ABSTRACT
Zeolite was shown to mitigate anaerobic digestion (AD) inhibition caused by several inhibitors such as long-chain fatty acids, ammonia, and phenolic compounds. In this paper, we verified the genericity of zeolite's mitigating effect against other types of inhibitors found in AD such as salts, antibiotics, and pesticides. The impacts of inhibitors and zeolite were assessed on AD performance and microbial dynamics. While sodium chloride and erythromycin reduced methane production rates by 34% and 32%, zeolite mitigated the inhibition and increased methane production rates by 72% and 75%, respectively, compared to conditions without zeolite in the presence of these two inhibitors. Noticeably, zeolite also enhanced methane production rate by 51% in the uninhibited control condition. Microbial community structure was analyzed at two representative dates corresponding to the hydrolysis/fermentation and methanogenesis stages through 16S rRNA gene sequencing. The microbial characteristics were further evidenced with common components analysis. Results revealed that sodium chloride and erythromycin inhibited AD by targeting distinct microbial populations, with more pronounced inhibitory effects during hydrolysis and VFAs degradation phases, respectively. Zeolite exhibited a generic effect on microbial populations in different degradation stages across all experimental conditions, ultimately contributing to the enhanced AD performance and mitigation of different inhibitions. Typically, hydrolytic and fermentative bacteria such as Cellulosilyticum, Sedimentibacter, and Clostridium sensu stricto 17, VFAs degraders such as Mesotoga, Syntrophomonas, and Syntrophobacter, and methanogens including Methanobacterium, Methanoculleus, and Methanosarcina were strongly favored by the presence of zeolite. These findings highlighted the promising use of zeolite in AD processes for inhibition mitigation in general.
Subject(s)
Zeolites , Anaerobiosis , Zeolites/pharmacology , Zeolites/chemistry , RNA, Ribosomal, 16S/genetics , Sodium Chloride , Bacteria/genetics , Erythromycin/metabolism , Methane , Bioreactors/microbiologyABSTRACT
As emerging pollutants, antibiotics were widely detected in water bodies and dietary sources. Recently, their obesogenic effects raised serious concerns. So far, it remained unclear whether their obesogenic effects would be influenced by water- and diet-borne exposure routes. In present study, Caenorhabditis elegans, nematodes free-living in air-water interface and feeding on bacteria, were exposed to water- and diet-borne erythromycin antibiotic (ERY). The statuses of the bacterial food, inactivated or alive, were also considered to explore their influences on the effects. Results showed that both water- and diet-borne ERY significantly stimulated body width and triglyceride contents. Moreover, diet-borne ERY's stimulation on the triglyceride levels was greater with alive bacteria than with inactivated bacteria. Biochemical analysis showed that water-borne ERY inhibited the activities of enzymes like adipose triglyceride lipase (ATGL) in fatty acid ß-oxidation. Meanwhile, diet-borne ERY inhibited the activities of acyl-CoA synthetase (ACS) and carnitine palmitoyl transferase (CPT) in lipolysis, while it stimulated the activities of fatty acid synthase (FAS) in lipogenesis. Gene expression analysis demonstrated that water-borne ERY with alive bacteria significantly upregulated the expressions of daf-2, daf-16 and nhr-49, without significant influences in other settings. Further investigation demonstrated that ERY interfered with bacterial colonization in the intestine and the permeability of the intestinal barrier. Moreover, ERY decreased total long-chained fatty acids (LCFAs) in bacteria and nematodes, while it decreased total short-chained fatty acids (SCFAs) in bacteria but increased them in nematodes. Collectively, the present study demonstrated the differences between water- and diet-borne ERY's obesogenic effects, and highlighted the involvement of insulin and nhr-49 signaling pathways, SCFAs metabolism and also the interaction between intestinal bacteria and the host.
