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.

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.

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.

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 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.

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.

Pseudaminobacter soli sp. nov., Isolated from Paddy Soil Contaminated with Heavy Metals.

A Gram stain-negative, aerobic, rod-shaped strain, designated HC19T, was isolated from heavy metals contaminated paddy soil. The 16S rRNA gene-based phylogenetic analysis indicated that strain HC19T belonged to the genus Pseudaminobacter, and shared 97.0% 16S rRNA gene sequence similarity with P. manganicus JH-7T, and less than 97% similarities with other type strains belonging to the genus. The major cellular fatty acids were C19:0 cyclo ω8c (55.0%) and C18: 1ω7c (18.7%). The major quinone was ubiquinone Q-10. The major polar lipids were phosphatidylmonomethylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine and phosphatidylethanolamine. The average nucleotide identity and digital DNA-DNA hybridization values between the genomes of HC19T and P. manganicus JH-7T were 68.0% and 22%, respectively. The G+C content of the genomic DNA was 63.3 mol%. On the basis of phenotypic, chemotaxonomic, and genotypic data, strain HC19T is considered as a novel species in the genus Pseudaminobacter, for which the name Pseudaminobacter. soli sp. nov. is proposed. The type strain is HC19T (= KCTC 82870T = CCTCC AB 2021107T).

Isolation and Characterization of the Wastewater Micropollutant Phenacetin-Degrading Bacterium Rhodococcus sp. Strain PNT-23.

Phenacetin, an antipyretic and analgesic drug, poses a serious health risk to both humans and aquatic organisms, which is of concern since this micropollutant is frequently detected in various aquatic environments. However, rare pure bacterial cultures have been reported to degrade phenacetin. Therefore, in this study, the novel phenacetin-degrading strain PNT-23 was isolated from municipal wastewater and identified as a Rhodococcus sp. based on its morphology and 16S rRNA gene sequencing. The isolated strain could completely degrade 100 mg/L phenacetin at an inoculum concentration of OD600 1.5 within 80 h, utilizing the micropollutant as its sole carbon source for growth. Strain PNT-23 exhibited optimal growth in LB medium at 37 °C and a pH of 7.0 with 1% NaCl, while the optimal degradation conditions in minimal medium were 30 °C and a pH of 7.0 with 1% NaCl. Two key intermediates were identified during phenacetin biodegradation by the strain PNT-23: N-acetyl-4-aminophenol and 4-aminophenol. This study provides novel insights into the biodegradation of phenacetin using a pure bacterium culture, expands the known substrate spectra of Rhodococcus strains and presents a potential new candidate for the microbial removal of phenacetin in a diverse range of environments.

Biodegradation of quinolinic acid by a newly isolated bacterium Alcaligenes faecalis strain JQ191.

Quinolinic acid (QA) is a pyridine derivative that can be found in many organisms and is widely used in the chemical industry. However, QA possesses excitotoxic properties. To date, the catabolism of QA mediated by microorganisms has rarely been reported. In this study, a QA-degrading strain (JQ191) was isolated from sewage sludge. Based on phenotypic and 16S rRNA gene phylogenetic analysis, the strain was identified as Alcaligenes faecalis. Strain JQ191 was able to utilize QA as the sole source of carbon and nitrogen for growth. QA-cultured cells of JQ191 completely degrade 200 mg/L QA within 2 days in a mineral salt medium, whereas the LB-cultured cells experienced a 2-day lag period before degrading QA, indicating that the catabolic enzymes involved in QA degradation were induced by QA. 6-Hydroxypicolinic acid (6HPA) was identified as an intermediate of QA degradation by strain JQ191. A 6HPA monooxygenase gene picB was cloned, genetically disrupted, and heterologously expressed, and the results show that picB was responsible for catalyzing 6HPA to 3,6DHPA in JQ191. A new QA mineralization pathway was proposed. This study identifies a new bacterium candidate that has a potential application prospect in the bioremediation of QA-polluted environment, as well as provides new insights into the bacterial catabolism of QA.

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.

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.