Phage-like Particle Research Articles

Structure and proposed DNA delivery mechanism of a marine roseophage

Tailed bacteriophages (order, Caudovirales) account for the majority of all phages. However, the long flexible tail of siphophages hinders comprehensive investigation of the mechanism of viral gene delivery. Here, we report the atomic capsid and in-situ structures of the tail machine of the marine siphophage, vB_DshS-R4C (R4C), which infects Roseobacter. The R4C virion, comprising 12 distinct structural protein components, has a unique five-fold vertex of the icosahedral capsid that allows genome delivery. The specific position and interaction pattern of the tail tube proteins determine the atypical long rigid tail of R4C, and further provide negative charge distribution within the tail tube. A ratchet mechanism assists in DNA transmission, which is initiated by an absorption device that structurally resembles the phage-like particle, RcGTA. Overall, these results provide in-depth knowledge into the intact structure and underlining DNA delivery mechanism for the ecologically important siphophages.

A Designer Nanoparticle Platform for Controlled Intracellular Delivery of Bioactive Macromolecules: Inhibition of Ubiquitin-Specific Protease 7 in Breast Cancer Cells.

Biological therapeutics represent an increasing and critical component of newly approved drugs; however, the inability to deliver biologics intracellularly in a controlled manner remains a major limitation. We have developed a semi-synthetic, tunable phage-like particle (PLP) platform derived from bacteriophage λ. The shell surface can be decorated with small-molecule, biological and synthetic moieties, alone or in combination and in defined ratios. Here, we demonstrate that the platform can be used to deliver biological macromolecules intracellularly and in a controlled manner. Ubiquitin-specific protease 7 (USP7) is a deubiquitinating enzyme that has been widely recognized as an ideal target for the treatment of a variety of cancers. Recently, UbV.7.2, a novel biologic derived from the ubiquitin scaffold, was developed for inhibition of USP7, but issues remain in achieving efficient and controlled intracellular delivery of the biologic. We have shown that decoration of PLPs with trastuzumab (Trz), a HER2-targeted therapeutic used in the treatment of various cancers, results in specific targeting and uptake of Trz-PLPs into HER2-overexpressing breast cancer cells. By simultaneously decorating PLPs with Trz and UbV.7.2, we now show that these particles are also internalized by HER2-positive cells, thus providing a means for intracellular delivery of the biologic in a controlled fashion. Internalized particles retain USP7 inhibition activity of UbV.7.2 and alter the metabolic and proteomic landscapes of these cells. This study demonstrates that the λ "designer nanoparticles" represent a powerful system for the intracellular delivery of biologics in a defined dose.

Phage-like particle vaccines are highly immunogenic and protect against pathogenic coronavirus infection and disease

The response by vaccine developers to the COVID-19 pandemic has been extraordinary with effective vaccines authorized for emergency use in the United States within 1 year of the appearance of the first COVID-19 cases. However, the emergence of SARS-CoV-2 variants and obstacles with the global rollout of new vaccines highlight the need for platforms that are amenable to rapid tuning and stable formulation to facilitate the logistics of vaccine delivery worldwide. We developed a “designer nanoparticle” platform using phage-like particles (PLPs) derived from bacteriophage lambda for a multivalent display of antigens in rigorously defined ratios. Here, we engineered PLPs that display the receptor-binding domain (RBD) protein from SARS-CoV-2 and MERS-CoV, alone (RBDSARS-PLPs and RBDMERS-PLPs) and in combination (hCoV-RBD PLPs). Functionalized particles possess physiochemical properties compatible with pharmaceutical standards and retain antigenicity. Following primary immunization, BALB/c mice immunized with RBDSARS- or RBDMERS-PLPs display serum RBD-specific IgG endpoint and live virus neutralization titers that, in the case of SARS-CoV-2, were comparable to those detected in convalescent plasma from infected patients. Further, these antibody levels remain elevated up to 6 months post-prime. In dose-response studies, immunization with as little as one microgram of RBDSARS-PLPs elicited robust neutralizing antibody responses. Finally, animals immunized with RBDSARS-PLPs, RBDMERS-PLPs, and hCoV-RBD PLPs were protected against SARS-CoV-2 and/or MERS-CoV lung infection and disease. Collectively, these data suggest that the designer PLP system provides a platform for facile and rapid generation of single and multi-target vaccines.

