Non-growing Cells Research Articles

Mechanistic study of a low-power bacterial maintenance state using high-throughput electrochemistry.

Mechanistic studies of life's lower metabolic limits have been limited due to a paucity of tractable experimental systems. Here, we show that redox-cycling of phenazine-1-carboxamide (PCN) by Pseudomonas aeruginosa supports cellular maintenance in the absence of growth with a low mass-specific metabolic rate of 8.7×10-4 W (g C)-1 at 25°C. Leveraging a high-throughput electrochemical culturing device, we find that non-growing cells cycling PCN tolerate conventional antibiotics but are susceptible to those that target membrane components. Under these conditions, cells conserve energy via a noncanonical, facilitated fermentation that is dependent on acetate kinase and NADH dehydrogenases. Across PCN concentrations that limit cell survival, the cell-specific metabolic rate is constant, indicating the cells are operating near their bioenergetic limit. This quantitative platform opens the door to further mechanistic investigations of maintenance, a physiological state that underpins microbial survival in nature and disease.

Growth Phase Contribution in Dictating Drug Transport and Subcellular Accumulation inside Escherichia coli.

Depending upon nutrient availability, bacteria transit to multiple growth phases. The transition from the active to nongrowing phase results in reduced drug efficacy and, in some cases, even multidrug resistance. However, due to multiple alterations in the cell envelope, probing the drug permeation kinetics during growth phases becomes perplexing, especially across the Gram-negative bacteria's complex dual membrane envelope. To advance the understanding of drug permeation during the life cycle of Gram-negative bacteria, we sought to address two underlying objectives: (a) how changes are occurring inside the bacterial envelope during growth and (b) how the drug permeation and accumulation vary across both the membranes and in subcellular compartments during growth. Both objectives are met with the help of nonlinear optical technique second-harmonic generation spectroscopy (SHG). Specifically, using SHG, we probed the transport kinetics and accumulation of a quaternary ammonium compound (QAC), malachite green, inside Escherichia coli in various growth phases. Further insight about another QAC molecule, propidium iodide, is accomplished using fluorescence microscopy. Results indicate that actively growing cells have faster drug transport and higher cytoplasmic accumulation than slow- or nongrowing cells. In this regard, the rpoS gene plays a crucial role in limiting drug transport across the saturation phase cultures. Moreover, within a particular growth phase, membrane permeability undergoes gradual changes much before the subsequent growth phase commences. These outcomes signify the importance of reporting the growth phase and rate in drug efficacy studies.

Oxidative Phosphorylation for Aerobic Survival, but Not for Growth: The Peculiar ‘Make-Accumulate-Consume’ Strategy in Zymomonas mobilis

Understanding the energy metabolism and its regulation is one of the clues to metabolic engineering of stress-resistant lignocellulose-converting microbial strains, also including the promising ethanologen Zymomonas mobilis. Z. mobilis is an obligately fermentative, facultatively anaerobic bacterium, carrying an active respiratory chain with low energy-coupling efficiency. Its respiration does not supply energy to aerobically growing cultures on sugary media, yet oxidative phosphorylation has been demonstrated in non-growing cells with ethanol. Here, we show, for the first time, that in respiring, non-growing Z. mobilis cells receiving regular small amounts of ethanol, oxidative phosphorylation significantly contributes to the maintenance of their viability. No improvement of viability is seen in the NADH dehydrogenase (ndh)-deficient respiratory mutant, which is unable to oxidize ethanol. The ethanol effect is also hampered by the protonophoric uncoupler CCCP, or the inhibitor of ATP synthase, DCCD. At higher concentrations (6% v/v), ethanol causes stress that slows down culture growth. By monitoring the activity of several respiratory gene promoters under ethanol stress with the green fluorescent protein reporter system, we demonstrate downregulation of these promoters, in particular the ndh promoter. We speculate that the decrease in respiratory chain activity in response to stress conditions mitigates the production of reactive oxygen species.

Mapping temperature-sensitive mutations at a genome scale to engineer growth switches in Escherichia coli.

