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See detailThe RNA Complement of Outer Membrane Vesicles From Salmonella enterica Serovar Typhimurium Under Distinct Culture Conditions
Malabirade, Antoine UL; Habier, Janine UL; Heintz-Buschart, Anna et al

in Frontiers in Microbiology (2018), 9

Bacterial outer membrane vesicles (OMVs), as well as OMV-associated small RNAs, have been demonstrated to play a role in host–pathogen interactions. The presence of larger RNA transcripts in OMVs has been ... [more ▼]

Bacterial outer membrane vesicles (OMVs), as well as OMV-associated small RNAs, have been demonstrated to play a role in host–pathogen interactions. The presence of larger RNA transcripts in OMVs has been less studied and their potential role in host–pathogen interactions remains largely unknown. Here we analyze RNA from OMVs secreted by Salmonella enterica serovar Typhimurium (S. Typhimurium) cultured under different conditions, which mimic host–pathogen interactions. S. Typhimurium was grown to exponential and stationary growth phases in minimal growth control medium (phosphate-carbon-nitrogen, PCN), as well as in acidic and phosphate-depleted PCN, comparable to the macrophage environment and inducing therefore the expression of Salmonella pathogenicity island 2 (SPI-2) genes. Moreover, Salmonella pathogenicity island 1 (SPI-1), which is required for virulence during the intestinal phase of infection, was induced by culturing S. Typhimurium to the stationary phase in Lysogeny Broth (LB). For each condition, we identified OMV-associated RNAs that are enriched in the extracellular environment relative to the intracellular space. All RNA classes could be observed, but a vast majority of rRNA was exported in all conditions in variable proportions with a notable decrease in LB SPI-1 inducing media. Several mRNAs and ncRNAs were specifically enriched in/on OMVs dependent on the growth conditions. Important to note is that some RNAs showed identical read coverage profiles intracellularly and extracellularly, whereas distinct coverage patterns were observed for other transcripts, suggesting a specific processing or degradation. Moreover, PCR experiments confirmed that distinct RNAs were present in or on OMVs as full-length transcripts (IsrB-1/2; IsrA; ffs; SsrS; CsrC; pSLT035; 10Sa; rnpB; STM0277; sseB; STM0972; STM2606), whereas others seemed to be rather present in a processed or degraded form. Finally, we show by a digestion protection assay that OMVs are able to prevent enzymatic degradation of given full-length transcripts (SsrS, CsrC, 10Sa, and rnpB). In summary, we show that OMV-associated RNA is clearly different in distinct culture conditions and that at least a fraction of the extracellular RNA is associated as a full-length transcripts with OMVs, indicating that some RNAs are protected by OMVs and thereby leaving open the possibility that those might be functionally active. [less ▲]

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See detailSmall RNA profiling of low biomass samples: identification and removal of contaminants
Heintz-Buschart, Anna; Yusuf, Dilmurat; Kaysen, Anne UL et al

in BMC Biology (2018), 16(52),

Background: Sequencing-based analyses of low-biomass samples are known to be prone to misinterpretation due to the potential presence of contaminating molecules derived from laboratory reagents and ... [more ▼]

Background: Sequencing-based analyses of low-biomass samples are known to be prone to misinterpretation due to the potential presence of contaminating molecules derived from laboratory reagents and environments. DNA contamination has been previously reported, however contamination with RNA is usually considered to be unlikely due to its inherent instability. Small RNAs (sRNAs) identified in tissues and bodily fluids such as blood plasma, have implications for physiology and pathology, and therefore the potential to act as disease biomarkers. Thus, the possibility for RNA contaminants demands a careful evaluation. Results: Here we report the presence of small RNA contaminants in widely used microRNA extraction kits and propose an approach for their depletion. We sequenced sRNAs extracted from human plasma samples and detected important levels of non-human (exogenous) sequences whose source could be traced to the microRNA extraction columns through a careful qPCR-based analysis of several laboratory reagents. Furthermore, we also detected the presence of artefactual sequences related to these contaminants in a range of published datasets, arguing for a re-evaluation of reports suggesting the presence of exogenous RNAs of microbial and dietary origins in blood plasma. To avoid artefacts in future experiments, we also devise several protocols of contaminant RNAs, define minimal amounts of starting material for artefact-free analyses, and confirm the reduction of contaminant levels for identification of bona fide sequences using ‘ultra-clean’ extraction kits. Conclusion: This is the first report of the presence of RNA molecules as contaminants in RNA extraction kits. The described protocols should be applied in the future to avoid confounding sRNA studies. [less ▲]

