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.

Pseudomonas gallaeciensis sp. nov., isolated from crude-oil-contaminated intertidal sand samples after the Prestige oil spill.

Strains V113T, V92 and V120 have been isolated from sand samples taken at the Atlantic intertidal shore in Galicia, Spain, after the Prestige oil spill. A preliminary analysis of the 16S rRNA and the partial rpoD gene sequences indicated that these strains belonged to the Pseudomonas genus, but they were distinct from any known Pseudomonas species. They were extensively characterized by a polyphasic taxonomic approach and phylogenetic data that confirmed that these strains belonged to the Pseudomonas pertucinogena group. Phylogenetic analysis of 16S rRNA, gyrB and rpoD gene sequences showed that the three strains were 99% similar and were closely related to members of the P. pertucinogena group, with less than 94% similarity to strains of established species; Pseudomonas pachastrellae was the closest relative. The Average Nucleotide Index based on blast values was 89.0% between V113T and the P. pachastrellae type strain, below the accepted species level (95%). The predominant cellular fatty acid contents and whole cell protein profiles determined by MALDI-TOF mass spectrometry also differentiated the studied strains from known Pseudomonas species. We therefore conclude that strains V113T, V92 and V120 represent a novel species of Pseudomonas, for which the name Pseudomonas gallaeciensis is proposed; the type strain is V113T (=CCUG 67583T=LMG 29038T).

Draft Genome Sequences of Thalassobacter Strains 1CONIMAR09 and 16PALIMAR09, Two Members of the Roseobacter Lineage Isolated from Coastal Areas of the Mediterranean Sea around Mallorca Island.

We report the draft genome sequence of two new members of the Roseobacter lineage, Thalassobacter strains 1CONIMAR09 and 16PALIMAR09, which were isolated from the seawater coast of Mallorca Island. Each genome harbored putative genes for obtaining energy by chemolithotrophy and making aerobic anoxygenic photosynthesis.

Comparative genomics of the protocatechuate branch of the ß-ketoadipate pathway in the Roseobacter lineage.

The protocatechuate branch of the ß-ketoadipate pathway is the most common pathway for degradation of monoaromatic compounds in the Roseobacter lineage. We analyzed 43 Roseobacter genomes in order to determine if they possessed all genetic elements for this pathway and if there were common patterns in gene organization. The eight genes of the pathway (pcaG, -H, -B, -C, -D, -I, -J, and -F), possible regulators, and genes encoding for proteins with related function (i.e. catabolism of 4-hydroxybenzoate, catechol, and meta-cleavage of protocatechuate) were predicted by sequence homology analysis. Most of the Roseobacters studied had putatively a complete protocatechuate branch of the ß-ketoadipate pathway while 11 of them would probably have an incomplete pathway. Thirty-one Roseobacters would be potentially able of transforming 4-hydroxybenzoate to protocatechuate, and 13 of them might transform catechol via ortho-cleavage, the starting reaction of the catechol branch of the ß-ketoadipate pathway. We observed variability in gene organization, with no clear relationship between gene order and Roseobacter taxonomy. Genes were usually organized in several gene clusters. One of the clusters (pcaRIJF) was not reported previously in Roseobacters. The presence of the putative regulator pcaR in these bacteria was also a novel finding. The conserved ORF (chp), encoding for a protein of family DUF849 whose functional role has been proven recently, was detected in 34 genomes. Sequence homology confirmed that proteins encoded by chp corresponded to putative BKACE G4 proteins, which are able to transform ß-ketoadipate. Therefore, most Roseobacters seemed to possess two different enzymes for transforming ß-ketoadipate. We also report two possible regulation mechanisms of gene pobA (encoding for the enzyme transforming 4-hydroxybenzoate to protocatechuate): via PcaQ, the regulator commonly found with pca genes, and via an independent regulator (PobR). The results of this study evidence the relevance of 4-hydroxybenzoate, protocatechuate and ß-ketoadipate degradation pathways in Roseobacters and provide a more complex view of possible regulation mechanisms.

Draft Genome Sequences of Two Isolates of the Roseobacter Group, Sulfitobacter sp. Strains 3SOLIMAR09 and 1FIGIMAR09, from Harbors of Mallorca Island (Mediterranean Sea).

