Characterization of Lactococcus lactis phage antigens.

Phage phi 197 is representative of a widespread lactococcal phage group characterized by a particular morphology (prolate head with a noncontractile tail). In order to develop an immunoenzymatic phage detection test, fusion proteins containing beta-galactosidase fused to epitopes of phage phi 197 structural proteins were constructed by cloning random DNA fragments from the phage genome upstream of a lacZ gene on a plasmid vector. Recombinant plasmids containing certain fragments encoded the synthesis of fusion proteins which react with polyclonal antibodies against the phage and confer a Lac+ phenotype on Escherichia coli. Three different epitopes were represented; phage-specific DNA fragments encoding these epitopes were mapped at three locations on the phage genome, and their nucleotide sequences were determined. Two fused phage antigens were conformational epitopes, whereas the phage epitope of protein encoded by the recombinant plasmid designated pOA17 was a denaturation-resistant epitope. This epitope was very immunogenic. Protein encoded by plasmid pOA17 was synthesized in large amounts from a strong promoter. Antibodies raised against this hybrid protein were used to identify the 46-kDa minor phage protein which provides the epitope. Antibody cross-reactivity of phages related to phi 197 showed that this epitope is well conserved in this genetic group.     

Mapping of linear epitopes recognized by monoclonal antibodies with gene-fragment phage display libraries

Epitope mapping with mono- or polyclonal antibodies has so far been done either by dissecting the antigens into overlapping polypeptides in the form of recombinantly expressed fusion proteins, or by synthesizing overlapping short peptides, or by a combination of both methods. Here, we report an alternative method which involves the generation of random gene fragments of approximately 50–200 by in length and cloning these into the 5 terminus of the protein III gene of fd phages. Selection for phages that bind a given monoclonal antibody and sequencing the DNA inserts of immunopositive phages yields derived amino acid sequences containing the desired epitope. A monoclonal antibody (mAb 215) directed against the largest subunit of Drosophila RNA polymerase II (RPB215) was used to map the corresponding epitope in a fUSE5 phage display library made of random DNA fragments from plasmid DNA containing the entire gene. After a single round of panning with this phage library, bacterial colonies were obtained which produced fd phages displaying the mAb 215 epitope. Sequencing of single-stranded phage DNA from a number of positive colonies (recognized by the antibody on colony immunoblots) resulted in overlapping sequences all containing the 15mer epitope determined by mapping with synthetic peptides. Similarly, we have localized the epitopes recognized by a mouse monoclonal antibody directed against the human p53 protein, and by a mouse monoclonal antibody directed against the human cytokeratin 19 protein. Identification of positive colonies after the panning procedure depends on the detection system used (colony immunoblot or ELISA) and there appear to be some restrictions to the use of linker-encoded amino acids for optimal presentation of epitopes. A comparison with epitope mapping by synthetic peptides shows that the phage display method allows one to map linear epitopes down to a size only slightly larger than the true epitope. In general, our phage display method is faster, easier, and cheaper than the construction of overlapping fusion proteins or the use of synthetic peptides, especially in cases where the antigen is a large polypeptide such as the 215 kDa subunit of eukaryotic RNA polymerase II.     

Phage display as a novel screening method to identify extracellular proteins

Extracellular proteins are involved in many diverse and essential cell functions and in pathogenic bacteria, and they may also serve as virulence factors. Therefore, there is a need for methods that identify the genes encoding this group of proteins in a bacterial genome. Here, we present such a method based on the phage display technology. A novel gene III-based phagemid vector, pG3DSS, was constructed that lacks the signal sequence which normally orientates the encoded fusion protein to the Escherichia coli cell membrane, where it is assembled into the phage particle. When randomly fragmented DNA is inserted into this vector, only phagemids containing an insert encoding a signal sequence will give rise to phage particles displaying a fusion protein. These phages also display an E-tag epitope in fusion with protein III, which enables isolation of phages displaying a fusion protein, using antibodies against the epitope. From a library constructed from Staphylococcus aureus chromosomal DNA, genes encoding secreted as well as transmembrane proteins were isolated, including adhesins, enzymes and transport proteins.     

Cloning and DNA sequence analysis of two abortive infection phage resistance determinants from the lactococcal plasmid pNP40.

