Mutational analysis of Escherichia coli GreA protein reveals new functional activity independent of antipause and lethal when overexpressed

There is a growing appreciation for the diverse regulatory consequences of the family of proteins that bind to the secondary channel of E. coli RNA polymerase (RNAP), such as GreA, GreB or DksA. Similar binding sites could suggest a competition between them. GreA is characterised to rescue stalled RNAP complexes due to its antipause activity, but also it is involved in transcription fidelity and proofreading. Here, overexpression of GreA is noted to be lethal independent of its antipause activity. A library of random GreA variants has been used to isolate lethality suppressors to assess important residues for GreA functionality and its interaction with the RNA polymerase. Some mutant defects are inferred to be associated with altered binding competition with DksA, while other variants seem to have antipause activity defects that cannot reverse a GreA-sensitive pause site in a fliC::lacZ reporter system. Surprisingly, apparent binding and cleavage defects are found scattered throughout both the coiled-coil and globular domains. Thus, the coiled-coil of GreA is not just a measuring stick ensuring placement of acidic residues precisely at the catalytic centre but also seems to have binding functions. These lethality suppressor mutants may provide valuable tools for future structural and functional studies

Temperature Dependent Control of the R27 Conjugative Plasmid Genes

Conjugation of R27 plasmid is thermoregulated, being promoted at 25°C and repressed at 37°C. Previous studies identified plasmid-encoded regulators, HtdA, TrhR and TrhY, that control expression of conjugation-related genes (tra). Moreover, the nucleoid-associated protein H-NS represses conjugation at non-permissive temperature. A transcriptomic approach has been used to characterize the effect of temperature on the expression of the 205 R27 genes. Many of the 35 tra genes, directly involved in plasmid-conjugation, were upregulated at 25°C. However, the majority of the non-tra R27 genes many of them with unknown function were more actively expressed at 37°C. The role of HtdA, a regulator that causes repression of the R27 conjugation by counteracting TrhR/TrhY mediated activation of tra genes, has been investigated. Most of the R27 genes are severely derepressed at 25°C in an htdA mutant, suggesting that HtdA is involved also in the repression of R27 genes other than the tra genes. Interestingly, the effect of htdA mutation was abolished at non-permissive temperature, indicating that the HtdA-TrhR/TrhY regulatory circuit mediates the environmental regulation of R27 gene expression. The role of H-NS in the proposed model is discussed

An improved and versatile methodology to quantify biofilms formed on solid surfaces and exposed to the air-liquid interphase

To study pellicle formation, a new method has been developed to quantify biofilm formed on solid surfaces and exposed to air-liquid interphase. It is a versatile system since different adherent material surfaces might be tested. The methodology is a robust and reproducible approach to quantify biofilm

Differential regulation of CsrC and CsrB by CRP-cAMP in Salmonella enterica

Post-transcriptional regulation mediated by regulatory small RNAs (sRNAs) has risen as a key player in fine-tuning gene expression in response to environmental stimuli. Here, we show that, in Salmonella enterica, the central metabolic regulator CRP-cAMP differentially regulates the sRNAs CsrB and CsrC in a growth phase-dependent manner. While CsrB expression remains unchanged during growth, CsrC displays a growth phase-dependent expression profile, being weakly expressed at the logarithmic growth phase and induced upon entry into stationary phase. We show that CRP-cAMP contributes to the expression pattern of CsrC by repressing its expression during the logarithmic growth phase. The CRP-cAMP mediated repression of CsrC is independent of SirA, a known transcriptional CsrB/CsrC activator. We further show that the sRNA Spot 42, which is derepressed in a Δcrp strain, upregulates CsrC during logarithmic growth. We propose a model where the growth-dependent regulation of CsrC is sustained by the CRP-cAMP-mediated repression of Spot 42. Together, our data point toward a differential regulation of the sRNAs CsrB and CsrC in response to environmental stimuli, leading to fine-tuning of gene expression via the sequestration of the RNA-binding protein CsrA