Temperature-sensitive (TS) mutants are a unique tool to perturb and engineer cellular systems. Here, we constructed a CRISPR library with 15,120 Escherichia coli mutants, each with a single amino acid change in one of 346 essential proteins. 1,269 of these mutants showed temperature-sensitive growth in a time-resolved competition assay. We reconstructed 94 TS mutants and measured their metabolism under growth arrest at 42°C using metabolomics. Metabolome changes were strong and mutant-specific, showing that metabolism of nongrowing E. coli is perturbation-dependent. For example, 24 TS mutants of metabolic enzymes overproduced the direct substrate metabolite due to a bottleneck in their associated pathway. A strain with TS homoserine kinase (ThrBF267D ) produced homoserine for 24 h, and production was tunable by temperature. Finally, we used a TS subunit of DNA polymerase III (DnaXL289Q ) to decouple growth from arginine overproduction in engineered E. coli. These results provide a strategy to identify TS mutants en masse and demonstrate their large potential to produce bacterial metabolites with nongrowing cells.

Combination drug strategies for biofilm eradication using synthetic and natural agents in KAPE pathogens.

Antibiotic resistance is a global threat caused by factors such as overuse of antibiotics, lack of awareness, development of biofilms etc. World Health Organization released a list of global priority pathogens which consisted of 12 species of bacteria categorized as expressing critical, high and medium resistance. Several Gram-negative and Gram-positive species are known to cause wide varieties of infections and have become multidrug or extremely drug resistant. Pathogens causing infections associated with invasive medical devices are biofilm producers and hence their treatment becomes difficult due to a structurally stable matrix which prevents antibiotics from penetrating the biofilm and thereby showing its effects. Factors contributing to tolerance are inhibition of penetration, restricted growth and activation of biofilm genes. Combination drug therapies has also shown potential to eradicate biofilm infections. A combination of inhaled Fosfomycin/tobramycin antibiotic strategy has been effective against Gram-negative as well as Gram positive organisms. Along with antibiotics, use of natural or synthetic adjuvants shows promising effects to treat biofilm infections. Fluroquinolone activity on biofilms is disrupted by low oxygen tension in the matrix, a strategy known as hyperbaric oxygen treatment that can enhance efficacy of antibiotics if well optimized. Adjuvants such as Ethylenediaminetetraacetic acid (EDTA), Sodium Dodecyl Sulphate (SDS) and chlorhexidine act by killing non-growing microbial cells aggregated on the inner layer of the biofilm. This review aims to list down current combination therapies used against Gram-negative and Gram-positive biofilm forming pathogens and brief about comparison of combination drugs and their efficacies.

Spatial cumulant models enable spatially informed treatment strategies and analysis of local interactions in cancer systems

Theoretical and applied cancer studies that use individual-based models (IBMs) have been limited by the lack of a mathematical formulation that enables rigorous analysis of these models. However, spatial cumulant models (SCMs), which have arisen from theoretical ecology, describe population dynamics generated by a specific family of IBMs, namely spatio-temporal point processes (STPPs). SCMs are spatially resolved population models formulated by a system of differential equations that approximate the dynamics of two STPP-generated summary statistics: first-order spatial cumulants (densities), and second-order spatial cumulants (spatial covariances). We exemplify how SCMs can be used in mathematical oncology by modelling theoretical cancer cell populations comprising interacting growth factor-producing and non-producing cells. To formulate model equations, we use computational tools that enable the generation of STPPs, SCMs and mean-field population models (MFPMs) from user-defined model descriptions (Cornell et al. Nat Commun 10:4716, 2019). To calculate and compare STPP, SCM and MFPM-generated summary statistics, we develop an application-agnostic computational pipeline. Our results demonstrate that SCMs can capture STPP-generated population density dynamics, even when MFPMs fail to do so. From both MFPM and SCM equations, we derive treatment-induced death rates required to achieve non-growing cell populations. When testing these treatment strategies in STPP-generated cell populations, our results demonstrate that SCM-informed strategies outperform MFPM-informed strategies in terms of inhibiting population growths. We thus demonstrate that SCMs provide a new framework in which to study cell-cell interactions, and can be used to describe and perturb STPP-generated cell population dynamics. We, therefore, argue that SCMs can be used to increase IBMs’ applicability in cancer research.