The lactococcal plasmid pNP40, from Lactococcus lactis subsp. lactis biovar diacetylactis DRC3, confers complete resistance to the prolate-headed phage phi c2 and the small isometric-headed phage phi 712 in L. lactis subsp. lactis MG1614. A 6.0-kb NcoI fragment of pNP40 cloned in the lactococcal Escherichia coli shuttle vector pAM401 was found to confer partial resistance to phi 712. Subcloning and deletion analysis of the recombinant plasmid pPG01 defined a 2.5-kb ScaIHpaI fragment as conferring phage insensitivity. Sequence analysis of this region confirmed the presence of two overlapping open reading frames (ORFs). Further subcloning of pNP40 to characterize the resistance determinant active against phi c2 identified a 5.6-kb EcoRV fragment of pNP40 which, when cloned in pAM401, conferred partial resistance to both phi c2 and phi 712. Subcloning and deletion analysis of the recombinant plasmid pCG1 defined a 3.7-kb EcoRV-XbaI fragment as encoding phage insensitivity. DNA sequence analysis of this region revealed the presence of a single complete ORF. The introduction of a frameshift mutation at the unique BglII site within this ORF disrupted the phage resistance phenotype, confirming that this ORF is responsible for the observed phage insensitivity. The mechanisms encoded by pPG01 and pCG1 in L. lactis subsp. lactis MG1614 conformed to the criteria defining abortive infection and were designated AbiE and AbiF, respectively. Analysis of the phage DNA content of phi 712-infected hosts containing AbiF demonstrated that it inhibited the rate of phage DNA replication, while AbiE had little effect on phage DNA replication, suggesting a later target of inhibition. The predicted protein product of abiF shows significant homology to the products of two other lactococcal abortive infection genes, abiD and abiD1.     

Phenogenetic characterization of a group of giant Phi KZ-like bacteriophages of Pseudomonas aeruginosa

A comparative study was made of a group of Pseudomonas aeruginosa virulent giant DNA bacteriophages similar to phage phi KZ in several genetic and phenotypic properties (particle size, particle morphology, genome size, appearance of negative colonies, high productivity, broad spectrum of lytic activity, ability to overcome the suppressing effect of plasmids, absence of several DNA restriction sites, capability of general transduction, pseudolysogeny). We have recently sequenced the phage phi KZ genome (288,334 bp) [J. Mol. Biol., 2002, vol. 317, pp. 1-19]. By DNA homology, the phages were assigned to three species (represented by phage phi KZ, Lin68, and EL, respectively) and two new genera (phi KZ and EL). Restriction enzyme analysis revealed the mosaic genome structure in four phages of the phi KZ species (phi KZ, Lin21, NN, and PTB80) and two phages of the EL species (EL and RU). Comparisons with respect to phage particle size, number of structural proteins, and the N-terminal sequences of the major capsid protein confirmed the phylogenetic relatedness of the phages belonging to the phi KZ genus. The origin and evolution of the phi KZ-like phages are discussed. Analysis of protein sequences encoded by the phage phi KZ genome made it possible to assume wide migration of the phi KZ-like phages (wandering phages) among various prokaryotes and possibly eukaryotes. Since the phage phi KZ genome codes for potentially toxic proteins, caution must be exercised in the employment of large bacteriophages in phage therapy.     

Cloning of restriction fragments of DNA from staphylococcal bacteriophage phi 11.

EcoRI fragments of Staphylococcus aureus bacteriophage phi 11 DNA were cloned in vector plasmid pSA2100 in S. aureus. The clones were analyzed in marker rescue experiments with suppressor- and temperature-sensitive mutants of phi 11 to correlate the genetic and physical map. Several mutants could be identified on the physical map, and a clone containing fragment EcoRI-B of phi 11 DNA expressed immunity to phage infection. In addition, it was found that recombinant plasmids containing phi 11 DNA sequences can be transferred by high-frequency transduction after phage phi 11 infection of host cells.     

In vivo genetic exchange of a functional domain from a type II A methylase between lactococcal plasmid pTR2030 and a virulent bacteriophage.

