Molecular characterization, structural analysis and determination of host range of a novel bacteriophage LSB-1
© Chai et al; licensee BioMed Central Ltd. 2010
Received: 12 July 2010
Accepted: 28 September 2010
Published: 28 September 2010
Bacteriophages (phages) are widespread in the environment and play a crucial role in the evolution of their bacterial hosts and the emergence of new pathogens.
LSB-1, a reference coliphage strain, was classified as a member of the Podoviridae family with a cystic form (50 ± 5 nm diameter) and short tail (60 ± 5 nm long). The double stranded DNA was about 30 kilobase pairs in length. We identified its host range and determined the gp17 sequences and protein structure using shotgun analysis and bioinformatics technology.
Coliphage LSB-1 possesses a tailspike protein with endosialidase activity which is probably responsible for its specific enteroinvasive E.coli host range within the laboratory.
Bacteriophages (phages) are widespread in the environment and play a crucial role in the evolution of their bacterial hosts and the emergence of new pathogens. They have enormous potential for the development of new drugs, therapies and environmental control technologies, such as natural, non-toxic alternatives for controlling bacterial pathogens. Recent interest in phages has been stimulated by studies that demonstrate the efficacy of phages in preventing and treating infections [1–3].
Phages are readily isolated from water samples in the environment. Some of the isolated phages have shown broad-host range interaction with the bacterial isolates and others have shown either species or strain level specificity. Both polyvalent phages and non-polyvalent phages are morphologically and genetically diverse [4, 5]. They are efficient at host recognition but there is no single method of adsorption, and different phages employ different strategies [6, 7]. Identification of the mechanisms of adsorption and the host ranges of different phages may allow genetic manipulation to alter the phage host binding profile artificially. To gain a better understanding of the biological properties of phages, we have sequenced the genome of the gp17 from phage LSB-1, which was isolated from sewage samples. We determined its host range and analyzed its 3-dimensional structure to identify possible functional domains.
Nucleic acid characterization
Preliminary overview of the coliphages genome
Genome relationship of LSB-1 and other phages
Gp17 DNA sequencing and function analysis
Parameters provided by different templates
Previous studies have suggested that gene 17 from the T7-like and K1F-like phage may be playing an important role in host range recognition processes [14, 15], but little work has been done on comparing their molecular characterization. K1F-like phages are known to possess tailspike proteins with endosialidase activity that degrades polySia with high substrate specificity. These tailspike-associated enzymatic activities enable the phages to penetrate the capsular layer and are important determinants of the bacteriophage host range . The T7-like phage encodes a tail fiber protein that specifically recognizes and binds to lipopolysaccharide  and recognizes E. coli B and many E. coli K-12 strains.
It was expected that molecular characterization would provide evidence for a host adsorbing mechanism of coliphage LSB-1. We found that the LSB-1 has some common features of the phage K1F supergroup. A tailspike protein with endosialidase activity is implicated in allowing a specific enteroinvasive E.coli host range. Insertion of such an endosialidase gene into a non-polyvalent virulent phage may artificially increase its host range to enteroinvasive E.coli. Using manipulation of the phage genome to kill pathogenic bacteria has broad implications for the welfare of both man and animals. Our results may inform new ways of genetic manipulation of phages to alter their host binding profile.
Materials and methods
Purification of phage particles
Bacteriophage LSB-1 was propagated in liquid culture. The EIEC strain 8401 was infected with phages at a multiplicity of infection of 0.1. Following complete lysis of the host cells, cell debris was removed by centrifugation at 4000 × g for 10 min, and phage particles in the supernatant were concentrated by adding polyethylene glycol 6000 and NaCl to achieve final concentrations of 10% and 1.0 M, respectively. Phage particles were collected by centrifugation at 8000 × g for 10 min. Pellets were re-suspended in 0.01× the original volume of sterile SM buffer (5.8 g sodium chloride, 2 g magnesium sulphate, 100 mg gelatin, 50 mL 1 mol·L-1 Tris (pH 7.5) and 945 mL distilled water). For isopycnic centrifugation, the phage suspension was placed on a cesium chloride gradient stepwise using three solutions whose densities were 1.45, 1.50, and 1.70, respectively. After centrifugation for 60 min at 150,000 × g, the phage band was withdrawn and dialyzed against 10 mM Tris-HCl (pH 7.5) containing 10 mM MgSO. The purified phage (approximately 1011PFU/ml) was stored at 4°C until use.
