Bacterial Communication Research Articles

Targeting Pseudomonas aeruginosa quorum sensing with sodium salicylate modulates immune responses in vitro and in vivo.

Chronic infections are a major clinical challenge in hard-to-heal wounds and implanted devices. Pseudomonas aeruginosa is a common causative pathogen that produces numerous virulence factors. Due to the increasing problem of antibiotic resistance, new alternative treatment strategies are needed. Quorum sensing (QS) is a bacterial communication system that regulates virulence and dampens inflammation, promoting bacterial survival. QS inhibition is a potent strategy to reduce bacterial virulence and alleviate the negative impact on host immune response. This study investigates how secreted factors from P. aeruginosa PAO1, cultured in the presence or absence of the QS inhibitor sodium salicylate (NaSa), influence host immune response. In vitro, THP-1 macrophages and neutrophil-like HL-60 cells were used. In vivo, discs of titanium were implanted in a subcutaneous rat model with local administration of P. aeruginosa culture supernatants. The host immune response to virulence factors contained in culture supernatants (+/-NaSa) was characterized through cell viability, migration, phagocytosis, gene expression, cytokine secretion, and histology. In vitro, P. aeruginosa supernatants from NaSa-containing cultures significantly increased THP-1 phagocytosis and HL-60 cell migration compared with untreated supernatants (-NaSa). Stimulation with NaSa-treated supernatants in vivo resulted in: (i) significantly increased immune cell infiltration and cell attachment to titanium discs; (ii) increased gene expression of IL-8, IL-10, ARG1, and iNOS, and (iii) increased GRO-α protein secretion and decreased IL-1β, IL-6, and IL-1α secretion, as compared with untreated supernatants. In conclusion, treating P. aeruginosa with NaSa reduces the production of virulence factors and modulates major immune events, such as promoting phagocytosis and cell migration, and decreasing the secretion of several pro-inflammatory cytokines.

New Preventive Strategy against Oral Biofilm Formation in Caries-Active Children: An In Vitro Study.

Quorum quenching (QQ) is the inhibition of bacterial communication, i.e., quorum sensing (QS). QS is a key mechanism in regulating biofilm formation and phenotype in complex bacterial communities, such as those found within cariogenic biofilms. Whereas QQ approaches were shown to effectively reduce biomass, knowledge of their impact on the taxonomic composition of oral polymicrobial biofilms remains scarce. Here, we investigate the effect of the QQ lactonase Aii20J on biomass production and taxonomical composition of biofilms. We collected supragingival plaque samples from 10 caries-free and 10 caries-active children and cultured them to generate in vitro biofilms. We describe significant biomass reductions upon Aii20J exposure, as assessed by crystal violet assays. Taxonomical profiling using 16S rRNA gene amplicon sequencing revealed no significant changes in bacterial composition at the genus level. Interestingly, at the species level Aii20J-treatment increased the abundance of Streptococcus cristatus and Streptococcus salivarius. Both S. cristatus and S. salivarius express pH-buffering enzymes (arginine deiminase and urease, respectively) that catalyze ammonia production, thereby potentially raising local pH and counteracting the biofilm's cariogenic potential. Within the limitations of the study, our findings provide evidence of the biofilm-modulating ability of QQ and offer novel insights into alternative strategies to restore homeostasis within dysbiotic ecosystems.

Molecular modeling study of micro and nanocurcumin with in vitro and in vivo antibacterial validation

Repurposing natural compounds as inhibitory targets to combat bacterial virulence is an important potential strategy to overcome resistance to traditional antibiotics, in the present study, the antibacterial activity of micro-curcumin and nano-sized curcumin was investigated against four predominant bacterial pathogens, namely, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Bacillus subtilis. Curcumin bactericidal susceptibility could be summarized as the order, P. aeruginosa > B. subtilis > S. aureus > E. coli. Molecular docking analysis was conducted to confirm the impact of curcumin on the most vital and positively identified quorum-sensing pathway signaling proteins SecA-SecY, LsrR, PqsR (MvfR), AgrA which act as key players in the bacterial communication systems. The in silico physicochemical properties revealed that curcumin as a nutraceutical can be classified as a drug-like compound. An in vivo infected wound model was employed in four groups of albino rats. Topical application of nano-curcumin lotion showed a marked reduction in wound area (98.8%) as well as nearly 100% reduction in total bacterial viable count compared to the control group, on the fifteenth day post-treatment post-injury. The obtained data suggested that curcumin nanoparticles exhibited superior antibacterial activity and may possess clinical utility as a novel topical antimicrobial and wound healing agent.

