A method for the direct isolation of IncH plasmid-dependent bacteriophages

Nuttall, D.; Maker, D.; Colleran, Emer

doi:10.1111/j.1472-765x.1987.tb01609.x

Cited by 13 publications

(9 citation statements)

References 15 publications

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“…The recipient strain was E. coli K-12 strain J53-1 (pro met F-Nal) (2). Bacteriophage Hgal (21) is an H-pilus-specific RNA phage, 18.7 nm in size (16), which adsorbs to the sides of the H-pilus shaft.…”

Section: Methodsmentioning

confidence: 99%

“…The elevated pilus levels associated with the R27::TnlacZ derivative make it a suitable test plasmid to study H-pilus assembly kinetics at permissive and nonpermissive conjugation temperatures. Two single-stranded RNA bacteriophage, pilHa and Hgal (10,16,21), adsorb uniformly along the H-pilus shaft. The goal of this study was to exploit phage adsorption, constitutive pilus production, and the thermosensitive conjugation system of R27::TnlacZ to examine several aspects of H-pilus assembly, stability, and morphology by transmission electron microscopy (TEM) and field emission scanning electron microscopy (FESEM).…”

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H-pilus assembly kinetics determined by electron microscopy

1993

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The kinetics of pilus outgrowth were examined for Escherichia coli containing pDT1942, a TnkacZ insertion derivative of the IncHIl plasmid R27, which was derepressed for transfer. IncHI1 plasmids are thermosensitive for transfer. The pili specified by pDT1942 were examined by transmission electron microscopy after the pilus had been labeled with the H-pilus-specific bacteriophage Hgal, which had been inactivated with RNase A. H pili were extended by extrusion from the cell surface and not by the addition of pilin subunits to the pilus tip. After pili were removed by vortexing, the outgrowth of full-length pili (2 ;Lm long) required 20 min. H pili expressed at the transfer optimal temperature (270C) remained stable after incubation at the transfer inhibitory temperature (370C), but the formation of mating aggregates was inhibited at 370C. Within 1 min of exposure of the host cell to a heat stimulus of 50'C, pili vanished. Pili were observed in straight and flexible forms with a field emission scanning electron microscope, which may indicate a dynamic role for the pilus in conjugation.H pili are conjugation appendages specified by incompatibility group HI and HII plasmids of the family Enterobacteriaceae (for reviews of H plasmids, see references 15 and 27). With the exception of the rigid pilus specified by the HI3 plasmid MIP233 (7), all plasmids belonging to the HIl, HI2, and HII groups specify thick, flexible pili (3). Thick, flexible pili are also specified by plasmids of the incompatibility groups FI, FII, FV, C, D, J, T, V, and X (3, 5, 6). Members of these plasmid incompatibility groups transfer to recipient cells with equal proficiency by both liquid and surface mating (9). HI group plasmids differ from those in the other groups in being thermosensitive for conjugation, i.e., transferring optimally below 30'C and negligibly (<10-8) at 370C (30). The basis for thermosensitive conjugation is not understood. H-pilus biochemistry is also unknown, and the basic pilin subunit(s) has not been characterized.Plasmid pDT1942, also known as R27::TnlacZ (16) Two single-stranded RNA bacteriophage, pilHa and Hgal (10, 16, 21), adsorb uniformly along the H-pilus shaft. The goal of this study was to exploit phage adsorption, constitutive pilus production, and the thermosensitive conjugation system of R27::TnlacZ to examine several aspects of H-pilus assembly, stability, and morphology by transmission electron microscopy (TEM) and field emission scanning electron microscopy (FESEM). * Corresponding author. MATERIALS AND METHODSBacterial strains, plasmid, and bacteriophage. The donor strain, Escherichia coli DT1944, is a rifampin-resistant mutant of JE2571 (leu thr str fla pit) (4) harboring plasmid pDT1942 (R27::TnlacZ) (16). This derivative of the tetracycline-resistant HIU plasmid R27 (28) mediates constitutive pilus production and kanamycin resistance. The recipient strain was E. coli K-12 strain J53-1 (pro met F-Nal) (2). Bacteriophage Hgal (21) is an H-pilus-specific RNA phage, 18.7 nm in size (16), which adsorbs to ...