The conjugative plasmid pTR2030 confers bacteriophage resistance to lactococci by two independent mechanisms, an abortive infection mechanism (Hsp+) and a restriction and modification system (R+/M+). pTR2030 transconjugants of lactococcal strains are used in the dairy industry to prolong the usefulness of mesophilic starter cultures. One bacteriophage which has emerged against a pTR2030 transconjugant is not susceptible to either of the two defense systems encoded by the plasmid. Phage nck202.50 (phi 50) is completely resistant to restriction by pTR2030. A region of homology between pTR2030 and phi 50 was subcloned, physically mapped, and sequenced. A region of 1,273 bp was identical in both plasmid and phage, suggesting that the fragment had recently been transferred between the two genomes. Sequence analysis confirmed that the transferred region encoded greater than 55% of the amino domain of the structural gene for a type II methylase designated LlaI. The LlaI gene is 1,869 bp in length and shows organizational similarities to the type II A methylase FokI. In addition to the amino domain, upstream sequences, possibly containing the expression signals, were present on the phage genome. The phage phi 50 fragment containing the methylase amino domain, designated LlaPI, when cloned onto the shuttle vector pSA3 was capable of modifying another phage genome in trans. This is the first report of the genetic exchange between a bacterium and a phage which confers a selective advantage on the phage. Definition of the LlaI system on pTR2030 provides the first evidence that type II systems contribute to restriction and modification phenotypes during host-dependent replication of phages in lactococci.     

MAPPING OF HIV-1 GAG EPITOPES RECOGNIZED BY POLYCLONAL ANTIBODIES USING GENE-FRAGMENT PHAGE DISPLAY SYSTEM

Phage display has emerged as a powerful technique for mapping epitopes recognised by monoclonal and polyclonal antibodies. We have recently developed a simple gene-fragment phage display system and have shown its utility in mapping epitope recognised by a monoclonal antibody. In the present study, we have employed this system in mapping epitopes recognised by polyclonal antibodies raised against HIV-1 capsid protein, p24 which is derived from proteolytic cleavage of Gag polyprotein. HIV-1 gag DNA was fragmented by DNase I and the fragments (50–250 bp) were cloned into gene-fragment phage display vector to construct a library of phages displaying peptides. This phage library was used for affinity selection of phages displaying epitopes recognised by rabbit anti-p24 polyclonal antibodies. Selected phages contained sequences from two discrete regions of p24, demonstrating the presence of two antigenic regions.

The DNA sequences encoding these regions were also cloned and expressed as GST fusion proteins. The immunoreactivity of these epitopes as GST fusion proteins, or as phage-displayed peptides, was comparable in ELISA system using same anti-p24 polyclonal antibodies. The results indicate that the gene-fragment based phage display system can be used efficiently to identify epitopes recognised by polyclonal antibodies, and phage displayed epitopes can be directly employed in ELISA to detect antibodies.     

Mapping of hiv-1 Gag epitopes recognized by polyclonal antibodies using gene-fragment phage display system.

Phage display has emerged as a powerful technique for mapping epitopes recognised by monoclonal and polyclonal antibodies. We have recently developed a simple gene-fragment phage display system and have shown its utility in mapping epitope recognised by a monoclonal antibody. In the present study, we have employed this system in mapping epitopes recognised by polyclonal antibodies raised against HIV-1 capsid protein, p24 which is derived from proteolytic cleavage of Gag polyprotein. HIV-1 gag DNA was fragmented by DNase I and the fragments (50-250 bp) were cloned into gene-fragment phage display vector to construct a library of phages displaying peptides. This phage library was used for affinity selection of phages displaying epitopes recognised by rabbit anti-p24 polyclonal antibodies. Selected phages contained sequences from two discrete regions of p24, demonstrating the presence of two antigenic regions. The DNA sequences encoding these regions were also cloned and expressed as GST fusion proteins. The immunoreactivity of these epitopes as GST fusion proteins, or as phage-displayed peptides, was comparable in ELISA system using same anti-p24 polyclonal antibodies. The results indicate that the gene-fragment based phage display system can be used efficiently to identify epitopes recognised by polyclonal antibodies, and phage displayed epitopes can be directly employed in ELISA to detect antibodies.     

Overproduction and purification of the connector protein of Bacillus subtilis phage phi 29.

A phi 29 DNA fragment containing genes 10 and 11, coding for the connector protein and the lower collar protein, respectively, has been cloned in the pBR322 derivative plasmid pKC30 under the control of the PL promoter of phage lambda. Two polypeptides with the electrophoretic mobility of proteins p10 and p11 were labelled with 35S-methionine after heat induction. The proteins were characterized as p10 and p11 by radioimmunoassay and they represented about 10% and 7%, respectively, of the total E. coli protein after 4 hours of induction. These proteins represent less than 1% of the B. subtilis protein in phi 29-infected cells. Protein p10 has been highly purified from the E. coli cells carrying the recombinant plasmid. Antibodies raised against the purified protein p10 reacted with the connector protein produced in phi 29-infected B. subtilis.     

