Estudio de la expresión de genes de Salmonella enterica y su efecto en la interacción con plantas.

Salmonella enterica es una bacteria patógena Gram negativas que causa enfermedades como la gastroenteritis o la fiebre tifoidea en diversos animales. Para desarrollar el proceso infeccioso en animales S. enterica se sirve de dos sistemas de secreción de tipo 3 (T3SS-1 y T3SS-2) los cuales inyectan al interior de la célula hospedadora una serie de proteínas de virulencia conocidas como efectores. Además de los T3SSs y los efectores que translocan, existen otras proteínas que intervienen en los procesos de virulencia como son las adhesinas y las fimbrias, encargadas del reconocimiento y adhesión a la célula hospedadora, así como la metilasa Dam, la cual regula la expresión de muchos de los factores de virulencia al metilar motivos GATC en los promotores. De la mayoría se estos factores se ha observado que presentan heterogeneidad fenotípica, formando poblaciones biestables en los que una parte de la población está expresando un determinado gen mientras que otra parte de población no lo está expresando. Recientemente se ha observado la capacidad de S. enterica de colonizar plantas y que en este proceso podrían estar implicados los mismos factores que son responsables de la virulencia en animales, que podrían presentar también heterogeneidad fenotípica. En el presente trabajo nos hemos propuesto estudiar tanto la heterogeneidad fenotípica de diversos genes de S. enterica implicados en su interacción con plantas, como el efecto que estos genes pueden tener en dicha interacción. Para ello nos servimos tanto de fusiones transcripcionales a fluoróforos como de mutantes nulos en los genes de interés, que serán estudiados en los medios LB, TM (extracto de hoja de tomate) y HIM (medio que emula las condiciones del apoplasto de las hojas de plantas) así como en plantas de tomate (crecimiento en la superficie de la hoja y posible formación de biofilm) y Arabidopsis (crecimiento en apoplasto).Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Salmonella Heterogeneously Expresses Flagellin During Colonization of Plants

Minimally processed or fresh fruits and vegetables are unfortunately linked to an increasing number of food-borne diseases, such as salmonellosis. One of the relevant virulence factors during the initial phases of the infection process is the bacterial flagellum. Although its function is well studied in animal systems, contradictory results have been published regarding its role during plant colonization. In this study, we tested the hypothesis that Salmonella's flagellin plays a versatile function during the colonization of tomato plants. We have assessed the persistence in plant tissues of a Salmonella enterica wild type strain, and of a strain lacking the two flagellins, FljB and FliC. We detected no differences between these strains concerning their respective abilities to reach distal, non-inoculated parts of the plant. Analysis of flagellin expression inside the plant, at both the population and single cell levels, shows that the majority of bacteria down-regulate flagellin production, however, a small fraction of the population continues to express flagellin at a very high level inside the plant. This heterogeneous expression of flagellin might be an adaptive strategy to the plant environment. In summary, our study provides new insights on Salmonella adaption to the plant environment through the regulation of flagellin expression.España, Ministerio de Ciencia, Innovación y Universidades (MCIU, Spain, RTI2018-095069-B-100

Expression of flagellar and type III secretion systems is under stochastic and deterministic regulation in Pseudomonas syringae.

We previously described bistability and phenotypic heterogeneity of the type III secretion system (T3SS) of Pseudomonas syringae (Rufián et al., 2016). First example of such phenomenon in a plant pathogen. Here, we describe heterogenous flagellar expression leads to phenotypic heterogeneity within P. syringae populations. We find that although as reported flagellin is downregulated inside the plant, it is still expressed by a part of the bacterial population that maintains high expression levels during colonization of the plant apoplast. We demonstrate that expression of the T3SS and flagellar systems undergo counter regulation that is displayed at a single-cell level as T3SSON/FlagellaOFF and T3SSOFF/FlagellaON subpopulations. Despite this counter regulation, T3SSON/FlagellaON and T3SSOFF/FlagellaOFF bacteria can also be found within the apoplast at significant levels. Genetic analysis of the elements involved shows that counter-regulation is reciprocal: altered levels of T3SS transcriptional activator HrpL affect flagellar expression and altered levels of flagellar master regulator FleQ affect T3SS gene expression. But it also shows that the heterogeneity of each of these systems arises through independent mechanisms and display different dynamics. The regulatory loops involved in establishing T3SS and flagellar heterogeneity in P. syringae are different to those described for these systems in animal pathogen, suggesting convergent evolution of heterogeneity. Finally, we analyze the biological implications of heterogeneity and propose that, through a division of labor strategy, heterogeneity may provide adaptive value to this pathogen. This is one of the few examples where phenotypic heterogeneity is analyzed in natural conditions within the context of host colonization.Proyecto PID2021-127245OB-I00; MCIN/ AEI/10.13039/501100011033/ Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Phenotypically heterogeneous loci in the plant pathogen Pseudomonas syringae.

