Phage Tail-like Particles Research Articles

Producing Tailocins from Phages Using Osmotic Shock and Benzalkonium Chloride.

In the light of the worldwide antimicrobial resistance crisis, new substitutes to antibiotics are urgently needed. Tailocins or phage tail-like bacteriocin particles, produced by bacteria for environmental competition, are a potential antimicrobial alternative to antibiotic treatment. Yet, the availability of characterized Tailocins is limited. We explored the possibility to produce new Tailocins from phage particles, using osmotic shock or chemical treatment by the ammonium quaternary compound benzalkonium chloride on Ackermannviridae phage S117 and using Straboviridae phage T4 as control. We report that phage S117 was resistant to such treatment, while successful production of Tailocins by osmotic shock was achieved for phage T4. Finally, chemical treatment with benzalkonium chloride was inefficient on phage S117 but successfully inactivated phage T4 without production of Tailocins. Further studies are needed to implement such treatments of phages for producing Tailocins with killing activity.

Intracellular Phage Tail-Like Nanostructures Affect Susceptibility of Streptomyces lividans to Osmotic Stress.

Contractile injection systems (CISs) are a large group of phage tail-like nanostructures conserved among bacteria. Despite their wide distribution, the biological significance of CISs in bacteria remains largely unclear except for a few unicellular bacteria. Here, we show that Streptomyces lividans-a model organism of filamentous Gram-positive bacteria with highly conserved CIS-related gene clusters-produces intracellular CIS-like nanostructures (Streptomyces phage tail-like particles [SLPs]) that affect phenotypes of this bacterium under hyperosmotic conditions. In contrast to typical CISs released from the cells, SLPs are localized in the cytoplasm of S. lividans. In addition, loss of SLPs leads to (i) delayed erection of aerial mycelia on hyperosmotic solid medium and (ii) decreased growth during the transition from exponential growth phase to stationary phase in hyperosmotic liquid medium. Localization of fluorescent protein-tagged SLPs showed partial correlation with cell wall synthesis-related proteins, including MreB, an actin-like cytoskeleton protein. Our pulldown assay and subsequent quantitative proteome analysis also suggest that 30S ribosomal proteins and cell wall-related proteins, including MreB, are coeluted with SLPs. Furthermore, an interaction assay using the recombinant proteins revealed a direct interaction between a sheath protein of SLP and ribosomal protein S16. Results of cross-linking experiments show indirect interactions between SLPs and translation elongation factors. These findings collectively suggest that SLPs are directly or indirectly associated with a protein interaction network within the cytoplasm of S. lividans and that SLP loss ultimately affects the susceptibility of the bacterium to certain stress conditions. IMPORTANCE Recent bioinformatic analyses have revealed that CIS-related gene clusters are highly conserved in Gram-positive actinomycetes, especially members of the genus Streptomyces known for their ability to produce therapeutic antibiotics. While typical CISs are released from the cells and can act as protein translocation systems that inject effector proteins into the target cells, our results indicate the unique intracellular localization of SLPs, CIS-related nanostructures produced by S. lividans. In addition, the direct and indirect interactions of SLPs with cytoplasmic proteins and SLP localization within specific regions of mycelia suggest that the biological significance of SLPs is related to intracellular processes. Further, SLP loss leads to increased susceptibility of S. lividans to osmotic stress, suggesting that production of these phage tail-like nanostructures ultimately affects the fitness of the bacterium under certain stress conditions. This work will provide new insight into the phage tail-like nanostructures highly conserved in Streptomyces species.

