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17β-estradiol (E2) is a natural endocrine disruptor that is frequently detected in surface and groundwater sources, thereby threatening ecosystems and human health. The newly isolated E2-degrading strain Sphingomonas colocasiae C3-2 can degrade E2 through both the 4,5-seco pathway and the 9,10-seco pathway; the former is the primary pathway supporting the growth of this strain and the latter is a branching pathway. The novel gene cluster ean was found to be responsible for E2 degradation through the 4,5-seco pathway, where E2 is converted to estrone (E1) by EanA, which belongs to the short-chain dehydrogenases/reductases (SDR) superfamily. A three-component oxygenase system (including the P450 monooxygenase EanB1, the small iron-sulfur protein ferredoxin EanB2, and the ferredoxin reductase EanB3) was responsible for hydroxylating E1 to 4-hydroxyestrone (4-OH-E1). The enzymatic assay showed that the proportion of the three components is critical for its function. The dioxygenase EanC catalyzes ring A cleavage of 4-OH-E1, and the oxidoreductase EanD is responsible for the decarboxylation of the ring A-cleavage product of 4-OH-E1. EanR, a TetR family transcriptional regulator, acts as a transcriptional repressor of the ean cluster. The ean cluster was also found in other reported E2-degrading sphingomonads. In addition, the novel two-component monooxygenase EanE1E2 can open ring B of 4-OH-E1 via the 9,10-seco pathway, but its encoding genes are not located within the ean cluster. These results refine research on genes involved in E2 degradation and enrich the understanding of the cleavages of ring A and ring B of E2. [ABSTRACT FROM AUTHOR]

The worldwide use of the carbamate insecticide carbofuran has caused considerable concern about its environmental fate. Degradation of carbofuran by Sphingobium sp. strain CFD-1 is initiated via the hydrolysis of its ester bond by carbamate hydrolase CehA to form carbofuran phenol. In this study, another carbofuran-degrading strain, Sphingobium sp. CFD-2, was isolated. Subsequently, a cfd gene cluster responsible for the catabolism of carbofuran phenol was predicted by comparing the genomes of strains CFD-1, CFD-2, and Novosphingobium sp. strain KN65.2. The key genes verified to be involved in the catabolism of carbofuran phenol within the cfd cluster include the hydroxylase gene cfdC, epoxide hydrolase gene cfdF, and ring cleavage dioxygenase gene cfdE and are responsible for the successive conversion of carbofuran phenol, resulting in complete ring cleavage. These carbofuran-catabolic genes (cehA and the cfd cluster) are distributed on two plasmids in strain CFD-1 and are highly conserved among the carbofuran-degrading sphingomonad strains. The mobile genetic element IS6100 flanks cehA and the cfd gene cluster, indicating the importance of horizontal gene transfer in the formation of carbofuran degradation gene clusters. The elucidation of the molecular mechanism of carbofuran catabolism provides insights into the evolutionary scenario of the conserved carbofuran catabolic pathway. IMPORTANCE Owing to the extensive use of carbofuran over the past 50 years, bacteria have evolved catabolic pathways to mineralize this insecticide, which plays an important role in eliminating carbofuran residue in the environment. In this study, the cfd gene cluster, responsible for the catabolism of carbofuran phenol, was predicted by comparing sphingomonad genomes. The function of key enzymatic genes in this gene cluster was identified. Furthermore, the carbamate hydrolase gene cehA and the cfd gene cluster are highly conserved in different carbofuran-degrading strains. Additionally, the horizontal gene transfer elements flanking the cfd gene cluster were investigated. These findings help elucidate the molecular mechanism of microbial carbofuran degradation and enhance our understanding of the evolutionary mechanism of the carbofuran catabolic pathway. [ABSTRACT FROM AUTHOR]

Phenazines are an important class of secondary metabolites and are primarily named for their heterocyclic phenazine cores, including phenazine-1-carboxylic acid (PCA) and its derivatives, such as phenazine-1-carboxamide (PCN) and pyocyanin (PYO). Although several genes involved in the degradation of PCA and PYO have been reported so far, the genetic foundations of PCN degradation remain unknown. In this study, a PCN-degrading bacterial strain, Sphingomonas histidinilytica DS-9, was isolated. The gene pcnH, encoding a novel amidase responsible for the initial step of PCN degradation, was cloned by genome comparison and subsequent experimental validation. PcnH catalyzed the hydrolysis of the amide bond of PCN to produce PCA, which shared low identity (only 26 to 33%) with reported amidases. The Km and kcat values of PcnH for PCN were 33.22 6 5.70 mM and 18.71 6 0.52 s21, respectively. PcnH has an Asp-Lys-Cys motif, which is conserved among amidases of the isochorismate hydrolase-like (IHL) superfamily. The replacement of Asp37, Lys128, and Cys163 with alanine in PcnH led to the complete loss of enzymatic activity. Furthermore, the genes pcaA1A2A3A4 and pcnD were found to encode PCA 1,2-dioxygenase and 1,2-dihydroxyphenazine (2OHPC) dioxygenase, which were responsible for the subsequent degradation steps of PCN. The PCN-degradative genes were highly conserved in some bacteria of the genus Sphingomonas, with slight variations in the sequence identities. [ABSTRACT FROM AUTHOR]

