Pilotins are mobile T3SS components involved in assembly and substrate specificity of the bacterial type III secretion system.

In animal pathogens, assembly of the type III secretion system injectisome requires the presence of so-called pilotins, small lipoproteins that assist the formation of the secretin ring in the outer membrane. Using a combination of functional assays, interaction studies, proteomics, and live-cell microscopy, we determined the contribution of the pilotin to the assembly, function, and substrate selectivity of the T3SS and identified potential new downstream roles of pilotin proteins. In absence of its pilotin SctG, Yersinia enterocolitica forms few, largely polar injectisome sorting platforms and needles. Accordingly, most export apparatus subcomplexes are mobile in these strains, suggesting the absence of fully assembled injectisomes. Remarkably, while absence of the pilotin all but prevents export of early T3SS substrates, such as the needle subunits, it has little effect on secretion of late T3SS substrates, including the virulence effectors. We found that although pilotins interact with other injectisome components such as the secretin in the outer membrane, they mostly localize in transient mobile clusters in the bacterial membrane. Together, these findings provide a new view on the role of pilotins in the assembly and function of type III secretion injectisomes.

The Function of Two Radical-SAM Enzymes, HcgA and HcgG, in the Biosynthesis of the [Fe]-Hydrogenase Cofactor.

In the biosynthesis of the iron-guanylylpyridinol (FeGP) cofactor, 6-carboxymethyl-5-methyl-4-hydroxy-2-pyridinol (1) is 3-methylated to form 2, then 4-guanylylated to form 3, and converted into the full cofactor. HcgA-G proteins catalyze the biosynthetic reactions. Herein, we report the function of two radical S-adenosyl methionine enzymes, HcgA and HcgG, as uncovered by in vitro complementation experiments and the use of purified enzymes. In vitro biosynthesis using the cell extract from the Methanococcus maripaludis ΔhcgA strain was complemented with HcgA or precursors 1, 2 or 3. The results suggested that HcgA catalyzes the biosynthetic reaction that forms 1. We demonstrated the formation of 1 by HcgA using the 3 kDa cell extract filtrate as the substrate. Biosynthesis in the ΔhcgG system was recovered by HcgG but not by 3, which indicated that HcgG catalyzes the reactions after the biosynthesis of 3. The data indicated that HcgG contributes to the formation of CO and completes biosynthesis of the FeGP cofactor.

An Easy-to-Use Plasmid Toolset for Efficient Generation and Benchmarking of Synthetic Small RNAs in Bacteria.

Synthetic biology approaches life from the perspective of an engineer. Standardized and de novo design of genetic parts to subsequently build reproducible and controllable modules, for example, for circuit design, is a key element. To achieve this, natural systems and elements often serve as a blueprint for researchers. Regulation of protein abundance is controlled at DNA, mRNA, and protein levels. Many tools for the activation or repression of transcription or the destabilization of proteins are available, but easy-to-handle minimal regulatory elements on the mRNA level are preferable when translation needs to be modulated. Regulatory RNAs contribute considerably to regulatory networks in all domains of life. In particular, bacteria use small regulatory RNAs (sRNAs) to regulate mRNA translation. Slowly, sRNAs are attracting the interest of using them for broad applications in synthetic biology. Here, we promote a "plug and play" plasmid toolset to quickly and efficiently create synthetic sRNAs to study sRNA biology or their application in bacteria. We propose a simple benchmarking assay by targeting the acrA gene of Escherichia coli and rendering cells sensitive toward the ß-lactam antibiotic oxacillin. We further highlight that it may be necessary to test multiple seed regions and sRNA scaffolds to achieve the desired regulatory effect. The described plasmid toolset allows quick construction and testing of various synthetic sRNAs based on the user's needs.

In Vitro Biosynthesis of the [Fe]-Hydrogenase Cofactor Verifies the Proposed Biosynthetic Precursors.

In the FeGP cofactor of [Fe]-hydrogenase, low-spin FeII is in complex with two CO ligands and a pyridinol derivative; the latter ligates the iron with a 6-acylmethyl substituent and the pyridinol nitrogen. A guanylylpyridinol derivative, 6-carboxymethyl-3,5-dimethyl-4-guanylyl-2-pyridinol (3), is produced by the decomposition of the FeGP cofactor under irradiation with UV-A/blue light and is also postulated to be a precursor of FeGP cofactor biosynthesis. HcgC and HcgB catalyze consecutive biosynthesis steps leading to 3. Here, we report an in vitro biosynthesis assay of the FeGP cofactor using the cell extract of the ΔhcgBΔhcgC strain of Methanococcus maripaludis, which does not biosynthesize 3. We chemically synthesized pyridinol precursors 1 and 2, and detected the production of the FeGP cofactor from 1, 2 and 3. These results indicated that 1, 2 and 3 are the precursors of the FeGP cofactor, and the carboxy group of 3 is converted to the acyl ligand.

AdipoAtlas: A reference lipidome for human white adipose tissue.

Obesity, characterized by expansion and metabolic dysregulation of white adipose tissue (WAT), has reached pandemic proportions and acts as a primer for a wide range of metabolic disorders. Remodeling of WAT lipidome in obesity and associated comorbidities can explain disease etiology and provide valuable diagnostic and prognostic markers. To support understanding of WAT lipidome remodeling at the molecular level, we provide in-depth lipidomics profiling of human subcutaneous and visceral WAT of lean and obese individuals. We generate a human WAT reference lipidome by performing tissue-tailored preanalytical and analytical workflows, which allow accurate identification and semi-absolute quantification of 1,636 and 737 lipid molecular species, respectively. Deep lipidomic profiling allows identification of main lipid (sub)classes undergoing depot-/phenotype-specific remodeling. Previously unanticipated diversity of WAT ceramides is now uncovered. AdipoAtlas reference lipidome serves as a data-rich resource for the development of WAT-specific high-throughput methods and as a scaffold for systems medicine data integration.

