RpoS Plays a Central Role in the SOS Induction by Sub-Lethal Aminoglycoside Concentrations in Vibrio cholerae

Bacteria encounter sub-inhibitory concentrations of antibiotics in various niches, where these low doses play a key role for antibiotic resistance selection. However, the physiological effects of these sub-lethal concentrations and their observed connection to the cellular mechanisms generating genetic diversification are still poorly understood. It is known that, unlike for the model bacterium Escherichia coli, sub-minimal inhibitory concentrations (sub-MIC) of aminoglycosides (AGs) induce the SOS response in Vibrio cholerae. SOS is induced upon DNA damage, and since AGs do not directly target DNA, we addressed two issues in this study: how sub-MIC AGs induce SOS in V. cholerae and why they do not do so in E. coli. We found that when bacteria are grown with tobramycin at a concentration 100-fold below the MIC, intracellular reactive oxygen species strongly increase in V. cholerae but not in E. coli. Using flow cytometry and gfp fusions with the SOS regulated promoter of intIA, we followed AG-dependent SOS induction. Testing the different mutation repair pathways, we found that over-expression of the base excision repair (BER) pathway protein MutY relieved this SOS induction in V. cholerae, suggesting a role for oxidized guanine in AG-mediated indirect DNA damage. As a corollary, we established that a BER pathway deficient E. coli strain induces SOS in response to sub-MIC AGs. We finally demonstrate that the RpoS general stress regulator prevents oxidative stress-mediated DNA damage formation in E. coli. We further show that AG-mediated SOS induction is conserved among the distantly related Gram negative pathogens Klebsiella pneumoniae and Photorhabdus luminescens, suggesting that E. coli is more of an exception than a paradigm for the physiological response to antibiotics sub-MIC.     

Genetic evidence for inhibition of bacterial division protein FtsZ by berberine

Background

Berberine is a plant alkaloid that is widely used as an anti-infective in traditional medicine. Escherichia coli exposed to berberine form filaments, suggesting an antibacterial mechanism that involves inhibition of cell division. Berberine is a DNA ligand and may induce filamentation through induction of the SOS response. Also, there is biochemical evidence for berberine inhibition of the cell division protein FtsZ. Here we aimed to assess possible berberine mechanism(s) of action in growing bacteria using genetics tools.

Methodology/Principal Findings

First, we tested whether berberine inhibits bacterial growth through DNA damage and induction of the SOS response. The SOS response induced by berberine was much lower compared to that induced by mitomycin C in an SOS response reporter strain. Also, cell filamentation was observed in an SOS-negative E. coli strain. To test whether berberine inhibits FtsZ, we assessed its effects on formation of the cell division Z-rings, and observed a dramatic reduction in Z-rings in the presence of berberine. We next used two different strategies for RNA silencing of ftsZ and both resulted in sensitisation of bacteria to berberine, visible as a drop in the Minimum Inhibitory Concentration (MIC). Furthermore, Fractional Inhibitory Concentration Indices (FICIs) showed a high level of synergy between ftsZ silencing and berberine treatment (FICI values of 0.23 and 0.25 for peptide nucleic acid- and expressed antisense RNA-based silencing of ftsZ, respectively). Finally, over-expression of ftsZ led to a mild rescue effect in berberine-treated cells.

Conclusions

The results argue against DNA binding as the primary mechanism of action of berberine and support the hypothesis that its antibacterial properties are due to inhibition of the cell division protein FtsZ. In addition, the genetic approach used here provides a means to rapidly test the activity of other putative FtsZ inhibitors.     

