Widespread and active piezotolerant microorganisms mediate phenolic compound degradation under high hydrostatic pressure in hadal trenches.

Phenolic compounds, as well as other aromatic compounds, have been reported to be abundant in hadal trenches. Although high-throughput sequencing studies have hinted at the potential of hadal microbes to degrade these compounds, direct microbiological, genetic and biochemical evidence under in situ pressures remain absent. Here, a microbial consortium and a pure culture of Pseudomonas, newly isolated from Mariana Trench sediments, efficiently degraded phenol under pressures up to 70 and 60 MPa, respectively, with concomitant increase in biomass. By analyzing a high-pressure (70 MPa) culture metatranscriptome, not only was the entire range of metabolic processes under high pressure generated, but also genes encoding complete phenol degradation via ortho- and meta-cleavage pathways were revealed. The isolate of Pseudomonas also contained genes encoding the complete degradation pathway. Six transcribed genes (dmpKLMNOPsed) were functionally identified to encode a multicomponent hydroxylase catalyzing the hydroxylation of phenol and its methylated derivatives by heterogeneous expression. In addition, key catabolic genes identified in the metatranscriptome of the high-pressure cultures and genomes of bacterial isolates were found to be all widely distributed in 22 published hadal microbial metagenomes. At microbiological, genetic, bioinformatics, and biochemical levels, this study found that microorganisms widely found in hadal trenches were able to effectively drive phenolic compound degradation under high hydrostatic pressures. This information will bridge a knowledge gap concerning the microbial aromatics degradation within hadal trenches. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-024-00224-2.

Semi-rational design of nitroarene dioxygenase for catalytic ability toward 2,4-dichloronitrobenzene.

Rieske non-heme dioxygenase family enzymes play an important role in the aerobic biodegradation of nitroaromatic pollutants, but no active dioxygenases are available in nature for initial reactions in the degradation of many refractory pollutants like 2,4-dichloronitrobenzene (24DCNB). Here, we report the engineering of hotspots in 2,3-dichloronitrobenzene dioxygenase from Diaphorobacter sp. strain JS3051, achieved through molecular dynamic simulation analysis and site-directed mutagenesis, with the aim of enhancing its catalytic activity toward 24DCNB. The computationally predicted activity scores were largely consistent with the detected activities in wet experiments. Among them, the two most beneficial mutations (E204M and M248I) were obtained, and the combined mutant reached up to a 62-fold increase in activity toward 24DCNB, generating a single product, 3,5-dichlorocatechol, which is a naturally occurring compound. In silico analysis confirmed that residue 204 affected the substrate preference for meta-substituted nitroarenes, while residue 248 may influence substrate preference by interaction with residue 295. Overall, this study provides a framework for manipulating nitroarene dioxygenases using computational methods to address various nitroarene contamination problems.IMPORTANCEAs a result of human activities, various nitroaromatic pollutants continue to enter the biosphere with poor degradability, and dioxygenation is an important kickoff step to remove toxic nitro-groups and convert them into degradable products. The biodegradation of many nitroarenes has been reported over the decades; however, many others still lack corresponding enzymes to initiate their degradation. Although rieske non-heme dioxygenase family enzymes play extraordinarily important roles in the aerobic biodegradation of various nitroaromatic pollutants, prediction of their substrate specificity is difficult. This work greatly improved the catalytic activity of dioxygenase against 2,4-dichloronitrobenzene by computer-aided semi-rational design, paving a new way for the evolution strategy of nitroarene dioxygenase. This study highlights the potential for using enzyme structure-function information with computational pre-screening methods to rapidly tailor the catalytic functions of enzymes toward poorly biodegradable contaminants.

Combined effects of azoxystrobin and oxytetracycline on rhizosphere microbiota of Arabidopsis thaliana.

The rhizosphere is one of the key determinants of plant health and productivity. Mixtures of pesticides are commonly used in intensified agriculture. However, the combined mechanisms underlying their impacts on soil microbiota remain unknown. The present study revealed that the rhizosphere microbiota was more sensitive to azoxystrobin and oxytetracycline, two commonly used pesticides, than was the microbiota present in bulk soil. Moreover, the rhizosphere microbiota enhanced network complexity and stability and increased carbohydrate metabolism and xenobiotic biodegradation as well as the expression of metabolic genes involved in defence against pesticide stress. Co-exposure to azoxystrobin and oxytetracycline had antagonistic effects on Arabidopsis thaliana growth and soil microbial variation by recruiting organic-degrading bacteria and regulating ABC transporters to reduce pesticide uptake. Our study explored the composition and function of soil microorganisms through amplicon sequencing and metagenomic approaches, providing comprehensive insights into the synergistic effect of plants and rhizosphere microbiota on pesticides and contributing to our understanding of the ecological risks associated with pesticide use.

