The fold change (FC) and differences in gene expression between two samples were calculated by DEseq2 [73]. 2012;78(17):6295301. PC1 and Cupriavidus sp. Malik VS, Vining LC. Overnight cultures (100 mL LB supplemented with 50 g/mL kanamycin and 34 g/mL chloramphenicol) were inoculated from freshly transformed BL21(DE3) pLysS expressing sfGFP, MTH1 or Neil3 in either the standard pET28a or pET28a-TIR-2+T7p CONS plasmid. [74]. PC1, and Cupriavidus sp. In general, PNB can be mineralized via PCA cleavage pathways in aerobic microbes [46, 47]. PubMed Swenson CE, Sadikot RT. 2012;82(3):70312. 6 ac). 6 ac). ), Pigmentiphaga sp. The biotransformation of CAP by isolated strains in co-culture. 2020;187:116397. PNB5, and Achromobacter sp. Terms and Conditions, Besides, 62-47 sp. DCA was predicted to be transformed to glyoxylate via dichlorination catalyzed by a 2-haloacid dehalogenase (dehII or dhlB) [39], followed by being metabolized to malate and oxaloacetate via successive catalysis of malate synthase (glcB) and malate dehydrogenase (mdh) [40] (Fig. Zhang J, Zhao R, Cao L, Lei Y, Liu J, Feng J, et al. Nitrobenzene compounds are ingredients for the biosynthesis of azoxybenzenes in microorganisms [49]. This study demonstrates the capacity of chloramphenicol to select the expression of an active efflux . 1c). PeerJ. These MAGs provided crucial clues in the elucidation of comprehensive metabolic pathways and biotransformation mechanisms of CAP as well as the functional interactions among the microorganisms in consortium CL. 1995;9(1):414. CAP-1 [14], Geobacter metallireducens GS-15 [29], Escherichia fergusonii I-10-CHL [30], Haemophilus influenzae Rd KW20 [31], Streptomyces sp. 3 ik), indicating that it cannot utilize CAP as the sole carbon or nitrogen source to support its growth. The taxonomies of all genomes including NAGs and MAGs were classified by GTDB-Tk (v1.0.2) according to 120 single-copy marker proteins [22]. Isolation, identification and characterization of human intestinal bacteria with the ability to utilize chloramphenicol as the sole source of carbon and energy. 2016;10(8):196783. As the core CAP degraders, Sphingomonas sp., Caballeronia sp., and Cupriavidus sp. Consortium CL was cultured and passaged in a mineral salt culture medium (MSM) with 120 mg/L CAP and 30 mg/L NH4Cl for about 1.5 years. Environ Sci Technol. Front Microbiol. Caballeronia sp. Article 6b). CLC6 were talented degraders of DCA. . Chloramphenicol is a broad-spectrum antibiotic that typically stops bacterial growth by stopping the production of proteins. At 21 h, the concentration of CAP was reduced to 11.9%, and the concentration of most metabolites began to decline from the peak level, which indicated the later stage (stage 4) of CAP biotransformation. Though no intermediate products of PNB were detected in this study, a metabolic pathway was established according to the metagenomic and metatranscriptomic information (module 5 of Figs. This batch test was conducted in duplicate. O'Brien RW, Morris JG. PLoS Biol. pLys comes in two flavorspLysS and pLysEthe difference being that the latter provides tighter control of basal expression. Expression vectors can drive expression in various cell types (mammalian, yeast, bacterial, etc. This was the first time to reveal the temporal transcriptional expression profiles of the key members of a CAP-degrading consortium utilizing CAP as the sole carbon and energy source, which could provide important clues for the decryption of CAP biotransformation mechanisms as well as interactions among functional microorganisms. CLC6, Sphingomonas sp. Google Scholar. It is also a rare cause of aplastic anemia. Hara H, Masai E, Miyauchi K, Katayama Y, Fukuda M. Characterization of the 4-carboxy-4-hydroxy-2-oxoadipate aldolase gene and operon structure of the protocatechuate 4,5-cleavage pathway genes in Sphingomonas paucimobilis SYK-6. The raw metatranscriptomic data were filtered by fastp (v0.20.1) [56]. 