The MocR family transcriptional regulator DnfR has multiple binding sites and regulates Dirammox gene transcription in Alcaligenes faecalis JQ135.

Microbial ammonia oxidation is vital to the nitrogen cycle. A biological process, called Dirammox (direct ammonia oxidation, NH3 →NH2 OH→N2 ), has been recently identified in Alcaligenes ammonioxydans and Alcaligenes faecalis. However, its transcriptional regulatory mechanism has not yet been fully elucidated. The present study characterized a new MocR-like transcription factor DnfR that is involved in the Dirammox process in A. faecalis strain JQ135. The entire dnf cluster was composed of 10 genes and transcribed as five transcriptional units, that is, dnfIH, dnfR, dnfG, dnfABCDE and dnfF. DnfR activates the transcription of dnfIH, dnfG and dnfABCDE genes, and represses its own transcription. The intact 1506-bp dnfR gene was required for activation of Dirammox. Electrophoretic mobility shift assays and DNase I footprinting analyses showed that DnfR has one binding site in the dnfH-dnfR intergenic region and two binding sites in the dnfG-dnfA intergenic region. Three binding sites of DnfR shared a 6-bp repeated conserved sequence 5'-GGTCTG-N17 -GGTCTG-3' which was essential for the transcription of downstream target genes. Cysteine and glutamate act as possible effectors of DnfR to activate the transcription of transcriptional units of dnfG and dnfABCDE, respectively. This study provided new insights in the transcriptional regulation mechanism of Dirammox by DnfR in A. faecalis JQ135.

Genetic Foundations of Direct Ammonia Oxidation (Dirammox) to N2 and MocR-Like Transcriptional Regulator DnfR in Alcaligenes faecalis Strain JQ135.

Ammonia oxidation is an important process in both the natural nitrogen cycle and nitrogen removal from engineered ecosystems. Recently, a new ammonia oxidation pathway termed Dirammox (direct ammonia oxidation, NH3→NH2OH→N2) has been identified in Alcaligenes ammonioxydans. However, whether Dirammox is present in other microbes, as well as its genetic regulation, remains unknown. In this study, it was found that the metabolically versatile bacterium Alcaligenes faecalis strain JQ135 could efficiently convert ammonia into N2 via NH2OH under aerobic conditions. Genetic deletion and complementation results suggest that dnfABC is responsible for the ammonia oxidation to N2 in this strain. Strain JQ135 also employs aerobic denitrification, mainly producing N2O and trace amounts of N2, with nitrite as the sole nitrogen source. Deletion of the nirK and nosZ genes, which are essential for denitrification, did not impair the capability of JQ135 to oxidize ammonia to N2 (i.e., Dirammox is independent of denitrification). Furthermore, it was also demonstrated that pod (which encodes pyruvic oxime dioxygenase) was not involved in Dirammox and that AFA_16745 (which was previously annotated as ammonia monooxygenase and is widespread in heterotrophic bacteria) was not an ammonia monooxygenase. The MocR-family transcriptional regulator DnfR was characterized as an activator of the dnfABC operon with the binding motif 5'-TGGTCTGT-3' in the promoter region. A bioinformatic survey showed that homologs of dnf genes are widely distributed in heterotrophic bacteria. In conclusion, this work demonstrates that, besides A. ammonioxydans, Dirammox occurs in other bacteria and is regulated by the MocR-family transcriptional regulator DnfR. IMPORTANCE Microbial ammonia oxidation is a key and rate-limiting step of the nitrogen cycle. Three previously known ammonia oxidation pathways (i.e., nitrification, anaerobic ammonia oxidation [Anammox], and complete ammonia oxidation [Comammox]) are mediated by autotrophic microbes. However, the genetic foundations of ammonia oxidation by heterotrophic microorganisms have not been investigated in depth. Recently, a previously unknown pathway, termed direct ammonia oxidation to N2 (Dirammox), has been identified in the heterotrophic bacterium Alcaligenes ammonioxydans HO-1. This paper shows that, in the metabolically versatile bacterium Alcaligenes faecalis JQ135, the Dirammox pathway is mediated by dnf genes, which are independent of the denitrification pathway. A bioinformatic survey suggests that homologs of dnf genes are widely distributed in bacteria. These findings enhance the understanding of the molecular mechanisms of heterotrophic ammonia oxidation to N2.

