Conditioned mediums modulate expression of the RcsB-regulon genes in S. typhimurium

The Rcs is an unusual phosphorelay system composed of the inner membrane proteins RcsC and RcsD as sensors, and the response regulator RcsB. At the present, the signals that lead to activate the Rcs phosphorelay system remain unknown. Even though, a wide range of conditions have been described as Rcs system activation state. The growth at low temperature or on solid surface; the polymyxin B exposition; the DjlA overproduction, the rcsC11 constitutive mutation; mutations that affect cell envelope like tolB and pmrA, or rcsB overexpression are some examples of this activation states. Previously, we reported that the rcsB gene is transcribed from two promoters: i) PrcsDB located upstream of rcsD, and ii) PrcsB located within the rcsD coding region. The first promoter is induced during the exponential growth phase while the last one it does at lower levels in stationary phase. We also reported that the RcsB overproduction repressed the rcsD expression by directly binding to the PrcsDB promoter. The repression, resulting in a differential rate of rcsD and rcsB genes expression levels, was also observed using rcsC11 mutant or polymyxin B to induce the system. Under these conditions an increased level of RcsB was observed when the bacteria reach the stationary growth phase and the regulator began to be also transcribed from PrcsB. In addition, the PrcsB is physiologically required to maintain the repression on swarming behavior. The subject of the present work was to determine if an Rcs stimulation factor is excreted to the supernatant of tolB and pmrA mutant culture. This finding would allow us to identify the Rcs system signal. Here, the supernatant obtained from the above mutants cultures, as ?conditioned mediums?, was used to determine the reporter expression levels. The cps and flhDC operons were the reporter of the factor presence in the conditioned mediums. We demonstrated that the cps and flhDC operons were modulated under the growth on these mediums. As RcsB regulator is expressed exponential and stationary phase and the above reporters are exponentially controlled, we looking for reporter RcsB-depended gene that are transcribed in stationary phase like bapA gene. Here we report that asr is a new member of the RcsB regulon, which was reported as an stationary phase expressed gene. Additionally, we studied the expression modulation of asr, bap, cps and flhDC using conditioned medium harvested from both different growth phase in order to relate with expression of RcsB regulator. Under this condition we observed a growth phase-dependent expression mediated by RcsB. These results increase the Rcs system knowledge on regulon and ligand identification issues.Fil: Pescaretti, María de Las Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Lopez, Fabian Enrique. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Morero, Roberto Dionisio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Delgado, Monica Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaVI Congreso Argentina de Microbiología GeneralVilla Carlos PazArgentinaSociedad Argentina de Microbiología Genera

Identification of genes involved in the Salmonella typhimurium RcsCDB activation pathway

The Rcs system consists of the sensor protein RcsC, the cognate response regulator RcsB, and the histidin-containing phosphotransfer protein RcsD, which serve as an intermediary in the phosphoryl transfer from RcsC to RcsB. This system controls the biosynthesis of the colanic acid and flagella, and virulence genes. Although the real signal that leads to activation of this system is unknown, numerous membrane stress conditions are known to induce the Rcs system. In order to determine the system signal, we studied the effect of different compounds that affect the membrane on activation cascade system, through the expression of reporter genes. As several two-component systems respond to acetyl phosphate, we investigated the effect of ack and pta mutations in the activation of Rcs because they lead to its accumulation. Due to that in different conditions the glucose modulates the system, we analyzed the effect of mutations in genes involved in the pathway of glucose-6-phosphate required for the formation of enterobacterial common antigen (ECA). Our results contribute to understanding the relationship between membrane stresses with the metabolism of glucose to the activation of RcsCDB system.Fil: Pescaretti, María de Las Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Lopez, Fabian Enrique. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Morero, Roberto Dionisio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Delgado, Monica Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaXIV Congreso Latinoamericano de GenéticaViña del MarChileAsociación Latinoamericana de GenéticaSociedad de Genética de ChileAsociación Latinoamericana de Mutagénesis, Carcinogénesis y Teratogénesis AmbientalSociedad Argentina de Genétic

Identification of a new promoter for the response regulator rcsB expression in Salmonella enterica serovar Typhimurium

