First Isolation and Characterization of Bacteria from the Core's Cooling Pool of an Operating Nuclear Reactor.

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.

Characterization of the mechanism of bile salt hydrolase substrate specificity by experimental and computational analyses.

Bile salt hydrolases (BSHs) are currently being investigated as target enzymes for metabolic regulators in humans and as growth promoters in farm animals. Understanding structural features underlying substrate specificity is necessary for inhibitor design. Here, we used a multidisciplinary workflow including mass spectrometry, mutagenesis, molecular dynamic simulations, machine learning, and crystallography to demonstrate substrate specificity in Lactobacillus salivarius BSH, the most abundant enzyme in human and farm animal intestines. We show the preference of substrates with a taurine head and a dehydroxylated sterol ring for hydrolysis. A regression model that correlates the relative rates of hydrolysis of various substrates in various enzyme mutants with the residue-substrate interaction energies guided the identification of structural determinants of substrate binding and specificity. In addition, we found T208 from another BSH protomer regulating the hydrolysis. The designed workflow can be used for fast and comprehensive characterization of enzymes with a broad range of substrates.

Dichloromethane Degradation Pathway from Unsequenced Hyphomicrobium sp. MC8b Rapidly Explored by Pan-Proteomics.

Several bacteria are able to degrade the major industrial solvent dichloromethane (DCM) by using the conserved dehalogenase DcmA, the only system for DCM degradation characterised at the sequence level so far. Using differential proteomics, we rapidly identified key determinants of DCM degradation for Hyphomicrobium sp. MC8b, an unsequenced facultative methylotrophic DCM-degrading strain. For this, we designed a pan-proteomics database comprising the annotated genome sequences of 13 distinct Hyphomicrobium strains. Compared to growth with methanol, growth with DCM induces drastic changes in the proteome of strain MC8b. Dichloromethane dehalogenase DcmA was detected by differential pan-proteomics, but only with poor sequence coverage, suggesting atypical characteristics of the DCM dehalogenation system in this strain. More peptides were assigned to DcmA by error-tolerant search, warranting subsequent sequencing of the genome of strain MC8b, which revealed a highly divergent set of dcm genes in this strain. This suggests that the dcm enzymatic system is less strongly conserved than previously believed, and that substantial molecular evolution of dcm genes has occurred beyond their horizontal transfer in the bacterial domain. Our study showed the power of pan-proteomics for quick characterization of new strains belonging to branches of the Tree of Life that are densely genome-sequenced.

Proteotyping Environmental Microorganisms by Phylopeptidomics: Case Study Screening Water from a Radioactive Material Storage Pool.

The microbial diversity encompassed by the environmental biosphere is largely unexplored, although it represents an extensive source of new knowledge and potentially of novel enzymatic catalysts for biotechnological applications. To determine the taxonomy of microorganisms, proteotyping by tandem mass spectrometry has proved its efficiency. Its latest extension, phylopeptidomics, adds a biomass quantitation perspective for mixtures of microorganisms. Here, we present an application of phylopeptidomics to rapidly and sensitively screen microorganisms sampled from an industrial environment, i.e., a pool where radioactive material is stored. The power of this methodology is demonstrated through the identification of both prokaryotes and eukaryotes, whether as pure isolates or present as mixtures or consortia. In this study, we established accurate taxonomical identification of environmental prokaryotes belonging to the Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria phyla, as well as eukaryotes from the Ascomycota phylum. The results presented illustrate the potential of tandem mass spectrometry proteotyping, in particular phylopeptidomics, to screen for and rapidly identify microorganisms.

High-throughput proteotyping of bacterial isolates by double barrel chromatography-tandem mass spectrometry based on microplate paramagnetic beads and phylopeptidomics.

Tandem mass spectrometry-based proteotyping of microorganisms presents several advantages over whole-cell MALDI-TOF mass spectrometry: because a larger number of signals are recorded with better accuracy and precision, the approach allows for the identification of microorganisms at more resolved taxonomic levels, and can easily manage complex samples. Additionally, the use of SP3 paramagnetic beads for cell lysis and protein cleanup simplifies sample preparation for proteotyping. Based on these features, we have developed and tested a 96-well plate platform for high-throughput proteotyping of a large variety of bacteria. We evaluated the performance of the platform in terms of bacterial load and found no cross-contamination between wells. Likewise, phylopeptidomics analysis revealed no alteration in the relative quantifications of microorganisms. Finally, we applied this new format for rapid proteotyping of a large set of dental isolates using double-barrel chromatography coupled to tandem mass spectrometry, which maximizes the number of spectra per unit of time. The procedure allowed us to establish whether these isolates were pure strains or mixtures of strains and to identify the microorganisms at the most resolved taxonomic level. SIGNIFICANCE: The rapid and accurate identification of microorganisms is a clinical priority in medical diagnostics; however, specimens containing mixtures of microorganisms are difficult to analyze and the procedure is time-consuming. Tandem mass spectrometry proteotyping allows the fast identification of complex mixtures of microorganisms, known or unknown, and can also establish the biomass ratio of each component. We describe here a new workflow for preparing microbial samples in a 96-well-plate format for tandem mass spectrometry proteotyping and document its advantages and limitations. We demonstrate that this new format coupled to a highly efficient double-barrel LC-MS/MS system allows proteotyping of 96 isolates in 55 h.

Evaluation of Sample Preparation Methods for Fast Proteotyping of Microorganisms by Tandem Mass Spectrometry.

Tandem mass spectrometry-based proteotyping allows characterizing microorganisms in terms of taxonomy and is becoming an important tool for investigating microbial diversity from several ecosystems. Fast and automatable sample preparation for obtaining peptide pools amenable to tandem mass spectrometry is necessary for enabling proteotyping as a high-throughput method. First, the protocol to increase the yield of lysis of several representative bacterial and eukaryotic microorganisms was optimized by using a long and drastic bead-beating setting with 0.1 mm silica beads, 0.1 and 0.5 mm glass beads, in presence of detergents. Then, three different methods to obtain greater digestion yield from these extracts were tested and optimized for improve efficiency and reduce application time: denaturing electrophoresis of proteins and in-gel proteolysis, suspension-trapping filter-based approach (S-Trap) and, solid-phase-enhanced sample preparation named SP3. The latter method outperforms the other two in terms of speed and delivers also more peptides and proteins than with the in-gel proteolysis (2.2 fold for both) and S-trap approaches (1.3 and 1.2 fold, respectively). Thus, SP3 directly improves tandem mass spectrometry proteotyping.