Characterization of cotinine degradation in a newly isolated Gram-negative strain Pseudomonas sp. JH-2.

Cotinine, the primary metabolite of nicotine in the human body, is an emerging pollutant in aquatic environments. It causes environmental problems and is harmful to the health of humans and other mammals; however, the mechanisms of its biodegradation have been elucidated incompletely. In this study, a novel Gram-negative strain that could degrade and utilize cotinine as a sole carbon source was isolated from municipal wastewater samples, and its cotinine degradation characteristics and kinetics were determined. Pseudomonas sp. JH-2 was able to degrade 100 mg/L (0.56 mM) of cotinine with high efficiency within 5 days at 30 ℃, pH 7.0, and 1% NaCl. Two intermediates, 6-hydroxycotinine and 6-hydroxy-3-succinoylpyridine (HSP), were identified by high-performance liquid chromatography and liquid chromatograph mass spectrometer. The draft whole genome sequence of strain JH-2 was obtained and analyzed to determine genomic structure and function. No homologs of proteins predicted in Nocardioides sp. JQ2195 and reported in nicotine degradation Pyrrolidine pathway were found in strain JH-2, suggesting new enzymes that responsible for cotinine catabolism. These findings provide meaningful insights into the biodegradation of cotinine by Gram-negative bacteria.

Ruixingdingia sedimenti gen. nov., sp. nov., a novel taxon within the family Paracoccaceae isolated from sediment of Qiantang River.

A Gram-stain-negative bacterium, designated LG-4T, was isolated from sediment of Qiantang River in Zhejiang Province, PR China. Cells were strictly aerobic, non-spore-forming, non-motile and short-rod-shaped (1.0-1.2 µm long and 0.7-0.8 µm wide). Growth occurred at 15-42 °C (optimum, 30 °C), at pH 5.0-9.0 (pH 7.0) and at 0-2.0 % (w/v) NaCl (optimum, 0.5 % NaCl). Strain LG-4T showed 95.75-96.90 % 16S rRNA gene sequence similarity to various type strains of the genera Tabrizicola, Pseudotabrizicola, Phaeovulum, Rhodobacter and Wagnerdoeblera of the family Paracoccaceae, and the most closely related strain was Tabrizicola soli ZQBWT (96.90 % similarity). The phylogenomic tree showed that strain LG-4T clustered in the family Paracoccaceae and was positioned outside of the clade composed of the genera Wagnerdoeblera and Falsigemmobacter. The average nucleotide identity and digital DNA-DNA hybridization values between strain LG-4T and the related type strains were in the range of 74.19-77.56 % and 16.70-25.80 %, respectively. The average amino acid identity (AAI) values between strain LG-4T and related type strains of the family Paracoccaceae were 60.94-69.73 %, which are below the genus boundary (70 %). The evolutionary distance (ED) values between LG-4T and the related genera of the family Paracoccaceae were 0.21-0.34, which are within the recommended standard (≥0.21-0.23) for defining a novel genus in the family Paracoccaceae. The predominant cellular fatty acids were C18 : 1 ω7c, C19 : 0 cyclo ω8c, C18 : 0 and C16 : 0, the isoprenoid quinone was Q-10, and the major polar lipids were phospholipid, phosphatidylglycerol, phosphatidylcholine, aminolipid and two unknown polar lipids. The genome size was 4.7 Mb with 68.6 mol% G+C content. On the basis of distinct phylogenetic relationships, low AAI values and high ED values, and differential phenotypic, physiological and biochemical characteristics, strain LG-4T represents a novel species of a new genus in the family Paracoccaceae, for which the name Ruixingdingia sedimenti gen. nov., sp. nov. is proposed. The type strain is LG-4T (=MCCC 1K08849T=KCTC 8136T).

Nocardioides imazamoxiresistens sp. nov. Isolated from the Activated Sludge.