We present the draft genome sequences of two isolates of the Roseobacter lineage, 3SOLIMAR09 and 1FIGIMAR09, which were obtained from harbors of Mallorca Island, Spain, and are affiliated with the Sulfitobacter genus. Both isolates harbor the complete gene set for protocatechuate catabolism and incomplete pathways for several additional monoaromatic compounds.

Proteomics meets blue biotechnology: a wealth of novelties and opportunities.

Blue biotechnology, in which aquatic environments provide the inspiration for various products such as food additives, aquaculture, biosensors, green chemistry, bioenergy, and pharmaceuticals, holds enormous promise. Large-scale efforts to sequence aquatic genomes and metagenomes, as well as campaigns to isolate new organisms and culture-based screenings, are helping to push the boundaries of known organisms. Mass spectrometry-based proteomics can complement 16S gene sequencing in the effort to discover new organisms of potential relevance to blue biotechnology by facilitating the rapid screening of microbial isolates and by providing in depth profiles of the proteomes and metaproteomes of marine organisms, both model cultivable isolates and, more recently, exotic non-cultivable species and communities. Proteomics has already contributed to blue biotechnology by identifying aquatic proteins with potential applications to food fermentation, the textile industry, and biomedical drug development. In this review, we discuss historical developments in blue biotechnology, the current limitations to the known marine biosphere, and the ways in which mass spectrometry can expand that knowledge. We further speculate about directions that research in blue biotechnology will take given current and near-future technological advancements in mass spectrometry.

Pipelines for New Chemicals: a strategy to create new value chains and stimulate innovation-based economic revival in Southern European countries.

Countries of Southern Europe are currently suffering from severe socio-economic pain resulting from high debt levels and austerity measures which constrain investment in innovation-based recovery strategies that are essential for entry into a long-term sustainable period of increasing employment and wealth creation. Young university-educated people are particularly innovative, and hence vital to the development of such strategies, but employment opportunities are poor and many are forced to seek employment that neither profits from their training nor satisfies their justified career expectations, or to emigrate. They are the 'lost generation'. A strategy is proposed here for the creation of Pipelines for New Chemicals, national centre-network partnerships for the discovery-synthesis of new chemicals obtained though harvesting new biological diversity, and their exploitation to develop new medicines, agrochemicals, materials, and other products and applications. The goal is to create new regional motors of economic growth and development, by harnessing the knowledge, motivation and innovation potential of the excellently educated young people of Europe to catalyse the development of new small, medium and large enterprises centred around novel chemicals, and the value chains that will evolve with them, and thereby develop a powerful sector of sustainable growth in employment and social and economic prosperity in Southern Europe.

Draft Genome Sequence of Pseudomonas stutzeri Strain B1SMN1, a Nitrogen-Fixing and Naphthalene-Degrading Strain Isolated from Wastewater.

Pseudomonas stutzeri strain B1SMN1 is a naphthalene-degrading and simultaneously nitrogen-fixing strain isolated from a wastewater sample taken at a lagooning treatment plant in Menorca (Balearic Islands, Spain). Here we report the draft genome sequence of P. stutzeri B1SMN1. It is composed of a chromosome of an estimated size of 5.2 Mb and two plasmids of 44,324 bp and 56,118 bp.

Draft Genome of Pseudomonas stutzeri Strain NF13, a Nitrogen Fixer Isolated from the Galapagos Rift Hydrothermal Vent.

Pseudomonas stutzeri strain NF13 was isolated from a water sample taken at a hydrothermal vent in the Galapagos rift. It was selected for its ability to metabolize sulfur compounds and to grow diazotrophically. Here, we report the first draft genome of a member of genomovar 19 of the species.

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.

Complete genome sequence of the naphthalene-degrading bacterium Pseudomonas stutzeri AN10 (CCUG 29243).

Pseudomonas stutzeri AN10 (CCUG 29243) can be considered a model strain for aerobic naphthalene degradation. We report the complete genome sequence of this bacterium. Its 4.71-Mb chromosome provides insights into other biodegradative capabilities of strain AN10 (i.e., benzoate catabolism) and suggests a high number of horizontal gene transfer events.

Genome sequence of Pseudomonas stutzeri strain JM300 (DSM 10701), a soil isolate and model organism for natural transformation.

