Downloaded from www.microbiologyresearch.org by IP: 54.70.40.11 On: Wed, 05 Dec 2018 04:05:50 CRISPR-Cas system presents multiple transcriptional units including antisense RNAs that are expressed in minimal medium and upregulated by pH in Salmonella enterica serovar Typhi Liliana Medina-Aparicio, Javier E. Rebollar-Flores, Am erica A. Beltr an-Luviano, Alejandra V azquez, Rosa M. Guti errez-Ríos, Leticia Olvera, Edmundo Calva and Ismael Hern andez-Lucas* Abstract The CRISPR-Cas system is involved in bacterial immunity, virulence, gene regulation, biofilm formation and sporulation. In Salmonella enterica serovar Typhi, this system consists of five transcriptional units including antisense RNAs. It was determined that these genetic elements are expressed in minimal medium and are up-regulated by pH. In addition, a transcriptional characterization of cas3 and ascse2-1 is included herein. INTRODUCTION Prokaryotes have developed strategies to survive phage invasion, including the CRISPR-Cas ( clustered regularly interspaced short palindromic repeats/ CRISPR- associated proteins) system that provides specific heritable immunity against these invaders [1, 2]. The expression of CRISPR-cas generates small RNAs (crRNAs) that, by homology, recog- nize genetic material of phage and facilitate cleavage of the invader by Cas proteins [3, 4]. These systems are classified according to the presence of signature Cas proteins [5]. The hallmark of the CRISPR-Cas Type I system is the presence of Cas3, which contains an N-terminal ssDNA nuclease and a C-terminal DExH helicase domains [6, 7]. This protein is involved in cleavage of exogenous target nucleic acids [7, 8]. The Type II system requires Cas9 and a trans-activating CRISPR RNA (tracrRNA) for DNA recognition and degrad- ation [9]. Based on its few requirements, this system has been used for genomic engineering in prokaryotes and eukaryotes [10]. The Type III system uses a complex com- posed of the RAMP proteins and Cas10 nuclease to silence the invader [11, 12]. The CRISPR-Cas systems are widely distributed in bacteria [13] and are also involved in virulence [14, 15], fruiting body development [16], swarming motility, biofilm formation [17] and response to stress conditions such as the perturbation of the bacterial envelope [18–20] or exposure to g-radiation [21]. Salmonella enterica serovar Typhi (S. Typhi) IMSS-1, a clin- ical strain that causes typhoid fever, contains a Type I-E CRISPR-Cas cluster composed of cas3, cse1-cse2-cas7-cas5- cas6e-cas1-cas2, an 84 bp leader sequence, seven 29 bp repeats and six 32 bp spacers [22]. The conserved genetic organization of the cas genes in some Salmonella serovars is consistent with its having a biological function in these bac- teria [23]. Interestingly, the expression of cas7 (STY3068) has been detected when S. Typhi infects macrophages [24]. Additionally, we previously reported the transcriptional expression of cse1 from this bacterium in N-minimal medium (N-MM) [22], which promotes the expression of the Salmonella pathogenicity island 2 genes [25]. Further- more, the S. Typhi CRISPR-Cas locus is regulated by LeuO, H-NS and LRP [22, 26], which are involved in pathogenesis [27–32]. A role of H-NS and LeuO in the transcriptional regulation of the Type I-E CRISPR-Cas system from Escherichia coli K12 has also been reported [33, 34]. Since some evidence for the activity of the CRISPR-Cas sys- tem in virulence has been reported, we evaluated the expres- sion of the S. Typhi IMSS-1 CRISPR-Cas locus in N-MM, Received 8 August 2016; Accepted 17 December 2016 Author affiliation: Departamento de Microbiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de M exico, Av. Universidad 2001, Cuernavaca, Morelos 62210, Mexico. *Correspondence: Ismael Hern andez-Lucas, ismaelh@ibt.unam.mx Keywords: S. Typhi; CRISPR-Cas; regulation; pH; antisense RNA. Abbreviations: Cas, CRISPR-associated; CAT, chloramphenicol acetyltransferase; CRISPR, clustered regularly interspaced short palindromic repeat; CRP, cAMP receptor protein; DTNB, 5,5¢-dithiobis(2-nitrobenzoic acid); EMSA, electrophoretic mobility shift assays; MA, medium A; N-MM, N-minimal medium; RT-PCR, reverse transcriptase PCR; S. Typhi, Salmonella enterica serovar Typhi. Two supplementary tables and two supplementary figures are available with the online Supplementary Material. RESEARCH ARTICLE Medina-Aparicio et al., Microbiology 2017;163:253–265 DOI 10.1099/mic.0.000414 000414 ã 2017 The Authors 253