Subject(s)
Anti-Bacterial Agents , Caenorhabditis elegans , Animals , Caenorhabditis elegans/metabolism , Anti-Bacterial Agents/pharmacology , Erythromycin/metabolism , Erythromycin/pharmacology , Fatty Acids , Triglycerides/metabolism , Triglycerides/pharmacology , WaterABSTRACT
Actinomyces are gram-positive bacteria known for their valuable secondary metabolites. Redirecting metabolic flux towards desired products in actinomycetes requires precise and dynamic regulation of gene expression. In this study, we integrated the CRISPR interference (CRISPRi) system with a cumate-inducible promoter to develop an inducible gene downregulation method in Saccharopolyspora erythraea, a prominent erythromycin-producing actinobacterium. The functionality of the cumate-inducible promoter was validated using the gusA gene as a reporter, and the successful inducible expression of the dCas9 gene was confirmed. The developed inducible CRISPRi strategy was then employed to downregulate the expression of target genes rppA in the wild-type strain NRRL2338 and sucC in the high erythromycin-producing strain E3. Through dynamic control of sucC expression, a significant enhancement in erythromycin production was achieved in strain E3. This study demonstrated the effectiveness of an inducible gene downregulation approach using CRISPRi and a cumate-inducible promoter, providing valuable insights for optimizing natural product production in actinomyces.
Subject(s)
Saccharopolyspora , Saccharopolyspora/genetics , Saccharopolyspora/metabolism , Clustered Regularly Interspaced Short Palindromic Repeats , Erythromycin/metabolism , Promoter Regions, Genetic/genetics , Gene Expression RegulationABSTRACT
Macrolides, lincosamides, and streptogramin B (MLS) are structurally distinct molecules that are among the safest antibiotics for prophylactic use and for the treatment of bacterial infections. The family of erythromycin resistance methyltransferases (Erm) invariantly install either one or two methyl groups onto the N6,6-adenosine of 2058 nucleotide (m6A2058) of the bacterial 23S rRNA, leading to bacterial cross-resistance to all MLS antibiotics. Despite extensive structural studies on the mechanism of Erm-mediated MLS resistance, how the m6A epitranscriptomic mark affects ribosome function and bacterial physiology is not well understood. Here, we show that Staphylococcus aureus cells harboring m6A2058 ribosomes are outcompeted by cells carrying unmodified ribosomes during infections and are severely impaired in colonization in the absence of an unmodified counterpart. The competitive advantage of m6A2058 ribosomes is manifested only upon antibiotic challenge. Using ribosome profiling (Ribo-Seq) and a dual-fluorescence reporter to measure ribosome occupancy and translational fidelity, we found that specific genes involved in host interactions, metabolism, and information processing are disproportionally deregulated in mRNA translation. This dysregulation is linked to a substantial reduction in translational capacity and fidelity in m6A2058 ribosomes. These findings point to a general "inefficient translation" mechanism of trade-offs associated with multidrug-resistant ribosomes.
Subject(s)
Adenine/analogs & derivatives , Anti-Bacterial Agents , Staphylococcus aureus , Staphylococcus aureus/genetics , Staphylococcus aureus/metabolism , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/metabolism , Lincosamides , Erythromycin/metabolism , Macrolides , Microbial Sensitivity TestsABSTRACT
Introduction: Oxidative stress and inflammation have proven to be key factors contributing to the occurrence of BPD. Erythromycin has been shown to be effective in treating the redox imbalance seen in many non-bacterial infectious chronic inflammatory diseases. Methods: Ninety-six premature rats were randomly divided into air + saline chloride group, air + erythromycin group, hyperoxia + saline chloride group and hyperoxia + erythromycin group. Lung tissue specimens were collected from 8 premature rats in each group on days 1, 7 and 14, respectively. Results: Pulmonary pathological changes in premature rats after hyperoxia exposure were similar to those of BPD. Hyperoxia exposure induced high expression of GSH, TNF-α, and IL-1ß. Erythromycin intervention caused a further increase in GSH expression and a decrease in TNF-α and IL-1ß expression. Conclusion: GSH, TNF-α and IL-1ß are all involved in the development of BPD. Erythromycin may alleviate BPD by enhancing the expression of GSH and inhibiting the release of inflammatory mediators.
