The complete genome sequence and genetic analysis of ΦCA82 a novel uncultured microphage from the turkey gastrointestinal system
© Zsak et al; licensee BioMed Central Ltd. 2011
Received: 25 March 2011
Accepted: 29 June 2011
Published: 29 June 2011
The genomic DNA sequence of a novel enteric uncultured microphage, ΦCA82 from a turkey gastrointestinal system was determined utilizing metagenomics techniques. The entire circular, single-stranded nucleotide sequence of the genome was 5,514 nucleotides. The ΦCA82 genome is quite different from other microviruses as indicated by comparisons of nucleotide similarity, predicted protein similarity, and functional classifications. Only three genes showed significant similarity to microviral proteins as determined by local alignments using BLAST analysis. ORF1 encoded a predicted phage F capsid protein that was phylogenetically most similar to the Microviridae ΦMH2K member's major coat protein. The ΦCA82 genome also encoded a predicted minor capsid protein (ORF2) and putative replication initiation protein (ORF3) most similar to the microviral bacteriophage SpV4. The distant evolutionary relationship of ΦCA82 suggests that the divergence of this novel turkey microvirus from other microviruses may reflect unique evolutionary pressures encountered within the turkey gastrointestinal system.
Keywordsmicrophage microviridae turkey enteric metagenomics
Metagenomics analyses have lead to the discovery of a variety of microbial nucleotide sequences from environmental samples . These techniques have also allowed for the discovery of uncultured viral nucleotide sequences that are commonly from bacteriophages [2–4] that has also resulted in the discovery of useful enzymes for molecular biology . There has been a resurgent interest in bacteriophage biology and their use or use of phage gene products as antibacterial agents [6–8]. Bacteriophages are thought to be the most abundant life form as a group  and the importance of phage to bacterial evolution [10, 11], the role of phage or prophage encoded virulence factors that contribute to bacterial infectious diseases [12–14] and their contribution to horizontal gene transfer  cannot be over stated. Additionally, the contribution to microbial ecology  and to agricultural production [17, 18] is also extremely important.
Enteric diseases are an important economic production problem for the poultry industry worldwide. One of the major economically important enteric diseases for the poultry industry are the poult enteritis complex (PEC) and poult enteritis mortality syndrome (PEMS) in turkeys and a runting-stunting syndrome (RSS) in broiler chickens . Consequently, studies have been ongoing to identify novel enteric viruses among poultry species at our laboratory. In a recent study, we utilized the Roche/454 Life Sciences GS-FLX platform to compile an RNA virus metagenome from turkey flocks experiencing enteric disease . This approach yielded numerous sequences homologous to viruses in the BLAST nr protein database, many of which have not been described in turkeys.
Additionally, we have successfully applied a random PCR-based method for detection of unknown microorganisms from enteric samples of turkeys that resulted in identification of genomic sequences and subsequent determination of the full-length genome from a previously uncultured parvovirus . During these ongoing investigations to further characterize the turkey gut microbiome and identify novel viral pathogens of poultry, bacteriophage genomic sequences have also been identified. Herein we report the complete genomic sequence of a putative novel member of the Microviridae obtained from turkey gastrointestinal DNA samples utilizing metagenomics approaches. The protein sequences of ΦCA82 were most similar to those of Chlamydia phages.
