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A DNA fragment containing gene VII of bacteriophage M13 has been transcribed and the nucleotide sequence of this 169-nucleotides long transcript was determined by RNA sequencing methods. Additionally, the nucleotide sequence of this gene and parts of its neighbouring genes V and VIII has been determined by the dimethylsulphate-hydrazine technique. The reading frame of gene VII has been established by determining the nucleotide changes occurring in the transcripts of two amber mutants of this gene. From these combined data it is apparent that gene VII is only 99 nucleotides long and is immediately followed by the termination codon UGA. Its initiation codon AUG is separated from gene V by only a single nucleotide. It was noted that between the UGA termination codon of gene VII and the initiation codon of the next gene (gene VIII) there is space for another, hitherto unknown gene. This gene (IX) most probably codes for the small polypeptide ("C-protein") present in mature M13 phage particles.
Nanovaccines co-assemble antigens and adjuvants to elicit robust immune responses but often require complex synthesis and post-modification procedures. Here, a programmable nanovaccine platform based on the M13 bacteriophage is developed for the scalable production of vaccines and single-step modular engineering of adjuvanticity, length, and antigen density. By reprogramming the sequence and size of the noncoding phage genome, the Toll-like receptor 9 activation and the length of the phage are precisely controlled. With a novel molecular engineering approach, the antigen density is tuned from 13.6% to 70.3%. A systematic modulation reveals an optimal adjuvanticity at a constant antigen density for maximum anti-tumor CD8+ T cell response, and vice versa, using the model antigen SIINFEKL. The M13 phage-based nanovaccine induces durable memory immunity lasting over a year. In addition, a 24-fold increase in neoantigen-specific CD8+ T cell frequency is achieved when increasing both the adjuvanticity and antigen density. Furthermore, when combined with anti-PD-1 therapy, the M13 phage-based personalized vaccine eradicates established MC-38 tumors in 75% of treated animals and they develop 100% resistance against tumor invasion when challenged 5 months after treatment. These findings establish M13 phage as a powerful and versatile nanovaccine platform with transformative potential for personalized cancer immunotherapy.
A DNA region of 2750 base pairs encompassing the genes III, VI and I of bacteriophage M13 has been sequenced by the Maxam-Gilbert procedure. By establishing the nucleotide changes introduced by several amber mutations, the coding region and the regulatory signals of each gene have been deduced. The genes appear to span 1275 base pairs (gene III; mol.wt. 44,748) 339 base pairs (gene VI; mol.wt. 12,264) and 1047 base pairs (gene I; mol.wt. 39,500). Their separating non-codogenic regions are extremely short, namely two and one base pair, respectively. The C-terminal end of gene I, however, intrudes 23 nucleotides into gene IV. From the nucleotide sequence it appears that the minor capsid protein of the phage, which is encoded by gene III, is synthesized in a precursor form containing 18 extra amino acids at its N-terminal end. Furthermore, in this capsid protein two clusters of a fourfold repeat of the sequence Glu-Gly-Gly-Gly-Ser are apparent. Gene VI appears to code for a small, extremely hydrophobic polypeptide. Its total hydrophobic amino acids content of 51% suggests that this protein can only function in the host cell membrane.
The interaction of gene V protein from bacteriophage M13 with the self-complementary tetranucleotide d(pC-G-C-G) was studied by 1H and 31P nuclear magnetic resonance. It is shown, using the hydrogen-bonded proton resonances of the Watson-Crick base pairs as a probe, that the protein is able to unwind the small double-helical fragment even at 0 degrees C. Binding of the tetranucleotide causes changes in the aromatic part of the 1H NMR spectrum of the complex, suggesting that aromatic residues, most likely tyrosines, take part in the protein.nucleic-acid interaction. From the 31P NMR spectra of the protein.nucleic-acid complex it follows that the pK value of the 5'-terminal phosphate is lower than for the free nucleic acid species. Moreover, it could be shown that the exchange of the protein between nucleic acid substrates is fast. Combination of these measurements has led us to derive a mechanism of unwinding on the tetranucleotide level. To a large extent the unwinding is determined by fluctuations in the double-helical DNA structure.
It is shown that photo-CIDNP effects (CIDNP, chemically induced dynamic nuclear polarization) can be generated in the 360-MHz proton NMR spectrum of gene-5 protein from bacteriophage M13. This technique is used to determine the number of tyrosyl residues at the surface of the protein and to assign the resonances from the 3,5-ring protons of these residues. The DNA-binding site of the protein is investigated by formation of complexes with oligonucleotides. Complex formation leads to shifting and/or quenching of the photo-CIDNP emission signals of the surface tyrosines, implying that they are involved in DNA-protein interaction. These experiments are complemented by studying the complex formation of Lys-Tyr-Lys to poly(A).
It is demonstrated that after infection of the appropriate minicell-producing strain of Escherichia coli with the filamentous bacteriophage M13, its replicative form DNA is segregated into minicells. Consequently these minicells have acquired the capability to direct the synthesis of phage-specific RNA and protein. Comparision of the electrophoretic mobilities of phage-specific RNA species made in vitro with those made in M13 replicative form DNA harbouring minicells, have indicated that almost all in vitro synthesized G-start RNAs have an equivalent among the in vivo synthesized RNA products. Furthermore it could be demonstrated that in M13 replicative form DNA harbouring minicells the phage-specific proteins encoded by genes III, IV, V and VIII are made. In addition the synthesis of a phage-specific polypeptide (molecular weight approx. 3000) co-migrating with the recently discovered capsid protein (designated C-protein) could be demonstrated. The meaning of these results for the resolution of the regulatory mechanisms operative during the life cycle of this phage will be discussed.