Rational selection of reverse phase columns for high throughput LC-MS lipidomics.

Natural lipidomes are characterized by extremely high complexity and dynamic range of lipid concentrations. Furthermore, high diversity of lipid physicochemical properties requires high resolving powers for both chromatographic and mass spectrometric analytical platforms. Reverse-phase chromatography coupled with data-dependent MS/MS acquisition is one of the most popular techniques in untargeted lipidomics. Optimal method should provide good chromatographic separation and resolution, reproducibility, selectivity and sensitivity. Here, we developed and set-up a RPLC-MS/MS workflow capable of resolving complex mixtures of lipids in 32 min of analysis. Human blood plasma was chosen as a representative complex natural lipidome with large variance of lipid classes, species and lipid concentrations. Lipids were separated by RPLC on five different reverse phase columns with different types of stationary phase particles, size and chemistry. High mass accuracy MS analysis and data-dependent MS/MS analysis were performed using a Q Exactive™ HF Hybrid Quadrupole-Orbitrap™ Mass Spectrometer to identify individual lipid molecular species. This workflow was applied to evaluate the separation capability of each column and to identify the lipidomics profile in highly complex biological samples. As a result, we report more than 600 lipid species covering 18 lipid classes in human blood plasma and provide suggestions to the selection of the appropriate reverse phase column for the analysis of specific lipidomes.

LPPtiger software for lipidome-specific prediction and identification of oxidized phospholipids from LC-MS datasets.

Oxidized phospholipids (oxPLs) have been recently recognized as important mediators of various and often controversial cellular functions and stress responses. Due to the low concentrations in vivo, oxPL detection is mostly performed by targeted mass spectrometry. Although significantly improving the sensitivity, this approach does not provide a comprehensive view on oxPLs required for understanding oxPL functional activities. While capable of providing information on the diversity of oxPLs, the main challenge of untargeted lipidomics is the absence of bioinformatics tools to support high-throughput identification of previously unconsidered, oxidized lipids. Here, we present LPPtiger, an open-source software tool for oxPL identification from data-dependent LC-MS datasets. LPPtiger combines three unique algorithms to predict oxidized lipidome, generate oxPL spectra libraries, and identify oxPLs from tandem MS data using parallel processing and a multi-scoring identification workflow.

LipidHunter Identifies Phospholipids by High-Throughput Processing of LC-MS and Shotgun Lipidomics Datasets.

Lipids are dynamic constituents of biological systems, rapidly responding to any changes in physiological conditions. Thus, there is a large interest in lipid-derived markers for diagnostic and prognostic applications, especially in translational and systems medicine research. As lipid identification remains a bottleneck of modern untargeted lipidomics, we developed LipidHunter, a new open source software for the high-throughput identification of phospholipids in data acquired by LC-MS and shotgun experiments. LipidHunter resembles a workflow of manual spectra annotation. Lipid identification is based on MS/MS data analysis in accordance with defined fragmentation rules for each phospholipid (PL) class. The software tool matches product and neutral loss signals obtained by collision-induced dissociation to a user-defined white list of fatty acid residues and PL class-specific fragments. The identified signals are tested against elemental composition and bulk identification provided via LIPID MAPS search. Furthermore, LipidHunter provides information-rich tabular and graphical reports allowing to trace back key identification steps and perform data quality control. Thereby, 202 discrete lipid species were identified in lipid extracts from rat primary cardiomyocytes treated with a peroxynitrite donor. Their relative quantification allowed the monitoring of dynamic reconfiguration of the cellular lipidome in response to mild nitroxidative stress. LipidHunter is available free for download at https://bitbucket.org/SysMedOs/lipidhunter .

Functional characterisation of long intergenic non-coding RNAs through genetic interaction profiling in Saccharomyces cerevisiae.

BACKGROUND: Transcriptome studies have revealed that many eukaryotic genomes are pervasively transcribed producing numerous long non-coding RNAs (lncRNAs). However, only a few lncRNAs have been ascribed a cellular role thus far, with most regulating the expression of adjacent genes. Even less lncRNAs have been annotated as essential hence implying that the majority may be functionally redundant. Therefore, the function of lncRNAs could be illuminated through systematic analysis of their synthetic genetic interactions (GIs). RESULTS: Here, we employ synthetic genetic array (SGA) in Saccharomyces cerevisiae to identify GIs between long intergenic non-coding RNAs (lincRNAs) and protein-coding genes. We first validate this approach by demonstrating that the telomerase RNA TLC1 displays a GI network that corresponds to its well-described function in telomere length maintenance. We subsequently performed SGA screens on a set of uncharacterised lincRNAs and uncover their connection to diverse cellular processes. One of these lincRNAs, SUT457, exhibits a GI profile associating it to telomere organisation and we consistently demonstrate that SUT457 is required for telomeric overhang homeostasis through an Exo1-dependent pathway. Furthermore, the GI profile of SUT457 is distinct from that of its neighbouring genes suggesting a function independent to its genomic location. Accordingly, we show that ectopic expression of this lincRNA suppresses telomeric overhang accumulation in sut457Δ cells assigning a trans-acting role for SUT457 in telomere biology. CONCLUSIONS: Overall, our work proposes that systematic application of this genetic approach could determine the functional significance of individual lncRNAs in yeast and other complex organisms.