The unique salt bridge network in GlacPETase: a key to its stability.

The extensive accumulation of polyethylene terephthalate (PET) has become a critical environmental issue. PET hydrolases can break down PET into its building blocks. Recently, we identified a glacial PET hydrolase GlacPETase sharing less than 31% amino acid identity with any known PET hydrolases. In this study, the crystal structure of GlacPETase was determined at 1.8 Å resolution, revealing unique structural features including a distinctive N-terminal disulfide bond and a specific salt bridge network. Site-directed mutagenesis demonstrated that the disruption of the N-terminal disulfide bond did not reduce GlacPETase's thermostability or its catalytic activity on PET. However, mutations in the salt bridges resulted in changes in melting temperature ranging from -8°C to +2°C and the activity on PET ranging from 17.5% to 145.5% compared to the wild type. Molecular dynamics simulations revealed that these salt bridges stabilized the GlacPETase's structure by maintaining their surrounding structure. Phylogenetic analysis indicated that GlacPETase represented a distinct branch within PET hydrolases-like proteins, with the salt bridges and disulfide bonds in this branch being relatively conserved. This research contributed to the improvement of our comprehension of the structural mechanisms that dictate the thermostability of PET hydrolases, highlighting the diverse characteristics and adaptability observed within PET hydrolases.IMPORTANCEThe pervasive problem of polyethylene terephthalate (PET) pollution in various terrestrial and marine environments is widely acknowledged and continues to escalate. PET hydrolases, such as GlacPETase in this study, offered a solution for breaking down PET. Its unique origin and less than 31% identity with any known PET hydrolases have driven us to resolve its structure. Here, we report the correlation between its unique structure and biochemical properties, focusing on an N-terminal disulfide bond and specific salt bridges. Through site-directed mutagenesis experiments and molecular dynamics simulations, the roles of the N-terminal disulfide bond and salt bridges were elucidated in GlacPETase. This research enhanced our understanding of the role of salt bridges in the thermostability of PET hydrolases, providing a valuable reference for the future engineering of PET hydrolases.

AlmA involved in the long-chain n-alkane degradation pathway in Acinetobacter baylyi ADP1 is a Baeyer-Villiger monooxygenase.

Many Acinetobacter species can grow on n-alkanes of varying lengths (≤C40). AlmA, a unique flavoprotein in these Acinetobacter strains, is the only enzyme proven to be required for the degradation of long-chain (LC) n-alkanes, including C32 and C36 alkanes. Although it is commonly presumed to be a terminal hydroxylase, its role in n-alkane degradation remains elusive. In this study, we conducted physiological, biochemical, and bioinformatics analyses of AlmA to determine its role in n-alkane degradation by Acinetobacter baylyi ADP1. Consistent with previous reports, gene deletion analysis showed that almA was vital for the degradation of LC n-alkanes (C26-C36). Additionally, enzymatic analysis revealed that AlmA catalyzed the conversion of aliphatic 2-ketones (C10-C16) to their corresponding esters, but it did not conduct n-alkane hydroxylation under the same conditions, thus suggesting that AlmA in strain ADP1 possesses Baeyer-Villiger monooxygenase (BVMO) activity. These results were further confirmed by bioinformatics analysis, which revealed that AlmA was closer to functionally identified BVMOs than to hydroxylases. Altogether, the results of our study suggest that LC n-alkane degradation by strain ADP1 possibly follows a novel subterminal oxidation pathway that is distinct from the terminal oxidation pathway followed for short-chain n-alkane degradation. Furthermore, our findings suggest that AlmA catalyzes the third reaction in the LC n-alkane degradation pathway.IMPORTANCEMany microbial studies on n-alkane degradation are focused on the genes involved in short-chain n-alkane (≤C16) degradation; however, reports on the genes involved in long-chain (LC) n-alkane (>C20) degradation are limited. Thus far, only AlmA has been reported to be involved in LC n-alkane degradation by Acinetobacter spp.; however, its role in the n-alkane degradation pathway remains elusive. In this study, we conducted a detailed characterization of AlmA in A. baylyi ADP1 and found that AlmA exhibits Baeyer-Villiger monooxygenase activity, thus indicating the presence of a novel LC n-alkane biodegradation mechanism in strain ADP1.

