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Antimicrobial resistance is a critical public health issue that requires a thorough understanding of the factors that influence the selection and spread of antibiotic-resistant bacteria. Biocides, which are widely used in cleaning and disinfection procedures in a variety of settings, may contribute to this resistance by inducing similar defense mechanisms in bacteria against both biocides and antibiotics. However, the strategies used by bacteria to adapt and develop cross-resistance remain poorly understood, particularly within biofilms -a widespread bacterial habitat that significantly influences bacterial tolerance and adaptive strategies. Using a combination of adaptive laboratory evolution experiments, genomic and RT-qPCR analyses, and biofilm structural characterization using confocal microscopy, we investigated in this study how Escherichia coli biofilms adapted after 28 days of exposure to three biocidal active substances and the effects on cross-resistance to antibiotics. Interestingly, polyhexamethylene biguanide (PHMB) exposure led to an increase of gentamicin resistance (GenR) phenotypes in biofilms formed by most of the seven E. coli strains tested. Nevertheless, most variants that emerged under biocidal conditions did not retain the GenR phenotype after removal of antimicrobial stress, suggesting a transient adaptation (adaptive resistance). The whole genome sequencing of variants with stable GenR phenotypes revealed recurrent mutations in genes associated with cellular respiration, including cytochrome oxidase ( cydA , cyoC ) and ATP synthase ( atpG ). RT-qPCR analysis revealed an induction of gene expression associated with biofilm matrix production (especially curli synthesis), stress responses, active and passive transport and cell respiration during PHMB exposure, providing insight into potential physiological responses associated with adaptive crossresistance. In addition, confocal laser scanning microscopy (CLSM) observations demonstrated a global effect of PHMB on biofilm architectures and compositions formed by most E. coli strains, with the appearance of dense cellular clusters after a 24h-exposure. In conclusion, our results showed that the PHMB exposure stimulated the emergence of an adaptive cross-resistance to gentamicin in biofilms, likely induced through the activation of physiological responses and biofilm structural modulations altering gradients and microenvironmental conditions in the biological edifice., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Charron, Lemée, Huguet, Minlong, Boulanger, Houée, Soumet, Briandet and Bridier.)

Cytochrome bd oxidase is a bacterial terminal oxygen reductase that was suggested to enable adaptation to different environments and to confer resistance to stress conditions. An electrocatalytic study of the cyt bd oxidases from Escherichia coli, Corynebacterium glutamicum and Geobacillus thermodenitrificans gives evidence for a different reactivity towards oxygen. An inversion of the redox potential values of the three hemes is found when comparing the enzymes from different bacteria. This inversion can be correlated with different protonated glutamic acids as evidenced by reaction induced FTIR spectroscopy. The influence of the microenvironment of the hemes on the reactivity towards oxygen is discussed., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Understanding and predicting how microbes respond to environmental fluctuations is a central challenge in present-day microbiology. Erickson et al. have proposed a quantitative and (kinetic) parameters-free model of Escherichia coli growth that successfully anticipates changes in gene expression and biomass accumulation in response to nutrients up- and down-shifts., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Understanding the interplay between genotype and fitness is a core question in evolutionary biology. Here, we address this challenge in the context of microbial adaptation to environmental stressors. This study explores the role of epistasis in bacterial adaptation by examining genetic and phenotypic changes in silver-adapted Escherichia coli populations, focusing on the role of beneficial mutations in two-component response systems (TCRS). To do this, we measured 24-hour growth assays and conducted whole-genome DNA and RNA sequencing on E. coli mutants that confer resistance to ionic silver. We showed recently that the R15L cusS mutation is central to silver resistance, primarily through upregulation of the cus efflux system. However, here we show that this mutation's effectiveness is significantly enhanced by epistatic interactions with additional mutations in regulatory genes such as ompR, rho, and fur. These interactions reconfigure global stress response networks, resulting in robust and varied resistance strategies across different populations. This study underscores the critical role of epistasis in bacterial adaptation, illustrating how interactions between multiple mutations and how genetic backgrounds shape the resistance phenotypes of E. coli populations. This work also allowed for refinement of our model describing the role TCRS genes play in bacterial adaptation by now emphasizing that adaptation to environmental stressors is a complex, context-dependent process, driven by the dynamic interplay between genetic and environmental factors. These findings have broader implications for understanding microbial evolution and developing strategies to combat antimicrobial resistance. [ABSTRACT FROM AUTHOR]

L-rhamnose, a naturally abundant sugar, plays diverse biological roles in bacteria, influencing biofilm formation and pathogenesis. This study investigates the global impact of L-rhamnose on the transcriptome and biofilm formation of PHL628 E. coli under various experimental conditions. We compared growth in planktonic and biofilm states in rich (LB) and minimal (M9) media at 28 °C and 37 °C, with varying concentrations of L-rhamnose or D-glucose as a control. Our results reveal that L-rhamnose significantly affects growth kinetics and biofilm formation, particularly reducing biofilm growth in rich media at 37 °C. Transcriptomic analysis through RNA-seq showed that L-rhamnose modulates gene expression differently depending on the temperature and media conditions, promoting a planktonic state by upregulating genes involved in rhamnose transport and metabolism and downregulating genes related to adhesion and biofilm formation. These findings highlight the nuanced role of L-rhamnose in bacterial adaptation and survival, providing insight into potential applications in controlling biofilm-associated infections and industrial biofilm management. [ABSTRACT FROM AUTHOR]

Antimicrobial resistance (AMR) is a significant global health threat, with multidrug-resistant (MDR) bacterial clones becoming a major concern. Polymyxins, especially colistin, have reemerged as last-resort treatments for MDR Gram-negative infections. However, colistin use in livestock has spread mobile colistin resistance (mcr) genes, notably mcr-1, impacting human health. In consequence, its livestock use was banned in 2017, originating a natural experiment to study bacterial adaptation. The aim of this work was to analyse the changes in the mcr-1 genetic background after colistin restriction across the world. This study analyses 3163 Escherichia coli genomes with the mcr-1 gene from human and livestock hosts, mainly from Asia (n = 2621) and Europe (n = 359). Genetic characterisation identifies IncI2 (40.4%), IncX4 (26.7%), and multidrug-resistant IncHI2 (18.8%) as the most common plasmids carrying mcr-1. There were differences in plasmids between continents, with IncX4 (56.6%) being the most common in Europe, while IncI2 (44.8%) was predominant in Asia. Promoter variants related to reduced fitness costs and ISApl1 showed a distinct pattern of association that appears to be associated with adaptation to colistin restriction, which differed between continents. Thus, after the colistin ban, Europe saw a shift to specialised mcr-1 plasmids as IncX4, while ISApl1 decreased in Asia due to changes in the prevalence of the distinct promoter variants. These analyses illustrate the evolution of mcr-1 adaptation following colistin use restrictions and the need for region-specific strategies against AMR following colistin restrictions. [ABSTRACT FROM AUTHOR]

