Characterization and genome sequencing of two Propionibacterium acnes phages displaying

Background: Propionibacterium acnes is a Gram positive rod inhabiting the human skin that also infects orthopaedic implants and is associated with acne vulgaris. Previously, one lytic bacteriophage, PA6, from P. acnes has been sequenced and partially characterized. We recently isolated several inducible phages from P. acnes classified as Siphoviruses based on morphology and partial genome sequencing. Results: In this study we sequenced the inducible P. acnes phages PAD20 and PAS50, isolated from deep infection and from skin, respectively. The genomes of PAD20 and PAS50 are 29,074 and 29,017 bp, respectively, compared with the 29,739 bp of PA6. The phage genomes have 87.3-88.7% nucleotide sequence identity. The genes are divided into clusters with different levels of similarity between the phages. PAD20 and PAS50 share four genes encoding identical amino acid sequences. Some deletions and insertions in the genomes have occurred, resulting in lack of genes, frame shifts, and possible regulatory differences. No obvious virulence factor gene candidates were found. The phages are inducible, but bacteria can be cured of phages by serial colony isolations and lose their phages during stationary phase, but are still sensitive to new phage infections. Construction of a phylogenetic tree based on more than 459 phage genomes, suggested that P. acnes phages represent a new lineage of Siphoviruses. Conclusions: The investigated P. acnes Siphovirus genomes share a high degree of homology to other P. acnes phages sequenced, but not to genomes of other phages isolated from Propionibacteria. The phage genomes are not integrated in the bacterial genome, but instead, most likely have a pseudolysogenic life cycle. Background Propionibacterium acnes is a Gram positive rod normally found on human skin [1], but has also been isolated from infections of orthopaedic implants [2,3], and has been suggested to be involved in the development of acne vulgaris. Recently, the first genome of a P. acnes strain was sequenced [4] and several putative virulence factors were identified [5]. The sequenced P. acnes genome only contains one cryptic prophage, but it has earlier been shown that some P. acnes strains are carriers of inducible phages [6]. Other groups have also studied Propionibacterium phages both in order to establish a tool to differentiate between serotypes, but also to further characterize phages mainly infecting bacteria important for the dairy industry [7-16]. Recently, Farrar et al, sequenced the first genome of a P. acnes phage, PA6 (GenBank accession no. DQ431235) [17]. The genome consists of 29,739 bp dsDNA with 48 putative coding genes. The phage was determined to be a lytic phage since no integrase or repressor could be detected in the genome. Eighteen of the genes were assigned predicted functions based on their similarities to other known phage genes. Recently, we isolated and characterized inducible phages from P. acnes isolated from both deep infections and from skin of healthy individuals [6]. By comparing the genomes of these different phages, a better understanding of how the phage influences its host and an insight in the great genetic diversity seen among phages, as shown * Correspondence: rolf.lood@med.lu. se Department of Clinical Sciences, Division of Infection Medicine, BMC-B14, Lund University, SE-221 84 Lund, Sweden Lood and Collin BMC Genomics 2011, 12:198 http://www.biomedcentral. com/1471-2164/12/198 © 2011 Lood and Collin; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. by other comparative studies on phage genomes can be achieved [18,19]. Phages can enter two principally different life cycles, e. g. a lytic life cycle or a lysogenic cycle. In the lysogenic cycle most phages integrate their genome into the host genome. This is most often regulated by a phage-borne integrase, but there is also a recent report of a filamentous phage, CTXj from Vibrio cholerae, that uses the host recombinases XerC and XerD to integrate into the host genome [20,21]. Furthermore, there are a few phages known to form stable linear or circular plasmids as a part of their integration mechanism [22-24], but most known temperate phages encode their own integrases. Other than those two well-defined phage life cycles, phages are also known to be able to interact with their host in other life cycles, termed as persistent infections, pseudolysogeny, carrier-state, and chronic infections [25,26]. Those phage life cycles, even though common, are less defined compared to the lytic and lysogenic life cycles. Common for all states, except the chronic infection, is that the bacteria carry the phage DNA, but the DNA is not integrated into the bacterial chromosome, but rather exists as a plasmid. The chronic infection is characterized by the release of phages from the bacteria without causing lysis [25]. In this study, we have sequenced the genome of one phage induced from a P. acnes strain isolated from deep infection (PAD20), and the genome of one phage induced from a P. acnes strain isolated from skin (PAS50), and we have compared the two genomes with the reported lytic phage PA6 [17]. The phages have earlier been shown to have dissimilarities in the genes encoding a putative major head protein and an amidase [6], but no other genes have been examined. This offers an opportunity to investigate if phages isolated from P. acnes contribute to the virulence seen in different strains of P. acnes, and to examine the genetic diversity seen among phages. Results and discussion Genomic organization and homology The genomic organization of the three examined phages are very similar, even though some genes differ (see Figure 1). Both phages have a 13 bp ssDNA extension at both ends of the genome, identical to that of phage PA6. These extensions enable the formation of potential concatamers and circularization of the phage genome. The genome of PAD20 is 29,074 bp and has a GC content of 54.1%, compared to the genome of PAS50 that is 29,017 bp and has a GC content of 53.9%. PAD20 has two larger deletions in the genome compared to PA6. The first is a 226 bp deletion resulting in the deletion of the main part of gp22 and thereby in a frame shift giving rise to a combination of the gene products Gp22 and Gp23 (Gp22.23, see additional file 1). The second larger deletion is a 378 bp deletion of the entire gp42 gene. PAS50 has two larger deletions and one insertion. The first deletion in PAS50 is a 207 bp deletion resulting in the deletion of the gp45 gene, and a second deletion of almost 500 bp in a non-coding region, corresponding to bp 27,932-28,419 in PA6. The insert in PAS50 is an 84 bp insertion in gp37 resulting in a 28 amino acid larger exonuclease. To further investigate the genomic organization of the different P. acnes phages, the genomes were screened for putative sigma70-dependent promoters and transcriptional terminators (see Figure 1 and Table 1). Several putative promoters were detected in all phages upstream of gp7, gp11 and gp46, as well as upstream of gp45 and gp30, potentially enabling the phage to transcribe different parts of the genome at different stages of infection. One of the promoters with the highest score was upstream of a hitherto unannotated ORF (3693-3301, gp50). However, Figure 1 Genomic organization of the P. acnes phages. Schematic representation of the genome organization of P. acnes phages PAD20, PA6 and PAS50 and some functional assignments. The arrows indicate the different ORFs and the direction of transcription. The colours of the ORFs indicate the homology of phage PAD20 and PAS50 to PA6. Putative sigma70-promoters and transcriptional terminators are indicated. Lood and Collin BMC Genomics 2011, 12:198 http://www.biomedcentral. com/1471-2164/12/198 Page 2 of 14