A widely distributed phosphate-insensitive phosphatase presents a route for rapid organophosphorus remineralization in the biosphere.

The regeneration of bioavailable phosphate from immobilized organophosphorus represents a key process in the global phosphorus cycle and is facilitated by enzymes known as phosphatases. Most bacteria possess at least one of three phosphatases with broad substrate specificity, known as PhoA, PhoX, and PhoD, whose activity is optimal under alkaline conditions. The production and activity of these phosphatases is repressed by phosphate availability. Therefore, they are only fully functional when bacteria experience phosphorus-limiting growth conditions. Here, we reveal a previously overlooked phosphate-insensitive phosphatase, PafA, prevalent in Bacteroidetes, which is highly abundant in nature and represents a major route for the regeneration of environmental phosphate. Using the enzyme from Flavobacterium johnsoniae, we show that PafA is highly active toward phosphomonoesters, is fully functional in the presence of excess phosphate, and is essential for growth on phosphorylated carbohydrates as a sole carbon source. These distinct properties of PafA may expand the metabolic niche of Bacteroidetes by enabling the utilization of abundant organophosphorus substrates as C and P sources, providing a competitive advantage when inhabiting zones of high microbial activity and nutrient demand. PafA, which is constitutively synthesized by soil and marine flavobacteria, rapidly remineralizes phosphomonoesters releasing bioavailable phosphate that can be acquired by neighboring cells. The pafA gene is highly diverse in plant rhizospheres and is abundant in the global ocean, where it is expressed independently of phosphate availability. PafA therefore represents an important enzyme in the context of global biogeochemical cycling and has potential applications in sustainable agriculture.

Beyond oil degradation: enzymatic potential of Alcanivorax to degrade natural and synthetic polyesters.

Pristine marine environments are highly oligotrophic ecosystems populated by well-established specialized microbial communities. Nevertheless, during oil spills, low-abundant hydrocarbonoclastic bacteria bloom and rapidly prevail over the marine microbiota. The genus Alcanivorax is one of the most abundant and well-studied organisms for oil degradation. While highly successful under polluted conditions due to its specialized oil-degrading metabolism, it is unknown how they persist in these environments during pristine conditions. Here, we show that part of the Alcanivorax genus, as well as oils, has an enormous potential for biodegrading aliphatic polyesters thanks to a unique and abundantly secreted alpha/beta hydrolase. The heterologous overexpression of this esterase proved a remarkable ability to hydrolyse both natural and synthetic polyesters. Our findings contribute to (i) better understand the ecology of Alcanivorax in its natural environment, where natural polyesters such as polyhydroxyalkanoates (PHA) are produced by a large fraction of the community and, hence, an accessible source of carbon and energy used by the organism in order to persist, (ii) highlight the potential of Alcanivorax to clear marine environments from polyester materials of anthropogenic origin as well as oils, and (iii) the discovery of a new versatile esterase with a high biotechnological potential.

Early Colonization of Weathered Polyethylene by Distinct Bacteria in Marine Coastal Seawater.

Plastic debris in aquatic environments is rapidly colonized by a diverse community of microorganisms, often referred to as the "Plastisphere." Given that common plastics are derived from fossil fuels, one would expect that Plastispheres should be enriched with obligate hydrocarbon-degrading bacteria (OHCB). So far, though, different polymer types do not seem to exert a strong effect on determining the composition of the Plastisphere, and putative biodegrading bacteria are only found as rare taxa within these biofilms. Here, we show through 16S rRNA gene sequencing that the enrichment of a prominent OHCB member on weathered and non-weathered polyethylene only occurred at early stages of colonization (i.e., after 2 days of incubation in coastal marine water; 5.8% and 3.7% of relative abundance, respectively, vs. 0.6% on glass controls). As biofilms matured, these bacteria decreased in relative abundance on all materials (< 0.3% after 9 days). Apart from OHCB, weathered polyethylene strongly enriched for other distinct organisms during early stages of colonization, such as a specific member of the Roseobacter group and a member of the genus Aestuariibacter (median 26.9% and 1.8% of the community, respectively), possibly as a consequence of the availability of short-oxidized chains generated from weathering. Our results demonstrate that Plastispheres can vary in accordance with the weathering state of the material and that very early colonizing communities are enriched with taxa that can potentially degrade hydrocarbons. Given the lack of persistent enrichment and overall community convergence between materials over time, common non-hydrolysable polymers might not serve as an important source of carbon for mature Plastispheres once the labile substrates generated from weathering have been depleted.

