Study of robustness of filamentous bacteriophages for industrial applications

The development of a whole new class of industrial agents, such as biologically based nanomaterials and viral vectors, has raised many challenges for their large-scale manufacture, principally due to the lack of essential physical data and bioprocessing knowledge. A new example is the promise of filamentous bacteriophages and their derivatives. As a result, there is now an increasing need for the establishment of strong biochemical engineering foundations to serve as a guide for future manufacture. This article investigates the effect of high-energy fluid flow on filamentous bacteriophage M13 to determine its robustness for large-scale processing. By the application of well-understood ultra scale-down predictive techniques, the viability of bacteriophage M13 was studied as a measure of its robustness and as a function of energy dissipation rate and fluid conditions. These experiments suggested that despite being perceived as a relatively fragile molecule in the literature, bacteriophage M13 should tolerate processing conditions in existing large-scale equipment designs. No loss of viability was noted up to a maximum energy dissipation rate of 2.9 × 10(6) W kg(-1) . Furthermore, significant losses above this threshold only occurred over periods well in excess of the exposure times expected in a bioprocess environment. Filamentous bacteriophages may therefore be regarded as a viable process material for industrial applications.     

Shuffling of structural elements in filamentous bacteriophages

All class II filamentous bacteriophage coat proteins contain a conserved, 12-amino acid sequence highly homologous to the loop portion of the EF-hand Ca²⁺-binding motif. The Pf3 coat protein contains two regions of homology to this sequence. The 12-amino acid sequence corresponds to a region of the Pf1 coat protein whose structure is controversial. In some models of the virus structure, this region is α-helical. In others, it forms a loop that folds back on itself. The similarity of this region to the loop in the helix-loop-helix Ca²⁺-binding motif suggests that it takes on a loop structure in the virion. Each filamentous phage lacks at least one residue normally involved in Ca²⁺-coordination, consistent with the relatively weak Ca²⁺ binding properties of the filamentous phages. Consideration of the structure of the coat protein in the membrane and in the virus particle indicates that the protein may be more effective in binding cations in its membrane-bound form than in the virus particle. This suggests that release of cations from this loop may be an obligate step during assembly of the proteins into the virus particle. Proteins 27:405–409, 1997. © 1997 Wiley-Liss, Inc.     

欧文氏菌噬菌体的分离纯化及生物学特性研究

以欧文氏菌胡萝卜亚种为宿主菌,从环境污泥中分离到10株噬菌体.噬菌体热稳定性、pH稳定性分析结果表明噬菌体Erj2、Erb1、Erc2在温度-20～40℃、pH4.0～9.0之间均表现出良好的稳定性.噬菌体生物学特性分析显示,它们的最佳感染复数分别为0.0001、0.001、0.0001;核酸类型均为双链DNA;一步生...     

Characterization of filamentous bacteriophage PE226 infecting Ralstonia solanacearum strains

Aims: The aim of this study was to isolate and characterize new bacteriophages that infect a wide range of plant pathogenic Ralstonia solanacearum strains. Methods and Results: Fifteen bacteriophages were isolated from pepper, tomato and tobacco plant rhizospheres infected with R. solanacearum. A host specificity analysis of the isolated phages using nine strains of R. solanacearum indicated great phage diversity in a single soil. Two phages, PE226 and TM227, showed clear plaques on all nine bacterial hosts tested and were virtually identical in morphology and genome. PE226, an Inovirus, is a long, flexible, filamentous phage carrying a circular (+) sense single‐strand DNA genome of 5475 nucleotides. DNA sequences of PE226 exhibited nine open reading frames (ORF) that were not highly similar to those of other phages infecting R. solanacearum. The genome organization of PE226 was partially similar to that of p12J of Ralstonia pickettii. One ORF of PE226 showed identity to the zot gene encoding zonula occludens toxin of Vibrio cholera. Orf7 of PE226 was also present in the genome of R. solanacearum strain SL341. However, SL341, a highly virulent strain in tomato, was still sensitive to phage PE226. Conclusions: A new, flexible, filamentous phage PE226 infected wide range of R. solanacearum strains and carried unique circular single‐strand DNA genome with an ORF encoding Zot‐like protein. Significance and Impact of the Study: PE226 may be a new type of temperate phage, based on its lytic nature on a wide range of hosts and the presence of a zot homologue in a host bacterial genome.     

