Arch Virol DOI 10.1007/s00705-011-1218-2
ANNOTATED SEQUENCE RECORD
Complete genome sequence of the podoviral bacteriophage UCP24R, which is virulent for Clostridium perfringens Cesar A. Morales • Brian B. Oakley • Johnna K. Garrish • Gregory R. Siragusa Mary B. Ard • Bruce S. Seal
•
Received: 21 October 2011 / Accepted: 30 November 2011 Ó Springer-Verlag (outside the USA) 2011
Abstract Bacteriophage UCP24R was isolated from raw sewage from a waste treatment plant, and lytic activity was observed against a type A Clostridium perfringens isolate. Electron microscopy revealed a small virion (44-nmdiameter icosahedral capsid) with a short, non-contractile tail, indicative of a member of the family Podoviridae. The phage had a linear, double-stranded DNA genome of 18,919 base pairs (bp) with 41 bp inverted terminal repeats and a type B DNA polymerase, which are characteristics of members of the subfamily Picovirinae. Out of 22 predicted genes in the genome, ten had significant sequence similarity to proteins of known function. Three distinct genes with lytic domains were identified, including a zinc carboxypeptidase domain that has not been previously reported in viruses. The UCP24R genome described herein is
Nucleotide sequence data reported are available in the GenBank database under the accession number JN800508.
Electronic supplementary material The online version of this article (doi:10.1007/s00705-011-1218-2) contains supplementary material, which is available to authorized users. C. A. Morales (&) B. B. Oakley J. K. Garrish B. S. Seal Poultry Microbiological Safety Research Unit, Richard B. Russell Agricultural Research Center, Agricultural Research Service, USDA, 950 College Station Road, Athens, GA 30605, USA e-mail:
[email protected] G. R. Siragusa Danisco USA, W227 N752 Westmound Dr., Waukesha, WI 53186, USA M. B. Ard Department of Pathology, College of Veterinary Medicine, The University of Georgia, Athens, GA 30602, USA
only the second Clostridium perfringens podovirus genome reported to date.
Clostridium perfringens is a Gram-positive, spore-forming, anaerobic bacterium that is commonly present in the intestines of humans and animals. The bacterium is classified into one of five types (A, B, C, D, or E) based on toxin production [1]. Spores of the bacterium can persist in soil, feces, or the environment, and C. perfringens causes many severe infections, presenting both animal- and human-health issues [2, 3]. C. perfringens can cause food poisoning, gas gangrene (clostridial myonecrosis), enteritis necroticans, and non-food-borne gastrointestinal infections in humans, and it is a veterinary pathogen causing enteric diseases in both domestic and wild animals [3]. This organism is considered to be the cause of necrotic enteritis in chickens and has the potential to become a far greater problem for the poultry industry if antibiotics are withdrawn from animal feeds, as was done in the European Union [4]. Because of this possibility, there has been a renewed interest in bacteriophages and/or their lytic enzyme products as alternative antibacterial agents [5]. Bacteriophage UCP24R was isolated from filtered raw sewage from a human waste treatment facility (Athens, GA) and found to have lytic activity against a type A C. perfringens whole-carcass rinse broiler isolate previously designated as CP24 [6], producing clear plaques (1-2 mm diameter) with diffuse edges. The original isolation and subsequent propagation, purification and DNA isolation of UCP24R have been described previously [7]. An aliquot of purified phage was submitted to the Electron Microscopy Laboratory, University of Georgia (Athens, GA), for negative-stain transmission electron microscopy (JOEL JEM-1210). UCP24R DNA was fragmented with