Subject(s)
Bronchopulmonary Dysplasia , Hyperoxia , Lung Injury , Animals , Rats , Lung Injury/drug therapy , Lung Injury/etiology , Lung Injury/metabolism , Hyperoxia/complications , Hyperoxia/metabolism , Hyperoxia/pathology , Cytokines/metabolism , Cytokines/pharmacology , Erythromycin/pharmacology , Erythromycin/metabolism , Animals, Newborn , Tumor Necrosis Factor-alpha/metabolism , Chlorides/metabolism , Chlorides/pharmacology , Lung , Inflammation/pathology , Bronchopulmonary Dysplasia/complications , Bronchopulmonary Dysplasia/metabolism , Disease Models, AnimalABSTRACT
Motilin is an endogenous peptide hormone almost exclusively expressed in the human gastrointestinal (GI) tract. It activates the motilin receptor (MTLR), a class A G protein-coupled receptor (GPCR), and stimulates GI motility. To our knowledge, MTLR is the first GPCR reported to be activated by macrolide antibiotics, such as erythromycin. It has attracted extensive attention as a potential drug target for GI disorders. We report two structures of Gq-coupled human MTLR bound to motilin and erythromycin. Our structures reveal the recognition mechanism of both ligands and explain the specificity of motilin and ghrelin, a related gut peptide hormone, for their respective receptors. These structures also provide the basis for understanding the different recognition modes of erythromycin by MTLR and ribosome. These findings provide a framework for understanding the physiological regulation of MTLR and guiding drug design targeting MTLR for the treatment of GI motility disorders.
Subject(s)
Motilin , Receptors, Gastrointestinal Hormone , Humans , Motilin/metabolism , Erythromycin/pharmacology , Erythromycin/metabolism , Receptors, Gastrointestinal Hormone/chemistry , Receptors, Gastrointestinal Hormone/metabolism , Receptors, Neuropeptide/metabolismABSTRACT
Continuous and excessive usage of erythromycin results in serious environmental pollution and presents a health risk to humans. Biological treatment is considered as an efficient and economical method to remove it from the environment. In this study, a novel erythromycin-degrading bacterial strain, W7, isolated from sewage sludge was identified as Paracoccus versutus. Strain W7 degraded 58.5% of 50 mg/L erythromycin in 72 h under the optimal conditions of 35 °C, pH 7.0, and 0.1% sodium citrate with yeast powder in mineral salt medium. It completely eliminated erythromycin from erythromycin fermentation residue at concentrations of 100 and 300 mg/L within 36 and 60 h, respectively. Erythromycin esterase (EreA) was found to be involved in erythromycin metabolism in this strain and was expressed successfully. EreA could hydrolyze erythromycin, and its maximum activity occurred at pH 8.5 and 35 °C. Finally, six intermediates of erythromycin degraded by strain W7 were detected by high performance liquid chromatography mass spectrometry. Based on the novel intermediates and enzymes, we determined two possible pathways of erythromycin degradation by strain W7. This study broadened our understanding of the erythromycin catabolic processes of P. versutus and developed a feasible microbial strategy for removing erythromycin from erythromycin fermentation residue, wastewater, and other erythromycin-contaminated environments.
Subject(s)
Paracoccus , Humans , Paracoccus/metabolism , Erythromycin/metabolism , Sewage , Biodegradation, EnvironmentalABSTRACT
The rapid development of biotechnology has provided new perspectives to observe and helped to gradually understand the significance of genetic instability in Actinobacteria. High frequency deletions of extremities and abnormal methylation of chromosomes suggest there might be relevant between the two phenomena. With this suspicion, we used single molecule real-time (SMRT) sequencing to map the genome-level methylation of one branch of actinomycetes, Saccharopolyspora erythraea, which have ring-shaped chromosomes. S. erythraea used for analysis in this study shares the same highly unstable phenotypic traits, as evidenced by diverse spore morphology and fluctuating erythromycin production. Multiple amplification of genomic islands closes to the replication initiation site and 6-methyladenine (m6A) deletion in genomic islands suggest that the interaction between the restriction modification (R-M) system and transposable elements provides an explanation for the division of labor by genomic heterogeneity in actinomycetes.
Subject(s)
Actinobacteria , Saccharopolyspora , Actinobacteria/genetics , DNA Transposable Elements , Saccharopolyspora/genetics , Erythromycin/metabolism , ChromosomesABSTRACT
Intratumor microbiota is a dynamic cancer component that can be carried over by metastatic tumor cells to distal organs. This protocol was developed to genetically label Staphylococcus xylosus and trace the recombinant strain in vivo in the tumor. We optimized the recombination-based gene replacement protocol to insert a GFP-Erythromycin resistant protein (Erm) cassette. The inserted cassette facilitates the tracking of the recombinant strain, allowing a sensitive interrogation of microbial dynamics with high temporal and spatial resolution. For complete details on the use and execution of this protocol, please refer to Fu et al. (2022).
