Genetic basis for the biodegradation of a polyether-polyurethane-acrylic copolymer by a landfill microbial community inferred by metagenomic deconvolution analysis.

Plastic accumulation in the world amounts to approximately 8300 million tons. Polyurethanes (PU) account for 7.7 % of total plastics production worldwide, and their diverse chemical composition makes them highly recalcitrant to biodegradation. Several works have reported polyurethane-degrading microbial communities. However, it is still necessary to learn more about the chemical, biochemical, and genetic bases linked to the polyurethanolytic phenotype and the microbial taxonomic determinants responsible for metabolizing the PU polymer and its associated chemical additives. To shed light on this problem, we applied physical, chemical, biochemical, metagenomic, and bioinformatic analyses to explore the biodegradation capability and related biochemical and genetic determinants of the BP6 microbial community that can grow in PolyLack, a commercial coating containing a polyether polyurethane acrylate (PE-PU-A) copolymer and several additives, as sole carbon source. We observed complete additives (isopropanol, N-methyl-2-pyrrolidone, 2-butoxyethanol, alkyl glycol ethers) biodegradation and the appearance of released polymer components (toluene diisocyanate (TDI) and methylene diphenyl diisocyanate (MDI) derivatives), and multiple degradation products since early cultivation times. The Hi-C metagenomic analysis identified a complex microbiome with 35 deconvolved Metagenome-Assembled Genomes (MAGs) - several new species - and biodegradation markers that suggest the coexistence of hydrolytic, oxidative, and reductive metabolic strategies for degrading the additives and the PU copolymer. This work also provides evidence of the metabolic capability the BP6 community has for biodegrading polyether polyurethane foams. Based on these analyses, we propose a novel metabolic pathway for 4,4'-methylenedianiline (MDA), an initial biodegradation intermediate of MDI-based PU, encoded in the complex BP6 community metagenome and suggest that this community is a potential biotechnological tool for PU bio-recycling.

Concerted action of extracellular and cytoplasmic esterase and urethane-cleaving activities during Impranil biodegradation by Alicycliphilus denitrificans BQ1.

The concerted action of commercial esterases, proteases and amidases has been demonstrated to be relevant in polyurethane (PU) degradation by in vitro experiments. However, the spatial and temporal dynamics of these activities during PU biodegradation by PU-degrading bacteria have not been addressed. Here, we examined the capability of Alicycliphilus denitrificans BQ1 to biodegrade the polyester (PS)-PU Impranil, analyzed the temporal and spatial coordination between the extracellular and cytoplasmic esterase and urethane-cleaving activities, and their independent and combined effects on Impranil biodegradation. A. denitrificans BQ1 grew in Impranil, and its clearing was correlated with the cleavage of ester and urethane groups since early times, with decrements of some Impranil compounds and the appearance of biodegradation products. While extracellular esterase was active at early times with its maximum at 18 h, urethanase appeared at this time and increased up to the end of the analysis (48 h), with the cytoplasmic activities behaving similarly but with lower levels than the extracellular ones. Both enzymatic activities exhibited distinct substrate specificity depending on their cellular localization and cultivation times, suggesting they cleave differentially located groups. As the urethane cleavage occurred since early times, when no urethane-cleaving activity was detected, different proteins should be acting at early and late times. In vitro experiments with independent or combined cellular protein fractions supported the previous deduction and confirmed the concerted action of extracellular and cytoplasmic esterase and urethane-cleaving activities. A two-stage process for Impranil degradation by A. denitrificans BQ1 is proposed.

Exploring the polyurethanolytic activity and microbial composition of landfill microbial communities.

