Diel Cycle Proteomics: Illuminating Molecular Dynamics in Purple Bacteria for Optimized Biotechnological Applications.

Circadian rhythms, characterized by approximately 24 h cycles, play a pivotal role in enabling various organisms to synchronize their biological activities with daily variations. While ubiquitous in Eukaryotes, circadian clocks remain exclusively characterized in Cyanobacteria among Prokaryotes. These rhythms are regulated by a core oscillator, which is controlled by a cluster of three genes: kaiA, kaiB, and kaiC. Interestingly, recent studies revealed rhythmic activities, potentially tied to a circadian clock, in other Prokaryotes, including purple bacteria such as Rhodospirillum rubrum, known for its applications in fuel and plastic bioproduction. However, the pivotal question of how light and dark cycles influence protein dynamics and the expression of putative circadian clock genes remains unexplored in purple non-sulfur bacteria. Unraveling the regulation of these molecular clocks holds the key to unlocking optimal conditions for harnessing the biotechnological potential of R. rubrum. Understanding how its proteome responds to different light regimes-whether under continuous light or alternating light and dark cycles-could pave the way for precisely fine-tuning bioproduction processes. Here, we report for the first time the expressed proteome of R. rubrum grown under continuous light versus light and dark cycle conditions using a shotgun proteomic analysis. In addition, we measured the impact of light regimes on the expression of four putative circadian clock genes (kaiB1, kaiB2, kaiC1, kaiC2) at the transcriptional and translational levels using RT-qPCR and targeted proteomic (MRM-MS), respectively. The data revealed significant effects of light conditions on the overall differential regulation of the proteome, particularly during the early growth stages. Notably, several proteins were found to be differentially regulated during the light or dark period, thus impacting crucial biological processes such as energy conversion pathways and the general stress response. Furthermore, our study unveiled distinct regulation of the four kai genes at both the mRNA and protein levels in response to varying light conditions. Deciphering the impact of the diel cycle on purple bacteria not only enhances our understanding of their ecology but also holds promise for optimizing their applications in biotechnology, providing valuable insights into the origin and evolution of prokaryotic clock mechanisms.

Towards the discovery of novel molecular clocks in Prokaryotes.

Diel cycle is of enormous biological importance as it imposes daily oscillation in environmental conditions, which temporally structures most ecosystems. Organisms developed biological time-keeping mechanisms - circadian clocks - that provide a significant fitness advantage over competitors by optimising the synchronisation of their biological activities. While circadian clocks are ubiquitous in Eukaryotes, they are so far only characterised in Cyanobacteria within Prokaryotes. However, growing evidence suggests that circadian clocks are widespread in the bacterial and archaeal domains. As Prokaryotes are at the heart of crucial environmental processes and are essential to human health, unravelling their time-keeping systems provides numerous applications in medical research, environmental sciences, and biotechnology. In this review, we elaborate on how novel circadian clocks in Prokaryotes offer research and development perspectives. We compare and contrast the different circadian systems in Cyanobacteria and discuss about their evolution and taxonomic distribution. We necessarily provide an updated phylogenetic analysis of bacterial and archaeal species that harbour homologs of the main cyanobacterial clock components. Finally, we elaborate on new potential clock-controlled microorganisms that represent opportunities of ecological and industrial relevance in prokaryotic groups such as anoxygenic photosynthetic bacteria, methanogenic archaea, methanotrophs or sulphate-reducing bacteria.

Diel Protein Regulation of Marine Picoplanktonic Communities Assessed by Metaproteomics.

The diel cycle is of enormous biological importance in that it imposes temporal structure on ecosystem productivity. In the world's oceans, microorganisms form complex communities that carry out about half of photosynthesis and the bulk of life-sustaining nutrient cycling. How the functioning of microbial communities is impacted by day and night periods in surface seawater remains to be elucidated. In this study, we compared the day and night metaproteomes of the free-living and the particle-attached bacterial fractions from picoplanktonic communities sampled from the northwest Mediterranean Sea surface. Our results showed similar taxonomic distribution of free-living and particle-attached bacterial populations, with Alphaproteobacteria, Gammaproteobacteria and Cyanobacteria being the most active members. Comparison of the day and night metaproteomes revealed that free-living and particle-attached bacteria were more active during the day and the night, respectively. Interestingly, protein diel variations were observed in the photoautotroph Synechococcales and in (photo)-heterotrophic bacteria such as Flavobacteriales, Pelagibacterales and Rhodobacterales. Moreover, our data demonstrated that diel cycle impacts light-dependent processes such as photosynthesis and UV-stress response in Synechococcales and Rhodobacterales, respectively, while the protein regulation from the ubiquitous Pelagibacterales remained stable over time. This study unravels, for the first time, the diel variation in the protein expression of major free-living and particle-attached microbial players at the sea surface, totaling an analysis of eight metaproteomes.