Dissociation kinetics of small-molecule inhibitors in Escherichia coli is coupled to physiological state of cells

Bioactive small-molecule inhibitors represent a treasure chest for future drugs. In vitro high-throughput screening is a common approach to identify the small-molecule inhibitors that bind tightly to purified targets. Here, we investigate the inhibitor-target binding/unbinding kinetics in E. coli cells using a benzimidazole-derivative DNA inhibitor as a model system. We find that its unbinding rate is not constant but depends on cell growth rate. This dependence is mediated by the cellular activity, forming a feedback loop with the inhibitor’s activity. In accordance with this feedback, we find cell-to-cell heterogeneity in inhibitor-target interaction, leading to co-existence of two distinct subpopulations: actively growing cells that dissociate the inhibitors from the targets and non-growing cells that do not. We find similar heterogeneity for other clinical DNA inhibitors. Our studies reveal a mechanism that couples inhibitor-target kinetics to cell physiology and demonstrate the significant effect of this coupling on drug efficacy.

Functional Diversity of Gram-Negative Permeability Barriers Reflected in Antibacterial Activities and Intracellular Accumulation of Antibiotics.

Gram-negative bacteria are notoriously more resistant to antibiotics than Gram-positive bacteria, primarily due to the presence of the outer membrane and a plethora of active efflux pumps. However, the potency of antibiotics also varies dramatically between different Gram-negative pathogens, suggesting major mechanistic differences in how antibiotics penetrate permeability barriers. Two approaches are used broadly to analyze how permeability barriers affect intracellular accumulation of antibiotics. One compares the antibacterial activities of compounds, while the other measures the total intracellular concentrations of compounds in nongrowing cells, with both approaches using strains harboring wild-type or genetically modified efflux systems and permeability barriers. Whether the two assays provide similar mechanistic insights remains unclear. In this study, we analyzed the intracellular accumulation and antibacterial activities of antibiotics representative of major clinical classes in three Gram-negative pathogens of high clinical importance, Pseudomonas aeruginosa, Escherichia coli, and Acinetobacter baumannii. We found that both assays are informative about properties of permeability barriers, but there is no quantitative agreement between the assays. Our results show that the three pathogens differ dramatically in their permeability barriers, with the outer membrane playing the dominant role in E. coli and P. aeruginosa but efflux dominating in A. baumannii. However, even compounds of the same chemotype may use different permeation pathways depending on small chemical modifications. Accordingly, a classification analysis revealed limited conservation of molecular properties that define compound penetration into the three bacteria.

Assessing the relative importance of bacterial resistance, persistence and hyper-mutation for antibiotic treatment failure.

To curb the rising threat of antimicrobial resistance, we need to understand the routes to antimicrobial treatment failure. Bacteria can survive treatment by using both genetic and phenotypic mechanisms to diminish the effect of antimicrobials. We assemble empirical data showing that, for example, <i>Pseudomonas aeruginosa</i> infections frequently contain persisters, transiently non-growing cells unaffected by antibiotics (AB) and hyper-mutators, mutants with elevated mutation rates, and thus higher probability of genetic resistance emergence. Resistance, persistence and hyper-mutation dynamics are difficult to disentangle experimentally. Hence, we use stochastic population modelling and deterministic fitness calculations to investigate the relative importance of genetic and phenotypic mechanisms for immediate treatment failure and establishment of prolonged, chronic infections. We find that persistence causes 'hidden' treatment failure with very low cell numbers if antimicrobial concentrations prevent growth of genetically resistant cells. Persister cells can regrow after treatment is discontinued and allow for resistance evolution in the absence of AB. This leads to different mutational routes during treatment and relapse of an infection. By contrast, hyper-mutation facilitates resistance evolution during treatment, but rarely contributes to treatment failure. Our findings highlight the time and concentration dependence of different bacterial mechanisms to escape AB killing, which should be considered when designing 'failure-proof' treatments.

