Delegan, Y. A. | Search bioRxiv

bioRxiv2024

Quantitative assessment of strain isolates and microbiomes using fast MS/MS-free metaproteomics

BACKGROUNDMicrobial communities play a crucial role in human health and environmental regulation, but present an especial challenge for the analytical science due to their diversity and dynamic range. Tandem mass spectrometry provides functional insights on microorganisms life cycle, but still lacks throughput and sensitivity. MALDI TOF is widely used for ultrafast identification of species, but does not assess their functional activity. Development of ultrafast mass spectrometry methods and bioinformatic approaches applicable for both accurate identification and functional assessment of microbial communities based on their protein content is of high interest. RESULTSWe show for the first time that both identity and functional activity of microorganisms and their communities can be accurately determined in experiments as short as 7 minutes per sample, using the basic Orbitrap MS configuration without peptide fragmentation. The approach was validated using strain isolates, mock microbiomes composed of bacteria spiked at known concentrations and human fecal microbiomes. Our new bioinformatic algorithm identifies the bacterial species with an accuracy of 95 %, when no prior information on the sample is available. Microbiome composition was resolved at the genus level with the mean difference between the actual and identified components of 12 %. For mock microbiomes, Pearson coefficient of up to 0.97 was achieved in estimates of strain biomass change. By the example of Rhodococcus biodegradation of n-alkanes, phenols and its derivatives, we showed the accurate assessment of functional activity of strain isolates, compared with the standard label-free and label-based approaches. SIGNIFICANCEOur approach makes microbial proteomics fast, functional and insightful using the Orbitrap instruments even without employing peptide fragmentation technology. The approach can be applied to any microorganisms and can take a niche in routine functional assessment of microbial pathogens and consortiums in clinical diagnostics together with MALDI TOF MS and 16S rRNA gene sequencing.

microbiology↗

bioRxiv2023

Sulfur cycle microbiota in extremely contaminated Technosol with ongoing pedogenesis: culture-dependent and metagenomic approach

Understanding the microbial communities involved in the global sulfur cycle is crucial for comprehending key biogeochemical processes on Earth. However, most studies tend to focus on marine ecosystems, while investigations into the terrestrial sulfur cycle are scarce. In this study, we employed culture-dependent techniques and metagenomics to characterize sulfur-cycling microbiota in extremely contaminated soils. We analyzed shotgun and amplicon sequencing data to assess taxonomical diversity, metagenome-assembled genomes (MAGs) for functional diversity, and also calculated the most probable numbers (MPN) of sulfur-oxidizing and sulfate-reducing bacteria based on culture-dependent data. Our taxonomic profiling, using both shotgun and amplicon data, revealed a high diversity of sulfur cycle bacteria, which was found to be dependent on pH levels. Additionally, our findings confirmed recent modelling of specific taxa biogeographical distribution, such as the sulfur-reducing Mesotoga. Using a functional metagenomics approach, we identified non-canonical taxa involved in dissimilatory sulfur metabolism (e.g., sulfate-reducing acidobacteria and members of the Binatota phylum), and canonical taxa engaged in various oxidative, reductive, and organosulfur transformations (e.g., sulfur-oxidizing alpha-, beta-, and gammaproteobacteria). Furthermore, we discovered that multiple taxa in the studied Technosol encoded different enzymes capable of sulfite transformation and the removal of sulfite from various organosulfonate molecules, thus contributing to the cryptic cycling of sulfur compounds. Estimated MPNs of sulfur-oxidizing bacteria aligned with our shotgun and amplicon data, while those of sulfate-reducing bacteria contradicted functional metagenomic findings. Based on our overall analysis, we support the idea that sulfate-reducers belong to the rare biosphere in soil. We suggest that they behave differently in soils compared to aquatic habitats due to the high taxonomic diversity along with low absolute abundance. Our findings unveil a diverse and unique community of sulfur-metabolizing bacteria that has evolved in soil under severe technogenic pollution, high bulk sulfur content, and fluctuating redox states.

microbiology↗