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restriction enzymes HindIII and AccI (New England Biolabs, Ipswich, MA) and treated with Taq polymerase to be subsequently cloned into vector pCR-XL-TOPO (Invitrogen, Carlsbad, CA) [8]. Complete, 6-fold coverage of the UCP24R genome was obtained by sequencing the cloned fragments, followed by custom primer design and direct sequencing to fill gaps. Sequence assembly was performed using DNASTAR SeqMan software [9], and the final genome sequence was submitted to the IMG/ER pipeline for gene predictions and initial annotation [10]. Additional annotation of predicted proteins was conducted using BLASTp [11] and the CDD database, which also imports data from the SMART, Pfam, COGs, TIGRFAM, and PRK databases [12]. Similarity scores to putative homologs were determined by performing global pairwise protein sequence alignments using EMBOSS Needle (http://www. ebi.ac.uk/Tools/psa/emobss_needle). Gene predictions for UCP24R and their respective annotations, protein domains and coordinates are listed in Online Resource 1. A genome map with a GC plot was constructed using DNAPlotter [13]. Proteome comparisons with other podoviruses within the subfamily Picovirinae were made using CoreGenes3.0 [14]. Electron microscopy revealed UCP24R to be nonenveloped with an icosahedral capsid approximately 44-45 nm in diameter and a short, non-contractile tail of 31-32 nm in length (Fig. 1), placing the clostridial bacteriophage within the family Podoviridae of the order Caudovirales. The complete genome was determined to be 18,919 bp in length with a G?C content of 27.8%, and inverted terminal repeats (ITRs) of 41 bp were identified at
Fig. 1 Electron micrograph of UCP24R. Scale bar represents 100 nm
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each end of the genome. The ITRs represent the presumptive origin of phage replication and suggest the presence of a terminal protein [15]. The genome was predicted to contain 22 open reading frames (ORFs) carrying a total of 19 identified protein domains, and transcription was predicted to be predominantly unidirectional (Fig. 2). The characteristic virion and genome size, number of ORFs, and presence of ITRs suggest that UCP24R belongs to the subfamily Picovirinae, which includes the well-characterized Bacillus phage U29 [16, 17]. Ten out of the 22 predicted gene products of UCP24R had significant amino acid similarity (E-value \ 10-22) to genes of known function and are therefore considered to be orthologous genes with equivalent functions. Five ORFs were identified as structural protein genes, encoding products for the major capsid (ORF-10), tail (ORF-13), connector (ORF-16), collar (ORF-17) and pre-neck appendage (ORF-21) proteins. All putative structural proteins had highest similarity with Clostridium phage UCPV1 [7], ranging from 51.0 to 62.8% amino acid similarity. The pre-neck appendage proteins of UCP24R and UCPV1 have low global alignment similarity to their closest BLASTp hit (Bacillus phage GA-1, accession NP_073695, 28.1%), but they share a common protein domain architecture (Pfam domain PF12708) with the pre-neck appendages of six other podoviruses, including Bacillus phage U29. Enzymes identified within the UCP24R genome include a type B DNA polymerase (ORF-5), which, among bacteriophages, is only found in members of the subfamily Picovirinae and phages belonging to the family Tectiviridae [16, 18]. A DNA encapsidation protein (ORF-6) was detected as well, and both the encapsidation and polymerase proteins have highest similarity to those found in UCPV1 (59.3 and 47.8%, respectively). Three genes were identified carrying domains with potential lytic activity to digest bacterial cell walls. ORF-14 contained two domains, an N-acetylmuramoyl-L-alanine amidase domain (PF01520) and a peptidoglycan-binding domain (PF05036) that had similarity (71%) with the siphovirus Clostridium phage UCP34O amidase (accession no. AEI74506). ORF12 of UCP24R and its UCPV1 ortholog (59.4% similarity), identified as lysozyme-peptidase, is unique among members of the Podoviridae and has three domains within the reading frame. The first domain in the N-terminal portion of the protein has a partial alignment with a phage tail protein domain (PHA00380), suggesting a possible structural role for ORF-12. The second and third domains of ORF 12 are lysozyme (CL00222) and peptidase domains (PF01551), respectively, which are involved in peptidoglycan degradation [19]. The third gene of UCP24R with potential lytic activity, ORF-20, contains a zinc carboxypeptidase domain (PF00246) of the peptidase family M14, whose members hydrolyze C-terminal amino acids from
Clostridium perfringens bacteriophage UCP24R
