Characterization of copABCD operon from a copper-sensitive Pseudomonas putida strain

2005 ◽  
Vol 51 (3) ◽  
pp. 209-216 ◽  
Author(s):  
Vellaichamy Adaikkalam ◽  
Sanjay Swarup
Keyword(s):  
Pseudomonas Putida ◽  
Metal Binding ◽  
Copper Homeostasis ◽  
Sequencing Analysis ◽  
Copper Binding ◽  
Binding Domains ◽  
Expression Studies ◽  
Sensitivity Studies ◽  
Cop Operon ◽  
Gene Expression Studies

We describe an operon, copABCD, that encodes copper-binding and sequestering proteins for copper homeostasis in the copper-sensitive strain Pseudomonas putida PNL-MK25. This is the second operon characterized as being involved in copper homeostasis, in addition to a P1-type ATPase encoded by cueAR, which was previously shown to be active in the same strain. In this study, 3 copper-responsive mutants were obtained through mini-Tn5::gfp mutagenesis and were found to exhibit reduced tolerance to copper. Sequencing analysis of the transposon-tagged region in the 3 mutants revealed insertions in 2 genes of an operon homologous to the copABCD of P. syringae and pcoABCD of Escherichia coli. Gene expression studies demonstrated that the P. putida copABCD is inducible starting from 3 µmol/L copper levels. Copper-sensitivity studies revealed that the tolerance of the mutant strains was reduced only marginally (only 0.16-fold) in comparison to a 6-fold reduced tolerance of the cueAR mutant. Thus, the cop operon in this strain has a minimal role when compared with its role both in other copper-resistant strains, such as P. syringae pv. syringae, and in the cueAR operon of the same strain. We propose that the reduced function of the copABCD operon is likely to be due to the presence of fewer metal-binding domains in the encoded proteins.Key words: cop operon, copper-binding proteins, mini-Tn5::gfp mutagenesis, transition metal.

scholarly journals Wilson disease missense mutations in ATP7B affect metal-binding domain structural dynamics

BioMetals ◽  
2019 ◽  
Vol 32 (6) ◽  
pp. 875-885 ◽  
Author(s):  
Kumaravel Ponnandai Shanmugavel ◽  
Ranjeet Kumar ◽  
Yaozong Li ◽  
Pernilla Wittung-Stafshede
Keyword(s):  
Structural Dynamics ◽  
Metal Binding ◽  
Wilson Disease ◽  
Copper Transport ◽  
Missense Mutations ◽  
Copper Binding ◽  
Structural Dynamic ◽  
Binding Domains ◽  
Metal Binding Domain ◽  
Dynamics Simulations

Abstract Wilson disease (WD) is caused by mutations in the gene for ATP7B, a copper transport protein that regulates copper levels in cells. A large number of missense mutations have been reported to cause WD but genotype–phenotype correlations are not yet established. Since genetic screening for WD may become reality in the future, it is important to know how individual mutations affect ATP7B function, with the ultimate goal to predict pathophysiology of the disease. To begin to assess mechanisms of dysfunction, we investigated four proposed WD-causing missense mutations in metal-binding domains 5 and 6 of ATP7B. Three of the four variants showed reduced ATP7B copper transport ability in a traditional yeast assay. To probe mutation-induced structural dynamic effects at the atomic level, molecular dynamics simulations (1.5 μs simulation time for each variant) were employed. Upon comparing individual metal-binding domains with and without mutations, we identified distinct differences in structural dynamics via root-mean square fluctuation and secondary structure content analyses. Most mutations introduced distant effects resulting in increased dynamics in the copper-binding loop. Taken together, mutation-induced long-range alterations in structural dynamics provide a rationale for reduced copper transport ability.

Functional characterization of the copper transcription factor AfMac1 from Aspergillus fumigatus

2017 ◽  
Vol 474 (14) ◽  
pp. 2365-2378 ◽  
Author(s):  
Yong-Sung Park ◽  
Tae-Hyoung Kim ◽  
Cheol-Won Yun
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Promoter Region ◽  
Functional Characterization ◽  
Copper Homeostasis ◽  
Binding Motif ◽  
Copper Binding ◽  
Binding Domains ◽  
Physiological Processes ◽  
Cellular Copper

