Project description:Using improved and robust biochemical methods, we present the first global analysis of RNA-binding proteins (RBPs) and RNA-binding domains (RBDs) in clinically relevant and multi-drug resistant Gram-positive bacteria. To validate our results in silico, we developed novel bioinformatics tools that compare RBDome data with ligand-binding site predictions generated by five different algorithms on a large number of S. aureus crystal structures. This revealed that the putative RBDs are highly enriched for predicted RNA-binding sites and basic and aliphatic amino acids, demonstrating the robustness of our approach. Surprisingly, we found that HTH-type DNA-binding and NAD and P-loop type Rossmann-fold proteins may also play a prominent role in post-transcriptional regulation in Gram-positive bacteria and we identified a common mode of RNA recognition for these domains. Subsequent in vivo validation studies showed that the HTH-type transcription factor CcpA, a master regulator of carbon metabolism in Gram-positive bacteria, is also a global post-transcriptional regulator and binds its RNA substrates at very specific distances from transcription terminators. Our novel experimental and computations tools are widely applicable, and our work provides an extremely valuable resource for groups studying post-transcriptional regulation and RNA-binding proteins in bacteria.
Project description:Using improved and robust biochemical methods, we present the first global analysis of RNA-binding proteins (RBPs) and RNA-binding domains (RBDs) in clinically relevant and multi-drug resistant Gram-positive bacteria. To validate our results in silico, we developed novel bioinformatics tools that compare RBDome data with ligand-binding site predictions generated by five different algorithms on a large number of S. aureus crystal structures. This revealed that the putative RBDs are highly enriched for predicted RNA-binding sites and basic and aliphatic amino acids, demonstrating the robustness of our approach. Surprisingly, we found that HTH-type DNA-binding and NAD and P-loop type Rossmann-fold proteins may also play a prominent role in post-transcriptional regulation in Gram-positive bacteria and we identified a common mode of RNA recognition for these domains. Subsequent in vivo validation studies showed that the HTH-type transcription factor CcpA, a master regulator of carbon metabolism in Gram-positive bacteria, is also a global post-transcriptional regulator and binds its RNA substrates at very specific distances from transcription terminators. Our novel experimental and computations tools are widely applicable, and our work provides an extremely valuable resource for groups studying post-transcriptional regulation and RNA-binding proteins in bacteria.
Project description:Objective: It is unclear whether the host response of gram-positive sepsis differs from gram-negative sepsis at a transcriptome level. Using microarray technology, we compared the gene-expression profiles of gram-positive sepsis and gram-negative sepsis in critically ill patients. Design: A prospective cross-sectional study. Setting: A 20-bed general intensive care unit of a tertiary referral hospital. Patients: Seventy-two patients admitted to the intensive care unit. Interventions: Intravenous blood was collected for leukocyte separation and RNA extraction. Microarray experiements were then performed examing the expression level of 19,232 genes in each sample. Measurements and Main Results: There was no difference in the expression profile between gram-positive and gram-negative sepsis. The finding remained unchanged even when genes with lower expression level were included or after statistical stringency was lowered. There were, however, ninety-four genes differentially expressed between sepsis and control patients. These genes included those involved in immune regulation, inflammation and mitochondrial function. Hierarchical cluster analysis confirmed that the difference in gene expression profile existed between sepsis and control patients, but not between gram-positive and gram-negative patients. Conclusion: Gram-positive and gram-negative sepsis share a common host response at a transcriptome level. These findings support the hypothesis that the septic response is non-specific and is designed to provide a more general response that can be elicited by a wide range of different micro-organisms. Keywords: disease state analysis, gram-positive sepsis, gram-negative sepsis
Project description:GAS is a highly virulent Gram-positive bacterium. For the successful infection GAS express many virulence factors, which are clustered together with transcriptional regulators in distinct genomic regions. Ralp3 is a central regulator of the ERES region. In this study, we investigated the role of Ralp3 in GAS pathogenesis. To characterize the Ralp3 regulatory function on the whole genome level, GAS M49 wild type and Äralp3 mutant strains were comprehensively compared by two colour microarray analysis. Samples were taken from cultures in the transition phase.
Project description:GAS is a highly virulent Gram-positive bacterium. For the successful infection GAS express many virulence factors, which are clustered together with transcriptional regulators in distinct genomic regions. Ralp3 is a central regulator of the ERES region. In this study, we investigated the role of Ralp3 in GAS pathogenesis. To characterize the Ralp3 regulatory function on the whole genome level, GAS M49 wild type and Äralp3 mutant strains were comprehensively compared by two colour microarray analysis. Samples were taken from cultures in the transition phase. One condition experiment, wild type vs Δralp3. Biological replicates: 4 wild type and 4 mutant replicates per array.
Project description:Clostridium acetobutylicum is a Gram-positive, endospore-forming bacterium that is considered as a strict anaerobe. It ferments sugars to the organic acids acetate and butyrate or shifts to formation of the solvents - ethanol, butanol and acetone. In most bacteria the major regulator of iron homeostasis is Fur (ferric uptake regulator). Analysis of the genome of Clostridium acetobutylicum has revealed three genes encoding Fur-like proteins. The amino acid sequece of one of them showed 70% similarity to the Fur protein of the closely related Bacillus subtilis.<br>Thus, to gain insight into the role of Fur and the mechanisms for maintenance of iron homeostasis in this strict anaerobic organism, we determined its transcriptional profile in response to iron limitation and inactivation of fur.
Project description:This SuperSeries is composed of the following subset Series: GSE33992: Streptomyces griseus transcriptome analysis in solid culture with delta adpA, encoding a global transcriptional regulator involved in morphological differentiation and secondary metabolism GSE33993: Streptomyces griseus transcriptome analysis in liquid culture with delta adpA, encoding a global transcriptional regulator involved in morphological differentiation and secondary metabolism GSE34036: Genome-wide distribution of AdpA, a global regulator for secondary metabolism and morphological differentiation in Streptomyces [liquid] GSE34037: Genome-wide distribution of AdpA, a global regulator for secondary metabolism and morphological differentiation in Streptomyces [solid] Refer to individual Series
Project description:Natural products represent a rich source for antibiotics addressing versatile cellular targets. The deconvolution of their targets via chemical proteomics is often challenged by the introduction of large photocrosslinkers. Here we select elegaphenone, a largely uncharacterized natural product antibiotic bearing a native benzophenone core scaffold, for affinity-based protein profiling (AfBPP) in Gram-positive and Gram-negative bacteria. This study utilizes the alkynylated natural product scaffold as a probe to uncover intriguing biological interactions with the transcriptional regulator AlgP. Furthermore, proteome profiling of a Pseudomonas aeruginosa AlgP transposon mutant revealed unique insights into the mode of action. Elegaphenone enhanced the killing of intracellular P. aeruginosa in macrophages exposed to sub-inhibitory concentrations of the fluoroquinolone antibiotic norfloxacin.