Lifelong cytomegalovirus and early‐LIFE irradiation synergistically potentiate age‐related defects in response to vaccination and infection

While whole‐body irradiation (WBI) can induce some hallmarks of immune aging, (re)activation of persistent microbial infection also occurs following WBI and may contribute to immune effects of WBI over the lifespan. To test this hypothesis in a model relevant to human immune aging, we examined separate and joint effects of lifelong latent murine cytomegalovirus (MCMV) and of early‐life WBI over the course of the lifespan. In late life, we then measured the response to a West Nile virus (WNV) live attenuated vaccine, and lethal WNV challenge subsequent to vaccination. We recently published that a single dose of non‐lethal WBI in youth, on its own, was not sufficient to accelerate aging of the murine immune system, despite widespread DNA damage and repopulation stress in hematopoietic cells. However, 4Gy sub‐lethal WBI caused manifest reactivation of MCMV. Following vaccination and challenge with WNV in the old age, MCMV‐infected animals experiencing 4Gy, but not lower, dose of sub‐lethal WBI in youth had reduced survival. By contrast, old irradiated mice lacking MCMV and MCMV‐infected, but not irradiated, mice were both protected to the same high level as the old non‐irradiated, uninfected controls. Analysis of the quality and quantity of anti‐WNV immunity showed that higher mortality in MCMV‐positive WBI mice correlated with increased levels of MCMV‐specific immune activation during WNV challenge. Moreover, we demonstrate that infection, including that by WNV, led to MCMV reactivation. Our data suggest that MCMV reactivation may be an important determinant of increased late‐life mortality following early‐life irradiation and late‐life acute infection.     

Repair on the Go: E. coli Maintains a High Proliferation Rate while Repairing a Chronic DNA Double-Strand Break

DNA damage checkpoints exist to promote cell survival and the faithful inheritance of genetic information. It is thought that one function of such checkpoints is to ensure that cell division does not occur before DNA damage is repaired. However, in unicellular organisms, rapid cell multiplication confers a powerful selective advantage, leading to a dilemma. Is the activation of a DNA damage checkpoint compatible with rapid cell multiplication? By uncoupling the initiation of DNA replication from cell division, the Escherichia coli cell cycle offers a solution to this dilemma. Here, we show that a DNA double-strand break, which occurs once per replication cycle, induces the SOS response. This SOS induction is needed for cell survival due to a requirement for an elevated level of expression of the RecA protein. Cell division is delayed, leading to an increase in average cell length but with no detectable consequence on mutagenesis and little effect on growth rate and viability. The increase in cell length caused by chronic DNA double-strand break repair comprises three components: two types of increase in the unit cell size, one independent of SfiA and SlmA, the other dependent of the presence of SfiA and the absence of SlmA, and a filamentation component that is dependent on the presence of either SfiA or SlmA. These results imply that chronic checkpoint induction in E. coli is compatible with rapid cell multiplication. Therefore, under conditions of chronic low-level DNA damage, the SOS checkpoint operates seamlessly in a cell cycle where the initiation of DNA replication is uncoupled from cell division.     

Recombinase and translesion DNA polymerase decrease the speed of replication fork progression during the DNA damage response in Escherichia coli cells

The SOS response is a DNA damage response pathway that serves as a general safeguard of genome integrity in bacteria. Extensive studies of the SOS response in Escherichia coli have contributed to establishing the key concepts of cellular responses to DNA damage. However, how the SOS response impacts on the dynamics of DNA replication fork movement remains unknown. We found that inducing the SOS response decreases the mean speed of individual replication forks by 30–50% in E. coli cells, leading to a 20–30% reduction in overall DNA synthesis. dinB and recA belong to a group of genes that are upregulated during the SOS response, and encode the highly conserved proteins DinB (also known as DNA polymerase IV) and RecA, which, respectively, specializes in translesion DNA synthesis and functions as the central recombination protein. Both genes were independently responsible for the SOS-dependent slowdown of replication fork progression. Furthermore, fork speed was reduced when each gene was ectopically expressed in SOS-uninduced cells to the levels at which they are expressed in SOS-induced cells. These results clearly indicate that the increased expression of dinB and recA performs a novel role in restraining the progression of an unperturbed replication fork during the SOS response.     

