Exposure to three herbicide mixtures influenced maize root-associated microbial community structure, function and the network complexity.

Herbicide mixtures are a new and effective agricultural strategy for managing suppress weed resistance and have been widely used in controlling weeding growth in maize fields. However, the potential ecotoxicological impact of these mixtures on the microbial community structure and function within various root-associated niches, remains inadequately understood. Here, the effects of nicosulfuron, mesotrione and atrazine on soil enzyme activity and microbial community structure and function were investigated when applied alone and in combination. The findings indicated that herbicide mixtures exhibit a prolonged half-life compared to single herbicides. Ecological niches are the major factor influencing the structure and functions of the microbial community, with the rhizosphere exhibiting a more intensive response to herbicide stress. Herbicides significantly inhibited the activities of soil functional enzymes, including dehydrogenase, urease and sucrose in the short-term. Single herbicide did not drastically influence the alpha or beta diversity of the soil bacterial community, but herbicide mixtures significantly increased the richness of the fungal community. Meanwhile, the key functional microbial populations, such as Pseudomonas and Enterobacteriaceae, were significantly altered by herbicide stress. Both individual and combined use of the three herbicides reduced the complexity and stability of the bacterial network but increased the interspecific cooperations of fungal community in the rhizosphere. Moreover, by quantification of residual herbicide concentrations in the soil, we showed that the degradation period of the herbicide mixture was longer than that of single herbicides. Herbicide mixtures increased the contents of NO3--N and NH4+-N in the soil in the short-term. Overall, our study provided a comprehensive insight into the response of maize root-associated microbial communities to herbicide mixtures and facilitated the assessment of the ecological risks posed by herbicide mixtures to the agricultural environment from an agricultural sustainability perspective.

Characterization of a new chlorimuron-ethyl-degrading strain Cedecea sp. LAM2020 and biodegradation pathway revealed by multiomics analysis.

The widespread use of the herbicide chlorimuron-methyl is hazard to rotational crops and causes soil degradation problems. Biodegradation is considered a promising way for removing herbicide residues from the environment. Here, a new isolated strain, Cedecea sp. LAM2020, enabled complete degradation of 100 mg/L chlorimuron-methyl within five days. Transcriptome analysis revealed that ABC transporters, atrazine degradation and purine metabolism were enriched in the KEGG pathway. Integrating GO and KEGG classification with related reports, we predict that carboxylesterases are involved in the biodegradation of chlorimuron-methyl by LAM2020. Heterologous expression of the carboxylesterase gene carH showed 26.67% degradation of 50 mg/L chlorimuron-methyl within 6 h. The intracellular potential biological response and extracellular degradation process of chlorimuron-ethyl were analyzed by the nontarget metabolomic and mass spectrometry respectively, and the biodegradation characteristics and complete mineralization pathway was revealed. The cleavage of the sulfonylurea bridge and the ester bond achieved the first step in the degradation of chlorimuron-methyl. Together, these results reveal the presence of acidolysis and enzymatic degradation of chlorimuron-methyl by strain LAM2020. Hydroponic corn experiment showed that the addition of strain LAM2020 alleviated the toxic effects of chlorimuron-ethyl on the plants. Collectively, strain LAM2020 may be a promising microbial agent for plants chlorimuron-ethyl detoxification and soil biofertilizer.

Oxygenases as Powerful Weapons in the Microbial Degradation of Pesticides.

Oxygenases, which catalyze the reductive activation of O2 and incorporation of oxygen atoms into substrates, are widely distributed in aerobes. They function by switching the redox states of essential cofactors that include flavin, heme iron, Rieske non-heme iron, and Fe(II)/α-ketoglutarate. This review summarizes the catalytic features of flavin-dependent monooxygenases, heme iron-dependent cytochrome P450 monooxygenases, Rieske non-heme iron-dependent oxygenases, Fe(II)/α-ketoglutarate-dependent dioxygenases, and ring-cleavage dioxygenases, which are commonly involved in pesticide degradation. Heteroatom release (hydroxylation-coupled hetero group release), aromatic/heterocyclic ring hydroxylation to form ring-cleavage substrates, and ring cleavage are the main chemical fates of pesticides catalyzed by these oxygenases. The diversity of oxygenases, specificities for electron transport components, and potential applications of oxygenases are also discussed. This article summarizes our current understanding of the catalytic mechanisms of oxygenases and a framework for distinguishing the roles of oxygenases in pesticide degradation.