In this investigation, we assessed the antibacterial properties of menthol using Escherichia coli strains, including a tetracycline-resistant (BN407) and a non-resistant reference (K12) strains. The disc-diffusion assay indicated remarkable antibacterial activity of menthol, warranting further exploration. The minimum inhibitory concentration (MIC) for menthol was determined to be 500 µg/ml for both strains. Despite identical MIC values, menthol exhibited different effects on the colony-forming units (CFUs) of the strains, reducing CFUs by 55% in E. coli K12 and 40% in E. coli BN407. Growth kinetics studies revealed that menthol extended the Lag phase by 50% and decreased the specific growth rate and mean generation time by nearly 50% for both strains. These findings illustrate menthol's significant impact on bacterial replication and adaptation processes. Additionally, menthol disrupted membrane-associated properties, as evidenced by reduced H+-flux through bacterial membranes, affecting both N,N-Dicyclohexylcarbodiimide (DCCD)-sensitive and non-sensitive proton flux rates. This indicates that menthol compromises the proton motive force critical for ATP synthesis and nutrient transport. In summary, menthol demonstrates potent antibacterial activity, influencing bacterial growth and survival. This activity is supposed to be due to the influence on membrane functionality. These effects are consistent across both tetracycline-resistant and non-resistant E. coli strains. [ABSTRACT FROM AUTHOR]

The airborne transmission of bacterial pathogens poses a significant challenge to public health, especially with the emergence of antibiotic-resistant strains. This study investigated environmental factors influencing the survival of airborne bacteria, focusing on the effects of different carbon dioxide (CO2) and dust concentrations. The experiments were conducted in an atmospheric simulation chamber using the non-resistant wild-type E. coli K12 (JM109) and a multi-resistant variant (JM109-pEC958). Different CO2 (100 ppm, 800 ppm, 3000 ppm) and dust concentrations (250 µg m−3, 500 µg m−3, 2000 µg m−3) were tested to encompass a wide range of CO2 and dust levels. The results revealed that JM109-pEC958 exhibited greater resilience to high CO2 and dust concentrations compared to its non-resistant counterpart. At 3000 ppm CO2, the survival rate of JM109 was significantly reduced, while the survival rate of JM109-pEC958 remained unaffected. At the dust concentration of 250 µg m−3, JM109 exhibited significantly reduced survival, whereas JM109-pEC958 did not. When the dust concentration was increased to 500 and 2000 µg m−3, even the JM109-pEC958 experienced substantially reduced survival rates, which were still significantly higher than those of its non-resistant counterpart at these concentrations. These findings suggest that multi-resistant E. coli strains possess mechanisms enabling them to endure extreme environmental conditions better than non-resistant strains, potentially involving regulatory genes or efflux pumps. The study underscores the importance of understanding bacterial adaptation strategies to develop effective mitigation approaches against antibiotic-resistant bacteria in atmospheric environments. Overall, this study provides valuable insights into the interplay between environmental stressors and bacterial survival, serving as a foundational step towards elucidating the adaptation mechanisms of multi-resistant bacteria and informing strategies for combating antibiotic resistance in the atmosphere. [ABSTRACT FROM AUTHOR]

Bacteria need to adjust their metabolism and protein synthesis simultaneously to adapt to changing nutrient conditions. It's still a grand challenge to predict how cells coordinate such adaptation due to the cross-regulation between the metabolic fluxes and the protein synthesis. Here we developed a dynamic Constrained Allocation Flux Balance Analysis method (dCAFBA), which integrates flux-controlled proteome allocation and protein limited flux balance analysis. This framework can predict the redistribution dynamics of metabolic fluxes without requiring detailed enzyme parameters. We reveal that during nutrient up-shifts, the calculated metabolic fluxes change in agreement with experimental measurements of enzyme protein dynamics. During nutrient down-shifts, we uncover a switch of metabolic bottleneck from carbon uptake proteins to metabolic enzymes, which disrupts the coordination between metabolic flux and their enzyme abundance. Our method provides a quantitative framework to investigate cellular metabolism under varying environments and reveals insights into bacterial adaptation strategies. • A dynamic model integrating protein allocation with flux balance analysis. • Model can predict transition kinetics of metabolic fluxes without enzymatic details. • Enzyme protein dynamics determine metabolic fluxes kinetics during nutrient upshift. • Switch of metabolic bottleneck during nutrient downshift. [ABSTRACT FROM AUTHOR]

Background CpxR is a critical regulator in bacterial adaptation to various harmful stresses, and is known to regulate bacterial resistance to commonly used antibiotics, such as aminoglycosides, β-lactams and polypeptides. However, the detailed study of functional residues of CpxR remains insufficient. Objectives To investigate the contribution of Lys219 to CpxR's function in regulating antibiotic resistance of Escherichia coli. Methods We performed sequence alignment and conservative analysis of the CpxR protein and constructed mutant strains. We then performed electrophoretic mobility shift assay, real-time quantitative PCR assay, determination of reactive oxygen species (ROS) levels, molecular dynamics simulation, conformational analysis and circular dichroism. Results All mutant proteins (K219Q, K219A and K219R) lost the cpxP DNA-binding ability. Additionally, the three complemented strains eK219A , eK219Q , and eK219R exhibited lower resistance to copper toxicity and alkaline pH toxicity than eWT. Molecular dynamics analysis revealed that mutation of Lys219 leads to looser and more unstable conformation of CpxR, leading to its decreased binding affinity with downstream genes. Moreover, the Lys219 mutation resulted in the down-regulation of efflux pump genes (acrD , tolC , mdtB and mdtA), leading to the accumulation of antibiotics inside the cells and an increase in ROS production, which significantly reduces antibiotic resistance. Conclusions The mutation of the key residue Lys219 causes a conformational change that results in the loss of regulatory ability of CpxR, which may potentially reduce to antibiotic resistance. Therefore, this study suggests that targeting the highly conserved sequence of CpxR could be a promising strategy for the development of new antibacterial drugs. [ABSTRACT FROM AUTHOR]

