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1.
《中国科学:生命科学英文版》2017,(9)
Increasing the self-resistance levels of Streptomyces is an effective strategy to improve the production of antibiotics.To increase the oxytetracycline(OTC) production in Streptomyces rimosus,we investigated the cooperative effect of three co-overexpressing OTC resistance genes:one gene encodes a ribosomal protection protein(otrA) and the other two express efflux proteins(otrB and otrC).Results indicated that combinational overexpression of otrA,otrB,and otrC(MKABC) exerted a synergetic effect.OTC production increased by 179%in the recombinant strain compared with that of the wild-type strain M4018.The resistance level to OTC was increased by approximately two-fold relative to the parental strain,thereby indicating that applying the cooperative effect of self-resistance genes is useful to improve OTC production.Furthermore,the previously identified cluster-situated activator OtcR was overexpressed in MKABC in constructing the recombinant strain MKRABC;such strain can produce OTC of approximately7.49 g L~((-1)),which represents an increase of 19%in comparison with that of the OtcR-overexpressing strain alone.Our work showed that the cooperative overexpression of self-resistance genes is a promising strategy to enhance the antibiotics production in Streptomyces. 相似文献
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土霉素是由龟裂链霉菌合成的一类广谱性抗生素,前期研究工作证明其生物合成受其自身途径特异性调控蛋白OtcR的直接调节,OtcR能够激活和促进土霉素合成基因簇的转录表达。在龟裂链霉菌M4018宿主内利用强启动子单独过表达OtcR蛋白,使土霉素的产量提高到原来产量的4倍;为了进一步提高土霉素产量,在M4108宿主内表达乙酰辅酶A羧化酶基因,提高其胞内土霉素合成的前体物丙二酸单酰辅酶A的含量。对出发菌株M4018进行工程改造,同时过表达途径特异性调控蛋白OtcR和乙酰辅酶A羧化酶,发酵检测改造后的重组工程菌株土霉素的产量由1.37g/L提高到9.09g/L,该研究策略对工程改造龟裂链霉菌提高土霉素的产量具有重要的指导意义。 相似文献
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The putative response regulator BaeR stimulates multidrug resistance of Escherichia coli via a novel multidrug exporter system,MdtABC 总被引:1,自引:0,他引:1
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Overproduction of the response regulator BaeR confers resistance to novobiocin and bile salts in a DeltaacrAB mutant by stimulating drug exporter gene expression. The mdtABC (multidrug transporter ABC, formerly known as yegMNO) genes, which encode a resistance-nodulation-cell division (RND) drug efflux system, are responsible for resistance. The MdtABC system comprises the transmembrane MdtB/MdtC heteromultimer and MdtA membrane fusion protein. MdtAC also confers bile salt, but not novobiocin, resistance. This indicates that the evolution from an MdtC homomultimer to an MdtBC heteromultimer contributed to extend the drug resistance spectrum. A BLAST search suggested that such a heteromultimer-type RND exporter constitutes a unique family among gram-negative organisms. 相似文献
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Some ABC transporters play a significant role in human health and illness because they confer multidrug resistance (MDR) through their overexpression. Compounds that inhibit the drug efflux mechanism can improve efficacy or reverse resistance. Of the eight described ABC transporter subfamilies, those proteins conferring MDR in humans are in subfamilies A, B, C, and G. In nematodes, transporters in subfamilies B and C are suggested to confer resistance to ivermectin. The Brugia malayi ABC transporter superfamily was examined to assess their potential to influence sensitivity to moxidectin. There was an increase in expression of ABC transporters in subfamilies A, B, C, and G following treatment. Co-administration of moxidectin with inhibitors of ABC transporter function did not enhance sensitivity to moxidectin in males; however, sensitivity was significantly enhanced in females and microfilariae. The work suggests that ABC transporters influence sensitivity to moxidectin and have a potential role in drug resistance. 相似文献
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Nobuyoshi Kobayashi Kunihiko Nishino Akihito Yamaguchi 《Journal of bacteriology》2001,183(19):5639-5644
In the Escherichia coli genome, five putative open reading frame (ORF) clusters, mdlAB, ybjYZ, yddA, yojHI, and yhiH, have been assumed to be possible genes for ABC drug efflux transporters (I. T. Paulsen, M. K. Sliwinski, and M. H. Saier, Jr., J. Mol. Biol. 277:573-592, 1998). We cloned all of these ORFs in multicopy plasmids and investigated the drug resistance of drug-supersensitive host cells lacking constitutive multidrug efflux transporter genes acrAB. Among them, only ybjYZ gave significant erythromycin resistance and significantly decreased the accumulation of [(14)C]erythromycin. Therefore, ybjYZ was renamed macAB (macrolide-specific ABC-type efflux carrier). Plasmids carrying both the macA and -B genes conferred resistance against macrolides composed of 14- and 15-membered lactones but no or weak resistance against 16-membered ones. Neither of the