Characterization of six bacteriophages of serratia liquefaciens CP6 isolated from the sugar beet phytosphere

Abstract

Six phages (PhiCP6-1 to PhiCP6-6) that are commonly found in the phytosphere of sugar beet (Beta vulgaris var. Amethyst) were investigated, and their relative impacts on their host (Serratia liquefaciens CP6) were compared. There were fundamental differences between the two most abundant predators of CP6 (PhiCP6-1 and PhiCP6-4). Like PhiCP6-2 and PhiCP6-5, PhiCP6-1 belonged to the family Siphoviridae, while PhiCP6-4 exhibited the morphology of the family Podoviridae. The other phages were members of the family Myoviridae. DNA-DNA cross-hybridization revealed that PhiCP6-1 and PhiCP6-4 had little common DNA, although all of the other phages exhibited some genetic similarity. Like PhiCP6-2, PhiCP6-3, and PhiCP6-5, PhiCP6-1 was capable of forming a lysogenic association with its host, while PhiCP6-4 and PhiCP6-6 appeared to be entirely virulent. Single-step growth curve experiments revealed that PhiCP6-4 had a much shorter latent period and a smaller burst size than PhiCP6-1. Also, PhiCP6-1 could transduce a number of host chromosomal markers with transfer frequencies of 2.9 x 10(-9) to 3.9 x 10(-7), whereas PhiCP6-4 could not transduce S. liquefaciens CP6 genes. When viewed in the context of the strikingly different temporal niches of these phages, our data provide an insight into how bacteriophage interactions with their hosts might reflect the natural ecology of bacteriophages. Our data also illustrate how the potential for gene transfer changes over time in an environment that supports several different phages.

FIG. 1

Plaque morphologies of phages ΦCP6-1 (A) and ΦCP6-4 (B) on overlay plates after 48 h of incubation at 15°C.

FIG. 2

Abilities of bacteriophages to visibly lyse lawns of S. liquefaciens CP6 of different ages. (A) Growth of the bacterial lawns over the 12-h experimental period. MSD, minimum significant difference. (B) Maximum time during which each of the six phages was still capable of forming visible zones of lysis on the lawns. Note that the two replicate experiments gave identical results.

FIG. 3

Contrasting morphologies of S. liquefaciens CP6 phages ΦCP6-1 to ΦCP6-6 (A to F, respectively), as determined by TEM.

FIG. 4

(A) EcoRI restriction digests of phages ΦCP6-1 to ΦCP6-6 (lanes 1 to 6, respectively) electrophoresed on a 0.7% agarose gel. (B) Southern blot of the same gel probed with DIG-labelled ΦCP6-3 DNA after 30 min of exposure to photographic film. (C) Southern blot shown in panel B after 24 h of exposure. Lanes λ contained a HindIII-cut lambda ladder.

FIG. 5

Summary of DNA-DNA cross-hybridization experiments. Each panel shows the results obtained with a different phage. The EcoRI-cut genome of each phage was separately probed with DNA from each of the six phages, including itself. Thus, in each panel there are six columns, each showing the band pattern produced when the digest was probed with a particular labelled phage. When a phage was probed with itself, the full digest pattern, as shown in Fig. 4A, was revealed.

FIG. 6

Transfer frequencies of generalized transducing lysates of all six phages. The following three phenotypic markers were investigated: kanamycin resistance (A), streptomycin resistance (B), and rifampin resistance (C). The symbols indicate the mean transfer frequencies (i.e., the numbers of transductants per recipient bacterium) recorded for each marker. The dashed lines show the limits of detection for each marker. MSD, minimum significant difference.

FIG. 7

Single-step growth curve obtained for phage ΦCP6-3 when it was incubated at 30°C (●) compared with the single-step growth curve obtained at 25°C (○). The Boltzman equation was used to produce the sigmoidal curves.