1887

Abstract

Mycobacteriophages that are specific to mycobacteria are sources of various effector proteins that are capable of eliciting bactericidal responses. We describe a genomics approach in combination with bioinformatics to identify mycobacteriophage proteins that are toxic to mycobacteria upon expression. A genomic library comprising phage genome collections was screened for clones capable of killing strain mc155. We identified four unique clones: clones 45 and 12N (from the mycobacteriophage D29) and clones 66 and 85 (from the mycobacteriophage Che12). The gene products from clones 66 and 45 were identified as Gp49 of the Che12 phage and Gp34 of the D29 phage, respectively. The gene products of the other two clones, 85 and 12N, utilized novel open reading frames (ORFs) coding for synthetic proteins. These four clones (clones 45, 66, 85 and 12N) caused growth defects in and upon expression. Clones with Gp49 and Gp34 also induced growth defects in , indicating that they target conserved host machineries. Their expression induced various morphological changes, indicating that they affected DNA replication and cell division steps. We predicted that Gp34 is a Xis protein that is required in phage DNA excision from the bacterial chromosome. Gp49 is predicted to have an HTH motif with DNA-bending/twisting properties. We suggest that this methodology is useful to identify new phage proteins with the desired properties without laboriously characterizing the individual phages. It is universal and could be applied to other bacteria–phage systems. We speculate that the existence of a virtually unlimited number of phages with unique gene products could offer a cheaper and less hazardous alternative to explore new antimicrobial molecules.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.000810
2019-07-01
2024-03-28
Loading full text...

Full text loading...

/deliver/fulltext/micro/165/7/722.html?itemId=/content/journal/micro/10.1099/mic.0.000810&mimeType=html&fmt=ahah

