A meet-up of acetyl phosphate and c-di-GMP modulates BldD activity for development and antibiotic production.

Actinobacteria are ubiquitous bacteria undergoing complex developmental transitions coinciding with antibiotic production in response to stress or nutrient starvation. This transition is mainly controlled by the interaction between the second messenger c-di-GMP and the master repressor BldD. To date, the upstream factors and the global signal networks that regulate these intriguing cell biological processes remain unknown. In Saccharopolyspora erythraea, we found that acetyl phosphate (AcP) accumulation resulting from environmental nitrogen stress participated in the regulation of BldD activity through cooperation with c-di-GMP. AcP-induced acetylation of BldD at K11 caused the BldD dimer to fall apart and dissociate from the target DNA and disrupted the signal transduction of c-di-GMP, thus governing both developmental transition and antibiotic production. Additionally, practical mutation of BldDK11R bypassing acetylation regulation could enhance the positive effect of BldD on antibiotic production. The study of AcP-dependent acetylation is usually confined to the control of enzyme activity. Our finding represents an entirely different role of the covalent modification caused by AcP, which integrated with c-di-GMP signal in modulating the activity of BldD for development and antibiotic production, coping with environmental stress. This coherent regulatory network might be widespread across actinobacteria, thus has broad implications.

Characterization of diverse lysine acylations in Bacillus thuringiensis: Substrate profiling and functional exploration.

Lysine acylation has been extensively investigated due to its regulatory role in a diverse range of biological functions across prokaryotic and eukaryotic species. In-depth acylomic profiles have the potential to enhance comprehension of the biological implications of organisms. However, the extent of research on global acylation profiles in microorganisms is limited. Here, four lysine acylomes were conducted in Bacillus thuringiensis by using the LC-MS/MS based proteomics combined with antibody-enrichment strategies, and a total of 3438 acetylated sites, 5797 propionylated sites, 1705 succinylated sites, and 925 malonylated sites were identified. The motif analysis of these modified proteins revealed a high conservation of glutamate in acetylation and propionylation, whereas such conservation was not observed in succinylation and malonylation modifications. Besides, conservation analysis showed that homologous acylated proteins in Bacillus subtilis and Escherichia coli were connected with ribosome and aminoacyl-tRNA biosynthesis. Further biological experiments showed that lysine acylation lowered the RNA binding ability of CodY and impaired the in vivo protein activity of MetK. In conclusion, our study expanded the current understanding of the global acylation in Bacillus, and the comparative analysis demonstrated that shared acylation proteins could play important roles in regulating both metabolism and RNA transcription progression.

Protein acylation links metabolism and the control of signal transduction, transcription regulation, growth, and pathogenicity in Actinobacteria.

Actinobacteria have a complex life cycle, including morphological and physiological differentiation which are often associated with the biosynthesis of secondary metabolites. Recently, increased interest in post-translational modifications (PTMs) in these Gram-positive bacteria has highlighted the importance of PTMs as signals that provide functional diversity and regulation by modifying proteins to respond to diverse stimuli. Here, we review the developments in research on acylation, a typical PTM that uses acyl-CoA or related metabolites as donors, as well as the understanding of the direct link provided by acylation between cell metabolism and signal transduction, transcriptional regulation, cell growth, and pathogenicity in Actinobacteria.

An Orthogonal CRISPR/dCas12a System for RNA Imaging in Live Cells.

CRISPR/Cas technology has made great progress in the field of live-cell imaging beyond genome editing. However, effective and easy-to-use CRISPR systems for labeling multiple RNAs of interest are still needed. Here, we engineered a CRISPR/dCas12a system that enables the specific recognition of the target RNA under the guidance of a PAM-presenting oligonucleotide (PAMmer) to mimic the PAM recognition mechanism for DNA substrates. We demonstrated the feasibility and specificity of this system for specifically visualizing endogenous mRNA. By leveraging dCas12a-mediated precursor CRISPR RNA (pre-crRNA) processing and the orthogonality of dCas12a from different bacteria, we further demonstrated the proposed system as a simple and versatile molecular toolkit for multiplexed imaging of different types of RNA transcripts in live cells with high specificity. This programmable dCas12a system not only broadens the RNA imaging toolbox but also facilitates diverse applications for RNA manipulation.

Biomimetic Electrochemical Sensor Based on Single-Atom Nickel Laccase Nanoenzyme for Quercetin Detection.

