菌体展示技术构建的重组噬菌体T7-LSB-gp17通过SDS-PAGE电泳显示在78 ku处有表达增强,对EIEC8401无感染、裂解作用,但对EIEC8401及其他试验菌有明显溶菌作用,宿主谱增加。通过重组技术获得的噬菌体LSB-1内溶素基因gp17的产物对LSB-1噬菌体原宿主具有明显的抑制效应。其中gp17表达的纯化蛋白具有明显的宿主专一性,重组噬菌体悬液有较宽种类的抗菌作用。这可能是因为gp17蛋白与噬菌体表面复杂空间结构的相互作用产生的生物效应。     

Cloning, overexpression and purification of the terminase proteins gp16 and gp17 of bacteriophage T4. Construction of a defined in-vitro DNA packaging system using purified terminase proteins

Terminases of double-stranded DNA bacteriophages are required for packaging and generation of terminii in replicated concatemeric DNA molecules. Genetic evidence suggests that these functions in phage T4 are carried out by the products of genes 16 and 17. We cloned these T4 genes into a heat-inducible cI repressor-lambda PL promoter vector system, and overexpressed them in Escherichia coli. We developed an in-vitro DNA packaging system, which, consistent with the genetic data, shows an absolute requirement for the terminase proteins. The overexpressed terminase proteins gp16 and gp17 appear to form a specific complex and an ATP binding site is present in the gp17 molecule. We purified the terminase proteins either as individual gp16 or gp17 proteins, or as a gp16-gp17 complex. The gp16 function of the terminase complex is dispensable for packaging mature DNA, whereas gp17 is essential for packaging DNA under any condition tested. We constructed a defined in-vitro DNA packaging system with the purified terminase proteins, purified proheads and a DNA-free phage completion gene products extract. All the components of this system can be stored at -90 degrees C without loss of packaging activity. The terminase proteins, therefore, may serve as useful reagents for mechanistic studies on DNA packaging, as well as to develop T4 as a packaging-cloning vector.     

T4 Phage and Its Head Surface Proteins Do Not Stimulate Inflammatory Mediator Production

Viruses are potent activators of the signal pathways leading to increased cytokine or ROS production. The effects exerted on the immune system are usually mediated by viral proteins. Complementary to the progress in phage therapy practice, advancement of knowledge about the influence of bacteriophages on mammalian immunity is necessary. Particularly, the potential ability of phage proteins to act like other viral stimulators of the immune system may have strong practical implications for the safety and efficacy of bacteriophage therapy. Here we present studies on the effect of T4 phage and its head proteins on production of inflammatory mediators and inflammation-related factors: IL-1α, IL-1β, IL-2, IL-6, IL-10, IL-12 p40/p70, IFN-γ, TNF-α, MCP-1, MIG, RANTES, GCSF, GM-CSF and reactive oxygen species (ROS). Plasma cytokine profiles in an in vivo mouse model and in human blood cells treated with gp23*, gp24*, Hoc and Soc were evaluated by cytokine antibody arrays. Cytokine production and expression of CD40, CD80, CD86 and MHC class II molecules were also investigated in mouse bone marrow-derived dendritic cells treated with whole T4 phage particle or the same capsid proteins. The influence of T4 and gp23*, gp24*, Hoc and Soc on reactive oxygen species generation was examined in blood cells using luminol-dependent chemiluminescence assay. In all performed assays, the T4 bacteriophage and its capsid proteins gp23*, gp24*, Hoc and Soc did not affect production of inflammatory-related cytokines or ROS. These observations are of importance for any medical or veterinary application of bacteriophages.     

Membrane Interaction of the Portal Protein gp20 of Bacteriophage T4

Assembly of the bacteriophage T4 head structure occurs at the cytoplasmic face of the inner membrane of Escherichia coli with the formation of proheads. The proheads contain an internal scaffolding core that determines the size and the structure of the capsid. In a mutant where the major shell protein gp23 was compromised, core structures without a shell had been detected. Such core structures were also found in the mutant T4am20am23. Since the mutation in gene 20 is at the N terminus of gp20, it was assumed that these core structures assemble in the absence of gp20. However, sequencing showed that the mutation introduces a new ribosome binding site that leads to a restart at codon 15. Although the mutant protein gp20s lacks the very N-terminal sequence, we found that it still binds to the membrane of the host cell and can initiate prohead assembly. This explains its activity to allow the assembly of core structures and proheads at the membrane surface. With a cross-linking approach, we show here that gp20 and gp20s are escorted by the chaperones DnaK, trigger factor, and GroEL and dock on the membrane at the membrane protein YidC.     