Effects of long-term exposure to the herbicide nicosulfuron on the bacterial community structure in a factory field.

This study aims to investigate the effects of long-term nicosulfuron residue on an herbicide factory ecosystem. High-throughput sequencing was used to investigate the environmental microbial community structure and interactions. The results showed that the main contributor to the differences in the microbial community structure was the sample type, followed by oxygen content, pH and nicosulfuron residue concentration. Regardless of the presence or absence of nicosulfuron, soil, sludge, and sewage were dominated by groups of Bacteroidetes, Actinobacteria, and Proteobacteria. Long-term exposure to nicosulfuron increased alpha diversity of bacteria and archaea but significantly decreased the abundance of Bacteroidetes and Acidobateria compared to soils without nicosulfuron residue. A total of 81 possible nicosulfuron-degrading bacterial genera, e.g., Rhodococcus, Chryseobacterium, Thermomonas, Stenotrophomonas, and Bacillus, were isolated from the nicosulfuron factory environmental samples through culturomics. The co-occurrence network analysis indicated that the keystone taxa were Rhodococcus, Stenotrophomonas, Nitrospira, Terrimonas, and Nitrosomonadaceae_MND1. The strong ecological relationship between microorganisms with the same network module was related to anaerobic respiration, the carbon and nitrogen cycle, and the degradation of environmental contaminants. Synthetic community (SynCom), which provides an effective top-down approach for the critical degradation strains obtained, enhanced the degradation efficiency of nicosulfuron. The results indicated that Rhodococcus sp. was the key genus in the environment of long-term nicosulfuron exposure.

Rhizosphere Microbial Community Diversity and Function Analysis of Cut Chrysanthemum During Continuous Monocropping.

As an ornamental flower crop, the long-term continuous monocropping of cut chrysanthemum causes frequent occurrence of diseases, seriously affecting the quality of cut chrysanthemum. The rhizosphere microbial community plays an important role in maintaining the healthy growth of plants, whereas the composition and dynamics of rhizosphere microbial community under continuous monocropping of cut chrysanthemum have not been fully revealed. In this study, the Illumina MiSeq high-throughput sequencing platform was used to monitor the dynamic changes of rhizosphere microbial communities in four varieties of cut chrysanthemum during 0-3 years of monocropping, and the soil physicochemical properties were also determined. Results showed that continuous monocropping significantly increased the fungal community richness and altered the profiles of the bacterial and fungal communities, leading to variation of community beta-diversity. With the increase of continuous cropping time, biocontrol bacteria decreased, while some plant pathogenic fungi were enriched in the rhizosphere of cut chrysanthemum. FAPROTAX-based functional prediction showed that the abundance of gene related to nitrogen and sulfur metabolism and chitin lysis was reduced in the rhizosphere of cut chrysanthemum. FUNGuild-based fungal function prediction showed that plant pathogenic fungal taxa were increasing in the rhizosphere of cut chrysanthemum, mainly Acremonium, Plectosphaerellaceae, Fusarium, and Cladosporium. Continuous cropping also reduced the content of ammonium nitrogen and increased soil salinity, resulting in deterioration of soil physical and chemical properties, which, together with the transformation of rhizosphere microbial community, became part of the reasons for the continuous cropping obstacle of cut chrysanthemum.

Metabolomics reveals the mechanism of tetracycline biodegradation by a Sphingobacterium mizutaii S121.