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See detailExtraction and Analysis of RNA Isolated from Pure Bacteria-Derived Outer Membrane Vesicles
Habier, Janine UL; May, Patrick UL; Heintz-Buschart, Anna et al

in Arluison, Véronique; Valverde, Claudio Valverde (Eds.) Bacterial Regulatory RNA (2018)

Outer membrane vesicles (OMVs) are released by commensal as well as pathogenic Gram-negative bacteria. These vesicles contain numerous bacterial components, such as proteins, peptidoglycans ... [more ▼]

Outer membrane vesicles (OMVs) are released by commensal as well as pathogenic Gram-negative bacteria. These vesicles contain numerous bacterial components, such as proteins, peptidoglycans, lipopolysaccharides, DNA, and RNA. To examine if OMV-associated RNA molecules are bacterial degradation products and/or are functionally active, it is necessary to extract RNA from pure OMVs for subsequent analysis. Therefore, we describe here an isolation method of ultrapure OMVs and the subsequent extraction of RNA and basic steps of RNA-Seq analysis. Bacterial culture, extracellular supernatant concentration, OMV purification, and the subsequent RNA extraction out of OMVs are described. Specific pitfalls within the protocol and RNA contamination sources are highlighted. [less ▲]

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See detailMechanisms of Persistence of the Ammonia-Oxidizing Bacteria Nitrosomonas to the Biocide Free Nitrous Acid.
Laloo, Andrew E.; Wei, Justin; Wang, Dongbo et al

in Environmental science & technology (2018), 52(9), 5386-5397

Free nitrous acid (FNA) exerts a broad range of antimicrobial effects on bacteria, although susceptibility varies considerably among microorganisms. Among nitrifiers found in activated sludge of ... [more ▼]

Free nitrous acid (FNA) exerts a broad range of antimicrobial effects on bacteria, although susceptibility varies considerably among microorganisms. Among nitrifiers found in activated sludge of wastewater treatment processes (WWTPs), nitrite-oxidizing bacteria (NOB) are more susceptible to FNA compared to ammonia-oxidizing bacteria (AOB). This selective inhibition of NOB over AOB in WWTPs bypasses nitrate production and improves the efficiency and costs of the nitrogen removal process in both the activated sludge and anaerobic ammonium oxidation (Anammox) system. However, the molecular mechanisms governing this atypical tolerance of AOB to FNA have yet to be understood. Herein we investigate the varying effects of the antimicrobial FNA on activated sludge containing AOB and NOB using an integrated metagenomics and label-free quantitative sequential windowed acquisition of all theoretical fragment ion mass spectra (SWATH-MS) metaproteomic approach. The Nitrosomonas genus of AOB, on exposure to FNA, maintains internal homeostasis by upregulating a number of known oxidative stress enzymes, such as pteridine reductase and dihydrolipoyl dehydrogenase. Denitrifying enzymes were upregulated on exposure to FNA, suggesting the detoxification of nitrite to nitric oxide. Interestingly, proteins involved in stress response mechanisms, such as DNA and protein repair enzymes, phage prevention proteins, and iron transport proteins, were upregulated on exposure to FNA. In addition enzymes involved in energy generation were also upregulated on exposure to FNA. The total proteins specifically derived from the NOB genus Nitrobacter was low and, as such, did not allow for the elucidation of the response mechanism to FNA exposure. These findings give us an understanding of the adaptive mechanisms of tolerance within the AOB Nitrosomonas to the biocidal agent FNA. [less ▲]

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See detailHuman Gut Microbiome: Function Matters.
Heintz-Buschart, Anna; Wilmes, Paul UL

in Trends in microbiology (2018), 26(7), 563-574

The human gut microbiome represents a complex ecosystem contributing essential functions to its host. Recent large-scale metagenomic studies have provided insights into its structure and functional ... [more ▼]