References

  1. Hendrix RW. Bacteriophages: evolution of the majority. Theor Popul Biol 2002; 61:471–480 [View Article]
    [Google Scholar]
  2. Chanishvili N. Phage therapy-history from Twort and d'Herelle through Soviet experience to current approaches. Adv Virus Res 2012; 83:3–40 [View Article]
    [Google Scholar]
  3. Merril CR, Scholl D, Adhya SL. The prospect for bacteriophage therapy in Western medicine. Nat Rev Drug Discov 2003; 2:489–497 [View Article]
    [Google Scholar]
  4. Liu J, Dehbi M, Moeck G, Arhin F, Bauda P et al. Antimicrobial drug discovery through bacteriophage genomics. Nat Biotechnol 2004; 22:185–191 [View Article]
    [Google Scholar]
  5. Projan S. Phage-inspired antibiotics?. Nat Biotechnol 2004; 22:167–168 [View Article]
    [Google Scholar]
  6. Hatfull GF. Mycobacteriophages: windows into tuberculosis. PLoS Pathog 2014; 10:e1003953 [View Article]
    [Google Scholar]
  7. Pope WH, Anders KR, Baird M, Bowman CA, Boyle MM et al. Cluster M mycobacteriophages Bongo, PegLeg, and Rey with unusually large repertoires of tRNA isotypes. J Virol 2014; 88:2461–2480 [View Article]
    [Google Scholar]
  8. Russell DA, Hatfull GF. PhagesDB: the actinobacteriophage database. Bioinformatics 2017; 33:784–786 [View Article]
    [Google Scholar]
  9. Hatfull GF. Mycobacteriophages. Microbiol Spectr 2018; 6: [View Article]
    [Google Scholar]
  10. Lorenz L, Lins B, Barrett J, Montgomery A, Trapani S et al. Genomic characterization of six novel Bacillus pumilus bacteriophages. Virology 2013; 444:374–383 [View Article]
    [Google Scholar]
  11. Pope WH, Bowman CA, Russell DA, Jacobs-Sera D, Asai DJ et al. Whole genome comparison of a large collection of mycobacteriophages reveals a continuum of phage genetic diversity. Elife 2015; 4:e06416 [View Article]
    [Google Scholar]
  12. Ford ME, Sarkis GJ, Belanger AE, Hendrix RW, Hatfull GF. Genome structure of mycobacteriophage D29: implications for phage evolution. J Mol Biol 1998; 279:143–164 [View Article]
    [Google Scholar]
  13. Hatfull GF, Pedulla ML, Jacobs-Sera D, Cichon PM, Foley A et al. Exploring the mycobacteriophage metaproteome: phage genomics as an educational platform. PLoS Genet 2006; 2:e92 [View Article]
    [Google Scholar]
  14. Pani B, Banerjee S, Chalissery J, Muralimohan A, Abishek M, Loganathan RM et al. Mechanism of inhibition of Rho-dependent transcription termination by bacteriophage P4 protein Psu. J Biol Chem 2006; 281:26491–26500 [View Article]
    [Google Scholar]
  15. Ghosh G, Reddy J, Sambhare S. SEN R. a bacteriophage capsid protein is an inhibitor of a conserved transcription terminator of various bacterial pathogens. J Bacteriol 2018; 200:
    [Google Scholar]
  16. Baulard A, Jourdan C, Mercenier A, Locht C. Rapid mycobacterial plasmid analysis by electroduction between Mycobacterium spp. and Escherichia coli. Nucleic Acids Res 1992; 20:4105 [View Article]
    [Google Scholar]
  17. Payne K, Sun Q, Sacchettini J, Hatfull GF. Mycobacteriophage lysin B is a novel mycolylarabinogalactan esterase. Mol Microbiol 2009; 73:367–381 [View Article]
    [Google Scholar]
  18. Schägger H. Tricine-SDS-PAGE. Nat Protoc 2006; 1:16–22 [View Article]
    [Google Scholar]
  19. Boratyn GM, Schäffer AA, Agarwala R, Altschul SF, Lipman DJ et al. Domain enhanced lookup time accelerated blast. Biol Direct 2012; 7:12 [View Article]
    [Google Scholar]
  20. Söding J, Biegert A, Lupas AN. The HHpred interactive server for protein homology detection and structure prediction. Nucleic Acids Res 2005; 33:W244–W248 [View Article]
    [Google Scholar]
  21. Yang J, Yan R, Roy A, Xu D, Poisson J et al. The I-TASSER suite: protein structure and function prediction. Nat Methods 2015; 12:7–8 [View Article]
    [Google Scholar]
  22. Bowie JU, Lüthy R, Eisenberg D. A method to identify protein sequences that fold into a known three-dimensional structure. Science 1991; 253:164–170 [View Article]
    [Google Scholar]
  23. Lüthy R, Bowie JU, Eisenberg D. Assessment of protein models with three-dimensional profiles. Nature 1992; 356:83–85 [View Article]
    [Google Scholar]
  24. Colovos C, Yeates TO. Verification of protein structures: patterns of nonbonded atomic interactions. Protein Sci 1993; 2:1511–1519 [View Article]
    [Google Scholar]
  25. Laskowski RA, Rullmannn JA, MacArthur MW, Kaptein R, Thornton JM. Aqua and PROCHECK-NMR: Programs for checking the quality of protein structures solved by NMR. J Biomol NMR 1996; 8:477–486 [View Article]
    [Google Scholar]
