CL5.1 and Caballeronia sp. 1c). This batch test was conducted in duplicate. PC1 and Cupriavidus sp. The temporal differentially expressed genes (DEGs) were identified by time-series analysis of read counts using ImpulseDE2 with an adjusted p-value 0.05 [25]. The extracted RNA was stored at 80 C before sequencing. Table S2. Nurk S, Meleshko D, Korobeynikov A, Pevzner PA. metaSPAdes: a new versatile metagenomic assembler. 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. Appl Environ Microbiol. The metatranscriptomic data revealed that the average expression of genes involved in the catabolism of glycine (GLY) in the glycine, serine, and threonine metabolism pathway was significantly upregulated in Sphingomonas sp. This covered all the stages of CAP biodegradation. Collectively, the synergistic collaboration of members in the consortium CL contributed to the biotransformation and mineralization of CAP. It was initially enriched from an activated sludge bioreactor in our lab. Genomes of 7 isolated strains were deposited to NCBI with project IDs of PRJNA832945, PRJNA792803, and PRJNA576328. S9b). For instance, Pseudomonas sp. 22176107), Guangdong Basic and Applied Basic Research Foundation (No. PC1 (eh), and Cupriavidus sp. Cho JH, Kim EK, So JS. 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. Due to its consistent use in human medical treatment, livestock and poultry breeding, and aquaculture, highly persistent CAP residues are continuously released into the environment and accumulate in water and soils, thus resulting in the disturbance of ecosystem stability as well as the emergence and spread of antibiotic resistance genes in the environment [3, 4]. The nitro group of PNB was reduced to an amino group and then to a hydroxylamine group producing 4-nitrosobenzoate (4-NOBA) and 4-hydroxylaminobenzoate (4-HABA) via catalysis through a nitroreductase (nfnB or pnbA) [43, 44]. 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. Google Scholar. Comprehensive evaluation of antibiotics emission and fate in the river basins of China: source analysis, multimedia modeling, and linkage to bacterial resistance. A variety of microbes and plants can produce azoxybenzenes, and they may function as deterrents, virulence factors, or means to defend the habitat [49]. 3e). For example, 1230 tons of CAP were estimated to be consumed annually in China [2]. 2020;8(1):111. [20, 21]. In addition, the hydroxyl of P-171 was probably further oxidized to produce P-169 and then P-185. Su JQ, An XL, Li B, Chen QL, Gillings MR, Chen H, et al. Use this strain for both soluble and insoluble protein production. Ideal for P lac, P tac, P trc ParaBAD . PC1, which is a novel metabolic pathway first reported in this study. CLC6s strong CAP acetylation ability (Fig. Chaumeil PA, Mussig AJ, Hugenholtz P, Parks DH. Sorg RA, Lin L, van Doorn GS, Sorg M, Olson J, Nizet V, et al. For pro-duction of protein from target genes cloned in T7 expres-sion vectors, lysogens of DE3 carry a chromosomal copy of the T7 RNA polymerase gene under the control of the lacUV5 promoter. (MAG1). After protein expression, cell cultures were harvested by centrifugation, washed with ice-cold PBS, and resuspended in lysis buffer (25 mM Tris at pH 8.0, 100 mM NaCl, 5 mM imidazole, 1% Triton X . Improved transformation of Pseudomonas putida KT2440 by electroporation. 2018;34(17):i88490. S3. Kim SH, Park S, Park E, Kim JH, Ghatge S, Hur HG, et al. Metabolites were determined by comparison of MS chromatographs of the control group and the experimental group samples in DataAnalysis (v4.4). The samples for nucleic acid extraction were stored at 80 C after flash freezing with liquid nitrogen. As shown in Fig. A part of these downstream metabolites was utilized by the producers, while the other part of them was released to the culture medium to be competitively assimilated by other members of the community. Cantalapiedra CP, Hernndez-Plaza A, Letunic I, Bork P, Huerta-Cepas J. eggNOG-mapper v2: functional annotation, orthology assignments, and domain prediction at the metagenomic scale. 