Virion-associated peptidoglycan hydrolases (VAPGH) in bacteriophages are potential antimicrobials. Xop411 is a syphophage infecting the Gram-negative Xanthomonas oryzae pv. oryzae that causes bacterial leaf blight in rice plants. The Xop411 gp21 protein was identified here as a peptidoglycan glycohydrolase by Western blotting and zymogram assay, and localized to the phage tail by immunogold-labelling electron microscopy. This protein showed an apparent molecular mass of 17 kDa in SDS-polyacrylamide gels, larger than that calculated from the amino acid sequence, 15 kDa with 130 residues. The recombinant gp21 expressed in Escherichia coli formed inclusion bodies, which gained enzyme activity after in-gel renaturation. In contrast, the secreted recombinant protein (s-gp21His) expressed in Pichia pastoris was soluble and enzymatically active. Plate assays showed that s-gp21His was capable of killing 3 species of Xanthomonas, a genus containing 27 closely related plant pathogenic species, as well as the opportunistic Pseudomonas aeruginosa and Stenotrophomonas maltophilia causing nosocomial infections. These results indicate that the Xop411 gp21 has possible wide applications as an antimicrobial against xanthomonads and at least 2 opportunistic bacteria. Several other VAPGH from Xanthomonas phages were also identified by bioinformatic analysis, with 1 being confirmed by Western blotting.

Journal Section:
Research Article
Keywords:
Xanthomonas, Phage, Tail protein, Peptidoglycan hydrolase
1.
Behlau F, Canteros BI, Minsavage GV, Jones JB, Graham JH: Molecular characterization of copper resistance genes from Xanthomonas citri subsp. citri and Xanthomonas alfalfae subsp. Citrumelonis. Appl Environ Microbiol 2011; 77: 4089–4096.
2.
Briers Y, Miroshnikov K, Chertkov O, Nekrasov A, Mesyanzhinov V, Volckaert G, Lavigne R: The structural peptidoglycan hydrolase gp181 of bacteriophage phiKZ. Biochem Biophys Res Commun 2008; 374: 747–751.
3.
Caldentey J, Bamford DH: The lytic enzyme of the Pseudomonas phage phi6: purification and biochemical characterization. Biochim Biophys Acta 1992; 1159: 44–50.
4.
Chang HC, Chen CR, Lin JW, Shen GH, Chang KM, Tseng YH, Weng SF: Isolation and characterization of novel giant Stenotrophomonas maltophilia phage phiSMA5. Appl Environ Microbiol 2005; 71: 1387–1393.
5.
Cregg JM, Barringer KJ, Hessler AY, Madden KR: Pichia pastoris as a host system for transformations. Mol Cell Biol 1985; 5: 3376–3385.
6.
Das M, Bhowmick TS, Ahern SJ, Young R, Gonzalez CF: Control of Pierce’s disease by phage. PLoS One 2015; 10:e0128902.
7.
Drulis-Kawa Z, Majkowska-Skrobek G, Maciejewska B: Bacteriophages and phage-derived proteins – application approaches. Curr Med Chem 2015; 22: 1757–1773.
8.
Frampton RA, Pitman AR, Fineran PC: Advances in bacteriophage-mediated control of plant pathogens. Int J Microbiol 2012; 2012: 326452.
9.
Gill JJ, Hollyer T, Sabour PM: Bacteriophages and phage-derived products as antibacterial therapeutics. Expert Opin Ther Pat 2007; 17: 1341–1350.
10.
Hayward AC: The hosts of Xanthomonas; in Swings JG, Civerolo EL (eds.): Xanthomonas. London, Chapman and Hall, 1993, pp 52–54.
11.
Hirst JM, Riche HH, Bascomb CL: Copper accumulation in the soils of apple orchards near Wisbech. Plant Pathol 1961; 10: 105–108.
12.
Hori M: Nippon Shin-noyaku Mongatri. Tokyo, Japan Plant Protection Association, 1973, pp 622.
13.
Inoue Y, Matsuura T, Ohara T: Bacteriophage OP1, lytic for Xanthomonas oryzae pv. oryzae, changes its host range by duplication and deletion of the small in the deduced tail fiber gene. J Gen Plant Pathol 2006a; 72: 111–118.
14.
Inoue Y, Matsuura T, Ohara T: Sequence analysis of the genome of OP2, a lytic bacteriophage of Xanthomonas oryzae pv. oryzae. J Gen Plant Pathol 2006b; 72: 104–110.
15.
Kuo TT, Huang TC, Wu RY, Yang CC: Characterization of three bacteriophages of Xanthomonas oryzae (Uyeda et Ishiyama) Dowson. Bot Bull Acad Sin 1967; 8: 246–254.
16.
Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680–685.
17.
Lee CN, Hu RM, Chow TY, Lin JW, Chen HY, Tseng YH, Weng SF: Comparison of genomes of three Xanthomonas oryzae bacteriophages. BMC Genomics 2007; 8: 442.
18.
Lee CN, Lin JW, Chow TY, Tseng YH, Weng SF: A novel lysozyme from Xanthomonas oryzae phage ΦXo411 active against Xanthomonas and Stenotrophomonas. Protein Expr Purif 2006; 50: 229–237.
