Genomic analysis and relatedness of P2-like phages of the Burkholderia cepacia complex

Abstract

Background: The Burkholderia cepacia complex (BCC) is comprised of at least seventeen Gram-negative species that cause infections in cystic fibrosis patients. Because BCC bacteria are broadly antibiotic resistant, phage therapy is currently being investigated as a possible alternative treatment for these infections. The purpose of our study was to sequence and characterize three novel BCC-specific phages: KS5 (vB_BceM-KS5 or vB_BmuZ-ATCC 17616), KS14 (vB_BceM-KS14) and KL3 (vB_BamM-KL3 or vB_BceZ-CEP511).

Results: KS5, KS14 and KL3 are myoviruses with the A1 morphotype. The genomes of these phages are between 32317 and 40555 base pairs in length and are predicted to encode between 44 and 52 proteins. These phages have over 50% of their proteins in common with enterobacteria phage P2 and so can be classified as members of the Peduovirinae subfamily and the "P2-like viruses" genus. The BCC phage proteins similar to those encoded by P2 are predominantly structural components involved in virion morphogenesis. As prophages, KS5 and KL3 integrate into an AMP nucleosidase gene and a threonine tRNA gene, respectively. Unlike other P2-like viruses, the KS14 prophage is maintained as a plasmid. The P2 E+E' translational frameshift site is conserved among these three phages and so they are predicted to use frameshifting for expression of two of their tail proteins. The lysBC genes of KS14 and KL3 are similar to those of P2, but in KS5 the organization of these genes suggests that they may have been acquired via horizontal transfer from a phage similar to λ. KS5 contains two sequence elements that are unique among these three phages: an ISBmu2-like insertion sequence and a reverse transcriptase gene. KL3 encodes an EcoRII-C endonuclease/methylase pair and Vsr endonuclease that are predicted to function during the lytic cycle to cleave non-self DNA, protect the phage genome and repair methylation-induced mutations.

Conclusions: KS5, KS14 and KL3 are the first BCC-specific phages to be identified as P2-like. As KS14 has previously been shown to be active against Burkholderia cenocepacia in vivo, genomic characterization of these phages is a crucial first step in the development of these and similar phages for clinical use against the BCC.

Figure 1

Transmission electron micrographs of KS5 (A), KS14 (B) and KL3 (C). Phages were stained with 2% phosphotungstic acid and viewed at 140,000-fold magnification. Scale bars represent 50 nm.

Figure 2

Genome maps of KS5, KS14 and KL3. Genes transcribed from the plus strand are shown above and genes transcribed from the minus strand are shown below. The scale (in kbp) is shown on the bottom. The prophage gene order is shown for KS5 and KL3. The gene order for KS14 (which is maintained as a plasmid prophage) was chosen based on alignment with the other two sequences. Legend: orange, recombinase; yellow, transcriptional or translational regulation; black, insertion sequence; purple, tail morphogenesis; red, DNA modification; light blue, lysis; dark blue, capsid morphogenesis and DNA packaging; green, reverse transcription; brown, replication and partitioning; gray, unknown function.

Figure 3

PROmer/MUMmer/Circos comparison of the KS5, KS14, KL3 and P2 prophages. Regions of similarity on the same strand are shown in green and regions of similarity on the opposite strand are shown in red. The scale (in kbp) is shown on the outside. The sequence start site for the KS14 prophage (which is maintained as a plasmid) was chosen based on alignment with the other three sequences. PROmer parameters: breaklen = 60, maxgap = 30, mincluster = 20, minmatch = 6.

Figure 4

Detection of lysogeny in KS14-resistant B. cenocepacia C6433 isolates [19]. Bacterial genomic DNA was amplified using KS14-specific primers. Lane 1: 1 Kb Plus DNA ladder (Invitrogen), lane 2: DNA-free control, lane 3: C6433 control, lane 4: KS14-resistant C6433 isolate I, lane 5: KS14-resistant C6433 isolate II, lane 6: KS14-resistant C6433 isolate III, lane 7: KS14-resistant C6433 isolate IV, lane 8: KS14-resistant C6433 isolate V. The size of the markers (in kbp) is shown on the left.

Figure 5

Isolation of the KS14 plasmid prophage. DNA was isolated using a QIAprep Spin Miniprep plasmid isolation kit (Qiagen) and digested with EcoRI (Invitrogen). Lane 1: 1 Kb Plus DNA ladder (Invitrogen), lane 2: B. cenocepacia C6433, lane 3: B. multivorans ATCC 17616, lane 4: B. cenocepacia CEP511 lane 5: B. cenocepacia K56-2, lane 6: blank, lane 7: 1 Kb Plus DNA ladder, lane 8: KS14-resistant C6433 isolate I, lane 9: KS14-resistant C6433 isolate II, lane 10: KS14-resistant C6433 isolate III, lane 11: KS14-resistant C6433 isolate IV, lane 12: KS14-resistant C6433 isolate V. The size of the markers (in kbp) is shown on the left. A KS14 EcoRI DNA digest and the size of the bands predicted for this digest (> 1 kbp in size) are shown on the far right.

Figure 6

Conservation of the P2 E/E+E' frameshift sequence in KS5, KS14 and KL3. For each phage, the DNA sequence is shown in the first line, the translation in the original frame is shown in the second line, the translation in the -1 frame is shown in the third line and the amino acid sequence of the frameshifted protein is shown in the fourth line. The conserved TTTTTTG frameshift sequence is underlined. The frameshift is predicted to occur after the terminal G in this sequence.

Figure 7

Organization of the lysBC genes in KS5, KS14 and KL3. R genes (KS5 30, KS14 24 and KL3 30) are shown in light gray, lysB genes (KS5 32, KS14 26 and KL3 32) are shown in dark gray and lysC genes (KS5 31, KS14 25 and KL3 31) are shown in white. The scale (in bp) is shown below.

Figure 8

Comparison of the ISBmu23 and ISBmu2 insertion sequences. A) Structure of ISBmu23. IR, inverted repeats. Relative positions of the inverted repeats and transposase gene (in bp) are shown below. B) Alignment of the ISBmu2 and ISBmu23 inverted repeats. Non-consensus bases are underlined. ISBmu2 sequences are from Ohtsubo et al. [72]. L, left repeat; Ri, right repeat inverted.