Highin vitroactivity of gold and silver nanoparticles from Solanum mammosum L. against SARS-CoV-2 surrogate Phi6 and viral model PhiX174.

The search for new strategies to curb the spread of the SARS-CoV-2 coronavirus, which causes COVID-19, has become a global priority. Various nanomaterials have been proposed as ideal candidates to inactivate the virus; however, because of the high level of biosecurity required for their use, alternative models should be determined. This study aimed to compare the effects of two types of nanomaterials gold (AuNPs) and silver nanoparticles (AgNPs), recognized for their antiviral activity and affinity with the coronavirus spike protein using PhiX174 and enveloped Phi6 bacteriophages as models. To reduce the toxicity of nanoparticles, a species known for its intermediate antiviral activity,Solanum mammosumL. (Sm), was used. NPs prepared with sodium borohydride (NaBH4) functioned as the control. Antiviral activity against PhiX174 and Phi6 was analyzed using its seed, fruit, leaves, and essential oil; the leaves were the most effective on Phi6. Using the aqueous extract of the leaves, AuNPs-Sm of 5.34 ± 2.25 nm and AgNPs-Sm of 15.92 ± 8.03 nm, measured by transmission electron microscopy, were obtained. When comparing NPs with precursors, both gold(III) acetate and silver nitrate were more toxic than their respective NPs (99.99% at 1 mg ml-1). The AuNPs-Sm were less toxic, reaching 99.30% viral inactivation at 1 mg ml-1, unlike the AgNPs-Sm, which reached 99.94% at 0.01 mg ml-1. In addition, cell toxicity was tested in human adenocarcinoma alveolar basal epithelial cells (A549) and human foreskin fibroblasts. Gallic acid was the main component identified in the leaf extract using high performance liquid chromatography with diode array detection (HPLC-DAD). The FT-IR spectra showed the presence of a large proportion of polyphenolic compounds, and the antioxidant analysis confirmed the antiradical activity. The control NPs showed less antiviral activity than the AuNPs-Sm and AgNPs-Sm, which was statistically significant; this demonstrates that both theS. mammosumextract and its corresponding NPs have a greater antiviral effect on the surrogate Phi bacteriophage, which is an appropriate model for studying SARS-CoV-2.

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.

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.

Effective Elimination and Biodegradation of Polycyclic Aromatic Hydrocarbons from Seawater through the Formation of Magnetic Microfibres.

Supramolecular aggregates formed between polycyclic aromatic hydrocarbons and either naphthalene or perylene-derived diimides have been anchored in magnetite magnetic nanoparticles. The high affinity and stability of these aggregates allow them to capture and confine these extremely carcinogenic contaminants in a reduced space. In some cases, the high cohesion of these aggregates leads to the formation of magnetic microfibres of several microns in length, which can be isolated from the solution by the direct action of a magnet. Here we show a practical application of bioremediation aimed at the environmental decontamination of naphthalene, a very profuse contaminant, based on the uptake, sequestration, and acceleration of the biodegradation of the formed supramolecular aggregate, by the direct action of a bacterium of the lineage Roseobacter (biocompatible with nanostructured receptors and very widespread in marine environments) without providing more toxicity to the environment.

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.

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.

Stutzerimonas decontaminans sp. nov. isolated from marine polluted sediments.

Strains 19SMN4T and ST27MN3 were isolated from marine sediments after enrichment with 2-methylnaphthalene and were classified as Pseudomonas stutzeri genomovar 4. Four other strains, BG 2, HT20, HT24, and A7, were isolated from sulphide-oxidizing bioreactors or activated sludge affiliated with the same clade in the 16S rRNA phylogenetic tree. P. stutzeri has been recently reclassified as a new genus, Stutzerimonas, and a preliminary analysis indicated that the strains in this study were distinct from any classified Stutzerimonas and are considered representatives of phylogenomic species 4 (pgs4). Strains 19SMN4T and ST27MN3 were extensively characterized with phenotypic, chemotaxonomic, genomic and phylogenomic data. Strain 19SMN4T had a well-characterized naphthalene degradative plasmid that has been compared with other plasmids, while in strain ST27MN3, the naphthalene degradative genes were detected in the chromosome sequence. Phylogenomic analysis of the core gene sequences showed that strains 19SMN4T and ST27MN3 shared 3,995 genes and were closely related to members of the species "Stutzerimonas songnenensis" and Stutzerimonas perfectomarina, as well as to the Stutzerimonas phylogenomic species, pgs9, pgs16 and pgs24. The aggregate average nucleotide identity (ANI) indicated that strains 19SMN4T and ST27MN3 belonged to the same genomic species, whereas the genomic indices with their closest-related type strains were below the accepted species threshold (95 %). We therefore conclude that strains 19SMN4T and ST27MN3 represent a novel species of Stutzerimonas, for which the name Stutzerimonas decontaminans is proposed; the type strain is 19SMN4T (=CCUG44593T = DSM6084T = LMG18521T).

