Convergence effect during spatiotemporal succession of lacustrine plastisphere: loss of priority effects and turnover of microbial species.

Succession is a fundamental aspect of ecological theory, but studies on temporal succession trajectories and ecological driving mechanisms of plastisphere microbial communities across diverse colonization environments remain scarce and poorly understood. To fill this knowledge gap, we assessed the primary colonizers, succession trajectories, assembly, and turnover mechanisms of plastisphere prokaryotes and eukaryotes from four freshwater lakes. Our results show that differences in microbial composition similarity, temporal turnover rate, and assembly processes in the plastisphere do not exclusively occur at the kingdom level (prokaryotes and eukaryotes), but also depend on environmental conditions and colonization time. Thereby, the time of plastisphere colonization has a stronger impact on community composition and assembly of prokaryotes than eukaryotes, whereas for environmental conditions, the opposite pattern holds true. Across all lakes, deterministic processes shaped the assembly of the prokaryotes, but stochastic processes influenced that of the eukaryotes. Yet, they share similar assembly processes throughout the temporal succession: species turnover over time causes the loss of any priority effect, which leads to a convergent succession of plastisphere microbial communities. The increase and loss of microbial diversity in different kingdoms during succession in the plastisphere potentially impact the stability of entire microbial communities and related biogeochemical cycles. Therefore, research needs to integrate temporal dynamics along with spatial turnovers of the plastisphere microbiome. Taking the heterogeneity of global lakes and the diversity of global climate patterns into account, we highlight the urgency to investigate the spatiotemporal succession mechanism of plastisphere prokaryotes and eukaryotes in more lakes around the world.

Environmental fate of microplastics in the world's third-largest river: Basin-wide investigation and microplastic community analysis.

Rivers have been recognized as major transport pathways for microplastics into the sea but large-scale quantitative data on the environmental fate of riverine microplastics remains limited, hindering proper risk assessment and development of regulatory measures. Microplastics in the whole Yangtze River Basin of China were systematically investigated by sampling the water, sediment, and soil. Microplastics were detected in all samples, with an average abundance of 1.27 items/L, 286.20 items/kg, and 338.09 items/kg for water, sediments, and soils, respectively, with polypropylene and polyethylene being the most abundant polymers. A generally increasing trend of microplastic abundance from upstream to downstream was identified, which were co-attributed by geographical and anthropogenic factors including elevation, longitude, distance from the nearest city, population density, urbanization rate, and land use. Microplastics in the sediments showed more prominent vertical migration than those in the soils, and the density and size of microplastics may be the key factors governing the migration of microplastics across different compartments. Community analysis showed that microplastics in different compartments were significantly different and highly correlated with geographical distance. Major cities at the middle and lower reaches were considered pivotal nodes of microplastic pollution in the Yangtze River Basin. Policy recommendations were also proposed towards better remediation of microplastic pollution involving riverine systems.

Assessing the exoproteome of marine bacteria, lesson from a RTX-toxin abundantly secreted by Phaeobacter strain DSM 17395.

Bacteria from the Roseobacter clade are abundant in surface marine ecosystems as over 10% of bacterial cells in the open ocean and 20% in coastal waters belong to this group. In order to document how these marine bacteria interact with their environment, we analyzed the exoproteome of Phaeobacter strain DSM 17395. We grew the strain in marine medium, collected the exoproteome and catalogued its content with high-throughput nanoLC-MS/MS shotgun proteomics. The major component represented 60% of the total protein content but was refractory to either classical proteomic identification or proteogenomics. We de novo sequenced this abundant protein with high-resolution tandem mass spectra which turned out being the 53 kDa RTX-toxin ZP_02147451. It comprised a peptidase M10 serralysin domain. We explained its recalcitrance to trypsin proteolysis and proteomic identification by its unusual low number of basic residues. We found this is a conserved trait in RTX-toxins from Roseobacter strains which probably explains their persistence in the harsh conditions around bacteria. Comprehensive analysis of exoproteomes from environmental bacteria should take into account this proteolytic recalcitrance.

Proteogenomic definition of biomarkers for the large Roseobacter clade and application for a quick screening of new environmental isolates.

