title: Mutational analysis of the mycobacteriophage BPs promoter PR reveals context-dependent sequences for mycobacterial gene expression.
abstract: The PR promoter of mycobacteriophage BPs directs early lytic gene expression and is under the control of the BPs repressor, gp33. Reporter gene fusions showed that PR has modest activity in an extrachromosomal context but has activity that is barely detectable in an integrated context, even in the absence of its repressor. Mutational dissection of PR showed that it uses a canonical -10 hexamer recognized by SigA, and mutants with mutations to the sequence 5'-TATAMT had the greatest activities. It does not contain a 5'-TGN-extended -10 sequence, although mutants with mutations creating an extended -10 sequence had substantially increased promoter activity. Mutations in the -35 hexamer also influenced promoter activity but were strongly context dependent, and similar substitutions in the -35 hexamer differentially affected promoter activity, depending on the -10 and extended -10 motifs. This warrants caution in the construction of synthetic promoters or the bioinformatic prediction of promoter activity. Combinations of mutations throughout PR generated a calibrated series of promoters for expression of stably integrated recombinant genes in both Mycobacterium smegmatis and M. tuberculosis, with maximal promoter activity being more than 2-fold that of the strong hsp60 promoter.
authors.affiliation: Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.
authors.forename: Lauren M
authors.initials: LM
authors.lastname: Oldfield
authors.affiliation: Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, USA gfh@pitt.edu.
authors.forename: Graham F
authors.initials: GF
authors.lastname: Hatfull
mesh_terms: D004269:DNA, Bacterial; D021901:DNA, Intergenic; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D015967:Gene Expression Regulation, Viral / Q000502:physiology*; D009154:Mutation; D009160:Mycobacteriophages / Q000235:genetics / Q000378:metabolism*; D020102:Mycobacterium smegmatis / Q000378:metabolism / Q000821:virology*; D011401:Promoter Regions, Genetic / Q000235:genetics*
chemical_list: D004269:DNA, Bacterial; D021901:DNA, Intergenic
references.citation: Nature. 1991 Jun 6;351(6326):456-60
references.pmid: 1904554
references.citation: Indian J Biochem Biophys. 1995 Dec;32(6):361-7
references.pmid: 8714205
references.citation: Metab Eng. 2013 Sep;19:98-106
references.pmid: 23876413
references.citation: J Bacteriol. 1996 Jun;178(12):3564-71
references.pmid: 8655555
references.citation: Mol Microbiol. 2002 Sep;45(6):1515-26
references.pmid: 12354222
references.citation: J Bacteriol. 2002 Jun;184(11):3034-43
references.pmid: 12003945
references.citation: Tuber Lung Dis. 1998;79(2):63-73
references.pmid: 10645443
references.citation: Gene. 2010 Nov 15;468(1-2):8-19
references.pmid: 20692326
references.citation: Mol Microbiol. 1993 Feb;7(3):395-405
references.pmid: 8459766
references.citation: J Bacteriol. 1996 Aug;178(16):4847-53
references.pmid: 8759847
references.citation: Mol Microbiol. 2013 May;88(3):577-89
references.pmid: 23560716
references.citation: Microbiology (Reading). 2009 Sep;155(Pt 9):2962-2977
references.pmid: 19556295
references.citation: Microbiol Spectr. 2014 Apr;2(2):
references.pmid: 26105821
references.citation: J Clin Microbiol. 2012 Apr;50(4):1362-9
references.pmid: 22278833
references.citation: Mol Microbiol. 2008 Jul;69(1):164-74
references.pmid: 18466296
references.citation: Nucleic Acids Res. 2006;34(15):4245-57
references.pmid: 16920742
references.citation: BMC Bioinformatics. 2011 Oct 12;12:395
references.pmid: 21991981
references.citation: Tuber Lung Dis. 1997;78(5-6):211-23
references.pmid: 10209675
references.citation: J Biochem Mol Biol. 2003 Nov 30;36(6):586-92
references.pmid: 14659078
references.citation: Tuberculosis (Edinb). 2009 Jan;89(1):12-6
references.pmid: 18801704
references.citation: Microbiology (Reading). 2008 Aug;154(Pt 8):2304-2314
references.pmid: 18667563
references.citation: PLoS One. 2012;7(3):e33279
references.pmid: 22442681
references.citation: Mol Microbiol. 1993 Apr;8(2):277-85
references.pmid: 8316080
references.citation: EMBO J. 1997 Oct 1;16(19):5914-21
references.pmid: 9312049
references.citation: Curr Opin Biotechnol. 2012 Dec;23(6):900-7
references.pmid: 22483201
references.citation: Appl Microbiol Biotechnol. 2011 Apr;90(2):615-23
references.pmid: 21243353
references.citation: Mol Microbiol. 2000 Dec;38(5):955-70
references.pmid: 11123671
references.citation: Tuberculosis (Edinb). 2013 Jan;93(1):60-74
references.pmid: 23017770
references.citation: FEMS Microbiol Lett. 2003 Aug 8;225(1):75-83
references.pmid: 12900024
references.citation: J Mol Biol. 1998 Mar 13;276(5):861-75
references.pmid: 9566192
references.citation: Mol Microbiol. 2000 Dec;38(5):971-85
references.pmid: 11123672
references.citation: Adv Virus Res. 2012;82:179-288
references.pmid: 22420855
references.citation: Microbiol Spectr. 2014 Feb;2(1):MGM2-0007-2013
references.pmid: 26082107
references.citation: FEBS J. 2010 Feb;277(3):605-26
references.pmid: 19951358
references.citation: Mol Cell. 2013 Jan 24;49(2):237-48
references.pmid: 23246436
references.citation: Mol Microbiol. 1995 Sep;17(6):1045-56
references.pmid: 8594325
references.citation: Appl Environ Microbiol. 2013 Jul;79(14):4484-92
references.pmid: 23686264
references.citation: Future Microbiol. 2006 Aug;1(2):177-84
references.pmid: 17661663
references.citation: Nat Methods. 2007 Feb;4(2):147-52
references.pmid: 17179933
references.citation: Microbiology (Reading). 2007 Aug;153(Pt 8):2711-2723
references.pmid: 17660435
references.citation: Microbiology (Reading). 1997 Jul;143 ( Pt 7):2267-2276
references.pmid: 9245815
references.citation: FEMS Microbiol Lett. 2010 Feb;303(2):190-6
references.pmid: 20041953
references.citation: Appl Environ Microbiol. 1998 Jan;64(1):82-7
references.pmid: 9435063
references.citation: J Bacteriol. 2002 Oct;184(19):5449-56
references.pmid: 12218033
references.citation: Mol Microbiol. 2008 Mar;67(5):1094-107
references.pmid: 18221264