The human gut microbiome represents a complex ecosystem contributing essential functions to its host. Recent large-scale metagenomic studies have provided insights into its structure and functional potential. However, the functional repertoire which is actually contributed to human physiology remains largely unexplored. Here, by leveraging recent omics datasets, we challenge current assumptions regarding key attributes of the functional gut microbiome, in particular with respect to its variability. We further argue that the closing of existing gaps in functional knowledge should be addressed by a most-wanted gene list, the development and application of molecular and cellular high-throughput measurements, the development and sensible use of experimental models, as well as the direct study of observable molecular effects in the human host. [less ▲]

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See detailIsolation of nucleic acids from low biomass samples: detection and removal of sRNA contaminants
Heintz-Buschart, Anna; Yusuf, Dilmurat; Kaysen, Anne UL et al

E-print/Working paper (2017)

Sequencing-based analyses of low-biomass samples are known to be prone to misinterpretation due to the potential presence of contaminating molecules derived from laboratory reagents and environments. Due ... [more ▼]

Sequencing-based analyses of low-biomass samples are known to be prone to misinterpretation due to the potential presence of contaminating molecules derived from laboratory reagents and environments. Due to its inherent instability, contamination with RNA is usually considered to be unlikely. Here we report the presence of small RNA (sRNA) contaminants in widely used microRNA extraction kits and means for their depletion. Sequencing of sRNAs extracted from human plasma samples was performed and significant levels of non-human (exogenous) sequences were detected. The source of the most abundant of these sequences could be traced to the microRNA extraction columns by qPCR-based analysis of laboratory reagents. The presence of artefactual sequences originating from the confirmed contaminants were furthermore replicated in a range of published datasets. To avoid artefacts in future experiments, several protocols for the removal of the contaminants were elaborated, minimal amounts of starting material for artefact-free analyses were defined, and the reduction of contaminant levels for identification of bona fide sequences using 'ultra-clean' extraction kits was confirmed. In conclusion, this is the first report of the presence of RNA molecules as contaminants in laboratory reagents. The described protocols should be applied in the future to avoid confounding sRNA studies. [less ▲]

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See detailSequential isolation of metabolites, RNA, DNA, and proteins from the same unique sample
Roume, Hugo UL; Heintz-Buschart, Anna; Muller, Emilie UL et al

in Methods in Enzymology (2013), 531

In microbial ecology, high-resolution molecular approaches are essential for characterizing the vast organismal and functional diversity and understanding the interaction of microbial communities with ... [more ▼]

In microbial ecology, high-resolution molecular approaches are essential for characterizing the vast organismal and functional diversity and understanding the interaction of microbial communities with biotic and abiotic environmental factors. Integrated omics, comprising genomics, transcriptomics, proteomics, and metabolomics allows conclusive links to be drawn between genetic potential and function. However, this requires truly systematic measurements. In this chapter, we first assess the levels of heterogeneity within mixed microbial communities, thereby demonstrating the need for analyzing biomolecular fractions obtained from a single and undivided sample to facilitate multi-omic analysis and meaningful data integration. Further, we describe a methodological workflow for the reproducible isolation of concomitant metabolites, RNA (optionally split into large and small RNA fractions), DNA, and proteins. Depending on the nature of the sample, the methodology comprises different (pre)processing and preservation steps. If possible, extracellular polar and nonpolar metabolites may first be extracted from cell supernatants using organic solvents. Cells are homogenized by cryomilling before small molecules are extracted with organic solvents. After cell lysis, nucleic acids and protein fractions are sequentially isolated using chromatographic spin columns. To prove the broad applicability of the methodology, we applied it to microbial consortia of biotechnological (biological wastewater treatment biomass), environmental (freshwater planktonic communities), and biomedical (human fecal sample) research interest. The methodological framework should be applicable to other microbial communities as well as other biological samples with a minimum of tailoring and represents an important first step in standardization for the emerging field of Molecular Eco-Systems Biology. [less ▲]

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