A Gram-staining-positive actinomycete named YZH12T was isolated from the sediment of the Yangtze River in Nanjing, Jiangsu province, China. Cells were aerobic, non-spore forming, non-motile, short rod (0.4-0.6 × 0.5-1.0 µm) or coccus (0.4-0.6 µm in diameter). Colonies were circular, smooth, and beige to yellowish. Growth occurred at 15-42 °C (optimal 28 °C), pH 5.0-9.0 (optimal 7.0), and 0-10% (w/v) NaCl (optimal 2%). The strain could tolerate 1500 mg/L of imazamox. Strain YZH12T showed 98.7% 16S rRNA gene sequence similarity Nocardioides zeae JM-1068T and less than 97% similarities with other type strains in the genus Nocardioides. Phylogenetic analysis based on genome and 16S rRNA gene sequences indicated that strain YZH12T was phylogenetically affiliated to the genus Nocardioides and formed a subclade with N. zeae JM-1068T and N. alkalitolerans DSM 16699T. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between YZH12T and closely related type strain N. zeae JM-1068T were 79.9% and 35.2%, respectively. The major fatty acids (> 5%) were C18: 1ω9c, iso-C16: 0, C16: 0, C17: 1ω8cand C18: 0; the major respiratory quinone was MK-8(H4); and the polar lipids profiles were diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), glycolipid (GL), two aminophospholipids (APL1, APL2), and an unknown polar lipid (L). The genomic DNA G + C content is 73.5%. Based on the phenotypic, chemotaxonomic, phylogenetic analyses, and genomic data, strain YZH12T represents a novel species of the genus Nocardioides, for which the name Nocardioides imazamoxiresistens YZH12T is proposed, with strain YZH12T (= KCTC 49964T = MCCC 1K0892T) as the type strain.

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.

PcaR, a GntR/FadR Family Transcriptional Repressor Controls the Transcription of Phenazine-1-Carboxylic Acid 1,2-Dioxygenase Gene Cluster in Sphingomonas histidinilytica DS-9.

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 -10 region of the promoter of A3-5205 operon and the -35 region and -10 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.

Unveiling the regulatory mechanisms of salicylate degradation gene cluster cehGHIR4 in Rhizobium sp. strain X9.

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.IMPORTANCEThe long-term residue of aromatic compounds in the environment has brought great threat to the environment and human health. Microbial degradation plays an important role in the elimination of aromatic compounds in the environment. Salicylate is a common intermediate metabolite in the degradation of various aromatic compounds. Recently, Rhizobium sp. strain X9, capable of degrading the pesticide carbaryl, was isolated from carbaryl-contaminated soil. Salicylate is the intermediate metabolite that appeared during the degradation of carbaryl, and a novel salicylate degradation pathway and the involved gene cluster cehGHIR4 have been identified. This study identified and characterized the IclR transcription regulator CehR4 that represses transcription of cehGHIR4 gene cluster. Additionally, the genetic arrangements of cehGH and cehIR4 and the binding sites of CehR4 were also found in other bacterial genera. This study provides insights into the biodegradation of salicylate and provides an application in the bioremediation of aromatic compound-contaminated environments.

Quinolinic acid catabolism is initiated by a novel four-component hydroxylase QuiA in Alcaligenes faecalis JQ191.

Quinolinic acid (QA) is an essential nitrogen-containing aromatic heterocyclic compounds in organisms and it also acts as an important intermediate in chemical industry, which has strong neurotoxicity and cytotoxicity. The wide range of sources and applications caused the release and accumulation of QA in the environment which might poses a hazard to ecosystems and human health. However, few research on the degradation of QA by microorganisms and toxicity of QA and its metabolites were reported. Alcaligenes faecalis JQ191 could degrade QA but the genetic foundation of QA degradation has not been studied. In this study, the gene cluster quiA1A2A3A4 was identified from A. faecalis JQ191, which was responsible for the initial catabolism step of QA. The quiA1A2A3A4 gene cluster encodes a novel cytoplasmic four-component hydroxylase QuiA. The 1H nuclear magnetic resonance indicated that QuiA catalyzed QA to 6-hydroxyquinolinic acid (6HQA) and the H218O-labeling analysis confirmed that the hydroxyl group incorporating into 6HQA was derived from water. Toxicity tests showed that the QA could approximately inhibit 20%-80% growth of Chlorella ellipsoidea, and 6HQA could relieve at least 50% QA growth inhibition of Chlorella ellipsoidea, indicating that the 6-hydroxylation of QA by QuiA is a detoxification process. This research provides new insights into the metabolism of QA by microorganism and potential application in the bioremediation of toxic pyridine derivatives-contaminated environments.