Identification of novel prophage regions in Xenorhabdus nematophila genome and gene expression analysis during phage-like particle induction.

BackgroundEntomopathogenic Xenorhabdus bacteria are endosymbionts of Steinernema nematodes and together they form an insecticidal mutualistic association that infects a wide range of insect species. Xenorhabdus produce an arsenal of toxins and secondary metabolites that kill the insect host. In addition, they can induce the production of diverse phage particles. A few studies have focused on one integrated phage responsible for producing a phage tail-like bacteriocin, associated with an antimicrobial activity against other Xenorhabdus species. However, very little is known about the diversity of prophage regions in Xenorhabdus species.MethodsIn the present study, we identified several prophage regions in the genome of Xenorhabdus nematophila AN6/1. We performed a preliminary study on the relative expression of genes in these prophage regions. We also investigated some genes (not contained in prophage region) known to be involved in SOS bacterial response (recA and lexA) associated with mitomycin C and UV exposure.ResultsWe described two integrated prophage regions (designated Xnp3 and Xnp4) not previously described in the genome of Xenorhabdus nematophila AN6/1. The Xnp3 prophage region appears very similar to complete Mu-like bacteriophage. These prophages regions are not unique to X. nematophila species, although they appear less conserved among Xenorhabdus species when compared to the previously described p1 prophage region. Our results showed that mitomycin C exposure induced an up-regulation of recA and lexA suggesting activation of SOS response. In addition, mitomycin C and UV exposure seems to lead to up-regulation of genes in three of the four integrated prophages regions.

Bacillus pumilus 15.1, a Strain Active against Ceratitis capitata, Contains a Novel Phage and a Phage-Related Particle with Bacteriocin Activity.

A 98.1 Kb genomic region from B. pumilus 15.1, a strain isolated as an entomopathogen toward C. capitata, the Mediterranean fruit fly, has been characterised in search of potential virulence factors. The 98.1 Kb region shows a high number of phage-related protein-coding ORFs. Two regions with different phylogenetic origins, one with 28.7 Kb in size, highly conserved in Bacillus strains, and one with 60.2 Kb in size, scarcely found in Bacillus genomes are differentiated. The content of each region is thoroughly characterised using comparative studies. This study demonstrates that these two regions are responsible for the production, after mitomycin induction, of a phage-like particle that packages DNA from the host bacterium and a novel phage for B. pumilus, respectively. Both the phage-like particles and the novel phage are observed and characterised by TEM, and some of their structural proteins are identified by protein fingerprinting. In addition, it is found that the phage-like particle shows bacteriocin activity toward other B. pumilus strains. The effect of the phage-like particles and the phage in the toxicity of the strain toward C. capitata is also evaluated.

Regulation of Rhodobacter capsulatus gene transfer agent production

Horizontal gene transfer (HGT) enables the spread of antimicrobial resistance, virulence, metabolic and other genes conferring an advantage to the organism. HGT is enhanced in biofilms because of increased cell-cell contact (conjugation), and eDNA in the biofilm matrix causing development of competence and providing material for transformation. Production of the Rhodobacter capsulatus gene transfer agent (RcGTA), another mechanism of HGT, could also increase in biofilm as high cell density increases the proportion of GTA particles produced that encounter a target cell. RcGTA is a phage-like particle that packages ∼4.5 kb pieces of random DNA from the producing cell’s genome and transfers it to a recipient cell. Five loci comprise the RcGTA genome: a 15kb cluster containing most of the RcGTA structural genes, a cell lysis locus, two structural loci encoding head spikes and tail fibres, and a maturation/regulation locus that includes that master regulator, gafA. I assayed RcGTA production using gene transfer bioassays and biofilm using a 96 well plate assay. I will present data showing that deletion of four GTA-related genes, including gafA itself, all lead to reduced biofilm production. All four gene knock-outs also strongly reduce GTA-mediated gene transfer, suggesting GTA production and biofilm are co-regulated. I will also present work to characterize GTA production in biofilms, for example by monitoring the transfer of fluorescent protein genes through confocal microscopy, and assessing how specific regulators control this process. Biofilms are ubiquitous in the environment so studying the spread of antimicrobial resistance genes by GTAs is important.

ZpdN, a Plasmid-Encoded Sigma Factor Homolog, Induces pBS32-Dependent Cell Death in Bacillus subtilis.