Phenotypic susceptibility testing of Escherichia (E.) coli is an essential tool to gain a better understanding of the potential impact of biocide selection pressure on antimicrobial resistance. We, therefore, determined the biocide and antimicrobial susceptibility of 216 extended-spectrum β-lactamase-producing (ESBL) and 177 non-ESBL E. coli isolated from swine feces, pork meat, voluntary donors and inpatients and evaluated associations between their susceptibilities. Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of benzalkonium chloride, chlorhexidine digluconate (CHG), chlorocresol (PCMC), glutaraldehyde (GDA), isopropanol (IPA), octenidine dihydrochloride and sodium hypochlorite (NaOCl) showed unimodal distributions, indicating the absence of bacterial adaptation to biocides due to the acquisition of resistance mechanisms. Although MIC95 and MBC95 did not vary more than one doubling dilution step between isolates of porcine and human origin, significant differences in MIC and/or MBC distributions were identified for GDA, CHG, IPA, PCMC and NaOCl. Comparing non-ESBL and ESBL E. coli, significantly different MIC and/or MBC distributions were found for PCMC, CHG and GDA. Antimicrobial susceptibility testing revealed the highest frequency of resistant E. coli in the subpopulation isolated from inpatients. We observed significant but weakly positive correlations between biocide MICs and/or MBCs and antimicrobial MICs. In summary, our data indicate a rather moderate effect of biocide use on the susceptibility of E. coli to biocides and antimicrobials. [ABSTRACT FROM AUTHOR]

Simple Summary: When pathogenic bacteria contaminate food, they can harm humans, causing, for example, gastrointestinal disorders. This is a main concern, especially in the case of foods that are consumed fresh and/or are not cooked following common hygiene standards. Thus, it is important to understand how bacteria can survive and prosper once they have contaminated fruits and vegetables, regardless of the presence of defense mechanisms in these plant tissues. In this work, we investigated the ability of a harmless E. coli strain's ability to adapt to a tomato's external portion, where bacteria encounter harsh growth conditions. E. coli grown in tomatoes was isolated and compared with E. coli grown in standard laboratory conditions: this comparison allowed us to identify potential molecular determinants helping E. coli to survive in tomatoes. This investigation was conducted through sequencing of DNA and through assays proving that growth in tomatoes altered, at least in part, some features of this bacterium, such as its ability to withstand chemical compounds. Food contamination can be a serious concern for public health because it can be related to the severe spreading of pathogens. This is a main issue, especially in the case of fresh fruits and vegetables; indeed, they have often been associated with gastrointestinal outbreak events, due to contamination with pathogenic bacteria. However, little is known about the physiological adaptation and bacterial response to stresses encountered in the host plant. Thus, this work aimed to investigate the adaptation of a commensal E. coli strain while growing in tomato pericarp. Pre-adapted and non-adapted cells were compared and used to contaminate tomatoes, demonstrating that pre-adaptation boosted cell proliferation. DNA extracted from pre-adapted and non-adapted cells was sequenced, and their methylation profiles were compared. Hence, genes involved in cell adhesion and resistance against toxic compounds were identified as genes involved in adaptation, and their expression was compared in these two experimental conditions. Finally, pre-adapted and non-adapted E. coli were tested for their ability to resist the presence of toxic compounds, demonstrating that adaptation exerted a protective effect. In conclusion, this work provides new information about the physiological adaptation of bacteria colonizing the tomato fruit pericarp. [ABSTRACT FROM AUTHOR]

Pathogenic bacteria possess a remarkable ability to adapt to fluctuating host environments and cause infection. Disturbing bacterial central metabolism through inhibition of 1-deoxy-d-xylulose 5-phosphate synthase (DXPS) has the potential to hinder bacterial adaptation, representing a new antibacterial strategy. DXPS functions at a critical metabolic branchpoint to produce the metabolite DXP, a precursor to pyridoxal-5-phosphate (PLP), thiamin diphosphate (ThDP) and isoprenoids presumed essential for metabolic adaptation in nutrient-limited host environments. However, specific roles of DXPS in bacterial adaptations that rely on vitamins or isoprenoids have not been studied. Here we investigate DXPS function in an adaptation of uropathogenic E. coli (UPEC) to d-serine (d-Ser), a bacteriostatic host metabolite that is present at high concentrations in the urinary tract. UPEC adapt to d-Ser by producing a PLP-dependent deaminase, DsdA, that converts d-Ser to pyruvate, pointing to a role for DXPS-dependent PLP synthesis in this adaptation. Using a DXPS-selective probe, butyl acetylphosphonate (BAP), and leveraging the toxic effects of d-Ser, we reveal a link between DXPS activity and d-Ser catabolism. We find that UPEC are sensitized to d-Ser and produce sustained higher levels of DsdA to catabolize d-Ser in the presence of BAP. In addition, BAP activity in the presence of d-Ser is suppressed by β-alanine, the product of aspartate decarboxylase PanD targeted by d-Ser. This BAP-dependent sensitivity to d-Ser marks a metabolic vulnerability that can be exploited to design combination therapies. As a starting point, we show that combining inhibitors of DXPS and CoA biosynthesis displays synergy against UPEC grown in urine where there is increased dependence on the TCA cycle and gluconeogenesis from amino acids. Thus, this study provides the first evidence for a DXPS-dependent metabolic adaptation in a bacterial pathogen and demonstrates how this might be leveraged for development of antibacterial strategies against clinically relevant pathogens. [ABSTRACT FROM AUTHOR]