Investigation of some basil genotypes in terms of their effect on bacterial communication system, and antimicrobial activity

The exponential growth of multiresistant bacterial strains creates the need to explore new or combined strategies to combat bacterial resistance. Medicinal plant-derived compounds against pathogenic bacteria may provide new, simple approaches to developing more environmentally friendly antimicrobial agents. Many researchers focus on exploring novel or combined strategies for combating bacterial resistance. Aromatic plants containing essential oils, such as basil, are often used as therapeutic agents in the pharmaceutical industry. Recent research has shown that basil is effective against certain harmful food phytopathogenic bacteria and has antimicrobial and anti-quorum sensing properties, which were investigated in this study. Our results have shown that the essential oil and ethanol extract of basil exhibits both antibacterial activity and anti-quorum sensing activity against some Gram-negative and Gram-positive bacterial species. It has also been found to have antifungal effects on C. albicans. Among the tested microorganisms, the genotypes of PI 531396, PI 296390, PI 414199, PI 253157, PI 296391, PI 652071, midnight, and Dino cultivars have been found to be more effective than other genotypes. The highest effect on quorum sensing system was found in Moonlight and Dino cultivars, PI 296391, PI 414199, PI 652070, PI 172997 and PI 190100 genotypes. Dendrogram analysis has shown that there is a relationship between different genotypes depending on microorganisms and anti-quorum sensing activity. Ames 29184, PI 207498, and PI 379412 genotypes were in the same group. Biplot analyses were performed to determine the relationship between the studied properties, and the results showed that more than 47% of the total variation was in all forms.

A Brief Review of In Situ and Operando Electrochemical Analysis of Bacteria by Scanning Probes.

Bacteria are similar to social organisms that engage in critical interactions with one another, forming spatially structured communities. Despite extensive research on the composition, structure, and communication of bacteria, the mechanisms behind their interactions and biofilm formation are not yet fully understood. To address this issue, scanning probe techniques such as atomic force microscopy (AFM), scanning electrochemical microscopy (SECM), scanning electrochemical cell microscopy (SECCM), and scanning ion-conductance microscopy (SICM) have been utilized to analyze bacteria. This review article focuses on summarizing the use of electrochemical scanning probes for investigating bacteria, including analysis of electroactive metabolites, enzymes, oxygen consumption, ion concentrations, pH values, biofilms, and quorum sensing molecules to provide a better understanding of bacterial interactions and communication. SECM has been combined with other techniques, such as AFM, inverted optical microscopy, SICM, and fluorescence microscopy. This allows a comprehensive study of the surfaces of bacteria while also providing more information on their metabolic activity. In general, the use of scanning probes for the detection of bacteria has shown great promise and has the potential to provide a powerful tool for the study of bacterial physiology and the detection of bacterial infections.

Quorum sensing gene regulation in Staphylococcus epidermidis reduces the attraction of Aedes aegypti (L.) (Diptera: Culicidae).

Identifying mechanisms regulating mosquito attraction to hosts is key to suppressing pathogen transmission. Historically, the ecology of the host microbial community and its influence on mosquito attraction, specifically, whether bacterial communication through quorum sensing (QS) modulates VOC production that affects mosquito behavior have not been extensively considered. Behavioral choice assays were applied along with volatile collection, followed by GC-MS and RNA transcriptome analyses of bacteria with and without a quorum-sensing inhibitor, furanone C-30. Utilizing the quorum-sensing inhibitor on a skin-inhabiting bacterium, Staphylococcus epidermidis, we disrupted its interkingdom communication with adult Aedes aegypti and mitigated their attraction to a blood-meal by 55.1%. One potential mechanism suppressing mosquito attraction could be the reduction (31.6% in our study) of bacterial volatiles and their associated concentrations by shifting S. epidermidis metabolic (12 of 29 up regulated genes) and stress (5 of 36 down regulated genes) responses. Manipulating the quorum-sensing pathways could serve as a mechanism to reduce mosquito attraction to a host. Such manipulations could be developed into novel control methods for pathogen-transmitting mosquitoes and other arthropods.

A Streptomyces tendae Specialized Metabolite Inhibits Quorum Sensing in Group A Streptococcus.

