Paraflavisolibacter caeni gen. nov., sp. nov., a novel taxon within the family Chitinophagaceae isolated from sludge.

A Gram-stain-negative bacterium, designated strain LB-8T, was isolated from an activated sludge sample collected from a factory in Binzhou city, Shandong province, PR China. Cells of strain LB-8T were strictly aerobic, non-motile and rod-shaped. Growth occurred at 15-40 °C (optimum, 30 °C), at pH 6.0-9.0 (optimum, pH 7.0) and at 0-7.5 % (w/v) NaCl (optimum, 0.5 % NaCl). Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain LB-8T formed a distinct phyletic branch within the family Chitinophagaceae and was most closely related to members of the genera Flavisolibacter, Cnuella and Paracnuella with 92.7-93.3 % 16S rRNA gene sequence similarities. The average amino acid identity values between strain LB-8T and its closed phylogenetic neighbours Flavisolibacter, Cnuella and Paracnuella were below 70 % supporting that strain LB-8T was a member of a novel genus. The predominant cellular fatty acids of LB-8T were iso-C15 : 0, anteiso-C15 : 0, summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c) and iso-C17 : 0 3-OH, and the only isoprenoid quinone was menaquinone-7 (MK-7). The major polar lipids of strain LB-8T were phosphatidylethanolamine, four unidentified aminolipids and two unidentified lipids. The genome size of strain LB-8T was 7.01 Mbp with 41.2 mol% G+C content. On the basis of the evidence presented in this study, strain LB-8T represents a novel species of a new genus in the family Chitinophagaceae, for which the name Paraflavisolibacter caeni gen. nov., sp. nov. (type strain LB-8T=GDMCC 1.3631T=KCTC 92688T) is proposed.

Precise Regulation of Differential Transcriptions of Various Catabolic Genes by OdcR via a Single Nucleotide Mutation in the Promoter Ensures the Safety of Metabolic Flux.

Synergistic regulation of the expression of various genes in a catabolic pathway is crucial for the degradation, survival, and adaptation of microorganisms in polluted environments. However, how a single regulator accurately regulates and controls differential transcriptions of various catabolic genes to ensure metabolic safety remains largely unknown. Here, a LysR-type transcriptional regulator (LTTR), OdcR, encoded by the regulator gene odcR, was confirmed to be essential for 3,5-dibromo-4-hydroxybenozate (DBHB) catabolism and simultaneously activated the transcriptions of a gene with unknown function, orf419, and three genes, odcA, odcB, and odcC, involved in the DBHB catabolism in Pigmentiphaga sp. strain H8. OdcB further metabolized the highly toxic intermediate 2,6-dibromohydroquinone, which was produced from DBHB by OdcA. The upregulated transcriptional level of odcB was 7- to 9-fold higher than that of orf419, odcA, or odcC in response to DBHB. Through an electrophoretic mobility shift assay and DNase I footprinting assay, DBHB was found to be the effector and essential for OdcR binding to all four promoters of orf419, odcA, odcB, and odcC. A single nucleotide mutation in the regulatory binding site (RBS) of the promoter of odcB (TAT-N11-ATG), compared to those of odcA/orf419 (CAT-N11-ATG) and odcC (CAT-N11-ATT), was identified and shown to enable the significantly higher transcription of odcB. The precise regulation of these genes by OdcR via a single nucleotide mutation in the promoter avoided the accumulation of 2,6-dibromohydroquinone, ensuring the metabolic safety of DBHB. IMPORTANCE Prokaryotes use various mechanisms, including improvement of the activity of detoxification enzymes, to cope with toxic intermediates produced during catabolism. However, studies on how bacteria accurately regulate differential transcriptions of various catabolic genes via a single regulator to ensure metabolic safety are scarce. This study revealed a LysR-type transcriptional activator, OdcR, which strongly activated odcB transcription for the detoxification of the toxic intermediate 2,6-dibromohydroquinone and slightly activated the transcriptions of other genes (orf419, odcA, and odcC) for 3,5-dibromo-4-hydroxybenozate (DBHB) catabolism in Pigmentiphaga sp. strain H8. Interestingly, the differential transcription/expression of the four genes, which ensured the metabolic safety of DBHB in cells, was determined by a single nucleotide mutation in the regulatory binding sites of the four promoters. This study describes a new and ingenious regulatory mode of ensuring metabolic safety in bacteria, expanding our understanding of synergistic transcriptional regulation in prokaryotes.