Oxidation of organoarsenicals and antimonite by a novel flavin monooxygenase widely present in soil bacteria.

Arsenic can be biomethylated to form a variety of organic arsenicals differing in toxicity and environmental mobility. Trivalent methylarsenite (MAs(III)) produced in the methylation process is more toxic than inorganic arsenite (As(III)). MAs(III) also serves as a primitive antibiotic and, consequently, some environmental microorganisms have evolved mechanisms to detoxify MAs(III). However, the mechanisms of MAs(III) detoxification are not well understood. In this study, we identified an arsenic resistance (ars) operon consisting of three genes, arsRVK, that contribute to MAs(III) resistance in Ensifer adhaerens ST2. ArsV is annotated as an NADPH-dependent flavin monooxygenase with unknown function. Expression of arsV in the arsenic hypersensitive Escherichia coli strain AW3110Δars conferred resistance to MAs(III) and the ability to oxidize MAs(III) to MAs(V). In the presence of NADPH and either FAD or FMN, purified ArsV protein was able to oxidize both MAs(III) to MAs(V) and Sb(III) to Sb(V). Genes with arsV-like sequences are widely present in soils and environmental bacteria. Metagenomic analysis of five paddy soils showed the abundance of arsV-like sequences of 0.12-0.25 ppm. These results demonstrate that ArsV is a novel enzyme for the detoxification of MAs(III) and Sb(III) and the genes encoding ArsV are widely present in soil bacteria.

Roles of Two Glutathione-Dependent 3,6-Dichlorogentisate Dehalogenases in Rhizorhabdus dicambivorans Ndbn-20 in the Catabolism of the Herbicide Dicamba.

The herbicide dicamba is initially demethylated to 3,6-dichlorosalicylate (3,6-DCSA) in Rhizorhabdus dicambivorans Ndbn-20 and is subsequently 5-hydroxylated to 3,6-dichlorogentisate (3,6-DCGA). In the present study, two glutathione-dependent 3,6-DCGA dehalogenases, DsmH1 and DsmH2, were identified in strain Ndbn-20. DsmH2 shared a low identity (only 31%) with the tetrachlorohydroquinone (TCHQ) dehalogenase PcpC from Sphingobium chlorophenolicum ATCC 39723, while DsmH1 shared a high identity (79%) with PcpC. In the phylogenetic tree of related glutathione S-transferases (GSTs), DsmH1 and DsmH2, together with PcpC and the 2,5-dichlorohydroquinone dehalogenase LinD, formed a separate clade. DsmH1 and DsmH2 were synthesized in Escherichia coli BL21 and purified as His-tagged enzymes. Both enzymes required glutathione (GSH) as a cofactor and could 6-dechlorinate 3,6-DCGA to 3-chlorogentisate in vitro DsmH2 had a significantly higher catalytic efficiency toward 3,6-DCGA than DsmH1. Transcription and disruption analysis revealed that DsmH2 but not DsmH1 was responsible for the 6-dechlorination of 3,6-DCGA in strain Ndbn-20 in vivo Furthermore, we propose a novel eta class of GSTs to accommodate the four bacterial dehalogenases PcpC, LinD, DsmH1, and DsmH2.IMPORTANCE Dicamba is an important herbicide, and its use and leakage into the environment have dramatically increased since the large-scale planting of genetically modified (GM) dicamba-resistant crops in 2015. However, the complete catabolic pathway of dicamba has remained unknown, which limits ecotoxicological studies of this herbicide. Our previous study revealed that 3,6-DCGA was an intermediate of dicamba degradation in strain Ndbn-20. In this study, we identified two glutathione-dependent 3,6-DCGA dehalogenases, DsmH1 and DsmH2, and demonstrated that DsmH2 is physiologically responsible for the 6-dechlorination of 3,6-DCGA in strain Ndbn-20. GSTs play an important role in the detoxification and degradation of a variety of endogenous and exogenous toxic compounds. On the basis of their sequence identities, phylogenetic status, and functions, the four bacterial GSH-dependent dehalogenases (PcpC, LinD, DsmH1, and DsmH2) were reclassified as a new eta class of GSTs. This study helps us to elucidate the microbial catabolism of dicamba and enhances our understanding of the diversity and functions of GSTs.

