Prokaryotes have developed multiple strategies to survive phage attack and invasive DNA. Recently, a novel genetic program denominated the CRISPR/Cas system was demonstrated to have a role in these biological processes providing genetic immunity. This defense mechanism is widespread in the Archaea and Bacteria, suggesting an ancient origin. In the last few years, progress has been made regarding the functionality of the CRISPR/Cas system; however, many basic aspects of the system remain unknown. For instance, there are few studies about the conditions and regulators involved in its transcriptional control. In this work, we analyzed the transcriptional organization of the CRISPR/Cas system as well as the positive and negative regulators involved in its genetic expression in Salmonella enterica serovar Typhi. The results obtained show that in S. Typhi the CRISPR/Cas system is a LeuO-dependent operon silenced by the global regulator LRP, in addition to the previously known nucleoidassociated protein H-NS; both LRP and H-NS bind upstream and downstream of the transcriptional start site of casA. In this study, relevant nucleotides of the casA regulatory region that mediate its LeuO transcriptional activation were identified. Interestingly, specific growth conditions (N-minimal medium) were found for the LeuOindependent expression of the CRISPR/Cas system in S. Typhi. Thus, our work provides evidence that there are multiple modulators involved in the genetic expression of this immune system in S. Typhi IMSS-1.

In nature, microorganisms are constantly exposed to multiple viral infections and thus have developed many strategies to survive phage attack and invasion by foreign DNA. One of such strategies is the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) bacterial immunological system. This defense mechanism is widespread in prokaryotes including several families such as Enterobacteriaceae. Much knowledge about the CRISPR-Cas system has been generated, including its biological functions, transcriptional regulation, distribution, utility as a molecular marker and as a tool for specific genome editing. This review focuses on these aspects and describes the state of the art of the CRISPR-Cas system in the Enterobacteriaceae bacterial family.

SummaryA characterization of the LtrR regulator, an S. Typhi protein belonging to the LysR family is presented. Proteomics, outer membrane protein profiles and transcriptional analyses demonstrated that LtrR is required for the synthesis of OmpR, OmpC and OmpF. DNA-protein interaction analysis showed that LtrR binds to the regulatory region of ompR and then OmpR interacts with the ompC and ompF promoters inducing porin synthesis. LtrR-dependent and independent ompR promoters were identified, and both promoters are involved in the synthesis of OmpR for OmpC and OmpF production. To define the functional role of the ltrR-ompR-ompC-ompF genetic network, mutants in each gene were obtained. We found that ltrR, ompR, ompC and ompF were involved in the control of bacterial transformation, while the two regulators and ompC are necessary for the optimal growth of S. Typhi in the presence of one of the major bile salts found in the gut, sodium deoxycholate. The data presented establish the pivotal role of LtrR in the regulatory network of porin synthesis and reveal new genetic strategies of survival and cellular adaptation to the environment used by Salmonella.

The CRISPR-Cas cluster is found in many prokaryotic genomes including those of the Enterobacteriaceae family. Salmonella enterica serovar Typhi (S. Typhi) harbors a Type I-E CRISPR-Cas locus composed of cas3, cse1, cse2, cas7, cas5, cas6e, cas1, cas2, and a CRISPR1 array. In this work, it was determined that, in the absence of cas5 or cas2, the amount of the OmpC porin decreased substantially, whereas in individual cse2, cas6e, cas1, or cas3 null mutants, the OmpF porin was not observed in an electrophoretic profile of outer membrane proteins. Furthermore, the LysR-type transcriptional regulator LeuO was unable to positively regulate the expression of the quiescent OmpS2 porin, in individual S. Typhi cse2, cas5, cas6e, cas1, cas2, and cas3 mutants. Remarkably, the expression of the master porin regulator OmpR was dependent on the Cse2, Cas5, Cas6e, Cas1, Cas2, and Cas3 proteins. Therefore, the data suggest that the CRISPR-Cas system acts hierarchically on OmpR to control the synthesis of outer membrane proteins in S. Typhi.

The assT gene encodes an arylsulfate sulfotransferase, an enzyme that catalyzes sulfuryl transfer from phenolic sulfate to a phenolic acceptor. In Salmonella enterica serovar Typhi IMSS-1, the assT gene is located upstream of the dsbL and dsbI genes, which are involved in a disulfide bond formation required for its activation. The assT-dsbL-dsbI gene cluster forms an operon transcribed by a LeuO-dependent promoter, in rich medium A (MA). Interestingly, in the absence of cloned leuO and in a ⌬leuO background, two transcription start sites were detected for assT and two for dsbL-dsbI in minimal medium. The H-NS nucleoid protein repressed the expression of the assT-dsbL-dsbI LeuO-dependent operon, as well as of the assT transcriptional units. Thus, the expression of the assT-dsbL-dsbI gene cluster depends on the global regulatory proteins LeuO and H-NS, as well as on specific growth conditions. T he assT gene encodes an arylsulfate sulfotransferase that is present in several organisms, including mammals and a wide spectrum of microbial genomes (2, 25, 55). Mammalian sulfotransferases are located in the cytosol and in the Golgi apparatus membrane; their activities have been detected in liver, brain, kidney, and intestinal epithelial cells (53,55). These sulfotransferases use 3=-phosphoadenosine-5=-phosphosulfate (PAPS) as sulfate donor and are involved in a wide variety of biological processes such as cell communication, growth, development, defense, and detoxification (6,19,43,63,65). Unlike mammals, bacterial sulfotransferases are periplasmic enzymes that require phenolic sulfate esters as donor substrates, and it has been suggested that the intestinal microflora uses this enzyme to detoxify phenolic compounds (31,32,34,35).In Salmonella enterica serovar Typhi, the assT gene is located upstream of dsbL and dsbI, a couple of genes encoding a reduction-oxidation (red-ox) pair of proteins involved in the disulfide bond formation required for AssT activation in the periplasm (25,40,44). The nomenclature was adopted from the orthologous genes previously described in uropathogenic Escherichia coli (UPEC) and Salmonella enterica serovar Typhimurium (25,40). In contrast to the highly conserved assT, the genomic organization of the assT-dsbL-dsbI gene cluster is only conserved in Enterobacter, Yersinia, Citrobacter, and Escherichia strains, as well as within the Salmonella genus (5,12,25,40,41,62).The crystal structures for AssT and DsbL proteins have been solved. AssT forms a homodimer, whose Cys-418 and Cys-424 are involved in disulfide bond formation and its His-436 undergoes the transient sulfurylation necessary for the ping-pong reaction mechanism (44). DsbL is a periplasmic monomer, forming a disulfide bond between Cys-29 and Cys-32, which is oxidized by the transmembrane protein DsbI (25). DsbI contains four predicted transmembrane helixes with two cysteine pairs .In vivo, assT, dsbL, and dsbI expression has been detected in macrophages infected with Salmonella; additionally, a transcriptome assay showed assT and ds...