Isolation and characterization of the 2,5-pyridinedicarboxylic acid-degrading bacterium Agrobacterium sp. strain YJ-5.

2,5-Pyridinedicarboxylic acid (2,5-PDA), a natural N-heterocyclic compound and a substitute for production in plastics, was widely distributed in industrial wastewater. However, the biodegradation of 2,5-PDA has been rarely reported. In this study, strain YJ-5, which could utilize 2,5-PDA as the sole carbon source for growth was isolated from pesticide-contaminated soil. Based on the comparative analysis of the 16S rRNA gene sequence, strain YJ-5 was identified as Agrobacterium sp. 2,5-PDA was completely degraded within 7 d and the optimal growth conditions of temperature, pH, and substrate concentration were 30°C, 7.0, and 0.6 mmol-1, respectively. A new intermediate 6-hydroxy-2,5-PDA was determined by UV/VIS spectroscopy and liquid chromatograph coupled time of flight mass spectrometry. When the electron acceptor (2,6-dichlorophenolindophenol) was employed, the 2,5-PDA could be converted by cell extracts of strain YJ-5 cells into 6-hydroxy-2,5-PDA. These results provided new insights for biodegradation on pyridine dicarboxylate.

Unveiling the CoA mediated salicylate catabolic mechanism in Rhizobium sp. X9.

Salicylate is a typical aromatic compound widely distributed in nature. Microbial degradation of salicylate has been well studied and salicylate hydroxylases play essential roles in linking the peripheral and ring-cleavage catabolic pathways. The direct hydroxylation of salicylate catalyzed by salicylate-1-hydroxylase or salicylate-5-hydroxylase has been well studied. However, the CoA mediated salicylate 5-hydroxylation pathway has not been characterized in detail. Here, we elucidate the molecular mechanism of the reaction in the conversion of salicylate to gentisate in the carbaryl-degrading strain Rhizobium sp. X9. Three enzymes (salicylyl-CoA ligase CehG, salicylyl-CoA hydroxylase CehH and gentisyl-CoA thioesterase CehI) catalyzed the conversion of salicylate to gentisate via a route, including CoA thioester formation, hydroxylation and thioester hydrolysis. Further analysis indicated that genes cehGHI are also distributed in other bacteria from terrestrial environment and marine sediments. These genomic evidences highlight the role of this salicylate degradation pathway in the carbon cycle of soil organic compounds and marine sediments. Our findings of this three-step strategy enhanced the current understanding of CoA mediated degradation of salicylate.

Functional analysis, diversity, and distribution of carbendazim hydrolases MheI and CbmA, responsible for the initial step in carbendazim degradation.

Strains Rhodococcus qingshengii djl-6 and Rhodococcus jialingiae djl-6-2 both harbour the typical carbendazim degradation pathway with the hydrolysis of carbendazim to 2-aminobenzimidazole (2-AB) as the initial step. However, the enzymes involved in this process are still unknown. In this study, the previous reported carbendazim hydrolase MheI was found in strain djl-6, but not in strain djl-6-2, then another carbendazim hydrolase CbmA was obtained by a four-step purification strategy from strain djl-6-2. CbmA was classified as a member of the amidase signature superfamily with conserved catalytic site residues Ser157, Ser181, and Lys82, while MheI was classified as a member of the Abhydrolase superfamily with conserved catalytic site residues Ser77 and His224. The catalytic efficiency (kcat /Km ) of MheI (24.0-27.9 µM-1  min-1 ) was 200 times more than that of CbmA (0.032-0.21 µM-1  min-1 ). The mheI gene (plasmid encoded) was highly conserved (>99% identity) in the strains from different bacterial genera and its plasmid encoded flanked by mobile genetic elements. The cmbA gene was highly conserved only in strains of the genus Rhodococcus and it was chromosomally encoded. Overall, the function, diversity, and distribution of carbendazim hydrolases MheI and CbmA will provide insights into the microbial degradation of carbendazim.