The microbial composition of polyurethane degrading communities has been barely addressed, and it is unknown if microenvironmental conditions modify its composition, affecting its biodegradative capacity. The polyurethanolytic activity and taxonomic composition of five microbial communities, selected by enrichment in the polyether-polyurethane-acrylic (PE-PU-A) coating PolyLack®, from deteriorated PU foams collected at different microenvironments in a municipal landfill (El Bordo Poniente, BP) were explored. All BP communities grew similarly in PolyLack® as the sole carbon source, although BP1, BP4, and BP5 showed better performance than BP2 and BP7. FTIR spectroscopy showed that ester, urethane, ether, aromatic and aliphatic groups, and the acrylate component were targets of the biodegradative activity. Extracellular esterase activity was higher at 5 days of cultivation and decreased at 21 days, while urease activity showed the opposite. Microbial composition analysis, assessed by 16S rDNA V3 region PCR-DGGE, revealed a preponderance of Rhizobiales and Micrococcales. The reported PU-degrading genera Paracoccus, Acinetobacter, and Pseudomonas were identified. In contrast, Advenella, Bordetella, Microbacterium, Castellaniella, and Populibacterium, some of them xenobiotics degraders, can be considered potentially PU-degrading genera. Correspondence analysis identified independent groups for all communities, except the BP4 and BP5. Although partial taxonomic redundancy was detected, unique OTUs were identified, e.g., three members of the Weeksellaceae family were present only in the BP4/BP5 group. These results suggest that the microenvironmental conditions where the landfill microbial communities were collected shaped their taxonomical composition, impacting their PE-PU biodegradative capacities. These BP communities represent valuable biological material for the treatment of PU waste and other xenobiotics. KEY POINTS: • Landfill microbial communities display slightly different capacities for growing in polyether-polyurethane-acrylic. • Ester, urethane, ether, aromatic, aliphatic, and acrylate groups were attacked. • Esterase activity was more significant at early culture times while urease activity at latter. • Landfill microenvironments shape partial taxonomical redundancy in the communities. • Best communities' performance seems to be related to unique members' composition.

Biodegradation of polyacrylic and polyester polyurethane coatings by enriched microbial communities.

Microbial communities are more effective in degrading natural polymers and xenobiotics than pure cultures. Biodegradation of polyacrylic and polyurethane polymers by bacterial and fungal strains has been addressed, but limited information about their biodegradation by microbial communities exists. The aim of this work was to evaluate the ability of three enriched microbial communities (BP1h, BP3h, and BP7h), selected from deteriorated foam pieces collected in a landfill, to biodegrade the polyacrylic component of the 2K-PU coating Bayhydrol® A2470 and the polyester polyurethane coating NeoRez™ R-9637. Two communities were further selected to quantify extracellular esterase, protease, and urease activities, to identify their taxonomic composition, and to analyze the ability of their isolated members to grow in those polymers. The growth of the three communities was larger in polyester polyurethane than in polyacrylic and their biodegradative activities affected ester, urethane, ether, aromatic, and aliphatic groups of the compounds present in the coatings. From all the communities growing in polyacrylic or in polyester polyurethane, two and five different types of colonies were isolated, respectively. In polyacrylic, extracellular esterase and protease activities were at their maximum level at 7 days of culture, whereas in polyester polyurethane, protease and urease were greatest at 21 days. All the isolated community members were identified as xenobiotics degraders. The complete communities grew better in media with the polymers than the isolated members. This is one of the few studies reporting biodegradation of synthetic polymers by microbial communities and serves as basis for developing synthetic consortia with enhanced degradative abilities.

Degradation of Recalcitrant Polyurethane and Xenobiotic Additives by a Selected Landfill Microbial Community and Its Biodegradative Potential Revealed by Proximity Ligation-Based Metagenomic Analysis.

Polyurethanes (PU) are the sixth most produced plastics with around 18-million tons in 2016, but since they are not recyclable, they are burned or landfilled, generating damage to human health and ecosystems. To elucidate the mechanisms that landfill microbial communities perform to attack recalcitrant PU plastics, we studied the degradative activity of a mixed microbial culture, selected from a municipal landfill by its capability to grow in a water PU dispersion (WPUD) as the only carbon source, as a model for the BP8 landfill microbial community. The WPUD contains a polyether-polyurethane-acrylate (PE-PU-A) copolymer and xenobiotic additives (N-methylpyrrolidone, isopropanol and glycol ethers). To identify the changes that the BP8 microbial community culture generates to the WPUD additives and copolymer, we performed chemical and physical analyses of the biodegradation process during 25 days of cultivation. These analyses included Nuclear magnetic resonance, Fourier transform infrared spectroscopy, Thermogravimetry, Differential scanning calorimetry, Gel permeation chromatography, and Gas chromatography coupled to mass spectrometry techniques. Moreover, for revealing the BP8 community structure and its genetically encoded potential biodegradative capability we also performed a proximity ligation-based metagenomic analysis. The additives present in the WPUD were consumed early whereas the copolymer was cleaved throughout the 25-days of incubation. The analysis of the biodegradation process and the identified biodegradation products showed that BP8 cleaves esters, C-C, and the recalcitrant aromatic urethanes and ether groups by hydrolytic and oxidative mechanisms, both in the soft and the hard segments of the copolymer. The proximity ligation-based metagenomic analysis allowed the reconstruction of five genomes, three of them from novel species. In the metagenome, genes encoding known enzymes, and putative enzymes and metabolic pathways accounting for the biodegradative activity of the BP8 community over the additives and PE-PU-A copolymer were identified. This is the first study revealing the genetically encoded potential biodegradative capability of a microbial community selected from a landfill, that thrives within a WPUD system and shows potential for bioremediation of polyurethane- and xenobiotic additives-contamitated sites.