Redundant and scattered genetic determinants for coumarin biodegradation in Pseudomonas sp. strain NyZ480.

Coumarin (COU) is both a naturally derived phytotoxin and a synthetic pollutant which causes hepatotoxicity in susceptible humans. Microbes have potentials in COU biodegradation; however, its underlying genetic determinants remain unknown. Pseudomonas sp. strain NyZ480, a robust COU degrader, has been isolated and proven to grow on COU as its sole carbon source. In this study, five homologs of xenobiotic reductase A scattered throughout the chromosome of strain NyZ480 were identified, which catalyzed the conversion of COU to dihydrocoumarin (DHC) in vitro. Phylogenetic analysis indicated that these COU reductases belong to different subgroups of the old yellow enzyme family. Moreover, two hydrolases (CouB1 and CouB2) homologous to the 3,4-dihydrocoumarin hydrolase in the fluorene degradation were found to accelerate the generation of melilotic acid (MA) from DHC. CouC, a new member from the group A flavin monooxygenase, was heterologously expressed and purified, catalyzing the hydroxylation of MA to produce 3-(2,3-dihydroxyphenyl)propionate (DHPP). Gene deletion and complementation of couC indicated that couC played an essential role in the COU catabolism in strain NyZ480, considering that the genes involved in the downstream catabolism of DHPP have been characterized (Y. Xu and N. Y. Zhou, Appl Environ Microbiol 86:e02385-19, 2020) and homologous catabolic cluster exists in strain NyZ480. This study elucidated the genetic determinants for complete degradation of COU by Pseudomonas sp. strain NyZ480.IMPORTANCECoumarin (COU) is a phytochemical widely distributed in the plant kingdom and also artificially produced as an ingredient for personal care products. Hence, the environmental occurrence of COU has been reported in different places. Toxicologically, COU was proven hepatotoxic to individuals with mutations in the CYP2A6 gene and listed as a group 3 carcinogen by the International Agency for Research on Cancer and thus has raised increasing concerns. Until now, different physicochemical methods have been developed for the removal of COU, whereas their practical applications were hampered due to high cost and the risk of secondary contamination. In this study, genetic evidence and biochemical characterization of the COU degradation by Pseudomonas sp. strain NyZ480 are presented. With the gene and strain resources provided here, better managements of the hazards that humans face from COU could be achieved, and the possible microbiota-plant interaction mediated by the COU-utilizing rhizobacteria could also be investigated.

Glacier as a source of novel polyethylene terephthalate hydrolases.

Polyethylene terephthalate (PET) is a major component of microplastic contamination globally, which is now detected in pristine environments including Polar and mountain glaciers. As a carbon-rich molecule, PET could be a carbon source for microorganisms dwelling in glacier habitats. Thus, glacial microorganisms may be potential PET degraders with novel PET hydrolases. Here, we obtained 414 putative PET hydrolase sequences by searching a global glacier metagenome dataset. Metagenomes from the Alps and Tibetan glaciers exhibited a higher relative abundance of putative PET hydrolases than those from the Arctic and Antarctic. Twelve putative PET hydrolase sequences were cloned and expressed, with one sequence (designated as GlacPETase) proven to degrade amorphous PET film with a similar performance as IsPETase, but with a higher thermostability. GlacPETase exhibited only 30% sequence identity to known active PET hydrolases with a novel disulphide bridge location and, therefore may represent a novel PET hydrolases class. The present work suggests that extreme carbon-poor environments may harbour a diverse range of known and novel PET hydrolases for carbon acquisition as an environmental adaptation mechanism.

A moderately thermophilic origin of a novel family of marine group II euryarchaeota from deep ocean.

