Burkholderia thailandensis E125 harbors a temperate bacteriophage specific for Burkholderia mallei

Abstract

Burkholderia thailandensis is a nonpathogenic gram-negative bacillus that is closely related to Burkholderia mallei and Burkholderia pseudomallei. We found that B. thailandensis E125 spontaneously produced a bacteriophage, termed phiE125, which formed turbid plaques in top agar containing B. mallei ATCC 23344. We examined the host range of phiE125 and found that it formed plaques on B. mallei but not on any other bacterial species tested, including B. thailandensis and B. pseudomallei. Examination of the bacteriophage by transmission electron microscopy revealed an isometric head and a long noncontractile tail. B. mallei NCTC 120 and B. mallei DB110795 were resistant to infection with phiE125 and did not produce lipopolysaccharide (LPS) O antigen due to IS407A insertions in wbiE and wbiG, respectively. wbiE was provided in trans on a broad-host-range plasmid to B. mallei NCTC 120, and it restored LPS O-antigen production and susceptibility to phiE125. The 53,373-bp phiE125 genome contained 70 genes, an IS3 family insertion sequence (ISBt3), and an attachment site (attP) encompassing the 3' end of a proline tRNA (UGG) gene. While the overall genetic organization of the phiE125 genome was similar to lambda-like bacteriophages and prophages, it also possessed a novel cluster of putative replication and lysogeny genes. The phiE125 genome encoded an adenine and a cytosine methyltransferase, and purified bacteriophage DNA contained both N6-methyladenine and N4-methylcytosine. The results presented here demonstrate that phiE125 is a new member of the lambda supergroup of Siphoviridae that may be useful as a diagnostic tool for B. mallei.

FIG. 1.

Transmission electron micrograph of bacteriophage φE125 negatively stained with 1% phosphotungstic acid. Scale bar, 100 nm.

FIG. 2.

Immunoblot analysis of B. mallei LPS O antigens. Bacteria were washed, resuspended in SDS-PAGE sample buffer, boiled, treated with proteinase K, and subjected to SDS-PAGE. The LPS O antigens were blotted to a polyvinylidene difluoride membrane and reacted with the monoclonal antibody 3D11. (A) LPS O-antigen profiles of NCTC and ATCC B. mallei strains. (B) Comparative LPS O-antigen profiles of φE125-resistant and φE125-susceptible B. mallei strains. All strains form plaques with bacteriophage φE125 except NCTC 120, DB110795, and NCTC 120 (pBHR1).

FIG. 3.

Physical and genetic map of the bacteriophage φE125 genome. The locations and directions of transcription of genes are represented by arrows, and the gene names are shown below. The locations of HindIII endonuclease restriction sites are shown (H), and the insertion sequence ISBt3 is represented as a rectangle. The locations of the cohesive (cos) and bacteriophage attachment (attP) sites are shown above and below the φE125 genome, respectively. The putative functions of proteins encoded by φE125 genes are color coded.

FIG. 4.

Bacteriophage φE125 integrates into the proline tRNA (UGG) gene in B. mallei and B. thailandensis. (A) The nucleotide sequence of the proline tRNA (UGG) gene of B. mallei ATCC 23344 and B. thailandensis E125. The underlined sequence represents the 49-bp attachment site that is identical in the φE125 genome (attP), the B. mallei chromosome (attB), and the B. thailandensis chromosome (attB). The location of the anticodon in the proline tRNA gene is shown in bold. (B) Schematic representation of integration of the φE125 genome into the proline tRNA (UGG) gene of B. mallei and B. thailandensis. The φE125 genome is depicted as a circle, and the approximate locations of gene1, gene18, gene32, gene33, gene34, gene35, gene70, and the cos site are shown. The B. mallei and B. thailandensis chromosomes are represented as a line, and the location and direction of transcription of orfA and orfB are represented by arrows. The 5′ end of the proline tRNA (UGG) gene is shown as a thin white rectangle, and the 3′ end (the attachment site) is shown as a thin black rectangle. Following site-specific recombination (X), the orfA and orfB genes are separated by the integrated φE125 prophage.

FIG. 5.

Dot blot assay to detect genomic DNA methylation using rabbit primary antibodies specific for m6A or m4C. (A) Methylase dot blot assay using polyclonal antibodies specific for m6A. Bacteriophage 2 genomic DNA was used as a positive (+) control. (B) Methylase dot blot assay using polyclonal antibodies specific for m4C. E. coli DB24 genomic DNA methylated by M.RsaI served as a positive (+) control. The quantities of genomic DNAs spotted on each panel are shown.