Although the microbial degradation of the six isomers of dimethylphenol has been extensively studied, the genetic and biochemical mechanisms of 2,6-DMP degradation remain unclear. This study identified the genes responsible for the initial step in the 2,6-DMP catabolic pathway in M. neoaurum B5-4.
Due to the extensive use of chloroacetanilide herbicides over the past 60 years, bacteria have evolved catabolic pathways to mineralize these compounds. In the upstream catabolic pathway, chloroacetanilide herbicides are transformed into the two common metabolites 2-methyl-6-ethylaniline (MEA) and 2,6-diethylaniline (DEA) through -dealkylation and amide hydrolysis. The pathway downstream of MEA is initiated by the hydroxylation of aromatic rings, followed by its conversion to a substrate for ring cleavage after several steps. Most of the key genes in the pathway have been identified. However, the genes involved in the initial hydroxylation step of MEA are still unknown. As a special aniline derivative, MEA cannot be transformed by the aniline dioxygenases that have been characterized. DE-13 can completely degrade MEA and use it as a sole carbon source for growth. In this work, an MEA degradation-deficient mutant of DE-13 was isolated. MEA catabolism genes were predicted through comparative genomic analysis. The results of genetic complementation and heterologous expression demonstrated that the products of and are responsible for the initial step of MEA degradation in DE-13. MeaXY is a two-component flavoprotein monooxygenase system that catalyzes the hydroxylation of MEA and DEA using NADH and flavin mononucleotide (FMN) as cofactors. Nuclear magnetic resonance (NMR) analysis confirmed that MeaXY hydroxylates MEA and DEA at the -position. Transcription of was enhanced remarkably upon induction of MEA or DEA in DE-13. Additionally, and were highly conserved among other MEA-degrading sphingomonads. This study fills a gap in our knowledge of the biochemical pathway that carries out mineralization of chloroacetanilide herbicides in sphingomonads. Much attention has been paid to the environmental fate of chloroacetanilide herbicides used for the past 60 years. Microbial degradation is considered an important mechanism in the degradation of these compounds. Bacterial degradation of chloroacetanilide herbicides has been investigated in many recent studies. Pure cultures or consortia able to mineralize these herbicides have been obtained. The catabolic pathway has been proposed, and most key genes involved have been identified. However, the genes responsible for the initiation step (from MEA to hydroxylated MEA or from DEA to hydroxylated DEA) of the downstream pathway have not been reported. The present study demonstrates that a two-component flavin-dependent monooxygenase system, MeaXY, catalyzes the -hydroxylation of MEA or DEA in sphingomonads. Therefore, this work finds a missing link in the biochemical pathway that carries out the mineralization of chloroacetanilide herbicides in sphingomonads. Additionally, the results expand our understanding of the degradation of a special kind of aniline derivative.
A novel Gram-positive, fluoroglycofen-degrading bacterium, designated cmg86(T), was isolated from herbicide contaminated soil collected from Tongjing, Jiangsu province, China. Strain cmg86(T) was found to be aerobic, motile, endospore-forming rods. Phylogenetic analyses based on 16S rRNA gene sequences indicated that strain cmg86(T) belongs to the genus Lysinibacillus and showed the highest sequence similarity to Lysinibacillus meyeri DSM 25057(T) (97.9 %) and Lysinibacillus odysseyi KCTC 3961(T) (96.6 %). The cell-wall peptidoglycan type was determined to be A4α (L-Lys-D-Asp), which is consistent with the cell-wall characteristics of the genus Lysinibacillus. The predominant respiratory quinones were identified as menaquinone-7 (MK-7, 89.5 %) and meanaquinone-6 (MK-6, 8.9 %), and the major fatty acids were identified as iso-C15:0, anteiso-C15:0 and antesio-C17:0. The major polar lipids were found to be phosphatidylglycerol, diphosphatidylglycerol and phosphatidylethanolamine. The genomic DNA G+C content of strain cmg86(T) was determined to be 37.6 mol%. The results of this study support the conclusion that strain cmg86(T) represents a novel species of the genus Lysinibacillus for which the name and Lysinibacillus fluoroglycofenilyticus sp. nov. is proposed. The type strain is cmg86(T) (=KCTC 33183(T) = CCTCC AB 2013247(T)).