Plasticizer Degradation by Marine Bacterial Isolates: A Proteogenomic and Metabolomic Characterization.

Many commercial plasticizers are toxic endocrine-disrupting chemicals that are added to plastics during manufacturing and may leach out once they reach the environment. Traditional phthalic acid ester plasticizers (PAEs), such as dibutyl phthalate (DBP) and bis(2-ethyl hexyl) phthalate (DEHP), are now increasingly being replaced with more environmentally friendly alternatives, such as acetyl tributyl citrate (ATBC). While the metabolic pathways for PAE degradation have been established in the terrestrial environment, to our knowledge, the mechanisms for ATBC biodegradation have not been identified previously and plasticizer degradation in the marine environment remains underexplored. From marine plastic debris, we enriched and isolated microbes able to grow using a range of plasticizers and, for the first time, identified the pathways used by two phylogenetically distinct bacteria to degrade three different plasticizers (i.e., DBP, DEHP, and ATBC) via a comprehensive proteogenomic and metabolomic approach. This integrated multi-OMIC study also revealed the different mechanisms used for ester side-chain removal from the different plasticizers (esterases and enzymes involved in the ß-oxidation pathway) as well as the molecular response to deal with toxic intermediates, that is, phthalate, and the lower biodegrading potential detected for ATBC than for PAE plasticizers. This study highlights the metabolic potential that exists in the biofilms that colonize plastics-the Plastisphere-to effectively biodegrade plastic additives and flags the inherent importance of microbes in reducing plastic toxicity in the environment.

Marine Plastic Debris: A New Surface for Microbial Colonization.

Plastics become rapidly colonized by microbes when released into marine environments. This microbial community-the Plastisphere-has recently sparked a multitude of scientific inquiries and generated a breadth of knowledge, which we bring together in this review. Besides providing a better understanding of community composition and biofilm development in marine ecosystems, we critically discuss current research on plastic biodegradation and the identification of potentially pathogenic "hitchhikers" in the Plastisphere. The Plastisphere is at the interface between the plastic and its surrounding milieu, and thus drives every interaction that this synthetic material has with its environment, from ecotoxicity and new links in marine food webs to the fate of the plastics in the water column. We conclude that research so far has not shown Plastisphere communities to starkly differ from microbial communities on other inert surfaces, which is particularly true for mature biofilm assemblages. Furthermore, despite progress that has been made in this field, we recognize that it is time to take research on plastic-Plastisphere-environment interactions a step further by identifying present gaps in our knowledge and offering our perspective on key aspects to be addressed by future studies: (I) better physical characterization of marine biofilms, (II) inclusion of relevant controls, (III) study of different successional stages, (IV) use of environmentally relevant concentrations of biofouled microplastics, and (V) prioritization of gaining a mechanistic and functional understanding of Plastisphere communities.

Riding the wave of genomics to investigate aquatic coliphage diversity and activity.

Bacteriophages infecting Escherichia coli (coliphages) have been used as a proxy for faecal matter and water quality from a variety of environments. However, the diversity of coliphages that is present in seawater remains largely unknown, with previous studies largely focusing on morphological diversity. Here, we isolated and characterized coliphages from three coastal locations in the United Kingdom and Poland. Comparative genomics and phylogenetic analysis of phage isolates facilitated the identification of putative new species within the genera Rb69virus and T5virus and a putative new genus within the subfamily Tunavirinae. Furthermore, genomic and proteomic analysis combined with host range analysis allowed the identification of a putative tail fibre that is likely responsible for the observed differences in host range of phages vB_Eco_mar003J3 and vB_Eco_mar004NP2.

