fENko-Kae01 is a flagellum-specific jumbo phage infecting Klebsiella aerogenes.

BACKGROUND: Klebsiella aerogenes is an opportunistic pathogen that causes a wide variety of infections. Due to the rising problem of antibiotic resistance, novel antibiotics and strategies to combat bacterial infections are needed. Host-specific bacteriophages are natural enemies of bacteria and can be used in phage therapy as an alternative form of treatment against bacterial infections. Jumbo phages are defined as phages with genomes larger than 200 kb. Relatively few studies have been done on jumbo phages compared to smaller phages. RESULTS: A novel phage, fENko-Kae01, was isolated from a commercial phage cocktail. Genomic analysis revealed that fENko-Kae01 is a lytic jumbo phage with a 360 kb genome encoding 578 predicted genes. No highly similar phage genomes were identified and fENko-Kae01 may be a completely new genus representative. No known genes associated with lysogenic life cycle, bacterial virulence, or antibiotic resistance were identified. The phage had myovirus morphology and a narrow host range. Phage resistant bacterial mutants emerged under phage selection. Whole genome sequencing revealed that the biogenesis of the flagellum was affected in four mutants and the lack of functional flagellum was confirmed in motility assays. Furthermore, phage fENKo-Kae01 failed to adsorb on the non-motile mutants indicating that the bacterial flagellum is the phage-binding receptor. CONCLUSIONS: fENko-Kae01 is a novel jumbo bacteriophage that is considered safe for phage therapy. fENko-Kae01 uses the flagellum as the phage-binding receptor and may represent a completely novel genus.

Viral Hijack of Filamentous Surface Structures in Archaea and Bacteria.

The bacterial and archaeal cell surface is decorated with filamentous surface structures that are used for different functions, such as motility, DNA exchange and biofilm formation. Viruses hijack these structures and use them to ride to the cell surface for successful entry. In this review, we describe currently known mechanisms for viral attachment, translocation, and entry via filamentous surface structures. We describe the different mechanisms used to exploit various surface structures bacterial and archaeal viruses. This overview highlights the importance of filamentous structures at the cell surface for entry of prokaryotic viruses.

Campylobacter jejuni motility is required for infection of the flagellotropic bacteriophage F341.

Previous studies have identified a specific modification of the capsular polysaccharide as receptor for phages that infect Campylobacter jejuni. Using acapsular kpsM mutants of C. jejuni strains NCTC11168 and NCTC12658, we found that bacteriophage F341 infects C. jejuni independently of the capsule. In contrast, phage F341 does not infect C. jejuni NCTC11168 mutants that either lack the flagellar filaments (ΔflaAB) or that have paralyzed, i.e., nonrotating, flagella (ΔmotA and ΔflgP). Complementing flgP confirmed that phage F341 requires rotating flagella for successful infection. Furthermore, adsorption assays demonstrated that phage F341 does not adsorb to these nonmotile C. jejuni NCTC11168 mutants. Taken together, we propose that phage F341 uses the flagellum as a receptor. Phage-host interactions were investigated using fluorescence confocal and transmission electron microscopy. These data demonstrate that F341 binds to the flagellum by perpendicular attachment with visible phage tail fibers interacting directly with the flagellum. Our data are consistent with the movement of the C. jejuni flagellum being required for F341 to travel along the filament to reach the basal body of the bacterium. The initial binding to the flagellum may cause a conformational change of the phage tail that enables DNA injection after binding to a secondary receptor.

Identification and characterization of a novel flagellum-dependent Salmonella-infecting bacteriophage, iEPS5.

