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At least 19 recordsLinked to original sources
PubMed1979

Bacteriophage fd gene II-protein. II. Specific cleavage and relaxation of supercoiled RF from filamentous phages.

Bacteriophage fd gene II-protein was characterized as an endonuclease which specifically nicked supercoiled replicative form (RF) of filamentous phages in the viral strand. No other supercoiled DNAs tested were attacked by the enzyme, nor were doubly closed fd RF in the relaxed state nor phage fd single strands. Maximal activity was found at pH 8.5 and 80 mM KCl using fd RFI of physiological superhelicity. Mg2+, but no other cofactor, was required for the cleavage reaction. A sealing activity was found to be associated with the enzyme. At a higher concentration of Mg2+ up to 40% of the reaction products were found as doubly closed relaxed fd RF. The protein was not found to be tightly attached to the cleaved strand.

Coliphages↗
PubMed2025

Filamentous cheater phages drive bacterial and phage populations to lower fitness.

Many bacteria carry phage genome(s) in their chromosome, which intertwines the fitness of the bacterium and the phage. Most Pseudomonas aeruginosa strains carry filamentous phages called Pf that establish chronic infections and do not require host lysis to spread. However, spontaneous mutations in the Pf repressor gene (pf5r) can allow extreme phage production that slows bacterial growth and increases cell death, violating an apparent détente between bacterium and phage. We observed this paradoxical outcome in an evolution experiment with P. aeruginosa in media simulating nutrients from the cystic fibrosis airway. Bacteria containing pf5r mutant phage grow to a lower density but directly outcompete their ancestor and convert them into pf5r mutants via phage superinfection. Reduced fitness therefore spreads throughout the bacterial population, driven by weaponized Pf. Yet high intracellular phage replication facilitates another evolutionary conflict: "cheater miniphages" lacking capsid genes and the superinfection exclusion gene (pfsE) invade populations of full-length phages within cells. Although bacteria containing both full-length phages and miniphages are most immune to superinfection by limiting the Pf receptor, this hybrid vigor is extremely unstable, as a classic Tragedy of the Commons scenario ensues that causes complete prophage loss. The entire cycle - from phage hyperactivation to miniphage invasion to prophage loss - can occur within 24h, showcasing rapid coevolution between bacteria and their filamentous phages. This study demonstrates that P. aeruginosa, and potentially many other bacterial species that carry filamentous prophages, risk being exploited by these phages in a runaway process that reduces fitness of both host and virus.

Inoviridae↗
PubMed1979

The requirement of nonsense suppression for the development of several phages.

A spontaneous streptomycin-resistant Escherichia coli mutant which is temperature-sensitive for suppression of a nonsense codon was studied for its ability to propagate phages T2, T4D, T5, phi K, f2, MS2, R17, Q beta, lambda as well as filamentous phages fl, fd and M13. Of all phages tested, only the growth of Q beta, lambda, and filamentous phages is inhibited in the mutant at 42 degree C. This selective inhibition suggests that, like Q beta, lambda and filamentous phages also require a read-through proten(s) which results from suppression of a termination codon.

Coliphages↗
PubMed1979

Ultraviolet and laser Raman investigation of the buried tyrosines in fd phage.

The tyrosines of the filamentous phage fd have been found to be inaccessible to solvent by pH titration while monitoring the ultraviolet spectrum or the laser Raman spectrum. The uv spectra suggest that the tyrosines do not become deprotonated unless the phage becomes disrupted. One possible explanation of the Raman spectra is that the tyrosine OH groups are the recipients of hydrogen-bonded protons arising from fairly acidic donors, yet these acidic donors do not become titrated over the pH 7 to 12 range.

Coliphages↗
PubMed2025

Pseudomonas aeruginosa biofilm-deficient mutants undergo parallel adaptation during chronic infection.