Pivotal role of O-antigenic polysaccharide display in the sensitivity against phage tail-like particles in environmental Pseudomonas kin competition

Environmental pseudomonads colonize various niches including insect and plant environments. When invading these environments, bacteria are confronted with the resident microbiota. To oppose with closely related strains, they rely on narrow-spectrum weaponry such as tailocins, i.e., phage tail-like particles. Little is known about the receptors for these tailocins especially among phylogenetically closely related species. Here, we studied the interaction between an R-tailocin from Pseudomonas protegens CHA0 and a targeted kin, Pseudomonas protegens Pf-5. Using genome-wide transposon insertion sequencing, we identified that lipopolysaccharides are involved in the sensitivity of Pf-5 towards the tailocin of CHA0. By generating Pf-5 lipopolysaccharide mutants and exposing them to extracted tailocin, we specified the two O-antigenic polysaccharides (O-PS) targeted by the tailocin. We affirmed the role of these O-PS through competition assays in vitro as well as in insects. Further, we demonstrate that O-PS are double-edge swords that are responsible for the sensitivity of P. protegens towards tailocins and phages produced by their kin, but shield bacteria from the immune system of the insect. Our results shed light on the trade-off that bacteria are confronted with, where specific O-PS decorations can both be of benefit or disadvantage depending on the host environment and its bacterial inhabitants.

Phage tail-like nanostructures affect microbial interactions between Streptomyces and fungi

Extracellular contractile injection systems (eCISs) are structurally similar to headless phages and are versatile nanomachines conserved among diverse classes of bacteria. Herein, Streptomyces species, which comprise filamentous Gram-positive bacteria and are ubiquitous in soil, were shown to produce Streptomyces phage tail-like particles (SLPs) from eCIS-related genes that are widely conserved among Streptomyces species. In some Streptomyces species, these eCIS-related genes are regulated by a key regulatory gene, which is essential for Streptomyces life cycle and is involved in morphological differentiation and antibiotic production. Deletion mutants of S. lividans of the eCIS-related genes appeared phenotypically normal in terms of morphological differentiation and antibiotic production, suggesting that SLPs are involved in other aspects of Streptomyces life cycle. Using co-culture method, we found that colonies of SLP-deficient mutants of S. lividans were more severely invaded by fungi, including Saccharomyces cerevisiae and Schizosaccharomyces pombe. In addition, microscopic and transcriptional analyses demonstrated that SLP expression was elevated upon co-culture with the fungi. In contrast, co-culture with Bacillus subtilis markedly decreased SLP expression and increased antibiotic production. Our findings demonstrate that in Streptomyces, eCIS-related genes affect microbial competition, and the patterns of SLP expression can differ depending on the competitor species.

Membrane vesicle secretion and prophage induction in multidrug-resistant Stenotrophomonas maltophilia in response to ciprofloxacin stress.

Several bacterial species produce membrane vesicles (MVs) in response to antibiotic stress. However, the biogenesis and role of MVs in bacterial antibiotic resistance mechanisms have remained unclear. Here, we studied the effect of the fluoroquinolone ciprofloxacin on MV secretion by Stenotrophomonas maltophilia using a combination of electron microscopy and proteomic approaches. We found that in addition to the classical outer membrane vesicles (OMV), ciprofloxacin-stimulated cultures produced larger vesicles containing both outer and inner membranes termed outer-inner membrane vesicles (OIMV), and that such MVs are enriched with cytosolic proteins. Remarkably, OIMV were found to be decorated with filamentous structures identified as fimbriae. In addition, ciprofloxacin stress leads to the release of bacteriophages and phage tail-like particles. Prophage induction by ciprofloxacin has been linked to pathogenesis and horizontal gene transfer in several bacterial species. Together, our findings show that ciprofloxacin treatment of S. maltophilia leads to the secretion of a heterogeneous pool of MVs and the induction of prophages that are potentially involved in adverse side-effects during antibiotic treatment.

Induction and Purification of C. difficile Phage Tail-Like Particles.