A novel flagellatropic phage of Salmonella enterica serovar Typhimurium, called iEPS5, was isolated and characterized. iEPS5 has an icosahedral head and a long noncontractile tail with a tail fiber. Genome sequencing revealed a double-stranded DNA of 59,254 bp having 73 open reading frames (ORFs). To identify the receptor for iEPS5, Tn5 transposon insertion mutants of S. Typhimurium SL1344 that were resistant to the phage were isolated. All of the phage-resistant mutants were found to have mutations in genes involved in flagellar formation, suggesting that the flagellum is the adsorption target of this phage. Analysis of phage infection using the ΔmotA mutant, which is flagellated but nonmotile, demonstrated the requirement of flagellar rotation for iEPS5 infection. Further analysis of phage infection using the ΔcheY mutant revealed that iEPS5 could infect host bacteria only when the flagellum is rotating counterclockwise (CCW). These results suggested that the CCW-rotating flagellar filament is essential for phage adsorption and required for successful infection by iEPS5. In contrast to the well-studied flagellatropic phage Chi, iEPS5 cannot infect the ΔfliK mutant that makes a polyhook without a flagellar filament, suggesting that these two flagellatropic phages utilize different infection mechanisms. Here, we present evidence that iEPS5 injects its DNA into the flagellar filament for infection by assessing DNA transfer from SYBR gold-labeled iEPS5 to the host bacteria.

Complete genome sequence analysis of bacterial-flagellum-targeting bacteriophage chi.

Bacteriophage chi is a well-known phage that infects pathogens such as E. coli, Salmonella, and Serratia via bacterial flagella. To further understand its host-phage interaction and infection mechanism via host flagella, the genome was completely sequenced and analyzed. The phage genome contains 59,407-bp-length DNA with a GC content of 56.51 %, containing 75 open reading frames (ORFs) with no tRNA genes. Its annotation and functional analysis revealed that chi is evolutionarily very closely related to Enterobacter phage Enc34 and Providencia phage Redjac. However, most of the annotated genes encode hypothetical proteins, indicating that further genomic study of phage chi is required to elucidate the bacterial-flagellum-targeting infection mechanism of phage chi.

Minimum requirements of flagellation and motility for infection of Agrobacterium sp. strain H13-3 by flagellotropic bacteriophage 7-7-1.

The flagellotropic phage 7-7-1 specifically adsorbs to Agrobacterium sp. strain H13-3 (formerly Rhizobium lupini H13-3) flagella for efficient host infection. The Agrobacterium sp. H13-3 flagellum is complex and consists of three flagellin proteins: the primary flagellin FlaA, which is essential for motility, and the secondary flagellins FlaB and FlaD, which have minor functions in motility. Using quantitative infectivity assays, we showed that absence of FlaD had no effect on phage infection, while absence of FlaB resulted in a 2.5-fold increase in infectivity. A flaA deletion strain, which produces straight and severely truncated flagella, experienced a significantly reduced infectivity, similar to that of a flaB flaD strain, which produces a low number of straight flagella. A strain lacking all three flagellin genes is phage resistant. In addition to flagellation, flagellar rotation is required for infection. A strain that is nonmotile due to an in-frame deletion in the gene encoding the motor component MotA is resistant to phage infection. We also generated two strains with point mutations in the motA gene resulting in replacement of the conserved charged residue Glu98, which is important for modulation of rotary speed. A change to the neutral Gln caused the flagellar motor to rotate at a constant high speed, allowing a 2.2-fold-enhanced infectivity. A change to the positively charged Lys caused a jiggly motility phenotype with very slow flagellar rotation, which significantly reduced the efficiency of infection. In conclusion, flagellar number and length, as well as speed of flagellar rotation, are important determinants for infection by phage 7-7-1.

Alternative mechanism for bacteriophage adsorption to the motile bacterium Caulobacter crescentus.

2D and 3D cryo-electron microscopy, together with adsorption kinetics assays of Cb13 and CbK phage-infected Caulobacter crescentus, provides insight into the mechanisms of infection. Cb13 and CbK actively interact with the flagellum and subsequently attach to receptors on the cell pole. We present evidence that the first interaction of the phage with the bacterial flagellum takes place through a filament on the phage head. This contact with the flagellum facilitates concentration of phage particles around the receptor (i.e., the pilus portals) on the bacterial cell surface, thereby increasing the likelihood of infection. Phage head filaments have not been well characterized and their function is described here. Phage head filaments may systematically underlie the initial interactions of phages with their hosts in other systems and possibly represent a widespread mechanism of efficient phage propagation.