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Section: Methodsmentioning

confidence: 99%

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confidence: 99%

H-pilus assembly kinetics determined by electron microscopy

1993

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“…Phage C-1 was originally isolated from sewage samples in Pretoria, South Africa (55), and phage Hgal1 was also isolated from raw sewage samples collected from the sea outfall in Galway docks, Ireland (43). Since their initial isolation and characterization, no further work has been performed.…”

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Rethinking the Evolution of Single-Stranded RNA (ssRNA) Bacteriophages Based on Genomic Sequences and Characterizations of Two R-Plasmid-Dependent ssRNA Phages, C-1 and Hgal1

Kannoly¹,

Shao²,

Wang³

2012

J. Bacteriol.

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ABSTRACTWe have sequenced and characterized two R-plasmid-dependent single-stranded RNA bacteriophages (RPD ssRNA phages), C-1 and Hagl1. Phage C-1 requires a conjugative plasmid of the IncC group, while Hgal1 requires the IncH group. Both the adsorption rate constants and one-step growth curves are determined for both phages. We also empirically confirmed the lysis function of the predicted lysis genes. Genomic sequencing and phylogenetic analyses showed that both phages belong to theLevivirusgroup and are most closely related to another IncP-plasmid-dependent ssRNA phage, PRR1. Furthermore, our result strongly suggests that the stereotypical bauplans of genome organization found inLevivirusandAlloleviviruspredate phage specialization for conjugative plasmids, suggesting that the utilization of conjugative plasmids for cell attachment and entry comprises independent evolutionary events for these two main clades of ssRNA phages. Our result is also consistent with findings of a previous study, making theLevivirus-like genome organization ancestral and theAllolevivirus-like genome derived. To obtain a deeper insight into the evolution of ssRNA phages, more phages specializing for various conjugative plasmids and infecting different bacterial species would be needed.

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“…Phage PRR1 [16] which adsorbs specifically to IncP plasmid-encoded pili was the first such example, and later other phages specific for Inc group C [17], D [18], H [19,20], I [21], M [22] and T [23] plasmids followed. Phages PRR1, C-1 (IncC-specific) and Hgal1 (IncH-specific) have been sequenced [24,25] and phage PRR1 capsids have also been crystallized [26], but no research has been done on the other plasmid-specific phages since their isolation.…”

Section: Introductionmentioning

confidence: 99%

Diversity of pili-specific bacteriophages: genome sequence of IncM plasmid-dependent RNA phage M

Rumnieks

Tars

2012

BMC Microbiol

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BackgroundBacteriophages of the Leviviridae family are small RNA viruses with linear, positive-sense, single-stranded RNA genomes that encode only four proteins. All phages of this family require bacterial pili to attach to and infect cells. Leviviridae phages utilizing F-pili for this purpose have been extensively studied. RNA phages specific for conjugative plasmid-encoded pili other than that of plasmid F have been isolated, but are much less understood and their relation to the F-pili-specific phages in many cases is not known.ResultsPhage M has the smallest known Leviviridae genome to date and has the typical genome organization with maturation, coat and replicase genes in the 5′ to 3′ direction. The lysis gene is located in a different position than in other known Leviviridae phages and completely overlaps with the replicase gene in a different reading frame. It encodes a 37 residue long polypeptide that contains a transmembrane helix like the other known lysis proteins of leviviruses. Sequence identities of M proteins to those of other phages do not exceed 25% for maturation protein, 51% for coat protein and 41% for replicase. Similarities in protein sequences and RNA secondary structures at the 3′ untranslated region place phage M together with phages specific for IncP, IncC and IncH, but not IncF plasmid-encoded pili. Phylogenetic analysis using the complete genome sequences and replicase proteins suggests that phage M represents a lineage that branched off early in the course of RNA phage specialization on different conjugative plasmids.ConclusionsThe genome sequence of phage M shows that it is clearly related to other conjugative pili-specific leviviruses but has an atypical location of the lysis gene. It provides a better view on the remarkable diversification of the plasmid-specific RNA phages.

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A method for the direct isolation of IncH plasmid-dependent bacteriophages

Cited by 13 publications

References 15 publications

H-pilus assembly kinetics determined by electron microscopy

H-pilus assembly kinetics determined by electron microscopy

Rethinking the Evolution of Single-Stranded RNA (ssRNA) Bacteriophages Based on Genomic Sequences and Characterizations of Two R-Plasmid-Dependent ssRNA Phages, C-1 and Hgal1

Diversity of pili-specific bacteriophages: genome sequence of IncM plasmid-dependent RNA phage M

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