Whole-cell, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry has become a routine and reliable method for microbial characterization due to its simplicity, low cost, and high reproducibility. The identification of microbial isolates relies on the spectral resemblance of low-molecular-weight proteins to already-existing isolates within the databases. This is a gold standard for clinicians who have a finite number of well-defined pathogenic strains but represents a problem for environmental microbiologists with an overwhelming number of organisms to be defined. Here we set a milestone for implementing whole-cell MALDI-TOF mass spectrometry to identify isolates from the biosphere. To make this technique accessible for environmental studies, we propose to (i) define biomarkers that will always show up with an intense m/z signal in the MALDI-TOF spectra and (ii) create a database with all the possible m/z values that these biomarkers can generate to screen new isolates. We tested our method with the relevant marine Roseobacter lineage. The use of shotgun nanoLC-MS/MS proteomics on the small proteome fraction of nine Roseobacter strains and the proteogenomic toolbox helped us to identify potential biomarkers in terms of protein abundance and low variability among strains. We show that the DNA binding protein, HU, and the ribosomal proteins, L29 and L30, are the most robust biomarkers within the Roseobacter clade. The molecular weights of these three biomarkers, as for other conserved homologous proteins, vary due to sequence variation above the genus level. Therefore, we calculated the m/z values expected for each one of the known Roseobacter genera and tested our strategy during an extensive screening of natural marine isolates obtained from coastal waters of the Western Mediterranean Sea. The use of this technique versus standard sequencing methods is discussed.

MiniUIB, a novel minitransposon-based system for stable insertion of foreign DNA into the genomes of Gram-negative and Gram-positive bacteria.

Transposition of the insertion sequence (IS) ISPpu12 is actively induced after conjugative interaction. The transposase of this IS can act in trans on structures flanked by inverted repeats similar to those of the transposon. Based on that fact, an ISPpu12-based minitransposon, miniUIB, has been constructed in order to biotechnologically exploit the self-regulation of ISPpu12 and its increased activity after conjugative interaction. Mobilization of the miniUIB structure into the genome of Pseudomonas stutzeri AN10 after conjugative interaction was demonstrated. A single gene, i.e., the kanamycin resistance determinant, or large genetic structures of >12 kb, i.e., alkBFGHJKL and alkST operons of Pseudomonas putida TF4-1L (GPo1), have been easily integrated in P. stutzeri AN10 by an RP4-based delivery system. Therefore, the integration of the alk determinants by use of the miniUIB system has extended the biodegradation capabilities of this strain. Plasmid pJOC100, containing the transposase and regulator genes of ISPpu12 adjacent to the miniUIB structure, was constructed in order to extend the host range of this biotechnologically useful genetic tool to other model and real-world bacteria. The effectiveness of the system for random mutagenesis in a phylogenetic wide range of bacteria and for the insertion of novel functions has been demonstrated, even in successive steps.

Shotgun nanoLC-MS/MS proteogenomics to document MALDI-TOF biomarkers for screening new members of the Ruegeria genus.

The identification of bacteria by means of matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry directly using whole cells has become a standard method in clinical diagnosis due to its rapidity and simplicity. Nevertheless, the analysis of environmental samples with this approach still represents a challenge due to the enormous microbial diversity existing on earth and the lack of a comprehensive database. Most of the environmentally relevant species comprise only one unique strain, while pathogens such as Escherichia coli, with 667 described strains, are well documented. In such case, identification of the proteins responsible for the peak signals within MALDI-TOF spectra can give crucial information for species discrimination. To give higher confidence in MALDI-TOF biomarker description we exploited information from proteins identified by shotgun nanoLC-MS/MS, consisting of the identification and quantification of low-molecular-weight proteins after SDS-PAGE, in-gel trypsin proteolysis and analysis of tryptic peptides. We also proposed the standardization of the inclusion of internal calibrants in the bacterial sample to improve the accuracy of the MALDI-TOF measurements. In this way, nine candidate biomarkers were tentatively proposed for Ruegeria lacuscaerulensis ITI-1157. The conserved biomarkers were theoretically deduced for all other Ruegeria strains whose genomes have been sequenced and their corresponding m/z MALDI-TOF signals were estimated. Among these, DNA-binding protein, HU, and ribosomal proteins, L29, L30, L32 and S17, were shown experimentally to be also the most prominent and conserved signals in the other strain tested, Ruegeria pomeroyi DSS-3. Thus, we suggested that these five biomarkers, which give rise to 10 m/z peak signals derived from the mono- and doubly protonated proteins, are the best candidates for identifying bacteria belonging to the Ruegeria genus, and quickly assessed their phylogenetic proximity to described species. As an application of these biomarkers, we quickly screened 30 seawater bacterial isolates by MALDI-TOF and found one belonging to the Ruegeria genus, as further confirmed by 16S RNA sequencing. Due to its simplicity and effectiveness, this technique could be of immense value in monitoring bacteria in the environment in the near future.