Exploitation of a new flagellatropic phage of Erwinia for positive selection of bacterial mutants attenuated in plant virulence: towards phage therapy.

AIMS: To isolate and characterize novel bacteriophages for the phytopathogen, Erwinia carotovora ssp. atroseptica (Eca), and to isolate phage-resistant mutants attenuated in virulence. METHODS AND RESULTS: A novel flagellatropic phage was isolated on the potato-rotting bacterial species, Eca, and characterized using electron microscopy and restriction analysis. The phage, named PhiAT1, has an icosahedral head and a long, contractile tail; it belongs to the Myoviridae family. Partial sequencing revealed the presence of genes with homology to those of coliphages T4, T7 and Mu. Phage-resistant transposon mutants of Eca were isolated and studied in vitro for a number of virulence-related phenotypes; only motility was found to be affected. In vivo tuber rotting assays showed that these mutants were attenuated in virulence, presumably because the infection is unable to spread from the initial site of inoculation. CONCLUSIONS: The Eca flagellum can act as a receptor for PhiAT1 infection, and resistant mutants are enriched for motility and virulence defects. SIGNIFICANCE AND IMPACT OF THE STUDY: PhiAT1 is the first reported flagellatropic phage found to infect Eca and has enabled further study of the virulence of this economically important phytopathogen.

The ability of flagellum-specific Proteus vulgaris bacteriophage PV22 to interact with Campylobacter jejuni flagella in culture.

BACKGROUND: There has been a recent resurgent interest in bacteriophage biology. Research was initiated to examine Campylobacter jejuni-specific bacteriophage in the Russian Federation to develop alternative control measures for this pathogen. RESULTS: A C. jejuni flagellum-specific phage PV22 from Proteus vulgaris was identified in sewage drainage. This phage interacted with C. jejuni by attachment to flagella followed by translocation of the phage to the polar region of the bacterium up to the point of DNA injection. Electron microscopic examination revealed adsorption of PV22 on C. jejuni flagella after a five minute incubation of the phage and bacteria. A different phenomenon was observed after incubating the mix under the same conditions, but for twenty minutes or longer. Phage accumulated primarily on the surface of cells at sites where flagella originated. Interestingly, PV22 did not inject DNA into C. jejuni and PV22 did not produce lytic plaques on medium containing C. jejuni cells. The constant of velocity for PV22 adsorption on cells was 7 x 10(-9) ml/min. CONCLUSION: It was demonstrated that a bacteriophage that productively infects P. vulgaris was able to bind C. jejuni and by a spot test that the growth of C. jejuni was reduced relative to control bacteria in the region of phage application. There may be two interesting applications of this effect. First, it may be possible to test phage PV22 as an antimicrobial agent to decrease C. jejuni colonization of the chicken intestine. Second, the phage could potentially be utilized for investigating biogenesis of C. jejuni flagella.

Flagellar determinants of bacterial sensitivity to chi-phage.

Bacteriophage chi is known to infect motile strains of enteric bacteria by adsorbing randomly along the length of a flagellar filament and then injecting its DNA into the bacterial cell at the filament base. Here, we provide evidence for a "nut and bolt" model for translocation of phage along the filament: the tail fiber of chi fits the grooves formed by helical rows of flagellin monomers, and active flagellar rotation forces the phage to follow the grooves as a nut follows the threads of a bolt.

Isolation of three different bacteriophage from mesophilic Aeromonas sp. that use different types of monopolar flagella as their primary receptor.

Bacteriophage PM4, PM5 and PM6 were isolated on different mesophilic Aeromonas strains. These bacteriophage use the flagellum as their primary bacterial receptor since purified flagella from these strains are able to inactivate these bacteriophages, independently, and the phage-resistant mutants are aflagellate and nonmotile. Furthermore, we showed that these bacteriophage may be useful to initiate the serotyping of mesophilic Aeromonas for the H-antigen (flagellum).