2020;11:394. b The relative abundances of 18 MAGs recovered from metagenomic data. 5b, Additional file 2: Fig. Process Biochem. To identify the main microbial players involved in CAP degradation, we assembled 24 metagenomic datasets of consortium CL. Data are presented as mean values SD (n = 3), The proposed core genes involved in CAP biotransformation in cells and the interaction between members of the consortium in CAP metabolism. The accumulation of these products was observed during the stage of CAP rapid degradation (Fig. DadA can catalyze the oxidative deamination of D-amino acids or their analogs into corresponding oxoacids with broad substrate specificity [41]. Then, the product 4-HABA was transformed to protocatechuate (PCA) catalyzed by 4-hydroxylaminobenzoate lyase (pnbB) [45]. CL5.1 using the primers 5-GTCGACGGTATCGATTAAGGAGGTTTTCTAGTGCAAGATATTAGAACTAC-3 and 5-CAGGAATTCGATATCTCAGTGGCTTCTTCGGATCA-3. JZ, BL, and HL curated the data. Besides, P-209, P179, P-195a, and P-195b were detected to be produced by Caballeronia sp. Clusters of orthologous group (COG) terms of CDSs were annotated by eggNOG-mapper (v2) [70]. Schofield CJ. statement and Chin J Antibiot. Appl Microbiol Biotechnol. Zhang J, Gan W, Zhao R, Yu K, Lei H, Li R, et al. 2018;46(20):e119. 3 and Additional file 2: Fig. The gene expression was presented as Log2(FC) (n = 3). First, the raw metagenomic data were filtered by fastp (v0.20.1) [56] and then were assembled using metaSPAdes (v3.13.2) [58]. These include the cleavage of C1/C2 and the hydrolysis of the amide bond. Guo YY, Li ZH, Xia TY, Du YL, Mao XM, Li YQ. Degradation pathways of 2- and 4-nitrobenzoates in Cupriavidus sp. 2020;11:604926. Here, we took advantage of a microbial consortium (named CL) which was enriched from an activated sludge bioreactor with CAP as the sole carbon source and continuously passaged for 1.5 years. Environ Sci Technol. and Pigmentiphaga sp. Herein, this study successfully provides the first demonstration of obtaining a comprehensive view of the biotransformation mechanisms and the bacterial interactions in a bacterial community feeding on CAP via an integrated multi-omics approach and cultivation-based techniques, which afforded desirable strain and enzyme resources and a theoretical foundation for developing enhanced bioremediation of CAP-contaminated hotspot sites such as hospital wastewater, pharmaceutical wastewater, and livestock and poultry breeding wastewater. The missing step is understanding CAP biotransformation by microbial consortia, which is especially relevant for full-scale wastewater treatment plants where many processes such as aerobic biodegradation and anaerobic digestion rely on microbial interactions rather than the performance of single strains or species. Immobilized metal affinity chromatography (IMAC) with a polyhistidine tag (usually six consecutive histidine residues) has emerged as the most common and convenient method for purifying recombinant proteins. 2018;128:38392. J Hazard Mater. To confirm the function of this candidate CAP oxidoreductase encoding gene capO, it was cloned and heterologously expressed in Pseudomonas putida KT2440. When CAP was supplied as the sole carbon or nitrogen source, the CAP degradation efficiency was less than 5.0%, and the biomass of Cupriavidus sp. The protein extraction and tandem mass tag (TMT) labeling proteomic sequencing were performed as the method described in the previous study [38]. T7 expression. Environ Chem Lett. Article MAGs de-replication was performed using drep (v2.0.0) [64]. (Cup. Provided by the Springer Nature SharedIt content-sharing initiative. D-CAP was also transformed to DD-CAP via dehydrogenation and dehydration. 2017;8:297. 2017;5(1):84. Qubit 2.0 Fluorometer (Life Technologies, USA) was used to quantify the constructed library. Figure S7. CL5.1, which further confirmed the proposed metabolic processes of PNB (Additional file 1: Table S3). CL5.1 was about 52.2% after 10-day cultivation (Additional file 2: Fig. ISME J. They converted CAP into RS-CAP, O-CAP, dD-CAP, DD-CAP, and A-CAP via isomerization, oxidization, and acetylation and thus detoxified CAP for other members of the community (Fig. Nitroaromatic compounds, from synthesis to biodegradation. 2021;49(W1):W2936. PC1 (Fig. Pasolli E, Asnicar F, Manara S, Zolfo M, Karcher N, Armanini F, et al. As a widely used broad-spectrum antibiotic, chloramphenicol is prone to be released into environments, thus resulting in the disturbance of ecosystem stability as well as the emergence of antibiotic resistance genes. Chloramphenicol is a substrate for a novel nitroreductase pathway in Haemophilus influenzae. Google Scholar. . 22176107), Guangdong Basic and Applied Basic Research Foundation (No. