Alteration of tail fiber protein gp38 enables T2 phage to infect Escherichia coli O157:H7

Artificial control of phage specificity may contribute to practical applications, such as the therapeutic use of phages and the detection of bacteria by their specific phages. To change the specificity of phage infection, gene products (gp) 37 and 38, expressed at the tip of the long tail fiber of T2 phage, were exchanged with those of PP01 phage, an Escherichia coli O157:H7 specific phage. Homologous recombination between the T2 phage genome and a plasmid encoding the region around genes 37-38 of PP01 occurred in transformant E. coli K12 cells. The recombinant T2 phage, named T2ppD1, carried PP01 gp37 and 38 and infected the heterogeneous host cell E. coli O157:H7 and related species. On the other hand, T2ppD1 could not infect E. coli K12, the original host of T2, or its derivatives. The host range of T2ppD1 was the same as that of PP01. Infection of T2ppD1 produced turbid plaques on a lawn of E. coli O157:H7 cells. The binding affinity of T2ppD1 to E. coli O157:H7 was weaker than that of PP01. The adsorption rate constant (ka) of T2ppD1 (0.17 x 10(-9)(ml CFU(-1) min(-1)) was almost 1/6 that of PP01 (1.10 x 10(-9)(ml CFU(-1) min(-1))). In addition to the tip of the long tail fiber, exchange of gene products expressed in the short tail fiber may be necessary for tight binding of recombinant phage.     

Identification of a unique specificity determinant of the colicin E3 immunity protein.

Plasmid immunity to a nuclease-type colicin is defined by the specific binding of an immunity (or inhibitor) protein, Imm, to the C-terminal nuclease domain, T2A, of the colicin molecule. Whereas most regions of colicin operons exhibit extensive sequence identity, the small plasmid region encoding T2A and Imm is exceptionally varied. Since immunity is essential for the survival of the potentially lethal colicin plasmid (Col), we inferred that T2A and Imm must have co-evolved, retaining their mutual binding specificities. To evaluate this co-evolution model for the col and imm genes of ColE3 and ColE6, we attempted to obtain a stabilized clone from a plasmid which had been destabilized with a non-cognate immunity gene. A hybrid Col, in which the immE3 gene of the ColE3 was replaced with immE6 from ColE6, was lethal to the host cells upon SOS induction. From among this suicidal cell population, we isolated a stabilized, i.e., evolved, clone which produced colicin E3 (E3) stably and exhibited immunity to E3. This change arose from only a single mutation in ImmE6, from Trp48 to Cys, the same residue as in the ImmE3 sequence. In addition, we constructed a series of chimeric genes through homologous recombination between immE3 and immE6. Characterization of these chimeric immunity genes confirmed the above finding that colicins E3 and E6 are mostly distinguished by only Cys48 of the ImmE3 protein.     

Control of gene expression in plant cells using a 434:VP16 chimeric protein

The herpes simplex virus type 1 VP16 polypeptide is a potent trans-activator of viral gene expression. We have tested the ability of the VP16 activation domain to activate gene expression in plant cells. A plasmid encoding a translational fusion between the full-length 434 repressor and the C-terminal 80 amino acids of VP16, was constructed. When expressed in Escherichia coli, the chimeric protein binds efficiently to 434-binding motifs (operators). For expression in plant cells, the chimeric activator gene was placed between the cauliflower mosaic virus (CaMV) 35S promoter and nos terminator sequences in a pUC-based plasmid. The 434 operators were placed upstream of a minimal CaMV 35S promoter linked to the E. coli gus reporter gene. This reporter-expression cassette was then incorporated into the same plasmid as the 434 cI/VP16 activator-expression cassette. Two control plasmids were also constructed, one encoding the 434 protein with no activator domain and the second a chimeric activator with no DNA-binding domain. The chimeric activator was tested for its ability to activate gene expression in a tobacco protoplast transient assay system. Results are presented to show that we can obtain in plant cells significant activation of gene expression that is dependent on both DNA-binding and the presence of the activator domain.     