  26. Parikh A, Kumar D, Chawla Y, Kurthkoti K, Khan S et al. Development of a new generation of vectors for gene expression, gene replacement, and protein-protein interaction studies in mycobacteria. Appl Environ Microbiol 2013; 79:1718–1729 [View Article]
    [Google Scholar]
  27. Hatfull GF. Mycobacteriophages: genes and genomes. Annu Rev Microbiol 2010; 64:331–356 [View Article]
    [Google Scholar]
  28. Dedrick RM, Mavrich TN, Ng WL, Hatfull GF. Expression and evolutionary patterns of mycobacteriophage D29 and its temperate close relatives. BMC Microbiol 2017; 17:225 [View Article]
    [Google Scholar]
  29. Dziadek J, Madiraju MVVS, Rutherford SA, Atkinson MAL, Rajagopalan M. Physiological consequences associated with overproduction of Mycobacterium tuberculosis FtsZ in mycobacterial hosts. Microbiology 2002; 148:961–971 [View Article]
    [Google Scholar]
  30. Plocinski P, Arora N, Sarva K, Blaszczyk E, Qin H et al. Mycobacterium tuberculosis CwsA interacts with CrgA and Wag31, and the CrgA-CwsA complex is involved in peptidoglycan synthesis and cell shape determination. J Bacteriol 2012; 194:6398–6409 [View Article]
    [Google Scholar]
  31. Rybniker J, Plum G, Robinson N, Small PL, Hartmann P. Identification of three cytotoxic early proteins of mycobacteriophage L5 leading to growth inhibition in Mycobacterium smegmatis . Microbiology 2008; 154:2304–2314 [View Article]
    [Google Scholar]
  32. Guzman LM, Belin D, Carson MJ, Beckwith J. Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol 1995; 177:4121–4130 [View Article]
    [Google Scholar]
  33. Ansari AZ, Chael ML, O'Halloran TV. Allosteric underwinding of DNA is a critical step in positive control of transcription by Hg-MerR. Nature 1992; 355:87–89 [View Article]
    [Google Scholar]
  34. Singh S, Plaks JG, Homa NJ, Amrich CG, Héroux A et al. The structure of xis reveals the basis for filament formation and insight into DNA bending within a mycobacteriophage intasome. J Mol Biol 2014; 426:412–422 [View Article]
    [Google Scholar]
  35. Glasgow AC, Bruist MF, Simon MI. DNA-binding properties of the Hin recombinase. J Biol Chem 1989; 264:10072–10082
    [Google Scholar]
  36. Feng JA, Johnson RC, Dickerson RE. Hin recombinase bound to DNA: the origin of specificity in major and minor groove interactions. Science 1994; 263:348–355 [View Article]
    [Google Scholar]
  37. Chiu TK, Sohn C, Dickerson RE, Johnson RC. Testing water-mediated DNA recognition by the Hin recombinase. EMBO J 2002; 21:801–814 [View Article]
    [Google Scholar]
  38. Lupas A, Van Dyke M, Stock J. Predicting coiled coils from protein sequences. Science 1991; 252:1162–1164 [View Article]
    [Google Scholar]
  39. Shibayama Y, Dabbs ER. Phage as a source of antibacterial genes multiple inhibitory products encoded by Rhodococcus phage YF1. Bacteriophage 2011; 4:195–197
    [Google Scholar]
  40. Chuzel L, Ganatra MB, Rapp E, Henrissat B, Taron CH. Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156). J Biol Chem 2018; 293:18138–18150 [View Article]
    [Google Scholar]
  41. Schmitz JE, Daniel AC, Schuch, R M, Fischetti VA, Collin M. Rapid DNA library construction for functional genomic and metagenomic screening. applied and Enviorn. Micro 2008; 74:1649–1652
    [Google Scholar]
  42. Schmelcher M, Donovan DM, Loessner MJ. Bacteriophage endolysins as novel antimicrobials. Future Microbiol 2012; 7:1147–1171 [View Article]
    [Google Scholar]
  43. Ko CC, Hatfull GF. Mycobacteriophage Fruitloop gp52 inactivates Wag31 (DivIVA) to prevent heterotypic superinfection. Mol Microbiol 2018; 108:443–460 [View Article]
    [Google Scholar]
  44. Hinton DM. Transcriptional control in the prereplicative phase of T4 development. Virol J 2010; 7:289 [View Article]
    [Google Scholar]
  45. Mekler V, Minakhin L, Sheppard C, Wigneshweraraj S, Severinov K. Molecular mechanism of transcription inhibition by phage T7 GP2 protein. J Mol Biol 2011; 413:1016–1027 [View Article]
    [Google Scholar]
  46. Santangelo TJ, Artsimovitch I. Termination and antitermination: RNA polymerase runs a stop sign. Nat Rev Microbiol 2011; 9:319–329 [View Article]
    [Google Scholar]
  47. Bahar AA, Ren D. Antimicrobial peptides. Pharmaceuticals 2013; 6:1543–1575 [View Article]
    [Google Scholar]
  48. Jenssen H, Hamill P, Hancock REW. Peptide antimicrobial agents. Clin Microbiol Rev 2006; 19:491–511 [View Article]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.000810
Loading
/content/journal/micro/10.1099/mic.0.000810
Loading

Data & Media loading...

Supplements

Supplementary material 1

PDF
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error