Prophages are phage genomes stably integrated into the host bacterium's chromosome and less frequently are maintained as extrachromosomal plasmids. Here we report that the extrachromosomal plasmid pBS32 of Bacillus subtilis encodes a prophage that, when activated, kills the host. pBS32 also encodes both the sigma factor homolog ZpdN that is necessary and sufficient for prophage induction and the protein ComI, which is a potent inhibitor of DNA uptake by natural transformation. We provide evidence that the entire pBS32 sequence may be part of the prophage and thus that competence inhibition may be linked to lysogeny.

Metastatic prostate cancer cell-specific phage-like particles as a targeted gene-delivery system

BackgroundOne of the cardinal requirements for effective therapeutic management of tumors is the selective delivery of cancer drugs to the right site by ligand-decorated nanomedicines. Screening of 2 × 109 clone landscape phage library provides a reliable avenue for generating protein ligands specific for tumor cells. It was shown that selective phage proteins derived from landscape phage libraries against breast and prostate cancer cells are able to navigate drug or siRNA loaded liposomes to corresponding cancer cells with minimal toxicity to non-neoplastic cells. In an alternative platform, glioma cell-specific phage proteins were used for assembling in vivo cancer-specific phage-like particles, named ‘phagemid infective particles’ as targeted gene-delivery vehicles.MethodsTo extend the panel of anticancer cell phages, we have screened a 2 × 109 clone landscape phage library f8/8 to select phage clones specific for metastatic prostate cancer cell PC-3M. The phage clones were characterized for their selective interaction with PC-3M cells using phage capture assay, immunofluorescence microscopy and electron microscopy. A prostate cancer selective phage was converted to phage-like particles harboring emerald green fluorescent protein.ResultsPhage clone EPTHSWAT (designated by the sequence of inserted peptide) was found to be most selective for PC-3M cells and was observed to internalize PC-3M cells as revealed by immunofluorescence microscopy and electron microscopy. Conversion of this phage to phage-like particles harboring emerald green fluorescent protein and the expression of emerald green fluorescent protein in the phage-like particles treated PC-3M cells showed potential of adoption of this phage-like particle in prostate cancer therapeutic gene delivery.ConclusionSuccessful employment of phage-like particles expressing emerald green fluorescent protein genes targeted to prostate cancer cells PC-3M confirms a prospect of their use for targeted delivery of therapeutic genes to cancer cells.

DNA packaging bias and differential expression of gene transfer agent genes within a population during production and release of the Rhodobacter capsulatus gene transfer agent, RcGTA

Rhodobacter capsulatus produces a gene transfer agent (GTA) called RcGTA. RcGTA is a phage-like particle that packages R. capsulatus DNA and transfers it to other R. capsulatus cells. We quantified the relative frequency of packaging for each gene in the genome by hybridization of DNA from RcGTA particles to an R. capsulatus microarray. All genes were found within the RcGTA particles. However, the genes encoding the RcGTA particle were under-packaged compared with other regions. Gene transfer bioassays confirmed that the transfer of genes within the RcGTA structural cluster is reduced relative to those of other genes. Single-cell expression analysis, by flow cytometry analysis of cells containing RcGTA-reporter gene fusion constructs, demonstrated that RcGTA gene expression is not uniform within a culture. This phenomenon was accentuated when the constructs were placed in a strain lacking a putative lysis gene involved in RcGTA release; a small subpopulation was found to be responsible for ∼ 95% of RcGTA activity. We propose a mechanism whereby high levels of RcGTA gene transcription in the most active RcGTA-producing cells cause a reduction in their packaging frequency. This subpopulation's role in producing and releasing the RcGTA particles explains the lack of observed cell lysis in cultures.

Proteomic Analysis and Identification of the Structural and Regulatory Proteins of the Rhodobacter capsulatus Gene Transfer Agent

The gene transfer agent of Rhodobacter capsulatus (GTA) is a unique phage-like particle that exchanges genetic information between members of this same species of bacterium. Besides being an excellent tool for genetic mapping, the GTA has a number of advantages for biotechnological and nanoengineering purposes. To facilitate the GTA purification and identify the proteins involved in GTA expression, assembly and regulation, in the present work we construct and transform into R. capsulatus Y262 a gene coding for a C-terminally His-tagged capsid protein. The constructed protein was expressed in the cells, assembled into chimeric GTA particles inside the cells and excreted from the cells into surrounding medium. Transmission electron micrographs of phosphotungstate-stained, NiNTA-purified chimeric GTA confirm that its structure is similar to normal GTA particles, with many particles composed both of a head and a tail. The mass spectrometric proteomic analysis of polypeptides present in the GTA recovered outside the cells shows that GTA is composed of at least 9 proteins represented in the GTA gene cluster including proteins coded for by Orf's 3, 5, 6-9, 11, 13, and 15.