Although copper functions as a cofactor in many physiological processes, copper overload leads to harmful effects in living cells. Thus, copper homeostasis is tightly regulated. However, detailed copper metabolic pathways have not yet been identified in filamentous fungi. In this report, we investigated the copper transcription factor AfMac1 (Aspergillus fumigatusMac1 homolog) and identified its regulatory mechanism in A. fumigatus. AfMac1 has domains homologous to the DNA-binding and copper-binding domains of Mac1 from Saccharomyces cerevisiae, and AfMac1 efficiently complemented Mac1 in S. cerevisiae. Expression of Afmac1 resulted in CTR1 up-regulation, and mutation of the DNA-binding domain of Afmac1 failed to activate CTR1 expression in S. cerevisiae. The Afmac1 deletion strain of A. fumigatus failed to grow in copper-limited media, and its growth was restored by introducing ctrC. We found that AfMac1 specifically bound to the promoter region of ctrC based on EMSA. The AfMac1-binding motif 5′-TGTGCTCA-3′ was identified from the promoter region of ctrC, and the addition of mutant ctrC lacking the AfMac1-binding motif failed to up-regulate ctrC in A. fumigatus. Furthermore, deletion of Afmac1 significantly reduced strain virulence and activated conidial killing activity by neutrophils and macrophages. Taken together, these results suggest that AfMac1 is a copper transcription factor that regulates cellular copper homeostasis in A. fumigatus.

scholarly journals Assessment of common housekeeping genes as reference for gene expression studies using RT-qPCR in mouse choroid plexus

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kim Hoa Ho ◽  
Annarita Patrizi
Keyword(s):  
Gene Expression ◽  
Choroid Plexus ◽  
Sensory Deprivation ◽  
Housekeeping Genes ◽  
Housekeeping Gene ◽  
Experimental Conditions ◽  
Brain Repair ◽  
Expression Studies ◽  
Testing Algorithms ◽  
Gene Expression Studies

AbstractChoroid plexus (ChP), a vascularized secretory epithelium located in all brain ventricles, plays critical roles in development, homeostasis and brain repair. Reverse transcription quantitative real-time PCR (RT-qPCR) is a popular and useful technique for measuring gene expression changes and also widely used in ChP studies. However, the reliability of RT-qPCR data is strongly dependent on the choice of reference genes, which are supposed to be stable across all samples. In this study, we validated the expression of 12 well established housekeeping genes in ChP in 2 independent experimental paradigms by using popular stability testing algorithms: BestKeeper, DeltaCq, geNorm and NormFinder. Rer1 and Rpl13a were identified as the most stable genes throughout mouse ChP development, while Hprt1 and Rpl27 were the most stable genes across conditions in a mouse sensory deprivation experiment. In addition, Rpl13a, Rpl27 and Tbp were mutually among the top five most stable genes in both experiments. Normalisation of Ttr and Otx2 expression levels using different housekeeping gene combinations demonstrated the profound effect of reference gene choice on target gene expression. Our study emphasized the importance of validating and selecting stable housekeeping genes under specific experimental conditions.

scholarly journals Validation of Reference Genes for Studying Different Abiotic Stresses in oat (Avena sativa L.) by RT-qPCR

Plants ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1272
Author(s):  
Judit Tajti ◽  
Magda Pál ◽  
Tibor Janda
Keyword(s):  
Gene Expression ◽  
Avena Sativa ◽  
Abiotic Stresses ◽  
Reference Genes ◽  
Nutritional Value ◽  
Tissue Type ◽  
Future Research ◽  
Experimental Conditions ◽  
Expression Studies ◽  
Gene Expression Studies

Oat (Avena sativa L.) is a widely cultivated cereal with high nutritional value and it is grown mainly in temperate regions. The number of studies dealing with gene expression changes in oat continues to increase, and to obtain reliable RT-qPCR results it is essential to establish and use reference genes with the least possible influence caused by experimental conditions. However, no detailed study has been conducted on reference genes in different tissues of oat under diverse abiotic stress conditions. In our work, nine candidate reference genes (ACT, TUB, CYP, GAPD, UBC, EF1, TBP, ADPR, PGD) were chosen and analysed by four statistical methods (GeNorm, Normfinder, BestKeeper, RefFinder). Samples were taken from two tissues (leaves and roots) of 13-day-old oat plants exposed to five abiotic stresses (drought, salt, heavy metal, low and high temperatures). ADPR was the top-rated reference gene for all samples, while different genes proved to be the most stable depending on tissue type and treatment combinations. TUB and EF1 were most affected by the treatments in general. Validation of reference genes was carried out by PAL expression analysis, which further confirmed their reliability. These results can contribute to reliable gene expression studies for future research in cultivated oat.

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