Fig. 2 Genomic map of Clostridium perfringens phage UCP24R with %GC plot. The %GC plot displays regions (500-bp window) above and below the average GC content. Open reading frames (ORFs) are depicted as arrows in the predicted direction of transcription. ORFs in grey scale represent those with unknown function. Dark grey ORFs have significant similarity to predicted genes of other phages as determined by BLASTp (E-value \ 0.01), while those in light grey contain no significant sequence similarity to proteins in GenBank. Genes colored in green are noted with their
predicted function. Identified putative protein domains are listed in the legend with their respective ORF designation, and their degree of similarity to conserved domains in the CDD database is indicated by asterisks. A single asterisk (*) denotes an E-value between 0.05 and 0.001, a double asterisk (**) denotes an E-value between 1e-3 and 1e-10, and a triple asterisk (***) denotes an E-value less than 1e-10. Vertical bars represent the inverted terminal repeat (ITR) sequences. Additional annotation information can be found in Online Resource 1
polypeptide chains [20]. The closest homolog of ORF-20 is a zinc carboxypeptidase gene from Staphylococcus epidermis strain VCU116 (accession no. EGS38303), which shares 32.3% similarity. To our knowledge, there is no record of this domain existing in viruses. Among the remaining predicted genes in UCP24R, five showed significant similarity (E-value \ 10-6) to hypothetical proteins, and seven ORFs were found to have no significant similarity (E-value [ 0.05) to proteins across multiple databases. ORF-1 had 71.4% similarity to a hypothetical protein of Fusobacterium varium (accession no. ZP_08694390) and 58.5% similarity to a hypothetical protein in Lactococcus phage ul36 (accession no. NP_663675). ORF-4 had 41.0% similarity to a hypothetical protein of UCPV1, though syntenous genome architecture with most, but not all, picoviruses suggests that it may be the terminal protein required for protein-primed DNA replication [15]. ORF-7 was found to have 40.7% similarity with a hypothetical protein from Streptococcus phage Dp-1 (accession no. YP_004306958), a siphovirus, and no significant BLASTp hits with other podoviruses. ORF-11 appears to be a possible paralog of ORF-10 (major capsid) as it contains a phage head protein domain (PHA00144) and has its highest similarity (45.6%) with ORF-10. Two distinct capsid genes within a single bacteriophage have
been reported previously in Salmonella podovirus phage 7-11, although their function has not been confirmed [21]. ORF-15, immediately downstream of the predicted amidase, shares 70.2% similarity with a UCPV1 protein that is believed to be a holin, which are often transcriptionally linked with their respective amidase [22]. Online Resource 2 displays the phylogenetic relationship of UCP24R’s DNA polymerase to related bacteriophages. However, homologous proteome analysis has been proposed as a means for classifying bacteriophages and has been successfully demonstrated to be consistent with taxonomic relationships established by the International Committee on Taxonomy of Viruses (ICTV) for phages in the family Podoviridae [16]. Using the same technique, UCP24R was found to share the highest number of putative homologs with UCPV1 (55%, 12 out of 22 gene products), and both of these phages are lytic for C. perfringens. UCP24R only has 29% and 26% gene products in common, respectively, with members of the two genera in the subfamily Picovirinae that have been accepted by the ICTV, AHJD-like and U29-like viruses, which is below the 40% homologous protein content threshold proposed for classification into an established genus. Therefore, we propose that UCP24R be considered an unassigned member of the subfamily Picovirinae until further ICTV classification.
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C. A. Morales et al. Acknowledgments This work was supported by ARS-USDA project number 6612-3200-060-00D. We thank Manju Amin, Susan Brooks, and Susan Mize for their technical assistance on this project. Conflict of interest of interest.
The authors declare that they have no conflict
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