In vitro assembly of a prohead-like structure of the Rhodobacter capsulatus gene transfer agent

The gene transfer agent (GTA) is a phage-like particle capable of exchanging double-stranded DNA fragments between cells of the photosynthetic bacterium Rhodobacter capsulatus. Here we show that the major capsid protein of GTA, expressed in E. coli, can be assembled into prohead-like structures in the presence of calcium ions in vitro. Transmission electron microscopy (TEM) of uranyl acetate staining material and thin sections of glutaraldehyde-fixed material demonstrates that these associates have spherical structures with diameters in the range of 27–35 nm. The analysis of scanning TEM images revealed particles of mass ∼ 4.3 MDa, representing 101 ± 11 copies of the monomeric subunit. The establishment of this simple and rapid method to form prohead-like particles permits the GTA system to be used for genome manipulation within the photosynthetic bacterium, for specific targeted drug delivery, and for the construction of biologically based distributed autonomous sensors for environmental monitoring.

Host physiological status determines phage-like particle distribution in the lysate

Bacteriophage morphotype diversity and latent period duration upon induction were correlated with the host population growth. The prophages of the lysogenic Vibrio sp. (DSM14379) were induced with mitomycin C in a batch culture with different salinity, substrate concentration or composition, and at different temperatures. Under all experimental conditions, phages were induced and a population of different complete and incomplete phage-like particles was observed in the lysate. Under favorable growth conditions, the phage-like particle community in the lysate was overpopulated with phage tail-like rigid rods. The number of rods was reduced in samples with low organic carbon concentration, samples with 8% and 10% NaCl, and samples induced at 40 and 43 degrees C. Although all lysates contained all phage-like particle-size fractions, their relative abundances varied. Up to a fivefold difference in phage-like particle size was observed in lysates. Size distribution of phage-like particles changed along temperature, salinity and organic carbon gradients. Results also indicated that the latent period of the induced phage-like particle population converged to approximately 90 min above a growth rate of 1.0 h(-1). At lower host growth rates, the latent period generally increased. However, at 40 and 43 degrees C and at low peptone-yeast extract concentration in the growth medium, the latent period remained short. We propose that different host physiological conditions influence organic matter composition upon prophage induction and may thus affect virus-controlled flow of the energy and carbon in the ecosystem.

Complete nucleotide sequence and molecular characterization of the temperate staphylococcal bacteriophage phiPVL carrying Panton-Valentine leukocidin genes.

The staphylococcal Panton-Valentine leukocidin (PVL) genes, [lukS-PV-lukF-PV], exist on the genome of a temperate bacteriophage phiPVL isolated from mitomycin C-induced Staphylococcus aureus V8 (ATCC 49775) (Kaneko, J., Kimura, T., Kawakami, Y., Tomita, T., Kamio, Y., 1997b. Panton-Valentine leukocidin genes in phage-like particle isolated from mitomycin C-treated Staphylococcus aureus V8 (ATCC 49775). Biosci. Biotechnol. Biochem. 61, 1960-1962). In this study, the complete nucleotide sequence of the phiPVL genome was analyzed, and the att sites (attL, attR, and attB) required for site-specific integration of phiPVL into the host chromosome were also determined. The linear double-stranded phiPVL genome comprised 41401bp with 3' staggered cohesive ends (cos) of nine bases and contained 63 ORFs, among which the regulatory proteins involved in DNA replication, structural proteins, a holin, a lysin, an integrase, and dUTPase, were tentatively identified by the comparison of the deduced amino acid sequences and by the analysis of the proteins isolated from phiPVL particles. The [lukS-PV-lukF-PV], attP, and int (integrase gene) of phiPVL were all located very close to one another within a 4.0-kb segment on the genome in the order given, and this segment was located at the center from the left and the right cos sites. In addition, the attP region contained five direct repeat sequences that showed a high degree of homology with the recombinase-binding sites of some other S. aureus bacteriophages. The phiPVL genome was found to integrate into an ORF encoding an unknown protein comprising 725 amino acid residues with two leucine zipper-like motifs.

