New insights into xenobiotic tolerance of Antarctic bacteria: transcriptomic analysis of Pseudomonas sp. TNT3 during 2,4,6-trinitrotoluene biotransformation.

The xenobiotic 2,4,6-trinitrotoluene (TNT) is a highly persistent environmental contaminant, whose biotransformation by microorganisms has attracted renewed attention. In previous research, we reported the discovery of Pseudomonas sp. TNT3, the first described Antarctic bacterium with the ability to biotransform TNT. Furthermore, through genomic analysis, we identified distinctive features in this isolate associated with the biotransformation of TNT and other xenobiotics. However, the metabolic pathways and genes active during TNT exposure in this bacterium remained unexplored. In the present transcriptomic study, we used RNA-sequencing to investigate gene expression changes in Pseudomonas sp. TNT3 exposed to 100 mg/L of TNT. The results showed differential expression of 194 genes (54 upregulated and 140 downregulated), mostly encoding hypothetical proteins. The most highly upregulated gene (> 1000-fold) encoded an azoreductase enzyme not previously described. Other significantly upregulated genes were associated with (nitro)aromatics detoxification, oxidative, thiol-specific, and nitrosative stress responses, and (nitro)aromatic xenobiotic tolerance via efflux pumps. Most of the downregulated genes were involved in the electron transport chain, pyrroloquinoline quinone (PQQ)-related alcohol oxidation, and motility. These findings highlight a complex cellular response to TNT exposure, with the azoreductase enzyme likely playing a crucial role in TNT biotransformation. Our study provides new insights into the molecular mechanisms of TNT biotransformation and aids in developing effective TNT bioremediation strategies. To the best of our knowledge, this report is the first transcriptomic response analysis of an Antarctic bacterium during TNT biotransformation.

Comparative Genomic Analysis of Antarctic Pseudomonas Isolates with 2,4,6-Trinitrotoluene Transformation Capabilities Reveals Their Unique Features for Xenobiotics Degradation.

The nitroaromatic explosive 2,4,6-trinitrotoluene (TNT) is a highly toxic and persistent environmental pollutant. Since physicochemical methods for remediation are poorly effective, the use of microorganisms has gained interest as an alternative to restore TNT-contaminated sites. We previously demonstrated the high TNT-transforming capability of three novel Pseudomonas spp. isolated from Deception Island, Antarctica, which exceeded that of the well-characterized TNT-degrading bacterium Pseudomonas putida KT2440. In this study, a comparative genomic analysis was performed to search for the metabolic functions encoded in the genomes of these isolates that might explain their TNT-transforming phenotype, and also to look for differences with 21 other selected pseudomonads, including xenobiotics-degrading species. Comparative analysis of xenobiotic degradation pathways revealed that our isolates have the highest abundance of key enzymes related to the degradation of fluorobenzoate, TNT, and bisphenol A. Further comparisons considering only TNT-transforming pseudomonads revealed the presence of unique genes in these isolates that would likely participate directly in TNT-transformation, and others involved in the ß-ketoadipate pathway for aromatic compound degradation. Lastly, the phylogenomic analysis suggested that these Antarctic isolates likely represent novel species of the genus Pseudomonas, which emphasizes their relevance as potential agents for the bioremediation of TNT and other xenobiotics.

Biotransformation of 2,4,6-Trinitrotoluene by Pseudomonas sp. TNT3 isolated from Deception Island, Antarctica.

2,4,6-Trinitrotoluene (TNT) is a nitroaromatic explosive, highly toxic and mutagenic for organisms. In this study, we report for the first time the screening and isolation of TNT-degrading bacteria from Antarctic environmental samples with potential use as bioremediation agents. Ten TNT-degrading bacterial strains were isolated from Deception Island. Among them, Pseudomonas sp. TNT3 was selected as the best candidate since it showed the highest tolerance, growth, and TNT biotransformation capabilities. Our results showed that TNT biotransformation involves the reduction of the nitro groups. Additionally, Pseudomonas sp. TNT3 was capable of transforming 100 mg/L TNT within 48 h at 28 °C, showing higher biotransformation capability than Pseudomonas putida KT2440, a known TNT-degrading bacterium. Functional annotation of Pseudomonas sp. TNT3 genome revealed a versatile set of molecular functions involved in xenobiotic degradation pathways. Two putative xenobiotic reductases (XenA_TNT3 and XenB_TNT3) were identified by means of homology searches and phylogenetic relationships. These enzymes were also characterized at molecular level using homology modelling and molecular dynamics simulations. Both enzymes share different levels of sequence similarity with other previously described TNT-degrading enzymes and with their closest potential homologues in databases.

Odd Loop Regions of XenA and XenB Enzymes Modulate Their Interaction with Nitro-explosives Compounds and Provide Structural Support for Their Regioselectivity.