Genetic bioaugmentation with triclocarban-catabolic plasmid effectively removes triclocarban from wastewater.

Triclocarban, one of the emerging pollutants, has been accumulating, and it is frequently detected in wastewater. Due to its toxicity and persistence, the efficient removal of triclocarban from wastewater systems is challenging. Genetic bioaugmentation with transferable catabolic plasmids has been considered to be a long-lasting method to clean up pollutants in continuous flow wastewater treatment systems. In this study, bioaugmentation with Pseudomonas putida KT2440, harboring the transferrable triclocarban-catabolic plasmid pDCA-1-gfp-tccA2, rapidly converted 50 µM triclocarban in wastewater into 3,4-dichloroaniline and 4-chloroaniline, which are further mineralized more easily. RT-qPCR results showed that the ratio of the copy number of pDCA-1-gfp-tccA2 to the cell number of strain KT2440 gradually increased during genetic bioaugmentation, suggesting horizontal transfer and proliferation of the plasmid. By using DNA stable isotope probing (SIP) and amplicon sequencing, OTU86 (Escherichia-Shigella), OTU155 (Citrobacter), OTU5 (Brucella), and OTU15 (Enterobacteriaceae) were found to be the potential recipients of the plasmid pDCA-1-gfp-tccA2 in the wastewater bacterial community. Furthermore, three transconjugants in the genera of Escherichia, Citrobacter, and Brucella showing triclocarban-degrading abilities were isolated from the wastewater. This study develops a new method for removing triclocarban from wastewater and provides insights into the environmental behavior of transferrable catabolic plasmids in bacterial community in wastewater systems.

A two-component monooxygenase for continuous denitration and dechlorination of chlorinated 4-nitrophenol in Ensifer sp. strain 22-1.

The environmental fates of chlorinated 4-nitrophenols, 2,6-dichloro-4-nitrophenol (2,6-DCNP) and 2-chloro-4-nitrophenol (2C4NP), mediated via microbial catabolism have attracted great attention due to their high toxicity and persistence in the environment. In this study, a strain of Ensifer sp. 22-1 that was capable of degrading both 2,6-DCNP and 2C4NP was isolated from a halogenated aromatic-contaminated soil sample. A gene cluster cnpBADCERM was predicted to be involved in the catabolism of 2,6-DCNP and 2C4NP based on genome sequence analysis. A two-component monooxygenase CnpAB, composed of an oxygenase component (CnpA) and a reductase component (CnpB), was confirmed to catalyze the continuous denitration and dechlorination of 2,6-DCNP and 2C4NP to 6-chlorohydroxyquinol (6-CHQ) and hydroxyquinol (HQ), respectively. Knockout of cnpA resulted in the complete loss of the capacity for strain 22-1 to degrade 2,6-DCNP and 2C4NP. Homologous modeling and docking showed that Val155~Ala159, Phe206~Pro209 and Phe446~Arg461 of CnpA participated in the formation of the FAD-binding pocket, and Arg101, Val155 and Asn447 formed hydrogen bonds with 2,6-DCNP/2C4NP in the substrate-binding pocket. This work characterized a new two-component monooxygenase for 2,6-DCNP and 2C4NP, and enriched our understanding of the degradation mechanism of chlorinated nitrophenols (CNPs) by microorganisms.

Pedobacter helvus sp. nov., isolated from farmland soil.