Panton-valentine leukocidin genes in a phage-like particle isolated from mitomycin C-treated Staphylococcus aureus V8 (ATCC 49775).

The staphylococcal Panton-Valentine leukocidin (PVL) genes [lukS-PV-lukF-PV] existed in a hexagonal phage-like particle (phi PVL) isolated from mitomycin C-induced Staphylococcus aureus V8 (ATCC 49775). The genome packed in phi PVL was a linear double-stranded 40-kb DNA with single-stranded cohesive ends (cos). The [lukS-PV-lukS-PV], attP, and int (integrase gene) of phi PVL were all located very close to one another within a 4.0 kb-segment on the genome in the order given, and the segment is located at the center from the left and the right cos sites. In addition, the [lukS-PV-lukF-PV]-attP-int region contains 5 direct repeat sequences that show high similarity with the recombinase-binding sites of bacteriophages of S. aureus.

Interaction of virulent and non-virulentRhodococcus equihuman isolates with phagocytes, fibroblast- and epithelial-derived cells

Rhodococcus equi is a facultative, intracellular, Gram-positive coccobacillus, increasingly reported in pneumonia of AIDS-infected patients. We investigated killing resistance properties of human R. equi virulent and avirulent human strains. Avirulent beta-lactam-susceptible strains had lower intracellular colony forming units after 45 min incubation in murine macrophages J774 and human monocyte-macrophage TPH-1 than those of virulent strains. Only virulent beta-lactam-resistant strains persisted within macrophages for at least 18 min only. A beta-lactam-resistant mutant was obtained from a beta-lactam-susceptible strain after selection in a penicillin G-containing culture medium. This mutant strain, like the natural virulent strains, persisted within macrophages, harboured cell-associated appendages, produced phage-like particles and induced, after its intravenous inoculation, a chronic infection in BALB/c nude mice. Supernatant culture of virulent strains transferred partial macrophage-killing resistance properties to avirulent strains. The same supernatant was toxic for L-929, HeLa and Vero cell cultures. These supernatant effects were heat-inactivated, trypsin-inactivated and did not seem to be linked to phage-like particle presence. These data argue that virulence, beta-lactam-resistance, and macrophage-killing resistance are associated in human R. equi isolates. Moreover, only virulent strains produced uncharacterized toxic factors.

Correlation between antibiotic resistance, phage-like particle presence, and virulence in Rhodococcus equi human isolates

Rhodococcus equi is a gram-positive coccobacillus that appears to be emerging as a pulmonary pathogen in AIDS patients. In four human clinical isolates, two antibiotic resistance phenotypes were found to coexist: one beta-lactam resistant and the other beta-lactam susceptible. In vitro, beta-lactam-resistant mutants were obtained at a frequency of 1 x 10(-5) to 5 x 10(-5) from beta-lactam-susceptible strains on cephalothin-containing plates. Neither beta-lactamase nor plasmid DNA was detected in beta-lactam-resistant or -susceptible strains. The penicillin-binding protein patterns for the two types of strains were identical. Electron microscopy revealed that the beta-lactam-resistant strains possessed cell-surface-associated appendages and produced phage-like particles. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of total cell protein showed at least three additional bands of 42, 39, and 30 kDa found only in the beta-lactam-resistant strains. Testing for virulence in Swiss mice revealed that (i) phage-like-particle-producing strains had lower 50% lethal doses when injected intravenously in euthymic and nude mice than the non-phage-like-particle-producing strains did and (ii) intravenous inoculation of a sublethal dose (5 x 10(6) CFU) in nude mice led to chronic infection by the phage-like-particle-producing bacteria only. Finally, in vitro growth curves indicated that the phage-like-particle-producing strains possessed an ecological selection advantage. These results suggest that, among R. equi human isolates, the antibiotic resistance phenotype is associated with virulence and may be phage mediated.