The RcsCDB (Rcs) phosphorelay system regulates capsule synthesis,flagella production and other cellular activities in several enteric bacteria. This system consists of three proteins: the sensor RcsC, the cognate response regulator RcsB and the histidine-containing phosphotransfer protein RcsD (YojN), which is hypothesized to act as an intermediary in the phosphotransfer from RcsC to RcsB. The rcsC gene is convergently transcribed toward rcsB, which follows rcsD in what appears to be a two-gene operon. Here, it is reported that the overproduction of the rcsB gene represses rcsD transcription, but has a weak effect on its own expression. We demonstrated that the differential rcsD and rcsB expression is due to the activity of two promoters to transcribe the rcsB gene: (1) PrcsDB located upstream of rcsD and (2) PrcsB located within the rcsD coding region. In addition,here it was demonstrated that in Salmonella typhimurium, PrcsB is important to activate the rcsB expression during the stationary growth phase.Fil: Pescaretti, María de Las Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Morero, Roberto Dionisio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Delgado, Monica Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentin

Characterization of a bacteriocin produced by a clinical isolate of Shigella flexneri 2

This study was conducted to determine the emergence of antibiotic resistant/producer strains, focusing the efforts in the search for new antimicrobial compounds. A total of 15,131 fecal samples (CI) were analyzed from patients from acute diarrhea (2015-2017 period) and observed that 1,794 were positive for either E. coli, Shigella or Salmonella presence. The ability of 388 Shigella isolates to produce antimicrobial peptides was determined by deferred-antagonism assay. Here, we observed that 9.02% of the Shigella isolates produced an antimicrobial agent able to inhibit the E. coli AB133 strain growth. The CI172 strain was selected as producer for its antimicrobial characterization. This antagonist compound (ShpCI172) was produced during exponential growth phase and displayed a restricted action spectrum. It is also thermo-resistant and has about 3 kDa molecular mass. The ShpCI172 can be classified as a microcin, since CI172 did not display cross immunity with other well-known microcins. This is the first report where the production of a microcin by a Shigella flexneri 2 strains is described. The use of ShpCI172 microcin may contribute to preventing or controlling diarrheal diseases. This finding represents an important contribution in the biotechnology field for its application in the development of new antibiotics and/or food preservatives agents.Fil: Torrez Lamberti, Monica Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Lopez, Fabian Enrique. Universidad Nacional de Chilecito; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Bianchi, A. M.. Hospital del Niño Jesús; ArgentinaFil: Pescaretti, María de Las Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Delgado, Monica Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentin

Cross-talk between the RcsCDB and RstAB systems to control STM1485 gene expression in Salmonella Typhimurium during acid-resistance response

Bacterial survive and respond to adverse changes in the environment by regulating gene transcription through two-component regulatory systems. In Salmonella Typhimurium the STM1485 gene expression is induced under low pH (4.5) during replication inside the epithelial host cell, but it is not involved in sensing or resisting to this condition. Since the RcsCDB system is activated under acidic conditions, we investigated whether this system is able to modulate STM1485 expression. We demonstrated that acid-induced activation of the RcsB represses STM1485 transcription by directly binding to the promoter. Under the same condition, the RstA regulator activates the expression of this gene. Physiologically, we observed that RcsB-dependent repression is required for the survival of bacteria when they are exposed to pancreatic fluids. We hypothesized that STM1485 plays an important role in Salmonella adaptation to pH changes, during transition in the gastrointestinal tract. We suggest that bacteria surviving the gastrointestinal environment invade the epithelial cells, where they can remain in vacuoles. In this new environment, acidity and magnesium starvation activate the expression of the RstA regulator in a PhoPQ-dependent manner, which in turn induces STM1485 expression. These levels of STM1485 allow increased bacterial replication within vacuoles to continue the course of infection.Fil: Torrez Lamberti, Monica Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Biológica; ArgentinaFil: Farizano, Juan Vicente. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Biológica; ArgentinaFil: Lopez, Fabian Enrique. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Biológica; Argentina. Universidad Nacional de Chilecito; ArgentinaFil: Martinez Zamora, Martin Gustavo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Biológica; ArgentinaFil: Pescaretti, María de Las Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Biológica; ArgentinaFil: Táquez Delgado, Mónica Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Biológica; Argentin

Genomic and proteomic characterization of two strains of Shigella flexneri 2 isolated from infants’ stool samples in Argentina

