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We have developed the GReedy Accumulated strategy for Protein Engineering (GRAPE) to improve enzyme stability across various applications, combining advanced computational methods with a unique clustering and greedy accumulation approach to efficiently explore epistatic effects with minimal experimental effort. To make this strategy accessible to nonexperts, we introduced GRAPE-WEB, an automated, user-friendly web server that allows the design, inspection, and combination of stabilizing mutations without requiring extensive bioinformatics knowledge. GRAPE-WEB's robust performance and accessibility provide a comprehensive and adaptable approach to protein thermostability design, suitable for both newcomers and experienced practitioners in the field. The web server is accessible at https://grape.wulab.xyz.

Compelling concerns about antimicrobial resistance and the emergence of multidrug-resistant pathogens call for novel strategies to address these challenges. Nanoparticles show promising antimicrobial activities; however, their actions are hindered primarily by the bacterial hydrophilic-hydrophobic barrier. To overcome this, we developed a method of electrochemically anchoring sodium dodecyl sulfate (SDS) coatings onto silver nanoparticles (AgNPs), resulting in improved antimicrobial potency. We then investigated the antimicrobial mechanisms and developed therapeutic applications. We demonstrated SDS-coated AgNPs with anomalous dispersive properties capable of dispersing in both polar and nonpolar solvents and, further, detected significantly higher bacteriostatic and bactericidal effects compared to silver ions (Ag⁺). Cellular assays suggested multipotent disruptions targeting the bacterial membrane, evidenced by increasing lactate dehydrogenase, protein and sugar leakage, and consistent with results from the transcriptomic analysis. Notably, the amphiphilic characteristics of the AgNPs maintained robust antibacterial activities for a year at various temperatures, indicating long-term efficacy as a potential disinfectant. In a murine model, the AgNPs showed considerable biocompatibility and could alleviate fatal Salmonella infections. Collectively, by gaining amphiphilic properties from SDS, we offer novel AgNPs against bacterial infections combined with long-term and cost-effective strategies.

Optical density (OD) is an important indicator of microbial density, and a commonly used variable in growth curves to express the growth of microbial culture. However, OD values show a linear relationship with bacterial concentration only at low concentrations. When the cell density is high, the relationship loses linearity, and serial dilution is needed to obtain readings of better accuracy. Here, we show that measuring OD values using shorter light paths is in close equivalence to measuring OD values of the cell culture with corresponding dilution. By measuring three different light paths simultaneously, accurate OD values can be easily obtained from low to high cell density. Using this method, growth curves of Escherichia coli, Staphylococcus aureus, and Pichia pastoris are measured with higher accuracy. To further simplify the process, an l-shaped cuvette and a corresponding turbidimeter are designed specifically for OD value measurement based on the multi-light path transmission method.

Ammonia-oxidizing archaea (AOA) play crucial roles in marine carbon and nitrogen cycles by fixing inorganic carbon and performing the initial step of nitrification. Evaluation of carbon and nitrogen metabolism popularly relies on functional genes such as amoA and accA. Increasing studies suggest that quorum sensing (QS) mainly studied in biofilms for bacteria may serve as a universal communication and regulatory mechanism among prokaryotes; however, this has yet to be demonstrated in marine planktonic archaea. To bridge this knowledge gap, we employed a combination of metabolic activity markers (amoA, accA, and grs) to elucidate the regulation of AOA-mediated nitrogen, carbon processes, and their interactions with the surrounding heterotrophic population. Through co-transcription investigations linking metabolic markers to potential key QS genes, we discovered that QS molecules could regulate AOA's carbon, nitrogen, and lipid metabolisms under different conditions. Interestingly, specific AOA ecotypes showed a preference for employing distinct QS systems and a distinct QS circuit involving a typical population. Overall, our data demonstrate that QS orchestrates nitrogen and carbon metabolism, including the exchange of organic metabolites between AOA and surrounding heterotrophic bacteria, which has been previously overlooked in marine AOA research.

Treatment of Mycobacterium abscessus (Mab) infections is very challenging due to its intrinsic resistance to most available drugs. Therefore, it is crucial to discover novel anti-Mab drugs. In this study, we explored an intrinsic resistance mechanism through which Mab resists echinomycin (ECH). ECH showed activity against Mab at a minimum inhibitory concentration (MIC) of 2 µg/ml. A ΔembC strain in which the embC gene was knocked out showed hypersensitivity to ECH (MIC: 0.0078-0.0156 µg/ml). The MICs of ECH-resistant strains screened with reference to ΔembC ranged from 0.25 to 1 µg/ml. Mutations in EmbB, including D306A, D306N, R350G, V555I, and G581S, increased the Mab's resistance to ECH when overexpressed in ΔembC individually (MIC: 0.25-0.5 µg/ml). These EmbB mutants, edited using the CRISPR/Cpf1 system, showed heightened resistance to ECH (MIC: 0.25-0.5 µg/ml). The permeability of these Mab strains with edited genes and overexpression was reduced, as evidenced by an ethidium bromide accumulation assay, but it remained significantly higher than that of the parent Mab. In summary, our study demonstrates that ECH exerts potent anti-Mab activity and confirms that EmbB and EmbC are implicated in Mab's sensitivity to ECH. Mutation in EmbB may partially compensate for a loss of EmbC function.