Silver‐based antibacterial nanoparticles have demonstrated indices of possible bacterial resistance after consecutive periods of time, limiting their application on antibacterial surfaces. In this work, we fabricate electrospun composites of polyethersulfone (PES) and silver nanoparticles (AgNPs), displaying highly‐persistent antibacterial behavior after 10 bacterial expositions. The herein‐reported membranes were synthesized by changing the Ag loading (0.05, 0.5, and 5 wt%), adding polyvinylpyrrolidone as a dispersion promoter, and the resulting composites were evaluated after a different number of bacterial expositions against Escherichia coli (E. coli) aiming to determine their bacterial tolerance. SEM micrographs revealed that the PES fiber diameters decreased as a function of the silver content from 798 to 398 nm, attributed to the conductivity and the viscosity improvement by AgNPs introduction in the polymer solution during the electrospun process. Moreover, the surface roughness (determined by atomic force microscopy) and the contact angle changed with the Ag content, increasing the hydrophilic behavior and promoting a better interaction with the bacteria suspended in the solution. Antibacterial activity kinetics of PES/AgNPs fibers showed 75% bacterial inhibition against E. coli in the composite with 0.5 wt% Ag after 3 h of exposition, and 75% after 1.5 h in composites with 5 wt% of Ag. The ca. 25% survival bacteria were used for consecutive bacterial tolerance assays, where the antibacterial behavior was constant after 10 expositions. Interestingly, electrochemical tests confirmed that PES fibers could control the silver‐releasing process. Thus, PES/AgNPs composites avoid the bacterial adaptation process, which makes them suitable as functional antimicrobial nanomaterials. [ABSTRACT FROM AUTHOR]

Experimental evolution studies have shown that weak antibiotic selective pressures (i.e. when the antibiotic concentrations are far below the minimum inhibitory concentration, MIC) can select resistant mutants, raising several unanswered questions. First, what are the lowest antibiotic concentrations at which selection for de novo resistance mutations can occur? Second, with weak antibiotic selections, which other types of adaptive mutations unrelated to the antibiotic selective pressure are concurrently enriched? Third, are the mutations selected under laboratory settings at subMIC also observed in clinical isolates? We addressed these questions using Escherichia coli populations evolving at subMICs in the presence of either of four clinically used antibiotics: fosfomycin, nitrofurantoin, tetracycline, and ciprofloxacin. Antibiotic resistance evolution was investigated at concentrations ranging from 1/4th to 1/2000th of the MIC of the susceptible strain (MICsusceptible). Our results show that evolution was rapid across all the antibiotics tested, and selection for fosfomycin- and nitrofurantoin-resistant mutants was observed at a concentration as low as 1/2000th of MICsusceptible. Several of the evolved resistant mutants showed increased growth yield and exponential growth rates, and outcompeted the susceptible ancestral strain in the absence of antibiotics as well, suggesting that adaptation to the growth environment occurred in parallel with the selection for resistance. Genomic analysis of the resistant mutants showed that several of the mutations selected under these conditions are also found in clinical isolates, demonstrating that experimental evolution at very low antibiotic levels can help in identifying novel mutations that contribute to bacterial adaptation during subMIC exposure in real-life settings. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) are critical elements of cell–cell communication. Here, we characterized the outer membrane vesicles (OMVs) released by specific clones of Escherichia coli isolated from the Long-Term Evolution Experiment after 50,000 generations (50K) of adaptation to glucose minimal medium. Compared with their ancestor, the evolved clones produce small OMVs but also larger ones which display variable amounts of both OmpA and LPS. Tracking ancestral, fluorescently labelled OMVs revealed that they fuse with both ancestral- and 50K-evolved cells, albeit in different proportions. We quantified that less than 2% of the cells from one 50K-evolved clone acquired the fluorescence delivered by OMVs from the ancestral strain but that one cell concomitantly fuses with several OMVs. Globally, our results showed that OMV production in E. coli is a phenotype that varies along bacterial evolution and question the contribution of OMVs-mediated interactions in bacterial adaptation. [ABSTRACT FROM AUTHOR]

Enteric bacteria have to adapt to environmental stresses in the human gastrointestinal tract such as acid and nutrient stress, oxygen limitation and exposure to antibiotics. Membrane lipid composition has recently emerged as a key factor for stress adaptation. The E. coli ravA-viaA operon is essential for aminoglycoside bactericidal activity under anaerobiosis but its mechanism of action is unclear. Here we characterise the VWA domain-protein ViaA and its interaction with the AAA+ ATPase RavA, and find that both proteins localise at the inner cell membrane. We demonstrate that RavA and ViaA target specific phospholipids and subsequently identify their lipid-binding sites. We further show that mutations abolishing interaction with lipids restore induced changes in cell membrane morphology and lipid composition. Finally we reveal that these mutations render E. coli gentamicin-resistant under fumarate respiration conditions. Our work thus uncovers a ravA-viaA-based pathway which is mobilised in response to aminoglycosides under anaerobiosis and engaged in cell membrane regulation. Membrane lipid composition is a key factor for bacterial stress adaptation. Here, authors show that ViaA and AAA+ ATPase RavA, alter lipid composition and modulate E. coli aminoglycoside sensitivity. [ABSTRACT FROM AUTHOR]

Many bacteria possess all the machineries required to grow on fatty acids (FA) as a unique source of carbon and energy. FA degradation proceeds through the β-oxidation cycle that produces acetyl-CoA and reduced NADH and FADH cofactors. In addition to all the enzymes required for β-oxidation, FA degradation also depends on sophisticated systems for its genetic regulation and for FA transport. The fact that these machineries are conserved in bacteria suggests a crucial role in environmental conditions, especially for enterobacteria. Bacteria also possess specific enzymes required for the degradation of FAs from their environment, again showing the importance of this metabolism for bacterial adaptation. In this review, we mainly describe FA degradation in the Escherichia coli model, and along the way, we highlight and discuss important aspects of this metabolism that are still unclear. We do not detail exhaustively the diversity of the machineries found in other bacteria, but we mention them if they bring additional information or enlightenment on specific aspects. [ABSTRACT FROM AUTHOR]

Magic Spot Nucleotides (MSN) regulate the stringent response, a highly conserved bacterial stress adaptation mechanism, enabling survival under adverse external challenges. In times of antibiotic crisis, a detailed understanding of stringent response is essential, as potentially new targets for pharmacological intervention could be identified. In this study, we delineate the MSN interactome in Escherichia coli and Salmonella typhimurium applying a family of trifunctional photoaffinity capture compounds. We introduce MSN probes covering a diverse phosphorylation pattern, such as pppGpp, ppGpp, and pGpp. Our chemical proteomics approach provides datasets of putative MSN receptors both from cytosolic and membrane fractions that unveil new MSN targets. We find that the activity of the non‐Nudix hydrolase ApaH is potently inhibited by pppGpp, which itself is converted to pGpp by ApaH. The capture compounds described herein will be useful to identify MSN interactomes across bacterial species. [ABSTRACT FROM AUTHOR]