Rapid detection of Listeria monocytogenes in dairy products by a novel chemilumonogenic approach

Routine monitoring of foodborne pathogens such as Listeria monocytogenes in food processing environments are time-consuming necessities to ensure food safety. Alternative rapid diagnostic methods for pathogen detection are increasingly used, but often demand specialized equipment, making them unsuitable for on-site testing. This short communication describes the successful demonstration of combining the sample preparation method Matrix-Lysis with a chemiluminescent based detection platform (AquaSpark™) for detection of L. monocytogenes in milk and yogurt. The proposed method was evaluated against qPCR resulting in 100% relative specificity for both foodstuffs and a relative sensitivity of 100% for milk as well as 96% for yogurt for bacterial levels >1 CFU/ml. Only at very low initial bacterial concentrations (<1 CFU/ml) diverging results were found highlighting the advantages and limitations of both methods. While being less susceptible to contamination and false positive results from non-growing or dead cells, qPCR had a slightly lower overall detection limit. However, it has to be pointed out that qPCR has an increased analytical cost and also requires an additional 24 h analysis time. This study demonstrates the first successful application of a chemilumonogenic detection approach for L. monocytogenes in food that has a high potential for on-site testing.

Roles of RodZ and class A PBP1b in the assembly and regulation of the peripheral peptidoglycan elongasome in ovoid-shaped cells of Streptococcus pneumoniae D39.

RodZ of rod-shaped bacteria functions to link MreB filaments to the Rod peptidoglycan (PG) synthase complex that moves circumferentially perpendicular to the long cell axis, creating hoop-like sidewall PG. Ovoid-shaped bacteria, such as Streptococcus pneumoniae (pneumococcus; Spn) that lack MreB, use a different modality for peripheral PG elongation that emanates from the midcell of dividing cells. Yet, S. pneumoniae encodes a RodZ homolog similar to RodZ in rod-shaped bacteria. We show here that the helix-turn-helix and transmembrane domains of RodZ(Spn) are essential for growth at 37°C. ΔrodZ mutations are suppressed by Δpbp1a, mpgA(Y488D), and ΔkhpA mutations that suppress ΔmreC, but not ΔcozE. Consistent with a role in PG elongation, RodZ(Spn) co-localizes with MreC and aPBP1a throughout the cell cycle and forms complexes and interacts with PG elongasome proteins and regulators. Depletion of RodZ(Spn) results in aberrantly shaped, non-growing cells and mislocalization of elongasome proteins MreC, PBP2b, and RodA. Moreover, Tn-seq reveals that RodZ(Spn), but not MreCD(Spn), displays a specific synthetic-viable genetic relationship with aPBP1b, whose function is unknown. We conclude that RodZ(Spn) acts as a scaffolding protein required for elongasome assembly and function and that aPBP1b, like aPBP1a, plays a role in elongasome regulation and possibly peripheral PG synthesis.

Bacteria grow swiftly and live thriftily

Bacteria have evolved numerous strategies to use resources efficiently. However, bacterial economies depend on both the physiological context of the organisms as well as their growth state - whether they are growing, non-growing or reinitiating growth. In this essay, we discuss some of the features that make bacteria efficient under these different conditions and during the transitions between them. We also highlight the many outstanding questions regarding the physiology of non-growing bacterial cells. Lastly, we examine how efficiency is apparent in both the mode and tempo of bacterial evolution.

Bacterial respiration during stationary phase induces intracellular damage that leads to delayed regrowth

SummaryBacterial survival is often challenged by nutrient-depleted conditions. Here, we show that Escherichia coli regrowth from prolonged stationary phase is heterogeneous. Some cells rejuvenated immediately, even after extended starvation, but others only restarted growth after a delay or not at all. The proportion of nongrowing cells increased with time spent in stationary phase, rather than time-dependent medium changes. Delayed regrowth was correlated with the dissolution of polar phase-bright foci likely representing damaged protein aggregates, and a deep learning algorithm distinguished cellular fates based on single images. Delayed regrowth initiated after upregulation of chaperones and DNA-repair enzymes, and deletion of a chaperone compromised stationary-phase morphology and increased the nongrowing cell proportion. Mathematical modeling of damage accumulation and division-mediated partitioning quantitatively predicted all rejuvenation statistics. Cells regrew immediately after starving in the absence of respiration. These findings reinforce the importance of intracellular damage control when nutrients are sparse, and repair when nutrients are plentiful.

Biological rhythms: The suspended animation clock

A circadian clock can be reconstituted in a test tube using three minimal components from cyanobacteria. A new study has extended this result to include output kinases and a transcription factor. One implication is that the circadian clock may have evolved to function even in a non-growing cell.