two genes produced resistance alone. The DNA sequence suggests that MacB is an integral membrane protein with four transmembrane segments and one nucleotide-binding domain, while MacA belongs to a membrane fusion protein (MFP) family with a signal-like sequence at its N terminus. The expression of the histidine-tagged proteins confirmed that MacB is an integral membrane protein and MacA is a peripheral membrane protein. In addition, MacAB required TolC for its function in a way similar to that of most of the MFP-dependent transporters in E. coli. MacB is thus a novel ABC-type macrolide efflux transporter which functions by cooperating with the MFP MacA and the multifunctional outer membrane channel TolC. This is the first case of an experimentally identified ABC antibiotic efflux transporter in gram-negative organisms. 相似文献
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The homodimeric ATP-binding cassette transporter LmrA mediates multidrug transport by an alternating two-site (two-cylinder engine) mechanism 总被引:11,自引:0,他引:11
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The bacterial LmrA protein and the mammalian multidrug resistance P-glycoprotein are closely related ATP-binding cassette (ABC) transporters that confer multidrug resistance on cells by mediating the extrusion of drugs at the expense of ATP hydrolysis. The mechanisms by which transport is mediated, and by which ATP hydrolysis is coupled to drug transport, are not known. Based on equilibrium binding experiments, photoaffinity labeling and drug transport assays, we conclude that homodimeric LmrA mediates drug transport by an alternating two-site transport (two-cylinder engine) mechanism. The transporter possesses two drug-binding sites: a transport-competent site on the inner membrane surface and a drug-release site on the outer membrane surface. The interconversion of these two sites, driven by the hydrolysis of ATP, occurs via a catalytic transition state intermediate in which the drug transport site is occluded. The mechanism proposed for LmrA may also be relevant for P-glycoprotein and other ABC transporters. 相似文献
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龟裂链霉菌zwf2基因阻断提高土霉素生物合成 总被引:2,自引:0,他引:2
葡萄糖-6-磷酸脱氢酶(G6PDH)是链霉菌磷酸戊糖途径中第一个酶("看家"酶),也是形成NADPH的关键酶,由zwf1和zwf2基因编码.以温敏型质粒pKC1139为基础构建了用于阻断龟裂链霉菌zwf2的重组质粒pKC1139-zwf2',通过大肠杆菌GM2929去甲基化pKC1139-zwf2'后电转至原始龟裂链霉菌M4018感受态细胞,筛选得到转化子.转化子进一步通过PCR鉴定和点杂交印迹分析鉴定,证明是zwf2基因阻断的阳性突变子命名为M4018-△zwf2.以原始菌株为对照,突变子摇瓶发酵结果表明:突变子的葡萄糖-6-磷酸脱氢酶酶活是原始菌的50%左右,但土霉素生物合成水平则提高了27%;在细胞生长方面,二者均在第4d进入生长稳定期而开始大量合成土霉素,发酵结束时细胞菌体浓度基本相同,但突变子的单位菌丝体土霉素生物合成能力则提高了31%.因此,zwf2的阻断有利于土霉素的生物合成,而对细胞生长没有明显影响. 相似文献
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An underlying mechanism for multi drug resistance (MDR) is up-regulation of the transmembrane ATP-binding cassette (ABC) transporter proteins. ABC transporters also determine the general fate and effect of pharmaceutical agents in the body. The three major types of ABC transporters are MDR1 (P-gp, P-glycoprotein, ABCB1), MRP1/2 (ABCC1/2) and BCRP/MXR (ABCG2) proteins. Flow cytometry (FCM) allows determination of the functional expression levels of ABC transporters in live cells, but most dyes used as indicators (rhodamine 123, DiOC(2)(3), calcein-AM) have limited applicability as they do not detect all three major types of ABC transporters. Dyes with broad coverage (such as doxorubicin, daunorubicin and mitoxantrone) lack sensitivity due to overall dimness and thus may yield a significant percentage of false negative results. We describe two novel fluorescent probes that are substrates for all three common types of ABC transporters and can serve as indicators of MDR in flow cytometry assays using live cells. The probes exhibit fast internalization, favorable uptake/efflux kinetics and high sensitivity of MDR detection, as established by multidrug resistance activity factor (MAF) values and Kolmogorov-Smirnov statistical analysis. Used in combination with general or specific inhibitors of ABC transporters, both dyes readily identify functional efflux and are capable of detecting small levels of efflux as well as defining the type of multidrug resistance. The assay can be applied to the screening of putative modulators of ABC transporters, facilitating rapid, reproducible, specific and relatively simple functional detection of ABC transporter activity, and ready implementation on widely available instruments. 相似文献
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Chen XJ Bauer BE Kuchler K Clark-Walker GD 《The Journal of biological chemistry》2000,275(20):14865-14872