Subject(s)
Erythromycin , Staphylococcus , Staphylococcus/genetics , Staphylococcus/metabolism , Erythromycin/metabolism , Proteins/metabolismABSTRACT
Erythromycin (ERY) is one of the most used antibiotics frequently detected in different aquatic environments and may bring burdens to aquatic ecosystems. However, the impacts of antibiotics on aquatic systems other than the antibiotic resistance genes remain largely unknown. In the present study, the responses to ERY exposure at the subcellular-organelle levels were for the first time investigated and imaged over 24 h. Exposure to ERY hampered the zebrafish (Danio rerio) cell growth and decreased the cell viability in a time-dependent mode. Meanwhile, exposure to a low concentration of ERY (73.4 µg L-1) induced reactive oxygen species (ROS) overproduction and lysosomal damage following lysosomal alkalization and swelling. In turn, the lysosomal stress was the major driver of altering the ROS level, superoxide dismutase (SOD) activity, and glutathione (GSH) content. Subsequently, mitochondria displayed dysfunction such as increased mitochondrial ROS, impaired mitophagy, and induced mitochondria-driven apoptosis, as well as impaired mitochondrial electron transport chain and loss of membrane potential. These results collectively demonstrated the subcellular sensitive machinery responses to ERY stress at environmentally relevant and slightly higher sub-lethal concentrations. ERY may induce switching from autophagy to apoptosis with corresponding changes in lysosomal activity, antioxidant activity, and mitochondrial activity. The findings provided important information on the physiological and subcellular responses of fish cells to ERY.
Subject(s)
Water Pollutants, Chemical , Zebrafish , Animals , Zebrafish/metabolism , Reactive Oxygen Species/metabolism , Erythromycin/toxicity , Erythromycin/metabolism , Antioxidants/metabolism , Anti-Bacterial Agents/pharmacology , Oxidative Stress , Ecosystem , Water Pollutants, Chemical/metabolism , Glutathione/metabolism , Superoxide Dismutase/metabolismABSTRACT
Microalgae-based biotechnology for antibiotic removal has received increasing attention as an economical and green method. This study investigated the removal mechanism of erythromycin by Chlorella pyrenoidosa and its correlation with the ecotoxic responses of microalgae. The degradation products (DPs) were identified, and their toxicity was predicted. The results indicated that only 4.04 %, 6.28 % and 23.53 % of erythromycin were left after 21-day microalgae treatment in 0.1, 1.0 and 10 mg/L treatments, respectively. Biodegradation contributed 48.62-67.01 %, 16.67-52.32 % and 6.42-24.82 %, while abiotic degradation contributed 8.76-29.61 %, 5.19-41.39 %, and 16.55-51.22 % to erythromycin attenuation in 0.1, 1.0, and 10 mg/L treatments, respectively. The growth and physiological-biochemical parameters of microalgae were slightly affected in low concentration treatment, which may be the main reason that biodegradation was the prominent removal mechanism. By contrast, oxidative damage in high concentration treatment inhibited the cell growth and chlorophyll content of microalgae, which hindered erythromycin biodegradation. In addition, eleven erythromycin degradation products (DPs) were identified during microalgae treatment of 21 days. Seven DPs including DP717, DP715, DP701A, DP701B, DP657, DP643, and DP557, represented higher toxicity to aquatic organisms than erythromycin.
Subject(s)
Chlorella , Microalgae , Water Pollutants, Chemical , Anti-Bacterial Agents/metabolism , Anti-Bacterial Agents/toxicity , Chlorophyll/metabolism , Erythromycin/metabolism , Erythromycin/toxicity , Microalgae/metabolism , Water Pollutants, Chemical/analysisABSTRACT
Macrophages serve an active role in the pathophysiology of chronic obstructive pulmonary disease (COPD). Erythromycin (EM) has been verified as an effective treatment for COPD. However, there are few studies on the effect of EM on the ultrastructure of macrophages exposed to cigarette smoke extract (CSE). In the present study, human macrophages were randomly divided into three groups: The control, CSE and the CSE+EM group, using electron microscopy, the effect of EM was evaluated by comparing the ultrastructural changes between these groups. The macrophages were additionally divided into a further four groups: The control, CSE, CSE+EM 24 h and CSE+EM 48 h groups. The generation of reactive oxygen species (ROS) in each group was evaluated by detecting fluorescence intensity. It was observed that the cellular ultrastructure of the CSE group exhibited abnormal changes, though this effect was reversed back to the level of the control in the CSE+EM group. Compared with the control group, the ROS expression level was significantly increased in the CSE group (P < .05); however, compared with the CSE group, the ROS concentration was decreased in the CSE+EM 24 h (P < .05) and CSE+EM 48 h groups (P < .05), though this was more apparent in the EM 48 h group. It was concluded that EM protects human macrophages against CSE. Moreover, it was hypothesized that EM may reduce the symptoms of patients with COPD by protecting the macrophage ultrastructure from the effects of CSE, resulting in the decreased generation of ROS, inhibiting autophagy and reducing endoplasmic reticulum stress.
