Neisseria gonorrhoeae Filamentous Phage NgoΦ6 Is Capable of Infecting a Variety of Gram-Negative Bacteria

Abstract

cWe constructed a phagemid consisting of the whole genome of the Neisseria gonorrhoeae bacteriophage Ngo⌽6 cloned into a pBluescript plasmid derivative lacking the f1 origin of replication (named pBS::⌽6). Escherichia coli cells harboring pBS::⌽6 were able to produce a biologically active phagemid, Ngo⌽6fm, capable of infecting, integrating its DNA into the chromosome of, and producing progeny phagemids in, a variety of taxonomically distant Gram-negative bacteria, including E. coli, Haemophilus influenzae, Neisseria sicca, Pseudomonas sp., and Paracoccus methylutens. A derivative of pBS::⌽6 lacking the phage orf7 gene, a positional homolog of filamentous phage proteins that mediate the interaction between the phage and the bacterial pilus, was capable of producing phagemid particles that were able to infect E. coli, Haemophilus influenzae, N. sicca, Pseudomonas sp., and Paracoccus methylutens, indicating that Ngo⌽6 infects cells of these species using a mechanism that does not involve the Orf7 gene product and that Ngo⌽6 initiates infection through a novel process in these species. We further demonstrate that the establishment of the lysogenic state does not require an active phage integrase. Since phagemid particles were capable of infecting diverse hosts, this indicates that Ngo⌽6 is the first broad-host-range filamentous bacteriophage described.

Characterization of MDAΦ, a temperate filamentous bacteriophage of Neisseria meningitidis

“…In 2007, Piekarowicz and colleagues described N. gonorrhoeae prophage sequences that belong to the dsDNA tailed group of bacteriophage and which are biologically active (visualization of phage particles in phage preparation) (Piekarowicz et al, 2007). In 2014, the same team constructed a phagemid (pBS : : W6) derived from the N. gonorrhoeae filamentous bacteriophage NgoW6 [Nf4-G2(inv)] of the Nf4 group of Kawai and colleagues (Kawai et al, 2005), and showed that isolated phagemid particles contained ssDNA that could infect and replicate in a variety of Gram-negative bacteria (Piekarowicz et al, 2014). This infection does not require piliation of the recipient strain.…”

“…The MDA island corresponded to subtype one, designated Nf1. Piekarowicz et al (2014) cloned into Escherichia coli the sequence corresponding to Nf4-G2(inv) and were able to show that it produces infective filamentous phage particles. In this work, we aimed to demonstrate that the MDA island in N. meningitidis encodes a functional prophage able to produce infectious particles.…”

The Genetics of Neisseria Species

“…However, the ease of transformation has made it unnecessary to use phages to generate knockouts and insertions or to cleanly backcross mutations. Although research on neisserial phages has been slow to yield genetic tools, recently the N. gonorrhoeae Ngoφ6 has been cloned as a phagemid and can function productively in a wide range of Gram-negative species, such as Escherichia coli, Haemophilus influenzae, N. sicca, and Pseudomonas species (122).…”

Oral Immunization of Rabbits with S. enterica Typhimurium Expressing Neisseria gonorrhoeae Filamentous Phage Φ6 Induces Bactericidal Antibodies Against N. gonorrhoeae

“…All N. gonorrhoeae genomes characterized to date encode several filamentous phage whose DNA and protein sequences show ~95% identity 27 28 . Because the filamentous phage replication cycle is conserved among most of these phage in other species 29 , this suggests that the assembly and structural proteins should be present on the surface of GC.…”

“…These facts suggest that filamentous phage proteins could be the basis of a gonococcal vaccine. Our discovery that N. gonorrhoeae filamentous phage can replicate and be stably maintained in different Gram-negative bacteria 27 suggested that this could allow for a novel way of delivering phage particles, using live non-pathogenic bacteria as the delivery vehicle.…”