Marine group II (MGII) is the most abundant planktonic heterotrophic archaea in the ocean. The evolutionary history of MGII archaea is elusive. In this study, 13 new MGII metagenome-assembled genomes were recovered from surface to the hadal zone in Challenger Deep of the Mariana Trench; four of them from the deep ocean represent a novel group. The optimal growth temperature (OGT) of the common ancestor of MGII has been estimated to be at about 60°C and OGTs of MGIIc, MGIIb, and MGIIa at 47°C-50ºC, 37°C-44ºC, and 30°C-37ºC, respectively, suggesting the adaptation of these species to different temperatures during evolution. The estimated OGT range of MGIIc was supported by experimental measurements of cloned ß-galactosidase that showed optimal enzyme activity around 50°C. These results indicate that MGIIc may have originated from a common ancestor that lived in warm or even hot marine environment, such as hydrothermal vents.

The presence of benzene ring activating CoA ligases for aromatics degradation in the ANaerobic MEthanotrophic (ANME) archaea.

Petroleum-source and black carbon-source aromatic compounds are present in the cold seep environments, where ANaerobic MEthanotrophic (ANME) archaea as the dominant microbial community mediates the anaerobic oxidation of methane to produce inorganic and organic carbon. Here, by predicting the aromatics catabolic pathways in ANME metagenome-assembled genomes, we provide genomic and biochemical evidences that ANME have the potential of metabolizing aromatics via the strategy of CoA activation of the benzene ring using phenylacetic acid and benzoate as the substrates. Two ring-activating enzymes phenylacetate-CoA ligase (PaaKANME) and benzoate-CoA ligase (BadAANME) are able to convert phenylacetate to phenylacetyl-CoA and benzoate to benzoyl-CoA in vitro, respectively. They are mesophilic, alkali resistance, and with broad substrate spectra showing different affinity with various substrates. An exploration of the relative gene abundance in ANME genomes and cold seep environments indicates that about 50% of ANME genomes contain PCL genes, and various bacteria and archaea contain PCL and BCL genes. The results provide evidences for the capability of heterotrophic metabolism of aromatic compounds by ANME. This has not only enhanced our understanding of the nutrient range of ANME but also helped to explore the additional ecological and biogeochemical significance of this ubiquitous sedimentary archaea in the carbon flow in the cold seep environments. IMPORTANCE ANaerobic MEthanotrophic (ANME) archaea is the dominant microbial community mediating the anaerobic oxidation of methane in the cold seep environments, where aromatic compounds are present. Then it is hypothesized that ANME may be involved in the metabolism of aromatics. Here, we provide genomic and biochemical evidences for the heterotrophic metabolism of aromatic compounds by ANME, enhancing our understanding of their nutrient range and also shedding light on the ecological and biogeochemical significance of these ubiquitous sedimentary archaea in carbon flow within cold seep environments. Overall, this study offers valuable insights into the metabolic capabilities of ANME and their potential contributions to the global carbon cycle.

A global atlas of marine antibiotic resistance genes and their expression.

Oceans serve as global reservoirs of antibiotic-resistant bacteria and antibiotic resistance genes (ARGs). However, little is known about the traits and expression of ARGs in response to environmental factors. We analyzed 347 metagenomes and 182 metatranscriptomes to determine the distribution, hosts, and expression of ARGs in oceans. Our study found that the diversity and abundance of ARGs varied with latitude and depth. The core marine resistome mainly conferred glycopeptide and multidrug resistance. The hosts of this resistome were mainly limited to the core marine microbiome, with phylogenetic barriers to the horizontal transfer of ARGs, transfers being more frequent within species than between species. Sixty-five percent of the marine ARGs identified were expressed. More than 90% of high-risk ARGs were more likely to be expressed. Anthropogenic activity might affect the expression of ARGs by altering nitrate and phosphate concentrations and ocean temperature. Machine-learning models predict >97% of marine ARGs will change expression by 2100. High-risk ARGs will shift to low latitudes and regions with high anthropogenic activity, such as the Pacific and Atlantic Oceans. Certain ARGs serve a dual role in antibiotic resistance and potentially participate in element cycling, along with other unknown functions. Determining whether changes in ARG expression are beneficial to ecosystems and human health is challenging without comprehensive understanding of their functions. Our study identified a core resistome in the oceans and quantified the expression of ARGs for the development of future control strategies under global change.

Biodegradation of additive-free polypropylene by bacterial consortia enriched from the ocean and from the gut of Tenebrio molitor larvae.