Subject(s)
Cigarette Smoking , Pulmonary Disease, Chronic Obstructive , Cigarette Smoking/adverse effects , Erythromycin/metabolism , Erythromycin/pharmacology , Humans , Macrophages/metabolism , Pulmonary Disease, Chronic Obstructive/metabolism , Reactive Oxygen Species/metabolism , Reactive Oxygen Species/pharmacology , Nicotiana/metabolismABSTRACT
Omics approaches have been applied to understand the boosted productivity of natural products by industrial high-producing microorganisms. Here, with the updated genome sequence and transcriptomic profiles derived from high-throughput sequencing, we exploited comparative omics analysis to further enhance the biosynthesis of erythromycin in an industrial overproducer, Saccharopolyspora erythraea HL3168 E3. By comparing the genome of E3 with the wild type NRRL23338, we identified fragment deletions inside 56 coding sequences and 255 single-nucleotide polymorphisms over the genome of E3. A substantial number of genomic variations were observed in genes responsible for pathways which were interconnected to the biosynthesis of erythromycin by supplying precursors/cofactors or by signal transduction. Furthermore, the transcriptomic data suggested that genes involved in the biosynthesis of erythromycin were significantly upregulated constantly, whereas some genes in biosynthesis clusters of other secondary metabolites contained nonsense mutations and were expressed at extremely low levels. Through comparative transcriptomic analysis, l-glutamine/l-glutamate and 2-oxoglutarate were identified as reporter metabolites. Around the node of 2-oxoglutarate, genomic mutations were also observed. Based on the omics association analysis, readily available strategies were proposed to engineer E3 by simultaneously overexpressing sucB (coding for 2-oxoglutarate dehydrogenase E2 component) and sucA (coding for 2-oxoglutarate dehydrogenase E1 component), which increased the erythromycin titer by 71% compared to E3 in batch culture. This study provides more promising molecular targets to engineer for enhanced production of erythromycin by the overproducer.
Subject(s)
Erythromycin , Saccharopolyspora , Bacterial Proteins/genetics , Erythromycin/metabolism , Genomics , Ketoglutarate Dehydrogenase Complex/genetics , Ketoglutarate Dehydrogenase Complex/metabolism , Ketoglutaric Acids/metabolism , Saccharopolyspora/genetics , Saccharopolyspora/metabolism , Transcriptome/geneticsABSTRACT
In the last two decades, the production of complex polyketides such as erythromycin and its precursor 6-deoxyerythronolide B (6-dEB) was demonstrated feasible in Escherichia coli. Although the heterologous production of polyketide skeleton 6-dEB has reached 210 mg l-1 in E. coli, the yield of its post-modification products erythromycins remains to be improved. Cytochrome P450EryF catalyses the C6 hydroxylation of 6-dEB to form erythronolide B (EB), which is the initial rate-limiting modification in a multi-step pathway to convert 6-dEB into erythromycin. Here, we engineered hydroxylase EryF to improve the production of heterologous polyketide EB in E. coli. By comparative analysis of various versions of P450EryFs, we confirmed the optimal SaEryF for the biosynthesis of EB. Further mutation of SaEryF based on the crystal structure of SaEryF and homology modelling of AcEryF and AeEryF afforded the enhancement of EB production. The designed mutant of SaEryF, I379V, achieved the yield of 131 mg l-1 EB, which was fourfold to that produced by wild-type SaEryF. Moreover, the combined mutagenesis of multiple residues led to further boost the EB concentration by another 41%, which laid the foundation for efficient heterologous biosynthesis of erythromycin or other complex polyketides.