Pseudomonas aeruginosa readily adapts to infection by acquiring stable and heritable mutations. Previously, we discovered that the first adaptations in a porcine wound model were rugose small-colony variants (RSCVs) caused by mutations in the wsp operon. These mutants overproduce Pel and Psl biofilm exopolysaccharides that improve defense against host responses. To identify other mechanisms of host adaptation that lead to hyperbiofilm phenotypes, we created a mutant with an activated wsp pathway but unable to produce these exopolysaccharides (ΔwspFΔpelAΔpslBCD). Porcine wounds were infected with this mutant and biopsies were sampled at days 7, 14 and 35. Small colony variants were isolated from the wound, and whole genome sequencing revealed these variants had acquired mutations in genes in lipopolysaccharide and type IV pili biosynthesis, with wzy and pilU genes being most commonly targeted. pilU mutants were associated with a hyperbiofilm phenotype that outcompeted the parental strain, and wzy mutants were associated with a hyperbiofilm phenotype and increased tolerance to host antimicrobial products. We further identified that several variants had acquired large genome deletions that spanned up to 320 consecutive genes and other variants with high copy numbers of Pf6 filamentous phage. Together our results suggest that the hyperbiofilm phenotype is adaptive in chronic infections and that P. aeruginosa has redundant and diverse pathways to generate this phenotype.

adaptation↗
PubMed1975

Asymmetric orientation of a phage coat protein in cytoplasmic membrane of Escherichia coli.

The coat protein of a filamentous phage (M13) enters the cytoplasmic membrane from two directions: from the outside upon infection and from the cell interior late in the viral life cycle prior to phage assembly and extrusion. Binding of 125I-labeled anti-coat protein antibody to spheroplasts or to inverted vesicles was used to assay the orientation of coat protein in the membrane. Both parental and newly synthesized coat protein were found to be exposed on the outer surface of the cytoplasmic membrane. Coat protein in intact infected cells is also accessible to external antibody. Thus two different processes of assembling a protein into membrane, each starting from a different membrane surface, appear to produce similar surface orientations.

Cell Membrane↗
PubMed1978

Isolation and characterization of transducing coliphage fd carrying a kanamycin resistance gene.

The DNA segment (Tn903) with a size of 3100 nucleotide pairs which carries a gene specifying kanamycin resistance derived from a chimeric plasmid pML21 (Hershfield et al., 1976) was transposed to various sites on the filamentous phage fd DNA. Wild type fd can be restored by excision of Tn903 from the resulting hybrid DNA molecule. The fd DNA carrying Tn903 when converted to the mature phage particle, was capable of transducing the kanamycin marker, and its replicative form DNA could be maintained in a bacterial cell like a plasmid.

Coliphages↗
PubMed2026

Mutations in filamentous bacteriophages spark eco-evolutionary feedbacks in Pseudomonas aeruginosa.

Microbial populations strongly shape their environment, which can re-route adaptation toward organism-generated fitness optima. However, the conditions that promote these eco-evolutionary feedbacks are unclear. Here, we used experimental evolution to test whether high population density, by strengthening niche construction, drives eco-evolutionary feedbacks in the bacterial pathogen Pseudomonas aeruginosa (Pa) MPAO1. We tested for adaptation to organism-modified environments by measuring the relative performance of ancestral and endpoint populations in filtrate generated by each evolutionary line sampled across generations. Contrary to expectations, we found that endpoint populations had higher performance than the ancestral strain in filtrate across nearly all evolutionary lines regardless of population density. This was caused by the emergence of hyperactive filamentous bacterio(phage) mutants during experimental passaging that inhibited the ancestral strain but not endpoint populations in modified media. Hyperactive phages emerged from one of two avirulent prophages in MPAO1's genome (Pf4 or Pf6). Hyperactive phages drove the evolution of phage resistance in bacterial populations via mutations in the type IV pilus (TIVP), the phage's binding receptor. In a follow-up experiment, we showed that these TIVP mutations pleiotropically reduced motility and conferred resistance to a TIVP-targeting virulent phage, both of which are important traits for Pa infection and treatment. Overall, this work suggests that filamentous phage evolution can drive eco-evolutionary feedbacks in bacterial populations, causing phenotypic and genetic changes that would not be anticipated from adaptation to the extrinsic environment alone.

Pseudomonas aeruginosa↗
PubMed2026

Dental wastewater reveals a hidden reservoir of oral bacteriophage diversity.

