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Because anthropogenic nitroaromatic compounds have entered the biosphere relatively recently, exploration of the recently evolved catabolic pathways can provide clues for adaptive evolutionary mechanisms in bacteria. The concept that nitroarene dioxygenases shared a common ancestor with naphthalene dioxygenase is well established. Diaphorobacter sp. strain JS3051 utilizes 2,3-dichloronitrobenzene (23DCNB), a toxic anthropogenic compound, as the sole carbon, nitrogen, and energy source for growth, but the metabolic pathway and its origins are unknown. Here, we establish that a gene cluster ( dcb ), encoding a Nag-like dioxygenase, is responsible for the initial oxidation of the 23DCNB molecule. The 2,3-dichloronitrobenzene dioxygenase system (DcbAaAbAcAd) catalyzes conversion of 23DCNB to 3,4-dichlorocatechol (34DCC). Site-directed mutagenesis studies indicated that residue 204 of DcbAc is crucial for the substrate specificity of 23DCNB dioxygenase. The presence of glutamic acid at position 204 of 23DCNB dioxygenase is unique among Nag-like dioxygenases. Genetic, biochemical, and structural evidence indicate that the 23DCNB dioxygenase is more closely related to 2-nitrotoluene dioxygenase from Acidovorax sp. strain JS42 than to the 34DCNB dioxygenase from Diaphorobacter sp. strain JS3050, which was isolated from the same site as strain JS3051. A gene cluster ( dcc ) encoding the enzymes for 34DCC catabolism, homologous to a clc operon in Pseudomonas knackmussii strain B13, is also on the chromosome at a distance of 2.5 Mb from the dcb genes. Heterologously expressed DccA catalyzed ring cleavage of 34DCC with high affinity and catalytic efficiency. This work not only establishes the molecular mechanism for 23DCNB mineralization, but also enhances the understanding of the recent evolution of the catabolic pathways for nitroarenes. IMPORTANCE Because anthropogenic nitroaromatic compounds have entered the biosphere relatively recently, exploration of the recently evolved catabolic pathways can provide clues for adaptive evolutionary mechanisms in bacteria. The concept that nitroarene dioxygenases shared a common ancestor with naphthalene dioxygenase is well established. But their phylogeny and how they evolved in response to novel nitroaromatic compounds are largely unknown. Elucidation of the molecular basis for 23DCNB degradation revealed that the catabolic pathways of two DCNB isomers in different isolates from the same site were derived from different recent origins. Integrating structural models of catalytic subunits and enzymatic activities data provided new insight about how recently modified enzymes were selected depending on the structure of new substrates. This study enhances understanding and prediction of adaptive evolution of catabolic pathways in bacteria in response to new chemicals.

Iron plaque is a strong adsorbent on rice roots, acting as a barrier to prevent metal uptake by rice. However, the role of root iron plaque microbes in governing metal redox cycling and metal bioavailability is unknown. In this study, the microbial community structure on the iron plaque of rice roots from an arsenic-contaminated paddy soil was explored using high-throughput next-generation sequencing. The microbial composition and diversity of the root iron plaque were significantly different from those of the bulk and rhizosphere soils. Using the aoxB gene as an identifying marker, we determined that the arsenite-oxidizing microbiota on the iron plaque was dominated by Acidovorax and Hydrogenophaga -affiliated bacteria. More importantly, the abundance of arsenite-oxidizing bacteria (AsOB) on the root iron plaque was significantly negatively correlated with the arsenic concentration in the rice root, straw and grain, indicating that the microbes on the iron plaque, particularly the AsOB, were actively catalyzing arsenic transformation and greatly influencing metal uptake by rice. This exploratory research represents a preliminary examination of the microbial community structure of the root iron plaque formed under arsenic pollution and emphasizes the importance of the root iron plaque environment in arsenic biogeochemical cycling compared with the soil-rhizosphere biotope.

ObjectivesCatalytic promiscuity, or the ability to catalyze a secondary reaction, provides new opportunities for industrial biocatalysis by expanding the range of biocatalytic reactions. Some nitrilases converting nitriles to amides, referred to as the secondary activity, show great potential for amides production. And our goal was exploiting the amide-forming potential of nitrilases.ResultsIn this study, we characterized and altered the secondary activity of nitrilase from Acidovorax facilis 72 W (Nit72W) towards different substrates. We increased the secondary activity of Nit72W towards 2-cyanopyridine by 196-fold and created activity toward benzonitrile and p-nitrophenylacetonitrile by modifying the active pocket. Surprisingly, the best mutant, W188M, completely converted 250 mM 2-cyanopyridine to more than 98% 2-picolinamide in 12 h with a specific activity of 90 U/mg and showed potential for industrial applications.ConclusionsNit72W was modified to increase its secondary activity for the amides production, especially 2-picolinamide.