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.

The Novel Amidase PcnH Initiates the Degradation of Phenazine-1-Carboxamide in Sphingomonas histidinilytica DS-9.

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 ± 5.70 µM and 18.71 ± 0.52 s-1, 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. IMPORTANCE Phenazines have been widely acknowledged as a natural antibiotic for more than 150 years, but their degradation mechanisms are still not completely elucidated. Compared with the studies on the degradation mechanism of PCA and PYO, little is known regarding PCN degradation by far. Previous studies have speculated that its initial degradation step may be catalyzed by an amidase, but no further studies have been conducted. This study identified a novel amidase, PcnH, that catalyzed the hydrolysis of PCN to PCA. In addition, the PCA 1,2-dioxygenase PcaA1A2A3A4 and 2OHPC dioxygenase PcnD were also found to be involved in the subsequent degradation steps of PCN in S. histidinilytica DS-9. And the genes responsible for PCN catabolism are highly conserved in some strains of Sphingomonas. These results deepen our understanding of the PCN degradation mechanism.

PicR as a MarR Family Transcriptional Repressor Multiply Controls the Transcription of Picolinic Acid Degradation Gene Cluster pic in Alcaligenes faecalis JQ135.

Picolinic acid (PA) is a natural toxic pyridine derivative as well as an important intermediate used in the chemical industry. In a previous study, we identified a gene cluster, pic, that responsible for the catabolism of PA in Alcaligenes faecalis JQ135. However, the transcriptional regulation of the pic cluster remains known. This study showed that the entire pic cluster was composed of 17 genes and transcribed as four operons: picR, picCDEF, picB4B3B2B1, and picT1A1A2A3T2T3MN. Deletion of picR, encoding a putative MarR-type regulator, greatly shortened the lag phase of PA degradation. An electrophoretic mobility shift assay and DNase I footprinting showed that PicR has one binding site in the picR-picC intergenic region and two binding sites in the picB-picT1 intergenic region. The DNA sequences of the three binding sites have the palindromic characteristics of TCAG-N4-CTNN: the space consists of four nonspecific bases, and the four palindromic bases on the left and the first two palindromic bases on the right are strictly conserved, while the last two bases on the right vary among the three binding sites. An in vivo ß-galactosidase activity reporter assay indicated that 6-hydroxypicolinic acid but not PA acted as a ligand of PicR, preventing PicR from binding to promoter regions and thus derepressing the transcription of the pic cluster. This study revealed the negative transcriptional regulation mechanism of PA degradation by PicR in A. faecalis JQ135 and provides new insights into the structure and function of the MarR-type regulator. IMPORTANCE The pic gene cluster was found to be responsible for PA degradation and widely distributed in Alpha-, Beta-, and Gammaproteobacteria. Thus, it is very necessary to understand the regulation mechanism of the pic cluster in these strains. This study revealed that PicR binds to three sites of the promoter regions of the pic cluster to multiply regulate the transcription of the pic cluster, which enables A. faecalis JQ135 to efficiently utilize PA. Furthermore, the study also found a unique palindrome sequence for binding of the MarR-type regulator. This study enhanced our understanding of microbial catabolism of environmental toxic pyridine derivatives.

The Novel Monooxygenase Gene dipD in the dip Gene Cluster of Alcaligenes faecalis JQ135 Is Essential for the Initial Catabolism of Dipicolinic Acid.