2003;69(5):278693. CL5.1 was supplied with ammonium chloride as an additional nitrogen source. Mol Biol Evol. PC1 and Cupriavidus sp. Nat Commun. Meanwhile, they competed for substrates for growth and reproduction resulting in differences in their population sizes. The ferredoxin-dependent reduction of chloramphenicol by Clostridium acetobutylicum. Three types of media, each supplemented with 120 mg/L CAP, were used for strain isolation via the spread plate method: Medium B was MSM with 30 mg/L NH4Cl and 500 mg/L sodium pyruvate. A Thermo Hypersil GOLD column (100 mm 2.1mm, 1.9 m) was used for chromatographic separation in HPLC. 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. Compared to the conventional pure culture isolation approach, metagenome assembly and binning could cover more strain genomes and even uncultured ones, which can provide full-spectrum genomic information for the complex microbial community [23]. Nat Biotechnol. 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. When used with the CE6 bacteriophage, the BL21 cells provide the tightest control of protein expression (see BL21(DE3) Strains and Protein . Total DNA extraction of consortium CL samples was performed using FastDNA Spin Kit for Soil (MP Biomedicals, USA) according to the manufacturers protocol. 2015;25(7):104355. The antimicrobial activity of the culture medium collected at various CAP biodegradation stages. Nobu MK, Narihiro T, Rinke C, Kamagata Y, Tringe SG, Woyke T, et al. PubMedGoogle Scholar. PMID: 11680726 DOI: 10.2144/01314pf01 Abstract The Rapid Translation System (RTS 500) (Roche Molecular Biochemicals) is a high-yield protein expression system that utilizes an enhanced E. coli lysate for an in vitro transcription/translation reaction. 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. S7), presenting a novel CAP metabolic pathway through the isomerization at C2 of CAP. The genomes obtained from pure culture isolation were superior to MAGs recovered from consortium CL in terms of completeness, the number of contigs, and N50 length (Additional file 2: Fig. In the present study, two types of gene clusters participated in the ortho-cleavage and meta-cleavage pathways of PCA in consortium CL (Additional file 2: Fig. CAP was rapidly reduced with continuous production and accumulation of metabolites at 7 h and 13 h, which was the CAP-degrading middle stage (stage 3). Microbes play a vital role in the decomposition of CAP in natural as well as engineered environments such as biological treatment systems for pharmaceutical wastewater treatment [8, 9]. The AAI and ANI similarities between NAGs and MAGs were calculated by CompareM (v0.0.23, https://github.com/dparks1134/CompareM) and pyani (v0.2.10, https://github.com/widdowquinn/pyani). NEB recommends using SHuffle Express strains for best performance. . The chiral chromatograms of four CAP stereoisomers standards and RS-CAP produced by Caballeronia sp. Guidelines for protein expression Choose 3-4 transformants when characterizing clones for protein expression because clones may exhibit differences in expression of the heterologous genes. 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). Nat Microbiol. Zhou Y, Sha J, Wu M, Liu J, Bi C, Lin J. 2018;128:38392. PC1. We hypothesize that P-225 was probably reduced to P-277a and P-277b by an uncertain ketol reductase in Sphingomonas sp. Look for a strain having a pLysS designa- . Nevertheless, synergism of the co-cultures Sphingomonas sp. Wu YW, Simmons BA, Singer SW. MaxBin 2.0: an automated binning algorithm to recover genomes from multiple metagenomic datasets. 1971;67(3):26571. PubMed These toxicities are thought to be related to mitochondrial dysfunction, since chloramphenicol inhibits mitochondrial protein synthesis. The expression of acetate symporter (actP) located at the genome of Cupriavidus sp. Total DNA was extracted using DNeasy UltraClean Microbial Kit (QIAGEN, USA) according to the manufacturers instructions. Clustering of orthologous groups (COGs) revealed that a large number of the temporal DEGs were involved in substrate transport and metabolism, energy production, and conversion during the biotransformation of CAP (Fig. Sphingomonas sp. CL5.1, Caballeronia sp. Consortium CL subsisting on CAP was originally enriched from the activated sludge of a local wastewater treatment plant, which had been described in detail in the previous study [19]. Food Addit Contam Part A Chem Anal Control Expo Risk Assess. Microbiome. J Nat Prod. 2017;27(4):60112. 3 and Additional file 2: Fig. The initial CAP concentration was 120 mg/L. were the keystone species and the primary degraders of CAP in the consortium CL. As shown in Fig. The plant. 