Tailspike Interactions with Lipopolysaccharide Effect DNA Ejection from Phage P22 Particles in Vitro

Initial attachment of bacteriophage P22 to the Salmonella host cell is known to be mediated by interactions between lipopolysaccharide (LPS) and the phage tailspike proteins (TSP), but the events that subsequently lead to DNA injection into the bacterium are unknown. We used the binding of a fluorescent dye and DNA accessibility to DNase and restriction enzymes to analyze DNA ejection from phage particles in vitro. Ejection was specifically triggered by aggregates of purified Salmonella LPS but not by LPS with different O-antigen structure, by lipid A, phospholipids, or soluble O-antigen polysaccharide. This suggests that P22 does not use a secondary receptor at the bacterial outer membrane surface. Using phage particles reconstituted with purified mutant TSP in vitro, we found that the endorhamnosidase activity of TSP degrading the O-antigen polysaccharide was required prior to DNA ejection in vitro and DNA replication in vivo. If, however, LPS was pre-digested with soluble TSP, it was no longer able to trigger DNA ejection, even though it still contained five O-antigen oligosaccharide repeats. Together with known data on the structure of LPS and phage P22, our results suggest a molecular model. In this model, tailspikes position the phage particles on the outer membrane surface for DNA ejection. They force gp26, the central needle and plug protein of the phage tail machine, through the core oligosaccharide layer and into the hydrophobic portion of the outer membrane, leading to refolding of the gp26 lazo-domain, release of the plug, and ejection of DNA and pilot proteins.     

Native folding of aggregation-prone recombinant proteins in Escherichia coli by osmolytes, plasmid- or benzyl alcohol-overexpressed molecular chaperones

When massively expressed in bacteria, recombinant proteins often tend to misfold and accumulate as soluble and insoluble nonfunctional aggregates. A general strategy to improve the native folding of recombinant proteins is to increase the cellular concentration of viscous organic compounds, termed osmolytes, or of molecular chaperones that can prevent aggregation and can actively scavenge and convert aggregates into natively refoldable species. In this study, metal affinity purification (immobilized metal ion affinity chromatography [IMAC]), confirmed by resistance to trypsin digestion, was used to distinguish soluble aggregates from soluble nativelike proteins. Salt-induced accumulation of osmolytes during induced protein synthesis significantly improved IMAC yields of folding-recalcitrant proteins. Yet, the highest yields were obtained with cells coexpressing plasmid-encoded molecular chaperones DnaK-DnaJ-GrpE, ClpB, GroEL-GroES, and IbpA/B. Addition of the membrane fluidizer heat shock-inducer benzyl alcohol (BA) to the bacterial medium resulted in similar high yields as with plasmid-mediated chaperone coexpression. Our results suggest that simple BA-mediated induction of endogenous chaperones can substitute for the more demanding approach of chaperone coexpression. Combined strategies of osmolyte-induced native folding with heat-, BA-, or plasmid-induced chaperone coexpression can be thought to optimize yields of natively folded recombinant proteins in bacteria, for research and biotechnological purposes.     

Biochemical characterization of an ATPase activity associated with the large packaging subunit gp17 from bacteriophage T4