Metal-induced bacterial interactions promote diversity in river-sediment microbiomes.

Anthropogenic metal contamination results in long-term environmental selective pressure with unclear impacts on bacterial communities, which comprise key players in ecosystem functioning. Since metal contamination poses serious toxicity and bioaccumulation issues, assessing their impact on environmental microbiomes is important to respond to current environmental and health issues. Despite elevated metal concentrations, the river sedimentary microbiome near the MetalEurop foundry (France) shows unexpected higher diversity compared with the upstream control site. In this work, a follow-up of the microbial community assembly during a metal contamination event was performed in microcosms with periodic renewal of the supernatant river water. Sediments of the control site were gradually exposed to a mixture of metals (Cd, Cu, Pb and Zn) in order to reach similar concentrations to MetalEurop sediments. Illumina sequencing of 16S rRNA gene amplicons was performed. Metal-resistant genes, czcA and pbrA, as well as IncP plasmid content, were assessed by quantitative PCR. The outcomes of this study support previous in situ observations showing that metals act as community assembly managers, increasing diversity. This work revealed progressive adaptation of the sediment microbiome through the selection of different metal-resistant mechanisms and cross-species interactions involving public good-providing bacteria co-occurring with the rest of the community.

Deciphering the Functioning of Microbial Communities: Shedding Light on the Critical Steps in Metaproteomics.

Unraveling the complex structure and functioning of microbial communities is essential to accurately predict the impact of perturbations and/or environmental changes. From all molecular tools available today to resolve the dynamics of microbial communities, metaproteomics stands out, allowing the establishment of phenotype-genotype linkages. Despite its rapid development, this technology has faced many technical challenges that still hamper its potential power. How to maximize the number of protein identification, improve quality of protein annotation, and provide reliable ecological interpretation are questions of immediate urgency. In our study, we used a robust metaproteomic workflow combining two protein fractionation approaches (gel-based versus gel-free) and four protein search databases derived from the same metagenome to analyze the same seawater sample. The resulting eight metaproteomes provided different outcomes in terms of (i) total protein numbers, (ii) taxonomic structures, and (iii) protein functions. The characterization and/or representativeness of numerous proteins from ecologically relevant taxa such as Pelagibacterales, Rhodobacterales, and Synechococcales, as well as crucial environmental processes, such as nutrient uptake, nitrogen assimilation, light harvesting, and oxidative stress response, were found to be particularly affected by the methodology. Our results provide clear evidences that the use of different protein search databases significantly alters the biological conclusions in both gel-free and gel-based approaches. Our findings emphasize the importance of diversifying the experimental workflow for a comprehensive metaproteomic study.

mPies: a novel metaproteomics tool for the creation of relevant protein databases and automatized protein annotation.

Metaproteomics allows to decipher the structure and functionality of microbial communities. Despite its rapid development, crucial steps such as the creation of standardized protein search databases and reliable protein annotation remain challenging. To overcome those critical steps, we developed a new program named mPies (metaProteomics in environmental sciences). mPies allows the creation of protein databases derived from assembled or unassembled metagenomes, and/or public repositories based on taxon IDs, gene or protein names. For the first time, mPies facilitates the automatization of reliable taxonomic and functional consensus annotations at the protein group level, minimizing the well-known protein inference issue, which is commonly encountered in metaproteomics. mPies' workflow is highly customizable with regards to input data, workflow steps, and parameter adjustment. mPies is implemented in Python 3/Snakemake and freely available on GitHub: https://github.com/johanneswerner/mPies/. REVIEWER: This article was reviewed by Dr. Wilson Wen Bin Goh.