Materials and Methods
Assembly of ΦCA82, a novel member of the Microviridae family
Forty-two complete intestinal tracts (from duodenum/pancreas to cloaca, including cecal tonsils) from a turkey farm in California, U.S.A. with histories of enteric disease problems were received at the Southeast Poultry Research Laboratory (SEPRL). The intestines were processed and pooled into a single sample, as previously described . A sequence-independent polymerase chain reaction (PCR) protocol was employed to amplify particle-associated nucleic acid (PAN) present in turkey intestinal homogenates, and has been described elsewhere in detail . Using this approach, a total of 576 clones were identified and sequenced with the M13 forward and reverse primers on an AB-3730 automated DNA sequencer. The sequenced clones were used as query sequences to search the GenBank non-redundant nucleotide and protein databases using the blastn and blastx algorithms . In total, the majority of clones with inserts had no hit in the databases using tblastx . However, 46% of the cloned DNA had homology to cellular DNA, bacterial DNA, bacteriophage DNA, and several eukaryotic viral DNA genomes. Twelve DNA clones had sequence similarity to single-stranded DNA microphages, which have also been identified predominantly in microbialites . A contig, CA82 with an average of eightfold coverage and length of 1962 nt was assembled from eight of those clones. This contig had no significant nucleotide similarity to database sequences, but the deduced amino acid sequence revealed significant similarity to the members of the family Microviridae. This initial contig was used to design PCR primers in the opposite orientation of the circular ssDNA to assemble into a contiguous ΦCA82 genome. The PCR amplification resulted in a 3.4 kb product that closed the gap between the CA82 contig and the rest of the circular genome. The final sequence was confirmed by sub-cloning and primer walking with primers resulting ~1 kb fragments containing 250 bp overlapping sequences across the genome. The circular DNA genome was assembled from contigs exhibiting 100% nucleotide identity within the overlapping regions.
The ΦCA82 genome and ORFs were aligned with selected microvirus sequences using ClustalW . Putative ORFs within the ΦCA82 genome were predicted using the FGENESV Trained Pattern/Markov chain-based viral gene prediction method from the Softberry website . Searches for conserved domains within the ΦCA82 genome were performed with the Conserved Domain Database (CDD) Search Service v2.17 at the National Center for Biotechnology Information (NCBI) website .
Comparative genomics of the Microviridae
Microviridae sequences used for comparative genomic analyses
NCBI Taxon ID
Genome Size (bp)
Chlamydia phage 1
Chlamydia phage 2
Chlamydia phage 3
Chlamydia phage 4
Chlamydia pneumoniae phage CPAR39
Enterobacteria phage G4
Enterobacteria phage St-1
Enterobacteria phage alpha3
Enterobacteria phage phiX174
Guinea pig Chlamydia phage
Spiroplasma phage 4
Genomic functional comparisons were based on pfam categories for each predicted gene as classified by the IMG annotation pipeline . A data table of pfam categories and gene counts for each genome was used to construct a similarity matrix and dendrogram in R. To determine which predicted genes were unique to ΦCA82 and those which were shared with other Microviridae members, the Microviridae pangenome was constructed as the union of all predicted genes from the 14 Microviridae genomes and compared to predicted genes for ΦCA82 using both CD-HIT and our data analysis pipeline as described above and blastp run with default parameters except for an e-value cutoff of 0.01.
Nucleotide accession number
The nucleotide sequence of ΦCA82 genome was deposited in GenBank under accession number HQ264138.
Results and Discussion
The ΦCA82 genome
Capsid proteins of ΦCA82
Putative ΦCA82 ORFs and amino acid (aa) homologies with members of Microviridae
No. of aa
Homologous protein (GenBank accession #)
% amino acid identity (homology)
major capsid protein
minor capsid protein
Recent studies using a comparative metagenomic analysis of viral communities associated with marine and freshwater microbialites indicated that identifiable sequences in these were dominated by single-stranded DNA microphages . Partial sequence analysis of the VP1 gene from these microphages showed that the similarity between metagenomic clones and cultured microphage capsid sequences ranged from 47.5 to 61.2% at the nucleic-acid level and from 37.2 to 69.3% at the protein level, respectively. Interestingly, the VP1 gene of ΦCA82 has a similarly high level of sequence similarity (69.1% at the amino acid level) with the seawater metagenomic phages within the same VP1 region (data not shown). This observation is consistent with an environmental origin of modern poultry phages that have since undergone significant host-specific evolutionary divergence in agricultural settings.
Chipman et al  predicted that the IN5 trimer structure in VP1 may function as a substitute for spike proteins of the ΦX174-like viruses, which are not found in SpV4 or the Chlamydia phages, and as such may be responsible for receptor recognition. It has also been suggested that the diverse sequence in this region is associated with host range of phages [36, 41, 43, 44]. The presence of a large insertion in ΦCA82 further supports that it is closer to the intracellular phage subfamily and the sequence dissimilarity within this region between the ΦCA82 and various other phages strongly indicates that this domain indeed may function as a host range determinant.