Inducible SOS response system of DNA repair and mutagenesis in Escherichia coli

Chromosomal DNA is exposed to continuous damage and repair. Cells contain a number of proteins and specific DNA repair systems that help maintain its correct structure. The SOS response was the first DNA repair system described in Escherichia coli induced upon treatment of bacteria with DNA damaging agents arrest DNA replication and cell division. Induction of the SOS response involves more than forty independent SOS genes, most of which encode proteins engaged in protection, repair, replication, mutagenesis and metabolism of DNA. Under normal growth conditions the SOS genes are expressed at a basal level, which increases distinctly upon induction of the SOS response. The SOS-response has been found in many bacterial species (e.g., Salmonella typhimurium, Caulobacter crescentus, Mycobacterium tuberculosis), but not in eukaryotic cells. However, species from all kingdoms contain some SOS-like proteins taking part in DNA repair that exhibit amino acid homology and enzymatic activities related to those found in E. coli. but are not organized in an SOS system. This paper presents a brief up-to-date review describing the discovery of the SOS system, the physiology of SOS induction, methods for its determination, and the role of some SOS-induced genes.     

Role of RecA and the SOS Response in Thymineless Death in Escherichia coli

Thymineless death (TLD) is a classic and enigmatic phenomenon, documented in bacterial, yeast, and human cells, whereby cells lose viability rapidly when deprived of thymine. Despite its being the essential mode of action of important chemotherapeutic agents, and despite having been studied extensively for decades, the basic mechanisms of TLD have remained elusive. In Escherichia coli, several proteins involved in homologous recombination (HR) are required for TLD, however, surprisingly, RecA, the central HR protein and activator of the SOS DNA–damage response was reported not to be. We demonstrate that RecA and the SOS response are required for a substantial fraction of TLD. We show that some of the Rec proteins implicated previously promote TLD via facilitating activation of the SOS response and that, of the roughly 40 proteins upregulated by SOS, SulA, an SOS–inducible inhibitor of cell division, accounts for most or all of how SOS causes TLD. The data imply that much of TLD results from an irreversible cell-cycle checkpoint due to blocked cell division. FISH analyses of the DNA in cells undergoing TLD reveal blocked replication and apparent DNA loss with the region near the replication origin underrepresented initially and the region near the terminus lost later. Models implicating formation of single-strand DNA at blocked replication forks, a SulA-blocked cell cycle, and RecQ/RecJ-catalyzed DNA degradation and HR are discussed. The data predict the importance of DNA damage-response and HR networks to TLD and chemotherapy resistance in humans.     

Construction of a functional network for common DNA damage responses in Escherichia coli

In this study, we aim to identify a common, general mode of toxic action in Escherichia coli when experiencing DNA damage, irrespective of the agents used. We conducted or collected 69 microarray data from seven different DNA damaging agents. In a quantitative manner, we constructed a probable DNA damage stress network, entitled the ‘Functional Linked Network (FLN)’, which consists of 399 significantly perturbed genes and the 1283 interactions among them. The SOS response related genes (LexA modules) were found to be dominantly activated by DNA damage, irrespective of the agents. Several minor, plausible modules were also implicated in this network, and appear to be related with the metabolic inhibition response to DNA damage or mediate the induction of SOS response. This systems and comparison approach across a variety of genotoxic agents may serve as a starting point to specify some of the unknown and common features of DNA damage responses in bacteria.     

