Transition metals serve as an important class of micronutrients that are indispensable for bacterial physiology but are cytotoxic when they are in excess. Bacteria have developed exquisite homeostatic systems to control the uptake, storage, and efflux of each of biological metals and maintain a thermodynamically balanced metal quota. However, whether the pathways that control the homeostasis of different biological metals cross-talk and render cross-resistance or sensitivity in the host-pathogen interface remains largely unknown. Here, we report that zinc (Zn) excess perturbs iron (Fe) and copper (Cu) homeostasis in Escherichia coli, resulting in increased Fe and decreased Cu levels in the cell. Gene expression analysis revealed that Zn excess transiently up-regulates Fe-uptake genes and down-regulates Fe-storage genes and thereby increases the cellular Fe quota. In vitro and in vivo protein-DNA binding assays revealed that the elevated intracellular Fe poisons the primary Cu detoxification transcription regulator CueR, resulting in dysregulation of its target genes copA and cueO and activation of the secondary Cu detoxification system CusSR-cusCFBA. Supplementation with the Fe chelator 2,2′-dipyridyl (DIP) or with the reducing agent GSH abolished the induction of cusCFBA during Zn excess. Consistent with the importance of this metal homeostatic network in cell physiology, combined metal treatment, including simultaneously overloading cells with both Zn (0.25 mm) and Cu (0.25 mm) and sequestering Fe with DIP (50 μm), substantially inhibited E. coli growth. These results advance our understanding of bacterial metallobiology and may inform the development of metal-based antimicrobial regimens to manage infectious diseases. Transition metals serve as an important class of micronutrients that are indispensable for bacterial physiology but are cytotoxic when they are in excess. Bacteria have developed exquisite homeostatic systems to control the uptake, storage, and efflux of each of biological metals and maintain a thermodynamically balanced metal quota. However, whether the pathways that control the homeostasis of different biological metals cross-talk and render cross-resistance or sensitivity in the host-pathogen interface remains largely unknown. Here, we report that zinc (Zn) excess perturbs iron (Fe) and copper (Cu) homeostasis in Escherichia coli, resulting in increased Fe and decreased Cu levels in the cell. Gene expression analysis revealed that Zn excess transiently up-regulates Fe-uptake genes and down-regulates Fe-storage genes and thereby increases the cellular Fe quota. In vitro and in vivo protein-DNA binding assays revealed that the elevated intracellular Fe poisons the primary Cu detoxification transcription regulator CueR, resulting in dysregulation of its target genes copA and cueO and activation of the secondary Cu detoxification system CusSR-cusCFBA. Supplementation with the Fe chelator 2,2′-dipyridyl (DIP) or with the reducing agent GSH abolished the induction of cusCFBA during Zn excess. Consistent with the importance of this metal homeostatic network in cell physiology, combined metal treatment, including simultaneously overloading cells with both Zn (0.25 mm) and Cu (0.25 mm) and sequestering Fe with DIP (50 μm), substantially inhibited E. coli growth. These results advance our understanding of bacterial metallobiology and may inform the development of metal-based antimicrobial regimens to manage infectious diseases.
Abstract Microorganisms commonly use two-component systems (TCSs) to detect specific environmental changes and respond accordingly for their own benefit. However, the regulatory mechanisms and physiological roles of a majority of TCSs are still elusive. In this study, we focused on a previously predicted TCS FleS/FleR in Pseudomonas aeruginosa to systematically investigate its regulation and physiological roles. Loss of fleS or fleR or both genes led to decreased biofilm formation and attenuated motility in PAO1, which could be restored by heterologously complementation of FleR but not FleS, confirming that the sensor kinase FleS and the response regulator FleR constitute a TCS pair. To determine the regulatory spectrum of this TCS, we conducted transcriptome sequencing and comparison between the wild-type strain and the fleR deletion mutant. The result showed that the TCS regulates about 440 genes including most of them are involved in the virulence-related pathways, e.g. siderophore biosynthesis, pyocyanin biosynthesis, type III/VI secretion systems, c-di-GMP metabolism, flagellar assembly etc . In addition to its roles in controlling biofilm formation and motility we have already shown, FleR was demonstrated to regulate the production of virulence factors such as pyocyanin and elastase, mediate stress response to SDS, and autoregulate its own expression. Moreover, EMSA assays revealed that FleR regulates flagellum biosynthesis genes flgBCDE , flgFGHIJKL , filC , which are essential for the bacterial motility, by directly interacting with their promoters. Taken together, these results expanded our understanding on the biological roles of FleS/FleR and provided new insights on its regulatory mechanisms.