Rep protein of ΦCA82
Full genome comparisons of ΦCA82 with other members of the Microviridae
These investigations were supported by ARS-USDA CRIS Project No. 6612-32000-054-00 "Epidemiology, Pathogenesis and Countermeasures to Prevent and Control Enteric Viruses of Poultry" at SEPRL and Project No. 6612-32000-055-00 "Molecular Characterization and Gastrointestinal Tract Ecology of Commensal Human Food-Borne Bacterial Pathogens in the Chicken" at PMSRU. The authors thank to Fenglan Li for excellent technical assistance and to the SEPRL sequencing facility for outstanding support.
- Handelsman J: Metagenomics: application of genomics to uncultured microorganisms. Microbiol Mol Biol Rev 2004, 68: 669-685. 10.1128/MMBR.68.4.669-685.2004PubMed CentralView ArticlePubMed
- Breitbart M, Salamon P, Andresen B, Mahaffy JM, Segall AM, Mead D, Azam F, Rohwer F: Genomic analysis of uncultured marine viral communities. Proc Natl Acad Sci USA 2002, 99: 14250-14255. 10.1073/pnas.202488399PubMed CentralView ArticlePubMed
- Edwards RA, Rohwer F: Viral metagenomics. Nat Rev Microbiol 2005, 3: 504-510. 10.1038/nrmicro1163View ArticlePubMed
- Kristensen DM, Mushegian AR, Dolja VV, Koonin EV: New dimensions of the virus world discovered through metagenomics. Trends Microbiol 2010, 18: 11-19. 10.1016/j.tim.2009.11.003PubMed CentralView ArticlePubMed
- Schoenfeld T, Liles M, Wommack KE, Polson SW, Godiska R, Mead D: Functional viral metagenomics and the next generation of molecular tools. Trends Microbiol 2010, 18: 20-29. 10.1016/j.tim.2009.10.001PubMed CentralView ArticlePubMed
- Fischetti VA: Bacteriophage lysins as effective antibacterials. Curr Opin Microbiol 2008, 11: 393-400. 10.1016/j.mib.2008.09.012PubMed CentralView ArticlePubMed
- Liu J, Dehbi M, Moeck G, Arhin F, Bauda P, Bergeron D, Callejo M, Ferretti V, Ha N, Kwan T, McCarty J, Srikumar R, Williams D, Wu JJ, Gros P, Pelletier J, DuBow M: Antimicrobial drug discovery through bacteriophage genomics. Nat Biotechnol 2004, 22: 185-191. 10.1038/nbt932View ArticlePubMed
- Merril CR, Scholl D, Adhya SL: The prospect for bacteriophage therapy in Western medicine. Nat Rev Drug Discov 2003, 2: 489-497. 10.1038/nrd1111View ArticlePubMed
- Suttle CA: Marine viruses--major players in the global ecosystem. Nat Rev Microbiol 2007, 5: 801-812. 10.1038/nrmicro1750View ArticlePubMed
- Ackermann HW: Bacteriophage observations and evolution. Res Microbiol 2003, 154: 245-251. 10.1016/S0923-2508(03)00067-6View ArticlePubMed
- Brüssow H, Canchaya C, Hardt WD: Phages and the evolution of bacterial pathogens: from genomic rearrangements to lysogenic conversion. Microbiol Mol Biol Rev 2004, 68: 560-602. 10.1128/MMBR.68.3.560-602.2004PubMed CentralView ArticlePubMed
- Boyd EF, Brüssow H: Common themes among bacteriophage-encoded virulence factors and diversity among the bacteriophages involved. Trends Microbiol 2002, 10: 521-529. 10.1016/S0966-842X(02)02459-9View ArticlePubMed
- Chen J, Novick RP: Phage-mediated intergeneric transfer of toxin genes. Science 2009, 323: 139-141. 10.1126/science.1164783View ArticlePubMed
- Wagner PL, Waldor MK: Bacteriophage control of bacterial virulence. Infect Immun 2002, 70: 3985-3993. 10.1128/IAI.70.8.3985-3993.2002PubMed CentralView ArticlePubMed
- Canchaya C, Fournous G, Chibani-Chennoufi S, Dillmann ML, Brüssow H: Phage as agents of lateral gene transfer. Curr Opin Microbiol 2003, 6: 417-424. 10.1016/S1369-5274(03)00086-9View ArticlePubMed
- Paul JH, Sullivan MB, Segall AM, Rohwer F: Marine phage genomics. Comp Biochem Physiol 2002, 133: 463-476. 10.1016/S1096-4959(02)00168-9View Article
- Brüssow H, Desiere F: Comparative phage genomics and the evolution of Siphoviridae: insights from dairy phages. Mol Microbiol 2001, 39: 213-222. 10.1046/j.1365-2958.2001.02228.xView ArticlePubMed
- Sturino JM, Klaenhammer TR: Bacteriophage defense systems and strategies for lactic acid bacteria. Adv Appl Microbiol 2004, 56: 331-378.View ArticlePubMed
- Barnes HJ, Guy JS, Barnes HJ, Glisson JR, Fadly AM, McDougald LR, Swayne DE: Poult enteritis mortality syndrome. In Diseases of Poultry. 11th edition. Edited by: Saif Y. Iowa State Press; 2003:1171-1180.