Mechanism of replication of bacteriophage phi X174. XXII. Site-specific mutagenesis of the A* gene reveals that A* protein is not essential for phi X174 DNA replication

The A and A* proteins of phage phi X174 are encoded in the same reading frame in the viral genome; the smaller A protein is the result of a translational start signal with the A gene. To differentiate their respective functions, oligonucleotide-directed site-specific mutagenesis was used to change the ATG start codon of the phi X 174 A* gene, previously cloned into pCQV2 under lambda repressor control, into a TAG stop codon. The altered A gene was then inserted back into phi X replicative form DNA to produce an amber mutant, phi XamA*. Two different Escherichia coli amber suppressor strains infected with this mutant produced viable progeny phage with only a slight reduction in yield. In Su+ cells infected with phi XamA*, phi X gene A protein, altered at one amino acid, was synthesized at normal levels; A* protein was not detectable. These observations indicate that the A* protein increases the replicative efficiency of the phage, perhaps by shutting down host DNA replication, but is not required for replication of phi X174 DNA or the packaging of the viral strand under the conditions tested.     

Molecular structure of hybrid phage phi80hy43

The phage hybrid phi80hy43 derived from a vegetative cross phi80cIhlambda x lambdacIc17 was constructed for discrimination phi80mono- and polylysogens. Molecular structure of this hybrid was established using heteroduplex analysis and restriction endonuclease EcoRI. It is found that the hybrid phi80hy43 represents a phage phi80 containing a foreign piece of DNA between genes cI and 0. The length of this piece of DNA comprises 0.7%, corresponding to the length of cy-cII region of th phage lambda. So it is believed that the hybrid phi80hy43 was formed due to insertion of the lambdacy region with the mutation c17 into phi80hlambda phage genome.     

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.     

The restriction mapping of c gene deletions in Streptomyces bacteriophage phi C31 and their use in cloning vector development

In addition to 20 previously mapped restriction sites in the DNA of phi C31, we have determined eight sites for SphI, four for EcoRV, and two for SstII; there are none for BglII or SstI. Nine sites were in a 12-kb segment of DNA containing no previously mapped sites. Deletions causing clear-plaque morphology were located in this part of the DNA, in a 3-kb interval between an EcoRV and an SphI site at the centre of the DNA molecule. One of the deletions (delta C3) was obtained in a previously described phi C31c+::vph (viomycin phosphotransferase) derivative containing two PstI sites separated by 3.9-kb of inessential DNA. After in vitro PstI treatment, plaque-forming phages lacking the 3.9-kb fragment were obtained from the c+ phage but not from its delta C3 derivative. Thus a 36.2-kb genome, but not one of 34.4 kb, was able to give infectious virions. PstI-generated DNA fragments of up to 8 kb can be inserted in vitro into the delta C3 derivative with retention of the vph selective marker. With the insertion of a 6.03-kb PstI fragment of plasmid SCP2, the latter phage became a potential vector (with loss of vph) for BamHI-generated DNA fragments of up to 9 kb. In the course of this work, several ClaI sites in phi C31::pBR322 bifunctional replicons were shown to be lost when the DNA was propagated in a dam+ Escherichia coli strain. This will allow the use of such replicons for the cloning of ClaI-generated DNA fragments of up to 6.7 kb.     

Construction and characterization of recombinant phage phi 105 d(Cmrmet) for cloning in Bacillus subtilis

A 1.6 kb fragment of DNA of plasmid pBD64, obtained after partial digestion with HpaII, carrying a chloramphenicol-resistance determinant and a single site for the enzyme Bg/II, was inserted into the genome of defective phage phi 105 d/ys. Two types of phage were subsequently isolated and both transduced cells of Bacillus subtilis to chloramphenicol resistance. One type contained 26 kb and the other 32 kb of DNA. Bacillus subtilis chromosomal DNA fragments generated by cleavage with Bg/II were ligated into the unique Bg/II site within the smaller phage genome. A specialized transducing phage was isolated which carried the metC gene on a 6 kb Bg/II fragment. This phage, denoted phi 105 d(Cmrmet), transduced B. subtilis strain MB79 pheA12 metC3 to Met+ and to chloramphenicol resistance, and the metC3 mutation was complemented in transductants.     