A strictly aerobic, Gram-stain-negative, rod-shaped, yellow, non-spore-forming bacterial strain, designated P-25T, was isolated from soil collected in Yantai, Shandong Province, PR China. The temperature, pH and NaCl concentration ranges for the growth of strain P-25T were 10-37 °C (optimum, 28-30 °C), pH 6.0-9.0 (optimum, pH 7.5-8.0) and 0-4 % (w/v) (optimum, 1 % w/v), respectively. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain P-25T was most closely related to Pedobacter xixiisoli S27T (98.1 % 16S rRNA gene sequence similarity), followed by Pedobacter chitinilyticus CM134L-2T (97.2 %) and Pedobacter ureilyticus THG-T11T (97.1 %). The genomic DNA G+C content of strain P-25T based on its draft genome sequence was 38.1 %. MK-7 was the major respiratory quinone, and iso-C15 : 0, C16 : 1ω7c and/or C16 : 1ω6c (summed feature 3) and iso-C17 : 0 3-OH were the major fatty acids. The major polar lipids were phosphatidylethanolamine, one unidentified aminophospholipid, one unidentified phospholipid, two unidentified lipids, five unidentified aminolipids and two unidentified glycolipids. Average nucleotide identity values for the draft genomes between strain P-25T and strains S27T, CM134L-2T and THG-T11T were 81.8, 77.6 and 81.2 %, respectively, and the digital DNA-DNA hybridization (dDDH) values were 30.0, 19.2 and 27.6 %, respectively. Based on their phylogenetic and phenotypic characteristics, chemotaxonomic data, and dDDH results, strain P-25T is considered to represent a novel species of the genus Pedobacter, for which the name Pedobacter helvus sp. nov. is proposed; the type strain is strain P-25T (KCTC 62821T=CCTCC AB 2018185T).

Pedobacter pollutisoli sp. nov., Isolated from Tetrabromobisphenol A-Contaminated Soil.

A Gram-stain negative, strictly aerobic, non-spore forming, non-motile, rod-shaped bacterium, designated TBBPA-24T, was isolated from tetrabromobisphenol A-contaminated soil in China. Phylogenetic analysis based on the 16S rRNA gene sequences showed that strain TBBPA-24T was most closely related to Pedobacter nanyangensis Q4T (96.5%) and Pedobacter 'zeaxanthinifaciens' TDMA-5T (96.1%). Chemotaxonomic analysis revealed that strain TBBPA-24T possessed MK-7 as the major respiratory quinone and lipid, aminolipid, phospholipid, phosphatidylethanolamine, and phosphoaminolipid as the major polar lipid. The major fatty acids were iso-C15:0 (40.2%), summed feature 3 (C16:1ω6c and/or C16:1ω7c, 25.6%) and iso-C17:0 3-OH (16.4%). The genomic DNA G+C content of strain TBBPA-24T was 43.9 mol%. Based on the phylogenetic, phenotypic characteristics, and chemotaxonomic data, strain TBBPA-24T is considered a novel species of the genus Pedobacter, for which the name Pedobacter pollutisoli sp. nov. is proposed. The type strain TBBPA-24T (= KCTC 62314T = CCTCC AB 2017244T) is proposed.

Comparative Transcriptome Analysis Reveals the Mechanism Underlying 3,5-Dibromo-4-Hydroxybenzoate Catabolism via a New Oxidative Decarboxylation Pathway.