Quorum sensing (QS) is a means of bacterial communication accomplished by microbe-produced signals and sensory systems. QS systems regulate important population-wide behaviors in bacteria, including secondary metabolite production, swarming motility, and bioluminescence. The human pathogen Streptococcus pyogenes (group A Streptococcus [GAS]) utilizes Rgg-SHP QS systems to regulate biofilm formation, protease production, and activation of cryptic competence pathways. Given their reliance on small-molecule signals, QS systems are attractive targets for small-molecule modulators that would then affect gene expression. In this study, a high-throughput luciferase assay was employed to screen an Actinobacteria-derived secondary metabolite (SM) fraction library to identify small molecule inhibitors of Rgg regulation. A metabolite produced by Streptomyces tendae D051 was found to be a general inhibitor of GAS Rgg-mediated QS. Herein, we describe the biological activity of this metabolite as a QS inhibitor. IMPORTANCE Streptococcus pyogenes, a human pathogen known for causing infections such as pharyngitis and necrotizing fasciitis, uses quorum sensing (QS) to regulate social responses in its environment. Previous studies have focused on disrupting QS as a means to control specific bacterial signaling outcomes. In this work, we identified and described the activity of a naturally derived S. pyogenes QS inhibitor. This study demonstrates that the inhibitor affects three separate but similar QS signaling pathways.

Extracellular vesicles of the Gram-positive gut symbiont Bifidobacterium longum induce immune-modulatory, anti-inflammatory effects

The gut microbiota is now well known to affect the host’s immune system. One way of bacterial communication with host cells is via the secretion of vesicles, small membrane structures containing various cargo. Research on vesicles secreted by Gram-positive gut bacteria, their mechanisms of interaction with the host and their immune-modulatory effects are still relatively scarce. Here we characterized the size, protein content, and immune-modulatory effects of extracellular vesicles (EVs) secreted by a newly sequenced Gram-positive human gut symbiont strain - Bifidobacterium longum AO44. We found that B. longum EVs exert anti-inflammatory effects, inducing IL-10 secretion from both splenocytes and dendritic cells (DC)-CD4+ T cells co-cultures. Furthermore, the EVs protein content showed enrichment in ABC transporters, quorum sensing proteins, and extracellular solute-binding proteins, which were previously shown to have a prominent function in the anti-inflammatory effect of other strains of B. longum. This study underlines the importance of bacterial vesicles in facilitating the gut bacterial immune-modulatory effects on the host and sheds light on bacterial vesicles as future therapeutics.

The Queen of Quorum Sensing: Scientist, Bacterial Linguist, Professor, Mentor

AbstractBonnie L. Bassler, Ph.D. is the 2022 recipient of the Wolf Prize in Chemistry, a highly honored, prestigious award given to the world's foremost scientists. Among her colleagues, students, and trainees, Bonnie is known for her innate curiosity, shrewd mind, and exuberant personality that collectively distinguish her in the scientific world. Those of us that have been taught by Bonnie – whether in the lab, the classroom, or at her unique seminars – have learned much about communication and coordination by bacteria through chemical language. We describe here our collective journeys with Bonnie, and how these have shaped our careers and perspectives in science, as well as, how in our opinion Bonnie's work and personality has inspired researchers across disciplines to become interested in Bacterial Chemical Communication.

Microbes as Communication & Decision-Making Networked Communities

Despite the simplicity of an individual cell, microbial communities exhibit complex behavior and play significant roles in many biological phenomena. Understanding the communication within and between bacterial species can illuminate the <italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">how</i> , as well as the <italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">why</i> of numerous interactions that enable their collective behavior. In this position paper, we first discuss bacterial molecular communication in multihop settings. We address this concept in the context of local and global quorum sensing (QS) within a colony and then consider induced QS at a distance between different, spatially separated communities. We then investigate <italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">how</i> the information is shared between cells when forming a quorum, particularly the intertwined relationship between cells’ observations, actions, and the state of their surrounding environment. Lastly, we extend to multi-species systems where all species coexist and interact, leveraging concepts from multiple-input-multiple-output (MIMO) communications. Community robustness and resiliency is also explored.