Manganese Oxide Biomineralization Provides Protection against Nitrite Toxicity in a Cell-Density-Dependent Manner.

Manganese biomineralization is a widespread process among bacteria and fungi. To date, there is no conclusive experimental evidence for how and if this process impacts microbial fitness in the environment. Here, we show how a model organism for manganese oxidation is growth inhibited by nitrite, and that this inhibition is mitigated in the presence of manganese. We show that such manganese-mediated mitigation of nitrite inhibition is dependent on the culture inoculum size, and that manganese oxide (MnOX) forms granular precipitates in the culture, rather than sheaths around individual cells. We provide evidence that MnOX protection involves both its ability to catalyze nitrite oxidation into (nontoxic) nitrate under physiological conditions and its potential role in influencing processes involving reactive oxygen species (ROS). Taken together, these results demonstrate improved microbial fitness through MnOX deposition in an ecological setting, i.e., mitigation of nitrite toxicity, and point to a key role of MnOX in handling stresses arising from ROS.IMPORTANCE We present here a direct fitness benefit (i.e., growth advantage) for manganese oxide biomineralization activity in Roseobacter sp. strain AzwK-3b, a model organism used to study this process. We find that strain AzwK-3b in a laboratory culture experiment is growth inhibited by nitrite in manganese-free cultures, while the inhibition is considerably relieved by manganese supplementation and manganese oxide (MnOX) formation. We show that biogenic MnOX interacts directly with nitrite and possibly with reactive oxygen species and find that its beneficial effects are established through formation of dispersed MnOX granules in a manner dependent on the population size. These experiments raise the possibility that manganese biomineralization could confer protection against nitrite toxicity to a population of cells. They open up new avenues of interrogating this process in other species and provide possible routes to their biotechnological applications, including in metal recovery, biomaterials production, and synthetic community engineering.

100 Days of marine Synechococcus-Ruegeria pomeroyi interaction: A detailed analysis of the exoproteome.

Marine phototroph and heterotroph interactions are vital in maintaining the nutrient balance in the oceans as essential nutrients need to be rapidly cycled before sinking to aphotic layers. The aim of this study was to highlight the molecular mechanisms that drive these interactions. For this, we generated a detailed exoproteomic time-course analysis of a 100-day co-culture between the model marine picocyanobacterium Synechococcus sp. WH7803 and the Roseobacter strain Ruegeria pomeroyi DSS-3, both in nutrient-enriched and natural oligotrophic seawater. The proteomic data showed a transition between the initial growth phase and stable-state phase that, in the case of the heterotroph, was caused by a switch in motility attributed to organic matter availability. The phototroph adapted to seawater oligotrophy by reducing its selective leakiness, increasing the acquisition of essential nutrients and secreting conserved proteins of unknown function. We also report a surprisingly high abundance of extracellular superoxide dismutase produced by Synechococcus and a dynamic secretion of potential hydrolytic enzyme candidates used by the heterotroph to cleave organic groups and hydrolase polymeric organic matter produced by the cyanobacterium. The time course dataset we present here will become a reference for understanding the molecular processes underpinning marine phototroph-heterotroph interactions.

Lost, but Found with Nile Red: A Novel Method for Detecting and Quantifying Small Microplastics (1 mm to 20 µm) in Environmental Samples.

Marine plastic debris is a global environmental problem. Surveys have shown that <5 mm plastic particles, known as microplastics, are significantly more abundant in surface seawater and on shorelines than larger plastic particles are. Nevertheless, quantification of microplastics in the environment is hampered by a lack of adequate high-throughput methods for distinguishing and quantifying smaller size fractions (<1 mm), and this has probably resulted in an underestimation of actual microplastic concentrations. Here we present a protocol that allows high-throughput detection and automated quantification of small microplastic particles (20-1000 µm) using the dye Nile red, fluorescence microscopy, and image analysis software. This protocol has proven to be highly effective in the quantification of small polyethylene, polypropylene, polystyrene, and nylon-6 particles, which frequently occur in the water column. Our preliminary results from sea surface tows show a power-law increase in small microplastics (i.e., <1 mm) with a decreasing particle size. Hence, our data help to resolve speculation about the "apparent" loss of this fraction from surface waters. We consider that this method presents a step change in the ability to detect small microplastics by substituting the subjectivity of human visual sorting with a sensitive and semiautomated procedure.

