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.

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.

Biodegradation of 3-Chloronitrobenzene and 3-Bromonitrobenzene by Diaphorobacter sp. Strain JS3051.

Halonitrobenzenes are toxic chemical intermediates used widely for industrial synthesis of dyes and pesticides. Bacteria able to degrade 2- and 4-chloronitrobenzene have been isolated and characterized; in contrast, no natural isolate has been reported to degrade meta-halonitrobenzenes. In this study, Diaphorobacter sp. strain JS3051, previously reported to degrade 2,3-dichloronitrobenzene, grew readily on 3-chloronitrobenzene and 3-bromonitrobenzene, but not on 3-fluoronitrobenzene, as sole sources of carbon, nitrogen, and energy. A Rieske nonheme iron dioxygenase (DcbAaAbAcAd) catalyzed the dihydroxylation of 3-chloronitrobenzene and 3-bromonitrobenzene, resulting in the regiospecific production of ring-cleavage intermediates 4-chlorocatechol and 4-bromocatechol. The lower activity and relaxed regiospecificity of DcbAaAbAcAd toward 3-fluoronitrobenzene is likely due to the higher electronegativity of the fluorine atom, which hinders it from interacting with E204 residue at the active site. DccA, a chlorocatechol 1,2-dioxygenase, converts 4-chlorocatechol and 4-bromocatechol into the corresponding halomuconic acids with high catalytic efficiency, but with much lower Kcat/Km values for fluorocatechol analogues. The results indicate that the Dcb and Dcc enzymes of Diaphorobacter sp. strain JS3051 can catalyze the degradation of 3-chloro- and 3-bromonitrobenzene in addition to 2,3-dichloronitrobenzene. The ability to utilize multiple substrates would provide a strong selective advantage in a habitat contaminated with mixtures of chloronitrobenzenes. IMPORTANCE Halonitroaromatic compounds are persistent environmental contaminants, and some of them have been demonstrated to be degraded by bacteria. Natural isolates that degrade 3-chloronitrobenzene and 3-bromonitrobenzene have not been reported. In this study, we report that Diaphorobacter sp. strain JS3051 can degrade 2,3-dichloronitrobenzene, 3-chloronitrobenzene, and 3-bromonitrobenzene using the same catabolic pathway, whereas it is unable to grow on 3-fluoronitrobenzene. Based on biochemical analyses, it can be concluded that the initial dioxygenase and lower pathway enzymes are inefficient for 3-fluoronitrobenzene and even misroute the intermediates, which is likely responsible for the failure to grow. These results advance our understanding of how the broad substrate specificities of catabolic enzymes allow bacteria to adapt to habitats with mixtures of xenobiotic contaminants.

Discovery of a Ni2+-dependent heterohexameric metformin hydrolase.

The biguanide drug metformin is a first-line blood glucose-lowering medication for type 2 diabetes, leading to its presence in the global environment. However, little is known about the fate of metformin by microbial catabolism. Here, we characterize a Ni2+-dependent heterohexameric enzyme (MetCaCb) from the ureohydrolase superfamily, catalyzing the hydrolysis of metformin into guanylurea and dimethylamine. Either subunit alone is catalytically inactive, but together they work as an active enzyme highly specific for metformin. The crystal structure of the MetCaCb complex shows the coordination of the binuclear metal cluster only in MetCa, with MetCb as a protein binder of its active cognate. An in-silico search and functional assay discover a group of MetCaCb-like protein pairs exhibiting metformin hydrolase activity in the environment. Our findings not only establish the genetic and biochemical foundation for metformin catabolism but also provide additional insights into the adaption of the ancient enzymes toward newly occurred substrate.

Optimized strategy for the control and prevention of newly emerging influenza revealed by the spread dynamics model.

No matching vaccine is immediately available when a novel influenza strain breaks out. Several nonvaccine-related strategies must be employed to control an influenza epidemic, including antiviral treatment, patient isolation, and immigration detection. This paper presents the development and application of two regional dynamic models of influenza with Pontryagin's Maximum Principle to determine the optimal control strategies for an epidemic and the corresponding minimum antiviral stockpiles. Antiviral treatment was found to be the most effective measure to control new influenza outbreaks. In the case of inadequate antiviral resources, the preferred approach was the centralized use of antiviral resources in the early stage of the epidemic. Immigration detection was the least cost-effective; however, when used in combination with the other measures, it may play a larger role. The reasonable mix of the three control measures could reduce the number of clinical cases substantially, to achieve the optimal control of new influenza.