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。     

Morphogenesis of the long tail fibers of bacteriophage T2 involves proteolytic processing of the polypeptide (gene product 37) constituting the distal part of the fiber

Gene 37 of phage T2 codes for a protein that, as a dimer, constitutes the most distal, receptor-recognizing part of its long tail fibers. It was found that, from a plasmid carrying a fragment of gene 37 that lacked a region of the gene encoding 87 CO2H-terminal amino acid residues, a protein was expressed that was slightly larger than that present in the phage. This size difference could not be accounted for. The missing region of gene 37 and also gene 38 (which codes for the auxiliary protein required for dimerization of protein 37) were cloned. Plasmids were constructed with gene 37, or gene 37 together with gene 38, under inducible control. Independent of the presence of the latter gene, a protein was produced that had the same size as protein 37 in the phage. A pulse-chase experiment revealed that a precursor of protein 37 is synthesized and processed such that approximately 120 amino acid residues, most likely CO2H-terminal, are removed. Therefore, the protein produced from the truncated gene was larger because it cannot be processed. This fact also solved an old puzzle: an amber fragment of protein 37 of phage T2 had been found to be larger than the mature protein. The amber codon could be located 24 codons away from the normal stop codon. Obviously, the fragment cannot be processed. The existence of this fragment demonstrates that processing occurs during phage maturation.     

Cloning and expression of a chloramphenicol acetyltransferase gene in cytosine-substituted T4 bacteriophage

We have developed an efficient method for transferring foreign genes into the T4 phage genome. Any foreign genes inserted into the T4 uvsY gene cloned on plasmids can be transferred into a cytosine-substituted T4dC(delta NB5060) phage genome by a replacement type of recombination. To achieve this, we constructed chimeric plasmids which had a chloramphenicol acetyltransferase gene (cat) derived from transposon Tn9 inserted into the Bg/II site within the T4 uvsY gene on pBR322. The cat gene was then transferred by in vivo recombination into the T4dC(delta NB5060) phage genome. Moreover, it was demonstrated that the cat gene in the hybrid T4dC phage was expressed upon phage infection and development.     

Synthesis, cloning, and expression in Escherichia coli of a spinach acyl carrier protein-I gene

A synthetic gene of 268 bp encoding the 82 amino acid spinach acyl carrier protein (ACP)-I was constructed based on the known amino acid sequence. Two gene fragments, one encoding the amino-terminal portion and the other the carboxy-terminal portion of the protein, were assembled from synthetic oligonucleotides and inserted into the phage M13mp19. These partial gene constructions were joined and inserted into the plasmid pTZ19R. DNA sequencing confirmed the accuracy of the constructions. The synthetic gene was then subcloned into the Escherichia coli expression vector pKK233-2, under the control of the trc promoter. Western blot analysis and radioimmunoassay indicated that E. coli cells carrying this plasmid produced up to 6 mg/liter of a protein which was immunologically cross-reactive and similar in electrophoretic mobility to authentic spinach acyl carrier protein. The bacterial cells were able to attach the phosphopantetheine prosthetic group to the synthetic plant gene product allowing it to be acylated in vitro by acyl-ACP synthetase.     

Chimeric pneumococcal cell wall lytic enzymes reveal important physiological and evolutionary traits.