The compound 3,5-dibromo-4-hydroxybenzoate (DBHB) is both anthropogenically released into and naturally produced in the environment, and its environmental fate is of great concern. Aerobic and anaerobic reductive dehalogenations are the only two reported pathways for DBHB catabolism. In this study, a new oxidative decarboxylation pathway for DBHB catabolism was identified in a DBHB-utilizing strain, Pigmentiphaga sp. strain H8. The genetic determinants underlying this pathway were elucidated based on comparative transcriptome analysis and subsequent experimental validation. A gene cluster comprising orf420 to orf426, with transcripts that were about 33- to 4,400-fold upregulated in DBHB-induced cells compared with those in uninduced cells, was suspected to be involved in DBHB catabolism. The gene odcA (orf420), which is essential for the initial catabolism of DBHB, encodes a novel NAD(P)H-dependent flavin monooxygenase that mediates the oxidative decarboxylation of DBHB to 2,6-dibromohydroquinone (2,6-DBHQ). The substrate specificity of the purified OdcA indicated that the 4-hydroxyl group and its ortho-halogen(s) are important for hydroxylation of the C-1 site carboxyl group by OdcA. 2,6-DBHQ is then ring cleaved by the dioxygenase OdcB (Orf425) to 2-bromomaleylacetate, which is finally transformed to ß-ketoadipate by the maleylacetate reductase OdcC (Orf426). These results provide a better understanding of the molecular mechanism underlying the catabolic diversity of halogenated para-hydroxybenzoates.IMPORTANCE Halogenated hydroxybenzoates (HBs), which are widely used synthetic precursors for chemical products and common metabolic intermediates from halogenated aromatics, exert considerable adverse effects on human health and ecological security. Microbial catabolism plays key roles in the dissipation of halogenated HBs in the environment. In this study, the discovery of a new catabolic pathway for 3,5-dibromo-4-hydroxybenzoate (DBHB) and clarification of the genetic determinants underlying the pathway broaden our knowledge of the catabolic diversity of halogenated HBs in microorganisms. Furthermore, the NAD(P)H-dependent flavin monooxygenase OdcA identified in Pigmentiphaga sp. strain H8 represents a novel 1-monooxygenase for halogenated para-HBs found in prokaryotes and enhances our knowledge of the decarboxylative hydroxylation of (halogenated) para-HBs.

Chitinophaga parva sp. nov., a new member of the family Chitinophagaceae, isolated from soil in a chemical factory.

A Gram-stain-negative bacterium, designated LY-1T, was isolated from the soil sample collected from a chemical factory in Fuyang city, Anhui province, China. Cells of strain LY-1T were strictly aerobic, non-motile and rod-shaped. Strain LY-1T grew optimally at pH 7.0 and at 30-35 °C. The taxonomic position was investigated using a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain LY-1T was a member of the genus Chitinophaga and showed the highest sequence similarity to Chitinophaga costaii A37T2T (97.5 %) and lower (<97.0 %) sequence similarity to other known Chitinophaga species. Chemotaxonomic analysis revealed that strain LY-1T possessed menaquinone-7 as the major isoprenoid quinone; and iso-C15 : 0 (46.4 %), C16 : 1ω5c (27.8 %) and iso-C17 : 0 3-OH (9.0 %) were the predominant fatty acids. The polar lipids of strain LY-1T consisted of phosphatidylethanolamine, three unidentified phosphoaminolipids, one unidentified phospholipid, four unidentified lipids, two unidentified aminolipids and two unidentified glycolipids. The genomic DNA G+C content of strain LY-1T was 52.4 mol% based on total genome calculations. The average nucleotide identity and the digital DNA-DNA hybridization value of the draft genomes between strain LY-1T and strain A37T2T were 76.8 and 19.8 %, respectively. Based on the phylogenetic and phenotypic characteristics, chemotaxonomic data, and DNA-DNA hybridization, strain LY-1T is considered a novel species of the genus Chitinophaga, for which the name Chitinophagaparva sp. nov. (type strain LY-1T=CCTCC AB 2018018T=KCTC 62444T) is proposed.

Shinella pollutisoli sp. nov., isolated from tetrabromobisphenol A-contaminated soil.

Strain AH-1T, a Gram-negative, aerobic, non-spore-forming, motile, rod-shaped bacterium, was isolated from tetrabromobisphenol A-contaminated soil in China. The taxonomic position was investigated using a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain AH-1T was a member of the genus Shinella and showed the highest sequence similarity to Shinella fusca DC-196T (97.7 %), Shinella granuli Ch06T (97.3 %), Shinella daejeonensis MJ02T (97.1 %) and Shinella yambaruensis MS4T (96.8 %), and lower (<96.7 %) sequence similarity to other known Shinella species. Chemotaxonomic analysis revealed that strain AH-1T possessed Q-10 as the major isoprenoid quinone; and summed feature 8 (C18 : 1ω6c/C18 : 1ω7c), C16 : 0, C12 : 0 aldehyde, C18 : 0, C19 : 0 cyclo ω8c and C18 : 0 3-OH were the predominant fatty acids. Strain AH-1T showed low DNA-DNA relatedness to S. fusca DC-196T (28.6±5.7 %), S. granuli Ch06T (43.7±3.8 %) and S. daejeonensis MJ02T (48.1±2.6 %). The DNA G+C content was 68.2 mol%. Based on the phylogenetic and phenotypic characteristics, chemotaxonomic data and DNA-DNA hybridization, strain AH-1T is considered a novel species of the genus Shinella, for which the name Shinella pollutisoli sp. nov. (type strain AH-1T=KCTC 52677T=CCTCC AB 2017242T) is proposed.