In a previous study, the novel gene cluster cehGHI was found to be involved in salicylate degradation through the CoA-mediated pathway in Rhizobium sp. strain X9 (Mol Microbiol 116:783-793, 2021). In this study, an IclR family transcriptional regulator CehR4 was identified. In contrast to other regulators involved in salicylate degradation, cehR4 forms one operon with the gentisyl-CoA thioesterase gene cehI, while cehG and cehH (encoding salicylyl-CoA ligase and salicylyl-CoA hydroxylase, respectively) form another operon. cehGH and cehIR4 are divergently transcribed, and their promoters overlap. The results of the electrophoretic mobility shift assay and DNase I footprinting showed that CehR4 binds to the 42-bp motif between genes cehH and cehI, thus regulating transcription of cehGH and cehIR4. The repeat sequences IR1 (5'-TTTATATAAA-3') and IR2 (5'-AATATAGAAA-3') in the motif are key sites for CehR4 binding. The arrangement of cehGH and cehIR4 and the conserved binding motif of CehR4 were also found in other bacterial genera. The results disclose the regulatory mechanism of salicylate degradation through the CoA pathway and expand knowledge about the systems controlled by IclR family transcriptional regulators. [ABSTRACT FROM AUTHOR]

In our previous study, the phenazine-1-carboxylic acid (PCA) 1,2-dioxygenase gene cluster (pcaA1A2A3A4 cluster) in Sphingomonas histidinilytica DS-9 was identified to be responsible for the conversion of PCA to 1,2-dihydroxyphenazine (Ren Y, Zhang M, Gao S, Zhu Q, et al. 2022. Appl Environ Microbiol 88:e00543-22). However, the regulatory mechanism of the pcaA1A2A3A4 cluster has not been elucidated yet. In this study, the pcaA1A2A3A4 cluster was found to be transcribed as two divergent operons: pcaA3-ORF5205 (named A3-5205 operon) and pcaA1A2-ORF5208-pcaA4-ORF5210 (named A1-5210 operon). The promoter regions of the two operons were overlapped. PcaR acts as a transcriptional repressor of the pcaA1A2A3A4 cluster, and it belongs to GntR/FadR family transcriptional regulator. Gene disruption of pcaR can shorten the lag phase of PCA degradation. The results of electrophoretic mobility shift assay and DNase I footprinting showed that PcaR binds to a 25-bp motif in the ORF5205-pcaA1 intergenic promoter region to regulate the expression of two operons. The 25-bp motif covers the 210 region of the promoter of A3-5205 operon and the 235 region and 210 region of the promoter of A1-5210 operon. The TNGT/ANCNA box within the motif was essential for PcaR binding to the two promoters. PCA acted as an effector of PcaR, preventing it from binding to the promoter region and repressing the transcription of the pcaA1A2A3A4 cluster. In addition, PcaR represses its own transcription, and this repression can be relieved by PCA. This study reveals the regulatory mechanism of PCA degradation in strain DS-9, and the identification of PcaR increases the variety of regulatory model of the GntR/FadR-type regulator. IMPORTANCE Sphingomonas histidinilytica DS-9 is a phenazine-1-carboxylic acid (PCA)- degrading strain. The 1,2-dioxygenase gene cluster (pcaA1A2A3A4 cluster, encoding dioxygenase PcaA1A2, reductase PcaA3, and ferredoxin PcaA4) is responsible for the initial degradation step of PCA and widely distributed in Sphingomonads, but its regulatory mechanism has not been investigated yet. In this study, a GntR/FadR-type transcriptional regulator PcaR repressing the transcription of pcaA1A2A3A4 cluster and pcaR gene was identified and characterized. The binding site of PcaR in ORF5205-pcaA1 intergenic promoter region contains a TNGT/ANCNA box, which is important for the binding. These findings enhance our understanding of the molecular mechanism of PCA degradation. [ABSTRACT FROM AUTHOR]

Previously, a LysR family transcriptional regulator, McbG, that activates the mcbBCDEF gene cluster involved in the upstream pathway (from carbaryl to salicylate) of carbaryl degradation in Pseudomonas sp. strain XWY-1 was identified by us (Z. Ke, Y. Zhou, W. Jiang, M. Zhang, et al., Appl Environ Microbiol 87:e02970-20, 2021, https://doi.org/10.1128/AEM.02970-20). In this study, we identified McbH and McbN, which activate the mcbIJKLM cluster (responsible for the midstream pathway, from salicylate to gentisate) and the mcbOPQ cluster (responsible for the downstream pathway, from gentisate to pyruvate and fumarate), respectively. They both belong to the LysR family of transcriptional regulators. Gene disruption and complementation study reveal that McbH is essential for transcription of the mcbIJKLM cluster in response to salicylate and McbN is indispensable for the transcription of the mcbOPQ cluster in response to gentisate. The results of electrophoretic mobility shift assay (EMSA) and DNase I footprinting showed that McbH binds to the 52-bp motif in the mcbIJKLM promoter area and McbN binds to the 58-bp motif in the mcbOPQ promoter area. The key sequence of McbH binding to the mcbIJKLM promoter is a 13-bp motif that conforms to the typical characteristics of the LysR family. However, the 12-bp motif that is different from the typical characteristics of the LysR family regulator binding site sequence is identified as the key sequence for McbN to bind to the mcbOPQ promoter. This study revealed the regulatory mechanisms for the midstream and downstream pathways of carbaryl degradation in strain XWY-1 and further our knowledge of (and the size of) the LysR transcription regulator family. [ABSTRACT FROM AUTHOR]

