1887

Abstract

Mycobacteriophage D29 is a lytic phage that infects various species of Mycobacterium including M. tuberculosis. Its genome has 77 genes distributed almost evenly between two converging operons designated as left and right. Transcription of the phage genome is negatively regulated by multiple copies of an operator-like element known as stoperator that acts by binding the phage repressor Gp71. The function of the D29 genes and their expression status are poorly understood and therefore we undertook a transcriptome analysis approach to address these issues. The results indicate that the average transcript intensity of the right arm genes was higher than of those on the left, at the early stage of infection. Moreover, the fold increase from early to the late stage was found to be less for the right arm genes than for the left. Both observations support the prediction that the right arm genes are expressed early whereas the left arm ones are expressed late. The analysis further revealed a break in the continuity of the right arm operon between 89, the first gene in it, and 88, the next. Gene 88 was found to be expressed from a newly identified promoter located between 88 and 89. Another new promoter was found upstream of 89. Thus, the promoter Pleft, identified earlier, is not the only one that drives expression of the right arm genes. All these promoters overlap with stoperators, with which they share a conserved sequence motif, TTGACA, commonly known as the −35 promoter element. We demonstrate mutually exclusive binding of RNA polymerase and Gp71 to the stoperator-promoters and conclude that stoperators can function as −35 promoter elements and that they can control gene expression not only negatively as was believed earlier but in many cases positively as well.

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2018-07-19
2019-12-07
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References