Due to their fast growth rate and robustness, some haloalkalitolerant methanotrophs from the genus Methylotuvimicrobium have recently become not only promising biocatalysts for methane conversion but also favorable materials for obtaining fundamental knowledge on methanotrophs. Here, to realize unmarked genome modification in Methylotuvimicrobium bacteria, a counterselectable marker (CSM) was developed based on pheS, which encodes the α-subunit of phenylalanyl-tRNA synthetase. Two-point mutations (T252A and A306G) were introduced into PheS in Methylotuvimicrobium buryatense 5GB1C, generating PheS AG , which can recognize p-chloro-phenylalanine (p-Cl-Phe) as a substrate. Theoretically, the expression of PheS AG in a cell will result in the incorporation of p-Cl-Phe into proteins, leading to cell death. The P tac promoter and the ribosome-binding site region of mmoX were employed to control pheS AG , producing the pheS AG-3 CSM. M. buryatense 5GB1C harboring pheS AG-3 was extremely sensitive to 0.5 mM p-Cl-Phe. Then, a positive and counterselection cassette, PZ (only 1.5 kb in length), was constructed by combining pheS AG-3 and the zeocin resistance gene. A PZ-and PCR-based strategy was used to create the unmarked deletion of glgA1 or the whole smmo operon in M. buryatense 5GB1C and Methylotuvimicrobium alcaliphilum 20Z. The positive rates were over 92%, and the process could be accomplished in as few as eight days.
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. Expected final online publication date for the Annual Review of Microbiology, Volume 76 is September 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
The bacterial hydrolytic dehalogenation of 4-chlorobenzoate (4CBA) is a CoA-activation type catabolic pathway that is usually a common part of the microbial mineralization of chlorinated aromatic compounds. Previous studies have shown that the transport and dehalogenation genes for 4CBA are typically clustered as an fcbBAT1T2T3C operon and are inducibly expressed in response to 4CBA. However, the associated molecular mechanism remains unknown. In this study, a gene (fcbR) adjacent to the fcb operon was predicted to encode a TetR-type transcriptional regulator in strain Comamonas sediminis CD-2. The fcbR knockout strain exhibited constitutive expression of the fcb cluster. In the host E. coli, the expression of the Pfcb-fused gfp reporter was repressed by the introduction of the fcbR gene, and genetic studies combining various catabolic genes suggest that the FcbR ligand may be an intermediate metabolite. Purified FcbR could bind to the Pfcb DNA probe in vitro, and the metabolite 4-chlorobenzyl-CoA (4CBA-CoA) prevented FcbR binding to the Pfcb DNA probe. Isothermal titration calorimetry (ITC) measurements showed that 4CBA-CoA could bind to FcbR at a 1:1 mole ratio. DNase I footprinting showed that FcbR protected a 42-bp DNA motif (5′-GGAAATCAATAGGTCCATAGAAAATCTATTGACTAATCGAAT-3′) that consists of two sequence repeats containing four pseudo-palindromic sequences (5’-TCNATNGA-3’). This binding motif overlaps with the -35 box of Pfcb and was proposed to prevent the binding of RNA polymerase. This study identified a transcriptional repressor and its ligand of the fcb operon, extending halogenated benzoyl-CoA as a member of known ligands of transcriptional regulators. Importance The bacterial hydrolytic dehalogenation of 4CBA is a special CoA-activation type catabolic pathway, which plays an important role in the biodegradation of polychlorinated biphenyls and many certain herbicides. With genetic and biochemical approaches, the present study identified the transcriptional repressor and its cognate effector of a 4CBA hydrolytic dehalogenation operon. This work extends halogenated benzoyl-CoA as a new member of CoA-derived effector compounds that mediate allosteric regulation of transcriptional regulators.
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