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. Goldberg K, Schroer K, Ltz S, Liese A. Biocatalytic ketone reduction--a powerful tool for the production of chiral alcohols-part II: whole-cell reductions. PC1. Clusters of orthologous group (COG) terms of CDSs were annotated by eggNOG-mapper (v2) [70]. strain HMH. Several studies investigated the metabolic pathways and mechanisms of CAP but mainly focused on the first biotransformation step. Sphingomonas sp. 6 a, b, c). The temporal DEGs were clustered to different expression patterns with the TPM table via Mfuzz [26]. Schofield CJ. Pilon: an integrated tool for comprehensive microbial variant detection and genome assembly improvement. 2021M691772). It is well-known that the C1 and C2 of chloramphenicol are chiral carbon atoms, and four stereoisomers exist, including 1R,2R-CAP (CAP, D-threo-chloramphenicol), 1S,2R-CAP (SR-CAP, D-erythro-chloramphenicol), 1R,2S-CAP (RS-CAP, L-erythro-chloramphenicol), and 1S,2S-CAP (SS-CAP, L-threo-chloramphenicol). was observably upregulated at stage 3 of CAP degradation (p < 0.001). The proteins predicted from the completed genome of strain CL5.1 were used as the database for MS spectra searching by Proteome Discoverer (v2.2), and a total of 3217 proteins were identified. Acetylated CAP is not able to bind to bacterial 50S ribosomal subunit, and acetylation is a common CAP bacterial resistance mechanism [37]. CL 5.1 and its co-culture with other strains. 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. Protein expression was . (Cup. 1978;32:31924. Privacy FastTree 2--approximately maximum-likelihood trees for large alignments. However, when coming across a difficult-to-express protein, things can get complicated. A series of enzymes in the glycine cleavage system respond to the high concentration of GLY. The filtered reads of each sample were mapped against MAGs using Bowtie2(v2.3.5.1) [65] to obtain genome coverage, and then the relative abundance of each MAG was calculated based on the output file of read mapping using the script calculate_breadth.py provided by Olm et al. in a sulfadiazine-degrading bioreactor [18]. However, when provided with both an additional carbon and nitrogen source, the strain could reproduce to a stationary phase in 2 days and degrade 87.3% of CAP within 10 days (Fig. [3] Chloramphenicol was discovered after being isolated from Streptomyces venezuelae in 1947. 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. Google Scholar. Integrated multi-omics analyses reveal the biochemical mechanisms and phylogenetic relevance of anaerobic androgen biodegradation in the environment. ISME J. Figure S8. The biotransformation of CAP by isolated strains in axenic culture. CL5.1, which further confirmed the proposed metabolic processes of PNB (Additional file 1: Table S3). and Caballeronia sp. Bacterial conversion of hydroxylamino aromatic compounds by both lyase and mutase enzymes involves intramolecular transfer of hydroxyl groups. The detailed metabolic pathways and biotransformation mechanisms will be discussed as follows. Environ Sci Technol. 2020;11:604926. 2009;57(21):1045765. S1). 2014;11(11):11446. D-CAP was also transformed to DD-CAP via dehydrogenation and dehydration. For best results, use BL21(DE3) competent cells to . 2020;36(7):22512. In addition, the C1-OH of D-CAP could be dehydrogenized to dD-CAP by Caballeronia sp. Nutrient supplementation significantly promoted the degradation of CAP by Caballeronia sp. 2017;5(1):84. These biotransformation processes were discovered in Sphingomonas sp. The authors declare that they have no competing interests. Bioinformatics. Genome Res. CheckM: assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes. Pigmentiphaga sp. Process Biochem. ), depending largely on which promoter is used to initiate transcription. 6 a and b). and Pimelobacter sp. Still, a comprehensive understanding of the metabolic pathways, mechanisms, and corresponding enzymes is lacking. in carbon utilization of PNB, which probably resulted in their low relative abundance in the consortium CL. Environ Chem Lett. CL5.1 (ad), Caballeronia sp. CL5.1 to degrade DCA converted from CAP and thus made great contributions to mineralization of CAP in co-cultures or consortia. Identical bacterial populations colonize premature infant gut, skin, and oral microbiomes and exhibit different in situ growth rates. was predicted to be capable of both ortho-cleavage and meta-cleavage of PCA. 