Proteome analysis of the UVB-resistant marine bacterium Photobacterium angustum S14

The proteome of the marine bacterium Photobacterium angustum S14 was exposed to UVB and analyzed by the implementation of both the post-digest ICPL labeling method and 2D-DIGE technique using exponentially growing cells. A total of 40 and 23 proteins were quantified in all replicates using either the ICPL or 2D-DIGE methods, respectively. By combining both datasets from 8 biological replicates (4 biological replicates for each proteomics technique), 55 proteins were found to respond significantly to UVB radiation in P. angustum. A total of 8 UVB biomarkers of P. angustum were quantified in all replicates using both methods. Among them, the protein found to present the highest increase in abundance (almost a 3-fold change) was RecA, which is known to play a crucial role in the so-called recombinational repair process. We also observed a high number of antioxidants, transport proteins, metabolism-related proteins, transcription/translation regulators, chaperonins and proteases. We also discuss and compare the UVB response and global protein expression profiles obtained for two different marine bacteria with trophic lifestyles: the copiotroph P. angustum and oligotroph Sphingopyxis alaskensis

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

Molecular insights into the powerful mucus-based adhesion of limpets (Patella vulgata L.).

Limpets (Patella vulgata L.) are renowned for their powerful attachments to rocks on wave-swept seashores. Unlike adult barnacles and mussels, limpets do not adhere permanently; instead, they repeatedly transition between long-term adhesion and locomotive adhesion depending on the tide. Recent studies on the adhesive secretions (bio-adhesives) of marine invertebrates have expanded our knowledge on the composition and function of temporary and permanent bio-adhesives. In comparison, our understanding of the limpets' transitory adhesion remains limited. In this study, we demonstrate that suction is not the primary attachment mechanism in P. vulgata; rather, they secrete specialized pedal mucus for glue-like adhesion. Through combined transcriptomics and proteomics, we identified 171 protein sequences from the pedal mucus. Several of these proteins contain conserved domains found in temporary bio-adhesives from sea stars, sea urchins, marine flatworms and sea anemones. Many of these proteins share homology with fibrous gel-forming glycoproteins, including fibrillin, hemolectin and SCO-spondin. Moreover, proteins with potential protein- and glycan-degrading domains could have an immune defence role or assist degrading adhesive mucus to facilitate the transition from stationary to locomotive states. We also discovered glycosylation patterns unique to the pedal mucus, indicating that specific sugars may be involved in transitory adhesion. Our findings elucidate the mechanisms underlying P. vulgata adhesion and provide opportunities for future studies on bio-adhesives that form strong attachments and resist degradation until necessary for locomotion

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

Novel functional insights into the microbiome inhabiting marine plastic debris: critical considerations to counteract the challenges of thin biofilms using multi-omics and comparative metaproteomics

Microbial functioning on marine plastic surfaces has been poorly documented, especially within cold climates where temperature likely impacts microbial activity and the presence of hydrocarbonoclastic microorganisms. To date, only two studies have used metaproteomics to unravel microbial genotype–phenotype linkages in the marine ‘plastisphere’, and these have revealed the dominance of photosynthetic microorganisms within warm climates. Advancing the functional representation of the marine plastisphere is vital for the development of specific databases cataloging the functional diversity of the associated microorganisms and their peptide and protein sequences, to fuel biotechnological discoveries. Here, we provide a comprehensive assessment for plastisphere metaproteomics, using multi-omics and data mining on thin plastic biofilms to provide unique insights into plastisphere metabolism. Our robust experimental design assessed DNA/protein co-extraction and cell lysis strategies, proteomics workflows, and diverse protein search databases, to resolve the active plastisphere taxa and their expressed functions from an understudied cold environment. Results For the first time, we demonstrate the predominance and activity of hydrocarbonoclastic genera (Psychrobacter, Flavobacterium, Pseudomonas) within a primarily heterotrophic plastisphere. Correspondingly, oxidative phosphorylation, the citrate cycle, and carbohydrate metabolism were the dominant pathways expressed. Quorum sensing and toxin-associated proteins of Streptomyces were indicative of inter-community interactions. Stress response proteins expressed by Psychrobacter, Planococcus, and Pseudoalteromonas and proteins mediating xenobiotics degradation in Psychrobacter and Pseudoalteromonas suggested phenotypic adaptations to the toxic chemical microenvironment of the plastisphere. Interestingly, a targeted search strategy identified plastic biodegradation enzymes, including polyamidase, hydrolase, and depolymerase, expressed by rare taxa. The expression of virulence factors and mechanisms of antimicrobial resistance suggested pathogenic genera were active, despite representing a minor component of the plastisphere community. Conclusion Our study addresses a critical gap in understanding the functioning of the marine plastisphere, contributing new insights into the function and ecology of an emerging and important microbial niche. Our comprehensive multi-omics and comparative metaproteomics experimental design enhances biological interpretations to provide new perspectives on microorganisms of potential biotechnological significance beyond biodegradation and to improve the assessment of the risks associated with microorganisms colonizing marine plastic pollution

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