Crystal structures of herbicide-detoxifying esterase reveal a lid loop affecting substrate binding and activity.

SulE, an esterase, which detoxifies a variety of sulfonylurea herbicides through de-esterification, provides an attractive approach to remove environmental sulfonylurea herbicides and develop herbicide-tolerant crops. Here, we determined the crystal structures of SulE and an activity improved mutant P44R. Structural analysis revealed that SulE is a dimer with spacious binding pocket accommodating the large sulfonylureas substrate. Particularly, SulE contains a protruding ß hairpin with a lid loop covering the active site of the other subunit of the dimer. The lid loop participates in substrate recognition and binding. P44R mutation altered the lid loop flexibility, resulting in the sulfonylurea heterocyclic ring repositioning to a relative stable conformation thus leading to dramatically increased activity. Our work provides important insights into the molecular mechanism of SulE, and establish a solid foundation for further improving the enzyme activity to various sulfonylurea herbicides through rational design.

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.

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.

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.

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.

Biodegradation of Quinoline by a Newly Isolated Salt-Tolerating Bacterium Rhodococcus gordoniae Strain JH145.

Quinoline is a typical nitrogen-heterocyclic compound with high toxicity and carcinogenicity which exists ubiquitously in industrial wastewater. In this study, a new quinoline-degrading bacterial strain Rhodococcus sp. JH145 was isolated from oil-contaminated soil. Strain JH145 could grow with quinoline as the sole carbon source. The optimum growth temperature, pH, and salt concentration were 30 °C, 8.0, and 1%, respectively. 100 mg/L quinoline could be completely removed within 28 h. Particularly, strain JH145 showed excellent quinoline biodegradation ability under a high-salt concentration of 7.5%. Two different quinoline degradation pathways, a typical 8-hydroxycoumarin pathway, and a unique anthranilate pathway were proposed based on the intermediates identified by liquid chromatography-time of flight mass spectrometry. Our present results provided new candidates for industrial application in quinoline-contaminated wastewater treatment even under high-salt conditions.

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.

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.

Characterization of a new heterotrophic nitrification bacterium Pseudomonas sp. strain JQ170 and functional identification of nap gene in nitrite production.

Heterotrophic nitrification bacteria play a critical role in nitrogen cycling and pollution removal. However, the underlying nitrification mechanisms are diverse and have rarely been investigated at the genetic level. In this study, the new heterotrophic nitrifier Pseudomonas sp. strain JQ170 was isolated. Strain JQ170 can utilize ammonia (NH4+-N), nitrite (NO2--N), or nitrate (NO3--N) as sole nitrogen sources, preferring NH4+-N. A ratio of 96.4% of 1.0 mM NH4+-N was removed in 24 h. The optimum pH, temperature, and carbon source for NH4+-N removal were pH 7.0, 30 °C, and citrate, at a C/N ratio of 9-18, respectively. During the NH4+-N removal process, only NO2--N but neither hydroxylamine, NO3--N, nor gaseous nitrogen were detected. A random transposon insertion mutagenesis library of strain JQ170 was constructed. Two NO2--N-production deficient mutants were screened and transposon insertion sites were located in nap genes (which encode periplasmic NO3--N reductase Nap). Further gene knockout and complementation of the napA gene confirmed nap as essential for NO2--N production. The following nitrification processes in strain JQ170 is proposed: NH4+-N to NO3--N in the cytoplasm; then NO3--N to NO2--N in the periplasmic space by Nap; finally, NO2--N secreted out of cells. Overall, this paper provides new insight towards understanding heterotrophic nitrification at the genetic level.

Rhodobacter kunshanensis sp. nov., a Novel Bacterium Isolated from Activated Sludge.

Strain HX-7-19T was isolated from the activated sludge collected from an abandoned herbicide manufacturing plant in Kunshan, China. Cells were Gram-reaction-negative, rod-shaped, and non-motile. The phylogenetic analysis based on 16S rRNA gene indicated that strain HX-7-19T formed a clade with Rhodobacter blasticus CGMCC 1.3365T (96.3% sequence similarity). The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between strain HX-7-19T and R. blasticus CGMCC 1.3365T were 76.2% and 20.3%, respectively. The genomic DNA G + C content of strain HX-7-19T was 65.9%. The major fatty acids (> 10% of the total fatty acids) were C18:1 ω7c and C18:1 ω7c 11-methyl. The major respiratory quinone was quinone Q-10. The major polar lipid profile consists of phosphatidylglycerol (PG), diphosphatidyl-glycerol (DPG), phosphatidylethanolamine (PE), and phosphatidylcholine (PC). Photosynthesis pigments bacteriochlorophyll a and carotenoids were formed and photosynthesis genes pufL and pufM were detected. On the basis of phenotypic and phylogenetic evidences, strain HX-7-19T is considered as a novel species in the genus Rhodobacter, for which the name Rhodobacter kunshanensis sp. nov. is proposed. The type strain is HX-7-19T (= KCTC 72471T = CCTCC AB 2020148T).

Cotinine Hydroxylase CotA Initiates Biodegradation of Wastewater Micropollutant Cotinine in Nocardioides sp. Strain JQ2195.

