Jean Armengaud

Microbial life can thrive in the most inhospitable places, such as nuclear facilities with high levels of ionizing radiation. Using direct meta-analyses, we have previously highlighted the presence of bacteria belonging to twenty-five different genera in the highly radioactive water of the cooling pool of an operating nuclear reactor core. In the present study, we further characterize this specific environment by isolating and identifying some of these microorganisms and assessing their radiotolerance and their ability to decontaminate uranium. This metal is one of the major radioactive contaminants of anthropogenic origin in the environment due to the nuclear and mining industries and agricultural practices. The microorganisms isolated when sampling was performed during the reactor operation consisted mainly of Actinobacteria and Firmicutes, whereas Proteobacteria were dominant when sampling was performed during the reactor shutdown. We investigated their tolerance to gamma radiation under different conditions. Most of the bacterial strains studied were able to survive 200 Gy irradiation. Some were even able to withstand 1 kGy, with four of them showing more than 10% survival at this dose. We also assessed their uranium uptake capacity. Seven strains were able to remove almost all the uranium from a 5 µM solution. Four strains displayed high efficiency in decontaminating a 50 µM uranium solution, demonstrating promising potential for use in bioremediation processes in environments contaminated by radionuclides.

A phyloprofile of Frankia genomes was carried out to identify those genes present in symbiotic strains of clusters 1, 1c, 2 and 3 and absent in non-infective strains of cluster 4. At a threshold of 50% AA identity, 108 genes were retrieved. Among these were known symbiosis-associated genes such as nif (nitrogenase), and genes which are not know as symbiosis-associated genes such as can (carbonic anhydrase, CAN). The role of CAN, which supplies carbonate ions necessary for carboxylases and acidifies the cytoplasm, was thus analyzed by staining cells with pH-responsive dyes; assaying for CO2 levels in N-fixing propionate-fed cells (that require a propionate-CoA carboxylase to yield succinate-CoA), fumarate-fed cells and N-replete propionate-fed cells; conducting proteomics on N-fixing fumarate and propionate-fed cells and direct measurement of organic acids in nodules and in roots. The interiors of both in vitro and nodular vesicles were found to be at a lower pH than that of hyphae. CO2 levels in N2-fixing propionate-fed cultures were lower than in N-replete ones. Proteomics of propionate-fed cells showed carbamoyl-phosphate synthase (CPS) as the most overabundant enzyme relative to fumarate-fed cells. CPS combines carbonate and ammonium in the first step of the citrulline pathway, something which would help manage acidity and NH4+. Nodules were found to have sizeable amounts of pyruvate and acetate in addition to TCA intermediates. This points to CAN reducing the vesicles’ pH to prevent the escape of NH3 and to control ammonium assimilation by GS and GOGAT, two enzymes that work in different ways in vesicles and hyphae. Genes with related functions (carboxylases, biotin operon and citrulline-aspartate ligase) appear to have undergone decay in non-symbiotic lineages.

Correct identification of the microorganisms present in a complex sample is a crucial issue. Proteotyping based on tandem mass spectrometry can help establish an inventory of organisms present in a sample. Evaluation of bioinformatics strategies and tools for mining the recorded datasets is essential to establish confidence in the results obtained and to improve these pipelines in terms of sensitivity and accuracy. Here, we propose several tandem mass spectrometry datasets recorded on an artificial reference consortium comprising 24 bacterial species. This assemblage of environmental and pathogenic bacteria covers 20 different genera and 5 bacterial phyla. The dataset comprises difficult cases, such as the Shigella flexneri species, which is closely related to Escherichia coli, and several highly sequenced clades. Different acquisition strategies simulate real-life scenarios: from rapid survey sampling to exhaustive analysis. We provide access to individual proteomes of each bacterium separately to provide a rational basis for evaluating the assignment strategy of MS/MS spectra when recorded from complex mixtures. This resource should provide an interesting common reference for developers who wish to compare their proteotyping tools and for those interested in evaluating protein assignment when dealing with complex samples, such as microbiomes.

Purpose Helcococcus kunzii is a skin commensal, Gram‐positive bacterium, mostly isolated from infected chronic wounds. This opportunistic pathogen is usually co‐isolated with Staphylococcus aureus . The present dataset explores the production and secretion of H. kunzii bacterial virulence interacting proteins in a growth medium mimicking chronic wounds in exponential and stationary growth phases. Experimental Design The H. kunzii cellular proteome and exoproteome were assessed by analyzing three biological replicates per condition tested. Samples were analyzed using a Q‐Exactive HF mass spectrometer. Comparative and functional analyses were performed to profile the identified protein set. Results The H. kunzii ’s cellular proteome encompassed 969 proteins, among which 64 and 53 were specifically identified in the exponential and stationary phase of growth, respectively. Its exoproteome comprised 58 proteins, among which 16 and 14 were characteristic of each growth stage. Metabolic differences between the two phases of growth are discussed. Besides, the production of previously shortlisted and novel putative H. kunzii targets involved in modulating the virulence of S. aureus is investigated. Conclusion and Clinical Relevance This work, pioneering the study of H . kunzii physiology in a chronic wound‐like environment, should assist future research on this opportunistic pathogen and the search for innovative approaches for wound management.