Characterization of bacteriophages facilitates better understanding of their biology, host specificity, genomic diversity, and adaptation to their bacterial hosts. This, in turn, is important for the exploitation of phages for therapeutic purposes, as the use of uncharacterized phages may lead to treatment failure. The present study describes the isolation and characterization of a bacteriophage effective against the important clinical pathogen Escherichia coli, which shows increasing accumulation of antibiotic resistance. Phage fEg-Eco19, which is specific for a clinical E. coli strain, was isolated from an Egyptian sewage sample. Phage fEg-Eco19 formed clear, sharp-edged, round plaques. Electron microscopy showed that the isolated phage is tailed and therefore belongs to the order Caudovirales, and morphologically, it resembles siphoviruses. The diameter of the icosahedral head of fEg-Eco19 is 68 ± 2 nm, and the non-contractile tail length and diameter are 118 ± 0.2 and 13 ± 0.6 nm, respectively. The host range of the phage was found to be narrow, as it infected only two out of 137 clinical E. coli strains tested. The phage genome is 45,805 bp in length with a GC content of 50.3% and contains 76 predicted genes. Comparison of predicted and experimental restriction digestion patterns allowed rough mapping of the physical ends of the phage genome, which was confirmed using the PhageTerm tool. Annotation of the predicted genes revealed gene products belonging to several functional groups, including regulatory proteins, DNA packaging and phage structural proteins, host lysis proteins, and proteins involved in DNA/RNA metabolism and replication. [ABSTRACT FROM AUTHOR]

Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) are standard indexes for determining disinfection effectiveness. Nevertheless, they are static values disregarding the kinetics at sub-MIC concentrations where adaptation, growth, stationary, and death phases can be observed. The understanding of these dynamic mechanisms is crucial to designing effective disinfection strategies. In this study, we studied the 48 h kinetics of Bacillus cereus and Escherichia coli cells exposed to sub-MIC concentrations of didecyldimethylammonium chloride (DDAC). Two mathematical models were employed to reproduce the experiments: the only-growth classical logistic model and a mechanistic model including growth and death dynamics. Although both models reproduce the lag, exponential and stationary phases, only the mechanistic model is able to reproduce the death phase and reveals the concentration dependence of the bactericidal/bacteriostatic activity of DDAC. This model could potentially be extended to study other antimicrobials and reproduce changes in optical density (OD) and colony-forming units (CFUs) with the same parameters and mechanisms of action. [ABSTRACT FROM AUTHOR]

Bacterial porin-encoding genes are often found under positive selection. Local recombination has also been identified in a few of them to facilitate bacterial rapid adaptation, although it remains unknown whether it is a common evolutionary mechanism for the porins or outer membrane proteins in Gram-negative bacteria. In this study, we investigated the beta-barrel (β-barrel) porin-encoding genes in Escherichia coli that were reported under positive Darwinian selection. Besides fhuA that was found with ingenic local recombination previously, we identified four other genes, i.e., lamB, ompA, ompC, and ompF, all showing the similar mosaic evolution patterns. Comparative analysis of the protein sequences disclosed a list of highly variable regions in each family, which are mostly located in the convex of extracellular loops and coinciding with the binding sites of bacteriophages. For each of the porin families, mosaic recombination leads to unique combinations of the variable regions with different sequence patterns, generating diverse protein groups. Structural modeling indicated a conserved global topology among the different porins, with the extracellular surface varying a lot due to individual or combinatorial variable regions. The conservation of global tertiary structure would ensure the channel activity, while the wide diversity of variable regions may represent selection to avoid the invasion of phages, antibiotics or immune surveillance factors. Our study identified multiple bacterial porin genes with mosaic evolution. We hypothesize that this could be generalized strategy for outer membrane proteins to both maintain normal life processes and evade the attack of unfavored factors rapidly. [ABSTRACT FROM AUTHOR]

The temporal change of phenotypes during the adaptive process remains largely unexplored, as do the genetic changes that affect these phenotypic changes. Here we focused on three mutations that rose to high frequency in the early stages of adaptation within 12 Escherichia coli populations subjected to thermal stress (42 °C). All the mutations were in the rpoB gene, which encodes the RNA polymerase beta subunit. For each mutation, we measured the growth curves and gene expression (mRNAseq) of clones at 42 °C. We also compared growth and gene expression with their ancestor under unstressed (37 °C) and stressed conditions (42 °C). Each of the three mutations changed the expression of hundreds of genes and conferred large fitness advantages, apparently through the restoration of global gene expression from the stressed toward the prestressed state. These three mutations had a similar effect on gene expression as another single mutation in a distinct domain of the rpoB protein. Finally, we compared the phenotypic characteristics of one mutant, I572L, with two high-temperature adapted clones that have this mutation plus additional background mutations. The background mutations increased fitness, but they did not substantially change gene expression. We conclude that early mutations in a global transcriptional regulator cause extensive changes in gene expression, many of which are likely under positive selection for their effect in restoring the prestress physiology.

Glutaraldehyde is a widely used biocide on the market for about 50 years. Despite its broad application, several reports on the emergence of bacterial resistance, and occasional outbreaks caused by poorly disinfection, there is a gap of knowledge on the bacterial adaptation, tolerance, and resistance mechanisms to glutaraldehyde. Here, we analyze the effects of the independent selection of mutations in the transcriptional regulator yqhC for biological replicates of Escherichia coli cells subjected to adaptive laboratory evolution (ALE) in the presence of glutaraldehyde. The evolved strains showed improved survival in the biocide (11–26% increase in fitness) as a result of mutations in the activator yqhC , which led to the overexpression of the yqhD aldehyde reductase gene by 8 to over 30-fold (3.1–5.2 log2FC range). The protective effect was exclusive to yqhD as other aldehyde reductase genes of E. coli , such as yahK , ybbO , yghA , and ahr did not offer protection against the biocide. We describe a novel mechanism of tolerance to glutaraldehyde based on the activation of the aldehyde reductase YqhD by YqhC and bring attention to the potential for the selection of such tolerance mechanism outside the laboratory, given the existence of YqhD homologs in various pathogenic and opportunistic bacterial species. [ABSTRACT FROM AUTHOR]