Characterisation of a repressor gene ( xre) and a temperature-sensitive allele from the Bacillus subtilis prophage, PBSX

The defective prophage of Bacillus subtilis 168, PBSX, is a chromosomally based element which encodes a non-infectious phage-like particle with bactericidal activity. PBSX is induced by agents which elicit the SOS response. In a PBSX thermoinducible strain which carries the xhi1479 mutation, PBSX is induced by raising the growth temperature from 37°C to 48°C. A 1.2-kb fragment has been cloned which complements the xhi1479 mutation. The nucleotide sequence of this fragment contains an open reading frame (ORF) which encodes a protein of 113 amino acids (aa). This aa sequence resembles that of other bacteriophage respressors and suggests that the N-terminal reion forms a helix-turn-helix motif, typical of the DNA-binding domain of many bacterial regulatory proteins. The ORF is preceded by four 15-bp direct repeats, each of which contains an internal palindromic sequence, and by sequences resembling a SigA-dependent promoter. The nt sequence of an equivalent fragment from the PBSX thermoinducible strain has also been determined. There are three aa differences within the ORF compared to the wild type, one of which lies within the helix-turn-helix segment. This ORF encodes a repressor protein of PBSX.

Plasmids associated with a phagelike particle and with a satellite inclusion in Bacillus thuringiensis ssp. israelensis.

Variants of Bacillus thuringiensis ssp. israelensis were isolated using a 42 degrees C plasmid curing method. Those that were missing the large toxic crystalline inclusion still possessed in their sporulated cells a small inclusion similar to the satellite inclusion of the wild type. These crystal-minus but satellite-positive variants, like the wild type, produced sporulation-dependent phagelike particles; variants lacking both the crystal and the satellite did not. These latter strains lacked both a 75 and a 68 megadalton plasmid present in the wild type. However, a crystal-positive, satellite-minus strain possessing the 75 but lacking the 68 megadalton plasmid produced the phagelike particles. Examination of recipient strains that were initially devoid of crystal, satellite, and plasmids revealed the satellite and phagelike producing characteristics could have been cotransformed with the 68 megadalton plasmid. Likewise the toxic crystal and phagelike particle producing characters could have been cotransformed into a recipient which had acquired the 75 megadalton plasmids already known to be associated with the synthesis of the crystal inclusion. Thus the gene coding for the satellite inclusion appears to reside in the 68 megadalton plasmid, while that coding for the phagelike particle can reside in either the 68 or 75 megadalton plasmid.

Morphogenesis of the tail of bacteriophage lambda: III. Morphogenetic pathway

Precursors of the tail of bacteriophage λ have been detected by measurements of in vitro complementation activities and serum blocking activity in sucrose gradients of lysates defective in tail genes. On the basis of these measurements, a pathway for the assembly of the λ tail is proposed: The morphogenesis of the λ tail starts from the tail fiber (product of gene J) located at the distal end of the tail, and proceeds to the proximal end. Gene J by itself produces a 15 S structure with serum blocking activity but without any detectable in vitro complementation activity, which may be the least advanced precursor of the λ tail or an abortive product. Functions of genes J, I, K, L are required for the formation of a 15 S precursor that has in vitro complementation activities with J −, I −, K − and L − lysates and serum blocking activity. If the products of genes G and H act on the latter 15 S precursor, a 25 S precursor is made, but this precursor seems either to be in equilibrium with the 15 S precursor or to degrade easily into the 15 S precursor. Gene M has a function of stabilizing the 25 S precursor. After the action of gene M product, the 25 S precursor is ready to serve as a nucleus on which the product of gene V (the major tail protein) assembles. However, gene U product is also necessary at this step for the correct assembly of the major tail protein on the 25 S precursor. Without gene U product the assembly of the major tail protein does not stop at the correct length and a polytail is formed instead of a morphologically normal tail. Finally, gene Z product acts on the morphologically normal tail and makes it a biologically active tail. Without the action of gene Z product, the defective tail binds to a head and forms a phage-like particle which is only very weakly infectious. (The position of gene T in the pathway is not determined, because no sus mutant is available in gene T.) Two abnormal, less efficient pathways are also present in vitro. (1) If gene U product acts on a polytail in an U − lysate, the polytail finally binds to a head and forms a phage particle with an extra long tail which is infectious to a small extent. (2) The function of gene K seems to be bypassed to some extent: K − lysates accumulate particles which sediment as fast as normal phage and which are complemented by other tail − lysates.

Characterization of a phage-like particle from cells of Nitrobacter. II. Structure and size (author's transl)

Characterization of a phage-like particle from cells of Nitrobacter. II. Structure and size (author's transl)