Laccase, a member of the copper oxidase family, has been used as a green catalyst in the environmental and biochemical industries. However, laccase nanoenzymes are limited to materials with copper as the active site, and noncopper laccase nanoenzymes have been scarcely reported. In this study, inspired by the multiple copper active sites of natural laccase and the redox Cu2+/Cu+ electron transfer pathway, a novel nitrogen/nickel single-atom nanoenzyme (N/Ni SAE) with high laccase-like activity was prepared by inducing Ni and dopamine precipitation through a controllable water/ethanol interface reaction. Compared with that of laccase, the laccase activity simulated by N/Ni SAE exhibited excellent stability and reusability. The N/Ni SAE exhibited a higher efficiency toward the degradation of 2,4-dichlorophenol, hydroquinone, bisphenol A, and p-aminobenzene. In addition, a sensitive electrochemical biosensor was constructed by leveraging the laccase-like activity of N/Ni SAE; this sensor offered unique advantages in terms of catalytic activity, selectivity, stability, and repeatability. Its detection ranges for quercetin were 0.01-0.1 and 1.0-100 µM, and the detection limit was 3.4 nM. It was also successfully used for the quantitative detection of quercetin in fruit juices. Therefore, the single-atom biomimetic nanoenzymes prepared in this study promote the development of a new electrochemical strategy for the detection of various bioactive molecules and show great potential for practical applications.

Cholesterol-autoxidation metabolites in host defense against infectious diseases.

Cholesterol plays essential roles in biological processes, including cell membrane stability and myelin formation. Cholesterol can be metabolized to oxysterols by enzymatic or nonenzymatic ways. Nonenzymatic cholesterol metabolites, also called cholesterol-autoxidation metabolites, are formed dependent on the oxidation of reactive oxygen species (ROS) such as OH• or reactive nitrogen species, such as ONOO- . Cholesterol-autoxidation metabolites are abundantly produced in diseases such as inflammatory bowel disease and atherosclerosis, which are associated with oxidative stress. Recent studies have shown that cholesterol-autoxidation metabolites can further regulate the immune system. Here, we review the literature and summarize how cholesterol-autoxidation metabolites, such as 25-hydroxycholesterol (25-OHC), 7α/ß-OHC, and 7-ketocholesterol, deal with the occurrence and development of infectious diseases through pattern recognition receptors, inflammasomes, ROS production, nuclear receptors, G-protein-coupled receptor 183, and lipid availability. In addition, we include the research regarding the roles of these metabolites in COVID-19 infection and discuss our viewpoints on the future research directions.

Engineering of a TetR family transcriptional regulator BkdR enhances heterologous spinosad production in Streptomyces albus B4 chassis.

Precursor supply plays a significant role in the production of secondary metabolites. In Streptomyces bacteria, propionyl-, malonyl-, and methylmalonyl-CoA are the most common precursors used for polyketide biosynthesis. Although propionyl-CoA synthetases participate in the propionate assimilation pathway and directly convert propionate into propionyl-CoA, malonyl- and methylmalonyl-CoA cannot be formed using common acyl-CoA synthetases. Therefore, both acetyl- and propionyl-CoA carboxylation, catalyzed by acyl-CoA carboxylases, should be considered when engineering a microorganism chassis to increase polyketide production. In this study, we identified a transcriptional regulator of the TetR family, BkdR, in Streptomyces albus B4, which binds directly to the promoter region of the neighboring pccAB operon. This operon encodes acetyl/propionyl-CoA carboxylase and negatively regulates its transcription. In addition to acetate and propionate, the binding of BkdR to pccAB is disrupted by acetyl- and propionyl-CoA ligands. We identified a 16-nucleotide palindromic BkdR-binding motif (GTTAg/CGGTCg/TTAAC) in the intergenic region between pccAB and bkdR. When bkdR was deleted, we found an enhanced supply of malonyl- and methylmalonyl-CoA precursors in S. albus B4. In this study, spinosad production was detected in the recombinant strain after introducing the entire artificial biosynthesized gene cluster into S. albus B4. When supplemented with propionate to provide propionyl-CoA, the novel bkdR-deleted strain produced 29.4% more spinosad than the initial strain in trypticase soy broth (TSB) medium. IMPORTANCE: In this study, we describe a pccAB operon involved in short-chain acyl-CoA carboxylation in S. albus B4 chassis. The TetR family regulator, BkdR, represses this operon. Our results show that BkdR regulates the precursor supply needed for heterologous spinosad biosynthesis by controlling acetyl- and propionyl-CoA assimilation. The deletion of the BkdR-encoding gene exerts an increase in heterologous spinosad yield. Our research reveals a regulatory mechanism in short-chain acyl-CoA metabolism and suggests new possibilities for S. albus chassis engineering to enhance heterologous polyketide yield.