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. To identify the main microbial players involved in CAP degradation, we assembled 24 metagenomic datasets of consortium CL. (Ach.). Swenson CE, Sadikot RT. 1b). Boer et al. 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. 2019;7:e7359. Expression vectors can drive expression in various cell types (mammalian, yeast, bacterial, etc. S13g). They carry the nfnB/pnbA and pnbB genes involved in the reductive conversion of PNB to PCA (Fig. CAP-1. Occurrence, toxicity and adsorptive removal of the chloramphenicol antibiotic in water: a review. Nucleic Acids Res. ), Cupriavidus sp. 2019;7(1):16. was conducive to the decrease of toxic stress of CAP on the microbial community in the consortium [11]. Molecular mechanism of azoxy bond formation for azoxymycins biosynthesis. Strains CL5.1, PC1, CLC6, RCL7, CLB6.2, PNB5, and CLB4 clustered with MAG1, MAG2, MAG3, MAG4, MAG5, MAG6, and MAG7, respectively, which could indicate their extensive homology (Fig. P-277a and P-277b were stereoisomers with the same mass and identical fragment ion spectra, but with different retention times. Hyatt D, Chen GL, Locascio PF, Land ML, Larimer FW, Hauser LJ. Water Res. KofamKOALA: KEGG Ortholog assignment based on profile HMM and adaptive score threshold. Similarly, there were 101 significantly up-regulated and 200 significantly down-regulated proteins in CT72 vs. the control group. 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. Staphylococcus aureus ATCC 25923 after overnight incubation was inoculated into the mixed culture medium in 96-well plates. The KEGG enrichment analysis of DEGs and correlation analysis of DEGs with CAP as well as its metabolites. 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. Thus, upregulation of this gene implied the assimilation of DCA by Cupriavidus sp., which promoted its predominant role in the community. d Proposed substrate cross-feeding among key members in consortium CL. CL5.1 and Caballeronia sp. were expressed in a reverse-U pattern, implying that they indeed mineralized PCA in the corresponding pathway. CL5.1 and Caballeronia sp. PC1. 2017;8:297. Basu S, Pal Chowdhury P, Deb S, Dutta TK. Catabolism and interactions of uncultured organisms shaped by eco-thermodynamics in methanogenic bioprocesses. 4 (module 3, module 4, and module 6), nitrobenzene products (Ar-NO2) including P-179, P-195b, and PNB supposedly formed into nitrosobenzene compounds (Ar-NO) first and then into hydroxylaminobenzene compounds (Ar-NHOH) under the catalysis of the nitroreductase (nfnB) based on the nitroaromatic compound degradation pathway reported previously [47]. Engineered E. coli K12 to promote disulfide bond formation in the cytoplasm. P-185 was inferred to be hydrolyzed into GLY and DCA by an uncertain hydrolase. 4 module 1). The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. Langmead B, Salzberg SL. GLY can be converted to serine, CO2, and NH4+ via the glycine cleavage system coupled with hydroxymethyltransferase (glyA) (Additional file 2: Fig. CAP-degrading features of seven isolated strains were investigated under different nutritional conditions. Caballeronia sp. In the current study, the detailed CAP biotransformation pathways, the key metabolic enzymes involved, and the interspecies interactions of members in this consortium were comprehensively elucidated using an integrated multi-omics (i.e., metagenomics, metatranscriptomics, and proteomics) and cultivation-based approach. 2019;176(3):64962. Figure S4. MAGs were recovered from the assemblies using BASALT (https://github.com/EMBL-PKU/BASALT) which integrates Maxbin2 (v2.2.7) [59], MetaBAT2 (v2.12.1) [60], and CONCOCT (v1.1.0) [61]. In the presence of additional carbon and nitrogen sources, 105.3 mg/L A-CAP was detected in the culture medium, which exhibits Cupriavidus sp. Then, the PCR product with the correct size was purified and confirmed by DNA sequencing. These include the cleavage of C1/C2 and the hydrolysis of the amide bond. ), Chryseobacterium sp. These are crucial for the exploitation in bioremediation applications. Price MN, Dehal PS, Arkin AP. and Pigmentiphaga sp. The cells of strain CL5.1 in the CAP-treatment group were collected at 34 h (CT34) and 72 h (CT72) when about 66.6% and 99.8% of CAP were degraded, respectively. Unfortunately, in this system, target protein expression gradually declines and is often undetectable following 3 to 5 subcultures. The BL21(DE3)pLysS competent cells provide tighter control of protein expression for expression of toxic proteins and are resistant to chloramphenicol. The relative abundance of all remaining MAGs was lower than 1.0%. 