Due to the inherent limitations of conventional antibiotics for the treatment of C. difficile infection (CDI), there is a growing interest in the development of alternative treatment strategies. Both bacteriophages and R-type bacteriocins, also known as phage tail-like particles (PTLPs), show promise as potential antibacterial alternatives for treating CDI. Similar to bacteriophages, but lacking a viral capsid and genome, PTLPs remain capable of killing target bacteria. Here we describe our experience in the induction and purification of C. difficile PTLPs. These methods have been optimized to allow production of concentrated, non-contractile, and non-aggregated samples for both sensitivity testing and structural electron microscopy studies.

Phage tail-like particles kill Clostridium difficile and represent an alternative to conventional antibiotics

Current Clostridium difficile infection (CDI) antibiotic regimens have become increasingly ineffective at achieving cure and preventing recurrence. A recently developed alternative to conventional antibiotics are phage tail-like particles (PTLPs), which are proteins that are morphologically similar to bacteriophages and are produced by C difficile. This study examines the in vitro killing spectrum of a previously unreported PTLP isolated from a clinical isolate of C difficile. Using patient-derived samples from an institutional review board-approved C difficile tissue bank, a ribotype 078 C difficile isolate was anaerobically incubated on blood agar plates that were preswabbed with norfloxacin to induce the production of PTLPs. Concentrated PTLP populations were confirmed using transmission electron microscopy. Using a standard lawn spot approach, bactericidal activity was assessed as indicated by a clearing within the bacterial lawn. The PTLP genomic cluster was also fully sequenced and open reading frames were annotated according to predicted function. PTLPs were assessed using 64 patient-derived C difficile isolates of varying ribotypes. PTLPs demonstrated complete bactericidal activity in 21 of 25 ribotype 027 isolates with partial activity in 2 of the 25. Complete bactericidal activity was not demonstrated against any other ribotype or non-difficile bacteria, suggesting a species and ribotype specificity. Functional genes, which may be necessary for killing, were identified within the PTLP genetic locus. PTLPs demonstrate capability in eradicating C difficile in vitro, and with further development, may represent an organism-specific, microbiome-sparing therapy for CDI.

Novel High-Molecular-Weight, R-Type Bacteriocins of Clostridium difficile

Clostridium difficile causes one of the leading nosocomial infections in developed countries, and therapeutic choices are limited. Some strains of C. difficile produce phage tail-like particles upon induction of the SOS response. These particles have bactericidal activity against other C. difficile strains and can therefore be classified as bacteriocins, similar to the R-type pyocins of Pseudomonas aeruginosa. These R-type bacteriocin particles, which have been purified from different strains, each have a different C. difficile-killing spectrum, with no one bacteriocin killing all C. difficile isolates tested. We have identified the genetic locus of these "diffocins" (open reading frames 1359 to 1376) and have found them to be common among the species. The entire diffocin genetic locus of more than 20 kb was cloned and expressed in Bacillus subtilis, and this resulted in production of bactericidal particles. One of the interesting features of these particles is a very large structural protein of ~200 kDa, the product of gene 1374. This large protein determines the killing spectrum of the particles and is likely the receptor-binding protein. Diffocins may provide an alternate bactericidal agent to prevent or treat infections and to decolonize individuals who are asymptomatic carriers.

Diverse temperate bacteriophage carriage in Clostridium difficile 027 strains.