Dual engineered bacteria improve inflammatory bowel disease in mice.

Currently, there are many different therapies available for inflammatory bowel disease (IBD), including engineered live bacterial therapeutics. However, most of these studies focus on producing a single therapeutic drug using individual bacteria, which may cause inefficacy. The use of dual drugs can enhance therapeutic effects. However, expressing multiple therapeutic drugs in one bacterial chassis increases the burden on the bacterium and hinders good secretion and expression. Therefore, a dual-bacterial, dual-drug expression system allows for the introduction of two probiotic chassis and enhances both therapeutic and probiotic effects. In this study, we constructed a dual bacterial system to simultaneously neutralize pro-inflammatory factors and enhance the anti-inflammatory pathway. These bacteria for therapy consist of Escherichia coli Nissle 1917 that expressed and secreted anti-TNF-α nanobody and IL-10, respectively. The oral administration of genetically engineered bacteria led to a decrease in inflammatory cell infiltration in colon and a reduction in the levels of pro-inflammatory cytokines. Additionally, the administration of engineered bacteria did not markedly aggravate gut fibrosis and had a moderating effect on intestinal microbes. This system proposes a dual-engineered bacterial drug combination treatment therapy for inflammatory bowel disease, which provides a new approach to intervene and treat IBD. KEY POINTS: • The paper discusses the effects of using dual engineered bacteria on IBD • Prospects of engineered bacteria in the clinical treatment of IBD.

Universal DNA-Based Sensing Toolbox for Programming Cell Functions.

By recombining natural cell signaling systems and further reprogramming cell functions, use of genetically engineered cells and bacteria as therapies is an innovative emerging concept. However, the inherent properties and structures of the natural signal sensing and response pathways constrain further development. We present a universal DNA-based sensing toolbox on the cell surface to endow new signal sensing abilities for cells, control cell states, and reprogram multiple cell functions. The sensing toolbox contains a triangular-prismatic-shaped DNA origami framework and a sensing core anchored inside the internal confined space to enhance the specificity and efficacy of the toolbox. As a proof of principle, the sensing toolbox uses the customizable sensing core with signal sensing switches and converters to recognize unconventional signal inputs, deliver functional components to cells, and then control cell responses, including specific tumor cell death, immune cell disinhibition and adhesion, and bacterial expression. This work expands the diversity of cell sensing signals and reprograms biological functions by constructing nanomechanical-natural hybrid cells, providing new strategies for engineering cells and bacteria in diagnosis and treatment applications.

Biological Sentinel: An Efficient Engineered Bacterial System for Monitoring and Tracing Regulated Hazardous Chemicals.

Monitoring and tracing of regulated hazardous chemicals is a public security issue of global concern. However, accurately recording historical exposure remains challenging. Here, we designed a Biological Sentinel System (BOSS) for in situ and long-term monitoring of hazardous chemical exposure using a chemical-induced base-editing system that activates antibiotic resistance screening, producing an obvious colorimetric signal. Exposure events can be written into an inheritable genomic DNA sequence, which can be read using gene sequencing. As a proof of concept, we demonstrated the accurate detection of cocaine and 2,4-dinitrotoluene using BOSS under simulated application scenarios. In addition, we integrated alternative biosensors to illustrate the modularity and extensibility of this monitoring platform. This work provides a promising paradigm for developing engineered microorganisms as an alternative to electronic monitors for regulated hazardous chemicals.

RNase H-Driven crRNA Switch Circuits for Rapid and Sensitive Detection of Various Analytical Targets.

The clustered regularly interspaced short palindromic repeats (CRISPR/Cas12a) system has exhibited great promise in the rapid and sensitive molecular diagnostics for its trans-cleavage property. However, most CRISPR/Cas system-based detection methods are designed for nucleic acids and require target preamplification to improve sensitivity and detection limits. Here, we propose a generic crRNA switch circuit-regulated CRISPR/Cas sensor for the sensitive detection of various targets. The crRNA switch is engineered and designed in a blocked state but can be activated in the presence of triggers, which are target-induced association DNA to initiate the trans-cleavage activity of Cas12a for signal reporting. Additionally, RNase H is introduced to specifically hydrolyze RNA duplexed with the DNA trigger, resulting in the regeneration of the trigger to activate more crRNA switches. Such a combination provides a generic and sensitive strategy for the effective sensing of the p53 sequence, thrombin, and adenosine triphosphate. The design is incorporated with nucleic acid nanotechnology and extensively broadens the application scope of the CRISPR technology in biosensing.