Urinary tract infections (UTIs) are a common bacterial infectious disease in humans, and strains of uropathogenic Escherichia coli (UPEC) are the most frequent cause of UTIs. During infection, UPEC must cope with a variety of stressful conditions in the urinary tract. Here, we demonstrate that the small RNA (sRNA) RyfA of UPEC strains is required for resistance to oxidative and osmotic stresses. Transcriptomic analysis of the ryfA mutant showed changes in expression of genes associated with general stress responses, metabolism, biofilm formation and genes coding for cell surface proteins. Inactivation of ryfA in UPEC strain CFT073 decreased urinary tract colonization in mice and the ryfA mutant also had reduced production of type 1 and P fimbriae (pili), adhesins which are known to be important for UTI. Furthermore, loss of ryfA also reduced UPEC survival in human macrophages. Thus, ryfA plays a key regulatory role in UPEC adaptation to stress, which contributes to UTI and survival in macrophages. Author summary: Small regulatory RNAs play a critical role in bacterial adaptation, physiology, and metabolism. We investigated the role of the small RNA ryfA in uropathogenic E. coli, by using genetic, molecular and cell culture approaches, and mouse infection models. We demon-strate that RyfA is important for resistance to oxidative and osmotic stresses and affects the global transcription of multiple responses to stress. Deletion of ryfA also reduced production of type 1 and P fimbriae (pili) and increased swimming motility. RyfA also plays an important role in urinary tract infection (UTI) in the mouse model and for survival in human primary macrophages and cell lines. Elucidating a role for RyfA in adaptation to stress response and virulence provides insight into new regulatory mechanisms important for virulence in pathogenic E. coli and likely other Enterobacteria that may lead to development of targeted therapeutic or prophylactic approaches against these pathogens. [ABSTRACT FROM AUTHOR]

With rising antibiotic resistance, alternative treatments for communicable diseases are increasingly relevant. One possible alternative for some types of infections is honey, used in wound care since before 2000 BCE and more recently in licensed, medical‐grade products. However, it is unclear whether medical application of honey results in the evolution of bacterial honey resistance and whether this has collateral effects on other bacterial traits such as antibiotic resistance. Here, we used single‐step screening assays and serial transfer at increasing concentrations to isolate honey‐resistant mutants of Escherichia coli. We only detected bacteria with consistently increased resistance to the honey they evolved in for two of the four tested honey products, and the observed increases were small (maximum twofold increase in IC90). Genomic sequencing and experiments with single‐gene knockouts showed a key mechanism by which bacteria increased their honey resistance was by mutating genes involved in detoxifying methylglyoxal, which contributes to the antibacterial activity of Leptospermum honeys. Crucially, we found no evidence that honey adaptation conferred cross‐resistance or collateral sensitivity against nine antibiotics from six different classes. These results reveal constraints on bacterial adaptation to different types of honey, improving our ability to predict downstream consequences of wider honey application in medicine. [ABSTRACT FROM AUTHOR]

Many motile bacteria are propelled by the rotation of flagellar filaments. This rotation is driven by a membrane protein known as the stator-complex, which drives the rotor of the bacterial flagellar motor. Torque generation is powered in most cases by proton transit through membrane protein complexes known as stators, with the next most common ionic power source being sodium. Sodium-powered stators can be studied through the use of synthetic chimeric stators that combine parts of sodium- and proton-powered stator proteins. The most well studied example is the use of the sodium-powered PomA-PotB chimeric stator unit in the naturally proton-powered Escherichia coli. Here we designed a fluidics system at low cost for rapid prototyping to separate motile and non-motile populations of bacteria while varying the ionic composition of the media and thus the sodium-motive force available to drive this chimeric flagellar motor. We measured separation efficiencies at varying ionic concentrations and confirmed using fluorescence that our device delivered eightfold enrichment of the motile proportion of a mixed population. Furthermore, our results showed that we could select bacteria from reservoirs where sodium was not initially present. Overall, this technique can be used to implement the selection of highly motile fractions from mixed liquid cultures, with applications in directed evolution to investigate the adaptation of motility in bacterial ecosystems. [ABSTRACT FROM AUTHOR]

Acquisition of new prophages that are able to increase the bacterial fitness by the lysogenic conversion is believed to be an important strategy of bacterial adaptation to the changing environment. However, in contrast to the factors determining the range of bacteriophage lytic activity, little is known about the factors that define the lysogenization host range. Bacteriophage phi24B is the paradigmal model of Stx-converting phages, encoding the toxins of the Shiga-toxigenic E. coli (STEC). This virus has been shown to lysogenize a wide range of E. coli strains that is much broader than the range of the strains supporting its lytic growth. Therefore, phages produced by the STEC population colonizing the small or large intestine are potentially able to lysogenize symbiotic E. coli in the hindgut, and these secondary lysogens may contribute to the overall patient toxic load and to lead to the emergence of new pathogenic STEC strains. We demonstrate, however, that O antigen effectively limit the lysogenization of the wild E. coli strains by phi24B phage. The lysogens are formed from the spontaneous rough mutants and therefore have increased sensitivity to other bacteriophages and to the bactericidal activity of the serum if compared to their respective parental strains. [ABSTRACT FROM AUTHOR]

