APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
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2022
·Vol. 106
·期 19
·6551-6566
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IF 5.56
Khan Sabir,Xu Xueqi,Song Jialei,Wu Panpan,Liu Xiaobin,Liu Jing,Chen Ketao,Xu Zhenyue,Wu Hang,Zhang Buchang
Abstract TetR family transcriptional regulators (TFRs) are widespread in actinomycetes, which exhibit diverse regulatory modes in antibiotic biosynthesis. Nitrogen regulators play vital roles in modulation of primary and secondary metabolism. However, crosstalk between TFR and nitrogen regulator has rarely been reported in actinomycetes. Herein, we demonstrated that a novel TFR, SACE_4839, was negatively correlated with erythromycin yield in Saccharopolyspora erythraea A226. SACE_4839 indirectly suppressed erythromycin synthetic gene eryAI and resistance gene ermE and directly inhibited its adjacent gene SACE_4838 encoding a homologue of nitrogen metabolite repression (NMR) regulator NmrA (herein named NmrR). The SACE_4839-binding sites within SACE_4839-nmrR intergenic region were identified. NmrR positively controlled erythromycin biosynthesis by indirectly stimulating eryAI and ermE and directly repressing SACE_4839 . NmrR was found to affect growth viability under the nitrogen source supply. Furthermore, NmrR directly repressed glutamine and glutamate utilization-related genes SACE_1623 , SACE_5070 and SACE_5979 but activated nitrate utilization-associated genes SACE_1163 , SACE_4070 and SACE_4912 as well as nitrite utilization-associated genes SACE_1476 and SACE_4514 . This is the first reported NmrA homolog for modulating antibiotic biosynthesis and nitrogen metabolism in actinomycetes. Moreover, combinatorial engineering of SACE_4839 and nmrR in the high-yield S. erythraea WB resulted in a 68.8% increase in erythromycin A production. This investigation deepens the understanding of complicated regulatory network for erythromycin biosynthesis. Key points
SACE_4839 and NmrR had opposite contributions to erythromycin biosynthesis.
NmrR was first identified as a homolog of another nitrogen regulator NmrA.
Cross regulation between SACE_4839 and NmrR was revealed.
研究领域:
微生物学
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分子生物学
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生物技术
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PubMed
高影响因子
MICROCHIMICA ACTA
·
2025
·Vol. 192
·期 9
·1-13
·
IF 5.3
Bi Huaixiu,Zhang Cheng,Zhao Chen,Zhang Xi,Li Chenyu,Wang Shang,Yang Xiaobo,Qiu Zhigang,Wang Jingfeng,Xue Bin,Shen Zhiqiang
The overuse of tetracycline (TC) a novel reverse ELISA assay integrating quantum dots (QDs) with an allosteric transcription factor (TetR) was developed for rapid tetracycline (TC) detection. Specifically, biotin-modified double-stranded DNA (dsDNA) was immobilized on a streptavidin-coated 96-well plate, after which QD-TetR conjugates were added. In the presence of TC, the QD-TetR conjugate binds TC and undergoes allosteric changes that result in its dissociation from the dsDNA. The developed QD-TetR-based reverse ELISA achieved quantitative TC detection within a linear dynamic range of 0.05–100 μM in just 5 min, with a method detection limit of 0.02 μM. The recoveries ranged from 93.43% to 106.6% for actual samples (seawater, sewage, milk, and honey) detection. Furthermore, on-site semi-quantitative detection of TC in actual samples was achieved across a concentration range 0.5–500 μM. The results were validated with commercial ELISA kits, indicating strong potential for monitoring TC residues in environmental and food samples. Graphical abstract
研究领域:
分析化学
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环境监测
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食品安全
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生物传感器
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分子生物学
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PubMed
高影响因子
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
·
2021
·Vol. 87
·期 6
·
IF 4.792
Minggen Cheng,Dongmei Pei,Xiangrong He,Yongchuang Liu,Pingping Zhu,Xin Yan
The bacterial hydrolytic dehalogenation of 4-chlorobenzoate (4CBA) is a coenzyme A (CoA)-activation-type catabolic pathway that is usually a common part of the microbial mineralization of chlorinated aromatic compounds. Previous studies have shown that the transport and dehalogenation genes for 4CBA are typically clustered as an fcbBAT1T2T3C operon and inducibly expressed in response to 4CBA. However, the associated molecular mechanism remains unknown. In this study, a gene (fcbR) adjacent to the fcb operon was predicted to encode a TetR-type transcriptional regulator in Comamonas sediminis strain CD-2. The fcbR knockout strain exhibited constitutive expression of the fcb cluster. In the host Escherichia coli, the expression of the Pfcb-fused green fluorescent protein (gfp) reporter was repressed by the introduction of the fcbR gene, and genetic studies combining various catabolic genes suggest that the ligand for FcbR may be an intermediate metabolite. Purified FcbR could bind to the Pfcb DNA probe in vitro, and the metabolite 4-chlorobenzyl-CoA (4CBA-CoA) prevented FcbR binding to the Pfcb DNA probe. Isothermal titration calorimetry (ITC) measurements showed that 4CBA-CoA could bind to FcbR at a 1:1 molar ratio. DNase I footprinting showed that FcbR protected a 42-bp DNA motif (5′-GGAAATCAATAGGTCCATAGAAAATCTATTGACTAATCGAAT-3′) that consists of two sequence repeats containing four pseudopalindromic sequences (5′-TCNATNGA-3′). This binding motif overlaps with the −35 box of Pfcb and was proposed to prevent the binding of RNA polymerase. This study characterizes a transcriptional repressor of the fcb operon, together with its ligand, thus identifying halogenated benzoyl-CoA as belonging to the class of ligands of transcriptional regulators.IMPORTANCE The bacterial hydrolytic dehalogenation of 4CBA is a special CoA-activation-type catabolic pathway that plays an important role in the biodegradation of polychlorinated biphenyls and some herbicides. With genetic and biochemical approaches, the present study identified the transcriptional repressor and its cognate effector of a 4CBA hydrolytic dehalogenation operon. This work extends halogenated benzoyl-CoA as a new member of CoA-derived effector compounds that mediate allosteric regulation of transcriptional regulators.