Dual-Recognition Triggered Proximity Ligation Combined with a Rolling Circle Amplification Strategy for Analysis of Exosomal Protein-Specific Glycosylation.

Exosomal surface glycan reveals the biological function and molecular information on the protein, especially in indicating the pathogenesis of certain diseases through monitoring of specific protein glycosylation accurately. However, in situ and nondestructive measurement techniques for certain Exosomal glycoproteins are still lacking. In this work, combined with on-chip purification, we designed a proximity ligation assay-induced rolling circle amplification (RCA) strategy for highly sensitive identification of Exosomal protein-specific glycosylation based on a couple of proximity probes to target Exosomal protein and the protein-specific glycosylation site. Benefiting from efficient separation, scalable dual-recognition, and proximity-triggered RCA amplification, the proposed strategy could convert different protein-specific glycan levels to prominent changes in absorbance signals, resulting in accurate quantification of specific glycosylated Exosomal protein. When detecting the glycosylated PD-L1 on MDA-MB-231 exosomes and glycosylated PTK7 on HepG2 exosomes, the detection limits were calculated to be as low as 1.04 × 104 and 2.759 × 103 particles/mL, respectively. In addition, we further expand the dual-recognition site to investigate the potential correlation of Exosomal glycosylation with polarization of THP-1 cells toward the tumor-suppressive M1 phenotype. Overall, this strategy provides a universal tool for multiple analyses of diverse protein-specific glycosylated exosomes, exhibiting enormous potential to explore exosome function and search for new early diagnosis markers.

A DNA Nanodevice-Based Platform with Diverse Capabilities.

Social biotic colonies often perform intricate tasks by interindividual communication and cooperation. Inspired by these biotic behaviors, a DNA nanodevice community is proposed as a universal and scalable platform. The modular nanodevice as the infrastructure of platform contains a DNA origami triangular prism framework and a hairpin-swing arm machinery core. By coding and decoding a signal domain on the shuttled output strand in different nanodevices, an orthogonal inter-nanodevice communication network is established to connect multi-nanodevices into a functional platform. The nanodevice platform enables implementation of diverse tasks, including signal cascading and feedback, molecular input recording, distributed logic computing, and modeling of simulation for virus transmission. The nanodevice platform with powerful compatibility and programmability presents an elegant example of the combination of the distributed operation of multiple devices and the complicated interdevice communication network, and may become a new generation of intelligent DNA nanosystems.

Acetylation of K188 and K192 inhibits the DNA-binding ability of NarL to regulate Salmonella virulence.

Salmonella infection significantly increases nitrate levels in the intestine, immune cells, and immune organs of the host, and it can exploit nitrate as an electron acceptor to enhance its growth. In the presence of nitrate or nitrite, NarL, a regulatory protein of the Nar two-component system, is activated and regulates a number of genes involved in nitrate metabolism. However, research on NarL at the post-translational level is limited. In this study, we demonstrate that the DNA-binding sites K188 and 192 of NarL can be acetylated by bacterial metabolite acetyl phosphate and that the degree of acetylation has a considerable influence on the regulatory function of NarL. Specifically, acetylation of NarL negatively regulates the transcription of narG, narK, and napF, which affects the utilization of nitrate in Salmonella. Besides, both cell and mouse models show that acetylated K188 and K192 result in attenuated replication in RAW 264.7 cells, as well as impaired virulence in mouse model. Together, this research identifies a novel NarL acetylation mechanism that regulates Salmonella virulence, providing a new insight and target for salmonellosis treatment.IMPORTANCESalmonella is an important intracellular pathogen that can cause limited gastroenteritis and self-limiting gastroenteritis in immunocompetent humans. Nitrate, the highest oxidation state form of nitrogen, is critical in the formation of systemic infection in Salmonella. It functions as a signaling molecule that influences Salmonella chemotaxis, in addition to acting as a reduced external electron acceptor for Salmonella anaerobic respiration. NarL is an essential regulatory protein involved in nitrate metabolism in Salmonella, and comprehending its regulatory mechanism is necessary. Previous research has linked NarL phosphorylation to the formation of its dimer, which is required for NarL to perform its regulatory functions. Our research demonstrated that acetylation also affects the regulatory function of NarL. We found that acetylation affects Salmonella pathogenicity by weakening the ability of NarL to bind to the target sequence, further refining the mechanism of the anaerobic nitrate respiration pathway.