3-nitrotoluene dioxygenase (3NTDO) from Diaphorobacter sp. strain DS2 catalyses the conversion of 3-nitrotoluene (3NT) into a mixture of 3- and 4-methylcatechols with release of nitrite. We report here, X-ray crystal structures of oxygenase and ferredoxin components of 3NTDO at 2.9 Å and 2.4 Å, respectively. The residues responsible for nitrite release in 3NTDO were further probed by four single and two double mutations in the catalytic site of α-subunit of the dioxygenase. Modification of Val 350 to Phe, Ile 204 to Ala, and Asn258 to Val by site directed mutagenesis resulted in inactive enzymes revealing the importance of these residues in catalysis. Docking studies of meta nitrotoluene to the active site of 3NTDO suggested possible orientations of binding that favor the formation of 3-methylcatechol (3MC) over 4-methylcatechol energetically. The electron transfer pathway from ferredoxin subunit to the active site of the oxygenase subunit is also proposed.

Ten phenol-degrading bacterial strains were isolated from three geographically distant environments. Five of them, identified as Diaphorobacter, Acidovorax, Acinetobacter (two strains), and Corynebacterium, could additionally transform pyridine, through the transcription of phenol hydroxylase genes induced both by phenol and pyridine. HPLC-UV and LC-MS analyses indicated that one metabolite (m/e = 96.07) with the same molecular weight as monohydroxylated pyridine was produced from the five phenol-degrading strains, when pyridine was the sole carbon source. Phenol (50 mg l⁻¹) could initially inhibit and later stimulate the pyridine transformation. In addition, heterologous expression of the phenol hydroxylase gene (pheKLMNOP) resulted in the detection of monohydroxylated pyridine, which confirmed the phenol hydroxylase could catalyze pyridine hydroxylation. Phylogeny of the phenol hydroxylase genes revealed that the genes from the five pyridine-hydroxylating strains form a clade with each other and with those catalyzing the hydroxylation of phenol, BTEX (acronym of benzene, toluene, ethylbenzene, and xylene), and trichloroethylene. These results suggest that pyridine transformation via hydroxylation by phenol hydroxylase may be prevalent in environments than expected.

Nitrilase is the member of carbon–nitrogen hydrogen hydrolase superfamily, which has been widely used for the hydrolysis of nitriles into corresponding carboxylic acids. But most nitrilases are plagued by product inhibition in the industrial application. In this study, a “super nitrilase mutant” of nitrilase with high activity, thermostability and improved product tolerance from Acidovorax facilis ZJB09122 was characterized. Then, an efficient process was developed by employing the whole cell of recombinant E. coli for the conversion of high concentration of 1-cyanocyclohexylacetonitrile-to-1-cyanocyclohexaneacetic acid, an important intermediate of gabapentin. Under the optimized conditions, the higher substrate concentrations such as 1.3 M, 1.5 M and 1.8 M could be hydrolyzed by 13.58 g DCW/L with outstanding productivity (> 740 g/L/day). This study developed a highly efficient bioprocess for the preparation of 1-cyanocyclohexaneacetic acid which has the great potential for industrial application.

Ramlibacter tataouinensis TTB310(T) (strain TTB310), a betaproteobacterium isolated from a semi-arid region of South Tunisia (Tataouine), is characterized by the presence of both spherical and rod-shaped cells in pure culture. Cell division of strain TTB310 occurs by the binary fission of spherical \"cyst-like\" cells (\"cyst-cyst\" division). The rod-shaped cells formed at the periphery of a colony (consisting mainly of cysts) are highly motile and colonize a new environment, where they form a new colony by reversion to cyst-like cells. This unique cell cycle of strain TTB310, with desiccation tolerant cyst-like cells capable of division and desiccation sensitive motile rods capable of dissemination, appears to be a novel adaptation for life in a hot and dry desert environment. In order to gain insights into strain TTB310's underlying genetic repertoire and possible mechanisms responsible for its unusual lifestyle, the genome of strain TTB310 was completely sequenced and subsequently annotated. The complete genome consists of a single circular chromosome of 4,070,194 bp with an average G+C content of 70.0%, the highest among the Betaproteobacteria sequenced to date, with total of 3,899 predicted coding sequences covering 92% of the genome. We found that strain TTB310 has developed a highly complex network of two-component systems, which may utilize responses to light and perhaps a rudimentary circadian hourglass to anticipate water availability at the dew time in the middle/end of the desert winter nights and thus direct the growth window to cyclic water availability times. Other interesting features of the strain TTB310 genome that appear to be important for desiccation tolerance, including intermediary metabolism compounds such as trehalose or polyhydroxyalkanoate, and signal transduction pathways, are presented and discussed.