N-Terminal-oriented proteogenomics of the marine bacterium roseobacter denitrificans Och114 using N-Succinimidyloxycarbonylmethyl)tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP) labeling and diagonal chromatography.

Given the ease of whole genome sequencing with next-generation sequencers, structural and functional gene annotation is now purely based on automated prediction. However, errors in gene structure are frequent, the correct determination of start codons being one of the main concerns. Here, we combine protein N termini derivatization using (N-Succinimidyloxycarbonylmethyl)tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP Ac-OSu) as a labeling reagent with the COmbined FRActional DIagonal Chromatography (COFRADIC) sorting method to enrich labeled N-terminal peptides for mass spectrometry detection. Protein digestion was performed in parallel with three proteases to obtain a reliable automatic validation of protein N termini. The analysis of these N-terminal enriched fractions by high-resolution tandem mass spectrometry allowed the annotation refinement of 534 proteins of the model marine bacterium Roseobacter denitrificans OCh114. This study is especially efficient regarding mass spectrometry analytical time. From the 534 validated N termini, 480 confirmed existing gene annotations, 41 highlighted erroneous start codon annotations, five revealed totally new mis-annotated genes; the mass spectrometry data also suggested the existence of multiple start sites for eight different genes, a result that challenges the current view of protein translation initiation. Finally, we identified several proteins for which classical genome homology-driven annotation was inconsistent, questioning the validity of automatic annotation pipelines and emphasizing the need for complementary proteomic data. All data have been deposited to the ProteomeXchange with identifier PXD000337.

Proteomics of the Roseobacter clade, a window to the marine microbiology landscape.

Oceans are powered by metabolically-active microorganisms which are main drivers of global biogeochemical cycles on Earth. A decade ago, marine microbiology was boosted with next-generation sequencing capacities and the launch of large metagenomics surveys. High-performing proteomics is now comprehensive enough for reaching genome-wide and systems-wide scales. It is highly complementary to transcriptomics in order to analyze functional dynamics of marine microbes and microbial complex systems. Next-generation proteomics allows new perspectives for better understanding microbial lifestyles and uncovering the complexity of microbial communities. Here, we review the proteomics approaches and outcomes of recent work carried out on one of the most thoroughly studied marine generalist microorganisms, i.e. the Roseobacter clade, as pivotal examples. We also discuss how the study of the proteome of these organisms has helped in the understanding of the ecological strategy and lifestyle of this relevant marine clade, not only in laboratory cultures but also in its natural environment.

"You produce while I clean up", a strategy revealed by exoproteomics during Synechococcus-Roseobacter interactions.

Most of the energy that is introduced into the oceans by photosynthetic primary producers is in the form of organic matter that then sustains the rest of the food web, from micro to macro-organisms. However, it is the interactions between phototrophs and heterotrophs that are vital to maintaining the nutrient balance of marine microbiomes that ultimately feed these higher trophic levels. The primary produced organic matter is mostly remineralized by heterotrophic microorganisms but, because most of the oceanic dissolved organic matter is in the form of biopolymers, and microbial membrane transport systems operate with molecules <0.6 kDa, it must be hydrolyzed outside the cell before a microorganism can acquire it. As a simili of the marine microbiome, we analyzed, using state-of-the-art proteomics, the exoproteomes obtained from synthetic communities combining specific Roseobacter (Ruegeria pomeroyi DSS-3, Roseobacter denitrificans OCh114, and Dinoroseobacter shibae DFL-12) and Synechococcus strains (WH7803 and WH8102). This approach identified the repertoire of hydrolytic enzymes secreted by Roseobacter, opening up the black box of heterotrophic transformation/remineralization of biopolymers generated by marine phytoplankton. As well as highlighting interesting exoenzymes this strategy also allowed us to infer clues on the molecular basis of niche partitioning.