Temporal evolution of master regulator Crp identifies pyrimidines as catabolite modulator factors

The evolution of microorganisms often involves changes of unclear relevance, such as transient phenotypes and sequential development of multiple adaptive mutations in hotspot genes. Previously, we showed that ageing colonies of an E. coli mutant unable to produce cAMP when grown on maltose, accumulated mutations in the crp gene (encoding a global transcription factor) and in genes involved in pyrimidine metabolism such as cmk; combined mutations in both crp and cmk enabled fermentation of maltose (which usually requires cAMP-mediated Crp activation for catabolic pathway expression). Here, we study the sequential generation of hotspot mutations in those genes, and uncover a regulatory role of pyrimidine nucleosides in carbon catabolism. Cytidine binds to the cytidine regulator CytR, modifies the expression of sigma factor 32 (RpoH), and thereby impacts global gene expression. In addition, cytidine binds and activates a Crp mutant directly, thus modulating catabolic pathway expression, and could be the catabolite modulating factor whose existence was suggested by Jacques Monod and colleagues in 1976. Therefore, transcription factor Crp appears to work in concert with CytR and RpoH, serving a dual role in sensing both carbon availability and metabolic flux towards DNA and RNA. Our findings show how certain alterations in metabolite concentrations (associated with colony ageing and/or due to mutations in metabolic or regulatory genes) can drive the evolution in non-growing cells.

Scan4CFU: Low-cost, open-source bacterial colony tracking over large areas and extended incubation times

A hallmark of bacterial populations cultured in vitro is their homogeneity of growth, where the majority of cells display identical growth rate, cell size and content. Recent insights, however, have revealed that even cells growing in exponential growth phase can be heterogeneous with respect to variables typically used to measure cell growth. Bacterial heterogeneity has important implications for how bacteria respond to environmental stresses, such as antibiotics. The phenomenon of antimicrobial persistence, for example, has been linked to a small subpopulation of cells that have entered into a state of dormancy where antibiotics are no longer effective. While methods have been developed for identifying individual non-growing cells in bacterial cultures, there has been less attention paid to how these cells may influence growth in colonies on a solid surface. In response, we have developed a low-cost, open-source platform to perform automated image capture and image analysis of bacterial colony growth on multiple nutrient agar plates simultaneously. The descriptions of the hardware and software are included, along with details about the temperature-controlled growth chamber, high-resolution scanner, and graphical interface to extract and plot the colony lag time and growth kinetics. Experiments were conducted using a wild type strain of Escherichia coli K12 to demonstrate the feasibility and operation of our setup. By automated tracking of bacterial growth kinetics in colonies, the system holds the potential to reveal new insights into understanding the impact of microbial heterogeneity on antibiotic resistance and persistence.

A Comparative Study of Re-Referencing EEGs With Non-Unipolar to Unipolar Reference by REST

The combination of biological and electrochemical techniques enhances the bioremediation efficiency of treating oil-contaminated water. In this study a non-growing fungal whole cell biocatalyst (BC; Aspergillus brasiliensis attached to perlite) pretreated with an electric field (EF), was used to degrade a hydrocarbon blend (hexadecane-phenanthrene-pyrene; 100:1:1 w/w) in an airlift bioreactor (ALB). During hydrocarbon degradation, all mass transfer resistances (internal and external) and sorption capacity were experimentally quantified. Internal mass transfer resistances were evaluated through BC effectiveness factor analysis as a function of the Thiele modulus (using first order reaction kinetics, assuming a spherical BC, five particle diameters). External (interfacial) mass transfer resistances were evaluated by kLa determination. EF pretreatment during BC production promoted surface changes in BC and production of an emulsifier protein in the ALB. The BC surface modifications enhanced the affinity for hydrocarbons, improving hydrocarbon uptake by direct contact. The resulting emulsion was associated with decreased internal and external mass transfer resistances. EF pretreatment effects can be summarized as: a combined uptake mechanism (direct contact dominant followed by emulsified form dominant) diminishing mass transfer limitations, resulting in a non-specific hydrocarbon degradation in blend. The pretreated BC is a good applicant for oil-contaminated water remediation.