Do microplastics impair male dominance interactions in fish? A test of the vector hypothesis

Microplastics (MPs) are widespread in aquatic environments and have become a critical environmental issue in recent years due to their adverse impacts on the physiology, reproduction, and survival of aquatic animals. Exposure to MPs also has the potential to induce sub‐lethal behavioral changes that can affect individual fitness, but these effects are understudied. Many plastic additives introduced during the manufacture of MPs are known endocrine‐disrupting chemicals (EDCs) that mimic the action of natural hormones, alter sexual and competitive behavior, and impair reproductive success in fish. In addition, EDCs and other aquatic contaminants may adhere to MPs in the environment, the latter of which may serve as transport vectors for these compounds (i.e., the vector hypothesis). In this study, we staged territorial contests between control males, and males exposed to virgin MP particles or to MPs previously immersed in one of two environmentally relevant concentrations of 17‐alpha ethinyl estradiol (EE2; 5 ng/L and 25 ng/L) to evaluate the independent and synergistic effects of exposure to MPs and a common environmental estrogen on male–male aggression and competitive territory acquisition in a freshwater fish, Pimephales promelas. Short‐term (30 days) dietary exposure to MPs did not impair the ability of males to successfully compete for and obtain a breeding territory. Overall levels of aggression in control and exposed males were also similar across trial series. These results help to fill a critical knowledge gap regarding the direct and indirect (vector‐borne) effects of MPs on the reproductive behavior of aquatic vertebrates in freshwater systems.     

High‐efficiency delivery of CRISPR‐Cas9 by engineered probiotics enables precise microbiome editing

Antibiotic resistance threatens our ability to treat infectious diseases, spurring interest in alternative antimicrobial technologies. The use of bacterial conjugation to deliver CRISPR‐cas systems programmed to precisely eliminate antibiotic‐resistant bacteria represents a promising approach but requires high in situ DNA transfer rates. We have optimized the transfer efficiency of conjugative plasmid TP114 using accelerated laboratory evolution. We hence generated a potent conjugative delivery vehicle for CRISPR‐cas9 that can eliminate > 99.9% of targeted antibiotic‐resistant Escherichia coli in the mouse gut microbiota using a single dose. We then applied this system to a Citrobacter rodentium infection model, achieving full clearance within four consecutive days of treatment.     

Trimethylamine modulates dauer formation,neurodegeneration, and lifespan through tyra‐3/daf‐11 signaling in Caenorhabditis elegans

In the nematode Caenorhabditis elegans, signals derived from bacteria in the diet, the animal''s major nutrient source, can modulate both behavior and healthspan. Here we describe a dual role for trimethylamine (TMA), a human gut flora metabolite, which acts as a nutrient signal and a neurotoxin. TMA and its associated metabolites are produced by the human gut microbiome and have been suggested to serve as risk biomarkers for diabetes and cardiovascular diseases. We demonstrate that the tyramine receptor TYRA‐3, a conserved G protein‐coupled receptor (GPCR), is required to sense TMA and mediate its responses. TMA activates guanylyl cyclase DAF‐11 signaling through TYRA‐3 in amphid neurons (ASK) and ciliated neurons (BAG) to mediate food‐sensing behavior. Bacterial mutants deficient in TMA production enhance dauer formation, extend lifespan, and are less preferred as a food source. Increased levels of TMA lead to neural damage in models of Parkinson''s disease and shorten lifespan. Our results reveal conserved signaling pathways modulated by TMA in C. elegans that are likely to be relevant for its effects in mammalian systems.     

A multifaceted cellular damage repair and prevention pathway promotes high‐level tolerance to β‐lactam antibiotics

Bactericidal antibiotics are powerful agents due to their ability to convert essential bacterial functions into lethal processes. However, many important bacterial pathogens are remarkably tolerant against bactericidal antibiotics due to inducible damage repair responses. The cell wall damage response two‐component system VxrAB of the gastrointestinal pathogen Vibrio cholerae promotes high‐level β‐lactam tolerance and controls a gene network encoding highly diverse functions, including negative control over multiple iron uptake systems. How this system contributes to tolerance is poorly understood. Here, we show that β‐lactam antibiotics cause an increase in intracellular free iron levels and collateral oxidative damage, which is exacerbated in the ∆vxrAB mutant. Mutating major iron uptake systems dramatically increases ∆vxrAB tolerance to β‐lactams. We propose that VxrAB reduces antibiotic‐induced toxic iron and concomitant metabolic perturbations by downregulating iron uptake transporters and show that iron sequestration enhances tolerance against β‐lactam therapy in a mouse model of cholera infection. Our results suggest that a microorganism''s ability to counteract diverse antibiotic‐induced stresses promotes high‐level antibiotic tolerance and highlights the complex secondary responses elicited by antibiotics.     