Contamination by tetracycline residues has adverse influences on the environment and is considered a pressing issue. Biodegradation is regarded as a promising way to treat tetracycline residues in the environment. Here, strain Sphingobacterium mizutaii S121, which could degrade 20 mg/L tetracycline completely within 5 days, was isolated from contaminated soil. The characteristics of tetracycline degradation by strain S121 were investigated under various culture conditions. Response surface methodology was used to predict the maximum tetracycline degradation ratio, which can be obtained under the following conditions: 31.36 °C, pH of 7.15, and inoculum volume of 5.5% (v/v). Furthermore, extracellular tetracycline biodegradation products and intracellular metabolic pathways of S121 were detected by ultraperformance liquid chromatography-quadrupole-time-of-flight-mass spectrometry (UPLC-Q-TOF-MS) and UHPLC-quadrupole electrospray (QE)-MS, respectively. The results identified eight possible degradation products, and three putative degradation pathways were proposed. In addition, exposure to tetracycline produced significant influences on metabolic pathways such as pyrimidine, purine, taurine and hypotaurine metabolism and lysine degradation. Consequently, the intracellular metabolic pathway response of S121 in the presence of tetracycline was proposed. These findings are presented for the first time, which will facilitate a comprehensive understanding of the mechanism of tetracycline degradation. Moreover, strain S121 can be a promising bacterium for tetracycline bioremediation.

Biodegradation of Tetracycline Antibiotics by the Yeast Strain Cutaneotrichosporon dermatis M503.

In this study, the Cutaneotrichosporon dermatis strain M503 was isolated and could efficiently degrade tetracycline, doxycycline, and chlorotetracyline. The characteristics of tetracycline degradation were investigated under a broad range of cultural conditions. Response surface methodology (RSM) predicted that the highest degradation rate of tetracycline could be obtained under the following conditions: 39.69 °C, pH of 8.79, and inoculum dose of 4.0% (v/v, ~3.5 × 106 cells/mL in the medium). In accordance with the five identified degradation products of tetracycline, two putative degradation pathways, which included the shedding of methyl and amino groups, were proposed. Moreover, the well diffusion method showed that the strain of M503 decreases the antibacterial potency of tetracycline, doxycycline, and chlorotetracycline. These findings proposed a putative mechanism of tetracycline degradation by a fungus strain and contributed to the estimation of the fate of tetracycline in the aquatic environment.

TaxisPy: A Python-based software for the quantitative analysis of bacterial chemotaxis.

Several species of bacteria are able to modify their swimming behavior in response to chemical attractants or repellents. Methods for the quantitative analysis of bacterial chemotaxis such as quantitative capillary assays are tedious and time-consuming. Computer-based video analysis of swimming bacteria represents a valuable method to directly assess their chemotactic response. Even though multiple studies have used this approach to elucidate various aspects of bacterial chemotaxis, to date, no computer software for such analyses is freely available. Here, we introduce TaxisPy, a Python-based software for the quantitative analysis of bacterial chemotaxis. The software comes with an intuitive graphical user interface and can be accessed easily through Docker on any operating system. Using a video of freely swimming cells as input, TaxisPy estimates the culture's average tumbling frequency over time. We demonstrate the utility of the software by assessing the effect of different concentrations of the attractant shikimate on the swimming behavior of Pseudomonas putida F1 and by capturing the adaptation process that Escherichia coli undergoes after being exposed to l-aspartate.

Bacterial catabolism of nicotine: Catabolic strains, pathways and modules.

Nicotine, the major alkaloid in tobacco, is a toxic, carcinogenic, and addictive compound. In recent years, nicotine catabolism in prokaryotes, including the catabolic pathways for its degradation and the catabolic genes that encode the enzymes of these pathways, have been systemically investigated. In this review, the three known pathways for nicotine catabolism in bacteria are summarized: the pyridine pathway, the pyrrolidine pathway, and a variation of the pyridine and pyrrolidine pathway (VPP pathway). The three nicotine catabolic pathways appear to have evolved separately in three distantly related lineages of bacteria. However, the general mechanism for the breakdown of the nicotine molecule in all three pathways is conserved and can be divided into six major enzymatic steps or catabolic modules that involve hydroxylation of the pyridine ring, dehydrogenation of the pyrrolidine ring, cleavage of the side chain, cleavage of the pyridine ring, dehydrogenation of the side chain, and deamination of pyridine ring-lysis products. In addition to summarizing our current understanding of nicotine degradation pathways, we identified several potential nicotine-degrading bacteria whose genome sequences are in public databases by comparing the sequences of conserved catabolic enzymes. Finally, several uncharacterized genes that are colocalized with nicotine degradation genes and are likely to be involved in nicotine catabolism, including regulatory genes, methyl-accepting chemotaxis protein genes, transporter genes, and cofactor genes are discussed. This review provides a comprehensive overview of the catabolism of nicotine in prokaryotes and highlights aspects of the process that still require additional research.