The nuclear gene encoding the Sit4 protein phosphatase was identified in the budding yeast Kluyveromyces lactis. K. lactis cells carrying a disrupted sit4 allele are resistant to oligomycin, antimycin, ketoconazole, and econazole but hypersensitive to paromomycin, sorbic acid, and 4-nitroquinoline-N-oxide (4-NQO). Overexpression of SIT4 leads to an elevation in resistance to paromomycin and to lesser extent tolerance to sorbic acid, but it has no detectable effect on resistance to 4-NQO. These observations suggest that the Sit4 protein phosphatase has a broad role in modulating multidrug resistance in K. lactis. Expression or activity of a membrane transporter specific for paromomycin and the ABC pumps responsible for 4-NQO and sorbic acid would be positively regulated by Sit4p. In contrast, the function of a Pdr5-type transporter responsible for ketoconazole and econazole extrusion, and probably also for efflux of oligomycin and antimycin, is likely to be negatively regulated by the phosphatase. Drug resistance of sit4 mutants was shown to be mediated by ABC transporters as efflux of the anionic fluorescent dye rhodamine 6G, a substrate for the Pdr5-type pump, is markedly increased in sit4 mutants in an energy-dependent and FK506-sensitive manner. 相似文献
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Multiple transporters are involved in natamycin efflux in Streptomyces chattanoogensis L10
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Tan‐Jun Wang Yi‐Ming Shan Han Li Wei‐Wang Dou Xin‐Hang Jiang Xu‐Ming Mao Shui‐Ping Liu Wen‐Jun Guan Yong‐Quan Li 《Molecular microbiology》2017,103(4):713-728
Antibiotic‐producing microorganisms have evolved several self‐resistance mechanisms to prevent auto‐toxicity. Overexpression of specific transporters to improve the efflux of toxic antibiotics has been found one of the most important and intrinsic resistance strategies used by many Streptomyces strains. In this work, two ATP‐binding cassette (ABC) transporter‐encoding genes located in the natamycin biosynthetic gene cluster, scnA and scnB, were identified as the primary exporter genes for natamycin efflux in Streptomyces chattanoogensis L10. Two other transporters located outside the cluster, a major facilitator superfamily transporter Mfs1 and an ABC transporter NepI/II were found to play a complementary role in natamycin efflux. ScnA/ScnB and Mfs1 also participate in exporting the immediate precursor of natamycin, 4,5‐de‐epoxynatamycin, which is more toxic to S. chattanoogensis L10 than natamycin. As the major complementary exporter for natamycin efflux, Mfs1 is up‐regulated in response to intracellular accumulation of natamycin and 4,5‐de‐epoxynatamycin, suggesting a key role in the stress response for self‐resistance. This article discusses a novel antibiotic‐related efflux and response system in Streptomyces, as well as a self‐resistance mechanism in antibiotic‐producing strains. 相似文献
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《Journal of molecular biology》2021,433(16):167005
Infectious diseases present a major threat to public health globally. Pathogens can acquire resistance to anti-infectious agents via several means including transporter-mediated efflux. Typically, multidrug transporters feature spacious, dynamic, and chemically malleable binding sites to aid in the recognition and transport of chemically diverse substrates across cell membranes. Here, we discuss recent structural investigations of multidrug transporters involved in resistance to infectious diseases that belong to the ATP-binding cassette (ABC) superfamily, the major facilitator superfamily (MFS), the drug/metabolite transporter (DMT) superfamily, the multidrug and toxic compound extrusion (MATE) family, the small multidrug resistance (SMR) family, and the resistance-nodulation-division (RND) superfamily. These structural insights provide invaluable information for understanding and combatting multidrug resistance. 相似文献
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Nidhi Arya Hadiar Rahman Andrew Rudrow Manuel Wagner Lutz Schmitt Suresh V. Ambudkar John Golin 《Molecular microbiology》2019,112(4):1131-1144
Resistance to antimicrobial and chemotherapeutic agents is a significant clinical problem. Overexpression of multidrug efflux pumps often creates broad‐spectrum resistance in cancers and pathogens. We describe a mutation, A666G, in the yeast ABC transporter Pdr5 that shows greater resistance to most of the tested compounds than does an isogenic wild‐type strain. This mutant exhibited enhanced resistance without increasing either the amount of protein in the plasma membrane or the ATPase activity. In fluorescence quenching transport assays with rhodamine 6G in purified plasma membrane vesicles, the initial rates of rhodamine 6G fluorescence quenching of both the wild type and mutant showed a strong dependence on the ATP concentration, but were about twice as high in the latter. Plots of the initial rate of fluorescence quenching versus ATP concentration exhibited strong cooperativity that was further enhanced in the A666G mutant. Resistance to imazalil sulfate was about 3–4x as great in the A666G mutant strain as in the wild type. When this transport substrate was used to inhibit the rhodamine 6G transport, the A666G mutant inhibition curves also showed greater cooperativity than the wild‐type strain. Our results suggest a novel and important mechanism: under selection, Pdr5 mutants can increase drug resistance by improving cooperative interactions between drug transport sites. 相似文献
