Bacteriophage phiYeO3-12, specific for Yersinia enterocolitica serotype O:3, is related to coliphages T3 and T7

Abstract

Bacteriophage phiYeO3-12 is a lytic phage of Yersinia enterocolitica serotype O:3. The phage receptor is the lipopolysaccharide O chain of this serotype that consists of the rare sugar 6-deoxy-L-altropyranose. A one-step growth curve of phiYeO3-12 revealed eclipse and latent periods of 15 and 25 min, respectively, with a burst size of about 120 PFU per infected cell. In electron microscopy phiYeO3-12 virions showed pentagonal outlines, indicating their icosahedral nature. The phage capsid was shown to be composed of at least 10 structural proteins, of which a protein of 43 kDa was predominant. N-terminal sequences of three structural proteins were determined, two of them showing strong homology to structural proteins of coliphages T3 and T7. The phage genome was found to consist of a double-stranded DNA molecule of 40 kb without cohesive ends. A physical map of the phage DNA was constructed using five restriction enzymes. The phage infection could be effectively neutralized using serum from a rabbit immunized with whole phiYeO3-12 particles. The antiserum also neutralized T3 infection, although not as efficiently as that of phiYeO3-12. phiYeO3-12 was found to share, in addition to the N-terminal sequence homology, several common features with T3, including morphology and nonsubjectibility to F exclusion. The evidence conclusively indicated that phiYeO3-12 is the first close relative of phage T3 to be described.

FIG. 1

Electron micrograph of negatively stained φYeO3-12 virions.

FIG. 2

Characterization of the φYeO3-12 genome. (A) Agarose gel electrophoresis of φYeO3-12 DNA after single and double digestions with restriction enzymes BclI, EcoRV, PmlI, SnaBI, and XbaI. (B) Restriction map of phage φYeO3-12 DNA (scale in kilobases) constructed on the basis of the data in panel A. The restriction fragments are indicated by double-headed arrows, below which the fragment sizes in kilobases are given.

FIG. 2

Characterization of the φYeO3-12 genome. (A) Agarose gel electrophoresis of φYeO3-12 DNA after single and double digestions with restriction enzymes BclI, EcoRV, PmlI, SnaBI, and XbaI. (B) Restriction map of phage φYeO3-12 DNA (scale in kilobases) constructed on the basis of the data in panel A. The restriction fragments are indicated by double-headed arrows, below which the fragment sizes in kilobases are given.

FIG. 3

One-step growth curve of bacteriophage φYeO3-12 on Y. enterocolitica serotype O:3 strain 6471/76-c at RT. Shown are the PFU per infected cell in chloroform-treated cultures (□) and in untreated cultures (■) at different time points. Each data point is a mean from three experiments.

FIG. 4

Characterization of the rabbit antiserum specific for φYeO3-12 particles. The sensitivity of the antiserum in detection of immobilized native phage particles was assessed using EIA. Shown are the absorbance values obtained using antiserum dilutions of 1:10,000 (□) and 1:2,000 (■). Each data point is a mean from three experiments.

FIG. 5

SDS-PAGE of the φYeO3-12 and T3 particles and immunoblot of the φYeO3-12 proteins developed with rabbit antiserum. The phage proteins discussed in the text are indicated by arrows at the right. Protein molecular size markers are shown at the left.

FIG. 6

Neutralization efficiency of the rabbit antiserum specific for φYeO3-12 particles. Shown are obtained PFU values of φYeO3-12 at different antiserum dilutions. Each data point is a mean from one to five experiments.