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

Lynch KH, Stothard P, Dennis JJ - BMC Genomics (2010)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological Sciences, University of Alberta, Edmonton, Alberta T6G 2E9, Canada. jon.dennis@ualberta.ca

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.

Show MeSH

Related in: MedlinePlus

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3091744&req=5

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.

Mentions: KS14 is different from other P2-like phages in that it does not encode a tyrosine integrase. Most temperate phages use a tyrosine recombinase (or, in rare cases, a serine recombinase) to facilitate recombination between the phage attP site and the host attB site [40]. KS14 encodes a serine recombinase (gp6), but this protein is unlikely to mediate prophage integration for three reasons. First, gp6 is more closely related to invertases such as Mu Gin (49% identity, E-value: 8e-44) and P1 Cin (49% identity, E-value: 7e-43) than to integrases such as those from Streptomyces lividans phage ϕC31 (29% identity, E-value: 1.2) and Mycobacterium smegmatis phage Bxb1 (29% identity, E-value: 3e-4) [41-44]. Second, gp6 lacks the conserved cysteine-rich and leucine/isoleucine/valine/methionine-rich regions found in other serine integrases [45]. Third, gp6 is only 225 aa in length, which is substantially smaller than the serine integrases that are typically between 450-600 aa in length [45]. We did not believe KS14 to be obligately lytic because it encodes a putative repressor protein (gp5) and because previously collected KS14-resistant C6433 isolates were predicted to be lysogenized based on PCR-positivity with KS14-specific primers (Figure 4) [19].


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

Lynch KH, Stothard P, Dennis JJ - BMC Genomics (2010)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3091744&req=5

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.
Mentions: KS14 is different from other P2-like phages in that it does not encode a tyrosine integrase. Most temperate phages use a tyrosine recombinase (or, in rare cases, a serine recombinase) to facilitate recombination between the phage attP site and the host attB site [40]. KS14 encodes a serine recombinase (gp6), but this protein is unlikely to mediate prophage integration for three reasons. First, gp6 is more closely related to invertases such as Mu Gin (49% identity, E-value: 8e-44) and P1 Cin (49% identity, E-value: 7e-43) than to integrases such as those from Streptomyces lividans phage ϕC31 (29% identity, E-value: 1.2) and Mycobacterium smegmatis phage Bxb1 (29% identity, E-value: 3e-4) [41-44]. Second, gp6 lacks the conserved cysteine-rich and leucine/isoleucine/valine/methionine-rich regions found in other serine integrases [45]. Third, gp6 is only 225 aa in length, which is substantially smaller than the serine integrases that are typically between 450-600 aa in length [45]. We did not believe KS14 to be obligately lytic because it encodes a putative repressor protein (gp5) and because previously collected KS14-resistant C6433 isolates were predicted to be lysogenized based on PCR-positivity with KS14-specific primers (Figure 4) [19].

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological Sciences, University of Alberta, Edmonton, Alberta T6G 2E9, Canada. jon.dennis@ualberta.ca

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.

Show MeSH
Related in: MedlinePlus