Dipicolinic acid (DPA), an essential pyridine derivative biosynthesized in Bacillus spores, constitutes a major proportion of global biomass carbon pool. Alcaligenes faecalis strain JQ135 could catabolize DPA through the "3HDPA (3-hydroxydipicolinic acid) pathway." However, the genes involved in this 3HDPA pathway are still unknown. In this study, a dip gene cluster responsible for DPA degradation was cloned from strain JQ135. The expression of dip genes was induced by DPA and negatively regulated by DipR. A novel monooxygenase gene, dipD, was crucial for the initial hydroxylation of DPA into 3HDPA and proposed to encode the key catalytic component of the multicomponent DPA monooxygenase. The heme binding protein gene dipF, ferredoxin reductase gene dipG, and ferredoxin genes dipJ/dipK/dipL were also involved in the DPA hydroxylation and proposed to encode other components of the multicomponent DPA monooxygenase. The 18O2 stable isotope labeling experiments confirmed that the oxygen atom in the hydroxyl group of 3HDPA came from dioxygen molecule rather than water. The protein sequence of DipD exhibits no significant sequence similarities with known oxygenases, suggesting that DipD was a new member of oxygenase family. Moreover, bioinformatic survey suggested that the dip gene cluster was widely distributed in many Alpha-, Beta-, and Gammaproteobacteria, including soil bacteria, aquatic bacteria, and pathogens. This study provides new molecular insights into the catabolism of DPA in bacteria. IMPORTANCE Dipicolinic acid (DPA) is a natural pyridine derivative that serves as an essential component of the Bacillus spore. DPA accounts for 5 to 15% of the dry weight of spores. Due to the huge number of spores in the environment, DPA is also considered to be an important component of the global biomass carbon pool. DPA could be decomposed by microorganisms and enter the global carbon cycling; however, the underlying molecular mechanisms are rarely studied. In this study, a DPA catabolic gene cluster (dip) was cloned and found to be widespread in Alpha-, Beta-, and Gammaproteobacteria. The genes responsible for the initial hydroxylation of DPA to 3-hydroxyl-dipicolinic acid were investigated in Alcaligenes faecalis strain JQ135. The present study opens a door to elucidate the mechanism of DPA degradation and its possible role in DPA-based carbon biotransformation on earth.

The TetR Family Repressor HpaR Negatively Regulates the Catabolism of 5-Hydroxypicolinic Acid in Alcaligenes faecalis JQ135 by Binding to Two Unique DNA Sequences in the Promoter of Hpa Operon.

5-Hydroxypicolinic acid (5HPA), an important natural pyridine derivative, is microbially degraded in the environment. Previously, a gene cluster, hpa, responsible for 5HPA degradation, was identified in Alcaligenes faecalis JQ135. However, the transcription regulation mechanism of the hpa cluster is still unknown. In this study, the transcription start site and promoter of the hpa operon was identified. Quantitative reverse transcription-PCR and promoter activity analysis indicated that the transcription of the hpa operon was negatively regulated by a TetR family regulator, HpaR, whereas the transcription of hpaR itself was not regulated by HpaR. Electrophoretic mobility shift assay and DNase I footprinting revealed that HpaR bound to two DNA sequences, covering the -35 region and -10 region, respectively, in the promoter region of the hpa operon. Interestingly, the two binding sequences are partially palindromic, with 3 to 4 mismatches and are complementary to each other. 5HPA acted as a ligand of HpaR, preventing HpaR from binding to promoter region and derepressing the transcription of the hpa operon. The study revealed that HpaR binds to two unique complementary sequences of the promoter of the hpa operon to negatively regulate the catabolism of 5HPA. IMPORTANCE This study revealed that the transcription of the hpa operon was negatively regulated by a TetR family regulator, HpaR. The binding of HpaR to the promoter of the hpa operon has the following unique features: (i) HpaR has two independent binding sites in the promoter of the hpa operon, covering -35 region and -10 region, respectively; (ii) the palindrome sequences of the two binding sites are complementary to each other; and (iii) both of the binding sites include a 10-nucleotide partial palindrome sequence with 3 to 4 mismatches. This study provides new insights into the binding features of the TetR family regulator with DNA sequences.

Two LysR Family Transcriptional Regulators, McbH and McbN, Activate the Operons Responsible for the Midstream and Downstream Pathways, Respectively, of Carbaryl Degradation in Pseudomonas sp. Strain XWY-1.

