Transcription factor shapes chromosomal conformation and regulates gene expression in bacterial adaptation.

Genomic mutations allow bacteria to adapt rapidly to adverse stress environments. The three-dimensional conformation of the genome may also play an important role in transcriptional regulation and environmental adaptation. Here, using chromosome conformation capture, we investigate the high-order architecture of the Zymomonas mobilis chromosome in response to genomic mutation and ambient stimuli (acetic acid and furfural, derived from lignocellulosic hydrolysate). We find that genomic mutation only influences the local chromosome contacts, whereas stress of acetic acid and furfural restrict the long-range contacts and significantly change the chromosome organization at domain scales. Further deciphering the domain feature unveils the important transcription factors, Ferric uptake regulator (Fur) proteins, which act as nucleoid-associated proteins to promote long-range (>200 kb) chromosomal communications and regulate the expression of genes involved in stress response. Our work suggests that ubiquitous transcription factors in prokaryotes mediate chromosome organization and regulate stress-resistance genes in bacterial adaptation.

The escape of CRISPR-mediated gene editing in Zymomonas mobilis.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems have been widely applied for gene or genome editing. Adequate checking is important to screen mutants after CRISPR-mediated editing events. Here, we report gene escape cases after the knockout by Type I-F native CRISPR system in Zymomonas mobilis. Through amplifying both the gene of interest and its flanking homologous arms, followed by curing the editing plasmid, we found different destinies for gene-editing events. Some genes were readily knocked out and followed by the easy plasmid curing. In some other cases, however, the editing plasmid was difficult to remove from the cell, or the deleted genes were transferred into the editing plasmid. For example, the targeted region of fur can be integrated into the editing plasmid after the knockout, resulting in a spurious editing event. We supposed that the transfer of the gene may be attributed to bacterial insertion sequences. Searching for literatures on the gene knockout using CRISPR in bacteria reveals that the escape event is likely underestimated due to inadequate validation in other microbes. Hence, several strategies are proposed to enhance gene knockout and plasmid curing.

Cascade i-motifs-dependent reversibleelectrochemical impedance strategy-oriented pH and terminal deoxynucleotidyl transferase biosensing.

In this study, we develop a novel and reversibleelectrochemical impedance strategy for pH and terminal deoxynucleotide transferase (TdT) analysis based on the TdT-assisted generation of long enough cytosine (C)-rich DNAs. The formation of this special DNA is rationally designed on 5'-thiol DNA modified Au electrode surface, and TdT can catalyze the extension of this 3'-OH end to form a long C-rich DNA in the presence of deoxycytidine triphosphate (dCTP). Here, we discover a reversible process, in which the TdT-generated C-rich DNA maintains an irregular single chain state under neutral conditions and some stable DNA i-motifs (cascade i-motifs) are formed due to the partial protonation of C under acidic conditions. More importantly, the electrochemical impedance spectroscopy (EIS) response varies with the configuration change of the TdT-mediated C-rich DNA under different pH conditions. In view of this, a unique EIS switch ("on-off-on") is constructed faithfully with the configuration change, thus achieving pH analysis well. Additionally, the TdT activity can be also detected well by recording the EIS response, because it can catalyze the DNA tailing process up to hundreds of cytosines; on the contrary, if its inhibitor exists, TdT-based extension and formation of cascade i-motifs will not occur. Using this strategy, the detection of limit for TdT is 0.79 × 10-5 U/mL (pH 7.0) and 0.25 × 10-5 U/mL (pH 5.8) (S/N = 3), respectively. All the above features make our biosensor a promising assay for in situ monitoring of pH and TdT in complex clinical diagnosis.

Coenzyme A-aptamer-facilitated label-free electrochemical stripping strategy for sensitive detection of histone acetyltransferase activity.

Abnormal histone acetyltransferases (HAT) activity gives rise to all kinds of cellular diseases. Herein, we first report a coenzyme A (CoA)-aptamer-facilitated label-free electrochemical stripping biosensor for sensitive detection of HAT activity via square wave voltammetry (SWV) technique. The presence of HAT can lead to the transfer of the acetyl group from acetyl coenzyme A (Ac-CoA) to lysine residues of substrate peptide, thus generating CoA molecule. Later, CoA, which acts as an initiator, can embrace its aptamer via the typical target-aptamer interaction, then arousing deoxynucleotide terminal transferase (TdT)-induced silver nanoclusters (AgNCs) as signal output. Under optimized conditions, the resultant aptasensor shows obvious electrochemical stripping signal and is employed for HAT p300 analysis in a wide concentration range from 0.01 to 100 nM with a very low detection limit of 0.0028 nM (3δ/slope). The good analytical performances of the biosensor depend on the strong interaction of CoA and its aptamer and abundant stripping resource rooted from AgNCs. Next, the proposed biosensor is used for screening HAT's inhibitors and the practical HAT detection with satisfactory results. Therefore, the new, simple and sensitive HAT biosensor presents a promising direction for HAT-targeted drug discovery and epigenetic research.