Functional distinctness in the exoproteomes of marine Synechococcus.

The exported protein fraction of an organism may reflect its life strategy and, ultimately, the way it is perceived by the outside world. Bioinformatic prediction of the exported pan-proteome of Prochlorococcus and Synechococcus lineages demonstrated that (i) this fraction of the encoded proteome had a much higher incidence of lineage-specific proteins than the cytosolic fraction (57% and 73% homologue incidence respectively) and (ii) exported proteins are largely uncharacterized to date (54%) compared with proteins from the cytosolic fraction (35%). This suggests that the genomic and functional diversity of these organisms lies largely in the diverse pool of novel functions these organisms export to/through their membranes playing a key role in community diversification, e.g. for niche partitioning or evading predation. Experimental exoproteome analysis of marine Synechococcus showed transport systems for inorganic nutrients, an interesting array of strain-specific exoproteins involved in mutualistic or hostile interactions (i.e. hemolysins, pilins, adhesins), and exoenzymes with a potential mixotrophic goal (i.e. exoproteases and chitinases). We also show how these organisms can remodel their exoproteome, i.e. by increasing the repertoire of interaction proteins when grown in the presence of a heterotroph or decrease exposure to prey when grown in the dark. Finally, our data indicate that heterotrophic bacteria can feed on the exoproteome of Synechococcus.

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.

Assessing microbial plastic degradation requires robust methods.

Plastics are versatile materials that have the potential to propel humanity towards circularity and ultimate societal sustainability. However, the escalating concern surrounding plastic pollution has garnered significant attention, leading to widespread negative perceptions of these materials. Here, we question the role microbes may play in plastic pollution bioremediation by (i) defining polymer biodegradability (i.e., recalcitrant, hydrolysable and biodegradable polymers) and (ii) reviewing best practices for evaluating microbial biodegradation of plastics. We establish recommendations to facilitate the implementation of rigorous methodologies in future studies on plastic biodegradation, aiming to push this field towards the use of isotopic labelling to confirm plastic biodegradation and further determine the molecular mechanisms involved.

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.

Microbial communities colonising plastics during transition from the wastewater treatment plant to marine waters.

BACKGROUND: Plastics pollution and antimicrobial resistance (AMR) are two major environmental threats, but potential connections between plastic associated biofilms, the 'plastisphere', and dissemination of AMR genes are not well explored. RESULTS: We conducted mesocosm experiments tracking microbial community changes on plastic surfaces transitioning from wastewater effluent to marine environments over 16 weeks. Commonly used plastics, polypropylene (PP), high density polyethylene (HDPE), low density polyethylene (LDPE) and polyethylene terephthalate (PET) incubated in wastewater effluent, river water, estuarine water, and in the seawater for 16 weeks, were analysed via 16S rRNA gene amplicon and shotgun metagenome sequencing. Within one week, plastic-colonizing communities shifted from wastewater effluent-associated microorganisms to marine taxa, some members of which (e.g. Oleibacter-Thalassolituus and Sphingomonas spp., on PET, Alcanivoracaceae on PET and PP, or Oleiphilaceae, on all polymers), were selectively enriched from levels undetectable in the starting communities. Remarkably, microbial biofilms were also susceptible to parasitism, with Saprospiraceae feeding on biofilms at late colonisation stages (from week 6 onwards), while Bdellovibrionaceae were prominently present on HDPE from week 2 and LDPE from day 1. Relative AMR gene abundance declined over time, and plastics did not become enriched for key AMR genes after wastewater exposure. CONCLUSION: Although some resistance genes occurred during the mesocosm transition on plastic substrata, those originated from the seawater organisms. Overall, plastic surfaces incubated in wastewater did not act as hotspots for AMR proliferation in simulated marine environments.

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.

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.

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.