Copper and superoxide are used by the phagocytes to kill bacteria. Copper is a host effector encountered by uropathogenic Escherichia coli (UPEC) during urinary tract infection in a non-human primate model, and in humans. UPEC is exposed to higher levels of copper in the gut prior to entering the urinary tract. Effects of pre-exposure to copper on bacterial killing by superoxide has not been reported. We hypothesized that copper-replete E. coli is more sensitive to killing by superoxide in vitro, and in activated macrophages. We utilized wild-type UPEC strain CFT073, and its isogenic mutants lacking copper efflux systems, superoxide dismutases (SODs), regulators of a superoxide dismutase, and complemented mutants to address this question. Surprisingly, our results reveal that copper protects UPEC against killing by superoxide in vitro. This copper-dependent protection was amplified in the mutants lacking copper efflux systems. Increased levels of copper and manganese were detected in UPEC exposed to sublethal concentration of copper. Copper activated the transcription of sodA in a SoxR- and SoxS-dependent manner resulting in enhanced levels of SodA activity. Importantly, pre-exposure to copper increased the survival of UPEC within RAW264.7 and bone marrow-derived murine macrophages. Loss of SodA, but not SodB or SodC, in UPEC obliterated copper-dependent protection from superoxide in vitro, and from killing within macrophages. Collectively, our results suggest a model in which sublethal levels of copper trigger the activation of SodA and SodC through independent mechanisms that converge to promote the survival of UPEC from killing by superoxide. A major implication of our findings is that bacteria colonizing copper-rich milieus are primed for efficient detoxification of superoxide. Author summary: Copper and superoxide are used by the host immune cells to limit bacterial growth during infection. We investigated the interplay between copper and superoxide in killing uropathogenic E. coli, the causative agent of urinary tract infection, by utilizing bacteriological, genetic, molecular biology, and cell culture approaches. Our findings reveal that exposure to sublethal levels of copper helps E. coli to survive better in the presence of superoxide by activating superoxide dismutases. Importantly, pre-exposure to copper protects E. coli from killing by macrophages, an important subset of immune cells that control bacterial growth during infection. By shedding light on a novel copper-dependent protection from superoxide in E. coli, findings of this report have major ramifications for bacterial adaptation to survival under superoxide stress. [ABSTRACT FROM AUTHOR]

The initial adaptive responses to nutrient depletion in bacteria often occur at the level of gene expression. Hfq is an RNAbinding protein present in diverse bacterial lineages that contributes to many different aspects of RNA metabolism during gene expression. Using photoactivated localization microscopy and single-molecule tracking, we demonstrate that Hfq forms a distinct and reversible focus-like structure in Escherichia coli specifically experiencing long-term nitrogen starvation. Using the ability of T7 phage to replicate in nitrogen-starved bacteria as a biological probe of E. coli cell function during nitrogen starvation, we demonstrate that Hfq foci have a role in the adaptive response of E. coli to long-term nitrogen starvation. We further show that Hfq foci formation does not depend on gene expression once nitrogen starvation has set in and occurs independently of the transcription factor N-regulatory protein C, which activates the initial adaptive response to N starvation in E. coli. These results serve as a paradigm to demonstrate that bacterial adaptation to long-term nutrient starvation can be spatiotemporally coordinated and can occur independently of de novo gene expression during starvation. [ABSTRACT FROM AUTHOR]

Efflux pumps (EPs) are present in all living cells and represent a large and important group of transmembrane proteins involved in transport processes. In bacteria, multidrug resistance efflux pumps (MDR EPs) confer resistance to antibiotics at different levels and are deeply implicated in the fast and dramatic emergence of antibiotic resistance. Recently, several reports have outlined the great versatility of MDR EPs in exporting a large variety of compounds other than antibiotics, thus promoting bacterial adaptation to a wide range of habitats. In several bacterial pathogens, MDR EPs contribute to increase the virulence potential and are directly involved in the crosstalk with host cells. In this work, we have investigated the possible role of MDR EPs in the infectious process of the adherent-invasive Escherichia coli (AIEC), a group of pathogenic E. coli that colonize the ileal mucosa of Crohn disease (CD) patients causing a strong intestinal inflammation. The results we have obtained indicate that, with the exception of mdtM , all MDR-EPs encoding genes present in E.coli K12 are conserved in the AIEC prototype strain LF82. The analysis of MDR EP expression during LF82 infection of macrophages and epithelial cells reveals that their transcription is highly modulated during the bacterial intracellular life. Notably, some EP genes are regulated in a cell-type specific manner, strongly suggesting that their function is required for LF82 successful infection. AIEC are able to adhere to and invade intestinal epithelial cells and, importantly, to survive and multiply within macrophages. Thus, we further investigated the role of EPs specifically induced by macrophage environment. We present evidence indicating that deletion of mdtEF genes, encoding an MDR EP belonging to the resistance nodulation division (RND) family, significantly impairs survival of LF82 in macrophages and that the wild type phenotype can be restored by trans-complementation with functional MdtEF pump. Altogether, our results indicate a strong involvement of MDR EPs in host pathogen interaction also in AIEC and highlight the contribution of MdtEF to the fitness of LF82 in the macrophage environment. [ABSTRACT FROM AUTHOR]

Escherichia coli is mostly a commensal of birds and mammals, including humans, where it can act as an opportunistic pathogen. It is also found in water and sediments. We investigated the phylogeny, genetic diversification, and habitat-association of 1,294 isolates representative of the phylogenetic diversity of more than 5,000 isolates from the Australian continent. Since many previous studies focused on clinical isolates, we investigated mostly other isolates originating from humans, poultry, wild animals and water. These strains represent the species genetic diversity and reveal widespread associations between phylogroups and isolation sources. The analysis of strains from the same sequence types revealed very rapid change of gene repertoires in the very early stages of divergence, driven by the acquisition of many different types of mobile genetic elements. These elements also lead to rapid variations in genome size, even if few of their genes rise to high frequency in the species. Variations in genome size are associated with phylogroup and isolation sources, but the latter determine the number of MGEs, a marker of recent transfer, suggesting that gene flow reinforces the association of certain genetic backgrounds with specific habitats. After a while, the divergence of gene repertoires becomes linear with phylogenetic distance, presumably reflecting the continuous turnover of mobile element and the occasional acquisition of adaptive genes. Surprisingly, the phylogroups with smallest genomes have the highest rates of gene repertoire diversification and fewer but more diverse mobile genetic elements. This suggests that smaller genomes are associated with higher, not lower, turnover of genetic information. Many of these genomes are from freshwater isolates and have peculiar traits, including a specific capsule, suggesting adaptation to this environment. Altogether, these data contribute to explain why epidemiological clones tend to emerge from specific phylogenetic groups in the presence of pervasive horizontal gene transfer across the species. Author summary: Previous large scale studies on the evolution of E. coli focused on clinical isolates emphasizing virulence and antibiotic resistance in medically important lineages. Yet, most E. coli strains are either human commensals or not associated with humans at all. Here, we analyzed a large collection of non-clinical isolates of the species to assess the mechanisms of gene repertoire diversification in the light of isolation sources and phylogeny. We show that gene repertoires evolve so rapidly by the high turnover of mobile genetic elements that epidemiologically indistinguishable strains can be phenotypically extremely heterogeneous, illustrating the velocity of bacterial adaptation and the importance of accounting for the information on the whole genome at the epidemiological scale. Phylogeny and habitat shape the genetic diversification of E. coli to similar extents. Surprisingly, freshwater strains seem specifically adapted to this environment, breaking the paradigm that E. coli environmental isolates are systematically fecal contaminations. As a consequence, the evolution of this species is also shaped by environmental habitats, and it may diversify by acquiring genes and mobile elements from environmental bacteria (and not just from gut bacteria). This may facilitate the acquisition of virulence factors and antibiotic resistance in the strains that become pathogenic. [ABSTRACT FROM AUTHOR]