Screening and identification of receptor antagonist for shiga toxin from random peptides displayed on filamentous bacteriophages

The bacteriophage clones which can bind with shiga toxin B subunit (StxB) and inhibit cytotoxicity of shiga toxin were obtained by using antibody capturing method from a 15-mer random peptide library displayed on the surface of bacteriophage fd. Among them, one peptide encoded by the random DNA region of a selected bacteriophage (A12) was synthesized and tested in vitro and in vivo, where the peptide competed with the receptor of shiga toxin to bind StxB, and inhibited the cytotoxicity and enterotoxicity of shiga toxin. The peptide can also block other apparently unrelated StxB binding bacteriophage (A3), which suggests that there are overlapping StxB interaction sites for those ligands with different sequences. The results provide a demonstration of bacteriophage display to screen peptide ligands for a small and/or unable biotinylated molecule by antibodies-capturing strategy, and take the lead for the development of receptor antagonists for shiga toxin.     

Isolation and characterization of bacteriophages specific for Campylobacter jejuni

Human infection by Campylobacter jejuni is mainly through the consumption of contaminated poultry products, which results in gastroenteritis and, rarely, bacteremia and polyneuropathies. In this study, six C. jejuni -specific bacteriophages (CPS1–6) were isolated by the spot-on-the-lawn technique from chicken samples in Korea and characterized for potential use as biocontrol agents. All isolated bacteriophages exhibited a high specificity, being able to lyse only C. jejuni , but not other Gram–negative bacteria, including C. coli , Escherichia coli , Salmonella spp., and Gram–positive bacteria. Bacteriophages contain an icosahedral head and a contractile tail sheath in transmission electron microscopy, and possess ds-DNA with an average genome size of approximately 145 kb; therefore, all bacteriophages are categorized into the Myoviridae family. Bacterial lysis studies in liquid media revealed that CPS2 could be used to control the growth of C. jejuni .     

An improved method for large-scale purification of recombinant human glucagon

Glucagon was expressed inEscherichia coli as a fusion protein including the glucagon sequence [Ishizakiet al. (1992),Appl. Microbiol. Biotechnol.36, 483–486]. The high-level expression of a protein inE. coli often results in an insoluble aggregate called an inclusion body containing a fusion protein. In our previous report [Yoshikawaet al. (1992),J. Protein Chem. 11, 517–525], we solubilized this inclusion body by using guanidinium chloride. However, the existence of denaturant caused problems such as a low proteolytic activity for transforming the fusion protein into glucagon and complicated purification methods. We tried to improve the method to enable large-scale purification. At alkaline pH, the inclusion body could be solubilized to a high concentration and cleaved by amino acid-specific endopeptidases. By utilizing isoelectric precipitations as a new economical purification method for glucagon from intermediates, the glucagon obtained was shown to be over 99.5% pure by analytical RP-HPLC. The yield was almost equal that of our previous method, and the glucagon produced was chemically and biochemically equivalent to natural glucagon.     

Delineating the site of interaction on the pIII protein of filamentous bacteriophage fd with the F-pilus of Escherichia coli

The minor coat protein pIII at one end of the filamentous bacteriophage fd, mediates the infection of Escherichia coli cells displaying an F-pilus. pIII has three domains (D1, D2 and D3), terminating with a short hydrophobic segment at the C-terminal end. Domain D2 binds to the tip of F-pilus, which is followed by retraction of the pilus and penetration of the E. coli cell membrane, the latter involving an interaction between domain D1 and the TolA protein in the membrane. Surface residues on the D2 domain of pIII were replaced systematically with alanine. Mutant virions were screened for D2-pilus interaction in vivo by measuring the release of infectious virions from E. coli F(+) cells infected with the mutants. A competitive ELISA was developed to measure in vitro the ability of mutant phages to bind to purified pili. This allowed the identification of amino acid residues involved in binding to F and to EDP208 pili. These residues were found to cluster on the outer rim of the 3D structure of the D2 domain, unexpectedly identifying this as the F-pilus binding region on the pIII protein.     

Protein purification by chemo‐selective precipitation using thermoresponsive polymers

A recoverable and thermoresponsive polymer‐protein bioconjugate is synthesized and employed in the purification of protein with free sulfhydryl groups. Initiator with disulphide was modified on the cysteine residue of the target protein. Poly(N‐isopropylacrylamide) exhibiting a lower critical solution temperature was grown from the protein. The resulting protein–polymer conjugate was successfully thermoprecipitated and separated from other proteins. The approach was demonstrated with bovine serum albumin with the recycling yield of 76.4%. Enzyme activity test with papain verified the reversible polymer modification protected protein under extreme environments without affecting the functionality of the protein. This study implies the favorable potential of chemo‐selective enriching and purification of proteins.     