Novel Metabolic Pathway for N-Methylpyrrolidone Degradation in Alicycliphilus sp. Strain BQ1.

The molecular mechanisms underlying the biodegradation of N-methylpyrrolidone (NMP), a widely used industrial solvent that produces skin irritation in humans and is teratogenic in rats, are unknown. Alicycliphilus sp. strain BQ1 degrades NMP. By studying a transposon-tagged mutant unable to degrade NMP, we identified a six-gene cluster (nmpABCDEF) that is transcribed as a polycistronic mRNA and encodes enzymes involved in NMP biodegradation. nmpA and the transposon-affected gene nmpB encode an N-methylhydantoin amidohydrolase that transforms NMP to γ-N-methylaminobutyric acid; this is metabolized by an amino acid oxidase (NMPC), either by demethylation to produce γ-aminobutyric acid (GABA) or by deamination to produce succinate semialdehyde (SSA). If GABA is produced, the activity of a GABA aminotransferase (GABA-AT), not encoded in the nmp gene cluster, is needed to generate SSA. SSA is transformed by a succinate semialdehyde dehydrogenase (SSDH) (NMPF) to succinate, which enters the Krebs cycle. The abilities to consume NMP and to utilize it for growth were complemented in the transposon-tagged mutant by use of the nmpABCD genes. Similarly, Escherichia coli MG1655, which has two SSDHs but is unable to grow in NMP, acquired these abilities after functional complementation with these genes. In wild-type (wt) BQ1 cells growing in NMP, GABA was not detected, but SSA was present at double the amount found in cells growing in Luria-Bertani medium (LB), suggesting that GABA is not an intermediate in this pathway. Moreover, E. coli GABA-AT deletion mutants complemented with nmpABCD genes retained the ability to grow in NMP, supporting the possibility that γ-N-methylaminobutyric acid is deaminated to SSA instead of being demethylated to GABA.IMPORTANCEN-Methylpyrrolidone is a cyclic amide reported to be biodegradable. However, the metabolic pathway and enzymatic activities for degrading NMP are unknown. By developing molecular biology techniques for Alicycliphilus sp. strain BQ1, an environmental bacterium able to grow in NMP, we identified a six-gene cluster encoding enzymatic activities involved in NMP degradation. These findings set the basis for the study of new enzymatic activities and for the development of biotechnological processes with potential applications in bioremediation.

Biodegradative Activities of Selected Environmental Fungi on a Polyester Polyurethane Varnish and Polyether Polyurethane Foams.

UNLABELLED: Polyurethane (PU) is widely used in many aspects of modern life because of its versatility and resistance. However, PU waste disposal generates large problems, since it is slowly degraded, there are limited recycling processes, and its destruction may generate toxic compounds. In this work, we isolated fungal strains able to grow in mineral medium with a polyester PU (PS-PU; Impranil DLN) or a polyether PU (PE-PU; Poly Lack) varnish as the only carbon source. Of the eight best Impranil-degrading strains, the six best degraders belonged to the Cladosporium cladosporioides complex, including the species C. pseudocladosporioides, C. tenuissimum, C. asperulatum, and C. montecillanum, and the two others were identified as Aspergillus fumigatus and Penicillium chrysogenum The best Impranil degrader, C. pseudocladosporioides strain T1.PL.1, degraded up to 87% after 14 days of incubation. Fourier transform infrared (FTIR) spectroscopy analysis of Impranil degradation by this strain showed a loss of carbonyl groups (1,729 cm(-1)) and N-H bonds (1,540 and 1,261 cm(-1)), and gas chromatography-mass spectrometry (GC-MS) analysis showed a decrease in ester compounds and increase in alcohols and hexane diisocyanate, indicating the hydrolysis of ester and urethane bonds. Extracellular esterase and low urease, but not protease activities were detected at 7 and 14 days of culture in Impranil. The best eight Impranil-degrading fungi were also able to degrade solid foams of the highly recalcitrant PE-PU type to different extents, with the highest levels generating up to 65% of dry-weight losses not previously reported. Scanning electron microscopy (SEM) analysis of fungus-treated foams showed melted and thinner cell wall structures than the non-fungus-treated ones, demonstrating fungal biodegradative action on PE-PU. IMPORTANCE: Polyurethane waste disposal has become a serious problem. In this work, fungal strains able to efficiently degrade different types of polyurethanes are reported, and their biodegradative activity was studied by different experimental approaches. Varnish biodegradation analyses showed that fungi were able to break down the polymer in some of their precursors, offering the possibility that they may be recovered and used for new polyurethane synthesis. Also, the levels of degradation of solid polyether polyurethane foams reported in this work have never been observed previously. Isolation of efficient polyurethane-degrading microorganisms and delving into the mechanisms they used to degrade the polymer provide the basis for the development of biotechnological processes for polyurethane biodegradation and recycling.