There are six major types of CRISPR-Cas systems that provide adaptive immunity in bacteria and archaea against invasive genetic elements. The discovery of CRISPR-Cas systems has revolutionized the field of genetics in many organisms. In the past few years, exploitations of the most abundant class 1 type I CRISPR-Cas systems have revealed their great potential and distinct advantages to achieve gene editing and regulation in diverse microorganisms in spite of their complicated structures. The widespread and diversified type I CRISPR-Cas systems are becoming increasingly attractive for the development of new biotechnological tools, especially in genetically recalcitrant microbial strains. In this review article, we comprehensively summarize recent advancements in microbial gene editing and regulation by utilizing type I CRISPR-Cas systems. Importantly, to expand the microbial host range of type I CRISPR-Cas-based applications, these structurally complicated systems have been improved as transferable gene-editing tools with efficient delivery methods for stable expression of CRISPR-Cas elements, as well as convenient gene-regulation tools with the prevention of DNA cleavage by obviating deletion or mutation of the Cas3 nuclease. We envision that type I CRISPR-Cas systems will largely expand the biotechnological toolbox for microbes with medical, environmental and industrial importance.
Introduction Sleep quality has a significant impact on the health-related quality of life, particularly among college students. This study proposes a framework for identifying factors that influence college students’ sleep quality, including stress, self-control, bedtime habits, and neighborhood environment. Methods The study employed a cross-sectional analytical approach on a convenience sample of 255 medical students from a private university in China during the 2021/2022 academic year, of which 80.39% (205) were women. Two complementary methodologies, partial least squares-structural equation modeling (PLS-SEM), and fuzzy sets qualitative comparative analysis (fsQCA), were utilized in the study. Results The results of the PLS-SEM analysis suggest that Stress and Self-control act as mediating variables in the model, with Bedtime habits and Neighborhood environment influencing sleep quality through these variables. Additionally, the fsQCA analysis reveals that Bedtime habits and Neighborhood environment can combine with Stress and Self-control, respectively, to influence sleep quality. Discussion These findings provide insight into how multiple factors, such as Stress, Self-control, Bedtime habits, and Neighborhood environment, can impact college students’ sleep quality, and can be used to develop intervention programs aimed at improving it. Moreover, the use of both methodologies enables the expansion of new methodological approaches that can be applied to different contexts.
Antibiotic resistance, one of the major medical threats worldwide, can be selected and induced by metals through multiple mechanisms such as co-resistance, cross-resistance, and co-regulation. Compared with co-resistance and cross-resistance which are attributed to the physically or functionally linked metal and antibiotic resistance genes, co-regulation of antibiotic resistance genes by metal-responsive regulators and pathways is much more complex and elusive. Here, we discussed the main mechanisms by which antibiotic resistance is regulated in response to metals and showed recent attempts to combat antibiotic resistance by interfering with metal-based signalling pathways. Further efforts to depict the intricate metal-based regulatory network of antibiotic resistance will provide tremendous opportunities for the discovery of novel anti-resistance targets, and blocking or rewiring the metal-based signalling pathways is emerging as a promising stratagem to reverse bacterial resistance to antibiotics and rejuvenate the efficacy of conventional antibiotics.