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. IMPORTANCE The enzyme-encoding genes involved in the complete degradation pathway of carbaryl in Pseudomonas sp. strain XWY-1 include mcbABCDEF, mcbIJKLM, and mcbOPQ. Previous studies demonstrated that the mcbA gene, responsible for hydrolysis of carbaryl to 1-naphthol, is constitutively expressed and that the transcription of mcbBCDEF was regulated by McbG. However, the transcription regulation mechanisms of mcbIJKLM and mcbOPQ have not been investigated yet. In this study, we identified two LysR-type transcriptional regulators, McbH and McbN, which activate the mcbIJKLM cluster (responsible for the degradation of salicylate to gentisate) and the mcbOPQ cluster (responsible for the degradation of gentisate to pyruvate and fumarate), respectively. The 13-bp motif is critical for McbH to bind to the promoter of mcbIJKLM, and 12-bp motif different from the typical characteristics of the LysR-type transcriptional regulator (LTTR) binding sequence affects the binding of McbN to the promoter. These findings help to expand the understanding of the regulatory mechanism of microbial degradation of carbaryl.

Comparative Genomic Analysis of Carbofuran-Degrading Sphingomonads Reveals the Carbofuran Catabolism Mechanism in Sphingobium sp. Strain CFD-1.

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.

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.

Degradation of dimethachlon by a newly isolated bacterium Paenarthrobacter sp. strain JH-1 relieves its toxicity against Chlorella ellipsoidea.

Dimethachlon, a broad-spectrum dicarboximide fungicide, poses a hazard to the safety of human and ecosystem due to its residue in the environment. A high-efficient dimethachlon degrading bacteria JH-1 belonging to Paenarthrobacter sp. was isolated and characterized. Strain JH-1 can utilize high concentration of dimethachlon as sole carbon source for growth and degrade 98.53% of 300 mg·L-1 dimethachlon within 72 h. Crude enzyme of strain JH-1 could degrade 99.76% of 100 mg·L-1 dimethachlon within 2 h. The optimum degradation condition of dimethachlon by strain JH-1 was at 35 °C and pH 7.0. Dimethachlon was degraded in Paenarthrobacter sp. JH-1 as following: it was firstly converted to 4-(3,5-dichloroanilino)-4-oxobutanoic acid and then subjected to the hydrolysis to 3,5-dichloroaniline and succinic acid, the latter was further degraded. Dimethachlon inhibited the growth of Chlorella ellipsoidea, while Paenarthrobacter sp. JH-1 could degrade dimethachlon to relieve its toxicity. This work facilitates our knowledge of the degradation mechanism of dimethachlon and offers potential resource of microbial strains for the bioremediation of dimethachlon-contaminated environments in the future.

Anaerobic biodegradation and detoxification of chloroacetamide herbicides by a novel Proteiniclasticum sediminis BAD-10T.

Chloroacetamide herbicides (CAAHs) are important herbicides that were widely used to control agricultural weeds. However, their mass applications have seriously contaminated environment, and they are toxic to living beings. CAAHs are easy to enter anoxic environments such as subsoil, wetland sediment, and groundwater, where CAAHs are mainly degraded by anaerobic organisms. To date, there are no research on the anaerobic degradation of CAAHs by pure isolate and toxicity of anaerobic metabolites of CAAHs. In this study, the anaerobic degradation kinetics and metabolites of CAAHs by an anaerobic isolate BAD-10T and the toxicity of anaerobic metabolites were studied. Isolate BAD-10T could degrade alachlor, acetochlor, propisochlor, butachlor, pretilachlor and metolachlor with the degradation kinetics fitting the pseudo-first-order kinetics equation. The degradation rates of CAAHs were significantly affected by the length of N-alkoxyalkyl groups, the shorter the N-alkoxyalkyl groups, the higher the degradation rates. Four metabolites 2-ethyl-6-methyl-N-(ethoxymethyl)-acetanilide (EMEMA), N-(2-methyl-6-ethylphenyl)-acetamide (MEPA), N-2-ethylphenyl acetamide and 2-ethyl-N-carboxyl aniline were identified during acetochlor degradation, and an anaerobic catabolic pathway of acetochlor was proposed. The toxicity of EMEMA and EMPA for zebrafish, Arabidopsis and Chlorella ellipsoidea were obviously lower than that of acetochlor, indicating that the anaerobic degradation of acetochlor by isolate BAD-10T is a detoxification process. The work reveals the anaerobic degradation kinetics and catabolic pathway of CAAHs and highlights a potential application of Proteiniclasticum sediminis BAD-10T for bioremediation of CAAHs residue-contaminated environment.