In situ monitoring and quantitative determination of R27 plasmid conjugation

Horizontal gene transfer (HGT) by plasmid conjugation is a major driving force in the spread of antibiotic resistance among Enterobacteriaceae. Most of the conjugation studies are based on calculation of conjugation ratios (number of transconjugants/number of donors) after viable counting of transconjugant and donor cells. The development of robust, fast and reliable techniques for in situ monitoring and quantification of conjugation ratios might accelerate progress in understanding the impact of this cellular process in the HGT. The IncHI1 plasmids, involved in multiresistance phenotypes of relevant pathogens such as Salmonella and E. coli, are distinguished by the thermosensitivity of their conjugative transfer. Conjugation mediated by IncHI1 plasmids is more efficient at temperatures lower than 30 °C, suggesting that the transfer process takes place during the environmental transit of the bacteria. In this report, we described a methodology to monitor in situ the conjugation process during agar surface matings of the IncHI1 plasmid R27 and its derepressed derivative drR27 at different temperatures. A three-color-labeling strategy was used to visualize the spatial distribution of transconjugants within the heterogeneous environment by epifluorescence and confocal microscopy. Moreover, the fluorescent labelling was also used to quantify conjugation frequencies in liquid media by flow cytometry

Mutaciones en ppGpp Y DksA afectan la secreción de proteínas efectoras de la isla de patogenicidad 1 de Salmonella enterica serovar Typhimurium

PublishedEl género Salmonella, perteneciente a la familia Enterobacteriaceae, está compuesto por bacterias Gram negativas, no esporuladas, en forma de bacilo. Salmonella tiene importante relevancia a nivel de salud pública ya que es uno de los principales patógenos entéricos tanto en países desarrollados como en vías de desarrollo. Diferentes serovares pueden cau-sar dos patologías claramente diferenciadas, salmonelosis y fiebres tifoideas. La salmonelosis es una gastroenteritis cuyos síntomas son diarrea, fiebre, vómito y dolor abdominal, causada por toda una serie de serovares no tifoideos, que también pueden infectar un amplio rango de animales. Los serovares más relevantes son Typhimurium y Enteritidis, (Ohl & Miller, 2001; Gordon, 2008). En Colombia, Salmonella enterica se-rovarTyphimurium es el serovar más prevalente. El último reporte del Instituto Nacional de Salud mostró que, de los 23 serovares incidentes en el país, el serovar Typhimurium representa el 30% del total de los aislamientos que se realizaron entre los años 1997 y 2016

Gre factors-mediated control of hilD transcription is essential for the invasion of epithelial cells by Salmonella enterica serovar Typhimurium

The invasion of epithelial cells by Salmonella enterica serovar Typhimurium is a very tightly regulated process. Signaling cascades triggered by different environmental and physiological signals converge to control HilD, an AraC regulator that coordinates the expression of several virulence factors. The expression of hilD is modulated at several steps of the expression process. Here, we report that the invasion of epithelial cells by S. Typhimurium strains lacking the Gre factors, GreA and GreB, is impaired. By interacting with the RNA polymerase secondary channel, the Gre factors prevent backtracking of paused complexes to avoid arrest during transcriptional elongation. Our results indicate that the Gre factors are required for the expression of the bacterial factors needed for epithelial cell invasion by modulating expression of HilD. This regulation does not occur at transcription initiation and depends on the capacity of the Gre factors to prevent backtracking of the RNA polymerase. Remarkably, genetic analyses indicate that the 3'-untranslated region (UTR) of hilD is required for Gre-mediated regulation of hilD expression. Our data provide new insight into the complex regulation of S. Typhimurium virulence and highlight the role of the hilD 3'-UTR as a regulatory motif

Growth phase-dependent control of R27 conjugation is mediated by the interplay between the plasmidencoded regulatory circuit TrhR/TrhY-HtdA and the cAMP regulon