Accumulation of highly recalcitrant PP wastes has caused a serious environmental pollution. We evaluated the biodegradation of two types of additive-free PP polymers by microbial degraders from different environments. Two bacterial consortia, designated as PP1M and PP2G, were enriched from the ocean and from the guts of Tenebrio molitor larvae. Both consortia were able to utilize each of two different additive-free PP plastics with relatively low molecular weights (low molecular weight PP powder and amorphous PP pellets) as the sole carbon source for growth. After a 30-day incubation, several plastic characterization methods, including high-temperature gel permeation chromatography, scanning electron microscopy, Fourier transform infrared spectroscopy, and differential scanning calorimetry, were used to characterize the PP samples. The bio-treated PP powder was covered with tight biofilms and extracellular secretions with significantly increased hydroxyl and carbonyl groups and slightly decreased methyl groups. This suggested that degradation and oxidation had occurred. The altered molecular weights and the increased melting enthalpy and average crystallinity of the bio-treated PP samples all suggested that both consortia preferred to depolymerize and degrade the fractions with molecular weights of ≤34 kDa and the amorphous phase fractions of the two types of PP. Furthermore, low molecular weight PP powder was more susceptible to bacterial degradation compared to amorphous PP pellets. This study provides a unique example of different types of additive-free PP degradation by different culturable bacteria from the ocean and insect guts as well as a feasibility of PP waste removal in different environments.

Elucidation of the coumarin degradation by Pseudomonas sp. strain NyZ480.

As a phytotoxin and synthetic chemical, coumarin (COU) is known for its hepatotoxicity and carcinogenicity. However, no thorough characterization of its microbial degradation has been reported. Here, Pseudomonas sp. strain NyZ480 was isolated for its capability of utilizing COU as the sole carbon source. Studies on its growth and degradation efficiency of COU under various conditions suggested that strain NyZ480 performed the optimum degradation at 30 â„ƒ, pH 7, and 0.5 mM COU was completely removed within 4 h with 1% inoculum. HPLC and LC-MS analyses indicated that dihydrocoumarin (DHC), melilotic acid (MA) and 3-(2,3-dihydroxyphenyl)propionate (DHPP) were the upstream biotransformation intermediates of COU. Enzyme assay established that the initial reaction transforming COU to DHC required an NAD(P)H-dependent reductase, followed by the hydrolysis of DHC to generate MA, and the third reaction catalyzing the monooxygenation of MA to DHPP utilized a strict NADH-dependent hydroxylase. Combining genomics and transcriptomics, we proposed that the COU downstream degradation (from DHPP) was catalyzed by enzymes encoded by a gene cluster homologous to the mhp cluster for 3(3-hydroxyphenyl)propionate degradation via DHPP in E. coli. This study thoroughly identified the intermediates from the COU catabolism, providing essential insights into the molecular evidences of its biodegradation pathway.

A cytochrome P450 system initiates 4-nitroanisole degradation in Rhodococcus sp. strain JS3073.

Nitroanisoles are used widely as synthetic intermediates and explosives. Although bacteria have been reported to degrade 4-nitroanisole (4NA) under aerobic conditions, the key enzymes and the catalytic mechanism have remained elusive. Rhodococcus sp. strain JS3073 was isolated for its ability to grow on 4NA as the sole carbon and energy source. In this study, whole cell biotransformation experiments indicated that 4NA degradation is initiated by O-demethylation to form 4-nitrophenol (PNP), which undergoes subsequent degradation by a previously established pathway involving formation of 1,2,4-benzenetriol and release of nitrite. Based on comparative transcriptomics and heterologous expression, a novel three-component cytochrome P450 system encoded by pnaABC initiates the O-demethylation of 4NA to yield formaldehyde and PNP. The pnaABC genes encode a phthalate dioxygenase type reductase (PnaA), a cytochrome P450 monooxygenase (PnaB), and an EthD family protein (PnaC) with putative function similar to ferredoxins. This unusual P450 system also has a broad substrate specificity for nitroanisole derivatives. Sequence analysis of PnaAB revealed high identity with multiple self-sufficient P450s of the CYP116B subfamily. The findings revealed the molecular basis of the catabolic pathway for 4NA initiated by an unusual O-demethylase PnaABC and extends the understanding of the diversity among P450s and their electron transport chains.