Double-stranded DNA-packaging in icosahedral bacteriophages is believed to be driven by a packaging "machine" constituted by the portal protein and the two packaging/terminase proteins assembled at the unique portal vertex of the empty prohead shell. Although ATP hydrolysis is evidently the principal driving force, which component of the packaging machinery functions as the translocating ATPase has not been elucidated. Evidence suggests that the large packaging subunit is a strong candidate for the translocating ATPase. We have constructed new phage T4 terminase recombinants under the control of phage T7 promoter and overexpressed the packaging/terminase proteins gp16 and gp17 in various configurations. The hexahistidine-tagged-packaging proteins were purified to near homogeneity by Ni(2+)-agarose chromatography and were shown to be highly active for packaging DNA in vitro. The large packaging subunit gp17 but not the small subunit gp16 exhibited an ATPase activity. Although gp16 lacked ATPase activity, it enhanced the gp17-associated ATPase activity by >50-fold. The gp16 enhancement was specific and was due to an increased catalytic rate for ATP hydrolysis. A phosphorylated gp17 was demonstrated under conditions of low catalytic rates but not under high catalytic rates in the presence of gp16. The data are consistent with the hypothesis that a weak ATPase is transformed into a translocating ATPase of high catalytic capacity after assembly of the packaging machine.     

Structure of the central hub of bacteriophage Mu baseplate determined by X-ray crystallography of gp44

Bacteriophage Mu is a double-stranded DNA phage that consists of an icosahedral head, a contractile tail with baseplate and six tail fibers, similar to the well-studied T-even phages. The baseplate of bacteriophage Mu, which recognizes and attaches to a host cell during infection, consists of at least eight different proteins. The baseplate protein, gp44, is essential for bacteriophage Mu assembly and the generation of viable phages. To investigate the role of gp44 in baseplate assembly and infection, the crystal structure of gp44 was determined at 2.1A resolution by the multiple isomorphous replacement method. The overall structure of the gp44 trimer is similar to that of the T4 phage gp27 trimer, which forms the central hub of the T4 baseplate, although these proteins share very little primary sequence homology. Based on these data, we confirm that gp44 exists as a trimer exhibiting a hub-like structure with an inner diameter of 25A through which DNA can presumably pass during infection. The molecular surface of the gp44 trimer that abuts the host cell membrane is positively charged, and it is likely that Mu phage interacts with the membrane through electrostatic interactions mediated by gp44.     

Bacteriophage attachment to the S-layer proteins of the mosquito-pathogenic strains ofBacillus sphaericus

The linearly arrayed surface layer proteins found on the mosquito-pathogenic strains ofBacillus sphaericus function as the site of bacteriophage attachment for the ten lytic bacteriophages used in a bacteriophage typing scheme. Attachment to the surface layer proteins was demonstrated by the ability to block bacteriophage binding with antisera and the ability of the purified proteins to neutralize bacteriophage. Bacteriophage-resistant mutants have modified surface proteins that are less able to neutralize bacteriophages than is the protein of the parent strain. No evidence was obtained that sugar residues play a part in bacteriophage attachment. Phage neutralization by surface proteins from strains that do not serve as host to the phage indicates that, although strains in each phage group have a unique surface protein, the proteins do not determine the phage groups.     

Bacterial stimulation upregulates the surface expression of the stress protein gp96 on B cells in the frog Xenopus

The presence of the soluble intracellular heat shock protein gp96 (an endoplasmic reticulum resident protein) at the surface of certain cell types is an intriguing phenomenon whose physiological significance has been unclear. We have shown that the active surface expression of gp96 by some immune cells is found throughout the vertebrate phylum including the Agnatha, the only vertebrate taxon whose members (lamprey, hagfish) lack an adaptive immune system. To determine whether gp96 surface expression can be modulated by pathogens, we investigated the effects of in vitro stimulation by purified lipopolysaccharide (LPS) and the heat-killed gram-negative bacteria, Escherichia coli and Aeromonas hydrophilia. Purified Xenopus B cells are readily activated and markedly proliferate in vitro in response to the heat-killed bacteria but not to purified LPS. Furthermore, messenger ribonucleic acid, and intracellular and surface protein expressions of both gp96 and immunoglobulin were upregulated only after activation of B cells by heat-killed bacteria. These data are consistent with an ancestral immunological role of gp96 as an antigen-presenting or danger-signaling molecule, or both, interacting directly with antigen-presenting cells, T cells, or natural killer cells, (or all), to trigger or amplify immune responses.     

