The paper described a label-free assay for the detection of single-nucleotide mismatches in which an unlabeled hairpin DNA probe and a MutS protein conjugate (His6-MutS-linker peptide-streptavidin binding peptide (HMLS)) are exploited for the detection of mismatches by electrochemical impedance spectroscopy (EIS). We demonstrate this method for eight single-nucleotide mismatches. Upon hybridization of the target strand with the hairpin DNA probe, the stem-loop structure is opened forming a duplex DNA. In duplexes containing a single nucleotide mismatch, the mismatch is present at the solvent exposed side, enabling more effective HMLS recognition and binding. The binding event is evaluated by EIS and analyzed with the help of Randles' equivalent circuits. The differences in the charge transfer resistance DeltaR(CT) before and after protein binding to the duplex DNA allows the unequivocal detection of all eight single-nucleotide mismatches. DeltaR(CT) allows the discrimination of a C-A mismatch with the concentration of the target strand as low as 100 pM.
Tuberculosis caused by the pathogen Mycobacterium tuberculosis (MTB), remains a significant threat to global health. Elucidating the mechanisms of essential MTB genes provides an important theoretical basis for drug exploitation. Gene mtsp17 is essential and is conserved in the Mycobacterium genus. Although Mtsp17 has a structure closely resembling typical steroidogenic acute regulatory protein-related lipid transfer (START) family proteins, its biological function is different. This study characterizes the transcriptomes of Mycobacterium smegmatis to explore the consequences of mtsp17 downregulation on gene expression. Suppression of the mtsp17 gene resulted in significant down-regulation of 3% and upregulation of 1% of all protein-coding genes. Expression of desA1, an essential gene involved in mycolic acid synthesis, and the anti-SigF antagonist MSMEG_0586 were down-regulated in the conditional Mtsp17 knockout mutant and up-regulated in the Mtsp17 over-expression strain. Trends in the changes of 70 of the 79 differentially expressed genes (Log2 fold change > 1.5) in the conditional Mtsp17 knockout strain were the same as in the SigF knockout strain. Our data suggest that Mtsp17 is likely an activator of desA1 and Mtsp17 regulates the SigF regulon by SigF regulatory pathways through the anti-SigF antagonist MSMEG_0586. Our findings indicate the role of Mtsp17 may be in transcriptional regulation, provide new insights into the molecular mechanisms of START family proteins, and uncover a new node in the regulatory network of mycobacteria.
Directed evolution in vitro is a powerful tool in the study and design of protein function. However, screening the desired mutants is a difficult task. To facilitate the screening, a method is proposed to eliminate wild type sequences and increase mutated DNA sequences, which is based on the preferential binding of MutS protein to heteroduplex DNA. Following error-prone PCR, amplified products are denatured and re-annealed to form heteroduplex and homoduplex DNA. Heteroduplexes are selectively bound to an engineered MutS protein and immobilized on a Strep-Tactin column. Homoduplexes are effectively removed by washing, and the final elution is enriched in mutated DNA sequences. One round of mutated DNA enrichment resulted in an about 2.3-fold of increase in mutation frequency compared to the control. The percentage of mutants rose from 44% in the control sample to 72% in the enrichment sample. Fluorescent assay by flow cytometry showed that the enrichment method increased the mutants with changed fluorescent activity by about 2.2-fold, which strongly justified the efficiency of enrichment in increasing mutants with functional changes. With reduced workload of screening and increased possibility of obtaining mutants with functional changes, the overall efficiency was improved by MutS-mediated enrichment of mutated DNA.
The MutS protein plays an important role in the DNA mismatch repair system. Mutations in the mutS gene can lead to genome instability and ultimately cell malfunction. Here we have established a method for identifying functional defective mutants of MutS by random mutation and rifampicin screening. Some novel functional sites in MutS were identified. The MutS mutant strains were analyzed using surface plasmon resonance, gel filtration and far-western methods to determine the molecular mechanisms behind the DNA mismatch repair function of MutS.
Background Tuberculosis caused by the pathogen Mycobacterium tuberculosis (MTB), remains a significant threat to global health. Elucidating the mechanism of essential MTB genes will provide a theoretical basis for drug exploitation. Gene mtsp17 is essential and conserved in the Mycobacterium genus. Although the structure of Mtsp17 exhibits as a typical steroidogenic acute regulatory protein-related lipid transfer (START) family protein, its biological function is different from other START proteins. This study characterized the transcriptomes of Mycobacterium smegmatis to address the problem how suppressing mtsp17 reduces bacterial growth rate. Results 3% down- and 1% up-regulated protein-coding genes were significantly differentiated expressed by suppressing mtsp17 gene. Among them, desA1, an essential gene involved in mycolic acid synthesis, was found to be significantly depressed (3.1-fold). Further analyses showed that mstp17 can activate desA1 to regulate the bacterial growth. In addition, the anti-anti-SigF gene was dramatically decreased (8.9-fold) and 70 of the 79 differentially expressed genes showed the same change trends in the SigF knockout strain. The data indicate that Mtsp17 regulates SigF regulon by modulating mRNA abundance of the anti-sigma factor antagonist. Conclusions Our findings discover the role of Mtsp17 in transcriptional regulation that was unknown before, which provides new insights into the molecular mechanisms of START family and introduces a new node to the regulatory network of mycobacteria.
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