Nano‐enabled Quenching of Bacterial Communications for the Prevention of Biofilm Formation

AbstractBiofilm formation is a major threat to industry, the environment and human health. While killing of embedded microbes in biofilms may inevitably lead to the evolution of antimicrobial resistance (AMR), catalytic quenching of bacterial communications by lactonase is a promising antifouling approach. Given the shortcomings of protein enzymes, it is attractive to engineer synthetic materials to mimic the activity of lactonase. Herein, an efficient lactonase‐like Zn−Nx−C nanomaterial was synthesized by tuning the coordination environment around zinc atoms to mimic the active domain of lactonase for catalytical interception of bacterial communications in biofilm formation. The Zn−Nx−C material could selectively catalyze 77.5 % hydrolysis of N‐acylated‐L‐homoserine lactone (AHL), a critical bacterial quorum sensing (QS) signal in biofilm construction. Consequently, AHL degradation downregulated the expression of QS‐related genes in antibiotic resistant bacteria and significantly prevented biofilm formation. As a proof of concept, Zn−Nx−C‐coated iron plates prevented 80.3 % biofouling after a month exposure in river. Overall, our study provides a nano‐enabled contactless antifouling insight to avoid AMR evolution by engineering nanomaterials for mimicking the key bacterial enzymes (e.g., lactonase) functioning in biofilm construction.

Nano-enabled Quenching of Bacterial Communications for the Prevention of Biofilm Formation.

Biofilm formation is a major threat to industry, the environment and human health. While killing of embedded microbes in biofilms may inevitably lead to the evolution of antimicrobial resistance (AMR), catalytic quenching of bacterial communications by lactonase is a promising antifouling approach. Given the shortcomings of protein enzymes, it is attractive to engineer synthetic materials to mimic the activity of lactonase. Herein, an efficient lactonase-like Zn-Nx -C nanomaterial was synthesized by tuning the coordination environment around zinc atoms to mimic the active domain of lactonase for catalytical interception of bacterial communications in biofilm formation. The Zn-Nx -C material could selectively catalyze 77.5 % hydrolysis of N-acylated-L-homoserine lactone (AHL), a critical bacterial quorum sensing (QS) signal in biofilm construction. Consequently, AHL degradation downregulated the expression of QS-related genes in antibiotic resistant bacteria and significantly prevented biofilm formation. As a proof of concept, Zn-Nx -C-coated iron plates prevented 80.3 % biofouling after a month exposure in river. Overall, our study provides a nano-enabled contactless antifouling insight to avoid AMR evolution by engineering nanomaterials for mimicking the key bacterial enzymes (e.g., lactonase) functioning in biofilm construction.

Two Permeases Associated with the Multifunctional CtaP Cysteine Transport System in Listeria monocytogenes Play Distinct Roles in Pathogenesis.

The soil-dwelling bacterium Listeria monocytogenes survives a multitude of conditions when residing in the outside environment and as a pathogen within host cells. Key to survival within the infected mammalian host is the expression of bacterial gene products necessary for nutrient acquisition. Similar to many bacteria, L. monocytogenes uses peptide import to acquire amino acids. Peptide transport systems play an important role in nutrient uptake as well as in additional functions that include bacterial quorum sensing and signal transduction, recycling of peptidoglycan fragments, adherence to eukaryotic cells, and alterations in antibiotic susceptibility. It has been previously described that CtaP, encoded by lmo0135, is a multifunctional protein associated with activities that include cysteine transport, resistance to acid, membrane integrity, and bacterial adherence to host cells. ctaP is located next to two genes predicted to encode membrane-bound permeases lmo0136 and lmo0137, termed CtpP1 and CtpP2, respectively. Here, we show that CtpP1 and CtpP2 are required for bacterial growth in the presence of low concentrations of cysteine and for virulence in mouse infection models. Taken together, the data identify distinct nonoverlapping roles for two related permeases that are important for the growth and survival of L. monocytogenes within host cells. IMPORTANCE Bacterial peptide transport systems are important for nutrient uptake and may additionally function in a variety of other roles, including bacterial communication, signal transduction, and bacterial adherence to eukaryotic cells. Peptide transport systems often consist of a substrate-binding protein associated with a membrane-spanning permease. The environmental bacterial pathogen Listeria monocytogenes uses the substrate-binding protein CtaP not only for cysteine transport but also for resistance to acid, maintenance of membrane integrity, and bacterial adherence to host cells. In this study, we demonstrate complementary yet distinct functional roles for two membrane permeases, CtpP1 and CtpP2, that are encoded by genes linked to ctaP and that contribute to bacterial growth, invasion, and pathogenicity.

Interkingdom Detection of Bacterial Quorum-Sensing Molecules by Mammalian Taste Receptors.