1970;16(3):1739. 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. Article Recovery of nearly 8,000 metagenome-assembled genomes substantially expands the tree of life. 2020;92:43746. The genetic modules controlling transcription and translation in these plasmids. Sphingomonas sp. 1a, CAP was not significantly degraded (p > 0.05) by consortium CL at 1 h (stage 2). CLC6, Pigmentiphaga sp. The integration analysis process of multi-omics data was shown in Additional file 2: Fig. Surprisingly, eight biotransformation products including P-314, P-298, P-286, P-282, P-272a, P-272b, P-270a, and P-270b produced by Sphingomonas sp. The dynamics of main CAP metabolites produced by isolated strains. Kanamycin, spectinomycin, gentamicin, and chloramphenicol were added into the medium for final concentrations of 20, 20, 2, and 10 g/mL, respectively. 2014;15(12):550. were hypothesized to compete for nutrients via improving cell motility. RCL7, Labrys sp. Most of these genes were upregulated in stages 2 or 3 of the CAP degradation process and had positive correlations with the dynamics of main CAP metabolites. CAP biotransformation capacity of Pseudomonas putida KT2440 harboring gene capO which was cloned from Sphingomonas sp. CL5.1 was inoculated into MSM containing 1 g/L sodium pyruvate and 30 mg/L NH4Cl. The ribosomal RNA was removed from RNA samples, and a strand-specific library was constructed using NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB, USA). . Figure S10. The first step is to lower the temperature to slow down protein production. Gene capO was amplified by PCR from the genomic DNA of Sphingomonas sp. Bioinformatics. 1995;9(1):414. Nguyen LM, Nguyen NTT, Nguyen TTT, Nguyen TT, Nguyen DTC, Tran TV. 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). Correspondence to Cupriavidus sp. Genomic characteristics of the seven isolated strains. CL5.1 (Additional file 2: Fig. (Pig. Chemically competent E. coli cells suitable for transformation and protein expression. An integrated meta-omics approach reveals substrates involved in synergistic interactions in a bisphenol A (BPA)-degrading microbial community. CL5.1 and Caballeronia sp. Overnight cultures were grown at 37 C with shaking at . PubMed Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. An expression vector can also include an enhancer sequence which increases the amount of protein or RNA produced. 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.) 3 mp). 1b). The parameters of HPLC and mass spectrometer were set as the previous description except for the HPLC flow rate which was modified to 0.2 mL/min [13]. Members belonging to the genera Caballeronia, Cupriavidus, and Achromobacter are highly motile via flagella [27, 28]. The protein function of CDSs was annotated by a blast search against the UniProtKB database. 2023 BioMed Central Ltd unless otherwise stated. Can J Microbiol. (Fig. Deciphering chloramphenicol biotransformation mechanisms and microbial interactions via integrated multi-omics and cultivation-dependent approaches, https://doi.org/10.1186/s40168-022-01361-5, Comprehensive metabolic pathways and biotransformation mechanisms of CAP deciphered via the integrated multi-omics and cultivation-dependent approaches, The synergistic interactions on CAP mineralization among functional microorganisms, http://creativecommons.org/licenses/by/4.0/, http://creativecommons.org/publicdomain/zero/1.0/. 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. CL5.1 could mineralize PCA via both ortho- and meta-cleavage pathways, while Caballeronia sp. CAS The co-culture of Sphingomonas sp. Sphingomonas sp. Genome Biol. 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. Figure S13. After incubation, 700 l lysis buffer containing 1% (v/v) -mercaptoethanol and an appropriate amount of 0.1 mm glass beads (BioSpec Products, USA) was added to the samples, and then, the lysate was homogenized with a homogenizer (JXFSTPRP-64, Shanghaijingxin Experimental Technology, China). E. coli. The gene catB encoding CAP acetyltransferase in Cupriavidus sp. 5c). 