Microbial Diversity Biased Estimation Caused by Intragenomic Heterogeneity and Interspecific Conservation of 16S rRNA Genes.

The 16S rRNA gene has been extensively used as a molecular marker to explore evolutionary relationships and profile microbial composition throughout various environments. Despite its convenience and prevalence, limitations are inevitable. Variable copy numbers, intragenomic heterogeneity, and low taxonomic resolution have caused biases in estimating microbial diversity. Here, analysis of 24,248 complete prokaryotic genomes indicated that the 16S rRNA gene copy number ranged from 1 to 37 in bacteria and 1 to 5 in archaea, and intragenomic heterogeneity was observed in 60% of prokaryotic genomes, most of which were below 1%. The overestimation of microbial diversity caused by intragenomic variation and the underestimation introduced by interspecific conservation were calculated when using full-length or partial 16S rRNA genes. Results showed that, at the 100% threshold, microbial diversity could be overestimated by as much as 156.5% when using the full-length gene. The V4 to V5 region-based analyses introduced the lowest overestimation rate (4.4%) but exhibited slightly lower species resolution than other variable regions under the 97% threshold. For different variable regions, appropriate thresholds rather than the canonical value 97% were proposed for minimizing the risk of splitting a single genome into multiple clusters and lumping together different species into the same cluster. This study has not only updated the 16S rRNA gene copy number and intragenomic variation information for the currently available prokaryotic genomes, but also elucidated the biases in estimating prokaryotic diversity with quantitative data, providing references for choosing amplified regions and clustering thresholds in microbial community surveys. IMPORTANCE Microbial diversity is typically analyzed using marker gene-based methods, of which 16S rRNA gene sequencing is the most widely used approach. However, obtaining an accurate estimation of microbial diversity remains a challenge, due to the intragenomic variation and low taxonomic resolution of 16S rRNA genes. Comprehensive examination of the bias in estimating such prokaryotic diversity using 16S rRNA genes within ever-increasing prokaryotic genomes highlights the importance of the choice of sequencing regions and clustering thresholds based on the specific research objectives.

Characterization of Two Marine Lignin-Degrading Consortia and the Potential Microbial Lignin Degradation Network in Nearshore Regions.

Terrestrial organic carbon such as lignin is an important component of the global marine carbon. However, the structural complexity and recalcitrant nature of lignin are deemed challenging for biodegradation. It has been speculated that bacteria play important roles in lignin degradation in the marine system. However, the extent of the involvement of marine microorganisms in lignin degradation and their contribution to the oceanic carbon cycle remains elusive. In this study, two bacterial consortia capable of degrading alkali lignin (a model compound of lignin), designated LIG-B and LIG-S, were enriched from the nearshore sediments of the East and South China Seas. Consortia LIG-B and LIG-S mainly comprised of the Proteobacteria phylum with Nitratireductor sp. (71.6%) and Halomonas sp. (91.6%), respectively. Lignin degradation was found more favorable in consortium LIG-B (max 57%) than in LIG-S (max 18%). Ligninolytic enzymes laccase (Lac), manganese peroxidase (MnP), and lignin peroxidase (LiP) capable of decomposing lignin into smaller fragments were all active in both consortia. The newly emerged low-molecular-weight aromatics, organic acids, and other lignin-derived compounds in biotreated alkali lignin also evidently showed the depolymerization of lignin by both consortia. The lignin degradation pathways reconstructed from consortium LIG-S were found to be more comprehensive compared to consortium LIG-B. It was further revealed that catabolic genes, involved in the degradation of lignin and its derivatives through multiple pathways via protocatechuate and catechol, are present not only in lignin-degrading consortia LIG-B and LIG-S but also in 783 publicly available metagenomic-assembled genomes from nine nearshore regions. IMPORTANCE Numerous terrigenous lignin-containing plant materials are constantly discharged from rivers and estuaries into the marine system. However, only low levels of terrigenous organic carbon, especially lignin, are detected in the global marine system due to the abundance of active heterotrophic microorganisms driving the carbon cycle. Simultaneously, the lack of knowledge on lignin biodegradation has hindered our understanding of the oceanic carbon cycle. Moreover, bacteria have been speculated to play important roles in the marine lignin biodegradation. Here, we enriched two bacterial consortia from nearshore sediments capable of utilizing alkali lignin for cell growth while degrading it into smaller molecules and reconstructed the lignin degradation network. In particular, this study highlights that marine microorganisms in nearshore regions mostly undergo similar pathways using protocatechuate and catechol as ring-cleavage substrates to drive lignin degradation as part of the oceanic carbon cycle, regardless of whether they are in sediments or water column.