Cloning of the uvrD gene of E. coli and identification of the product

The uvrD gene has been cloned from Escherichia coli chromosomal DNA into phage lambda, cosmid, and low-copy-number plasmid vectors. Comparison of the proteins encoded by the cloned fragments with those encoded by fragments in which the uvrD gene is inactivated by transposon insertion or by deletion shows that the uvrD gene product is a protein of Mr = 73000.     

Genetic analysis of chromosomal regions of Lactococcus lactis acquired by recombinant lytic phages

Recombinant phages are generated when Lactococcus lactis subsp. lactis harboring plasmids encoding the abortive type (Abi) of phage resistance mechanisms is infected with small isometric phages belonging to the P335 species. These phage variants are likely to be an important source of virulent new phages that appear in dairy fermentations. They are distinguished from their progenitors by resistance to Abi defenses and by altered genome organization, including regions of L. lactis chromosomal DNA. The objective of this study was to characterize four recombinant variants that arose from infection of L. lactis NCK203 (Abi(+)) with phage phi31. HindIII restriction maps of the variants (phi31.1, phi31.2, phi31.7, and phi31.8) were generated, and these maps revealed the regions containing recombinant DNA. The recombinant region of phage phi31.1, the variant that occurred most frequently, was sequenced and revealed 7.8 kb of new DNA compared with the parent phage, phi31. This region contained numerous instances of homology with various lactococcal temperate phages, as well as homologues of the lambda recombination protein BET and Escherichia coli Holliday junction resolvase Rus, factors which may contribute to efficient recombination processes. A sequence analysis and phenotypic tests revealed a new origin of replication in the phi31.1 DNA, which replaced the phi31 origin. Three separate HindIII fragments, accounting for most of the recombinant region of phi31.1, were separately cloned into gram-positive suicide vector pTRK333 and transformed into NCK203. Chromosomal insertions of each plasmid prevented the appearance of different combinations of recombinant phages. The chromosomal insertions did not affect an inducible prophage present in NCK203. Our results demonstrated that recombinant phages can acquire DNA cassettes from different regions of the chromosome in order to overcome Abi defenses. Disruption of these regions by insertion can alter the types and diversity of new phages that appear during phage-host interactions.     

Organization of the early region of bacteriophage phi 80. Genes and proteins

An EcoRI segment containing the early region of bacteriophage phi 80 DNA that controls immunity and lytic growth was identified as a segment whose presence on a plasmid prevented growth of infecting phi 80cI phage. The nucleotide sequence of the segment (EcoRI-F) and adjacent regions was determined. Based on the positions of amber mutations and the sizes of some gene products, the reading frames for five genes were identified. From the relative locations of these genes in the genome, the properties of some isolated gene products, and the analysis of the structures of predicted proteins, the following phi 80 to lambda analogies are deduced: genes cI and cII to their lambda namesakes; gene 30 to cro; gene 15 to O; and gene 14 to P. An amber mutation by which gene 16 was defined is a nonsense mutation in the frame for gene 15 protein, excluding the presence of gene 16. An amber mutation in gene 14 or 15 inhibits phage DNA synthesis, as is the case with their lambda analogues, gene O or P. Some characteristics of proteins from the early region predicted from their primary structures and their possible functions are discussed.     

Structural organization of pLP1, a cryptic plasmid from Lactobacillus plantarum CCM 1904

To construct shuttle vectors based on an endogenous replicon, we isolated a small cryptic plasmid (pLP1) from Lactobacillus plantarum CCM 1904. The nucleotide sequence (2093 bp, 38.25 GC mol%) revealed one major open reading frame encoding for a 317 amino acid protein (Rep). Comparisons with proteins encoded by other Gram-positive bacteria plasmids strongly suggest that the protein encoded by pLP1 has a replicative role. The presence of a consensus sequence including a tyrosine residue known to be the replication protein binding site to the DNA (in phage phi X174) strengthens this hypothesis. The DNA sequence contains also a sequence similar to the pC194 origin nick sequence, which initiates the plasmid replication at the plus origin, characteristic of plasmids which replicate following a rolling circle mechanism via single-stranded DNA intermediates. A set of 13 direct repeats of 17 bp could be involved in the expression of the incompatibility or in the copy number control as in the other plasmids. A promoter sequence located at the rep 5' region has been identified and is functional in Bacillus subtilis.