The complete genome sequence and genetic analysis of ΦCA82 a novel uncultured microphage from the turkey gastrointestinal system

Virology Journal The complete genome sequence and genetic analysis of FCA82 a novel uncultured microphage from the turkey gastrointestinal system Laszlo Zsak 0 J Michael Day 0 Brian B Oakley 1 Bruce S Seal 1 0 Southeast Poultry Research Laboratory, Agricultural Research Service, United States Department of Agriculture , 934 College Station Road, Athens, GA 30605 USA 1 Poultry Microbiological Safety Research Unit, Agricultural Research Service, United States Department of Agriculture , 950 College Station Road, Athens, GA 30605 USA The genomic DNA sequence of a novel enteric uncultured microphage, FCA82 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 FCA82 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 FMH2K member's major coat protein. The FCA82 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 FCA82 suggests that the divergence of this novel turkey microvirus from other microviruses may reflect unique evolutionary pressures encountered within the turkey gastrointestinal system. microphage; microviridae; turkey; enteric; metagenomics - Introduction Metagenomics analyses have lead to the discovery of a variety of microbial nucleotide sequences from environmental samples [1]. 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 [5]. 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 [9] 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 [15] cannot be over stated. Additionally, the contribution to microbial ecology [16] 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 [19]. 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 [20]. 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 [21]. 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 FCA82 were most similar to those of Chlamydia phages. Materials and Methods Assembly of FCA82, 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 [22]. 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 [22]. 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 [23]. In total, the majority of clones with inserts had no hit in the databases using tblastx [24]. 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 singlestranded DNA microphages, which have also been identified predominantly in microbialites [25]. 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 FCA82 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. Sequence analysis The FCA82 genome and ORFs were aligned with selected microvirus sequences using ClustalW [26]. Putative ORFs within the FCA82 genome were predicted using the FGENESV Trained Pattern/Markov chain-based viral gene prediction method from the Softberry website [27]. Searches for conserved domains within the FCA82 genome were performed with the Conserved Domain Database (CDD) Search Service v2.17 at the National Center for Biotechnology Information (NCBI) website [28]. Comparative genomics of the Microviridae The sequence of phage FCA82 was compared to 14 other members of the Microviridae (Table 1) obtaine (...truncated)