2016;94:73657. 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. 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. PLoS One. could reproduce rapidly due to their advantage in nutrient utilization and thus be dominant in the microbial community. CL5.1 in the presence of CAP was identified by proteomic sequencing. 6b). Zhang J, Zhao R, Cao L, Lei Y, Liu J, Feng J, et al. PC1, and Bosea sp. Olm MR, Brown CT, Brooks B, Banfield JF. Genome Res. Provided by the Springer Nature SharedIt content-sharing initiative. CLC6 removed 73.6% of soluble TOC after 3-day cultivation which was also markedly higher than the axenic culture (p < 0.05) (Additional file 2: Fig. Olm MR, Brown CT, Brooks B, Firek B, Baker R, Burstein D, et al. A significant characteristic of the r-protein gene expression in diploid strains was revealed . 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. The draft assemblies were improved by Medaka (v1.2.3) (https://github.com/nanoporetech/medaka), Rcon (v1.4.3) (https://github.com/isovic/racon), and Pilon (v1.23) [57] reference to the Illumina short reads to obtain high-quality Nanopore long read assemblies (NAGs) of strains. found that P-286 was a metabolite of CAP degradation in eye drops [51]. All batch experiments were conducted in triplicate. Protocols. Figure S12. [6] Its chemical structure was identified and it was first synthesized in 1949. 2017;39(10):155966. Google Scholar. In summary, different from the biotransformation studies using pure cultures, investigating the chloramphenicol biotransformation by microbial consortia could truly reflect the metabolic mechanisms without bias in the engineered or natural environments, where biotransformation processes are especially dependent on synergistic interactions and metabolite exchanges among microbes. To confirm the function of this candidate CAP oxidoreductase encoding gene capO, it was cloned and heterologously expressed in Pseudomonas putida KT2440. The antibiotics chloramphenicol (Cm), tetracycline, and erythromycin, which inhibit bacterial protein synthesis and are known to induce the cold shock response, unexpectedly enhance the heterologous expression of P450s and related proteins in Escherichia coli. Though Cupriavidus sp. Protein expression with this method occurs in an open reaction environment to which crude cell extracts containing an overexpressed tRNA/aa-RS pair are added along with the ncAA, which is perpetually accessible to the translation machinery. JZ, BL, UK, and TUB reviewed and edited the manuscript. Yu K, Yi S, Li B, Guo F, Peng X, Wang Z, et al. Dantas G, Sommer MO, Oluwasegun RD, Church GM. PLoS One. For the purpose of protein expression in L. lactis, the nisRK genes have been integrated into the pepN locus of the chromosome of a MG1363 derivative called NZ9000. b The KEGG annotation of temporal DEGs in each core species of consortium CL including Sphingomonas sp. 2021;38(12):58259. significantly promoted chloramphenicol mineralization, and the substrate exchange interaction network occurred actively among key microbes. Xu H, Xiao L, Zheng S, Zhang Y, Li J, Liu F. Reductive degradation of chloramphenicol by Geobacter metallireducens. It is also a rare cause of aplastic anemia. 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). To sum up, the transcriptional profiling results revealed that CAP significantly triggered and promoted the metabolic activities of the key dominant members of consortium CL. 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. 2020;54(12):7591600. 2007;76(2):24955. CLC6 could completely degrade 50 mg/L DCA within 24 h (Additional file 2: Fig. PC1. It was reported that antibiotic concentrations ranged from ng/L to mg/L levels in aquatic environments worldwide [5], especially in pharmaceutical wastewater, which could reach up to 115 mg/L [6]. CL5.1 was about 52.2% after 10-day cultivation (Additional file 2: Fig. Metatranscriptomic sequencing was performed on the Illumina NovaSeq platform (paired-end 150 bp) with a sequencing depth of about 10 Gb at Novogene (Tianjin, China). CAP-1, and it was consistent with our study that the expression of a GMC oxidoreductase protein homologous to capO was markedly upregulated by 3.20-fold in the presence of CAP [14]. (c). 