Two novel chimeric pneumococcal cell wall lytic enzymes, named LC7 and CL7, have been constructed by in vitro recombination of the lytA gene encoding the major autolysin (LYTA amidase) of Streptococcus pneumoniae, a choline-dependent enzyme, and the cpl7 gene encoding the CPL7 lysozyme of phage Cp-7, a choline-independent enzyme. In remarkable contrast with previous chimeric constructions, we fused here two genes that lack nucleotide homology. The CL7 enzyme, which contains the N-terminal domain of CPL7 and C-terminal domain of LYTA, exhibited a choline-dependent lysozyme activity. This experimental rearrangement of domains might mimic the process that have generated the choline-dependent CPL1 lysozyme of phage Cp-1 during evolution, providing additional support to the modular theory of protein evolution. The LC7 enzyme, built up by fusion of the N-terminal domain of LYTA and the C-terminal domain of CPL7, exhibited an amidase activity capable of degrading ethanolamine-containing cell walls. The chimeric amidase behaved as an autolytic enzyme when it was cloned and expressed in S. pneumoniae. The chimeric enzymes provided new insights on the mechanisms involved in regulation of the host pneumococcal autolysins and on the participation of these enzymes in the process of cell separation. Furthermore, our experimental approach confirmed the basic role of the C-terminal domains in substrate recognition and revealed the influence of these domains on the optimal pH for catalytic activity.     

Cloning of linear DNAs in vivo by overexpressed T4 DNA ligase: construction of a T4 phage hoc gene display vector.

A method was developed to clone linear DNAs by overexpressing T4 phage DNA ligase in vivo, based upon recombination deficient E. coli derivatives that carry a plasmid containing an inducible T4 DNA ligase gene. Integration of this ligase-plasmid into the chromosome of such E. coli allows standard plasmid isolation following linear DNA transformation of the strains containing high levels of T4 DNA ligase. Intramolecular ligation allows high efficiency recircularization of cohesive and blunt-end terminated linear plasmid DNAs following transformation. Recombinant plasmids could be constructed in vivo by co-transformation with linearized vector plus insert DNAs, followed by intermolecular ligation in the T4 ligase strains to yield clones without deletions or rearrangements. Thus, in vitro packaged lox-site terminated plasmid DNAs injected from phage T4 were recircularized by T4 ligase in vivo with an efficiency comparable to CRE recombinase. Clones that expressed a capsid-binding 14-aa N-terminal peptide extension derivative of the HOC (highly antigenic outer capsid) protein for T4 phage hoc gene display were constructed by co-transformation with a linearized vector and a PCR-synthesized hoc gene. Therefore, the T4 DNA ligase strains are useful for cloning linear DNAs in vivo by transformation or transduction of DNAs with nonsequence-specific but compatible DNA ends.     

Site-specific recombination between cloned attP and attB sites from the Haemophilus influenzae bacteriophage HP1 propagated in recombination-deficient Escherichia coli.

Plasmids were constructed which contain both attP and attB DNA segments derived from the insertion sites of the lysogenic bacteriophage HP1 and its host, Haemophilus influenzae. Similar plasmids containing the two junction segments (attL and attR regions) between the phage genome and the lysogenic host chromosome were also prepared. The formation of recombinant dimer plasmids was observed when attP-attB plasmids were propagated in Escherichia coli HB101 (recA), while plasmids containing the junction segments did not form recombinant dimers. Deletion of the phage DNA segment adjacent to the attP site from the attP-attB constructions eliminated detectable recombination, suggesting that this sequence contains the gene encoding the HP1 integrase. No plasmid recombination was observed in strains of E. coli defective in integration host factor. This suggests that integration host factor is important in the expression or activity of the system which produces the site-specific recombination of sequences derived from HP1 and H. influenzae. Further, it suggests that a protein functionally analogous to E. coli integration host factor may be present in H. influenzae.     

In vitro packaging into phage T4 particles and specific recircularization of phage lambda DNAs

Concatemeric phage lambda imm434 DNA packaged in vitro into phage T4 particles produced plaques on a selective host. Moreover, lambda DNA containing a pBR322 derivative flanked by the lambda attL and attR sites could be specifically recircularized by excisive lambda recombination to yield the pBR322 derivative. A host deficient in generalized recombination and containing a defective lambda c Its prophage which provided Int and Xis proteins was the recipient for this plasmid derivative carried by T4. Such a T4-lambda hybrid may potentially allow almost one T4 headful of donor DNA (166 kb) to be packaged and recircularized.     