Host range determination
Phage and bacterial strains used in the study and host range spectrum of the bacteriophages
EIEC ATCC 43893d
To examine phage LSB-1 morphology, 50 μl of purified viral stock solution was fixed by addition of 50 μl of 0.5% glutaraldehyde in 4% paraformaldehyde and a drop of this solution was placed on a carbon coated copper grid After waiting 30 min for settlement, excess liquid was removed and the grid was allowed to dry. A drop of 2% phosphotungstic acid was added for 2 min before excess was removed with filter paper before drying and then examination by TEM (Hitachi, model S-800) at an acceleration voltage of 45 kV.
Nucleic acid characterization
The method used was based upon that described by Sambrook and Russell . Bacteriophage from the concentrated solutions were lysed with the addition of EDTA (final concentration 20 mmol·L-1), proteinase K (final concentration 50 μg·mL-1) and SDS (final concentration 0.5%) and incubation at 56°C for 1 h. The nucleic acid was purified using phenol, phenol/chloroform and chloroform extraction. The final aqueous phase was dialyzed overnight against Tris EDTA buffer (TE). In this method, nucleic acid yield was estimated by agarose gel electrophoresis and comparison of ethidium bromide stain intensity with known DNA standards (Hind III cut lambda phage DNA, New England Biolabs Inc, USA).
The nucleic acid extracts were diluted to a standard concentration of ~20 ng·μL-1. Approximately 250ng of each extract was subjected to digestion with DNase I (Sigma Aldrich), RNase A (Sigma Aldrich) and S1 nuclease (Promega). All reactions were terminated with the addition of EDTA (10 mmol·L-1 final concentration) and analyzed using 0.8% agarose gel electrophoresis at 5 V cm-1.
Gp17 DNA sequencing and analysis
The coliphage genome was sequenced by the shotgun method. Genomic DNA was sheared by sonication, cloned into pUC18 and sequenced with an ABI 3700 automated DNA sequencer, to give 13-fold coverage of the genome. Sequences were assembled into contigs, and gaps linked using a primer-walking technique (Kaczorowski and Szybalski, 1998) . Potential open reading frames (ORFs) were predicted using ORF Finder http://www.ncbi.nlm.nih.gov/projects/gorf/ and manual correction. Translated ORFs were used in a BLAST search against the Swiss-Prot http://us.expasy.org/tools/blast and NCBI protein databases http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html. Protein secondary structures were predicted with SOPMA http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html.
The amino acid sequence of endosialidase was determined and compared with the protein structure family databases PDB , SCOP [24, 25], and PFAM  to identify the most suitable template structure. The eventual template structure was taken from PDB. Three-dimensional models were created using PHYRE http://www.sbg.bio.ic.ac.uk/~phyre/ by mapping the coordinates of the template structure with aligned residues of the endosialidase. Computer-generated three-dimensional models were viewed and analyzed using CN3 D 4.1 application software programs obtained from http://www.ncbi.nlm.nih.gov/Structure/CN3D/cn3d.shtml.
In summary, a typical Podoviridae morphology and the double-stranded nature of its DNA give the coliphage LSB-1 some common features with the phage K1F supergroup. It possesses a tailspike protein with endosialidase activity which is probably responsible for its specific enteroinvasive E.coli host range within the laboratory.
The authors would like to thank Jinxin Ke and Wenqi Huang for their help in electron microscopy. The project was supported by National Natural Science Foundation of China (Grant No.30571637), Scientific Research Project of the Chongqing China (Grant No. CSTC, 2008AC5005) and National Key Technology R&D Program of China (Grant No. 2008BAD96B06-05).