Biotransformation of the herbicide nicosulfuron residues in soil and seven sulfonylurea herbicides by Bacillus subtilis YB1: A climate chamber study.

Bacillus subtilis YB1 is a strain that can efficiently transform nicosulfuron. In order to study its remediation ability and effects on other microorganisms in the soil, indoor biological remediation experiments and rhizosphere microbial diversity analysis were performed. B. subtilis YB1 granules were prepared and applied to the nicosulfuron contaminated soil. The concentration of nicosulfuron was detected by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) and changes in the physiological indicators of wheat were measured. At the same time, the changes in the rhizosphere soil microbial diversity were determined by 16S RNA sequencing. Results showed that the YB1 granules made a contribution to the transformation of nicosulfuron (0.05 mg kg-1) in the soil within 55 days. The physiological indicators of wheat also showed consistent result about nicosulfuron transformation. Rhizosphere soil microbial diversity results indicated the relative abundance of Firmicutes decreased (3.0%-0.35%) and Acidobacteria first decreased (25.82%-22.38%) and then increased (22.3%-26.1%) with nicosulfuron added (N group). The relative abundance of Acidobacteria first decreased (25.8%-15.3%) and then increased (15.3%-21.7%) while Proteobacteria increased (26.5%-38.08%). At the same time, Firmicutes first increased (2.6%-12.3%) and then decreased to original level (12.3%-0.7%) in the N group with YB1 granules (NYB1 group). Members of the genus Bacillus initially increased and then decreased to the original level as the Control group, therefore, they did not become dominant in the rhizosphere soil. Alpha diversity analyses showed no obvious differences in species diversity among the N, NYB1 and Control groups. So YB1 did not have obvious influence on the rhizosphere microbial community structure during nicosulfuron transformation, which only had some effect on species abundance. This study revealed the successful indoor bioremediation of nicosulfuron in the soil, providing a potential strategy for solving the problem of nicosulfuron contamination.

Pathway and kinetics of malachite green biodegradation by Pseudomonas veronii.

Malachite green is a common environmental pollutant that poses a great threat to non-target organisms, including humans. This study reports the characterization of a bacterial strain, Pseudomonas veronii JW3-6, which was isolated from a malachite green enrichment culture. This strain degraded malachite green efficiently in a wide range of temperature and pH levels. Under optimal degradation conditions (32.4 °C, pH 7.1, and inoculum amount of 2.5 × 107 cfu/mL), P. veronii JW3-6 could degrade 93.5% of 50 mg/L malachite green within seven days. Five intermediate products from the degradation of malachite green were identified: leucomalachite green, 4-(dimethylamino) benzophenone, 4-dimethylaminophenol, benzaldehyde, and hydroquinone. We propose a possible degradation pathway based on these findings. The present study is the first to report the degradation of malachite green by P. veronii and the identification of hydroquinone as a metabolite in the degradation pathway.

Draft Genome Sequence of the Caffeine-Degrading Methylotroph Methylorubrum populi Pinkel.

A pink-pigmented facultative methylotroph, Methylorubrum populi Pinkel, was isolated from compost by selective enrichment with caffeine (3,5,7-trimethylxanthine) as the sole carbon, nitrogen, and energy source. We report here its high-quality draft genome sequence, assembled in 35 contigs totaling 5,630,907 bp. We identified 5,681 protein-coding sequences, including those putatively involved in caffeine degradation.