Phenotypic heterogeneity usually refers to the co-existance of different phenotypes within a population. Phenotypic differences may arise through genetic variation (genomic rearrangements or mutations) or through the response to differences in the stimuli as encountered within the microenvironment. But also, sometimes, the sources of variation may not be deterministic, i.e. directly related to stimuli, but a consequence of molecular noise in gene expression and/or a programmed event under genetic or epigenetic control. A particular example of phenotypic heterogeneity is bistability. Bistability occurs when a bacterial clonal population splits into two subpopulations showing distinct phenotypes. Phenotypic heterogeneity can be beneficial in fluctuating environments by allowing some individuals within the clonal population to survive sudden changes (risk-spreading). It can also benefit the entire population through cooperation between individuals displaying phenotypic differences (division of labour). These processes and their biological relevance have been described in some animal pathogens, but little is known about them in plant-pathosystems. Pseudomonas syringae is a plant-pathogenic bacterium whose virulence depends on the expression of a type III secretion system (T3SS). Our team has previously reported that T3SS expression is bistable under inducing conditions, generating two subpopulations (T3SSON/T3SSOFF) that show differences in virulence. We have identified other loci including genes related to motility, biofilm formation and DNA methylation that also display phenotypic heterogeneity to very different degrees and with different dynamics and are at different stages on their molecular and biological characterization. Our latest advances on this front will be presented and discussed.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Análisis estructural y funcional del elemento de inserción IS200

El descubrimiento de las secuencias de inserción bacterianas y de su capacidad de modificar la expresión génica, movilizar genes y promover reorganizaciones cromosómicas ha supuesto una importante contribución para acabar con el concepto del genomio com

IS200: um antigo e calmo transposon bacteriano

IS200 is a mobile element found in a variety of eubacterial genera, such as Salmonella, Escherichia, Shigella, Vibrio, Enterococcus, Clostridium, Helicobacter, and Actinobacillus. In addition, IS200-like elements are found in archaea. IS200 elements are very small (707-711 bp) and contain a single gene. Cladograms constructed with IS200 DNA sequences suggest that IS200 has not spread among eubacteria by horizontal transfer; thus it may be an ancestral component of the bacterial genome. Self-restraint may have favored this evolutionary endurance; in fact, unlike typical mobile elements, IS200 transposes rarely. Tight repression of transposase synthesis is achieved by a combination of mechanisms: inefficient transcription, protection from impinging transcription by a transcriptional terminator, and repression of translation by a stem-loop mRNA structure. A consequence of IS200 self-restraint is that the number and distribution of IS200 elements remain fairly constant in natural populations of bacteria. This stability makes IS200 a suitable molecular marker for epidemiological and ecological studies, especially when the number of IS200 copies is high. In Salmonella enterica, IS200 fingerprinting is extensively used for strain discriminationEspaña University of Seville was supported by a Spanish-Italian Collaborative Grant (Acción Integrada HI2001-0052

Repression of IS200 transposase synthesis by RNA secondary structures

The IS200 transposase, a 16 kDa polypeptide encoded by the single open reading frame (ORF) of the insertion element, has been identified using an expression system based on T7 RNA polymerase. In wild-type IS200, two sets of internal inverted repeats that generate RNA secondary structures provide two independent mechanisms for repression of transposase synthesis. The inverted repeat located near the left end of IS200 is a transcriptional terminator that terminates read-through transcripts before they reach the IS200 ORF. The terminator is functional in both directions and may terminate > 80% of transcripts. Another control operates at the translational level: transposase synthesis is inhibited by occlusion of the ribosome-binding site (RBS) of the IS200 ORF. The RBS (5'-AGGGG-3') is occluded by formation of a mRNA stem-loop structure whose 3' end is located only 3 nt upstream of the start codon. This mechanism reduces transposase synthesis ~ 10-fold. Primer extension experiments with AMV reverse transcriptase have provided evidence that this stem-loop RNA structure is actually formed. Tight repression of transposase synthesis, achieved through synergistic mechanisms of negative control, may explain the unusually low transposition frequency of IS200.Dirección General de Investigación Científica y Técnica PB93/64