- Day JM, Ballard LL, Duke MV, Scheffler BE, Zsak L: Metagenomic analysis of the turkey gut RNA virus community. Virol J 2010, 7: 313. 10.1186/1743-422X-7-313PubMed CentralView ArticlePubMed
- Day JM, Zsak L: Determination and analysis of the full-length chicken parvovirus genome. Virology 2010, 399: 59-64. 10.1016/j.virol.2009.12.027View ArticlePubMed
- Zsak L, Strother KO, Kisary J: Partial genome sequence analysis of parvoviruses associated with enteric disease in poultry. Avian Pathol 2008, 37: 435-441. 10.1080/03079450802210648View ArticlePubMed
- Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ: Basic local alignment search tool. J Mol Biol 1990, 215: 403-410.View ArticlePubMed
- Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ: Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 1997, 25: 3389-3402. 10.1093/nar/25.17.3389PubMed CentralView ArticlePubMed
- Desnues C, Rodriguez-Brito B, Rayhawk S, Kelley S, Tran T, Haynes M, Liu H, Furlan M, Wegley L, Chau B, Ruan Y, Hall D, Angly FE, Edwards RA, Li L, Thurber RV, Reid RP, Siefert J, Souza V, Valentine DL, Swan BK, Breitbart M, Rohwer F: Biodiversity and biogeography of phages in modern stromatolites and thrombolites. Nature 2008, 452: 340-343. 10.1038/nature06735View ArticlePubMed
- Thompson JD, Higgins DG, Gibson TJ: CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 1994, 22: 4673-4680. 10.1093/nar/22.22.4673PubMed CentralView ArticlePubMed
- Markov: Markov chain-based viral gene prediction.[http://linux1.softberry.com/berry.phtml?topic=virusgroup=programssubgroup=gfindv]
- Marchler-Bauer A, Anderson JB, Chitsaz F, Derbyshire MK, DeWeese-Scott C, Fong JH, Geer LY, Geer RC, Gonzales NR, Gwadz M, He S, Hurwitz DI, Jackson JD, Ke Z, Lanczycki CJ, Liebert CA, Liu C, Lu F, Lu S, Marchler GH, Mullokandov M, Song JS, Tasneem A, Thanki N, Yamashita RA, Zhang D, Zhang N, Bryant SH: CDD: specific functional annotation with the Conserved Domain Database. Nucleic Acids Res 2009, 37: 205-210. 10.1093/nar/gkn845View Article
- Markowitz VM, Korzeniewski F, Palaniappan K, Szeto E, Werner G, Padki A, Zhao X, Dubchak I, Hugenholtz P, Anderson I, Lykidis A, Mavromatis K, Ivanova N, Kyrpides NC: The integrated microbial genomes (IMG) system. Nucleic Acids Res 2006, 34: 344-348. 10.1093/nar/gkj024View Article
- Richter M, Rossello-Mora R: Shifting the genomic gold standard for the prokaryotic species definition. Proc Natl Acad Sci USA 2009, 106: 19126-19131. 10.1073/pnas.0906412106PubMed CentralView ArticlePubMed
- Team RDC: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria.2008. [http://www.R-project.org]
- Li W, Godzik A: Cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences. Bioinformatics 2006, 22: 1658-1659. 10.1093/bioinformatics/btl158View ArticlePubMed
- Rokyta DR, Abdo Z, Wichman HA: The genetics of adaptation for eight microvirid bacteriophages. J Mol Evol 2009, 69: 229-239. 10.1007/s00239-009-9267-9PubMed CentralView ArticlePubMed