Cotinine is a stable toxic contaminant, produced as a by-product of smoking. It is of emerging concern due to its global distribution in aquatic environments. Microorganisms have the potential to degrade cotinine; however, the genetic mechanisms of this process are unknown. Nocardioides sp. strain JQ2195 is a pure-culture strain that has been reported to degrade cotinine at micropollutant concentrations. This strain utilizes cotinine as its sole carbon and nitrogen source. In this study, a 50-kb gene cluster (designated cot), involved in cotinine degradation, was predicted based on genomic and transcriptomic analyses. A novel three-component cotinine hydroxylase gene (designated cotA1A2A3), which initiated cotinine catabolism, was identified and characterized. CotA from Shinella sp. strain HZN7 was heterologously expressed and purified and was shown to convert cotinine into 6-hydroxycotinine. H218O-labeling and electrospray ionization-mass spectrometry (ESI-MS) analysis confirmed that the hydroxyl group incorporated into 6-hydroxycotinine was derived from water. This study provides new molecular insights into the microbial metabolism of heterocyclic chemical pollutants. IMPORTANCE In the human body, cotinine is the major metabolite of nicotine, and 10 to 15% of generated cotinine is excreted in urine. Cotinine is a structural analogue of nicotine and is much more stable than nicotine. Increased tobacco consumption has led to high environmental concentrations of cotinine, which may have detrimental effects on aquatic ecosystems and human health. Nocardioides sp. strain JQ2195 is a unique cotinine-degrading bacterium. However, the underlying genetic and biochemical foundations of cotinine degradation are still unknown. In this study, a 50-kb gene cluster (designated cot) was identified by genomic and transcriptomic analyses as being involved in the degradation of cotinine. A novel three-component cotinine hydroxylase gene (designated cotA1A2A3) catalyzed cotinine to 6-hydroxy-cotinine. This study provides new molecular insights into the microbial degradation and enzymatic transformation of cotinine.

Rudanella paleaurantiibacter sp. nov., Isolated from Activated Sludge.

A Gram-stain-negative, orange-colored bacterium, designated HX-22-17T, was isolated from activated sludge of an agricultural chemical plant in Maanshan, Anhui province, China (118° 52' E 31° 68' N). The strain was strictly aerobic, non-endospore forming, non-motile, and ellipse. Growth of the strain was observed at 16-42 °C (optimum between 25 and 30 °C) at pH 6.0-9.0 (optimum at pH 7.0) and with 0-6.0% (w/v) NaCl (optimum at 1.0%). 16S rRNA gene sequence analysis showed that the strain was most closely related to Rudanella lutea KACC 12603T (99.5% similarity). The predominant cellular fatty acids were summed feature 3 (C16:1ω7c and/or C16:1ω6c), iso-C15:0, and anteiso-C15:0. The major polar lipids included posphatidylethanolamine (PE), aminolipid (AL), and phospholipids (PL). The genomic DNA G+C content of the strain was 54.1 mol%. The ANI and dDDH values obtained between the genomes of HX-22-17T and R. lutea KACC 12603T were 89.3% and 39.3%, respectively. The phenotypic, chemotaxonomic, and genotypic data clearly showed that strain HX-22-17T represents a novel species of the genus Rudanella, for which the name R. paleaurantiibacter sp. nov. is proposed. The type strain is HX-22-17T (=KCTC 72656T = CCTCC AB 2019347T).

Identification and Characterization of a Novel pic Gene Cluster Responsible for Picolinic Acid Degradation in Alcaligenes faecalis JQ135.

Picolinic acid (PA) is a natural toxic pyridine derivative. Microorganisms can degrade and utilize PA for growth. However, the full catabolic pathway of PA and its physiological and genetic foundation remain unknown. In this study, we identified a gene cluster, designated picRCEDFB4B3B2B1A1A2A3, responsible for the degradation of PA from Alcaligenes faecalis JQ135. Our results suggest that PA degradation pathway occurs as follows: PA was initially 6-hydroxylated to 6-hydroxypicolinic acid (6HPA) by PicA (a PA dehydrogenase). 6HPA was then 3-hydroxylated by PicB, a four-component 6HPA monooxygenase, to form 3,6-dihydroxypicolinic acid (3,6DHPA), which was then converted into 2,5-dihydroxypyridine (2,5DHP) by the decarboxylase PicC. 2,5DHP was further degraded to fumaric acid through PicD (2,5DHP 5,6-dioxygenase), PicE (N-formylmaleamic acid deformylase), PicF (maleamic acid amidohydrolase), and PicG (maleic acid isomerase). Homologous pic gene clusters with diverse organizations were found to be widely distributed in Alpha-, Beta-, and Gammaproteobacteria Our findings provide new insights into the microbial catabolism of environmental toxic pyridine derivatives.IMPORTANCE Picolinic acid is a common metabolite of l-tryptophan and some aromatic compounds and is an important intermediate in organic chemical synthesis. Although the microbial degradation/detoxification of picolinic acid has been studied for over 50 years, the underlying molecular mechanisms are still unknown. Here, we show that the pic gene cluster is responsible for the complete degradation of picolinic acid. The pic gene cluster was found to be widespread in other Alpha-, Beta-, and Gammaproteobacteria These findings provide a new perspective for understanding the catabolic mechanisms of picolinic acid in bacteria.