Hypophosphorylated pRb knock‐in mice exhibit hallmarks of aging and vitamin C‐preventable diabetes

Despite extensive analysis of pRB phosphorylation in vitro, how this modification influences development and homeostasis in vivo is unclear. Here, we show that homozygous Rb^∆K4 and Rb^∆K7 knock‐in mice, in which either four or all seven phosphorylation sites in the C‐terminal region of pRb, respectively, have been abolished by Ser/Thr‐to‐Ala substitutions, undergo normal embryogenesis and early development, notwithstanding suppressed phosphorylation of additional upstream sites. Whereas Rb^∆K4 mice exhibit telomere attrition but no other abnormalities, Rb^∆K7 mice are smaller and display additional hallmarks of premature aging including infertility, kyphosis, and diabetes, indicating an accumulative effect of blocking pRb phosphorylation. Diabetes in Rb^∆K7 mice is insulin‐sensitive and associated with failure of quiescent pancreatic β‐cells to re‐enter the cell cycle in response to mitogens, resulting in induction of DNA damage response (DDR), senescence‐associated secretory phenotype (SASP), and reduced pancreatic islet mass and circulating insulin level. Pre‐treatment with the epigenetic regulator vitamin C reduces DDR, increases cell cycle re‐entry, improves islet morphology, and attenuates diabetes. These results have direct implications for cell cycle regulation, CDK‐inhibitor therapeutics, diabetes, and longevity.     

Olive phenols preserve lamin B1 expression reducing cGAS/STING/NFκB‐mediated SASP in ionizing radiation‐induced senescence

Senescence occurs upon critical telomere shortening, or following DNA damage, oncogenic activation, hypoxia and oxidative stress, overall referred to stress‐induced premature senescence (SIPS). In response to DNA damage, senescent cells release cytoplasmic chromatin fragments (CCFs), and express an altered secretome, the senescence‐associated secretory phenotype (SASP), which contributes to generate a pro‐inflammatory and pro‐tumoral extracellular milieu. Polyphenols have gained significant attention owing to their anti‐inflammatory and anti‐tumour activities. Here, we studied the effect of oleuropein aglycone (OLE) and hydroxytyrosol (HT) on DNA damage, CCF appearance and SASP in a model of irradiation‐induced senescence. Neonatal human dermal fibroblasts (NHDFs) were γ‐irradiated and incubated with OLE, 5 µM and HT, 1 µM. Cell growth and senescence‐associated (SA)‐β‐Gal‐staining were used as senescence markers. DNA damage was evaluated by Comet assay, lamin B1 expression, release of CCFs, cyclic GMP‐AMP Synthase (cGAS) activation. IL‐6, IL‐8, MCP‐1 and RANTES were measured by ELISA assay. Our results showed that OLE and HT exerted a protective effect on 8 Gy irradiation‐induced senescence, preserving lamin B1 expression and reducing cGAS/STING/NFκB‐mediated SASP. The ability of OLE and HT to mitigate DNA damage, senescence status and the related SASP in normal cells can be exploited to improve the efficacy and safety of cancer radiotherapy.     