Comparative proteogenomics of twelve Roseobacter exoproteomes reveals different adaptive strategies among these marine bacteria.

Roseobacters are generalist bacteria abundantly found in the oceans. Because little is known on how marine microorganisms interact in association or competition, we focused our attention on the microbial exoproteome, a key component in their interaction with extracellular milieu. Here we present a comparative analysis of the theoretically encoded exoproteome of twelve members of the Roseobacter group validated by extensive comparative proteogenomics. In silico analysis revealed that 30% of the encoded proteome of these microorganisms could be exported. The ratio of the different protein categories varied in accordance to the ecological distinctness of each strain, a trait reinforced by quantitative proteomics data. Despite the interspecies variations found, the most abundantly detected proteins by shotgun proteomics were from transporter, adhesion, motility, and toxin-like protein categories, defining four different plausible adaptive strategies within the Roseobacter group. In some strains the toxin-secretion strategy was over-represented with repeats-in-toxin-like proteins. Our results show that exoproteomes strongly depend on bacterial trophic strategy and can slightly change because of culture conditions. Simulated natural conditions and the effect of the indigenous microbial community on the exoproteome of Ruegeria pomeroyi DSS-3 were also assayed. Interestingly, we observed a significant depletion of the toxin-like proteins usually secreted by R. pomeroyi DSS-3 when grown in presence of a natural community sampled from a Mediterranean Sea port. The significance of this specific fraction of the exoproteome is discussed.

Proteomic insights into the lifestyle of an environmentally relevant marine bacterium.

In terms of lifestyle, free-living bacteria are classified as either oligotrophic/specialist or opportunist/generalist. Heterogeneous marine environments such as coastal waters favour the establishment of marine generalist bacteria, which code for a large pool of functions. This is basically foreseen to cope with the heterogeneity of organic matter supplied to these systems. Nevertheless, it is not known what fraction of a generalist proteome is needed for house-keeping functions or what fraction is modified to cope with environmental changes. Here, we used high-throughput proteomics to define the proteome of Ruegeria pomeroyi DSS-3, a model marine generalist bacterium of the Roseobacter clade. We evaluated its genome expression under several natural environmental conditions, revealing the versatility of the bacterium to adapt to anthropogenic influence, poor nutrient concentrations or the presence of the natural microbial community. We also assayed 30 different laboratory incubations to increase proteome coverage and to dig further into the functional genomics of the bacterium. We established its core proteome and the proteome devoted to adaptation to general cellular physiological variations (almost 50%). We suggest that the other half of its theoretical proteome is the opportunist genetic pool devoted exclusively to very specific environmental conditions.

In-depth analysis of exoproteomes from marine bacteria by shotgun liquid chromatography-tandem mass spectrometry: the Ruegeria pomeroyi DSS-3 case-study.

Microorganisms secrete into their extracellular environment numerous compounds that are required for their survival. Many of these compounds could be of great interest for biotechnology applications and their genes used in synthetic biology design. The secreted proteins and the components of the translocation systems themselves can be scrutinized in-depth by the most recent proteomic tools. While the secretomes of pathogens are well-documented, those of non-pathogens remain largely to be established. Here, we present the analysis of the exoproteome from the marine bacterium Ruegeria pomeroyi DSS-3 grown in standard laboratory conditions. We used a shotgun approach consisting of trypsin digestion of the exoproteome, and identification of the resulting peptides by liquid chromatography coupled to tandem mass spectrometry. Three different proteins that have domains homologous to those observed in RTX toxins were uncovered and were semi-quantified as the most abundantly secreted proteins. One of these proteins clearly stands out from the catalogue, representing over half of the total exoproteome. We also listed many soluble proteins related to ABC and TRAP transporters implied in the uptake of nutrients. The Ruegeria pomeroyi DSS-3 case-study illustrates the power of the shotgun nano-LC-MS/MS strategy to decipher the exoproteome from marine bacteria and to contribute to environmental proteomics.