Reversion of Q beta RNA phage mutants by homologous RNA recombination.

Q beta phage RNAs with inactivating insertion (8-base) or deletion (17-base) mutations within their replicase genes were prepared from modified Q beta cDNAs and transfected into Escherichia coli spheroplasts containing Q beta replicase provided in trans by a resident plasmid. Replicase-defective (Rep-) Q beta phage produced by these spheroplasts were detected as normal-sized plaques on lawns of cells containing plasmid-derived Q beta replicase, but were unable to form plaques on cells lacking this plasmid. When individual Rep- phage were isolated and grown to high titer in cells containing plasmid-derived Q beta replicase, revertant (Rep+) Q beta phage were obtained at a frequency of ca. 10(-8). To investigate the mechanism of this reversion, a point mutation was placed into the plasmid-derived Q beta replicase gene by site-directed mutagenesis. Q beta mutants amplified on cells containing the resultant plasmid also yielded Rep+ revertants. Genomic RNA was isolated from several of the latter phage revertants and sequenced. Results showed that the original mutation (insertion or deletion) was no longer present in the phage revertants but that the marker mutation placed into the plasmid was now present in the genomic RNAs, indicating that recombination was one mechanism involved in the reversion of the Q beta mutants. Further experiments demonstrated that the 3' noncoding region of the plasmid-derived replicase gene was necessary for the reversion-recombination of the deletion mutant, whereas this region was not required for reversion or recombination of the insertion mutant. Results are discussed in terms of a template-switching model of RNA recombination involving Q beta replicase, the mutant phage genome, and plasmid-derived replicase mRNA.     

Rapid detection of Escherichia coli O157:H7 by using green fluorescent protein-labeled PP01 bacteriophage

A previously isolated T-even-type PP01 bacteriophage was used to detect its host cell, Escherichia coli O157:H7. The phage small outer capsid (SOC) protein was used as a platform to present a marker protein, green fluorescent protein (GFP), on the phage capsid. The DNA fragment around soc was amplified by PCR and sequenced. The gene alignment of soc and its upstream region was g56-soc.2-soc.1-soc, which is the same as that for T2 phage. GFP was introduced into the C- and N-terminal regions of SOC to produce recombinant phages PP01-GFP/SOC and PP01-SOC/GFP, respectively. Fusion of GFP to SOC did not change the host range of PP01. On the contrary, the binding affinity of the recombinant phages to the host cell increased. However, the stability of the recombinant phages in alkaline solution decreased. Adsorption of the GFP-labeled PP01 phages to the E. coli cell surface enabled visualization of cells under a fluorescence microscope. GFP-labeled PP01 phage was not only adsorbed on culturable E. coli cells but also on viable but nonculturable or pasteurized cells. The coexistence of insensitive E. coli K-12 (W3110) cells did not influence the specificity and affinity of GFP-labeled PP01 adsorption on E. coli O157:H7. After a 10-min incubation with GFP-labeled PP01 phage at a multiplicity of infection of 1,000 at 4 degrees C, E. coli O157:H7 cells could be visualized by fluorescence microscopy. The GFP-labeled PP01 phage could be a rapid and sensitive tool for E. coli O157:H7 detection.     

Construction and properties of a recombinant plasmid containing gene 32 of bacteriophage T4D

This paper describes the construction and characterization of a chimeric plasmid that encodes the single-stranded DNA-binding protein of bacteriophage T4D (the product of gene 32). The plasmid contains a 2·6 × 10³ base HindIII segment of T4 DNA that includes genes 59 and 32 as well as a portion of gene 33. Isolation of bacteria carrying the recombinant plasmid became possible when the segment of phage DNA contained an amber mutation in gene 32. This suggests that a functional gene 32 is deleterious to the cell. Using antibody to gene 32 protein, we have been able to demonstrate expression of the plasmid-borne gene 32 in uninfected bacteria. Deletion variants of the gene 32 plasmid have been constructed in vitro. These have been used to align the genetic map of the region with the restriction map and to study phage gene expression from the plasmid in both infected and uninfected cells. In phage-infected cells the level of functional gene 32 product regulates the efficiency of translation of its own messenger RNA. We also observe such self-regulation for gene 32 present on the plasmid.     

