Motility modulation by the small non-coding RNA SroC in Salmonella Typhimurium.

Bacterial regulatory networks of gene expression include the interaction of diverse types of molecules such as the small non-coding RNAs (sRNAs) and their cognate messenger RNAs (mRNAs). In this study, we demonstrated that the Salmonella Typhimurium sRNA SroC is significantly expressed between the late-exponential and stationary phase of growth in an rpoS-dependent manner. The expression of flagellar genes predicted as targets of this sRNA was quantitatively analyzed in both a ΔsroC mutant and a SroC-overexpressing (pSroC) strain. Deletion of sroC increased flagellar gene expression (i.e. flhBAE and fliE). Conversely, overexpression of SroC reduced flhBAE and fliE expression. These observations correlated with phenotypic evaluation of motility, where sroC deletion slightly increased motility, which in turn, was drastically reduced upon overexpression of SroC. The effects of deletion and overexpression of sroC in biofilm formation were also examined, where the ΔsroC and pSroC strains exhibited a reduced and increased ability to form biofilm, respectively. Furthermore, electron microscopy revealed that the wild-type strain overexpressing SroC had a non-flagellated phenotype. Taken together, our results showed that S. Typhimurium sRNA SroC modulates the flagellar synthesis by down-regulating the expression of flhBAE and fliE genes.

Microarray analysis of the Escherichia coli response to CdTe-GSH Quantum Dots: understanding the bacterial toxicity of semiconductor nanoparticles.

BACKGROUND: Most semiconductor nanoparticles used in biomedical applications are made of heavy metals and involve synthetic methods that require organic solvents and high temperatures. This issue makes the development of water-soluble nanoparticles with lower toxicity a major topic of interest. In a previous work our group described a biomimetic method for the aqueous synthesis of CdTe-GSH Quantum Dots (QDs) using biomolecules present in cells as reducing and stabilizing agents. This protocol produces nanoparticles with good fluorescent properties and less toxicity than those synthesized by regular chemical methods. Nevertheless, biomimetic CdTe-GSH nanoparticles still display some toxicity, so it is important to know in detail the effects of these semiconductor nanoparticles on cells, their levels of toxicity and the strategies that cells develop to overcome it. RESULTS: In this work, the response of E. coli exposed to different sized-CdTe-GSH QDs synthesized by a biomimetic protocol was evaluated through transcriptomic, biochemical, microbiological and genetic approaches. It was determined that: i) red QDs (5 nm) display higher toxicity than green (3 nm), ii) QDs mainly induce expression of genes involved with Cd+2 stress (zntA and znuA) and tellurium does not contribute significantly to QDs-mediated toxicity since cells incorporate low levels of Te, iii) red QDs also induce genes related to oxidative stress response and membrane proteins, iv) Cd2+ release is higher in red QDs, and v) QDs render the cells more sensitive to polymyxin B. CONCLUSION: Based on the results obtained in this work, a general model of CdTe-GSH QDs toxicity in E. coli is proposed. Results indicate that bacterial toxicity of QDs is mainly associated with cadmium release, oxidative stress and loss of membrane integrity. The higher toxicity of red QDs is most probably due to higher cadmium content and release from the nanoparticle as compared to green QDs. Moreover, QDs-treated cells become more sensitive to polymyxin B making these biomimetic QDs candidates for adjuvant therapies against bacterial infections.

The small RNA RyhB homologs from Salmonella typhimurium participate in the response to S-nitrosoglutathione-induced stress.

Typically, the expression of sRNAs is activated in response to environmental stimuli in order to regulate gene expression through post-transcriptional mechanisms. In the present work we show that the Salmonellatyphimurium paralogous sRNAs RyhB-1 and RyhB-2 are induced in response to the nitrosating agent S-nitrosoglutathione (GSNO). Inactivation of these sRNAs decreased S. typhimurium resistance to GSNO and increased the levels of nitrosylated proteins. These results prompted us to evaluate a possible role of these sRNAs in nitrosative stress resistance. RNA profiling was used as a screen to identify novel RyhB-1 and RyhB-2 regulated targets. A subset of genes was filtered based on their potential role in the response to nitrosative stress and their expression was analyzed by quantitative RT-PCR in wild type, single and double mutant strains (ΔryhB1, ΔryhB2 and ΔryhB1 ΔryhB2) treated with GSNO. In response to GSNO RyhB-1 and RyhB-2 negatively regulate the expression of the genes cyoABC (cytochrome bo oxidase), cydB (cytochrome bd oxidase), cybC (cytochrome b-562), and positively regulate the nirBCD operon (nitrite reductase system). Together, these results suggest that RyhB-1 and RyhB-2 finely tune the expression of genes coding for cytochrome oxidases and the nitrate reductase system, allowing the cell to cope with GSNO-induced stress.

Role of Salmonella Typhimurium small RNAs RyhB-1 and RyhB-2 in the oxidative stress response.

As part of the response to specific stress conditions, bacteria express small molecules of non-coding RNA which maintain cellular homeostasis by regulating gene expression, commonly at the post-transcriptional level. Among these, in Salmonella enterica sv. Typhimurium, the paralog small non-coding RNAs RyhB-1 and RyhB-2 play an important role in iron homeostasis. In addition, in the present work, we show that RyhB-1 and RyhB-2 also participate in the response to hydrogen peroxide (H2O2). Deletion of RyhB-1 and/or RyhB-2 resulted in increased levels of intracellular reactive oxygen species, protein carbonylation and an altered NADH/NAD(+) ratio. Analyses of the transcriptional profiles of ryhB-1 and ryhB-2 by northern blot and qRT-PCR showed that they are induced in response to H2O2 in an OxyR-dependent manner. By using lacZ-fusions and electrophoretic mobility shift assays, we confirmed the requirement of OxyR for inducing expression of both ryhB-1 and ryhB-2. Taken together, our results support a model in which, in response to peroxide treatment, ryhB-1 and ryhB-2 are upregulated by OxyR through direct interaction with their promoter region.