title: RcsB-BglJ-mediated activation of Cascade operon does not induce the maturation of CRISPR RNAs in E. coli K12.
abstract: Prokaryotic immunity against foreign nucleic acids mediated by clustered, regularly interspaced, short palindromic repeats (CRISPR) depends on the expression of the CRISPR-associated (Cas) proteins and the formation of small CRISPR RNAs (crRNAs). The crRNA-loaded Cas ribonucleoprotein complexes convey the specific recognition and inactivation of target nucleic acids. In E. coli K12, the maturation of crRNAs and the interference with target DNA is performed by the Cascade complex. The transcription of the Cascade operon is tightly repressed through H-NS-dependent inhibition of the Pcas promoter. Elevated levels of the LysR-type regulator LeuO induce the Pcas promoter and concomitantly activate the CRISPR-mediated immunity against phages. Here, we show that the Pcas promoter can also be induced by constitutive expression of the regulator BglJ. This activation is LeuO-dependent as heterodimers of BglJ and RcsB activate leuO transcription. Each transcription factor, LeuO or BglJ, induced the transcription of the Cascade genes to comparable amounts. However, the maturation of the crRNAs was activated in LeuO but not in BglJ-expressing cells. Studies on CRISPR promoter activities, transcript stabilities, crRNA processing and Cascade protein levels were performed to answer the question why crRNA maturation is defective in BglJ-expressing cells. Our results demonstrate that the activation of Cascade gene transcription is necessary but not sufficient to turn on the CRISPR-mediated immunity and suggest a more complex regulation of the type I-E CRISPR-Cas system in E. coli.
authors: Molecular Biology of Bacteria, Heinrich-Heine University, Düsseldorf, Germany.
authors: Zihni
authors: Z
authors: Arslan
authors: Thomas
authors: T
authors: Stratmann
authors: Reinhild
authors: R
authors: Wurm
authors: Rolf
authors: R
authors: Wagner
authors: Karin
authors: K
authors: Schnetz
authors: Ümit
authors: Ü
authors: Pul
mesh_terms: D064130:CRISPR-Associated Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D048168:Escherichia coli K12 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D009876:Operon; D011401:Promoter Regions, Genetic; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D015534:Trans-Activators / Q000737:chemistry / Q000378:metabolism*; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*
chemical_list: C103411:BglJ protein, E coli; D064130:CRISPR-Associated Proteins; D029968:Escherichia coli Proteins; C430340:LeuO protein, E coli; D012329:RNA, Bacterial; D016601:RNA-Binding Proteins; C489568:RcsB protein, E coli; D015534:Trans-Activators; D014157:Transcription Factors
keywords: CRISPR; Cas protein; Cascade; H-NS; LeuO; transcription regulation
references: PLoS One. 2012;7(4):e35888
references: 22558257
references: J Mol Biol. 2012 Oct 12;423(1):14-6
references: 22771574
references: Annu Rev Genet. 2012;46:311-39
references: 23145983
references: Biol Direct. 2012 Jul 31;7:24
references: 22849651
references: Mol Microbiol. 2010 Sep;77(6):1380-93
references: 20659289
references: Nat Struct Mol Biol. 2011 May;18(5):529-36
references: 21460843
references: Nature. 2011 Sep 21;477(7365):486-489
references: 21938068
references: Microbiology (Reading). 2005 Oct;151(Pt 10):3349-3359
references: 16207917
references: J Mol Evol. 2005 Feb;60(2):174-82
references: 15791728
references: J Mol Biol. 2010 Jan 15;395(2):270-81
references: 19891975
references: Nat Commun. 2012 Jul 10;3:945
references: 22781758
references: Nature. 2011 Mar 31;471(7340):602-7
references: 21455174
references: Trends Biochem Sci. 2009 Aug;34(8):401-7
references: 19646880
references: J Bacteriol. 2010 Dec;192(23):6291-4
references: 20889749
references: Microbiology (Reading). 2005 Aug;151(Pt 8):2551-2561
references: 16079334
references: Mol Microbiol. 2011 Feb;79(3):557-61
references: 21255103
references: J Bacteriol. 2002 Aug;184(15):4168-76
references: 12107134
references: Microbiology (Reading). 2012 Dec;158(12):2997-3004
references: 28206908
references: Microbiology (Reading). 2010 May;156(5):1351-1361
references: 28206910
references: Mol Microbiol. 2012 Mar;83(6):1109-23
references: 22295907
references: PLoS One. 2012;7(5):e38077
references: 22666452
references: Mol Microbiol. 2010 Sep;77(6):1341-5
references: 20860086
references: Proc Natl Acad Sci U S A. 2011 Jun 21;108(25):10098-103
references: 21646539
references: Genes Dev. 2005 Nov 1;19(21):2526-33
references: 16264189
references: Proc Natl Acad Sci U S A. 2011 Dec 13;108(50):20136-41
references: 22114197
references: Mol Microbiol. 2010 Sep;77(6):1367-79
references: 20624226
references: Science. 2007 Mar 23;315(5819):1709-12
references: 17379808
references: J Bacteriol. 2010 Dec;192(24):6456-64
references: 20952573
references: PLoS One. 2012;7(11):e50797
references: 23189210
references: Mol Microbiol. 2010 Mar;75(6):1495-512
references: 20132443
references: Mol Cell. 2012 Jun 8;46(5):595-605
references: 22521689
references: Mol Microbiol. 2011 Feb;79(3):584-99
references: 21255106
references: Mol Microbiol. 1998 May;28(3):641-53
references: 9632265
references: J Bacteriol. 2011 May;193(10):2460-7
references: 21421763
references: Science. 2008 Aug 15;321(5891):960-4
references: 18703739
references: Nucleic Acids Res. 2012 Jul;40(12):5569-76
references: 22402487
references: Nat Rev Microbiol. 2011 Jun;9(6):467-77
references: 21552286