Recent progress in quantitative technologies for the analysis of cancer-related exosome proteins.

Exosomes are a kind of extracellular vesicles, which play a significant role in intercellular communication and molecular exchange. Cancer-derived exosomes are potential and ideal biomarkers for the early diagnosis and treatment monitoring of cancers because of their abundant biological information and contribution to the interaction between cancer cells and the tumor microenvironment. However, there are a number of drawbacks, such as low sensitivity and tedious steps, in conventional detection techniques. Furthermore, exosome quantification is not enough to accurately distinguish cancer patients from healthy individuals. Therefore, developing efficient, accurate, and inexpensive exosome surface protein analysis techniques is necessary and critical. In recent years, a considerable number of researchers have presented novel detection strategies in this field. This review summarizes the recent progress in quantitative technologies for the analysis of cancer-related exosome proteins, mainly including the detection methods based on aptamers, nanomaterials, and antibodies, discusses a roadmap for future developments, and aims to offer an innovative perspective of exosome research.

Development of a "turn off-on" whole-cell biosensor for sulforaphane detection based on the ultrasensitive activator HrpRS.

Sulforaphane (SFN), a defense secondary metabolite, can be used to predict the health status of plants and also has pharmacological effects, including anticancer, antioxidant, and anti-inflammatory properties. The detection of SFN is therefore of great significance for the prevention and treatment of diseases. In this study, a "turn off" whole-cell biosensor that can rapidly and robustly respond to the presence of SFN was constructed based on the orthogonal genetic components (hrpR, hrpS, and PhrpL ) of Pseudomonas syringae (PS). The final optimized biosensor, p114(30R-30S), was able to inhibit 91.7% of the fluorescence intensity in the presence of 100-µM SFN. Subsequently, a HrpRS-regulated OFF-ON genetic switch was designed by reconstituting a reverse σ70 promoter on the σ54 -PhrpL promoter sequence; this was coupled with dual-color reporter genes to construct a "turn off-on" whole-cell SFN biosensor. The PhrpLB variant increased the expression of green fluorescence a factor of 11.9 and reduced the expression of red fluorescence by 85.8% compared with the system in the absence of SFN. Thus, a robust switching of signal output from "turn off" to "turn on" was realized. In addition, the biosensor showed good linearity in the SFN concentration ranges of 0.1-10 µM (R2  = 0.99429) and 10-100 µM (R2  = 0.99465) and a detection limit of ~0.1 µM.

A robust electrochemical sensor based on N,S-FeNi3/C for simultaneous detection of hydroquinone and arbutin in cosmetics.

For practical analysis and simultaneous detection of arbutin (AR) and hydrochinone (HQ) in cosmetics, an electrochemical sensor has been designed based on nitrogen and sulfur co-doped Fe-Ni alloy (N,S-FeNi3/C) nanoparticles. The N,S-FeNi3/C has been prepared for the first time via hydrothermal synthesis and high-temperature carbonization. N,S-FeNi3/C not only improves the charge transfer to the surface, but also provides rich active sites and fast ion diffusion rates owing to the iron and nickel bimetallic materials. In addition, the d-band structure of transition metals (nickel and iron) introduced by the N and S atoms exhibits an electronic structure similar to that of noble metal catalysts, thus enhancing electrocatalytic activity and increasing conductivity. Additionally, the double doping of S and N atoms significantly increases the active sites of carbon atoms; thus, N-S-FeNi3/C exhibits excellent electrochemical catalytic activity for the oxidation of AR and HQ. Further, the N,S-FeNi3/C sensor is used for the simultaneous determination of HQ and AR by square-wave pulse voltammetry. Distinct oxidation peaks of HQ and AR are observed at potentials of +0.028 V and +0.352 V (vs. SCE). The electrical signal increases linearly in the HQ concentration ranges of 0.1-100 µM and 0.05-70 µM for the simultaneous determination of AR and HQ with a detection limit as low as 0.0476 and 0.0135 µM (S/N = 3), respectively. Thus, rapid and accurate detection of AR and HQ in spiked cosmetics is successfully achieved, with a recovery ranging from 96.4 to 104.2%, and the relative standard deviation is lower than 3.8-4.0%.