2014;30(14):20689. During CAP-degrading batch experiments, the microbiome of consortium CL was sampled for metagenomic sequencing and metatranscriptomic sequencing at 0 (before CAP dosing, defined as stage 1), 1, 7, 13, 21, 25, 31, and 48 h after CAP dosing at an initial concentration of 120 mg/L. The GMC oxidoreductase superfamily harbors diverse enzymes with the function of catalyzing a wide variety of redox reactions such as the oxidation of an alcohol moiety to the corresponding aldehyde and the two-step oxidation of an alcohol moiety to the corresponding carboxylic acid [34]. The library was sequenced on the Illumina NovaSeq platform (paired-end 150 bp) with a sequencing depth of about 2 Gb at NextOmics (Wuhan, China). Carvalho IT, Santos L. Antibiotics in the aquatic environments: a review of the European scenario. Moreover, the protein expression profile of Sphingomonas sp. PC1 (Additional file 2: Fig. 2012;75(10):172934. The biotransformation of CAP by isolated strains in co-culture. 6 ac). S4b). ISME J. Deng Y, Wang Y, Mao Y, Zhang T. Partnership of Arthrobacter and Pimelobacter in aerobic degradation of sulfadiazine revealed by metagenomics analysis and isolation. Nevertheless, the lack of genes involved in PNB nitroreduction was a disadvantage for Bosea sp. FEMS Microbiol Ecol. The gene expression was presented as Log2(FC) (n = 3). P-226 was inferred to be further deaminized to P-225 via oxidative catalysis of a D-amino acid dehydrogenase (dadA) (Fig. Nat Microbiol. Xue J, Lei D, Zhao X, Hu Y, Yao S, Lin K, et al. (Cab. A high membership value indicates a high correlation of the gene expression with the cluster. PC1 removed 76.4% of soluble TOC in 2 days, which was significantly higher than that in axenic culture (p < 0.05) (Additional file 2: Fig. A phylogenetic tree of isolated strain genomes and MAGs relative to 2170 GTDB reference genomes was constructed to determine their phylogenetic relationship. The mineralization rate of CAP in the axenic culture of Sphingomonas sp. Sci China Technol Sci. The little squares in various colors indicated the gene expression difference at different sampling times compared to that at 0 h (before CAP dosing). However, Cm has severe side effects in eukaryotes of which the cause remains unknown. This study was funded by the National Key R&D Program of China (No. 2017;11(12):28648. Background The most widespread, efficient prokaryotic protein-producing system is one where the T7 phage polymerase recognizes the T7 phage promoter (T7 p/p system). The protein extraction and tandem mass tag (TMT) labeling proteomic sequencing were performed as the method described in the previous study [38]. The protein synthesis inhibitor chloramphenicol prevents the induction of KaiC and blocks phase shifting. 2018;12(3):83848. S4a). BL21 Competent E. coli is a widely used non-T7 expression E. coli strain and is suitable for transformation and protein expression. When CAP was supplied as the sole carbon source, the co-cultures did not perform better than axenic cultures regarding CAP biodegradation efficiency (Fig. Part of DNA for Oxford Nanopore sequencing was purified by 1% agarose gel electrophoresis and Monarch DNA Gel Extraction Kit (NEB, USA). S2c). 6 a, b). CL5.1. Novel pathway for chloramphenicol catabolism in the activated sludge bacterial isolate Sphingobium sp. V4.4 ) chromatograms of four CAP stereoisomers standards and RS-CAP produced by Caballeronia sp CAP oxidoreductase encoding gene which... Chloramphenicol mineralization, and oral microbiomes and exhibit different in situ growth rates: a review of genes in... Chromatographs of the heterologous genes, Brooks B, Baker R, Burstein D, R! Be dominant in the aquatic environments: a new versatile metagenomic assembler Bi C, Kamagata Y, Li,. That P-225 was probably further oxidized to produce P-169 and then P-185 azoxymycins biosynthesis (... To P-277a and P-277b were stereoisomers with the cluster ] its chemical structure identified! And mineralization of CAP was identified and it was cloned from Sphingomonas sp data was in! Insoluble protein production, Zheng S, Hur HG, et al Applied Basic Research Foundation No... Hypothesized to compete for nutrients via improving cell motility mg/L A-CAP was detected the. Dehydrogenized to DD-CAP via dehydrogenation and dehydration recover genomes from multiple metagenomic datasets of consortium CL ) used. 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