Flavobacterium zaozhuangense sp. nov., a new member of the family Flavobacteriaceae, isolated from metolachlor-contaminated soil.

Strain ZZ-8T, a Gram-negative, aerobic, non-spore-forming, non-motile, yellow-pigmented, rod-shaped bacterium, was isolated from metolachlor-contaminated soil in China. The taxonomic position was investigated using a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain ZZ-8T is a member of the genus Flavobacterium and shows high sequence similarity to Flavobacterium humicola UCM-46T (97.2%) and Flavobacterium pedocola UCM-R36T (97.1%), and lower (< 97%) sequence similarity to other known Flavobacterium species. Chemotaxonomic analysis revealed that strain ZZ-8T possessed MK-6 as the major respiratory quinone; and iso-C15:0 (28.5%), summed feature 9 (iso-C17:1 w9c/C16:0 10-methyl, 22.9%), iso-C17:0 3-OH (17.0%), iso-C15:0 3-OH (8.9%), iso-C15:1 G (8.6%) and summed feature 3 (C16:1 w7c/C16:1 w6c, 5.7%) as the predominant fatty acids. The polar lipids of strain ZZ-8T were determined to be lipids, a glycolipid, aminolipids and phosphatidylethanolamine. Strain ZZ-8T showed low DNA-DNA relatedness with F. pedocola UCM-R36T (43.23 ± 4.1%) and F. humicola UCM-46T (29.17 ± 3.8%). The DNA G+C content was 43.3 mol%. Based on the phylogenetic and phenotypic characteristics, chemotaxonomic data and DNA-DNA hybridization, strain ZZ-8T is considered a novel species of the genus Flavobacterium, for which the name Flavobacterium zaozhuangense sp. nov. (type strain ZZ-8T = KCTC 62315 T = CCTCC AB 2017243T) is proposed.

Paenibacillus shunpengii sp. nov., isolated from farmland soil.

A bacterial strain designated YYJ7-1T was isolated from farmland soil in China and characterized using a polyphasic taxonomic approach. Cells of strain YYJ7-1T were Gram-staining-positive, aerobic or facultatively anaerobic, rod-shaped, motile and endospore-forming. Growth occurred at 18-42 °C (optimum at 35 °C), at pH 6.0-8.0 (optimum at pH 7.5) and with 0.0-4.0 % NaCl (optimum with 0.5 %). Phylogenetic analysis based on 16S rRNA gene sequences showed that the strain belonged to the genus Paenibacillus and showed high levels of sequence similarity with respect to Paenibacillus provencensis 4401170T (98.6 %) and Paenibacillus urinalis 5402403T (98.4 %), while lower 16S rRNA gene sequence similarities were observed with all other type strains (97.0 %). However, strain YYJ7-1T showed low DNA-DNA relatedness with P. provencensis 4401170T 48.7±4.5 % (43.6±7.1 % in a reciprocal experiment), and P. urinalis 5402403T 38.9±5.7 % (35.6±6.8 %). The major cellular fatty acids (>10.0 %) of strain YYJ7-1T were anteiso-C15 : 0, iso-C16 : 0 and anteiso-C17 : 0. The polar lipid profile consisted of phospholipids, diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. The major isoprenoid quinone was MK-7. The DNA G+C content was 39.4 mol%. Based on these results, it is concluded that strain YYJ7-1T represents a novel species of the genus Paenibacillus, for which the name Paenibacillus shunpengii sp. nov. is proposed, with YYJ7-1T (=ACCC 19965T=KCTC 33849T) as the type strain.