Chemotaxis of Pseudomonas putida F1 to Alcohols Is Mediated by the Carboxylic Acid Receptor McfP.

Although alcohols are toxic to many microorganisms, they are good carbon and energy sources for some bacteria, including many pseudomonads. However, most studies that have examined chemosensory responses to alcohols have reported that alcohols are sensed as repellents, which is consistent with their toxic properties. In this study, we examined the chemotaxis of Pseudomonas putida strain F1 to n-alcohols with chain lengths of 1 to 12 carbons. P. putida F1 was attracted to all n-alcohols that served as growth substrates (C2 to C12) for the strain, and the responses were induced when cells were grown in the presence of alcohols. By assaying mutant strains lacking single or multiple methyl-accepting chemotaxis proteins, the receptor mediating the response to C2 to C12 alcohols was identified as McfP, the ortholog of the P. putida strain KT2440 receptor for C2 and C3 carboxylic acids. Besides being a requirement for the response to n-alcohols, McfP was required for the response of P. putida F1 to pyruvate, l-lactate, acetate, and propionate, which are detected by the KT2440 receptor, and the medium- and long-chain carboxylic acids hexanoic acid and dodecanoic acid. ß-Galactosidase assays of P. putida F1 carrying an mcfP-lacZ transcriptional fusion showed that the mcfP gene is not induced in response to alcohols. Together, our results are consistent with the idea that the carboxylic acids generated from the oxidation of alcohols are the actual attractants sensed by McfP in P. putida F1, rather than the alcohols themselves.IMPORTANCE Alcohols, released as fermentation products and produced as intermediates in the catabolism of many organic compounds, including hydrocarbons and fatty acids, are common components of the microbial food web in soil and sediments. Although they serve as good carbon and energy sources for many soil bacteria, alcohols have primarily been reported to be repellents rather than attractants for motile bacteria. Little is known about how alcohols are sensed by microbes in the environment. We report here that catabolizable n-alcohols with linear chains of up to 12 carbons serve as attractants for the soil bacterium Pseudomonas putida, and rather than being detected directly, alcohols appear to be catabolized to acetate, which is then sensed by a specific cell-surface chemoreceptor protein.

Hybrid Two-Component Sensors for Identification of Bacterial Chemoreceptor Function.

Soil bacteria adapt to diverse and rapidly changing environmental conditions by sensing and responding to environmental cues using a variety of sensory systems. Two-component systems are a widespread type of signal transduction system present in all three domains of life and typically are comprised of a sensor kinase and a response regulator. Many two-component systems function by regulating gene expression in response to environmental stimuli. The bacterial chemotaxis system is a modified two-component system with additional protein components and a response that, rather than regulating gene expression, involves behavioral adaptation and results in net movement toward or away from a chemical stimulus. Soil bacteria generally have 20 to 40 or more chemoreceptors encoded in their genomes. To simplify the identification of chemoeffectors (ligands) sensed by bacterial chemoreceptors, we constructed hybrid sensor proteins by fusing the sensor domains of Pseudomonas putida chemoreceptors to the signaling domains of the Escherichia coli NarX/NarQ nitrate sensors. Responses to potential attractants were monitored by ß-galactosidase assays using an E. coli reporter strain in which the nitrate-responsive narG promoter was fused to lacZ Hybrid receptors constructed from PcaY, McfR, and NahY, which are chemoreceptors for aromatic acids, tricarboxylic acid cycle intermediates, and naphthalene, respectively, were sensitive and specific for detecting known attractants, and the ß-galactosidase activities measured in E. coli correlated well with results of chemotaxis assays in the native P. putida strain. In addition, a screen of the hybrid receptors successfully identified new ligands for chemoreceptor proteins and resulted in the identification of six receptors that detect propionate.IMPORTANCE Relatively few of the thousands of chemoreceptors encoded in bacterial genomes have been functionally characterized. More importantly, although methyl-accepting chemotaxis proteins, the major type of chemoreceptors present in bacteria, are easily identified bioinformatically, it is not currently possible to predict what chemicals will bind to a particular chemoreceptor. Chemotaxis is known to play roles in biodegradation as well as in host-pathogen and host-symbiont interactions, but many studies are currently limited by the inability to identify relevant chemoreceptor ligands. The use of hybrid receptors and this simple E. coli reporter system allowed rapid and sensitive screening for potential chemoeffectors. The fusion site chosen for this study resulted in a high percentage of functional hybrids, indicating that it could be used to broadly test chemoreceptor responses from phylogenetically diverse samples. Considering the wide range of chemical attractants detected by soil bacteria, hybrid receptors may also be useful as sensitive biosensors.

