Campylobacter jejuni group III phage CP81 contains many T4-like genes without belonging to the T4-type phage group: implications for the evolution of T4 phages

Abstract

CP81 is a virulent Campylobacter group III phage whose linear genome comprises 132,454 bp. At the nucleotide level, CP81 differs from other phages. However, a number of its structural and replication/recombination proteins revealed a relationship to the group II Campylobacter phages CP220/CPt10 and to T4-type phages. Unlike the T4-related phages, the CP81 genome does not contain conserved replication and virion modules. Instead, the respective genes are scattered throughout the phage genome. Moreover, most genes for metabolic enzymes of CP220/CPt10 are lacking in CP81. On the other hand, the CP81 genome contains nine similar genes for homing endonucleases which may be involved in the attrition of the conserved gene order for the virion core genes of T4-type phages. The phage apparently possesses an unusual modification of C or G bases. Efficient cleavage of its DNA was only achieved with restriction enzymes recognizing pure A/T sites. Uncommonly, phenol extraction leads to a significant loss of CP81 DNA from the aqueous layer, a property not yet described for other phages belonging to the T4 superfamily.

Fig. 1.

Morphology and structural proteins of CP81. (A) Electron micrographs of negatively stained virions. The micrographs on the left and on the right show particles stained with phosphotungstate and ammonium molybdate, respectively (see Materials and Methods). The head diameter was measured between the sides parallel and diagonal to the tail. Both axes were identical within the error range. A collar structure as in T4 was not observed. (B) SDS-PAGE of structural CP81 proteins (27). As a marker, the unstained molecular mass marker of Fermentas (St. Leon-Rot, Germany) was used. Visible bands were excised from the gel and analyzed by mass spectrometry (53).

Fig. 2.

Restriction patterns of native CP81 DNA. The enzymes DraI, VspI, and SmiI recognize pure A/T sequences.

Fig. 3.

Analysis of native and amplified CP81 DNA. (A) Loss of native CP81 DNA after extraction with phenol. Native DNA of group II phage NCTC12684 and the amplified DNAs of both phages were not lost after phenol extraction. (B) Susceptibility of native and amplified CP81 DNA to cleavage by restriction endonucleases whose recognition sites are composed of A/T and G/C sequences.

Fig. 4.

Map of the CP81 genome. Putative genes are colored according to the predicted functions of their products. The positions of putative transcription terminators are indicated.

Fig. 5.

Relationship of CP81 to T4-type phages. Circular illustration of the CP81 genome and similarities of predicted products to proteins of the group II phages CP220/CPt10 and to phages belonging to the T4 superfamily. The inner circle (black) shows a plot of the G+C content deviation along the CP81 genome.

Fig. 6.

Virion structural modules of T4-type phages. The T-even, PseudoT-even, SchizoT-even, and ExoT-even subgroups are represented by T4/RB69, RB49/RB43, Aeh1/KVP40, and S-PM2, respectively (10, 14). Genes are denoted according to the nomenclature of T4 and colored on the basis of similarities of their products to other proteins.