Bitter and sweet taste G protein-coupled receptors (known as T2Rs and T1Rs, respectively) were originally identified in type II taste cells on the tongue, where they signal perception of bitter and sweet tastes, respectively. Over the past ~15 years, taste receptors have been identified in cells all over the body, demonstrating a more general chemosensory role beyond taste. Bitter and sweet taste receptors regulate gut epithelial function, pancreatic β cell secretion, thyroid hormone secretion, adipocyte function, and many other processes. Emerging data from a variety of tissues suggest that taste receptors are also used by mammalian cells to "eavesdrop" on bacterial communications. These receptors are activated by several quorum-sensing molecules, including acyl-homoserine lactones and quinolones from Gram-negative bacteria such as Pseudomonas aeruginosa, competence stimulating peptides from Streptococcus mutans, and D-amino acids from Staphylococcus aureus. Taste receptors are an arm of immune surveillance similar to Toll-like receptors and other pattern recognition receptors. Because they are activated by quorum-sensing molecules, taste receptors report information about microbial population density based on the chemical composition of the extracellular environment. This review summarizes current knowledge of bacterial activation of taste receptors and identifies important questions remaining in this field.

Phytochemicals: potential alternative strategy to fight Salmonella enterica serovar Typhimurium.

The rise of multidrug resistant (MDR) microorganisms is a great hazard worldwide and has made it difficult to treat many infectious diseases adequately. One of the most prevalent causes of outbreaks of foodborne illness worldwide is Salmonella. The ability of this and other harmful bacteria to withstand antibiotics has recently proven crucial to their effective control. Since the beginning of time, herbal medicines and phytochemicals have been employed for their potent antibacterial action and there is a growing trend toward the production of plant based natural products for the prevention and treatment of pathogenic infections. Numerous phytochemicals have been proven effective against the molecular determinants responsible for attaining drug resistance in pathogens like efflux pumps, membrane proteins, bacterial cell communications and biofilms. The medicinal plants having antibacterial activity and antibiotics combination with phytochemicals have shown synergetic activity against Salmonella enterica serovar Typhimurium. The inhibitory effects of tannins on rumen proteolytic bacteria can be exploited in ruminant nutrition. Improved control of the rumen ecology and practical use of this feed additive technology in livestock production will be made possible by a better knowledge of the modulatory effects of phytochemicals on the rumen microbial populations in combination with fermentation. This review focuses on the development of antibacterial resistance in Salmonella, the mechanism of action of phytochemicals and the use of phytochemicals against S. enterica serovar Typhimurium. The advances and potential future applications of phytochemicals in the fight against resistant are also discussed.

Quorum sensing system effectively enhances DegU-mediated degradation of pyrethroids by Bacillus subtilis

The contamination of the natural environment is a growing concern that threatens all life forms, including microorganisms. Bacteria protect themselves by initiating quorum sensing (QS), a bacterial cell-cell communication, to generate adaptive responses to these pollutants. Bacillus subtilis has a typical QS ComQXPA system that regulates the phosphorylation of the transcription factor DegU (DegU-P), and thus can mediate the expression of various downstream genes under different stress conditions. Herein, we found that cesB, a gene of Bacillus subtilis 168, plays a key role in pyrethroid degradation, and cesB-mediated degradation could be enhanced by coordinating with the ComX communication system. Using β-cypermethrin (β-CP) as a paradigm, we demonstrated that DegU-P increased upon exposure to β-CP, thus facilitating β-CP degradation by binding to the upstream regulatory regions of cesB, leading to the activation of the expression of cesB. Further, we showed that the expression of different levels of phosphorylated DegU in a degU deletion strain resulted in varying degrees of β-CP degradation efficiency, with phosphorylated DegUH12L achieving 78.39% degradation efficiency on the first day, surpassing the 56.27% degradation efficiency in the wild type strain. Consequently, based on the conserved regulatory mechanism of ComQXPA system, we propose that DegU-P-dependent regulation serves as a conserved defense mechanism owing to its ability to fine-tune the expression of genes involved in the degradation of pollutants upon exposure to different pesticides.

Bacteria separation and enrichment using viscoelastic flows in a straight microchannel

Bacteria occupy a large domain of prokaryotic microorganisms on earth and play a significant role in public health. In this study, we achieve continuous size-based bacteria separation using viscoelastic flows in a microchannel. Three types of bacteria that differ in size, including Lactobacillus rhamnosus (L. rhamnosus), Staphylococcus epidermidis (S. epidermidis) and Escherichia coli (E. coli) are employed. First, the separation of L. rhamnosus and S. epidermidis is investigated. Under the combined effects of inertial lift and elastic forces, the larger L. rhamnosus cells are driven toward the channel centre while the smaller S. epidermidis cells flow in narrow streams along the channel sidewalls. The results show that the separation performance of the viscoelastic microfluidic device is affected by four main factors: channel length, total flow rate (TFR), sheath-to-sample flow rate ratio (FRR) and polyethylene oxide (PEO) concentration. Furthermore, the separation of L. rhamnosus and E. coli is demonstrated. This work overcomes the issues like shape irregularity and size variation in bacterial samples and achieves the separation of two types of bacteria in a size-dependent manner. We expect that this work will facilitate the future studies of bacterial interspecies communication and interactions as well as the development of biology, biomedicine and biotechnology.