Aerobic Degradation of the Antidiabetic Drug Metformin by Aminobacter sp. Strain NyZ550.

Metformin is becoming one of the most common emerging contaminants in surface and wastewater. Its biodegradation generally leads to the accumulation of guanylurea in the environment, but the microorganisms and mechanisms involved in this process remain elusive. Here, Aminobacter sp. strain NyZ550 was isolated and characterized for its ability to grow on metformin as a sole source of carbon, nitrogen, and energy under oxic conditions. This isolate also assimilated a variety of nitrogenous compounds, including dimethylamine. Hydrolysis of metformin by strain NyZ550 was accompanied by a stoichiometric accumulation of guanylurea as a dead-end product. Based on ion chromatography, gas chromatography-mass spectrometry, and comparative transcriptomic analyses, dimethylamine was identified as an additional hydrolytic product supporting the growth of the strain. Notably, a microbial mixture consisting of strain NyZ550 and an engineered Pseudomonas putida PaW340 expressing a guanylurea hydrolase was constructed for complete elimination of metformin and its persistent product guanylurea. Overall, our results not only provide new insights into the metformin biodegradation pathway, leading to the commonly observed accumulation of guanylurea in the environment, but also open doors for the complete degradation of the new pollutant metformin.

Molecular Basis and Evolutionary Origin of 1-Nitronaphthalene Catabolism in Sphingobium sp. Strain JS3065.

Nitrated polycyclic aromatic hydrocarbons (nitro-PAHs) enter the environment from natural sources and anthropogenic activities. To date, microorganisms able to mineralize nitro-PAHs have not been reported. Here, Sphingobium sp. strain JS3065 was isolated by selective enrichment for its ability to grow on 1-nitronaphthalene as the sole carbon, nitrogen, and energy source. Analysis of the complete genome of strain JS3065 indicated that the gene cluster encoding 1-nitronaphthalene catabolism (nin) is located on a plasmid. Based on the genetic and biochemical evidence, the nin genes share an origin with the nag-like genes encoding naphthalene degradation in Ralstonia sp. strain U2. The initial step in degradation of 1-nitronaphthalene is catalyzed by a three-component dioxygenase, NinAaAbAcAd, resulting in formation of 1,2-dihydroxynaphthalene which is also an early intermediate in the naphthalene degradation pathway. Introduction of the ninAaAbAcAd genes into strain U2 enabled its growth on 1-nitronaphthalene. Phylogenic analysis of NinAc suggested that an ancestral 1-nitronaphthalene dioxygenase was an early step in the evolution of nitroarene dioxygenases. Based on bioinformatic analysis and enzyme assays, the subsequent assimilation of 1,2-dihydroxynaphthalene seems to follow the well-established pathway for naphthalene degradation by Ralstonia sp. strain U2. This is the first report of catabolic pathway for 1-nitronaphthalene and is another example of how expanding the substrate range of Rieske type dioxygenase enables bacteria to grow on recalcitrant nitroaromatic compounds. IMPORTANCE Nitrated polycyclic aromatic hydrocarbons (nitro-PAHs) have been widely detected in the environment and they are more toxic than their corresponding parent PAHs. Although biodegradation of many PAHs has been extensively described at genetic and biochemical levels, little is known about the microbial degradation of nitro-PAHs. This work reports the isolation of a Sphingobium strain growing on 1-nitronaphthalene and the genetic basis for the catabolic pathway. The pathway evolved from an ancestral naphthalene catabolic pathway by a remarkably small modification in the specificity of the initial dioxygenase. Data presented here not only shed light on the biochemical processes involved in the microbial degradation of globally important nitrated polycyclic aromatic hydrocarbons, but also provide an evolutionary paradigm for how bacteria evolve a novel catabolic pathway with minimal alteration of preexisting pathways for natural organic compounds.

Degradation of polyvinyl chloride by a bacterial consortium enriched from the gut of Tenebrio molitor larvae.

