Discovery, purification, and characterization of a temperate transducing bacteriophage for Bordetella avium

Abstract

We discovered and characterized a temperate transducing bacteriophage (Ba1) for the avian respiratory pathogen Bordetella avium. Ba1 was initially identified along with one other phage (Ba2) following screening of four strains of B. avium for lysogeny. Of the two phage, only Ba1 showed the ability to transduce via an allelic replacement mechanism and was studied further. With regard to host range, Ba1 grew on six of nine clinical isolates of B. avium but failed to grow on any tested strains of Bordetella bronchiseptica, Bordetella hinzii, Bordetella pertussis, or Bordetella parapertussis. Ba1 was purified by CsCl gradient centrifugation and was found to have an icosahedral head that contained a linear genome of approximately 46.5 kb (contour length) of double-stranded DNA and a contractile, sheathed tail. Ba1 readily lysogenized our laboratory B. avium strain (197N), and the prophage state was stable for at least 25 generations in the absence of external infection. DNA hybridization studies indicated the prophage was integrated at a preferred site on both the host and phage replicons. Ba1 transduced five distinctly different insertion mutations, suggesting that transduction was generalized. Transduction frequencies ranged from approximately 2 x 10(-7) to 1 x 10(-8) transductants/PFU depending upon the marker being transduced. UV irradiation of transducing lysates markedly improved transduction frequency and reduced the number of transductants that were lysogenized during the transduction process. Ba1 may prove to be a useful genetic tool for studying B. avium virulence factors.

FIG. 1

(A) Transmission electron micrograph of negatively stained Ba1 phage particles. One phage (arrowhead) appears to have an empty head. (Note its electron-darkened head and contracted tail sheath exposing the inner tail core.) (B) Transmission electron micrograph of Ba1 DNA prepared from purified phage as described in Materials and Methods. Arrowheads denote a single linear genome. The relaxed form of pBR322 DNA (4.36 kb) was used as a size standard (box).

FIG. 2

(A) An autoradiograph of NotI-digested chromosome and phage DNA that was hybridized with DIG-labeled vegetative Ba1 DNA. Lanes: 1, nonlysogenic strain (197N); 2, lysogenic strain (AP21); 3, vegetative Ba1. The numbers to the left denote the fragment sizes in kilobases of the bands lettered on the right. The letters (a to e) identify specific vegetative phage fragments. The c′ in the lysogen lane (2) denotes a new fragment in the lysogen not present in the vegetative phage. (B) An autoradiograph of NotI-digested chromosomal and phage DNA hybridized with a DIG-labeled EcoRI restriction endonuclease fragment internal to the vegetative Ba1 NotI c fragment. Lanes: 1, the nonlysogenic strain (197N); 2, lysogenic strain (AP21); 3, vegetative Ba1. The numbers to the left denote the molecular weights of the putative attachment site-containing bands (c in the vegetative phage, and c′ and c" in the lysogen) in kilobases.

FIG. 3

Autoradiograph showing EcoRI restriction endonuclease fragments of donor and recipient strains in transduction experiments using a neoR gene probe as described in Materials and Methods. Lanes: 1, mot::mini-Tn5lacZ donor strain (G146); 2, recipient strain (197N2); 3, mot::mini-Tn5lacZ transductant; 4, hag::mini-Tn5lacZ donor strain (G145); 5, recipient strain (197N2); 6, hag::mini-Tn5lacZ transductant. Arrowheads denote the size of the reacting bands.

FIG. 4

Effect of UV irradiation on transduction parameters: viable phage, transduction frequency, and transductant lysogeny. Solid squares (■) denote the percentage of viable Ba1 after UV irradiation of a crude lysate grown on strain G146. Also plotted are the percentage of mot::mini-Tn5lacZ nonlysogenized transductants (●) and the number of transductants emerging from the irradiated lysate at each UV dose (▵). Points represent the averages of two experiments. Vertical bars denote standard errors of the means.