A Novel Aerobic Degradation Pathway for Thiobencarb Is Initiated by the TmoAB Two-Component Flavin Mononucleotide-Dependent Monooxygenase System in Acidovorax sp. Strain T1.

Thiobencarb is a thiocarbamate herbicide used in rice paddies worldwide. Microbial degradation plays a crucial role in the dissipation of thiobencarb in the environment. However, the physiological and genetic mechanisms underlying thiobencarb degradation remain unknown. In this study, a novel thiobencarb degradation pathway was proposed in Acidovorax sp. strain T1. Thiobencarb was oxidized and cleaved at the C-S bond, generating diethylcarbamothioic S-acid and 4-chlorobenzaldehyde (4CDA). 4CDA was then oxidized to 4-chlorobenzoic acid (4CBA) and hydrolytically dechlorinated to 4-hydroxybenzoic acid (4HBA). The identification of catabolic genes suggested further hydroxylation to protocatechuic acid (PCA) and finally degradation through the protocatechuate 4,5-dioxygenase pathway. A novel two-component monooxygenase system identified in the strain, TmoAB, was responsible for the initial catabolic reaction. TmoA shared 28 to 32% identity with the oxygenase components of pyrimidine monooxygenase from Agrobacterium fabrum, alkanesulfonate monooxygenase from Pseudomonas savastanoi, and dibenzothiophene monooxygenase from Rhodococcus sp. TmoB shared 25 to 37% identity with reported flavin reductases and oxidized NADH but not NADPH. TmoAB is a flavin mononucleotide (FMN)-dependent monooxygenase and catalyzed the C-S bond cleavage of thiobencarb. Introduction of tmoAB into cells of the thiobencarb degradation-deficient mutant T1m restored its ability to degrade and utilize thiobencarb. A dehydrogenase gene, tmoC, was located 7,129 bp downstream of tmoAB, and its transcription was clearly induced by thiobencarb. The purified TmoC catalyzed the dehydrogenation of 4CDA to 4CBA using NAD+ as a cofactor. A gene cluster responsible for the complete 4CBA metabolic pathway was also cloned, and its involvement in thiobencarb degradation was preliminarily verified by transcriptional analysis.IMPORTANCE Microbial degradation is the main factor in thiobencarb dissipation in soil. In previous studies, thiobencarb was degraded initially via N-deethylation, sulfoxidation, hydroxylation, and dechlorination. However, enzymes and genes involved in the microbial degradation of thiobencarb have not been studied. This study revealed a new thiobencarb degradation pathway in Acidovorax sp. strain T1 and identified a novel two-component FMN-dependent monooxygenase system, TmoAB. Under TmoAB-mediated catalysis, thiobencarb was cleaved at the C-S bond, producing diethylcarbamothioic S-acid and 4CDA. Furthermore, the downstream degradation pathway of thiobencarb was proposed. Our study provides the physiological, biochemical, and genetic foundation of thiobencarb degradation in this microorganism.

Degradation of Diphenyl Ether in Sphingobium phenoxybenzoativorans SC_3 Is Initiated by a Novel Ring Cleavage Dioxygenase.

