GIL16, a new gram-positive tectiviral phage related to the Bacillus thuringiensis GIL01 and the Bacillus cereus pBClin15 elements

Abstract

One of the most notable characteristics of Tectiviridae resides in their double-layer coats: the double-stranded DNA is located within a flexible lipoprotein vesicle covered by a rigid protein capsid. Despite their apparent rarity, tectiviruses have an extremely wide distribution compared to other phage groups. Members of this family have been found to infect gram-negative (PRD1 and relatives) as well as gram-positive (Bam35, GIL01, AP50, and phiNS11) hosts. Several reports have shown that tectiviruses infecting gram-negative bacteria are closely related, whereas no information is currently available on the genetic relationship among those infecting gram-positive bacteria. The present study reports the sequence of GIL16, a new isolate originating from Bacillus thuringiensis, and a genetic comparison of this isolate with the tectiviral bacteriophages Bam35 and GIL01, which originated from B. thuringiensis serovars Alesti and Israelensis, respectively. In contrast to PRD1 and its relatives, these are temperate bacteriophages existing as autonomous linear prophages within the host cell. Mutations in a particular motif in both the GIL01 and GIL16 phages are also shown to correlate with a switch to the lytic cycle. Interestingly, both bacterial viruses displayed narrow, yet slightly different, host spectrums. We also explore the hypothesis that pBClin15, a linear plasmid hosted by the Bacillus cereus reference strain ATCC 14579, is also a prophage. Sequencing of its inverted repeats at both extremities and a comparison with GIL01 and GIL16 emphasize its relationship to the Tectiviridae.

FIG. 1.

Electron micrograph of GIL16 particles with a tail-like structure. Bar, 50 nm.

FIG. 2.

ITRs at the GIL16 extremities and comparison with those of GIL01. Conserved residues within the GIL16 ITRs are indicated by full vertical lines. Conserved residues between the GIL01 and GIL16 left and right ends are shown by dashed vertical lines.

FIG. 3.

Genetic map of the GIL16c and GIL01 bacteriophages and of pBClin15. Predicted ORFs are depicted by block arrows and ORFs sharing similarities with known proteins are shown in dark gray. DNA pol, DNA polymerase; LexA, LexA-like repressor; Pack, DNA-packaging protein. Similar ORFs are connected by vertical lines. ORFs shared by two genomes are shown in pale gray, while unique ORFs are shown in black.

FIG. 4.

Amino acid comparison between the putative DNA-packaging proteins of GIL16 (ORF12), GIL01 (ORF13), and pBClin15 (ORF12) and the P9 protein of PRD1. The ATP-binding motif (GXXGXGKXXXXXXXL) is framed, and conserved residues are indicated by asterisks. All residues of GIL16 that are present in at least two other ORFs are indicated by gray boxes.

FIG. 5.

Mutations observed in the ORF6-ORF7 region of GIL16 and GIL01 clear plaque (cp) mutants compared to the wt sequence. Motifs affected by the mutations are indicated by white letters over a black background (mismatches are indicated by gray boxes), and mutations observed in each cp mutant are indicated by black letters over a gray background.

FIG. 6.

PFGE patterns of undigested genomic DNAs from B. cereus and B. thuringiensis strains used in this study. The molecular size markers are the lambda 48.5-kb size marker (Sigma) (left) and a Gene Ruler mix (Fermentas) (right).

FIG. 7.

Inverted terminal repeats of pBClin15. Conserved nucleotides are indicated by vertical lines.