BackgroundThe hypervirulent Clostridium difficile ribotype 027 can be classified into subtypes, but it unknown if these differ in terms of severity of C. difficile infection (CDI). Genomic studies of C. difficile 027 strains have established that they are rich in mobile genetic elements including prophages. This study combined physiological studies, electron microscopy analysis and molecular biology to determine the potential role of temperate bacteriophages in disease and diversity of C. difficile 027.Methodology/Principal FindingsWe induced prophages from 91 clinical C. difficile 027 isolates and used transmission electron microscopy and pulsed-field gel electrophoresis to characterise the bacteriophages present. We established a correlation between phage morphology and subtype. Morphologically distinct tailed bacteriophages belonging to Myoviridae and Siphoviridae were identified in 63 and three isolates, respectively. Dual phage carriage was observed in four isolates. In addition, there were inducible phage tail-like particles (PT-LPs) in all isolates. The capacity of two antibiotics mitomycin C and norfloxacin to induce prophages was compared and it was shown that they induced specific prophages from C. difficile isolates. A PCR assay targeting the capsid gene of the myoviruses was designed to examine molecular diversity of C. difficile myoviruses. Phylogenetic analysis of the capsid gene sequences from eight ribotypes showed that all sequences found in the ribotype 027 isolates were identical and distinct from other C. difficile ribotypes and other bacteria species.Conclusion/SignificanceA diverse set of temperate bacteriophages are associated with C. difficile 027. The observed correlation between phage carriage and the subtypes suggests that temperate bacteriophages contribute to the diversity of C. difficile 027 and may play a role in severity of disease associated with this ribotype. The capsid gene can be used as a tool to identify C. difficile myoviruses present within bacterial genomes.

Morphological and Genetic Diversity of Temperate Phages in Clostridium difficile

Eight temperate phages were characterized after mitomycin C induction of six Clostridium difficile isolates corresponding to six distinct PCR ribotypes. The hypervirulent C. difficile strain responsible for a multi-institutional outbreak (NAP1/027 or QCD-32g58) was among these prophage-containing strains. Observation of the crude lysates by transmission electron microscopy (TEM) revealed the presence of three phages with isometric capsids and long contractile tails (Myoviridae family), as well as five phages with long noncontractile tails (Siphoviridae family). TEM analyses also revealed the presence of a significant number of phage tail-like particles in all the lysates. Southern hybridization experiments with restricted prophage DNA showed that C. difficile phages belonging to the family Myoviridae are highly similar and most likely related to previously described prophages phiC2, phiC5, and phiCD119. On the other hand, members of the Siphoviridae phage family are more genetically divergent, suggesting that they originated from distantly related ancestors. Our data thus suggest that there are at least three genetically distinct groups of temperate phages in C. difficile; one group is composed of highly related myophages, and the other two groups are composed of more genetically heterogeneous siphophages. Finally, no gene homologous to genes encoding C. difficile toxins or toxin regulators could be identified in the genomes of these phages using DNA hybridization. Interestingly, each unique phage restriction profile correlated with a specific C. difficile PCR ribotype.

Characterization of enterocoliticin, a phage tail-like bacteriocin, and its effect on pathogenic Yersinia enterocolitica strains.

Yersinia enterocolitica 29930 (biogroup 1A; serogroup O:7,8) produces a bacteriocin, designated enterocoliticin, that shows inhibitory activity against enteropathogenic strains of Y. enterocolitica belonging to serogroups O:3, O:5,27 and O:9. Enterocoliticin was purified, and electron micrographs of enterocoliticin preparations revealed the presence of phage tail-like particles. The particles did not contain nucleic acids and showed contraction upon contact with susceptible bacteria. Enterocoliticin addition to logarithmic-phase cultures of susceptible bacterial strains led to a rapid dose-dependent reduction in CFU. Calorimetric measurements of the heat output of cultures of sensitive bacteria showed a complete loss of cellular metabolic activity immediately upon addition of enterocoliticin. Furthermore, a dose-dependent efflux of K(+) ions into the medium was determined, indicating that enterocoliticin has channel-forming activity.

Formation of Spontaneously Developing Pocks and Production of Phage Taillike Particles in Thiostrepton-Producing Streptomyces laurentii ATCC 31255.

Formation of Spontaneously Developing Pocks and Production of Phage Taillike Particles in Thiostrepton-Producing Streptomyces laurentii ATCC 31255.

Phage taillike particles found in the pocks of Streptomyces coerulescens.

Etude chez les souches productrices d'antibiotiques, la formation de «pocks» presentant des inconvenients pour leur culture

Pock Formation of Streptomyces endus with Production of Phage Taillike Particles

[This corrects the article on p. 164 in vol. 44.].