Abstract Background The Organization for Economic Cooperation and Development emphasizes the importance of complex problem-solving (CPS) skills in the 21st century. CPS skills have been linked to academic performance, career development, and job competency training. Reflective learning, which includes journal writing, peer reflection, selfreflection, and group discussion, has been explored to improve critical thinking and problem-solving abilities. The development of various thinking modes and abilities, such as algorithmic thinking, creativity, and empathic concern, all affect problem-solving skills. However, there is a lack of an overall theory to relate variables to each other, which means that different theories need to be integrated to focus on how CPS skills can be effectively trained and improved. Methods Data from 136 medical students were analyzed using partial least square structural equation modeling (PLSSEM) and fuzzy set qualitative comparative analysis (fsQCA). A hypothesized model examining the associations between the CPS skills and influence factors was constructed. Results The evaluation of the structural model showed that some variables had significant influences on CPS skills, while others did not. After deleting the insignificant pathways, a structural model was built, which showed that mediating effects of empathic concern and critical thinking were observed, while personal distress only had a direct effect on CPS skills. The results of necessity showed that only cooperativity and creativity are necessary conditions for critical thinking. The fsQCA analysis provided clues for each different pathway to the result, with all consistency values being higher than 0.8, and most coverage values being between 0.240 and 0.839. The fsQCA confirmed the validity of the model and provided configurations that enhanced the CPS skills. Conclusions This study provides evidence that reflective learning based on multi-dimensional empathy theory and 21 stcentury skills theory can improve CPS skills in medical students. These results have practical implications for learning and suggest that educators should consider incorporating reflective learning strategies that focus on empathy and 21 stcentury skills to enhance CPS skills in their curricula.
Pseudomonas aeruginosa ATCC 27853 was isolated from a hospital blood specimen in 1971 and has been widely used as a model strain to survey antibiotics susceptibilities, biofilm development, and metabolic activities of Pseudomonas spp.. Although four draft genomes of P. aeruginosa ATCC 27853 have been sequenced, the complete genome of this strain is still lacking, hindering a comprehensive understanding of its physiology and functional genome.Here we sequenced and assembled the complete genome of P. aeruginosa ATCC 27853 using the Pacific Biosciences SMRT (PacBio) technology and Illumina sequencing platform. We found that accessory genes of ATCC 27853 including prophages and genomic islands (GIs) mainly contribute to the difference between P. aeruginosa ATCC 27853 and other P. aeruginosa strains. Seven prophages were identified within the genome of P. aeruginosa ATCC 27853. Of the predicted 25 GIs, three contain genes that encode monoxoygenases, dioxygenases and hydrolases that could be involved in the metabolism of aromatic compounds. Surveying virulence-related genes revealed that a series of genes that encode the B-band O-antigen of LPS are lacking in ATCC 27853. Distinctive SNPs in genes of cellular adhesion proteins such as type IV pili and flagella biosynthesis were also observed in this strain. Colony morphology analysis confirmed an enhanced biofilm formation capability of ATCC 27853 on solid agar surface compared to Pseudomonas aeruginosa PAO1. We then performed transcriptome analysis of ATCC 27853 and PAO1 using RNA-seq and compared the expression of orthologous genes to understand the functional genome and the genomic details underlying the distinctive colony morphogenesis. These analyses revealed an increased expression of genes involved in cellular adhesion and biofilm maturation such as type IV pili, exopolysaccharide and electron transport chain components in ATCC 27853 compared with PAO1. In addition, distinctive expression profiles of the virulence genes lecA, lasB, quorum sensing regulators LasI/R, and the type I, III and VI secretion systems were observed in the two strains.The complete genome sequence of P. aeruginosa ATCC 27853 reveals the comprehensive genetic background of the strain, and provides genetic basis for several interesting findings about the functions of surface associated proteins, prophages, and genomic islands. Comparative transcriptome analysis of P. aeruginosa ATCC 27853 and PAO1 revealed several classes of differentially expressed genes in the two strains, underlying the genetic and molecular details of several known and yet to be explored morphological and physiological potentials of P. aeruginosa ATCC 27853.
Active drug efflux constitutes an important mechanism of antibiotic and multidrug resistance in bacteria. Understanding the distribution, expression, and physiological functions of multidrug efflux pumps, especially under physiologically and clinically relevant conditions of the pathogens, is the key to combat drug resistance. In animal hosts, most wounded, infected and inflamed tissues display low oxygen tensions. In this article, we summarize research development on multidrug efflux pumps in the medicinally relevant microaerobic and anaerobic pathogens and their implications in the effort to combat drug-resistant infections.
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