Genome Comparison In Silico in Neisseria Suggests Integration of Filamentous Bacteriophages by their Own Transposase

We have identified filamentous prophages, Nf (Neisserial filamentousphages), during an in silico genome comparison in Neisseria.Comparison of three genomes of Neisseria meningitidis and oneof Neisseria gonorrhoeae revealed four subtypes of Nf. Elevenintact copies are located at different loci in the four genomes.Each intact copy of Nf is flanked by duplication of 5'-CT and,at its right end, carries a transposase homologue (pivNM/irg)of RNaseH/Retroviral integrase superfamily. The phylogeny ofthese putative transposases and that of phage-related proteinson Nfs are congruent. Following circularization of Nfs, a promoter-likesequence forms. The sequence at the junction of these predictedcircular forms (5'-atCTtatat) was found in a related plasmid(pMU1) at a corresponding locus. Several structural variantsof Nfs—partially inverted, internally deleted and truncated—werealso identified. The partial inversion seems to be a productof site-specific recombination between two 5'-CTtat sequencesthat are in inverse orientation, one at its end and the otherupstream of pivNM/irg. Formation of internally deleted variantsprobably proceeded through replicative transposition that alsoinvolved two 5'-CTtat sequences. We concluded that the PivNM/Irgtransposase on Nfs integrated their circular forms into thechromosomal 5'-CT-containing sequences and probably mediatedthe above rearrangements.     

Unlocking of the filamentous bacteriophage virion during infection is mediated by the C domain of pIII

Protein III (pIII) of filamentous phage is required for both the beginning and the end of the phage life cycle. The infection starts by binding of the N-terminal N2 and N1 domains to the primary and secondary host receptors, F pilus and TolA protein, respectively, whereas the life cycle terminates by the C-terminal domain-mediated release of the membrane-anchored virion from the cell. It has been assumed that the role of the C-terminal domain of pIII in the infection is that of a tether for the receptor-binding domains N1N2 to the main body of the virion. In a poorly understood process that follows receptor binding, the virion disassembles as its protein(s) become integrated into the host inner membrane, resulting in the phage genome entry into the bacterial cytoplasm. To begin revealing the mechanism of this process, we showed that tethering the functional N1N2 receptor-binding domain to the virion via termination-incompetent C domain abolishes infection. This infection defect cannot be complemented by in trans supply of the functional C domain. Therefore, the C domain of pIII acts in concert with the receptor-binding domains to mediate the post receptor binding events in the infection. Based on these findings, we propose a model in which binding of the N1 domain to the periplasmic portion of TolA, the secondary receptor, triggers in cis a conformational change in the C domain, and that this change opens or unlocks the pIII end of the virion, allowing the entry phase of infection to proceed. To our knowledge, this is the first virus that uses the same protein domain both for the insertion into and release from the host membrane.     

Precipitation of RNA impurities with high salt in a plasmid DNA purification process: use of experimental design to determine reaction conditions

The use of high salt solution to precipitate RNA in a pharmaceutical-grade plasmid DNA purification process was investigated. Five antichaotropic salts were tested for their potential to precipitate RNA. Calcium chloride was by far the best precipitant with high RNA removal in a very short incubation time. Calcium chloride precipitation conditions were investigated at two stages of a plasmid purification process using experimental design techniques. The effect of up to five factors on RNA precipitation and plasmid recovery was assessed by statistical modeling. Optimized conditions for calcium chloride precipitation were then introduced to the plasmid purification process resulting in the efficient removal of most impurities (RNA, chromosomal DNA, proteins, and endotoxins).     

Temperature-triggered purification of antibodies

In this article the unique capability of elastin-like protein (ELP) to reversibly precipitate was combined with the high affinity and specificity of antibody-binding domains such as Protein G, Protein L, or Protein LG as a general method for antibody purification that combines in a unique manner the simplicity and robustness of temperature-triggered precipitation with the selectivity of affinity interactions. In a single precipitation step, antibodies derived from different sources (animal sera or hybridoma cell cultures) were selectively recovered by a simple temperature trigger. Due to the versatility of the binding ligands toward different classes of antibodies, we believe that this technology will be useful as an economical, highly efficient, and universal platform for the purification of antibodies.     