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. These biotransformation processes were discovered in Sphingomonas sp. The mixture of MH broth and MSM without CAP was included as the control sample. Ideal for P lac, P tac, P trc ParaBAD . An integrated meta-omics approach reveals substrates involved in synergistic interactions in a bisphenol A (BPA)-degrading microbial community. Genome Biol. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Occurrence, toxicity and adsorptive removal of the chloramphenicol antibiotic in water: a review. For instance, CAP, D-CAP, dD-CAP, and DD-CAP could be cleaved into 4-nitrobenzaldehyde (PNBD) via the oxidative cleavage of C1 and C2 and could finally be oxidized to 4-nitrobenzoic acid (PNB) (Module 4). Seemann T. Prokka: rapid prokaryotic genome annotation. Sphingomonas sp. PC1 could degrade about 99.0% of CAP within 10 days (Fig. A high membership value indicates a high correlation of the gene expression with the cluster. strain HKI0708. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. S5, Additional file 3) and 23 metabolites deduced by cultivation-independent multi-omics. 7 Citations 14 Altmetric Metrics Abstract Several essential components of the electron transport chain, the major producer of ATP in mammalian cells, are encoded in the mitochondrial genome. The expression of acetate symporter (actP) located at the genome of Cupriavidus sp. A comprehensive CAP metabolism pathway, related functional genes, and enzymes were thoroughly revealed by the combined application of proteomics, metagenomics, and metatranscriptomics, as well as cultivation-dependent approaches. The temporal expression of genes involved in the glycine cleavage system of Sphingomonas sp. Figure S13. were the three remaining dominant MAGs in consortium CL with average relative abundances of 10.6 8.8%, 13.8 7.7%, and 3.6% 0.6%, respectively (Fig. Kanamycin, spectinomycin, gentamicin, and chloramphenicol were added into the medium for final concentrations of 20, 20, 2, and 10 g/mL, respectively. CL5.1 and Caballeronia sp. Anders S, Pyl PT, Huber W. HTSeq--a Python framework to work with high-throughput sequencing data. By using this website, you agree to our Antibiotic residue and toxicity assessment of wastewater during the pharmaceutical production processes. The MS/MS spectra of 27 detected metabolites and 5 available metabolite standards, including information about m/z values of parent and fragment ions and retention time, were listed in Additional file 3. Moreover, the conversion from CAP to O-CAP was also found in Sphingobium sp. 2019;176(3):64962. This finding implies the potential of biosynthesis of the azoxy bond on the base of nitrobenzene compounds, which probably opens a new route for azoxybenzenes biosynthesis. However, when coming across a difficult-to-express protein, things can get complicated. Cell Lysis (DAY 2; TIMING 4 hours) MetaBAT 2: an adaptive binning algorithm for robust and efficient genome reconstruction from metagenome assemblies. Xin Z, Fengwei T, Gang W, Xiaoming L, Qiuxiang Z, Hao Z, et al. Walker BJ, Abeel T, Shea T, Priest M, Abouelliel A, Sakthikumar S, et al. Besides, the subsequent biotransformation steps, corresponding catalyzing enzymes, and the microbial players responsible for each step were comprehensively deciphered via integrated multi-omic analysis and cultivation-dependent approaches. during CAP rapid-degrading stage 3. 1971;67(3):26571. JZ, HL, FG, KY, and CY performed investigation and analyses. Bioinformatics. Your privacy choices/Manage cookies we use in the preference centre. Cell. S13a). 4 module 6). In summary, the initial biotransformation steps involved in this comprehensive microbial metabolism pathway were the oxidization at the C1-OH and C3-OH groups, the isomerizationatC2, and the acetylation at C3-OHof CAP. These. 6 a, b, c). Article ac Differential expression of genes involved in the CAP biodegradation in MAG1 (Sphingomonas sp.) Zhou Y, Sha J, Wu M, Liu J, Bi C, Lin J. Nat Methods. CL 5.1 and its co-culture with other strains. The strain-level transcriptional profiles of seven isolated key species (Sphingomonas sp., Caballeronia sp., Cupriavidus sp., Pigmentiphaga sp., Chryseobacterium sp., Labrys sp., and Achromobacter sp.) 2022;20(3):192963. Despite a number of proven side effects, including aplastic anemia, marrow aplasia, visual impairment, and deafness in humans, it is still widely used in developing countries because of its low cost and excellent antibacterial properties against both gram-negative and gram-positive bacterial pathogens [1]. The assemblies of the filtered data were obtained using Flye (v2.6) [55]. Nat Biotechnol. For example, 1230 tons of CAP were estimated to be consumed annually in China [2]. 