  1. 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 [CrossRef][PubMed]
    [Google Scholar]
  2. Hatfull GF, Sarkis GJ. DNA sequence, structure and gene expression of mycobacteriophage L5: a phage system for mycobacterial genetics. Mol Microbiol 1993;7:395–405 [CrossRef][PubMed]
    [Google Scholar]
  3. Mediavilla J, Jain S, Kriakov J, Ford ME, Duda RL et al. Genome organization and characterization of mycobacteriophage Bxb1. Mol Microbiol 2000;38:955–970 [CrossRef][PubMed]
    [Google Scholar]
  4. Nesbit CE, Levin ME, Donnelly-Wu MK, Hatfull GF. Transcriptional regulation of repressor synthesis in mycobacteriophage L5. Mol Microbiol 1995;17:1045–1056 [CrossRef][PubMed]
    [Google Scholar]
  5. Brown KL, Sarkis GJ, Wadsworth C, Hatfull GF. Transcriptional silencing by the mycobacteriophage L5 repressor. Embo J 1997;16:5914–5921 [CrossRef][PubMed]
    [Google Scholar]
  6. Giri N, Bhowmik P, Bhattacharya B, Mitra M, Das Gupta SK. The mycobacteriophage D29 gene 65 encodes an early-expressed protein that functions as a structure-specific nuclease. J Bacteriol 2009;191:959–967 [CrossRef][PubMed]
    [Google Scholar]
  7. Samaddar S, Grewal RK, Sinha S, Ghosh S, Roy S et al. Dynamics of mycobacteriophage-mycobacterial host interaction: evidence for secondary mechanisms for host lethality. Appl Environ Microbiol 2016;82:124–133 [CrossRef][PubMed]
    [Google Scholar]
  8. Jain S, Kaushal D, Dasgupta SK, Tyagi AK. Construction of shuttle vectors for genetic manipulation and molecular analysis of mycobacteria. Gene 1997;190:37–44 [CrossRef][PubMed]
    [Google Scholar]
  9. Ganguly T, Bandhu A, Chattoraj P, Chanda PK, das M et al. Repressor of temperate mycobacteriophage L1 harbors a stable C-terminal domain and binds to different asymmetric operator DNAs with variable affinity. Virol J 2007;4:64 [CrossRef][PubMed]
    [Google Scholar]
  10. van Kessel JC, Marinelli LJ, Hatfull GF. Recombineering mycobacteria and their phages. Nat Rev Microbiol 2008;6:851–857 [CrossRef][PubMed]
    [Google Scholar]
  11. Ghosh S, Samaddar S, Kirtania P, das Gupta SK. A DinB ortholog enables mycobacterial growth under dTTP-limiting conditions induced by the expression of a mycobacteriophage-derived ribonucleotide reductase gene. J Bacteriol 2015;198:352–362 [CrossRef][PubMed]
    [Google Scholar]
  12. Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc 2008;3:1101–1108 [CrossRef][PubMed]
    [Google Scholar]
  13. Benito A, Feliu JX, Villaverde A. Beta-galactosidase enzymatic activity as a molecular probe to detect specific antibodies. J Biol Chem 1996;271:21251–21256 [CrossRef][PubMed]
    [Google Scholar]
  14. Chatterjee S, Patra MM, Samaddar S, Basu A, das Gupta SK. Mutual interaction enables the mycobacterial plasmid pAL5000 origin binding protein RepB to recruit RepA, the plasmid replicase, to the origin. Microbiology 2017;163:595–610 [CrossRef][PubMed]
    [Google Scholar]
  15. Basu A, Chatterjee S, Chatterjee S, das Gupta SK. Evolutionary link between the mycobacterial plasmid pAL5000 replication protein RepB and the extracytoplasmic function family of σ factors. J Bacteriol 2012;194:1331–1341 [CrossRef][PubMed]
    [Google Scholar]
  16. Banerjee R, Rudra P, Prajapati RK, Sengupta S, Mukhopadhyay J. Optimization of recombinant Mycobacterium tuberculosis RNA polymerase expression and purification. Tuberculosis 2014;94:397–404 [CrossRef][PubMed]
    [Google Scholar]
  17. Sambrook J, Russell DW. Isolation of DNA fragments from polyacrylamide gels by the crush and soak method. CSH Protoc 2006;2006:pdb.prot2936 [CrossRef][PubMed]
    [Google Scholar]
  18. Rudra P, Prajapati RK, Banerjee R, Sengupta S, Mukhopadhyay J. Novel mechanism of gene regulation: the protein Rv1222 of Mycobacterium tuberculosis inhibits transcription by anchoring the RNA polymerase onto DNA. Nucleic Acids Res 2015;43:5855–5867 [CrossRef][PubMed]
    [Google Scholar]
  19. Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 1994;22:4673–4680 [CrossRef][PubMed]
    [Google Scholar]
  20. Tamura K, Dudley J, Nei M, Kumar S. MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 2007;24:1596–1599 [CrossRef][PubMed]
    [Google Scholar]
  21. Dehaseth PL, Zupancic ML, Record MT. RNA polymerase-promoter interactions: the comings and goings of RNA polymerase. J Bacteriol 1998;180:3019–3025[PubMed]
    [Google Scholar]
  22. 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 [CrossRef][PubMed]
    [Google Scholar]
  23. Bandhu A, Ganguly T, Jana B, Chakravarty A, Biswas A et al. Biochemical characterization of L1 repressor mutants with altered operator DNA binding activity. Bacteriophage 2012;2:79–88 [CrossRef][PubMed]
    [Google Scholar]
  24. Peña CE, Stoner J, Hatfull GF. Mycobacteriophage D29 integrase-mediated recombination: specificity of mycobacteriophage integration. Gene 1998;225:143–151 [CrossRef][PubMed]
    [Google Scholar]
  25. Kamilla S, Jain V. Mycobacteriophage D29 holin C-terminal region functionally assists in holin aggregation and bacterial cell death. Febs J 2016;283:173–190 [CrossRef][PubMed]
    [Google Scholar]
  26. Payne KM, Hatfull GF. Mycobacteriophage endolysins: diverse and modular enzymes with multiple catalytic activities. PLoS One 2012;7:e34052 [CrossRef][PubMed]
    [Google Scholar]
  27. Pope WH, Ferreira CM, Jacobs-Sera D, Benjamin RC, Davis AJ et al. Cluster K mycobacteriophages: insights into the evolutionary origins of mycobacteriophage TM4. PLoS One 2011;6:e26750 [CrossRef][PubMed]
    [Google Scholar]
  28. 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 [CrossRef][PubMed]
    [Google Scholar]
  29. Cresawn SG, Pope WH, Jacobs-Sera D, Bowman CA, Russell DA et al. Comparative genomics of Cluster O mycobacteriophages. PLoS One 2015;10:e0118725 [CrossRef][PubMed]
    [Google Scholar]
  30. Dasgupta SK, Jain S, Kaushal D, Tyagi AK. Expression systems for study of mycobacterial gene regulation and development of recombinant BCG vaccines. Biochem Biophys Res Commun 1998;246:797–804 [CrossRef][PubMed]
    [Google Scholar]
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