Phenotype-centric modeling for elucidation of biological design principles.

A recently developed 'phenotype-centric' modeling strategy combines four innovations with the potential to advance our understanding of complex biological systems: (1) a rigorous mathematical definition of biochemical phenotypes, (2) a method for enumerating the phenotypic repertoire based on the biomolecular network architecture, (3) an integrated suite of computational algorithms for the efficient prediction of parameter values and analysis of the phenotypic repertoire, and (4) a user-focused environment for navigating the resulting space of phenotypes and identifying biologically relevant features and system design principles. These innovations will facilitate deterministic and stochastic simulations that require parameter values, will accelerate both hypothesis discrimination in systems biology and the design cycle in synthetic biology. Here we first review the fundamental definition of biochemical phenotype that enables this new modeling strategy and give an overview of the strategy using a simple system from phage λ to provide an example of a global design principle. Second, we illustrate this approach in more detail with an application to a common network architecture involving positive and negative feedback. We report system design principles related to the global tolerances of this system's phenotypes. Finally, we apply the phenotype-centric strategy to a logic network and compare the results with those obtained from a Boolean approach. Mechanistic and Boolean models have well-documented complementary advantages and disadvantages. Mechanistic models have the advantage of being biologically realistic; however, they also are limited by the large number of kinetic parameters whose values are largely unknown. Boolean models have the advantage of being parameter free; however, they also are limited by the absence of well-known physical and chemical constraints. We show that the phenotype-centric modeling strategy combines advantages of both.

Chemotaxis to Atypical Chemoattractants by Soil Bacteria.

Although the mechanism of bacterial chemotaxis has been extensively studied in enteric bacteria, the hunt for novel and atypical chemoeffectors (in enterics and distantly-related species alike) has necessitated the modification of classic chemotaxis assays to deal with recalcitrant and potentially toxic chemicals. Here, we describe detailed protocols for the quantitative and qualitative assessment of chemotaxis responses that are categorized into short-term direct population response assays and long-term metabolism-based assays that can be used to identify novel chemoeffector molecules and the specific chemoreceptors involved. We emphasize the importance of behavior-based assays to verify the biochemical and physiological relevance of newly identified chemoeffector-receptor pairs.

Genome Sequence of Pseudomonas putida Strain ASAD, an Acetylsalicylic Acid-Degrading Bacterium.

Pseudomonas putida strain ASAD was isolated from compost because of its ability to utilize aspirin (acetylsalicylic acid) as a carbon and energy source. We report the draft genome sequence of strain ASAD, with an estimated length of 6.9 Mb. Study of this isolate will provide insight into the aspirin biodegradation pathway.

Pseudomonas putida F1 uses energy taxis to sense hydroxycinnamic acids.