Background: Shigella specie is a globally important intestinal pathogen disseminated all over the world. In this study we analyzed the genome and the proteomic component of two Shigella flexneri 2a clinical isolates, collected from pediatric patients with gastroenteritis of the Northwest region of Argentina (NWA) in two periods of time, with four years of difference. Our goal was to determine putative changes at molecular levels occurred during these four years, that could explain the presence of this Shigella`s serovar as the prevalent pathogen in the population under study. Results: As previously reported, our findings support the idea of Shigella has a conserved “core” genome, since comparative studies of CI133 and CI172 genomes performed against 80 genomes obtained from the NCBI database, showed that there is a large number of genes shared among all of them. However, we observed that CI133 and CI172 harbors a small number of strain-specific genes, several of them present in mobile genetic elements, supporting the hypothesis that these isolates were established in the population by horizontal acquisition of genes. These differences were also observed at proteomic level, where it was possible to detect the presence of certain secreted proteins in a culture medium that simulates the host environment. Conclusion: Great similarities were observed between the CI133 and CI172 strains, confirming the high percentage of genes constituting the “core” genome of S. flexneri 2. However, numerous strain specific genes were also determined. The presence of the here identified molecular elements into other strain of our culture collation, is currently used to develop characteristic markers of local pathogens. In addition, the most outstanding result of this study was the first description of a S. flexneri 2 producing Colicin E, as one of the characteristics that allows S. flexneri 2 to persist in the microbial community. These findings could also contribute to clarify the mechanism and the evolution strategy used by this pathogen to specifically colonize, survive, and cause infection within the NWA population.Fil: Torrez Lamberti, Monica Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Teran, Lucrecia Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Lopez, Fabian Enrique. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Chilecito; ArgentinaFil: Pescaretti, María de Las Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Delgado, Monica Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentin

The RcsC/RcsD/RcsB system in Salmonella typhimurium: a bacterial two-hybrid analysis

The Rcs phosphorelay is a adaptive response system composed of three proteins: the sensor RcsC, the cognate response regulator RcsB; and the intermediary in the phosphoryl transfer RcsD. Has been described that the flow of phosphoryl groups through the Rcs phosphorelay can occur in following way RcsC/RcsD/RcsB. At present, the signal that activates the Rcs system has not been determined. We have demonstrated in a previous study that the overexpression of rcsB modulates the RcsB-dependent genes in an rcsC or rcsD mutant but not in the doble mutant. These results suggest that only RcsB-P, the RcsB phosphorylated form, is able to induce this modulation, and we postulated that the RcsB-P would occur by two or more different pathways. The goal of present work was study the possible phosphorelay pathways. In this work we used the two-hybrid assay (BACTH), consisting of the reconstitution of the adenylate cyclase activity in E. coli when two target proteins interacts in vivo in bacteria, measured by b-galactosidase activity. In this assay we probe the RcsC, RcsD and RcsB interaction to define the Rcs phosphorelay transduction mechanism.Fil: Pescaretti, María de Las Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Lopez, Fabian Enrique. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Morero, Roberto Dionisio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Delgado, Monica Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaXLV Reunión Anual de la Sociedad Argentina de Investigaciones en Bioquímica y Biología MolecularSan Miguel de TucumánArgentinaSociedad Argentina de Investigaciones en Bioquímica y Biología Molecula

A novel insight on signal transduction mechanism of RcsCDB system in Salmonella enterica serovar typhimurium.

The RcsCDB system of Salmonella enterica serovar Typhimurium is implicated in the control of capsule and flagella synthesis. The hybrid sensor RcsC, the phosphotransferase RcsD and the RcsB regulator, constitute the main components of the RcsCDB system. The proposed Rcs signaling cascade involves the autophosphorylation of RcsC and the transfer of the phosphate group to RcsB, mediated by RcsD. We previously reported that the overexpression of rcsB repress the transcription of rcsD by an autoregulation mechanism. Moreover, we demonstrated that during the rcsD repression, the RcsB-dependent flagellar modulation remained active. These results suggest that the Rcs phosphorelay mechanism occurs even in the absence of RcsD. In this work, we established the existence of two alternative phosphorelay pathways driving activation of this system. We demonstrated that RcsC and RcsD can act as histidine kinase proteins which, after autophosphorylated, are able to independently transfer the phosphate to RcsB. Our results suggest that these pathways could be activated by different environmental signals, leading different levels of RcsB-phosphorylated to produce a differential gene modulation. These findings contribute to a better understanding of the complexity and importance of the Rcs system activation, where more than one phosphate flow pathway increases the possibilities to exert gene regulation for a quick environmental changes response