Pock Formation of Streptomycetes endus with Production of Phage Taillike Particles

Plate (or slope) cultures of endomycin-producing Streptomyces endus (KCC S-0213) showed spontaneously developing pocks which increased in number during subculturing. Neither spore formation nor typical aerial hyphae formation was observed in the pocks, whereas formation of substrate hyphase was not inhibited. Almost all of the hyphae were broken or lysed in the pocks, and many phage tail tiplike particles were observed in the pocks. No self-replication activity was associated with the particles. The particles often formed a hexagonal crystal or a large crystal mass. The production of these particles did not occur in the liquid culture or in young or normal plate cultures having no pocks. These results were similar to those obtained from the plaque-making phenomenon, except for active phage production, in thiostrepton-producing Streptomyces azureus (ATCC 14921), which has been described previously.

R-Bodies in newly isolated free-living hydrogen-oxidizing bacteria

R-Bodies have been found in a recently isolated pseudomonas-like free-living hydrogen oxidizing bacterium. Their isolation, fine structure and chemical composition are described and compared with the R-bodies from the kappa particles (Caedobacter), obligate endosymbionts of Paramecium aurelia. The 2K 1 R-bodies exhibited essential characteristics of the kappa R-bodies; however, their size and some other structural aspects proved that they represent a new type of R-bodies. The presence of phage tail-like particles in cells induced with Mitomycin C is in favour of the hypothesis that the R-bodies might be coded by defective prophages, or by extrachromosomal elements.

Intracellular organization of bacteriocin particles in Proteus vulgaris.

The intracellular development of a bacteriocin in a strain of Proteus vulgaris was investigated. Particles which resemble the bacteriocin make their appearance in the cell after 60 min induction with mitomycin C. At this stage they appear as broad bands of tightly-packed phage tail-like structures. With longer periods of induction the bands are disrupted to liberate individual phage tail-like particles.

Inducible Phage Tail-like Particles ofClostridium saccharoperbutylacetonicumand Its Related Strains

Two new kinds of high molecular bacteriocin, named as clostocins O and M, were found in Clostridium saccharoperbutylacetonicum and its related strains. Production of both clostocins was inducible by mitomycin C (MC) or ultraviolet ray. The active clostocins appeared in the bacterial cells at about 105 min after MC-treatment, increased rapidly during next 75 min and then released into the medium with the cell lysis. The clear lysis of O- and M-producing cells was observed at 3.5 hr and 4 hr respectively after MC-treatment. Clostocin O killed M-producing strains, and clostocin M did O-producing strains. Electron microscopic observation revealed that clostocins O and M were phage tail-like particles with contractile sheath round a core. The particles resembled some pyocins and also the tail of phage HM 3 of Cl. saccharoperbutylacetonicum. It was also found that M-producing strains had simultaneously the ability of production of low molecular bacteriocin, named as clostocin D.

Inducible Phage Tail-like Particles of Clostridium saccharoperbutylacetonicum and Its Related Strains

Two new kinds of high molecular bacteriocin, named as clostocins O and M, were found in Clostridium saccharoperbutylacetonicum and its related strains. Production of both clostocins was inducible by mitomycin C (MC) or ultraviolet ray. The active clostocins appeared in the bacterial cells at about 105 min after MC-treatment, increased rapidly during next 75 min and then released into the medium with the cell lysis. The clear lysis of O- and M-producing cells was observed at 3.5 hr and 4 hr respectively after MC-treatment. Clostocin O killed M-producing strains, and clostocin M did O-producing strains. Electron microscopic observation revealed that clostocins O and M were phage tail-like particles with contractile sheath round a core. The particles resembled some pyocins and also the tail of phage HM 3 of Cl. saccharoperbutylacetonicum.It was also found that M-producing strains had simultaneously the ability of production of low molecular bacteriocin, named as clostocin D.