Bacteriophages (phages) are being explored as alternatives or complements to antibiotics because of their ability to selectively kill bacterial pathogens. However, phages that infect many oral bacteria remain undiscovered. Here, we discovered that dental wastewater harbors previously underexplored phage diversity. Viral particles concentrated from dental wastewater displayed diverse morphologies, including abundant filamentous phage-like particles. Deep long-read metagenomic sequencing of concentrated viral particles generated 7.4 billion bases of sequence data and yielded 255 medium- to high-quality viral operational taxonomic units (vOTUs), including 46 predicted complete genomes. Comparison with large phage databases revealed that 63 of these 255 vOTUs had no detectable match, indicating that extensive sequencing of dental wastewater substantially expands the number of potential bacteriophages associated with the human oral microbiome. Host prediction linked many vOTUs to oral-associated bacterial taxa, including species with few or no previously reported phages, such as Porphyromonas gingivalis, Tannerella forsythia, and Candidatus Saccharibacteria. Functional annotation identified diverse genes associated with antiphage defense systems within a subset of vOTUs, suggesting that oral phages may contribute to the movement of genes encoding bacterial immune functions within the oral microbiome. Together, these findings expand the known oral phageome and show that dental wastewater contains a largely untapped diversity of phages.IMPORTANCEThe human oral cavity contains a diverse microbial community, but the bacteriophages (phages) that infect many oral bacteria remain poorly characterized. This gap limits our understanding of how phages shape oral microbial communities. Here, we show that dental wastewater is an underexplored source of oral phage diversity. Deep long-read metagenomic sequencing revealed 255 medium- to high-quality phage operational taxonomic units, many of which are not present in existing oral phage databases. These genomes include predicted phages of periodontal disease-associated bacteria and other oral taxa with few or no known phages. Dental wastewater therefore expands the known human oral phageome and reveals candidate phages linked to bacteria associated with oral health and disease.

Bacteriophages↗
PubMed1978

Insertion of the Tn3 transposon into the genome of the single-stranded DNA phage M13.

The transposable genetic element Tn3, which carries an ampicillin (Ap) resistance determinant, has been translocated from a ColE1-Apr plasmid, RSF2124, to the genome of the filamentous single-stranded DNA phage M13. The site orientation of the inserted element has been determined for one such phage, M13::Tn3-15. The insertion is within the intergenic space separating genes 2 and 4 and containing both the viral strand and complementary strand origins. The lengths of both the filamentous phage and the duplex replicative form (RF) DNA are 1.7--1.8 times those of M13 phage and replicative form DNA. Both plaque formation and transduction of sensitive cells to ampicillin resistance by M13::Tn3-15 are sensitive to purified antibodies to the M13 major coat protein.

Ampicillin↗
PubMed1979

The smoB mutation suppressing cell filamentation and ability to support the multiplication of phage P22 in Salmonella typhimurium.

Isolation and properties of a Salmonella typhimurium mutant smoB are described. The mutation maps at unit 99 of the S. typhimurium chromosome between pyrB and deoC. It suppresses cell filamentation and temperature sensitivity of histidine-constitutive mutants, but does not restore the normal regulatory pattern to the histidine operon. Strains carrying the mutation have greatly reduced ability to support the growth of phage P22, but not of ES18 or Felix O.

Chromosome Mapping↗
PubMed2025

Functional integration of the bacteriophage T4 DNA replication complex: The multiple roles of the ssDNA binding protein (gp32).

Single-stranded DNA binding protein (gp32) serves as the central regulatory component of the multi-subunit T4 bacteriophage DNA replication system by coordinating the system's three functional sub-assemblies, resulting in phage DNA synthesis in T4-infected E. coli cells at the high speeds (~1,000 nts s-1) and the high fidelity (< 1 error per 107 nts) required for genomic function within this cellular eco-system. Gp32 proteins continuously bind to, slide as cooperatively-linked clusters on, and un-bind from transiently exposed single-stranded (ss) DNA templates to carry out their coordinating functions, as well as to protect genomic sequences from nuclease activity and block the formation of interfering secondary structures. The N-terminal domains (NTDs) of gp32 mediate cooperative interactions within ssb clusters, but the roles of the disordered C-terminal domains (CTD) in the nucleation of gp32-ssDNA filaments at ss-dsDNA junctions are less well understood. We here present microsecond-resolved single-molecule F&#xf6;rster resonance energy transfer studies of the initial steps of gp32 assembly on short oligo-deoxythymidine lattices of varying lattice length and polarity near model ss-dsDNA junctions. These data are analyzed to define the molecular steps and related free energy surfaces involved in initiating gp32 cluster formation, which show that the nucleation mechanisms and regulatory interactions driven by gp32 proteins at ss-dsDNA junctions are significantly directed by lattice polarity. We propose a model for the role of the CTDs in orienting gp32 monomers at lattice positions close to ss-dsDNA junctions that suggests how intrinsically disordered CTD domains might facilitate and control non-base-sequence-specific binding in both the nucleation and the dissociation of the gp32-ssDNA filaments involved in phage DNA replication and related processes.