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.

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.

A multi-OMIC characterisation of biodegradation and microbial community succession within the PET plastisphere.

BACKGROUND: Plastics now pollute marine environments across the globe. On entering these environments, plastics are rapidly colonised by a diverse community of microorganisms termed the plastisphere. Members of the plastisphere have a myriad of diverse functions typically found in any biofilm but, additionally, a number of marine plastisphere studies have claimed the presence of plastic-biodegrading organisms, although with little mechanistic verification. Here, we obtained a microbial community from marine plastic debris and analysed the community succession across 6 weeks of incubation with different polyethylene terephthalate (PET) products as the sole carbon source, and further characterised the mechanisms involved in PET degradation by two bacterial isolates from the plastisphere. RESULTS: We found that all communities differed significantly from the inoculum and were dominated by Gammaproteobacteria, i.e. Alteromonadaceae and Thalassospiraceae at early time points, Alcanivoraceae at later time points and Vibrionaceae throughout. The large number of encoded enzymes involved in PET degradation found in predicted metagenomes and the observation of polymer oxidation by FTIR analyses both suggested PET degradation was occurring. However, we were unable to detect intermediates of PET hydrolysis with metabolomic analyses, which may be attributed to their rapid depletion by the complex community. To further confirm the PET biodegrading potential within the plastisphere of marine plastic debris, we used a combined proteogenomic and metabolomic approach to characterise amorphous PET degradation by two novel marine isolates, Thioclava sp. BHET1 and Bacillus sp. BHET2. The identification of PET hydrolytic intermediates by metabolomics confirmed that both isolates were able to degrade PET. High-throughput proteomics revealed that whilst Thioclava sp. BHET1 used the degradation pathway identified in terrestrial environment counterparts, these were absent in Bacillus sp. BHET2, indicating that either the enzymes used by this bacterium share little homology with those characterised previously, or that this bacterium uses a novel pathway for PET degradation. CONCLUSIONS: Overall, the results of our multi-OMIC characterisation of PET degradation provide a significant step forwards in our understanding of marine plastic degradation by bacterial isolates and communities and evidences the biodegrading potential extant in the plastisphere of marine plastic debris. Video abstract.

Pili allow dominant marine cyanobacteria to avoid sinking and evade predation.

How oligotrophic marine cyanobacteria position themselves in the water column is currently unknown. The current paradigm is that these organisms avoid sinking due to their reduced size and passive drift within currents. Here, we show that one in four picocyanobacteria encode a type IV pilus which allows these organisms to increase drag and remain suspended at optimal positions in the water column, as well as evade predation by grazers. The evolution of this sophisticated floatation mechanism in these purely planktonic streamlined microorganisms has important implications for our current understanding of microbial distribution in the oceans and predator-prey interactions which ultimately will need incorporating into future models of marine carbon flux dynamics.

Determination of Antioxidant Activity by Oxygen Radical Absorbance Capacity (ORAC-FL), Cellular Antioxidant Activity (CAA), Electrochemical and Microbiological Analyses of Silver Nanoparticles Using the Aqueous Leaf Extract of Solanum mammosum L.