Pseudomonas stutzeri strain JM300 (DSM 10701) is a denitrifying soil isolate and a model organism for natural transformation in bacteria. Here we report the first complete genome sequence of JM300, the reference strain of genomovar 8 for the species.

Draft genome of Pseudomonas stutzeri strain ZoBell (CCUG 16156), a marine isolate and model organism for denitrification studies.

Pseudomonas stutzeri strain ZoBell, formerly a strain of Pseudomonas perfectomarina (CCUG 16156 = ATCC 14405), is a model organism for denitrification. It was isolated by ZoBell in 1944 from a marine sample, and here we report the first genome draft of a strain assigned to genomovar 2 of the species P. stutzeri.

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.

Pseudomonas diversity in crude-oil-contaminated intertidal sand samples obtained after the Prestige oil spill.

The Galicia seashore, in northwestern Spain, was one of the shorelines affected by the Prestige oil spill in November 2002. The diversity of autochthonous Pseudomonas populations present at two beaches (Carnota municipality) was analyzed using culture-independent and culture-dependent methods. The first analysis involved the screening of an rpoD gene library. The second involved the isolation of 94 Pseudomonas strains that were able to grow on selective media by direct plating or after serial enrichments on several carbon sources: biphenyl, gentisate, hexadecane, methylnaphthalene, naphthalene, phenanthrene, salicylate, xylene, and succinate. Eight denitrifying Pseudomonas strains were also isolated by their ability to grow anaerobically with nitrate. The calculated coverage index for Pseudomonas species was 89% when clones and isolates were considered together, and there were 29 phylospecies detected. The most abundant were members of the species P. stutzeri, P. putida, P. anguilliseptica, and P. oleovorans. Thirty-one isolates could not be identified at the species level and were considered representatives of 16 putative novel Pseudomonas species. One isolate was considered representative of a novel P. stutzeri genomovar. Concordant results were obtained when the diversities of the cloned DNA library and the cultured strains were compared. The clone library obtained by the rpoD PCR method was a useful tool for evaluating Pseudomonas communities and also for microdiversity studies of Pseudomonas populations.

Anthropogenic perturbations in marine microbial communities.

Human activities impact marine ecosystems at a global scale and all levels of complexity of life. Despite their importance as key players in ecosystem processes, the stress caused to microorganisms has been greatly neglected. This fact is aggravated by difficulties in the analysis of microbial communities and their high diversity, making the definition of patterns difficult. In this review, we discuss the effects of nutrient increase, pollution by organic chemicals and heavy metals and the introduction of antibiotics and pathogens into the environment. Microbial communities respond positively to nutrients and chemical pollution by increasing cell numbers. There are also significant changes in community composition, increases in diversity and high temporal variability. These changes, which evidence the modification of the environmental conditions due to anthropogenic stress, usually alter community functionality, although this aspect has not been explored in depth. Altered microbial communities in human-impacted marine environments can in turn have detrimental effects on human health (i.e. spread of pathogens and antibiotic resistance). New threats to marine ecosystems, i.e. related to climate change, could also have an impact on microbial communities. Therefore, an effort dedicated to analyse the microbial compartment in detail should be made when studying the impact of anthropogenic activities on marine ecosystems.

Halophilic archaea in the human intestinal mucosa.

The human gastrointestinal tract microbiota, despite its key roles in health and disease, remains a diverse, variable and poorly understood entity. Current surveys reveal a multitude of undefined bacterial taxa and a low diversity of methanogenic archaea. In an analysis of the microbiota in colonic mucosal biopsies from patients with inflammatory bowel disease we found 16S rDNA sequences representing a phylogenetically rich diversity of halophilic archaea from the Halobacteriaceae (haloarchaea), including novel phylotypes. As the human colon is not considered a salty environment and haloarchaea are described as extreme halophiles, we evaluated and further discarded the possibility that these sequences originated from pre-colonoscopy saline lavage solutions. Furthermore, aerobic enrichment cultures prepared from a patient biopsy at low salinity (2.5% NaCl) yielded haloarchaeal sequence types. Microscopic observation after fluorescence in situ hybridization provided evidence of the presence of viable archaeal cells in these cultures. These results prove the survival of haloarchaea in the digestive system and suggest that they may be members of the mucosal microbiota, even if present in low numbers in comparison with methanogenic archaea. Investigation of a potential physiological basis of this association may lead to new insights into gastrointestinal health and disease.