Staphylococcus aureus is a common cause of diverse infections, ranging from superficial to invasive, affecting both humans and animals. The widespread use of antibiotics in clinical treatments has led to the emergence of antibiotic-resistant strains and small colony variants. This surge presents a significant challenge in eliminating infections and undermines the efficacy of available treatments. The bacterial Save Our Souls (SOS) response, triggered by genotoxic stressors, encompasses host immune defenses and antibiotics, playing a crucial role in bacterial survival, invasiveness, virulence, and drug resistance. Accumulating evidence underscores the pivotal role of the SOS response system in the pathogenicity of S. aureus. Inhibiting this system offers a promising approach for effective bactericidal treatments and curbing the evolution of antimicrobial resistance. Here, we provide a comprehensive review of the activation, impact, and key proteins associated with the SOS response in S. aureus. Additionally, perspectives on therapeutic strategies targeting the SOS response for S. aureus, both individually and in combination with traditional antibiotics are proposed.

Fzf1 is a Saccharomyces cerevisiae transcription factor containing five zinc fingers (ZFs). It regulates the expression of at least five downstream genes, including SSU1, YHB1, DDI2/3, and YNR064c, by recognizing a consensus sequence, CS2, found in these gene promoters. These gene products are involved in cellular responses to various chemical stresses. For example, SSU1 encodes a sodium sulfite efflux protein that confers sulfite resistance. However, the underlying molecular mechanism through which Fzf1 responds to chemical stress and coordinates target gene activation remains elusive. Interestingly, several mutations in the fourth ZF (ZF4) of Fzf1 have previously been reported to confer either sulfite resistance or elevated basal-level expression of YHB1, indicating that ZF4 negatively impacts Fzf1 activity. Since ZF4 is dispensable for CS2 binding in vitro, we hypothesized that ZF4 is a negative regulator of Fzf1 and that chemically induced Fzf1-regulated gene expression occurs via de-repression. All five genes examined were cross-induced by corresponding chemicals in an Fzf1-dependent manner, and all three ZF4 mutations and a ZF4 deletion conferred increased basal-level expression and SSU1-dependent sulfite resistance. A ZF4 deletion did not alter the target DNA binding, consistent with the observed codominant phenotype. These observations collectively reveal that Fzf1 remains inactive by default at the target promoters and that its activation is at least partially achieved by self-derepression through chemical modification and/or a conformational change.

Salicylic acid (SA) plays an essential role in plant defense against biotrophic and semi-biotrophic pathogens. Following pathogen recognition, SA biosynthesis dramatically increases at the infection site of the host plant. The manner in which pathogens sense and tolerate the onslaught of SA stress to survive in the plant following infection remains to be understood. The objective of this work was to determine how the model phytopathogen Xanthomonas campestris pv. campestris (Xcc) senses and effluxes SA during infection inside host plants. First, RNA-Seq analysis identified an SA-responsive operon Xcc4167-Xcc4171, encoding a MarR family transcription factor HepR and an RND (resistance-nodulation-cell division) family efflux pump HepABCD in Xcc. Electrophoretic mobility shift assays and DNase I footprint analysis revealed that HepR negatively regulated hepABCD expression by specifically binding to an AT-rich region of the promoter of the hepRABCD operon, P_hep. Second, isothermal titration calorimetry and further genetic analysis suggest that HepR is a novel SA sensor. SA binding released HepR from its cognate promoter P_hep and then induced the expression of hepABCD. Third, the RND family efflux pump HepABCD was responsible for SA efflux. The hepRABCD cluster was also involved in the regulation of culture pH and quorum sensing signal diffusible signaling factor turnover. Finally, the hepRABCD cluster was transcribed during the XC1 infection of Chinese radish and was required for the full virulence of Xcc in Chinese radish and cabbage. These findings suggest that the ability of Xcc to co-opt the plant defense signal SA to activate the multidrug efflux pump may have evolved to ensure Xcc survival and virulence in susceptible host plants.

Earth's lower near space of 20-40 km above sea level with polyextreme conditions serves as a unique Mars analog for astrobiological research to investigate the limits of life on Earth and planetary protection considerations for Mars exploration. In this study, we exposed Mars-like desert regolith to near space at a float altitude of ~35 km and isolated four bacterial strains after exposure. In addition to stress tolerance to extreme environmental stressors, these strains represent a remarkable tolerance to perchlorate that is widespread in present-day Martian soils. These extremophilic bacterial strains screened through near-space exposure could serve as promising candidates for future astrobiological research in space stations or in laboratory-based planetary simulation environments.