Strobilurin fungicides are widely used in agricultural production due to their broad-spectrum and fungal mitochondrial inhibitory activities. However, their massive application has restrained the growth of eukaryotic algae and increased collateral damage in freshwater systems, notably harmful cyanobacterial blooms (HCBs). In this study, a strobilurin fungicide-degrading strain, Hyphomicrobium sp. strain DY-1, was isolated and characterized successfully. Moreover, a novel esterase gene, strH, responsible for the de-esterification of strobilurin fungicides, was cloned, and the enzymatic properties of StrH were studied. For trifloxystrobin, StrH displayed maximum activity at 50°C and pH 7.0. The catalytic efficiencies (kcat/Km) of StrH for different strobilurin fungicides were 196.32±2.30μM-1 · s-1 (trifloxystrobin), 4.64±0.05μM-1 · s-1 (picoxystrobin), 2.9460.02μM-1 · s-1 (pyraclostrobin), and (2.41±0.19)×10-2μM-1 · s-1 (azoxystrobin). StrH catalyzed the de-esterification of a variety of strobilurin fungicides, generating the corresponding parent acid to achieve the detoxification of strobilurin fungicides and relieve strobilurin fungicide growth inhibition of Chlorella. This research will provide insight into the microbial remediation of strobilurin fungicide-contaminated environments. [ABSTRACT FROM AUTHOR]

Although enzyme-encoding genes involved in the degradation of carbaryl have been reported in Pseudomonas sp. strain XWY-1, no regulator has been identified yet. In the mcbABCDEF cluster responsible for the upstream pathway of carbaryl degradation (from carbaryl to salicylate), the mcbA gene is constitutively expressed, while mcbBCDEF is induced by 1-naphthol, the hydrolysis product of carbaryl by McbA. In this study, we identified McbG, a transcriptional activator of the mcbBCDEF cluster. McbG is a 315-amino-acid protein with a molecular mass of 35.7 kDa. It belongs to the LysR family of transcriptional regulators and shows 28.48% identity to the pentachlorophenol (PCP) degradation transcriptional activation protein PcpR from Sphingobium chlorophenolicum ATCC 39723. Gene disruption and complementation studies reveal that mcbG is essential for transcription of the mcbBCDEF cluster in response to 1-naphthol in strain XWY-1. The results of the electrophoretic mobility shift assay (EMSA) and DNase I footprinting show that McbG binds to the 25-bp motif in the mcbBCDEF promoter area. The palindromic sequence TATCGATA within the motif is essential for McbG binding. The binding site is located between the -10 box and the transcription start site. In addition, McbG can repress its own transcription. The EMSA results show that a 25-bp motif in the mcbG promoter area plays an important role in McbG binding to the promoter of mcbG. This study reveals the regulatory mechanism for the upstream pathway of carbaryl degradation in strain XWY-1. The identification of McbG increases the variety of regulatory models within the LysR family of transcriptional regulators. [ABSTRACT FROM AUTHOR]

Methomyl {bis[1-methylthioacetaldehyde-O-(N-methylcarbamoyl)oximino] sulfide} is a highly toxic oxime carbamate insecticide. Several methomyl-degrading microorganisms have been reported so far, but the role of specific enzymes and genes in this process is still unexplored. In this study, a protein annotated as a carbamate C-N hydrolase was identified in the methomyl-degrading strain Aminobacter aminovorans MDW-2, and the encoding gene was termed ameH. A comparative analysis between the mass fingerprints of AmeH and deduced proteins of the strain MDW-2 genome revealed AmeH to be a key enzyme of the detoxification step of methomyl degradation. The results also demonstrated that AmeH was a functional homodimer with a subunit molecular mass of approximately 34 kDa and shared the highest identity (27%) with the putative formamidase from Schizosaccharomyces pombe ATCC 24843. AmeH displayed maximal enzymatic activity at 50°C and pH 8.5. Km and kcat of AmeH for methomyl were 87.5 μM and 345.2 s-1, respectively, and catalytic efficiency (kcat/Km) was 3.9 μM-1 s-1. Phylogenetic analysis revealed AmeH to be a member of the FmdA_AmdA superfamily. Additionally, five key amino acid residues (162, 164, 191, 193, and 207) of AmeH were identified by amino acid variations. [ABSTRACT FROM AUTHOR]