70 research outputs found
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
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/
Gel-Free Proteome Analysis Isotopic Labelling Vs. Label-Free Approaches for Quantitative Proteomics
Shedding light on the bacterial resistance to toxic UV filters: A comparative genomic study
UV filters are toxic to marine bacteria that dominate the marine biomass. Ecotoxicology often studies the organism response but rarely integrates the toxicity mechanisms at the molecular level. In this study, in silico comparative genomics between UV filters sensitive and resistant bacteria were conducted in order to unravel the genes responsible for a resistance phenotype. The genomes of two environmentally relevant Bacteroidetes and three Firmicutes species were compared through pairwise comparison. Larger genomes were carried by bacteria exhibiting a resistant phenotype, favoring their ability to adapt to environmental stresses. While the antitoxin and CRISPR systems were the only distinctive features in resistant Bacteroidetes, Firmicutes displayed multiple unique genes that could support the difference between sensitive and resistant phenotypes. Several genes involved in ROS response, vitamin biosynthesis, xenobiotic degradation, multidrug resistance, and lipophilic compound permeability were shown to be exclusive to resistant species. Our investigation contributes to a better understanding of UV filters resistance phenotypes, by identifying pivotal genes involved in key pathways
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
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
Toxicity of UV filters on marine bacteria: Combined effects with damaging solar radiation
Organic UV filters are of emerging concern due to their occurrence and persistence in coastal ecosystems. Because marine bacteria are crucial in the major biogeochemical cycles, there is an urgent need to understand to what extent these microorganisms are affected by those chemicals. This study deciphers the impact of five common sunscreen UV filters on twenty-seven marine bacteria, combining both photobiology and toxicity analysis on environmentally relevant species. Seven bacteria were sensitive to different organic UV filters at 1000 μg L−1, including octinoxate and oxybenzone. This is the first report demonstrating inhibition of bacterial growth from 100 μg L−1. None of the UV filters showed any toxicity at 1000 μg L−1 on stationary phase cells, demonstrating that physiological state was found to be a key parameter in the bacterial response to UV-filters. Indeed, non-growing bacteria were resistant to UV filters whereas growing cells exhibited UV filter dependent sensitivity. Octinoxate was the most toxic chemical at 1000 μg L−1 on growing cells. Interestingly, photobiology experiments revealed that the toxicity of octinoxate and homosalate decreased after light exposure while the other compounds were not affected. In terms of environmental risk characterization, our results revealed that the increasing use of sun blockers could have detrimental impacts on bacterioplanktonic communities in coastal areas. Our findings contribute to a better understanding of the impact of the most common UV filters on bacterial species and corroborate the importance to consider environmental parameters such as solar radiation in ecotoxicology studies
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
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
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