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Genomes and Characterization of Phages Bcep22 and Bcepil02, Founders of a Novel Phage Type in Burkholderia Cenocepaciaᰔ† Jason J

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Genomes and Characterization of Phages Bcep22 and Bcepil02, Founders of a Novel Phage Type in Burkholderia Cenocepaciaᰔ† Jason J View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Texas A&M University JOURNAL OF BACTERIOLOGY, Oct. 2011, p. 5300–5313 Vol. 193, No. 19 0021-9193/11/$12.00 doi:10.1128/JB.05287-11 Copyright © 2011, American Society for Microbiology. All Rights Reserved. Genomes and Characterization of Phages Bcep22 and BcepIL02, Founders of a Novel Phage Type in Burkholderia cenocepaciaᰔ† Jason J. Gill,1,2 Elizabeth J. Summer,1,2 William K. Russell,3 Stephanie M. Cologna,3 Thomas M. Carlile,1 Alicia C. Fuller,1 Kate Kitsopoulos,1 Leslie M. Mebane,1 Brandi N. Parkinson,1 David Sullivan,1 Lisa A. Carmody,4 Carlos F. Gonzalez,2,5 John J. LiPuma,4 and Ry Young1,2* Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843-21281; Center for Phage Technology, Texas A&M University, College Station, Texas 77843-21282; Department of Chemistry, Texas A&M University, College Station, Texas 77843-30123; Department of Pediatrics and Communicable Disease, University of Michigan Medical School, Downloaded from Ann Arbor, Michigan 481094; and Department of Plant Pathology and Microbiology, Texas A&M University, College Station, Texas 77843-21325 Received 19 May 2011/Accepted 18 July 2011 Within the Burkholderia cepacia complex, B. cenocepacia is the most common species associated with aggres- sive infections in the lungs of cystic fibrosis patients, causing disease that is often refractive to treatment by antibiotics. Phage therapy may be a potential alternative form of treatment for these infections. Here we describe the genome of the previously described therapeutic B. cenocepacia podophage BcepIL02 and its close relative, Bcep22. Phage Bcep22 was found to contain a circularly permuted genome of 63,882 bp containing 77 http://jb.asm.org/ genes; BcepIL02 was found to be 62,714 bp and contains 76 predicted genes. Major virion-associated proteins were identified by proteomic analysis. We propose that these phages comprise the founding members of a novel podophage lineage, the Bcep22-like phages. Among the interesting features of these phages are a series of tandemly repeated putative tail fiber genes that are similar to each other and also to one or more such genes in the other phages. Both phages also contain an extremely large (ca. 4,600-amino-acid), virion-associated, multidomain protein that accounts for over 20% of the phages’ coding capacity, is widely distributed among other bacterial and phage genomes, and may be involved in facilitating DNA entry in both phage and other mobile DNA elements. The phages, which were previously presumed to be virulent, show evidence of a temperate lifestyle but are apparently unable to form stable lysogens in their hosts. This ambiguity complicates on September 21, 2018 by guest determination of a phage lifestyle, a key consideration in the selection of therapeutic phages. Bacteria of the genus Burkholderia constitute an ecologically treatments, has ignited interest in the development of alterna- and metabolically diverse group of organisms, which includes tive treatment options. soil saprophytes, nitrogen-fixing rhizosphere inhabitants, bio- The use of bacteriophages for the treatment of bacterial logical control agents, and species that are pathogenic to infections, a practice commonly termed phage therapy, is an plants, animals, and humans (14). Within this genus, the Burk- old idea, first proposed by the phage codiscoverer Felix holderia cepacia complex is currently comprised of at least 17 d’Herelle (78). While phage therapy was largely abandoned in bacterial species, including several species recognized as op- the West with the discovery of chemical antibiotics, the prac- portunistic human pathogens capable of infecting immuno- tice continued in the former Soviet Union and its satellite compromised individuals (50, 85). Especially at risk from B. states and is still used routinely today in these regions as an cepacia complex infections are individuals with cystic fibrosis adjunct to antibiotics in human medicine (72). Phage therapy (CF) or chronic granulomatous disease. While all members of has been a topic of increasing research in the West over the the B. cepacia complex have been associated with infections in past several years, as new bacterial pathogens have emerged, the lungs of CF patients, B. cenocepacia is the most common antimicrobial resistance in important human pathogens has member associated with persistent and aggressive infections in become more widespread, and the development pipelines for these patients, causing necrotizing pneumonia, sepsis, and novel small-molecule antimicrobials have largely run dry (63). death. Clinical outcomes of B. cenocepacia infections in CF Phages that have utility as potential therapeutic agents should patients are typically poor, and this bacterium is often innately meet at least two minimal criteria: they should