Subject(s)
Escherichia coli , Polyketides , Erythromycin/analogs & derivatives , Erythromycin/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Polyketides/metabolismABSTRACT
An alternative method of electrochemical oxidation was employed to degrade persistent compounds in the form of antibiotics using strong oxidizing agents such as hydroxyl ions. A 24 factorial design was employed to check the effect of four factors namely pH, current density, electrolysis time and electrolyte concentration set at their high (+) and low (-) levels on the antibiotics (amoxicillin, ciprofloxacin and erythromycin) degradation in water. The response was obtained in the form of COD (chemical oxygen demand) removal. A prediction model was developed to predict the values of COD removal. Later the main effect, contribution and interactions were studied with Design Expert Software 7.0. About 89.5% COD removal was obtained when pH and time were set at their high level and the other two factors at their low level. It was determined that the pH when set at high level (pH 9) had the most effect (24.68) and contribution (43.6) in the degradation process and hence the removal of COD. This technology of electrochemical oxidation can be employed in industries to efficiently remove pharmaceuticals, paints, dyes and other organic compounds.
Subject(s)
Amoxicillin/analysis , Biological Oxygen Demand Analysis/methods , Ciprofloxacin/analysis , Electrochemical Techniques/methods , Erythromycin/analysis , Water/analysis , Amoxicillin/metabolism , Anti-Bacterial Agents/analysis , Anti-Bacterial Agents/metabolism , Ciprofloxacin/metabolism , Erythromycin/metabolism , Water/metabolism , Water Pollutants, Chemical/analysis , Water Pollutants, Chemical/metabolism , Water Purification/methodsABSTRACT
Macrolide antibiotics, such as erythromycin, bind to the nascent peptide exit tunnel (NPET) of the bacterial ribosome and modulate protein synthesis depending on the nascent peptide sequence. Whereas in vitro biochemical and structural methods have been instrumental in dissecting and explaining the molecular details of macrolide-induced peptidyl-tRNA drop-off and ribosome stalling, the dynamic effects of the drugs on ongoing protein synthesis inside live bacterial cells are far less explored. In the present study, we used single-particle tracking of dye-labeled tRNAs to study the kinetics of mRNA translation in the presence of erythromycin, directly inside live Escherichia coli cells. In erythromycin-treated cells, we find that the dwells of elongator tRNAPhe on ribosomes extend significantly, but they occur much more seldom. In contrast, the drug barely affects the ribosome binding events of the initiator tRNAfMet. By overexpressing specific short peptides, we further find context-specific ribosome binding dynamics of tRNAPhe, underscoring the complexity of erythromycin's effect on protein synthesis in bacterial cells.
Subject(s)
Anti-Bacterial Agents/pharmacology , Erythromycin/pharmacology , Escherichia coli/drug effects , Protein Biosynthesis/drug effects , Protein Synthesis Inhibitors/pharmacology , Ribosomes/drug effects , Amino Acid Sequence , Anti-Bacterial Agents/metabolism , Carbocyanines/chemistry , Codon , Erythromycin/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Fluorescent Dyes/chemistry , Peptides/chemistry , Peptides/genetics , Peptides/metabolism , Protein Binding , Protein Synthesis Inhibitors/metabolism , RNA, Bacterial/chemistry , RNA, Bacterial/genetics , RNA, Bacterial/metabolism , RNA, Transfer, Met/chemistry , RNA, Transfer, Met/genetics , RNA, Transfer, Met/metabolism , RNA, Transfer, Phe/chemistry , RNA, Transfer, Phe/genetics , RNA, Transfer, Phe/metabolism , Ribosomes/genetics , Ribosomes/metabolism , Ribosomes/ultrastructure , Single Molecule ImagingABSTRACT
Developing and optimizing small-molecule biosensors is a central goal of synthetic biology. Here we incorporate additional cellular components to improve biosensor sensitivity by preventing target molecules from diffusing out of cells. We demonstrate that trapping erythromycin within Escherichia coli through phosphorylation increases the sensitivity of its biosensor (MphR) by approximately 10-fold. When combined with prior engineering efforts, our optimized biosensor can detect erythromycin concentrations as low as 13 nM. We show that this strategy works with a range of macrolide substrates, enabling the potential usage of our optimized system for drug development and metabolic engineering. This strategy can be extended in future studies to improve the sensitivity of other biosensors. Our findings further suggest that many naturally evolved genes involved in resistance to multiple classes of antibiotics may increase intracellular drug concentrations to modulate their own expression, acting as a form of regulatory feedback.