19.
Lee CN, Lin JW, Weng SF, Tseng YH: Genomic characterization of the intron-containing T7-like phage phiL7 of Xanthomonas campestris. Appl Environ Microbiol 2009; 75: 7828–7837.
20.
Lee CW, Tseng YH, Deng FS, Lin JW, Tseng YH, Weng SF: Contribution of Phe-7 to Tat-dependent export of β-lactamase in Xanthomonas campestris. Antimicrob Agents Chemother 2012; 56: 3597–3602.
21.
Leyns F, De Cleene M, Swings J, De Ley J: The host range of the genus Xanthomonas. Bot Rev 1984; 50: 308–356.
22.
Li Z, Xiong F, Lin Q, D’Anjou M, Daugulis AJ, Yang DS, Hew CL: Low-temperature increases the yield of biologically active herring antifreeze protein in Pichia pastoris. Protein Expr Purif 2001; 21: 438–445.
23.
Mellroth P, Karlsson J, Steiner H: A scavenger function for a Drosophila peptidoglycan recognition protein. J Biol Chem 2003; 278: 7059–7064.
24.
Miller JH: Experiments in molecular genetics. Cold Spring Harbor, Cold Spring Harbor Laboratory, 1972.
25.
Moak M, Molineux IJ: Role of the Gp16 lytic transglycosylase motif in bacteriophage T7 virions at the initiation of infection. Mol Microbiol 2000; 37: 345–355.
26.
Moak M, Molineux IJ: Peptidoglycan hydrolytic activities associated with bacteriophage virions. Mol Microbiol 2004; 51: 1169–1183.
27.
Nakagawa H, Arisaka F, Ishii S: Isolation and characterization of the bacteriophage T4 tail-associated lysozyme. J Virol 1985; 54: 460–466.
28.
Nishima W, Kanamaru S, Arisaka F, Kitao A: Screw motion regulates multiple functions of T4 phage protein gene product 5 during cell puncturing. J Am Chem Soc 2011; 133: 13571–13576.
29.
Rodríguez-Rubio L, Martínez B, Donovan DM, Rodríguez A, García P: Bacteriophage virion-associated peptidoglycan hydrolases: potential new enzybiotics. Crit Rev Microbiol 2013; 39: 427–434.
30.
Rodríguez-Rubio L, Martínez B, Rodríguez A, Donovan DM, García P: Enhanced staphylolitic activity of the Staphylococcus aureus bacteriophage vB_SauS-phiIPLA88 virion associated peptidoglycan hydrolase: fusions, deletions and synergy with LysH5. Appl Environ Microbiol 2012; 78: 2241–2248.
31.
Sambrook J, Russell DW: Molecular Cloning: A Laboratory Manual, ed 3. Cold Spring Harbor, Cold Spring Harbor Laboratory, 2001.
32.
Smibert RM, Krieg NR: General characterization; in: Gerhardt P, Murray RGE, Costilow RN, Nester EW, Wood WA, Krieg NR, Phillips GB (eds): Manual of Methods for General Bacteriology. Washington, American Society for Microbiology, 1981, pp 409–443.
33.
Tagami Y, Mizukami T: Historical Review of the Researches on Bacterial Leaf Blight of Rice Caused by Xanthomonas oryzae (Uyeda et Ishiyama) Dowson. Special report of the plant diseases and insect pests forecasting service No. 10, Plant Protection Division, Ministry of Agriculture and Forestry, Tokyo, 1962, pp 112.
34.
Vieira J, Messing J: New pUC-derived cloning vectors with different selectable markers and DNA replication origins. Gene 1991; 100: 189–194.
35.
Weng SF, Lin JW, Chen CH, Chen YY, Tseng YH, Tseng YH: Constitutive expression of a chromosomal class A (BJM group 2) β-lactamase in Xanthomonas campestris. Antimicrob Agents Chemother 2004; 48: 209–215.
36.
Weng SF, Lin NT, Fan YF, Lin JW, Tseng YH: Characterization of the 1.8-kb plasmid pXV64 from Xanthomonas campestris pv. vesicatoria. Bot Bull Acad Sin 1996; 37: 93–98.
37.
Wu S, Letchworth GJ: High efficiency transformation by electroporation of Pichia pastoris pretreated with lithium acetate and dithiothreitol. Biotechniques 2004; 36: 152–154.
38.
Young R: Phage lysis: three steps, three choices, one outcome. J Microbiol 2014; 52: 243–258.
39.
Yuzenkova J, Nechaev S, Berlin J, Rogulja D, Kuznedelov K, Inman R, Mushegian A, Severinov K: Genome of Xanthomonas oryzae bacteriophage Xp10: an odd T-odd phage. J Mol Biol 2003; 330: 735–748.
40.
Zimmer M, Vukov N, Scherer S, Loessner MJ: The murein hydrolase of the bacteriophage Φ3626 dual lysis system is active against all tested Clostridium perfringens strains. Appl Environ Microbiol 2002; 68: 5311–5317.
© 2018 S. Karger AG, Basel
Copyright / Drug Dosage / Disclaimer
Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.
2018
You do not currently have access to this content.