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Prior to virus assembly, the major coat protein of coliphage M13 is an integral protein of the host cytoplasmic membrane. Coat protein synthesized in vitro is initially made with an NH2-terminal "leader peptide" of 23 amino acids and is termed "procoat." We now report that procoat is a biosynthetic precursor of coat protein in vivo. Conversion of procoat to coat occurs within 30 sec in cells infected with wild-type virus. This proteolytic processing is delayed in cells infected by M13 mutants (in genes 1, 5, or 7) that are defective in virus assembly. Pulse--chase experiments in combination with subcellular fractionation show that procoat is synthesized in a soluble form in the cytoplasm and is then incorporated into the cytoplasmic membrane, where it is converted to coat protein. This finding is supported by the observation that procoat is synthesized exclusively by polysomes that are not membrane bound. These results are interpreted in terms of the "membrane-triggered folding" hypothesis of membrane protein assembly.
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A functional dnaZ product, known to be essential for host DNA polymerization and for the synthesis of M13 and phiX174 parental replicative-form (RF) DNA, is required also for RF replication and single-strand synthesis by both of these phages. All three stages of M13 and phiX174DNA replication (parental RF formation, RF replication, and single-strand synthesis) are inhibited in dnazts mutants at elevated temperatures. In addition, the thermolabile step in M13 parental RF formation appears to occur after RNA priming;i.e., the synthesis of M13 RF DNA proceeded when a dnaZts mutant, infected at a nonpermissive temperature, was transferred to a permissive temperature in the presence of rifampin.
Previous studies have shown that M13 single-strand synthesis is inhibited at nonpermissive temperature in Escherichia coli polAexl, a temperature-sensitive mutant defective in the 5' leads to 3' exonuclease activity of polymerase I (T.-C. Chen and D. S. Ray, J. Mol. Biol. 106:589-604, 1976). Under these conditions the formation of covalently closed replicative form (RF) molecules is greatly reduced, and miniature forms of RF accumulate. We show here that the accumulation of mini-RFs is the consequence of a differential inhibition of the replication of unit-length phage and preexisting miniphage rather than a de novo production of miniphage. Mini-RFs do not accumulate even after as many as nine cycles of growth in the mutant host infected only with unit-length phage. Mixed infections of the mutant host with plaque-purified unit-length phage and a single cloned miniphage show that discontinuities in the mini-RFs are joined with higher efficiency than are those contained in unit-length RFs. After a shift to nonpermissive temperature during single-strand synthesis in cells infected with plaque-purified phage alone, M13 RFs are found largely as RFII molecules (RF form having one or more single-strand discontinuities) containing only a single discontinuity in the viral strand. The inability of the accumulated unit-length RFII molecules to actively replicate may reflect the presence of either a bound protein or RNA primer on the 5' terminus of the viral strand and provides further support for the existence of distinct initiation and termination events in the synthesis of the viral strand.
Bacteriophage M13-infected carrier cells were shown to be unstable to prolonged growth under all conditions. Carrier Hfr cells were transferred in dilute culture (10(3) to 10(4)/ml), where reinfection was impossible and the physiology of the cell was minimally altered. After an initial period of about 10 generations, during which all cells in the culture remained infected, there was exponential decay in the proportion of infected cells in the culture. Uninfected cells that appeared were M13 sensitive. Hfr and F' males were also transferred serially at high cell densities (10(7) to 10(9)/ml), where high levels of phage should permit reinfection. The proportion of phage-producing cells in the cultures remained constant for 7 to 15 generations and then dropped exponentially on further growth. Non-phage-producing cells appearing in the culture were refractory to infection by M13; in some cases cells scored as non-phage producers for 20 generations were observed to produce phage on further growth in liquid culture. F'trp+ males infected with M13 lost trp+ function almost immediately; this was not regained in these experiments. Infected cells grown in dilute culture or on plates remained infected longer, produced more PFU per cell for a longer period, and retained trp+ function in F'trp+ males for over 90 generations. Non-phage-producing cells that appeared were sometimes phage resistant, sometimes phage sensitive. The existence of a phage-related material accumulating at high cell densities and affecting expression of free episomes, episomal expression in Hfr males, and phage synthesis itself is suggested.
Screening of a collection of temperature-sensitive mutants of Escherichia coli for defects in phospholipid metabolism led to the isolation of a mutant deficient in cardiolipin synthesis. The defective gene, named cls, is closely linked to the trp marker and maps at about Minute 27 on the E. coli chromosome. After transfer of cls to a defined genetic background by transduction, the mutant has the following properties as compared to an isogenic wild type. Exponentially growing cells show a reduction in cardiolipin content by a factor of at least 15 (less than 0.2 mol % of the total phospholipids). A crude membrane fraction derived from the mutant is unable to synthesize cardiolipin from phosphatidylglycerol in vitro. The mutant has no distinctive phenotype regarding its growth properties, membrane-associated respiratory functions, or the ability to insert bacteriophage M13 coat protein into the cell envelope. The cls mutation confers a 5-times reduction in the turnover of the phosphate moiety of phosphatidylglycerol.
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