Subject(s)
Anti-Bacterial Agents/metabolism , Biosensing Techniques/methods , Erythromycin/metabolism , Escherichia coli Proteins/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Metabolic Engineering/methods , Transcription Factors/genetics , Drug Resistance, Bacterial/genetics , Escherichia coli Proteins/metabolism , Gene Expression , Gene Expression Regulation, Bacterial , Phosphorylation , Synthetic Biology/methods , Transcription Factors/metabolismABSTRACT
The effects of bioaugmentation with immobilized Penicillium restrictum on the removal efficiency of sulfamethoxazole (SMX), erythromycin (ERY) and tetracycline (TC) antibiotics as well as membrane biofouling was studied using hollow-fiber membrane bioreactor (HF-MBR). Bioaugmentation with P. restrictum led to a significant change in the antibiotic removal efficiency and relative abundance of aerobic microbial community, most probably as a result of its quorum quenching activity. Furthermore, in addition to its role in the increase of SMX and ERY removal efficiencies and the decrease of their sorption on solid phase, bioaugmentation significantly reduced the transmembrane pressure which in turn reduced membrane clogging. The most abundant phyla in sludge and biofilm samples in the presence of P. restrictum were observed to be Proteobacteria, Bacteroidetes and Firmicutes. Differences in bacterial compositions and their specificity in biodegradation of antibiotics in different reactors showed that bacteria were specifically selected under the pressure of antibiotics and growing fungus.
Subject(s)
Anti-Bacterial Agents/metabolism , Bioreactors/microbiology , Penicillium/physiology , Bacteria/metabolism , Biodegradation, Environmental , Biofilms , Biofouling , Erythromycin/metabolism , Membranes, Artificial , Microbiota , Quorum Sensing , Sulfamethoxazole/metabolism , Tetracycline/metabolism , Wastewater/microbiologyABSTRACT
Erythromycin pollution is an important risk to the ecosystem and human health worldwide. Thus, it is urgent to develop effective approaches to decontaminate erythromycin. In this study, we successfully isolated a novel erythromycin-degrading fungus from an erythromycin-contaminated site. The erythromycin biodegradation characteristics were investigated in mineral salt medium with erythromycin as the sole carbon and energy source. The metabolites of erythromycin degraded by fungus were identified and used to derive the degradation pathway. Based on morphological and phylogenetic analyses, the isolated strain was named Curvularia sp. RJJ-5 (MN759651). Optimal degradation conditions for strain RJJ-5 were 30°C, and pH 6.0 with 100 mg L-1 erythromycin substrate. The strain could degrade 75.69% erythromycin under this condition. The following metabolites were detected: 3-depyranosyloxy erythromycin A, 7,12-dyhydroxy-6-deoxyerythronolide B, 2,4,6,8,10,12-hexamethyl-3,5,6,11,12,13-hexahydroxy-9-ketopentadecanoic acid and cladinose. It was deduced that the erythromycin A was degraded to 3-depyranosyloxy erythromycin A by glycoside hydrolase in the initial reaction. These results imply that Curvularia sp. RJJ-5 is a novel erythromycin-degrading fungus that can hydrolyze erythromycin using a glycoside hydrolase and has great potential for removing erythromycin from mycelial dreg and the contaminated environment.
Subject(s)
Anti-Bacterial Agents/metabolism , Curvularia/metabolism , Erythromycin/metabolism , Anti-Bacterial Agents/chemistry , Biodegradation, Environmental , Curvularia/classification , Curvularia/genetics , Curvularia/isolation & purification , Erythromycin/chemistry , Phylogeny , Soil MicrobiologyABSTRACT
The residual erythromycin in fermentation waste can pollute the environment and threaten human health. However, there are no effective approaches to remedy this issue. In this study, an erythromycin-degrading bacterium named RJJ-61 was isolated and identified as a strain of Delftia lacustris based on morphological and phylogenetic analyses. The degradation ability of this strain was also evaluated; it could degrade 45.18% of erythromycin at 35°C in 120 h. Furthermore, the key degradation gene ereA was cloned from strain RJJ-61 and expressed in Escherichia coli BL21; the molecular weight of the expressed protein was ~45 kDa. The enzyme activity of EreA was 108.0 mU ml-1 at 35°C and pH 7.0. Finally, the EreA protein was used to degrade erythromycin from mycelial dregs and 50% diluted solution, and the removal rates in them were 41.42% and 69.78%, respectively. In summary, D. lacustris RJJ-61 is a novel erythromycin-degrading strain that has great potential to remove erythromycin pollutants from the environment.