Expression of chemically synthesized alpha-neo-endorphin gene fused to E. coli alkaline phosphatase.

An alpha-neo-endorphin (alpha NE) gene, which we previously synthesized chemically and inserted into E. coli beta-galactosidase gene of pK013 plasmid, has been excised and fused to E. coli alkaline phosphatase (APase) gene. One of the transformants was named E15/pA alpha NE1. Under the APase gene regulation, APase-alpha NE chimeric protein was expressed at 1.3 X 10(6) molecules per cell, and accounted for about 60% of total cellular proteins. The HPLC pattern of CNBr treated E15/pA alpha NE1 was very simple reflecting the high content of the chimeric protein and low numbers of methionine residues in it. A series of genes encoding APase-alpha NE chimeric proteins in which 30 to 94 C-terminal amino acid residues were replaced by (met)-alpha NE, was cloned in E. coli. Transportation of the chimeric proteins to periplasmic space was studied. All chimeric proteins were apparently processed by signal peptidase but few, if any, was transported to the periplasmic space.     

HIV—1 P24截短体与gp41截短体的融合蛋白在大肠杆菌中的表达、纯化和鉴定

用基因重组技术将截短的HIV－1 p24基因和gp41基因连接成嵌合基因,插入质粒pGEX-4T3,构建成重组表达质粒pGEX-F。将pGEX-F转化大肠杆菌BL21。经IPTG诱导表达,pGEX-F在大肠杆菌BL21中获得了高效表达。融合蛋白P24－gp41经Glutathione-Sepharose4B亲和层析纯化后,用间接ELISA和免疫印迹检测HIV抗体阳性血清和正常人血清,P24－gp41只与HIV抗体阳性血清反应,证明获得的融合蛋白P24－gp41有很强的抗原特异性和免疫反应性,具有较高的应用价值。     

Detection of Escherichia coli with fluorescent labeled phages that have a broad host range to E. coli in sewage water

Escherichia coli is used as an indicator microorganism in public health. The conventional way to detect E. coli requires several days to produce a result, because it requires incubation of cells. Therefore a rapid and sensitive detection method is needed. T4e-/GFP phage, characterized by suppression of lysozyme and fusion of GFP (green fluorescent protein) to its SOC (small outer capsid) protein, was constructed, and it was shown to be able to detect E. coli K12 sensitively within several hours. However, because the host range of T4 phage to E. coli present in sewage water and sea water is narrow, this phage cannot be used to detect E. coli in environmental water. Two phages named IP008 and IP052, which have a broad host range to E. coli present in sewage influent, were screened from sewage influent. Mixture of these two phages produced clear plaques on 50% of E. coli screened from sewage influent. To use these phages as a tool for detection of E. coli, gfp was inserted into gene e, which encodes a lytic enzyme, and thus lytic-activity-suppressed phages were constructed (IP008e-/GFP and IP052e-/GFP). However, the fluorescent intensity of E. coli cells infected with IP008e-/GFP and IP052e-/GFP was not enough for visualization of the cell. Therefore, in addition to the insertion of gfp into gene e, fusion of GFP to SOC of IP008e-/GFP and IP052e-/GFP was conducted to produce IP008e-/2xGFP and IP052e-/2xGFP. E. coli cells infected with IP008e-/2xGFP and IP052e-/2xGFP showed much stronger fluorescence intensity than E. coli cells infected by IP008e-/GFP and IP052e-/GFP. It is anticipated that, using these GFP-labeled phages, a broad range of E. coli present in sewage influent water can be detected rapidly.