In silico studies of bacterial quorum sensing during population dynamics: simulations by using COMSOL Multiphysics

In the present study, computing techniques are designated and employed in order to analyze “quorum sensing” as a special case of cell-to-cell bacterial communication attributed to the Pseudomonas bacterial genus. One of the challenges consists in predicting the concentration of key substances characterizing the “quorum level” during bacterial population dynamics. To estimate relevant characteristics of bacterial communication, we applied a deterministic approach. The mathematical model is formalized as an initial-boundary value problem for a system of semilinear partial differential equations supplemented by the procedure to specify the multiphase dynamics of bacterial populations. The finite element solution of the problem is obtained by COMSOL Multiphysics. The comparative numerical analysis for various types of space approximation of bacterial density is performed. A series of computational experiments were conducted to estimate changes in concentrations of chemical compounds during the Pseudomonas putida population dynamics.

TapA acts as specific chaperone in TasA filament formation by strand complementation

Studying mechanisms of bacterial biofilm generation is of vital importance to understanding bacterial cell-cell communication, multicellular cohabitation principles, and the higher resilience of microorganisms in a biofilm against antibiotics. Biofilms of the nonpathogenic, gram-positive soil bacterium Bacillus subtilis serve as a model system with biotechnological potential toward plant protection. Its major extracellular matrix protein components are TasA and TapA. The nature of TasA filaments has been of debate, and several forms, amyloidic and non-Thioflavin T-stainable have been observed. Here, we present the three-dimensional structure of TapA and uncover the mechanism of TapA-supported growth of nonamyloidic TasA filaments. By analytical ultracentrifugation and NMR, we demonstrate TapA-dependent acceleration of filament formation from solutions of folded TasA. Solid-state NMR revealed intercalation of the N-terminal TasA peptide segment into subsequent protomers to form a filament composed of β-sandwich subunits. The secondary structure around the intercalated N-terminal strand β0 is conserved between filamentous TasA and the Fim and Pap proteins, which form bacterial type I pili, demonstrating such construction principles in a gram-positive organism. Analogous to the chaperones of the chaperone-usher pathway, the role of TapA is in donating its N terminus to serve for TasA folding into an Ig domain-similar filament structure by donor-strand complementation. According to NMR and since the V-set Ig fold of TapA is already complete, its participation within a filament beyond initiation is unlikely. Intriguingly, the most conserved residues in TasA-like proteins (camelysines) of Bacillaceae are located within the protomer interface.

Spectroelectrochemical behavior of parallel arrays of single vertically oriented Pseudomonas aeruginosa cells

Pseudomonas aeruginosa is a Gram-negative opportunistic human pathogen responsible for a number of healthcare-associated infection. It is currently difficult to assess single cell behaviors of P. aeruginosa that might contribute to acquisition of antibiotic resistance, intercellular communication, biofilm development, or virulence, because mechanistic behavior is inferred from ensemble collections of cells, thus averaging effects over a population. Here, we develop and characterize a device that can capture and trap arrays of single P. aeruginosa cells in individual micropores in order to study their behaviors using spectroelectrochemistry. Focused ion beam milling is used to fabricate an array of micropores in a Au/dielectric/Au/SiO2-containing multilayer substrate, in which individual micropores are formed with dimensions that facilitate the capture of single P. aeruginosa cells in a predominantly vertical orientation. The bottom Au ring is then used as a working electrode to explore the spectroelectrochemical behavior of parallel arrays of individual P. aeruginosa cells. Application of step-potential or swept-potential waveforms produces changes in the fluorescence emission that can be imaged and correlated with applied potential. Arrays of P. aeruginosa cells typically exhibit three characteristic fluorescence behaviors that are sensitive to nutritional stress and applied potential. The device developed here enables the study of parallel collections of single bacterial cells with well-defined orientational order and should facilitate efforts to elucidate methods of bacterial communication and multidrug resistance at the single cell level.