PURPOSE: The importance of studying polyphenolic compounds as natural antioxidants has encouraged the search for new methods of analysis that are quick and simple. The synthesis of silver nanoparticles (AgNPs) using plant extracts has been presented as an alternative to determine the total polyphenolic content and its antioxidant activity. METHODS: In this study, aqueous leaf extract of Solanum mammosum, a species of plant endemic to South America, was used to produce AgNPs. The technique of oxygen radical absorption capacity using fluorescein (ORAC-FL) was used to measure antioxidant activity. The oxidation of the 2´,7´-dichlorodihydrofluorescein diacetate (DCFH2-DA) as fluorescent probe was used to measure cellular antioxidant activity (CAA). Electrochemical behavior was also examined using differential pulse voltammetry (DPV) and cyclic voltammetry (CV). Total polyphenolic content (TPH) was analyzed using the Folin-Ciocalteu method, and the major polyphenolic compound was analyzed by high performance liquid chromatography with diode array detector (HPLC/DAD). Finally, a microbial analysis was conducted using Escherichia coli and Bacillus sp. RESULTS: The average size of nanoparticles was 5.2 ± 2.3 nm measured by high-resolution transmission electron microscopy (HR-TEM). The antioxidant activity measured by ORAC-FL in the extract and nanoparticles were 3944 ± 112 and 637.5 ± 14.8 µM ET/g of sample, respectively. Cellular antioxidant activity was 14.7 ± 0.2 for the aqueous extract and 12.5 ± 0.2 for the nanoparticles. The electrochemical index (EI) was 402 µA/V for the extract and 324 µA/V for the nanoparticles. Total polyphenolic content was 826.6 ± 20.9 and 139.7 ± 20.9 mg EGA/100 g of sample. Gallic acid was the main polyphenolic compound present in the leaf extract. Microbiological analysis revealed that although leaf extract was not toxic for Escherichia coli and Bacillus sp., minor toxic activity for AgNPs was detected for both strains. CONCLUSION: It is concluded that the aqueous extract of the leaves of S. mammosum contains nontoxic antioxidant compounds capable of producing AgNPs. The methods using AgNPs can be used as a fast analytical tool to monitor the presence of water-soluble polyphenolic compounds from plant origin. Analysis and detection of new antioxidants from plant extracts may be potentially applicable in biomedicine.

Biology of Pseudomonas stutzeri.

Pseudomonas stutzeri is a nonfluorescent denitrifying bacterium widely distributed in the environment, and it has also been isolated as an opportunistic pathogen from humans. Over the past 15 years, much progress has been made in elucidating the taxonomy of this diverse taxonomical group, demonstrating the clonality of its populations. The species has received much attention because of its particular metabolic properties: it has been proposed as a model organism for denitrification studies; many strains have natural transformation properties, making it relevant for study of the transfer of genes in the environment; several strains are able to fix dinitrogen; and others participate in the degradation of pollutants or interact with toxic metals. This review considers the history of the discovery, nomenclatural changes, and early studies, together with the relevant biological and ecological properties, of P. stutzeri.

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).

Evaluation of the ability of erythromycin-resistant and -susceptible pharyngeal group A Streptococcus isolates from Spain to enter and persist in human keratinocytes.

The macrolide-resistance rate among group A Streptococcus (GAS) isolates has increased in some European countries. To investigate the reasons for this increase, the ability of 60 erythromycin-resistant and 61 erythromycin-susceptible, genetically unrelated, pharyngeal GAS isolates from Spain to enter and persist within human keratinocytes was evaluated. It was observed that erythromycin resistance was associated with the presence of prtF1, a gene related to invasiveness, whereas no association was observed with the ability to enter human keratinocytes. However, the ability to enter human keratinocytes was not associated with the presence of prtF1 or with the emm type, suggesting that interaction with keratinocytes depends on several characteristics of the isolate. Almost all strains (95.9 %) were capable of persisting within human keratinocytes. However, most of them (91.7 %) exhibited a decline in viability over time. Interestingly, the ability to persist within keratinocytes protected from the action of the beta-lactams was higher among erythromycin-resistant isolates and correlated with their ability to avoid the induction of cellular lysis. These observations suggest that if the carrier state results from intracellular GAS survival, the association between erythromycin resistance and intracellular persistence may represent a serious problem for the eradication of these isolates.