- Hermoso JL, García : Taking aim on bacterial pathogens: from phage therapy to enzybiotics. Curr Opin Microbiol 2007, 10: 461-472. 10.1016/j.mib.2007.08.002PubMedView Article
- Mann : The potential of phages to prevent MRSA infections. Res Microbiol 2008, 159: 400-405. 10.1016/j.resmic.2008.04.003PubMedView Article
- Sulakvelidze : Phage therapy: an attractive option for dealing with antibiotic-resistant bacterial infections. Drug Discov Today 2005, 10: 807-809. 10.1016/S1359-6446(05)03441-0PubMedView Article
- Hyman ST: Bacteriophage Host Range and Bacterial Resistance. Adv Appl Microbiol 2010, 70: 217-248. full_textPubMedView Article
- Song HY, Xu XM, Zhang : Isolation of a novel polyvalent virulent bacteriophage of E. coli. J Med Coll PLA 2007, 22: 261-267. 10.1016/S1000-1948(07)60054-9View Article
- Konopa KT: Isolation of coliphage lambda ghosts able to adsorb onto bacterial cells. Biochim Biophys Acta 1975, 399: 460-467.PubMedView Article
- Paranchych PM, Bradley : Stages in phage R17 infection. Virology 1970, 41: 465-473. 10.1016/0042-6822(70)90168-6PubMedView Article
- Duda : Icosahedral Tailed dsDNA Bacterial Viruses. Encyclopedia of Virology 2008, 30-37. full_textView Article
- Molineux : T7-Like Phages (Podoviridae). Encyclopedia of Virology 2004, 1722-1729. full_text
- Rothman-Denes : Enterobacteria Phage N4 (Podoviridae). Encyclopedia of Virology 2004, 450-454. full_text
- Bujnicki AE, Fischer LR: LiveBench-2: large-scale automated evaluation of protein structure prediction servers. Proteins 2001, (Suppl 5):184-191. 10.1002/prot.10039
- Bujnicki AE, Fischer LR: Structure prediction meta server. Bioinformatics 2001, 17: 750-751. 10.1093/bioinformatics/17.8.750PubMedView Article
- Cristobal AZ, Fischer , et al.: A study of quality measures for protein threading models. BMC Bioinformatics 2001, 2: 5. 10.1186/1471-2105-2-5PubMedPubMed CentralView Article
- Machida KM, Hattori SY, Kawase SI: Structure and function of a novel coliphage-associated sialidase. FEMS Microbiol Lett 2000, 182: 333-337. 10.1111/j.1574-6968.2000.tb08917.xPubMedView Article
- Schulz DS, Frank KS, Mühlenhoff AD, Gerardy-Schahn RF: Structural Basis for the Recognition and Cleavage of Polysialic Acid by the Bacteriophage K1F Tailspike Protein EndoNF. J Mol Biol 2010, 397: 341-351. 10.1016/j.jmb.2010.01.028PubMedView Article
- Bull ER, Molineux : A tale of tails: Sialidase is key to success in a model of phage therapy against K1-capsulated Escherichia coli. Virology 2010, 398: 79-86. 10.1016/j.virol.2009.11.040PubMedPubMed CentralView Article
- Steven BL, Maizel MU, Parry JS, Hainfeld FW: Molecular substructure of a viral receptor-recognition protein: The gp17 tail-fiber of bacteriophage T7. J Mol Biol 1988, 200: 351-365. 10.1016/0022-2836(88)90246-XPubMedView Article
- Champagne NG: The spot test method for the in-plant enumeration of bacteriophages with paired cultures of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus salivarius subsp. thermophilus. Int Dairy J 1995, 5: 417-425. 10.1016/0958-6946(95)00011-QView Article
- Sambrook DW: Molecular cloning. In A laboratory manual. CSHL Press. New York; 2001.
- Kaczorowski WS: Genomic DNA sequencing by SPEL-6 primer walking using hexamer ligation. Gene 1998, 223: 83-91. 10.1016/S0378-1119(98)00241-8PubMedView Article
- Heres DV: Phylogenetic analysis of the pathogenic bacteria Spiroplasma penaei based on multilocus sequence analysis. J Invertebr Pathol 2010, 103: 30-35. 10.1016/j.jip.2009.10.004PubMedView Article
- El Ossmani BB, Aboukhalid MB, Gazzaz DZ, Chafik JT: Assessment of phylogenetic structure of Berber-speaking population of Azrou using 15 STRs of Identifiler kit. Leg Med 2010, 12: 52-56. 22. Ossmani, B.B., boukhalid, M.B., Gazzaz, D.Z. & Chafik, J.T. (2010). Assessment of phylogenetic structure of Berber-speaking population of Azrou using 15 STRs of Identifiler kit. Leg Med. 12, 52-56. 10.1016/j.legalmed.2009.10.004View Article
- Berman KH, Nakamura : Announcing the worldwide Protein Data Bank. Nat Struct Biol 2003, 10: 980. 10.1038/nsb1203-980PubMedView Article
- Andreeva DH, Brenner : SCOP database in 2004: refinements integrate structure and sequence family data. Nucleic Acids Res 2004, 32: D226-D229. 10.1093/nar/gkh039PubMedPubMed CentralView Article
- Brenner CC, Hubbard AG: Understanding protein structure: using scop for fold interpretation. Methods Enzymol 1996, 266: 635-643. full_textPubMedView Article
- Bateman EB, Durbin : Pfam 3.1: 1313 multiple alignments and profile HMMs match the majority of proteins. Nucleic Acids Res 1999, 27: 260-262. 10.1093/nar/27.1.260PubMedPubMed CentralView Article
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.