be virulent resistant to many chemical antibiotics. The severity of these infections, coupled with the current lack of available effective (unable to form lysogens that would then be insensitive to further phage infections) and they must target the specific strains of the pathogen that they are intended to treat. While * Corresponding author. Mailing address: Department of Biochem- determination of the latter is relatively straightforward, the istry and Biophysics, Texas A&M University, 2128 TAMU, College issue of phage virulence is not always clear. By its nature, a Station, TX 77843-2128. Phone: (975) 845-2087. Fax: (979) 862-9718. temperate lifestyle is easy to prove (by isolation of stable ly- E-mail: [email protected]. sogens) but not as easily disproven, as failure to establish † Supplemental material for this article may be found at http://jb .asm.org/. lysogeny is not conclusive proof of virulence. ᰔ Published ahead of print on 29 July 2011. A number of phages capable of infecting clinically relevant 5300 VOL. 193, 2011 B. CENOCEPACIA PHAGES Bcep22 AND BcepIL02 5301 members of the B. cepacia complex have been characterized, wise protein sequence similarities were calculated by the Dice method, using and some have also been sequenced. Several of these phages, BLASTp determinations of amino acid identity. Proteomic analysis. such as NS1 and NS2 (57), DK1 and DK3 (41), KS4 and KS9 Phages were purified for proteomic analysis by two rounds of banding in a CsCl gradient, based on previously described methods (42, 67). (71), BcepMu (77), and KS10 (22), were isolated from super- Phage was then dialyzed thoroughly against gelatin-free SM buffer and concen- natants of lysogenic bacteria and are therefore temperate trated further using a 100-kDa molecular weight cutoff Amicon ultrafiltration phages. Based on their genome organization, the environmen- unit (Millipore). Phage proteins were analyzed by SDS-PAGE by boiling purified tally isolated phages Bcep781, Bcep1, Bcep43, and BcepB1A phage for 5 min in Laemmli sample buffer (62.5 mM Tris-HCl [pH 6.8], 2% SDS, 10% glycerol, 5% ␤-mercaptoethanol, 0.001% bromophenol blue), and the have been reported to be virulent phages (76). However, while equivalent of ϳ3 ϫ 1010 PFU was loaded per lane on a 10% SDS-PAGE gel by these phages appear to possess the desired characteristic of standard methods (40). Coomassie-stained bands were excised and subjected to virulence, they were not isolated on Bcc strains that are clini- reduction using dithiothreitol and alkylation with iodoacetamide followed by cally relevant for purposes of phage therapy. Recent work has tryptic digestion carried out robotically using a ProGest robotic device (Genomic shown that a lysogeny-defective mutant of the BcepMu-like Solutions, Ann Arbor, MI) suited for automated digestion protocols and tem- perature-controlled reactions. The resulting tryptic peptides were concentrated KS4, the temperate phage KS14, and the less-well-character- Downloaded from and desalted using C18 ZipTips (Millipore) and spotted robotically directly onto ized phage KS12 reduced B. cepacia complex-induced mortal- a matrix-assisted laser desorption ionization (MALDI) target (Genomic Solu- ity in a wax worm model (70). A repressorless virulent mutant tions) using a 7.5-mg/ml solution of ␣-cyano 4-hydroxycinnamic acid (CHCA). of phage KS9 has also been shown to reduce mortality in this Additionally, phage proteins were subjected to liquid chromatography-MALDI- tandem mass spectrometry (LC-MALDI-MS/MS) analysis. Phage samples were model (49). Here we describe two related B. cepacia complex denatured in 6 M urea and 1 mM dithiothreitol (DTT; Sigma) and subjected to phages, Bcep22 and BcepIL02. Both phages are able to infect 5 freeze-thaw cycles between a dry ice-ethanol bath and a 37°C heat block. The pathogenic clinical isolates of B. cenocepacia, and one of them, samples were centrifuged at 14,000 rpm for 10 min, and the supernatant was BcepIL02, has been shown to reduce the colonization of patho- subjected to solution-phase tryptic digestion. Briefly, 10 mM iodoacetamide was genic B. cenocepacia in a mouse lung model (10). added to the protein solution followed by addition of trypsin at a 1:50 (wt/wt) ratio and incubated at 37°C overnight. Approximately 1 ␮g of the tryptic diges- tion solution was further separated by loading onto a trap column (300 ␮mby1 http://jb.asm.org/ ␮ cm; C18 PepMap) coupled with a Vydac (150 mby15cm;C18) analytical MATERIALS AND METHODS column, and chromatography was carried out using an LC Packings Ultimate Strains and culture conditions. Phage Bcep22 was isolated from soil collected system (Dionex). The eluent from the analytical column was spotted robotically in the summer of 2000 from Orange County, NY, by enrichment against a B. onto the MALDI target via a ProBot (LC Packings) as described previously (65). cenocepacia strain (S198C28A) isolated from the same soil sample. Phage Bce- MALDI-MS and MS/MS analysis were carried out on a model 4700 Proteom- pIL02 was isolated from a corn field soil sample in Champaign County, IL, in the ics Analyzer (Applied Biosystems).
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