Soil bacteria such as pseudomonads are widely studied due to their diverse metabolic capabilities, particularly the ability to degrade both naturally occurring and xenobiotic aromatic compounds. Chemotaxis, the directed movement of cells in response to chemical gradients, is common in motile soil bacteria and the wide range of chemicals detected often mirrors the metabolic diversity observed. Pseudomonas putida F1 is a soil isolate capable of chemotaxis toward, and degradation of, numerous aromatic compounds. We showed that P. putida F1 is capable of degrading members of a class of naturally occurring aromatic compounds known as hydroxycinnamic acids, which are components of lignin and are ubiquitous in the soil environment. We also demonstrated the ability of P. putida F1 to sense three hydroxycinnamic acids: p-coumaric, caffeic and ferulic acids. The chemotaxis response to hydroxycinnamic acids was induced during growth in the presence of hydroxycinnamic acids and was negatively regulated by HcaR, the repressor of the hydroxycinnamic acid catabolic genes. Chemotaxis to the three hydroxycinnamic acids was dependent on catabolism, as a mutant lacking the gene encoding feruloyl-CoA synthetase (Fcs), which catalyzes the first step in hydroxycinnamic acid degradation, was unable to respond chemotactically toward p-coumaric, caffeic, or ferulic acids. We tested whether an energy taxis mutant could detect hydroxycinnamic acids and determined that hydroxycinnamic acid sensing is mediated by the energy taxis receptor Aer2.

Direct sensing and signal transduction during bacterial chemotaxis toward aromatic compounds in Comamonas testosteroni.

Micro-organisms sense and chemotactically respond to aromatic compounds. Although the existence of chemoreceptors that bind to aromatic attractants and subsequently trigger chemotaxis have long been speculated, such a chemoreceptor has not been demonstrated. In this report, we demonstrated that the chemoreceptor MCP2901 from Comamonas testosteroni CNB-1 binds to aromatic compounds and initiates downstream chemotactic signaling in addition to its ability to trigger chemotaxis via citrate binding. The function of gene MCP2901 was investigated by genetic deletion from CNB-1 and genetic complementation of the methyl-accepting chemotaxis protein (MCP)-null mutant CNB-1Δ20. Results showed that the expression of MCP2901 in the MCP-null mutant restored chemotaxis toward nine tested aromatic compounds and nine carboxylic acids. Isothermal titration calorimetry (ITC) analyses demonstrated that the ligand-binding domain of MCP2901 (MCP2901LBD) bound to citrate, and weakly to gentisate and 4-hydroxybenzoate. Additionally, ITC assays indicated that MCP2901LBD bound strongly to 2,6-dihydroxybenzoate and 2-hydroxybenzoate, which are isomers of gentisate and 4-hydroxybenzoate respectively that are not metabolized by CNB-1. Agarose-in-plug and capillary assays showed that these two molecules serve as chemoattractants for CNB-1. Through constructing membrane-like MCP2901-inserted Nanodiscs and phosphorelay activity assays, we demonstrated that 2,6-dihydroxybenzoate and 2-hydroxybenzoate altered kinase activity of CheA. This is the first evidence of an MCP binding to an aromatic molecule and triggering signal transduction for bacterial chemotaxis.

Substrate and Enzyme Specificity of the Kinetic Isotope Effects Associated with the Dioxygenation of Nitroaromatic Contaminants.

Compound-specific isotope analysis (CSIA) is a promising approach for tracking biotransformation of organic pollutants, but isotope fractionation associated with aromatic oxygenations is only poorly understood. We investigated the dioxygenation of a series of nitroaromatic compounds to the corresponding catechols by two enzymes, namely, nitrobenzene and 2-nitrotoluene dioxygenase (NBDO and 2NTDO) to elucidate the enzyme- and substrate-specificity of C and H isotope fractionation. While the apparent (13)C- and (2)H-kinetic isotope effects of nitrobenzene, nitrotoluene isomers, 2,6-dinitrotoluene, and naphthalene dioxygenation by NBDO varied considerably, the correlation of C and H isotope fractionation revealed a common mechanism for nitrobenzene and nitrotoluenes. Similar observations were made for the dioxygenation of these substrates by 2NTDO. Evaluation of reaction kinetics, isotope effects, and commitment-to-catalysis based on experiment and theory showed that rates of dioxygenation are determined by the enzymatic O2 activation and aromatic C oxygenation. The contribution of enzymatic O2 activation to the reaction rate varies for different nitroaromatic substrates of NBDO and 2NTDO. Because aromatic dioxygenation by nonheme iron dioxygenases is frequently the initial step of biodegradation, O2 activation kinetics may also have been responsible for the minor isotope fractionation reported for the oxygenation of other aromatic contaminants.