A 1-aminocyclopropane-1-carboxylate deaminase producing bacterium, designated GXGD002ᵀ, was isolated from the rhizosphere of banana plants cultivated in Guangxi province, China. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain GXGD002ᵀis a member of the genus Variovorax. High levels of 16S rRNA gene sequence similarity are found between strain GXGD002ᵀand Variovorax paradoxus DSM 30034ᵀ(99.4 %), Variovorax ginsengisoli KCTC 12583ᵀ(99.1 %), Variovorax boronicumulans KCTC 22010ᵀ(99.0 %), Variovorax soli DSM18216ᵀ(98.7 %), Variovorax defluvii DSM 27259ᵀ(98.1 %) and Variovorax dokdonensis KCTC 12544ᵀ(97.4 %) respectively. However, the DNA–DNA hybridization values between strain GXGD002ᵀand its closely related species V. paradoxus DSM 30034ᵀ, V. ginsengisoli KCTC 12583ᵀand V. boronicumulans KCTC 22010ᵀwere found to be 40.7, 30.9 and 23.7 %, respectively. The DNA G + C content of strain GXGD002ᵀwas found to be 67.8 mol%. The major fatty acids of strain GXGD002ᵀare C₁₆:₀(20.3 %), C₁₀:₀3OH (18.4 %), C₁₇:₀cyclo (18.9 %), C₁₈:₁w₇c (12.3 %) and summed feature 3 (13.9 %). The predominant respiratory quinone was identified as ubiquinone-8 (Q-8) and the major polar lipids as phosphatidylethanolamine and phosphatidylglycerol. The results of polyphasic taxonomic study including physiological and biochemical tests, whole-cell SDS-PAGE profiles and chemotaxonomic analysis allowed a clear differentiation of strain GXGD002ᵀfrom the other species in the genus Variovorax. Based on these results, a new species, Variovorax guangxiensis, is proposed. The type strain is GXGD002ᵀ(=DSM 27352ᵀ = ACCC 05911ᵀ).

Polyhydroxyalkanoate depolymerase derived from Variovorax sp. C34 (PhaZVₛ) was identified as the first enzyme that is capable of degrading isotactic P[67 mol% (R)-lactate(LA)-co-(R)-3-hydroxybutyrate(3HB)] [P(D-LA-co-D-3HB)]. This study aimed at analyzing the monomer sequence specificity of PhaZVₛ for hydrolyzing P(LA-co-3HB) in comparison with a P(3HB) depolymerase from Alcaligenes faecalis T1 (PhaZAf) that did not degrade the same copolymer. Degradation of P(LA-co-3HB) by action of PhaZVₛ generated dimers, 3HB-3HB, 3HB-LA, LA-3HB, and LA-LA, and the monomers, suggesting that PhaZVₛ cleaved the linkages between LA and 3HB units and between LA units. To provide a direct evidence for the hydrolysis of these sequences, the synthetic methyl trimers, 3HB-3HB-3HB, LA-LA-3HB, LA-3HB-LA, and 3HB-LA-LA, were treated with the PhaZs. Unexpectedly, not only PhaZVₛ but also PhaZAf hydrolyzed all of these substrates, namely PhaZAf also cleaved LA-LA linkage. Considering the fact that both PhaZs did not degrade P[(R)-LA] (PDLA) homopolymer, the cleavage capability of LA-LA linkage by PhaZs was supposed to depend on the length of the LA-clustering region in the polymer chain. To test this hypothesis, PDLA oligomers (6 to 40 mer) were subjected to the PhaZ assay, revealing that there was an inverse relationship between molecular weight of the substrates and their hydrolysis efficiency. Moreover, PhaZVₛ exhibited the degrading activity toward significantly longer PDLA oligomers compared to PhaZAf. Therefore, the cleaving capability of PhaZs used here toward the D-LA-based polymers containing the LA-clustering region was strongly associated with the substrate length, rather than the monomer sequence specificity of the enzyme.

Acidovorax sp. CHX100 has a remarkable ability for growth on short cycloalkanes (C5–C8) as a sole source of carbon and energy under aerobic conditions via an uncharacterized mechanism. Transposon mutagenesis of Acidovorax sp. CHX100 revealed a novel cytochrome P450 monooxygenase (CYP450chx) which catalyzed the transformation of cyclohexane to cyclohexanol. Primer walking methods categorized CYP450chx as cytochrome P450 class I taking into account its operon structure: monooxygenase, FAD oxidoreductase, and ferredoxin. CYP450chx was successfully cloned and expressed in Escherichia coli JM109. The activity of CYP450chx was demonstrated by means of the indole co-oxidation. Biotransformation capability of CYP450chx was confirmed through the catalysis of cycloalkanes (C5–C8) to their respective cyclic alcohols.