Plasmids of the incompatibility group HI1 (IncHI1) have been isolated from several Gram-negative pathogens and are associated with the spread of multidrug resistance. Their conjugation is tightly regulated and it is inhibited at temperatures higher than 30ºC, indicating that conjugation occurs outside warm-blooded hosts. Using R27, the prototype of IncHI1 plasmids, we report that plasmid transfer efficiency in E. coli strongly depends on the physiological state of the donor cells. Conjugation frequency is high when cells are actively growing, dropping sharply when cells enter the stationary phase of growth. Accordingly, our transcriptomic assays show significant downregulation of numerous R27 genes during the stationary phase, including several tra (transfer) genes. Growth phase-dependent regulation of tra genes transcription is independent of H-NS, a silencer of horizontal gene transfer, and ppGpp and RpoS, regulators of the stationary phase, but highly dependent on the plasmid-encoded regulatory circuit TrhR/TrhY-HtdA. The metabolic sensor cAMP, whose synthesis is chromosomally encoded, is also involved in the growth phase regulation of R27 conjugation by modulating htdA expression. Our data suggest that the involvement of regulators encoded by both chromosome and plasmid are required for efficient physiological control of IncHI1 plasmid conjugation

Crosstalk between bacterial conjugationa and motility is mediated by plasmid-borne regulators

Plasmid conjugation is a major horizontal gene transfer mechanism. The acquisition of a plasmid may cause a perturbation of the cell functions in addition to provide advantageous properties for the recipient cell, such as the gaining of antibiotic resistances. The interplay between plasmid and chromosomal functions has been studied using the IncHI1 plasmid R27. Plasmids of the incompatibility group HI1, isolated from several Gram-negative pathogens, are associated with the spread of multidrug resistance. Their conjugation is tightly regulated by temperature, being repressed at temperatures within the host (37 ºC). In this report, we described that at permissive temperature, when conjugation of plasmid R27 is prompted, a reduction in the motility of the cells is observed. This reduction is mediated by the plasmid-encoded regulators TrhR/TrhY, which together with HtdA form a plasmid -borne regulatory circuit controlling R27 conjugation. TrhR/TrhY, required to induce R27 conjugation, are responsible of the downregulation of the flagella synthesis and the consequent decrease in motility. TrhR/TrhYrepress, direct or indirectly, the expression of the specific flagellar sigma subunit FliA and, consequently, the expression of all genes located bellow in the flagellar expression cascade

Nutrient composition promotes switching between pellicle and bottom biofilm in Salmonella

Salmonella is one of the most frequently reported causes of foodborne illness worldwide. Non-typhoidal serovars cause gastroenteritis in humans. Salmonella can grow on surfaces forming biofilms, contributing to its persistence since biofilms are difficult to eradicate due to the high resistance to antimicrobials and disinfectants. It has been described that there are two crucial biofilm promoting factors in Salmonella: curli and cellulose. The expression of both factors is coordinately regulated by the transcriptional regulator CsgD. Most biofilm studies of Salmonella have been performed by growing bacteria in low osmolarity rich medium and low temperature (25°C). In such conditions, the biofilm is formed at the air-liquid interface (pellicle biofilm). Remarkably, when Salmonella grow in minimal medium, biofilm formation switches from the air-liquid interface to the solid-liquid interface (bottom biofilm). In this report, the switching between pellicle and bottom biofilm has been characterized. Our data indicate that curli, but not cellulose, is crucial for the formation of both kinds of biofilms. In minimal medium, conditions promoting formation of bottom biofilm, a high transcriptional expression of csgD and consequently of the genes involved in the synthesis of curli and cellulose was detected. The nutritional status of the cells seems to be pivotal for the spatial distribution of the biofilms formed. When bacteria is growing in minimal medium the addition of amino acids downregulates the expression of csgB and causes the switch between bottom and pellicle biofilm. The crosstalk between general metabolism and biofilm formation is also highlighted by the fact that the metabolic sensor cAMP modulates the type of biofilm generated by Salmonella. Moreover, cAMP regulates transcriptional expression of csgD and stimulates pellicle biofilm formation, suggesting that the physiological conditions define the type of biofilm formed by Salmonella. The consequences of the switching between pellicle and bottom biofilm during either infection or survival in natural environments remain undercover