Sphingobium phenoxybenzoativorans SC_3 degrades and utilizes diphenyl ether (DE) or 2-carboxy-DE as its sole carbon and energy source. In this study, we report the degradation of DE and 2-carboxy-DE initiated by a novel ring cleavage angular dioxygenase (diphenyl ether dioxygenase [Dpe]) in the strain. Dpe functions at the angular carbon and its adjacent carbon (C-1a, C-2) of a benzene ring in DE (or the 2-carboxybenzene ring in 2-carboxy-DE) and cleaves the C-1a-C-2 bond (decarboxylation occurs simultaneously for 2-carboxy-DE), yielding 2,4-hexadienal phenyl ester, which is subsequently hydrolyzed to muconic acid semialdehyde and phenol. Dpe is a type IV Rieske non-heme iron oxygenase (RHO) and consists of three components: a hetero-oligomer oxygenase, a [2Fe-2S]-type ferredoxin, and a glutathione reductase (GR)-type reductase. Genetic analyses revealed that dpeA1A2 plays an essential role in the degradation and utilization of DE and 2-carboxy-DE in S. phenoxybenzoativorans SC_3. Enzymatic study showed that transformation of 1 molecule of DE needs two molecules of oxygen and two molecules of NADH, supporting the assumption that the cleavage of DE catalyzed by Dpe is a continuous two-step dioxygenation process: DE is dioxygenated at C-1a and C-2 to form a hemiacetal-like intermediate, which is further deoxygenated, resulting in the cleavage of the C-1a-C-2 bond to form one molecule of 2,4-hexadienal phenyl ester and two molecules of H2O. This study extends our knowledge of the mode and mechanism of ring cleavage of aromatic compounds.IMPORTANCE Benzene ring cleavage, catalyzed by dioxygenase, is the key and speed-limiting step in the aerobic degradation of aromatic compounds. As previously reported, in the ring cleavage of DEs, the benzene ring needs to be first dihydroxylated at a lateral position and subsequently dehydrogenated and opened through extradiol cleavage. This process requires three enzymes (two dioxygenases and one dehydrogenase). In this study, we identified a novel angular dioxygenase (Dpe) in S. phenoxybenzoativorans SC_3. Under Dpe-mediated catalysis, the benzene ring of DE is dioxygenated at the angular position (C-1a, C-2), resulting in the cleavage of the C-1a-C-2 bond to generate a novel product, 2,4-hexadienal phenyl ester. This process needs only one angular dioxygenase, Dpe. Thus, the ring cleavage catalyzed by Dpe represents a novel mechanism of benzene ring cleavage.

A Tetrahydrofolate-Dependent Methyltransferase Catalyzing the Demethylation of Dicamba in Sphingomonas sp. Strain Ndbn-20.

UNLABELLED: Sphingomonas sp. strain Ndbn-20 degrades and utilizes the herbicide dicamba as its sole carbon and energy source. In the present study, a tetrahydrofolate (THF)-dependent dicamba methyltransferase gene, dmt, was cloned from the strain, and three other genes, metF, dhc, and purU, which are involved in THF metabolism, were found to be located downstream of dmt A transcriptional study revealed that the four genes constituted one transcriptional unit that was constitutively transcribed. Lysates of cells grown with glucose or dicamba exhibited almost the same activities, which further suggested that the dmt gene is constitutively expressed in the strain. Dmt shared 46% and 45% identities with the methyltransferases DesA and LigM from Sphingomonas paucimobilis SYK-6, respectively. The purified Dmt catalyzed the transfer of methyl from dicamba to THF to form the herbicidally inactive metabolite 3,6-dichlorosalicylic acid (DCSA) and 5-methyl-THF. The activity of Dmt was inhibited by 5-methyl-THF but not by DCSA. The introduction of a codon-optimized dmt gene into Arabidopsis thaliana enhanced resistance against dicamba. In conclusion, this study identified a THF-dependent dicamba methyltransferase, Dmt, with potential applications for the genetic engineering of dicamba-resistant crops. IMPORTANCE: Dicamba is a very important herbicide that is widely used to control more than 200 types of broadleaf weeds and is a suitable target herbicide for the engineering of herbicide-resistant transgenic crops. A study of the mechanism of dicamba metabolism by soil microorganisms will benefit studies of its dissipation, transformation, and migration in the environment. This study identified a THF-dependent methyltransferase, Dmt, capable of catalyzing dicamba demethylation in Sphingomonas sp. Ndbn-20, and a preliminary study of its enzymatic characteristics was performed. Introduction of a codon-optimized dmt gene into Arabidopsis thaliana enhanced resistance against dicamba, suggesting that the dmt gene has potential applications for the genetic engineering of herbicide-resistant crops.