Co-expression of gene 31 and 23 products of bacteriophage T4

Folding of the major capsid protein of bacteriophage T4 encoded by gene 23 is aided by Escherichia coli GroEL chaperonin and phage co-chaperonin gp31. In the absence of gene product (gp) 31, aggregates of recombinant gp23 accumulate in the cell similar to inclusion bodies. These aggregates can be solubilized with 6 M urea. However, the protein cannot form regular structures in solution. A system of co-expression of gp31 and gp23 under the control of phage T7 promoter in E. coli cells has been constructed. Folding of entire-length gp23 (534 amino acid residues) in this system results in the correctly folded recombinant gp23, which forms long regular structures (polyheads) in the cell.     

Inactivation of bacteriophages by protein E, a new major membrane protein isolated from an Escherichia coli mutant.

Pure protein E, obtained after diethylaminoethyl-cellulose chromatography of ethylenediaminetetraacetic acid-Triton X-100-solubilized outer membrane proteins of Escherichia coli strain JF694, inactivated bacteriophage K3. Lipopolysaccharide enhanced bacteriophage inactivation. Antibody prepared against purified protein E protected bacteriophage K3 from inactivation by protein E. Bacteriophage K3 used a major outer membrane protein, protein II*, as part of its receptor. We conclude that proteins E and II* have a common region which interacts with bacteriophage K3. Protein E also inactivated two recently described bacteriophages, TC45 and TC23, that use protein E as at least part of their receptor.     

Co-expression of the Plasmodium falciparum molecular chaperone, PfHsp70, improves the heterologous production of the antimalarial drug target GTP cyclohydrolase I, PfGCHI

Molecular chaperones have been used for the improved expression of target proteins within heterologous systems; however, the chaperone and target protein have seldom been matched in terms of origin. We have developed a heterologous co-expression system that allows independent expression of the plasmodial chaperone, PfHsp70, and a plasmodial target protein. In this study, the target was Plasmodium falciparum GTP cyclohydrolase I (PfGCHI), the first enzyme in the plasmodial folate pathway. The sequential expression of the molecular chaperone followed by the target protein increased the expression of soluble functional PfGCHI. His-tagged PfGCHI was successfully purified using nickel affinity chromatography, and the specific activity was determined by high performance liquid chromatography with spectrofluorometeric detection to be 5.93nmol/h/mg. This is the first report of a heterologous co-expression system in which a plasmodial chaperone is harnessed for the improved production and purification of a plasmodial target protein.     

hTNFR1在大肠杆菌中可溶性表达及纯化

将人源肿瘤坏死因子Ⅰ型受体（hTNFR1）基因克隆到pET-22b表达载体,成功构建了重组表达质粒pETH1,电转到Escherichia coli BL21（DE3）表达菌株中进行摇瓶发酵。实现了hTNFR1在大肠杆菌表达系统中的重组表达。但目的蛋白全部以包涵体的形式存在于沉淀中。为了提高hTNFR1在大肠杆菌中的可溶性表达,融合标签和分子伴侣两种策略被实施用于辅助hTNFR1的可溶性表达。结果表明,在hTNFR1的N端融合NusA标签后,hTNFR1的可溶性有一定提高;在NusA-hTNFR1基础上,过表达了7种分子伴侣,筛选出tig分子伴侣对hTNFR1蛋白可溶性表达有明显的促进作用,可溶性表达量约占总量的90％;对优化后的hTNFR1表达系统的可溶性蛋白进行Ni-NTA亲和层析纯化后,TEV蛋白酶酶切去除N端的NusA标签,结合Western blot分析鉴定,获得了大量高纯度的hTNFR1蛋白。研究结果为进一步研究hTNFR1的生理学活性及其在疾病治疗方面的应用奠定了良好基础。     

In vitro reconstitution of the bacteriophage T4 clamp loader complex (gp44/62)

The clamp loader complex (CLC) of bacteriophage T4 is essential for viability and has analogs in both prokaryotes and eukaryotes. The gp44 and gp62 subunits of the T4 CLC, in a 4:1 ratio, tightly associate such that the two proteins co-purify. Using transformed Escherichia coli, we were able to demonstrate for the first time purification of the unique protein gp62 in the absence of gp44. We experimentally determined the isoelectric point for the individual subunits. An in vitro physical interaction could be observed between the native subunits, which resulted in a reconstituted CLC that displayed the signature pattern of the ATPase functions of native CLC. Thus we demonstrate that the CLC forms via a self-assembly pathway rather than through a translational capture mechanism.     