Protein phosphorylation regulates in vitro spinach chloroplast petD mRNA 3'-untranslated region stability, processing, and degradation.

RNA-binding proteins (RNPs) participate in diverse processes of mRNA metabolism, and phosphorylation changes their binding properties. In spinach chloroplasts, 24RNP and 28RNP are associated with polynucleotide posphorylase forming a complex on charge of pre-mRNA 3'-end maturation. Here, we tested the hypothesis that the phosphorylation status of 24RNP and 28RNP, present in a spinach chloroplast mRNA 3'-UTR processing extract (CPE), controls the transition between petD precursor stabilization, 3'-UTR processing, and RNA degradation in vitro. The CPE processed or stabilized petD precursor depending on the ATP concentration present in an in vitro 3'-UTR processing (IVP) assay. These effects were also observed when ATP was pre-incubated and removed before the IVP assay. Moreover, a dephosphorylated (DP)-CPE degraded petD precursor and recovered 3'-UTR processing or stabilization activities in an ATP concentration dependent manner. To determine the role 24/28RNP plays in regulating these processes a 24/28RNP-depleted (Δ24/28)CPE was generated. The Δ24/28CPE degraded the petD precursor, but when it was reconstituted with recombinant non-phosphorylated (NP)-24RNP or NP-28RNP, the precursor was stabilized, whereas when Δ24/28CPE was reconstituted with phosphorylated (P)-24RNP or P-28RNP, it recovered 3'-UTR processing, indicating that 24RNP or 28RNP is needed to stabilize the precursor, have a redundant role, and their phosphorylation status regulates the transition between precursor stabilization and 3'-UTR processing. A DP-Δ24/28CPE reconstituted or not with NP-24/28RNP degraded petD precursor. Pre-incubation of DP-Δ24/28CPE with NP-24/28RNP plus 0.03 mM ATP recovered 3'-UTR processing activity, and its reconstitution with P-24/28RNP stabilized the precursor. However, pre-incubation of DP-Δ24/28CPE with 0.03 mM ATP, and further reconstitution with NP-24/28RNP or P-24/28RNP produced precursor stability instead of RNA degradation, and RNA processing instead of precursor stability, respectively. Moreover, in vitro phosphorylation of CPE showed that 24RNP, 28RNP, and other proteins may be phosphorylated. Altogether, these results reveal that phosphorylation of 24RNP, 28RNP, and other unidentified CPE proteins mediates the in vitro interplay between petD precursor stability, 3'-UTR processing, and degradation, and support the idea that protein phosphorylation plays an important role in regulating mRNA metabolism in chloroplast.

Purification of an Arabidopsis chloroplast extract with in vitro RNA processing activity on psbA and petD 3'-untranslated regions.

The mature 3'-end of many chloroplast mRNAs is generated by the processing of the 3'-untranslated region (3'-UTR), which is a mechanism that involves the removal of a segment located downstream an inverted repeat sequence that forms a stem-loop structure. Nuclear-encoded chloroplast RNA binding proteins associate with the stem-loop to process the 3'-UTR or to influence mRNA stability. A spinach chloroplast processing extract (CPE) has been previously generated and used to in vitro dissect the biochemical mechanism underlying 3'-UTR processing. Being Arabidopsis thaliana an important genetic model, the development of a CPE allowing to correlate 3'-UTR processing activity with genes encoding proteins involved in this process, would be of great relevance. Here, we developed a purification protocol that generated an Arabidopsis CPE able to correctly process a psbA 3'-UTR precursor. By UV crosslinking, we characterized the protein patterns generated by the interaction of RNA binding proteins with Arabidopsis psbA and petD 3'-UTRs, finding that each 3'-UTR bound specific proteins. By testing whether Arabidopsis CPE proteins were able to bind spinach ortholog 3'-UTRs, we also found they were bound by specific proteins. When Arabidopsis CPE 3'-UTR processing activity on ortholog spinach 3'-UTRs was assessed, stable products appeared: for psbA, a smaller size product than the expected mature 3'-end, and for petD, low amounts of the expected product plus several others of smaller sizes. These results suggest that the 3'-UTR processing mechanism of these chloroplast mRNAs might be partially conserved in Arabidopsis and spinach.