Polyvinyl chloride (PVC), a carbon backbone synthetic plastic containing chlorine element, is one of six widely used plastics accounting for 10% global plastics production. PVC wastes are recalcitrant to be broken down in the environment but release harmful chlorinated compounds, causing damage to the ecosystem. Although biodegradation represents a sustainable approach for PVC reduction, virtually no efficient bacterial degraders for additive-free PVC have been reported. In addition, PVC depolymerization by Tenebrio molitor larvae was suggested to be gut microbe-dependent, but to date no additive-free PVC degraders have been isolated from insect guts. In this study, a bacterial consortium designated EF1 was newly enriched from the gut of Tenebrio molitor larvae, which was capable of utilizing additive-free PVC for its growth with the PVC-mass reduction and dechlorination of PVC. PVC films inoculated with consortium EF1 for 30 d were analyzed by diverse polymer characterization methods including atomic force microscopy, scanning electron microscope, water contact angle, time-of-flight secondary ion mass spectrometry, Fourier transform infrared spectroscopy, differential scanning calorimetry, thermogravimetric analysis technique, and ion chromatography. It was found that bio-treated PVC films were covered with tight biofilms with increased -OH and -CC- groups and decreased chlorine contents, and erosions and cracks were present on their surfaces. Meanwhile, the hydrophilicity of bio-treated films increased, but their thermal stability declined. Furthermore, Mw, Mn and Mz values were reduced by 17.0%, 28.5% and 16.1% using gel permeation chromatography, respectively. In addition, three medium-chain aliphatic primary alcohols and their corresponding fatty acids were identified as PVC degradation intermediates by gas chromatography-mass spectrometry. Combing all above results, it is clear that consortium EF1 is capable of efficiently degrading PVC polymer, providing a unique example for PVC degradation by gut microbiota of insects and a feasibility for the removal of PVC wastes.

Negative effects of abamectin on soil microbial communities in the short term.

With the widespread use of abamectin in agriculture, there is increasing urgency to assess the effects of abamectin on soil microorganisms. Here, we treated plant-soil microcosms with abamectin at concentrations of 0.1 and 1.0 mg/kg and quantified the impacts of abamectin on bulk and rhizosphere soil microbial communities by shotgun metagenomics after 7 and 21 days of exposure. Although abamectin was reported to be easily degradable, it altered the composition of the soil microbial communities, disrupted microbial interactions, and decreased community complexity and stability after 7 days of exposure. After treatment with abamectin at a concentration of 1.0 mg/kg, some opportunistic human diseases, and soil-borne pathogens like Ralstonia were enriched in the soil. However, most ecological functions in soil, particularly the metabolic capacities of microorganisms, recovered within 21 days after abamectin treatment. The horizontal and vertical gene transfer under abamectin treatments increased the levels of antibiotic resistance genes dissemination. Overall, our findings demonstrated the negative effects of abamectin on soil ecosystems in the short-term and highlight a possible long-term risk to public and soil ecosystem health associated with antibiotic resistance genes dissemination.

Wide distribution of the sad gene cluster for sub-terminal oxidation in alkane utilizers.

Alkane constitutes major fractions of crude oils, and its microbial aerobic degradation dominantly follows the terminal oxidation and the sub-terminal pathways. However, the latter one received much less attention, especially since the related genes were yet to be fully defined. Here, we isolated a bacterium designated Acinetobacter sp. strain NyZ410, capable of growing on alkanes with a range of chain lengths and derived sub-terminal oxidation products. From its genome, a secondary alcohol degradation gene cluster (sad) was identified to be likely involved in converting the aliphatic secondary alcohols (the sub-terminal oxidation products of alkanes) to the corresponding primary alcohols by removing two-carbon unit. On this cluster, sadC encoded an alcohol dehydrogenase converting the aliphatic secondary alcohols to the corresponding ketones; sadD encoded a Baeyer-Villiger monooxygenase catalysing the conversion of the aliphatic ketones to the corresponding esters; SadA and SadB are two esterases hydrolyzing aliphatic esters to the primary alcohols and acetic acids. Bioinformatics analyses indicated that the sad cluster was widely distributed in the genomes of probable alkane degraders, apparently coexisting (64%) with the signature enzymes AlkM and AlmA for alkane terminal oxidation in 350 bacterial genomes. It suggests that the alkane sub-terminal oxidation may be more ubiquitous than previously thought.