- Rokyta DR, Burch CL, Caudle SB, Wichman HA: Horizontal gene transfer and the evolution of microvirid coliphage genomes. J Bacteriol 2006, 188: 1134-1142. 10.1128/JB.188.3.1134-1142.2006PubMed CentralView ArticlePubMed
- Garner SA, Everson JS, Lambden PR, Fane BA, Clarke IN: Isolation, molecular characterisation and genome sequence of a bacteriophage (Chp3) from Chlamydophila pecorum. Virus Genes 2004, 28: 207-214.View ArticlePubMed
- Brentlinger KL, Hafenstein S, Novak CR, Fane BA, Borgon R, McKenna R, Agbandje-McKenna M: Microviridae, a family divided: isolation, characterization, and genome sequence of φMH2K, a bacteriophage of the obligate intracellular parasitic bacterium Bdellovibrio bacteriovorus. J Bacteriol 2002, 184: 1089-1094. 10.1128/jb.184.4.1089-1094.2002PubMed CentralView ArticlePubMed
- McKenna R, Bowman BR, Ilag LL, Rossmann MG, Fane BA: Atomic structure of the degraded procapsid particle of the bacteriophage G4: induced structural changes in the presence of calcium ions and functional implications. J Mol Biol 1996, 256: 736-750. 10.1006/jmbi.1996.0121View ArticlePubMed
- Storey CC, Lusher M, Richmond SJ: Analysis of the complete nucleotide sequence of Chp1, a phage which infects avian Chlamydia psittaci. J Gen Virol 1989, 70: 3381-3390. 10.1099/0022-1317-70-12-3381View ArticlePubMed
- Renaudin J, Pascarel MC, Bove JM: Spiroplasma virus 4: nucleotide sequence of the viral DNA, regulatory signals, and proposed genome organization. J Bacteriol 1987, 169: 4950-4961.PubMed CentralPubMed
- Sait M, Livingstone M, Graham R, Inglis NF, Wheelhouse N, Longbottom D: Identification, sequencing and molecular analysis of Chp4, a novel chlamydiaphage of Chlamydophila abortus belonging to the family Microviridae. J Gen Virol 2011, 92: 1733-1737.View ArticlePubMed
- Hsia RC, Ting LM, Bavoil PM: Microvirus of Chlamydia psittaci strain guinea pig inclusion conjunctivitis: isolation and molecular characterization. Microbiology 2000, 46: 1651-1660.View Article
- Liu BL, Everson JS, Fane B, Giannikopoulou P, Vretou E, Lambden PR, Clarke IN: Molecular characterization of a bacteriophage (Chp2) from Chlamydia psittaci. J Virol 2000, 74: 3464-3469. 10.1128/JVI.74.8.3464-3469.2000PubMed CentralView ArticlePubMed
- Read TD, Brunham RC, Shen C, Gill SR, Heidelberg JF, White O, Hickey EK, Peterson J, Umayam L, Utterback T, Berry K, Bass S, Linher K, Weidman J, Khouri H, Craven B, Bowman C, Dodson R, Gwinn M, Nelson W, DeBoy R, Kolonay J, McClarty G, Salzberg SL, Eisen J, Fraser CM: Genome sequences of Chlamydia trachomatis MoPn and Chlamydia pneumoniae AR39. Nucleic Acids Res 2000, 28: 1397-1406. 10.1093/nar/28.6.1397PubMed CentralView ArticlePubMed
- Chipman PR, Agbandje-McKenna M, Renaudin J, Baker TS, McKenna R: Structural analysis of the Spiroplasma virus, SpV4: implications for evolutionary variation to obtain host diversity among the Microviridae. Structure 1998, 6: 135-145. 10.1016/S0969-2126(98)00016-1PubMed CentralView ArticlePubMed
This article is published under license to 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.