6 a, b, c). The pLysS presence allows tight control of expression. Nat Commun. However, the enzymes involved in these biotransformation processes were still unclear. 2017;34(6):95061. 2b). CLB4. 3e). found that P-286 was a metabolite of CAP degradation in eye drops [51]. Wongnate T, Chaiyen P. The substrate oxidation mechanism of pyranose 2-oxidase and other related enzymes in the glucose-methanol-choline superfamily. Science. This suggested that the genes belonging to clusters 5 and 6 might be involved in the biotransformation of CAP (Additional file 2: Fig. Gustavo Bodelon CINBIO (University of Vigo) For the BL21 (DE3)pLysS host strain, the overnight culture must contain chloramphenicol at a final concentration of 50 g/ml in addition to the. Price MN, Dehal PS, Arkin AP. PC1 was another degrader that could utilize CAP as the sole carbon and nitrogen source but with a significantly lower degradation efficiency compared to Sphingomonas sp. ISME J. The relative abundance of all remaining MAGs was lower than 1.0%. These biotransformation products were further cleaved into the downstream metabolites including PNB, DCA, azoxybenzenes, and dichloroacetyl compounds (P-171, P169, and P-185) by Sphingomonas sp. Chemically competent E. coli K12 cells suitable for T7 protein expression with enhanced capacity to correctly fold proteins with multiple disulfide bonds in the cytoplasm. Xue J, Lei D, Zhao X, Hu Y, Yao S, Lin K, et al. could utilize products of bisphenol A produced by Sphingonomas spp. In common, ketone reduction can convert prochiral ketones into chiral alcohols [42]. Ding L, Ndejouong Ble S, Maier A, Fiebig HH, Hertweck C. Elaiomycins D-F, antimicrobial and cytotoxic azoxides from Streptomyces sp. Letunic I, Bork P. Interactive Tree Of Life (iTOL) v5: an online tool for phylogenetic tree display and annotation. CL5.1 and Caballeronia sp. Langmead B, Salzberg SL. Food Addit Contam Part A Chem Anal Control Expo Risk Assess. The biomass of strains was quantified by a microplate reader Infinite M200 (Tecan, Switzerland). is responsible for CAP acetylation in consortium CL (Fig. Fischer DS, Theis FJ, Yosef N. Impulse model-based differential expression analysis of time course sequencing data. TO CONTINUE WITH PROTEIN PURIFICATION (DURING DAY 2) Add E/W to a total volume of 16 mL for a 400 or 800 mL expression, or 32 mL (or 216 mL) for a 1 or 2 L expression. Rimkus GG, Hoffmann D. Enantioselective analysis of chloramphenicol residues in honey samples by chiral LC-MS/MS and results of a honey survey. Inactivation of chloramphenicol and florfenicol by a novel chloramphenicol hydrolase. viciae. [20, 21]. was predicted to be capable of both ortho-cleavage and meta-cleavage of PCA. 2016;82(14):425363. A series of enzymes in the glycine cleavage system respond to the high concentration of GLY. Figure S3. The samples for nucleic acid extraction were stored at 80 C after flash freezing with liquid nitrogen. Chloramphenicol is an antibiotic that consistently suppresses the bone marrow and induces sideroblastic anemia. PC1 (Fig. CAP-degrading features of seven isolated strains were investigated under different nutritional conditions. After 17 days of cultivation, it could degrade 51.7% of CAP (Fig. RCL7, Labrys sp. Binning metagenomic contigs by coverage and composition. CLC6. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. New insights into thiamphenicol biodegradation mechanism by Sphingomonas sp. 20 . The dynamics of other CAP metabolites produced by isolated strains including Sphingomonas sp. PC1 (Additional file 2: Fig. PC1, and Cupriavidus sp. CL5.1 was upregulated by 2.74-fold (p < 0.001) at 34 h compared to that at 0 h (Additional file 1: Table S3). to enhance bisphenol A mineralization in a microbial consortium [17]. (Fig. The production of P-226 indicated the hydrolysis of the amide bond of metabolites in Module 1 (Fig. PubMed Central The metabolites in red color were deduced according to multi-omics rather than directly detected by HPLC-QTOF-MS. Pseudomonas putida KT2440 transformed with pBBR-capO could convert CAP into D-CAP and O-CAP with high efficiency, which was consistent with the above speculation (Additional file 2: Fig. Based on the community-wide effects, the predicted varying roles of the key strains