Adaptive laboratory evolution of microbial co‐cultures for improved metabolite secretion

Adaptive laboratory evolution has proven highly effective for obtaining microorganisms with enhanced capabilities. Yet, this method is inherently restricted to the traits that are positively linked to cell fitness, such as nutrient utilization. Here, we introduce coevolution of obligatory mutualistic communities for improving secretion of fitness‐costly metabolites through natural selection. In this strategy, metabolic cross‐feeding connects secretion of the target metabolite, despite its cost to the secretor, to the survival and proliferation of the entire community. We thus co‐evolved wild‐type lactic acid bacteria and engineered auxotrophic Saccharomyces cerevisiae in a synthetic growth medium leading to bacterial isolates with enhanced secretion of two B‐group vitamins, viz., riboflavin and folate. The increased production was specific to the targeted vitamin, and evident also in milk, a more complex nutrient environment that naturally contains vitamins. Genomic, proteomic and metabolomic analyses of the evolved lactic acid bacteria, in combination with flux balance analysis, showed altered metabolic regulation towards increased supply of the vitamin precursors. Together, our findings demonstrate how microbial metabolism adapts to mutualistic lifestyle through enhanced metabolite exchange.     

Purification and characterization of the receptor‐binding domain of SARS‐CoV‐2 spike protein from Escherichia coli

SARS‐CoV‐2 is responsible for a disruptive worldwide viral pandemic, and renders a severe respiratory disease known as COVID‐19. Spike protein of SARS‐CoV‐2 mediates viral entry into host cells by binding ACE2 through the receptor‐binding domain (RBD). RBD is an important target for development of virus inhibitors, neutralizing antibodies, and vaccines. RBD expressed in mammalian cells suffers from low expression yield and high cost. E. coli is a popular host for protein expression, which has the advantage of easy scalability with low cost. However, RBD expressed by E. coli (RBD‐1) lacks the glycosylation, and its antigenic epitopes may not be sufficiently exposed. In the present study, RBD‐1 was expressed by E. coli and purified by a Ni Sepharose Fast Flow column. RBD‐1 was structurally characterized and compared with RBD expressed by the HEK293 cells (RBD‐2). The secondary structure and tertiary structure of RBD‐1 were largely maintained without glycosylation. In particular, the major β‐sheet content of RBD‐1 was almost unaltered. RBD‐1 could strongly bind ACE2 with a dissociation constant (K_D) of 2.98 × 10^–8 M. Thus, RBD‐1 was expected to apply in the vaccine development, screening drugs and virus test kit.     

A Constitutively Expressed, Truncated umuDC Operon Regulates the recA-Dependent DNA Damage Induction of a Gene in Acinetobacter baylyi Strain ADP1

In response to environmentally caused DNA damage, SOS genes are up-regulated due to RecA-mediated relief of LexA repression. In Escherichia coli, the SOS umuDC operon is required for DNA damage checkpoint functions and for replicating damaged DNA in the error-prone process called SOS mutagenesis. In the model soil bacterium Acinetobacter baylyi strain ADP1, however, the content, regulation, and function of the umuDC operon are unusual. The umuC gene is incomplete, and a remnant of an ISEhe3-like transposase has replaced the middle 57% of the umuC coding region. The umuD open reading frame is intact, but it is 1.5 times the size of other umuD genes and has an extra 5′ region that lacks homology to known umuD genes. Analysis of a umuD::lacZ fusion showed that umuD was expressed at very high levels in both the absence and presence of mitomycin C and that this expression was not affected in a recA-deficient background. The umuD mutation did not affect the growth rate or survival after UV-induced DNA damage. However, the UmuD-like protein found in ADP1 (UmuDAb) was required for induction of an adjacent DNA damage-inducible gene, ddrR. The umuD mutation specifically reduced the DNA damage induction of the RecA-dependent DNA damage-inducible ddrR locus by 83% (from 12.9-fold to 2.3-fold induction), but it did not affect the 33.9-fold induction of benA, an unrelated benzoate degradation gene. These data suggest that the response of the ADP1 umuDC operon to DNA damage is unusual and that UmuDAb specifically regulates the expression of at least one DNA damage-inducible gene.