Bacteriophage P4282, a parasite of Ralstonia solanacearum, encodes a bacteriolytic protein important for lytic infection of its host

To enhance bacterial wilt resistance in tobacco expressing a foreign protein, we isolated the bacteriolytic gene from a bacteriophage that infects Ralstonia solanacearum. The bacteriolytic protein of phage P4282 isolated in Tochigi Prefecture was purified from a lysate of R. solanacearum M4S cells infected with the phage, and its bacteriolytic activity was assayed by following the decrease in the turbidity of suspensions of R. solancacearum M4S cells. The molecular weight of the bacteriolytic protein was approximately 71 kDa, and the sequence of the N-terminal 13 amino acids was determined. We used oligonucleotide probes based on this amino acid sequence to isolate the bacteriolytic gene from phage P4282 DNA. This gene of 2061 bp encodes a product of 687 amino acids, whose calaculated molecular weight was 70.12 kDa. The bacteriolytic gene was placed under the control of an inducible promoter. and the plasmid was transformed into Escherichia coli NM522. The soluble proteins extracted from E.coli NM522 cells harboring the plasmid with the bacteriolytic gene showed obvious bacteriolytic activities against several strains of R. solanacearum isolated in various districts in Japan. DNA fragments from five phages, isolated in Niigata, Aomori, Okinawa, Fukushima and Yamaguchi Prefectures, hybridized to the bacteriolytic gene of phage P4282. These observations indicate that the bacteriolytic protein shows nonspecific activity against R. solanacearum strains, and a sequence similar to that of the bacteriolytic gene is conserved in the DNA of other bacteriophages. These results indicate that the generation of transgenic (tobacco) plants expressing the bacteriolytic gene of phage P4282 might result in enhanced resistance to bacterial wilt in tobacco.     

INVESTIGATION OF SECONDARY STRUCTURES AND MACROMOLECULAR INTERACTIONS IN BACTERIOPHAGE P22 BY LASER RAMAN SPECTROSCOPY

Laser Raman spectra of the DNA bacteriophage P22 and of its precursor particles and related structures have been obtained using 514.5-nm excitation. The spectra show that P22 DNA exists in the B form both inside of the phage head and after extraction from the phage. The major coat protein (gp5) contains a secondary structure composed of 18% α-helix, 20% β-sheet and 62% irregular conformations. The scaffolding protein (gp8) in the phage prohead is substantially richer than gp5 in α-helical content. Among the amino acid residues which give prominent Raman lines, the spectra show that tryptophans are exposed to solvent and most tyrosines are hydrogen bonded to positive donor groups. The above features of phage DNA and protein structures are nearly invariant to changes in temperature up to 80°C, indicating a remarkable thermal stability of the phage head and its encapsulated DNA.     

Tail-associated structural protein gp61 of Staphylococcus aureus phage phi MR11 has bifunctional lytic activity.

A tailed bacteriophage, phi MR11 (siphovirus), was selected as a candidate therapeutic phage against Staphylococcus aureus infections. Gene 61, one of the 67 ORFs identified, is located in the morphogenic module. The gene product (gp61) has lytic domains homologous to CHAP (corresponding to an amidase function) at its N-terminus and lysozyme subfamily 2 (LYZ2) at its C-terminus. Each domain of gp61 was purified as a recombinant protein. Both the amidase [amino acids (aa) 1-150] and the lysozyme (aa 401-624) domains but not the linker domain (aa 151-400) caused efficient lysis of S. aureus. Immunoelectron microscopy localized gp61 to the tail tip of the phi MR11 phage. These data strongly suggest that gp61 is a tail-associated lytic factor involved in local cell-wall degradation, allowing the subsequent injection of phi MR11 DNA into the host cytoplasm. Staphylococcus aureus lysogenized with phi MR11 was also lysed by both proteins. Staphylococcus aureus strains on which phi MR11 phage can only produce spots but not plaques were also lysed by each protein, indicating that gp61 may be involved in 'lysis from without'. This is the first report of the presence of a tail-associated virion protein that acts as a lysin, in an S. aureus phage.