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MgMfs1, a major facilitator superfamily (MFS) gene from the wheat pathogenic fungus Mycosphaerella graminicola, was identified in expressed sequence tag (EST) libraries. The encoded protein has high homology to members of the drug:H(+) antiporter efflux family of MFS transporters with 14 predicted transmembrane spanners (DHA14), implicated in mycotoxin secretion and multidrug resistance. Heterologous expression of MgMfs1 in a hypersensitive Saccharomyces cerevisiae strain resulted in a strong decrease in sensitivity of this organism to a broad range of unrelated synthetic and natural toxic compounds. The sensitivity of MgMfs1 disruption mutants of M. graminicola to most of these compounds was similar when compared to the wild-type but the sensitivity to strobilurin fungicides and the mycotoxin cercosporin was increased. Virulence of the disruption mutants on wheat seedlings was not affected. The results indicate that MgMfs1 is a true multidrug transporter that can function as a determinant of pathogen sensitivity and resistance to fungal toxins and fungicides. 相似文献
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ATP-binding cassette (ABC) transporters are integral membrane proteins that couple ATP hydrolysis to the transport of various molecules across cellular membranes. Found in both prokaryotes and eukaryotes, a sub-group of these transporters are involved in the efflux of hydrophobic drugs and lipids, causing anti-microbial and chemotherapeutic multidrug resistance. In this review, we examine recent structural and functional analysis of the ABC transporter MsbA and implications on the mechanism of multidrug efflux. 相似文献
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A subset of the family of ATP-binding cassette (ABC) transporters has been in focus owing to their involvement in conferring multidrug resistance in cancer cells and among immune compromised individuals. Saccharomyces cerevisiae is protected against xenobiotics by similar machineries that are part of the pleitropic drug resistance (PDR) network. The ABC transporter Pdr5 is an important member of this PDR network in yeast and is involved in cellular detoxification by the efflux of a wide variety of drugs and substrates. In this review, we focus on the aspects of detergent effects and the degeneracy in conserved sequences that is observed in the nucleotide binding domains of Pdr5 and discuss their functional relevance. 相似文献
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Zwiers LH Stergiopoulos I Gielkens MM Goodall SD De Waard MA 《Molecular genetics and genomics : MGG》2003,269(4):499-507
We have studied the role of five ABC transporter genes (MgAtr to MgAtr5) from the wheat pathogen Mycosphaerella graminicola in multidrug resistance (MDR). Complementation of Saccharomyces cerevisiae mutants with the ABC transporter genes from M. graminicola showed that all the genes tested encode proteins that provide protection against chemically unrelated compounds, indicating that their products function as multidrug transporters with distinct but overlapping substrate specificities. Their substrate range in yeast includes fungicides, plant metabolites, antibiotics, and a mycotoxin derived from Fusarium graminearum (diacetoxyscirpenol). Transformants of M. graminicola in which individual ABC transporter genes were deleted or disrupted did not exhibit clear-cut phenotypes, probably due to the functional redundancy of transporters with overlapping substrate specificity. Independently generated MgAtr5 deletion mutants of M. graminicola showed an increase in sensitivity to the putative wheat defence compound resorcinol and to the grape phytoalexin resveratrol, suggesting a role for this transporter in protecting the fungus against plant defence compounds. Bioassays with antagonistic bacteria indicated that MgAtr2 provides protection against metabolites produced by Pseudomonas fluorescens and Burkholderia cepacia. In summary, our results show that ABC transporters from M. graminicola play a role in protection against toxic compounds of natural and artificial origin. 相似文献
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真菌的多向耐药性ABC转运蛋白(ATP-binding cassette transporters)是导致多药耐药性和抗真菌药物治疗效果明显下降的主要原因。文章对酿酒酵母(Saccharomyces cerevisiae)和主要致病真菌白色假丝酵母(Candida albicans)、新型隐球酵母(Cryptococcus neoformans)和烟曲霉(Aspergillus fumigatus)中的多向耐药性ABC转运蛋白的种类、药物外排机制以及基因表达调控网络的研究进展作一综述,为深入了解真菌的多向耐药性机制以及探讨克服多向耐药性的策略和提高药效提供参考。 相似文献