within the microbial consortium CL were investigated in microcosm experiments. Figure S10. The synergistic interactions of the isolated strains were investigated via co-culture experiments. Electrolytic and electro-irradiated technologies for the removal of chloramphenicol in synthetic urine with diamond anodes. Eighteen metagenome-assembled genomes (MAGs) including 13 high-quality genomes (completeness > 94%, contamination < 3%) were recovered from over 377 GB of metagenomic data via metagenome binning (Additional file 1: Table S1). The antimicrobial activity of CAP and its product residues in the culture medium was determined by an antimicrobial activity test using a CAP susceptible strain Staphylococcus aureus ATCC 25923. or Cupriavidus sp. The integration analysis process of multi-omics data was shown in Additional file 2: Fig. The MS/MS spectra of CAP and its products determined by HPLC-QTOF-MS. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. For instance, Pseudomonas sp. PC1 (p < 0.001) (Fig. Nat Microbiol. Huang L, Yuan H, Liu MF, Zhao XX, Wang MS, Jia RY, et al. Anyone you share the following link with will be able to read this content: Sorry, a shareable link is not currently available for this article. PLoS One. Novel pathway for chloramphenicol catabolism in the activated sludge bacterial isolate Sphingobium sp. 3 ad). (MAG8) was predicted to conduct ortho-cleavage of PCA (Additional file 2: Fig. The top histogram indicated the total DEG number (log10 transformation) in each KEGG term in consortium CL. The biotransformation of organics usually relies on the synergistic microbial metabolism to achieve desirable performance outcomes in engineered or natural environments [15, 16]. Caballeronia sp. Chloramphenicol (Cm) is a broad-spectrum classic antibiotic active against prokaryotic organisms. 2021M691772). in carbon utilization of PNB, which probably resulted in their low relative abundance in the consortium CL. Expression host strains in many different versions can be used with a variety of protein expression systems. CL5.1 and Caballeronia sp. The interactions between bacteria are complicated processes, and only limited knowledge regarding them can be acquired based on cultivation-based techniques, especially since only a few bacteria can be isolated [17, 54]. JZ wrote the original manuscript. CLC6 had the ability of PNB utilization (Additional file 2: Fig. 2021;38(12):58259. CL5.1 and Cupriavidus sp. 1a). Integrated multi-omics analyses reveal the biochemical mechanisms and phylogenetic relevance of anaerobic androgen biodegradation in the environment. Eluent A was Milli-Q water containing 5 mM ammonium acetate, and Eluent B was methanol. Can J Microbiol. The passage was conducted once a week. To elucidate the temporal transcriptional profile of consortium CL during CAP biotransformation, over 271 GB of time-series metatranscriptomic datasets were obtained from consortium CL. Expression, purification, and characterization of a membrane-bound D-amino acid dehydrogenase from Proteus mirabilis JN458. and Pimelobacter sp. CL5.1 was inoculated into MSM containing 1 g/L sodium pyruvate and 30 mg/L NH4Cl. 2021;297(4):101143. The initial concentrations of CAP, PNB, and DCA were all 50 mg/L, and 30 mg/L NH4Cl was supplied as the additional nitrogen source. Microbes play a vital role in the decomposition of chloramphenicol in the environment, and the biotransformation processes are especially dependent on synergistic interactions and metabolite exchanges among microbes. The recombinant vector pBBR1MCS-2 (pBBR) harboring the cloned capO (pBBR-capO) was constructed through homologous recombination using a seamless cloning kit (SparkJade Biotechnology Co., Ltd., Shandong, China). CAP and its metabolites were quantified using QuantAnalysis (v4.4) through the external standard method. 3l). J Agric Food Chem. Prodigal: prokaryotic gene recognition and translation initiation site identification. What if I don't see protein overexpression? PC1 (eh), and Cupriavidus sp. 2012;9(4):3579. Similarly, there were 101 significantly up-regulated and 200 significantly down-regulated proteins in CT72 vs. the control group. Kuprat T, Johnsen U, Ortjohann M, Schnheit P. Acetate metabolism in archaea: characterization of an acetate transporter and of enzymes involved in acetate activation and gluconeogenesis in Haloferax volcanii.

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