Anaerobic Degradation of Dicamba via a Novel Catabolic Pathway by a Consortium Enriched from Deep Paddy Soil.

Dicamba is widely used in the paddy field to control broadleaf weeds. Dicamba easily migrates to deep soil, which is anoxic; however, the anaerobic catabolism of dicamba in paddy soil is still unknown. In this study, an anaerobic dicamba-degrading consortium was enriched from deep paddy soil. The consortium completely degraded 0.83 mM dicamba within 7 days. Five metabolites were identified, one of which is a new metabolite, 2,5-dichlorophenol, and a novel anaerobic dicamba degradation pathway was proposed. 2.5 mM dicamba, 1.5-2.0% NaCl, and 20 mM electron acceptors Na2SO4, NaNO3, and FeCl3, and 0.5 mM or more of metabolites 3-CP and 2,5-DCP strongly inhibited the degradation efficiency. During enrichment, the microbial community of the consortium was significantly changed with OTU numbers, and diversity decreased. The study is valuable to elucidate the catabolism and ecotoxicology studies of dicamba in paddy soil and to facilitate the engineering application of anaerobic technology to treat dicamba-manufacturing wastewater.

Expression and Characterization of 3,6-Dihydroxy-picolinic Acid Decarboxylase PicC of Bordetella bronchiseptica RB50.

Picolinic acid (PA) is a typical mono-carboxylated pyridine derivative produced by human/animals or microorganisms which could be served as nutrients for bacteria. Most Bordetella strains are pathogens causing pertussis or respiratory disease in humans and/or various animals. Previous studies indicated that Bordetella strains harbor the PA degradation pic gene cluster. However, the degradation of PA by Bordetella strains remains unknown. In this study, a reference strain of genus Bordetella, B. bronchiseptica RB50, was investigated. The organization of pic gene cluster of strain RB50 was found to be similar with that of Alcaligenes faecalis, in which the sequence similarities of each Pic proteins are between 60% to 80% except for PicB2 (47% similarity). The 3,6-dihydroxypicolinic acid (3,6DHPA) decarboxylase gene (BB0271, designated as picCRB50) of strain RB50 was synthesized and over-expressed in E. coli BL21(DE3). The PicCRB50 showed 75% amino acid similarities against known PicC from Alcaligenes faecalis. The purified PicCRB50 can efficiently transform 3,6DHPA to 2,5-dihydroxypyridine. The PicCRB50 exhibits optimal activities at pH 7.0, 35 °C, and the Km and kcat values of PicCRB50 for 3,6DHPA were 20.41 ± 2.60 µM and 7.61 ± 0.53 S-1, respectively. The present study provided new insights into the biodegradation of PA by pathogens of Bordetella spp.

[Insights into the applications of 3D bioprinting for bioremediation technologies].

A plethora of organic pollutants such as pesticides, polycyclic and halogenated aromatic hydrocarbons, and emerging pollutants, such as flame retardants, is continuously being released into the environment. This poses a huge threat to the society in terms of environmental pollution, agricultural product quality, and general safety. Therefore, effective removal of organic pollutants from the environment has become an important challenge to be addressed. As a consequence of the recent and rapid developments in additive manufacturing, 3D bioprinting technology is playing an important role in the pharmaceutical industry. At the same time, an increasing number of microorganisms suitable for the production of biomaterials with complex structures and functions using 3D bioprinting technology, have been identified. This article briefly discusses the principles, advantages, and disadvantages of different 3D bioprinting technologies for pollutant removal. Furthermore, the feasibility and challenges of developing bioremediation technologies based on 3D bioprinting have also been discussed.