The nitro-explosive compounds 2,4,6-trinitrotoluene, 2,4,6-trinitrophenol, and 1,2,3-trinitroglycerol are persistent environmental contaminants. The presence of different functional groups in these molecules represents a great challenge to enzymatic catalysis. The chemical variety of these three substrates is such that they do not bind and interact with catalytic residues within an enzyme with the same affinity. In this context, two Xenobiotic Reductase enzymes produced by the bacteria Pseudomonas putida can catalyze the reduction of these compounds with different affinities and regioselectivity. The structural bases that support this substrate promiscuity and catalytic preferences are unknown. Therefore, through molecular dynamics simulations and free energy calculations, we explored the structural properties driving the specific interactions of these enzymes with their substrates and cofactor. Models of Xenobiotic Reductase A and B enzymes in complex with 2,4,6-trinitrotoluene, 2,4,6-trinitrophenol, or 1,2,3-trinitroglycerol were built, and the ligand enzyme interaction was simulated by molecular dynamics. The structural analysis of the molecular dynamics simulations shows that loops 3, 5, 7, 9, 11, and 13 of Xenobiotic Reductase B, and loops 4, 5, 7, 11, 13, and 15 Xenobiotic Reductase A, are in contact with the ligands during the first stages of the molecular recognition. These loops are the most flexible regions for both enzymes; however, Xenobiotic Reductase B presents a greater range of movement and a higher number of residues interacting with the ligands. Finally, the distance between the cofactor and the different reactive groups in the substrate reflects the regioselectivity of the enzymes, and the free energy calculations are consistent with the substrate specificity of both enzymes studied. The simulation shows a stable interaction between the aromatic ring of the substrates and Xenobiotic Reductase B. In contrast, a less stable interaction with the different nitro groups of the aromatic ligands was observed. In the case of 1,2,3-trinitroglycerol, Xenobiotic Reductase B interacts more closely with the nitro groups of carbon 1 or 3, while Xenobiotic Reductase A is more selective by nitro groups of carbon 2. The obtained results suggest that the flexibility of the loops in Xenobiotic Reductase B and the presence of polar and aromatic residues present in loops 5 and 7 are fundamental to determine the affinity of the enzyme with the different substrates, and they also contribute to the proper orientation of the ligands that directs the catalytic reaction.

Biotechnological applications of archaeal enzymes from extreme environments.

To date, many industrial processes are performed using chemical compounds, which are harmful to nature. An alternative to overcome this problem is biocatalysis, which uses whole cells or enzymes to carry out chemical reactions in an environmentally friendly manner. Enzymes can be used as biocatalyst in food and feed, pharmaceutical, textile, detergent and beverage industries, among others. Since industrial processes require harsh reaction conditions to be performed, these enzymes must possess several characteristics that make them suitable for this purpose. Currently the best option is to use enzymes from extremophilic microorganisms, particularly archaea because of their special characteristics, such as stability to elevated temperatures, extremes of pH, organic solvents, and high ionic strength. Extremozymes, are being used in biotechnological industry and improved through modern technologies, such as protein engineering for best performance. Despite the wide distribution of archaea, exist only few reports about these microorganisms isolated from Antarctica and very little is known about thermophilic or hyperthermophilic archaeal enzymes particularly from Antarctica. This review summarizes current knowledge of archaeal enzymes with biotechnological applications, including two extremozymes from Antarctic archaea with potential industrial use, which are being studied in our laboratory. Both enzymes have been discovered through conventional screening and genome sequencing, respectively.

Biotechnological applications of archaeal enzymes from extreme environments

To date, many industrial processes are performed using chemical compounds, which are harmful to nature. An alternative to overcome this problem is biocatalysis, which uses whole cells or enzymes to carry out chemical reactions in an environmentally friendly manner. Enzymes can be used as biocatalyst in food and feed, pharmaceutical, textile, detergent and beverage industries, among others. Since industrial processes require harsh reaction conditions to be performed, these enzymes must possess several characteristics that make them suitable for this purpose. Currently the best option is to use enzymes from extremophilic microorganisms, particularly archaea because of their special characteristics, such as stability to elevated temperatures, extremes of pH, organic solvents, and high ionic strength. Extremozymes, are being used in biotechnological industry and improved through modern technologies, such as protein engineering for best performance. Despite the wide distribution of archaea, exist only few reports about these microorganisms isolated from Antarctica and very little is known about thermophilic or hyperthermophilic archaeal enzymes particularly from Antarctica. This review summarizes current knowledge of archaeal enzymes with biotechnological applications, including two extremozymes from Antarctic archaea with potential industrial use, which are being studied in our laboratory. Both enzymes have been discovered through conventional screening and genome sequencing, respectively.

Cloning, overexpression, and characterization of a thermostable nitrilase from an Antarctic Pyrococcus sp.

Nitriles are important chemical building blocks for the synthesis of intermediates in fine chemical and pharmaceutical industries. Here, we report a new highly thermostable nitrilase from an Antarctic Pyrococcus sp. MC-FB, a hyperthermophilic archaeon. A gene that encoded a nitrilase was identified and subsequently cloned and overexpressed in Escherichia coli. The recombinant nitrilase, named NitMC-FB, is active as a homodimer (60 kDa) with an optimal temperature and pH of 90 °C and 7.0, respectively. NitMC-FB hydrolyzes preferentially aromatic nitriles, being the first aromatic nitrilase from an archaeon described so far. The K M and V max parameters were determined to be 13.9 mM and 3.7 µmol/min*mg, respectively, with 2-cyanopyridine as the substrate. Additionally, the recombinant nitrilase is highly thermostable with a half-life of 8 h at 90 °C.