Transcription coupled base excision repair in mammalian cells: So little is known and so much to uncover

Oxidized bases in the genome has been implicated in various human pathologies, including cancer, aging and neurological diseases. Their repair is initiated with excision by DNA glycosylases (DGs) in the base excision repair (BER) pathway. Among the five oxidized base-specific human DGs, OGG1 and NTH1 preferentially excise oxidized purines and pyrimidines, respectively, while NEILs remove both oxidized purines and pyrimidines. However, little is known about why cells possess multiple DGs with overlapping substrate specificities. Studies of the past decades revealed that some DGs are involved in repair of oxidized DNA base lesions in the actively transcribed regions. Preferential removal of lesions from the transcribed strands of active genes, called transcription-coupled repair (TCR), was discovered as a distinct sub-pathway of nucleotide excision repair; however, such repair of oxidized DNA bases had not been established until our recent demonstration of NEIL2’s role in TC-BER of the nuclear genome. We have shown that NEIL2 forms a distinct transcriptionally active, repair proficient complex. More importantly, we for the first time reconstituted TC-BER using purified components. These studies are important for characterizing critical requirement for the process. However, because NEIL2 cannot remove all types of oxidized bases, it is unlikely to be the only DNA glycosylase involved in TC-BER. Hence, we postulate TC-BER process to be universally involved in maintaining the functional integrity of active genes, especially in post-mitotic, non-growing cells. We further postulate that abnormal bases (e.g., uracil), and alkylated and other small DNA base adducts are also repaired via TC-BER. In this review, we have provided an overview of the various aspects of TC-BER in mammalian cells with the hope of generating significant interest of many researchers in the field. Further studies aimed at better understanding the mechanistic aspects of TC-BER could help elucidate the linkage of TC-BER deficiency to various human pathologies.

Sequestration of the exocytic SNARE Psy1 into multiprotein nodes reinforces polarized morphogenesis in fission yeast.

Polarized morphogenesis is achieved by targeting or inhibiting growth in distinct regions. Rod-shaped fission yeast cells grow exclusively at their ends by restricting exocytosis and secretion to these sites. This growth pattern implies the existence of mechanisms that prevent exocytosis and growth along nongrowing cell sides. We previously identified a set of 50–100 megadalton-sized node structures along the sides of fission yeast cells that contained the interacting proteins Skb1 and Slf1. Here, we show that Skb1–Slf1 nodes contain the syntaxin-like soluble N-ethylmaleimide-sensitive factor attachment protein receptor Psy1, which mediates exocytosis in fission yeast. Psy1 localizes in a diffuse pattern at cell tips, where it likely promotes exocytosis and growth, but is sequestered in Skb1–Slf1 nodes at cell sides where growth does not occur. Mutations that prevent node assembly or inhibit Psy1 localization to nodes lead to aberrant exocytosis at cell sides and increased cell width. Genetic results indicate that this Psy1 node mechanism acts in parallel to actin cables and Cdc42 regulation. Our work suggests that sequestration of syntaxin-like Psy1 at nongrowing regions of the cell cortex reinforces cell morphology by restricting exocytosis to proper sites of polarized growth.

Alkyl-Aryl-Vancomycins: Multimodal Glycopeptides with Weak Dependence on the Bacterial Metabolic State.

Resistance to last-resort antibiotics such as vancomycin for Gram-positive bacterial infections necessitates the development of new therapeutics. Furthermore, the ability of bacteria to survive antibiotic therapy through formation of biofilms and persister cells complicates treatment. Toward this, we report alkyl-aryl-vancomycins (AAVs), with high potency against vancomycin-resistant enterococci and staphylococci. Unlike vancomycin, the lead compound AAV-qC10 was bactericidal and weakly dependent on bacterial metabolism. This resulted in complete eradication of non-growing cells of MRSA and disruption of its biofilms. In addition to inhibiting cell wall biosynthesis like vancomycin, AAV-qC10 also depolarizes and permeabilizes the membrane. More importantly, the compound delocalized the cell division protein MinD, thereby impairing bacterial growth through multiple pathways. The potential of AAV-qC10 is exemplified by its superior efficacy against MRSA in a murine thigh infection model as compared to vancomycin. This work paves the way for structural optimization and drug development for combating drug-resistant bacterial infections.