Journal Article↗
PubMed1978

Expression of bacteriophage M13 DNA in vivo. Localization of the transcription initiation and termination signal of the mRNA coding for the major capsid protein.

During the infection cycle of the filamentous bacteriophage M13 a phage specific RNA species is made which selectively directs in vitro the synthesis of the precursor of the major capsid protein encoded by gene VIII. This RNA is unstable (its mean half-life is 11 min) and is made in amounts representing at least 2% of the newly synthesized RN. Nucleotide sequence analysis have indicated that the synthesis of this RNA species is initiated and terminated at the same promoter (G0.18) and termination signal (T0.25) of the M13 genome as the 8S RNA species made in vitro under the direction of M13 replicative form DNA.

Base Sequence↗
PubMed1979

High and low toxin production by a non-toxigenic strain of Clostridium botulinum type C following infection with type C phages of different passage history.

Toxin production in Clostridium botulinum types C and D is governed by specific bacteriophages. Prior passages of a phage controlling type C toxin production caused subsequently lysogenized bacteria to become variably toxigenic. This appears to be one of the causes of the decrease in toxigenicity which is common in some type C and D strains. The morphology of bacteria was also changed from rod-shaped to filamentous by infection with a successively propagated phage.

Bacteriophages↗
PubMed1979

Thermoresistant revertants of an Escherichia coli strain carrying tif-1 and ruv mutations: non-suppressibility of ruv by sfi.

Spontaneous thermoresistant revertants were isolated from Tif1 Ruv- and Tif1 Ruv+ strains of Escherichia coli K-12. They were divided into five groups; backmutants to tif+ and recA structural gene mutants accounted for at least two of these groups. Mutations with an unconditional RecA- phenyotype were detected at a higher frequency in the Tif1 Ruv- strains (65%) than in the Tif1 Ruv+ strains (25%). A third group consisted of revertants exhibiting a RecA- phenotype at low temperature. Revertants with normal recombination ability and UV resistance, but with a thermosensitive defect in propagating lambda bio11 phage, were also isolated (group 4). The alleles responsible for this property were cotransducible with the srl gene, suggesting that they are located at the recA locus. Other revertants, which might carry lex, LEXB, or zab mutations, were UV sensitive and were able to propagate lambda bio11 phage (group 5). The sfi mutation, which suppresses filamentation in the Tif1 and UV-sensitive Lon- strains, does not restore UV resistance of the Ruv- mutant.

Coliphages↗
PubMed1978

Expression of bacteriophage M13 dna in vivo. I. Synthesis of phage-specific RNA and protein in minicells.

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.

Bacterial Proteins↗
PubMed1978

Serological characteristics of pili determined by the plasmids R711b and F0lac.

The plasmids R711b (at present IncX) and F0lac (IncFV) both determine pili morphologically like those of F (IncFI), and confer sensitivity to the F-specific filamentous bacteriophages, but not to the F-specific isometric RNA phages. Detailed serological studies show that the two pilus types are unrelated, and that neither is related to any of the previously defined F pilus serotypes. Adsorption of the isometric RNA phage MS2 to R711b pili occurs in the presence but not in the absence of formalin, which presumably prevents elution of reversibly adsorbed virions. No adsorption occurs with F0lac pili. MS2 multiplication, as measured by titre increase tests in liquid medium, is found with neither plasmid. The two plasmids are not incompatible. These observations indicate that R744b and F0lac are different both from one another and from the plasmids belonging to the incompatibility groups IncFI--IV.

Adsorption↗