Nano-Biohybrid DNA Engager That Reprograms the T-Cell Receptor.

Although engineered T cells with transgenic chimeric antigen receptors (CARs) have made a breakthrough in cancer therapeutics, this approach still faces many challenges in the specificity, efficacy, and self-safety of genetic engineering. Here, we developed a nano-biohybrid DNA engager-reprogrammed T-cell receptor (EN-TCR) system to improve the specificity and efficacy, mitigate the excessive activation, and shield against risks from transgenesis, thus achieving a diversiform and precise control of the T-cell response. Utilizing modular assembly, the EN-TCR system can graft different specificities on T cells via antibody assembly. Besides, the designability of DNA hybridization enables precise target recognition by the library of multiantigen cell recognition circuits and allows gradual tuning of the T-cell activation level by the signaling switch and independent control over different types of T cells. Furthermore, we demonstrated the effectiveness of the system in tumor models. Together, this study provides a nongenetic T-cell engineering strategy to overcome major hindrances in T-cell therapy and may be extended to a general and convenient cell engineering strategy.

Rapid On-Chip Isolation of Cancer-Associated Exosomes and Combined Analysis of Exosomes and Exosomal Proteins.

Exosomes are lipid bilayer extracellular vesicles secreted by various types of cells and inherit abundant molecular information from parental cells. Tumor-derived exosomes have been widely recognized as noninvasive biomarkers for early cancer diagnosis and surveillance, but the separation of intact exosomes and detection of exosomal proteins remain challenging. Herein, we proposed a microfluidic chip for specific exosome isolation, integrated with sensitive quantification by a novel PTCDI-aptamer signal switch strategy. To enhance the capture efficiency, an alternating drop-shaped micropillar array was designed to assist the capture of tumor-derived exosomes by Tim4-modified magnetic beads (Tim4 beads) on the chip. Following capture, a chelating agent can easily elute intact exosomes which were further used for profiling exosomal surface proteins by the multiplexed fluorescence turn-on approach. Profiting from the efficient on-chip enrichment of the Tim4 beads and superior fluorescence signal transduction strategy, the detection limit of the analysis platform for HepG2 exosomes is as low as 8.69 × 103 particles/mL with a wide linear range spanning 6 orders of magnitude. Meanwhile, the proposed platform could recognize subtle changes in protein levels on the exosomal surface from various cell lines. More importantly, this strategy is successfully applied to analyze exosomes in human serum to distinguish liver cancer patients from healthy individuals. Combined analysis of different types of biomarkers on the exosomal membrane surface can greatly improve the accuracy of cancer type identification and disease monitoring. We hope that this convenient, rapid, and sensitive platform may become a powerful tool in the field of exosome analysis and early cancer screening.

De Novo Design of the ArsR Regulated Pars Promoter Enables a Highly Sensitive Whole-Cell Biosensor for Arsenic Contamination.

Whole-cell biosensors for arsenic contamination are typically designed based on natural bacterial sensing systems, which are often limited by their poor performance for precisely tuning the genetic response to environmental stimuli. Promoter design remains one of the most important approaches to address such issues. Here, we use the arsenic-responsive ArsR-Pars regulation system from Escherichia coli MG1655 as the sensing element and coupled gfp or lacZ as the reporter gene to construct the genetic circuit for characterizing the refactored promoters. We first analyzed the ArsR binding site and a library of RNA polymerase binding sites to mine potential promoter sequences. A set of tightly regulated Pars promoters by ArsR was designed by placing the ArsR binding sites into the promoter's core region, and a novel promoter with maximal repression efficiency and optimal fold change was obtained. The fluorescence sensor PlacV-ParsOC2 constructed with the optimized ParsOC2 promoter showed a fold change of up to 63.80-fold (with green fluorescence visible to the naked eye) at 9.38 ppb arsenic, and the limit of detection was as low as 0.24 ppb. Further, the optimized colorimetric sensor PlacV-ParsOC2-lacZ with a linear response between 0 and 5 ppb was used to perform colorimetric reactions in 24-well plates combined with a smartphone application for the quantification of the arsenic level in groundwater. This study offers a new approach to improve the performance of bacterial sensing promoters and will facilitate the on-site application of arsenic whole-cell biosensors.