Isolation, characterization of bacteriophages specific to Microlunatus phosphovorus and their application for rapid host detection

AIMS: To isolate and characterize lytic-bacteriophages specific to Microlunatus phosphovorus, and prepare fluorescently labelled phages (FLPs) for the rapid detection of the host bacterium in activated sludge. METHODS AND RESULTS: Isolation of bacteriophages lytic to M. phosphovorus was attempted by applying supernatants of activated sludge processes on the lawn of M. phosphovorus JCM9379 for plaque formation. Thirteen bacteriophage isolates were obtained. The restriction fragment length polymorphism analysis distinguished them into two different bacteriophages designated as phiMP1 and phiMP2. They were found to possess double-stranded DNA and host specificity. Morphological observations were done by electron microscopy. The bacteriophage particles stained by SYBR Green I was shown to be applicable to detect their host bacterial cells mixed with activated sludge. CONCLUSIONS: Two M. phosphovorus-specific bacteriophages were isolated and classified as Siphoviridae. FLPs of them were prepared, and successfully applied to detect the host bacterium added into the activated sludge. SIGNIFICANCE AND IMPACT OF THE STUDY: At least some of bacteria in activated sludge are susceptible to their related bacteriophages. Bacteriophages lytic to activated sludge bacteria could be affecting the bacterial population in activated sludge. The FLPs could be used for the easy-rapid detection of their host bacterium in activated sludge.     

链球菌噬菌体裂解酶在大肠杆菌中的表达、纯化及活性检测

噬菌体裂解酶是噬菌体产生的细胞壁水解酶,通过水解宿主菌细胞壁使子代噬菌体释放,在体外能高效且特异性地杀死细菌。本研究旨在克隆和表达链球菌噬菌体裂解酶PlyC,并测定其生物学活性。利用PCR方法扩增PlyC的2条肽链PlyCA和PlyCB,构建表达载体pET-32a(+)-PlyCA和pET-32a(+)-PlyCB,分别转化至大肠杆菌BL21(DE3)中,以0.7 mmol/L IPTG在30 oC诱导7 h实现了高效表达,SDS-PAGE分析表明PlyCA和PlyCB表达量均可达菌体总蛋白的30%以上。采用Ni2+-NTA亲和层析法纯化目的蛋白,其纯度大于95%。用透析复性方法得到目的产物重组链球菌噬菌体裂解酶PlyC,以浊度法和平板计数法检测其体外抗菌效果,扫描电子显微镜观察裂解酶作用前后细菌细胞形态变化。结果表明重组PlyC能特异性裂解化脓性链球菌(A组β-溶血性链球菌),以4μg/mL浓度作用于OD600为0.56的菌液60 min后杀菌率达99.6%,扫描电镜观察结果显示该酶作用于菌体后,链球菌细胞裂解,呈碎片状态。本研究为开发一种新型、高效的链球菌感染疾病治疗药物打下了基础。     

Molecular structure of fd (f1, M13) filamentous bacteriophage refined with respect to X-ray fibre diffraction and solid-state NMR data supports specific models of phage assembly at the bacterial membrane

Filamentous bacteriophage (Inovirus) is a simple and well-characterized model system. The phage particle, or virion, is about 60 angstroms in diameter and several thousand angstrom units long. The virions are assembled at the bacterial membrane as they extrude out of the host without killing it, an example of specific transport of nucleoprotein assemblages across membranes. The Ff group (fd, f1 and M13) has been especially widely studied. Models of virion assembly have been proposed based on a molecular model of the fd virion derived by X-ray fibre diffraction. A somewhat different model of the fd virion using solid-state NMR data has been proposed, not consistent with these models of assembly nor with the X-ray diffraction data. Here we show that reinterpreted NMR data are also consistent with the model derived from X-ray fibre diffraction studies, and discuss models of virion assembly.     

Precipitation of collagens by polyethylene glycols

Types I, II, and III collagens are readily precipitated at neutral pH by polyethylene glycols (PEG). As the molecular weight fraction of the polyethylene glycols increases, they become more effective as precipitants on a weight basis. The amount of PEG required for precipitation depends on the pH, the ionic strength, and the nature of the buffer or salts present. In tissue culture media, low concentrations of collagens and procollagens may be quantitatively precipitated and readily collected by low-speed centrifugation. Polyethylene glycol precipitation can be used to obtain collagens and procollagens from tissue culture media at either analytical or preparative scale, and since the polyethylene glycols do not bind to collagens, the precipitates may be further analyzed directly by chromatographic or electrophoretic methods.