研究领域:
微生物学
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分子生物学
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环境科学
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高影响因子
JOURNAL OF HAZARDOUS MATERIALS
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2023
·
IF 13.6
Shunlan Li,Dongdong Chen,Ziqing Liu,Shipin Tao,Tianyi Zhang,Yiwen Chen,Lingzhi Bao,Jie Ma,Yuee Huang,Shengmin Xu,Lijun Wu,Shaopeng Chen
Antibiotic abuse is the main reason for the drug resistance of pathogenic bacteria, posing a potential health risk. Antibiotic surveillance is critical for preventing antibiotic contamination. This study aimed to develop a sensitive and broad-spectrum whole-cell biosensor for tetracycline antibiotics (TCs) detection. Wild-type TCs-responsive biosensor was constructed by introducing a tetracycline operon into a sfGFP reporter plasmid. Using error-prone PCR, mutation libraries containing approximately 10 7 variants of the tetracycline repressor (TetR) gene were generated. The tigecycline-senstive mutants were isolated using high-throughput flow cytometric sorting. After 2 rounds of directed evolution, a mutant epS2–22 of TerR was isolated and assembled as a TCs biosensor. The epS2–22 biosensor was more sensitive and broad-spectrum than the wild-type biosensors. The detection limits of the epS2–22 biosensor for seven TCs were 4- to 62-fold lower than the wild-type biosensor (no response to tigecycline). Meanwhile, the epS2–22 biosensor had a shorter detection time and a stronger signal output than the wild type. In addition, the evolved epS2–22 biosensor showed excellent performance in detecting low traces of TCs in environmental water. These results suggest that directed evolution is a powerful tool for developing high-performance whole-cell biosensors, and the evolved epS2–22 biosensors have the potential for wider applications in real-world TCs detection.
研究领域:
生物技术
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微生物学
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环境科学
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产品关联
DOI
PubMed
高影响因子
ANALYTICA CHIMICA ACTA
·
2023
·
IF 6.2
Wan Qiu Xia,Jing Liu,Jian Ping Wang
Compared with antibody, the recognition spectrum of a receptor is broader, and its recognition ability can be improved using simple mutagenesis technique. Compared with conventional immunoassay , the magnetic bead based immunoassay is simpler and can be recycled. Compared with colorimetric and luminescent immunoassays, fluoroimmunoassay is simpler because it does not require a substrate. So a method combines these merits is desirable. In this study, two amino acids in the binding pocket of a natural Escherichia coli TetR protein were mutated to produce a mutant, and the molecular docking showed the binding energies and the numbers of contact acid for 10 tetracyclines all increased. The mutant was coupled with Fe 3 O 4 to synthesize a magnetic complex, and a fluorescent tracer was synthesized by coupling quantum dot and minocycline with bovine serum albumin . Under the assistance of 96-well bottom magnet, a semi-homogeneous method based on the two materials was developed on conventional microplate for determination of the 10 tetracyclines in milk. Results showed once assay was finished within 20 min, the limits of detection (drug concentration showing 10% inhibition) for the 10 drugs were in the range of 0.32–0.94 ng/mL, and the magnetic complex could be regenerated for 6 times. Furthermore, the sensitivities were improved for 4–6 folds in comparison with the use of natural TetR. Therefore, this method is simple, sensitive, time-saving and recyclable, and it can be used for routine screening of the 10 tetracyclines in milk.
研究领域:
分析化学
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生物化学
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食品科学
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分子生物学
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DOI
PubMed
高影响因子
Virulence
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2025
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IF 5.4
Hao-Jie Shen,Jun-Feng Wang,Zheng-Jie Xue,Jia-Qi Shi,Bing Chen,Dong-Min Chen,Ni-Pi Chen,Yu-Dong Li,Chao-Dong Qian
Antimicrobial resistance is one of the greatest threats to global health. A thorough comprehension of the underlying processes through which bacteria evolve the ability to withstand diverse stressors is vital for the development of efficacious strategies to address this challenge. This study aimed to elucidate the mechanisms underlying the adaptation of an acrB-deficient strain of Escherichia coli ATCC 35218 (35218m) to sanguinarine (SAN), the main component of phytobiotic derived from Macleaya cordata. Analysis of the in vitro-selected SAN-resistant clones of strain 35218m led to the identification of a functionally uncharacterized TetR regulator, EefR. The regulator represses its own expression and that of four neighboring genes forming the eefRABCD operon, encoding components of a tripartite RND-efflux pump (EefABC) and a putative MFS-type exporter (EefD). Overexpression of these pumps reduces the susceptibility of acrB-deficient E. coli to SAN and structurally diverse antibiotics. The eefR-eefABC-eefD cluster, widely distributed across Enterobacteriaceae genomes, is also found on plasmids in several E. coli isolates, indicating a significant risk for the rapid spread of this multidrug resistance mechanism.
研究领域:
微生物学
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分子生物学
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耐药机制
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细菌遗传学
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抗生素耐药性
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DOI
PubMed
高影响因子
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