Protein-mediated protection as the predominant mechanism for defining processed mRNA termini in land plant chloroplasts.

Most chloroplast mRNAs are processed from larger precursors. Several mechanisms have been proposed to mediate these processing events, including site-specific cleavage and the stalling of exonucleases by RNA structures. A protein barrier mechanism was proposed based on analysis of the pentatricopeptide repeat (PPR) protein PPR10: PPR10 binds two intercistronic regions and impedes 5'- and 3'-exonucleases, resulting in processed RNAs with PPR10 bound at the 5'- or 3'-end. In this study, we provide evidence that protein barriers are the predominant means for defining processed mRNA termini in chloroplasts. First, we map additional RNA termini whose arrangement suggests biogenesis via a PPR10-like mechanism. Second, we show that the PPR protein HCF152 binds to the immediate 5'- or 3'-termini of transcripts that require HCF152 for their accumulation, providing evidence that HCF152 defines RNA termini by blocking exonucleases. Finally, we build on the observation that the PPR10 and HCF152 binding sites accumulate as small chloroplast RNAs to infer binding sites of other PPR proteins. We show that most processed mRNA termini are represented by small RNAs whose sequences are highly conserved. We suggest that each such small RNA is the footprint of a PPR-like protein that protects the adjacent RNA from degradation.

Cytokinin promotes catalase and ascorbate peroxidase activities and preserves the chloroplast integrity during dark-senescence.

Increased oxidative stress displayed during dark-senescence of wheat leaves (Triticum aestivum L.) is caused not only by the increased levels of radicals but also by a loss of antioxidant capacity. Mature leaves were incubated in 6-benzylaminopurine (BAP 10(-4)M) or water (control) during 6d in the dark. The senescence-delaying effect of BAP was associated with the retention of the chloroplast structure, 60% of the initial content of chlorophyll (Chl) and 77% of the initial content of protein. BAP reduced the degradation of the light-harvesting chlorophyll a/b binding protein (LHCP-2), and the large (LSU) and small subunits (SSU) of Rubisco. Our results indicated that the presence of the NADPH:protochlorophyllide oxidoreductase (POR, EC.1.6.99.1) was not promoted by the cytokinin, leading to the conclusion that BAP maintains the level of Chl, preventing its degradation, rather than inducing Chl biosynthesis. The internal structure of chloroplasts was maintained in BAP-treated leaves for up to 6d, with well-organized grana thylakoids and small plastoglobuli; in contrast, chloroplasts of control leaves deteriorated rapidly from day 4 with disorganized internal membranes, and more and larger plastoglobuli. BAP increased the activities of catalase (CAT, EC 1.11.1.6) and ascorbate peroxidase (APX, EC 1.11.1.11) and reduced the level of H(2)O(2) in the delayed-senescence tissue. The present research indicates that BAP reduces levels of reactive oxygen species (ROS), and enhances the activity of antioxidant enzymes (CAT, APX). Our results suggest that BAP protects the cell membranes and the photosynthetic machinery from oxidative damage during delay of senescence in the dark.

Rubisco activase chaperone activity is regulated by a post-translational mechanism in maize leaves.

Rubisco activase (RCA) is a molecular chaperone present in maize as 43 kDa and 41 kDa polypeptides. They are encoded by two different genes comprising an identical ORF that corresponds to the 43 kDa RCA polypeptide, and their transcripts do not show putative splicing sites. To determine the origin of the 41 kDa polypeptide, leaf poly A(+) mRNA was in vitro translated. Results demonstrated de novo synthesis only for the 43 kDa RCA. Antibodies developed against peptides from either the carboxy- or the amino-terminal end of 43 kDa RCA showed by western blot that the 43 kDa polypeptide amino-terminal region is missing in the 41 kDa polypeptide, whereas both RCA polypeptides shared the carboxy-end region. Regulation of RCA polypeptide ratios was determined in plant leaves at different developmental stages and under stressing environmental conditions. Increased levels of 43/41 kDa RCA ratio were found in leaves under low light exposure, whereas this ratio declined under water stress. Measurements of chaperone activity either on each RCA polypeptide alone or in a mixture showed the functional relevance of different 43/41 kDa RCA polypeptide ratios. Greater chaperone activity was found for the 41 kDa than for the 43 kDa polypeptide. Taken together, these results indicate that 41 kDa RCA polypeptide formation is regulated by limited proteolysis of the 43 kDa RCA at its amino-terminal region. This pathway is sensitive to developmental and environmental signals, and seems to play a relevant function during plant stress.