Escherichia coli serotype O157:H7 is one of the major agents of pathogen outbreaks associated with fresh fruits and vegetables. Gaseous chlorine dioxide (ClO2) has been reported to be an effective intervention to eliminate bacterial contamination on fresh produce. Although remarkable positive effects of low doses of ClO2 have been reported, the genetic regulatory machinery coordinating the mechanisms of xenobiotic effects and the potential bacterial adaptation remained unclear. This study examined the temporal transcriptome profiles of E. coli O157:H7 during exposure to different doses of ClO2 in order to elucidate the genetic mechanisms underlying bacterial survival under such harsh conditions. Dosages of 1 μg, 5 μg, and 10 μg ClO2 per gram of tomato fruits cause different effects with dose-by-time dynamics. The first hour of exposure to 1 μg and 5 μg ClO2 caused only partial killing with significant growth reduction starting at the second hour, and without further significant reduction at the third hour. However, 10 μg ClO2 exposure led to massive bacterial cell death at 1 h with further increase in cell death at 2 and 3 h. The first hour exposure to 1 μg ClO2 caused activation of primary defense and survival mechanisms. However, the defense response was attenuated during the second and third hours. Upon treatment with 5 μg ClO2, the transcriptional networks showed massive downregulation of pathogenesis and stress response genes at the first hour of exposure, with decreasing number of differentially expressed genes at the second and third hours. In contrast, more genes were further downregulated with exposure to 10 μg ClO2 at the first hour, with the number of both upregulated and downregulated genes significantly decreasing at the second hour. A total of 810 genes were uniquely upregulated at the third hour at 10 μg ClO2, suggesting that the potency of xenobiotic effects had led to potential adaptation. This study provides important knowledge on the possible selection of target molecules for eliminating bacterial contamination on fresh produce without overlooking potential risks of adaptation. [ABSTRACT FROM AUTHOR]

Amino acid starvation in Escherichia coli activates the enzymatic activity of the stringent factor RelA, leading to accumulation of the alarmone nucleotide (p)ppGpp. The alarmone acts as an intercellular messenger to regulate transcription, translation and metabolism to mediate bacterial stress adaptation. The enzymatic activity of RelA is subject to multi-layered allosteric control executed both by ligands – such as "starved" ribosomal complexes, deacylated tRNA and pppGpp – and by individual RelA domains. The auto-regulation of RelA is proposed to act either in cis (inhibition of the enzymatic activity of the N-terminal region, NTD, by regulatory C-terminal region, CTD) or in trans (CTD-mediated dimerization leading to enzyme inhibition). In this report, we probed the regulatory roles of the individual domains of E. coli RelA and our results are not indicative of RelA dimerization being the key regulatory mechanism. First, at growth-permitting levels, ectopic expression of RelA CTD does not interfere with activation of native RelA, indicating lack of regulation via inhibitory complex formation in the cell. Second, in our biochemical assays, increasing RelA concentration does not decrease the enzyme activity, as would be expected in the case of efficient auto-inhibition via dimerization. Third, while high-level CTD expression efficiently inhibits the growth, the effect is independent of native RelA and is mediated by direct inhibition of protein synthesis, likely via direct interaction with the ribosomal A-site. Finally, deletion of the RRM domain of the CTD region leads to growth inhibition mediated by accumulation of (p)ppGpp, suggesting de-regulation of the synthetic activity in this mutant. [ABSTRACT FROM AUTHOR]

The benefits and detriments of recombination for adaptive evolution have been studied both theoretically and experimentally, with conflicting predictions and observations. Most pertinent experiments examine recombination's effects in an unchanging environment and do not study its genomewide effects. Here, we evolved six replicate populations of either highly recombining R+ or lowly recombining R− E. coli strains in a changing environment, by introducing the novel nutrients L-arabinose or indole into the environment. The experiment's ancestral strains are not viable on these nutrients, but 130 generations of adaptive evolution were sufficient to render them viable. Recombination conferred a more pronounced advantage to populations adapting to indole. To study the genomic changes associated with this advantage, we sequenced the genomes of 384 clones isolated from selected replicates at the end of the experiment. These genomes harbour complex changes that range from point mutations to large-scale DNA amplifications. Among several candidate adaptive mutations, those in the tryptophanase regulator tnaC stand out, because the tna operon in which it resides has a known role in indole metabolism. One of the highly recombining populations also shows a significant excess of large-scale segmental DNA amplifications that include the tna operon. This lineage also shows a unique and potentially adaptive combination of point mutations and DNA amplifications that may have originated independently from one another, to be joined later by recombination. Our data illustrate that the advantages of recombination for adaptive evolution strongly depend on the environment and that they can be associated with complex genomic changes. [ABSTRACT FROM AUTHOR]

Escherichia coli contains two cytochrome bd oxidases, bd- I and bd- II. The structure of both enzymes is highly similar, but they exhibit subtle differences such as the accessibility of the active site through a putative proton channel. Here, we demonstrate that the duroquinol:dioxygen oxidoreductase activity of bd- I increased with alkaline pH, whereas bd- II showed a broad activity maximum around pH 7. Likewise, the pH dependence of NO release from the reduced active site, an essential property of bd oxidases, differed between the two oxidases as detected by UV/vis spectroscopy. Both findings may be attributed to differences in the proton channel leading to the active site heme d. The channel comprises a titratable residue (Asp58B in bd -I and Glu58B in bd -II). Conservative mutations at this position drastically altered NO release demonstrating its contribution to the process. [Display omitted] • E. coli expresses two bd type oxidases. • Activity and NO binding of both oxidases show different pH profiles. • Reduced Cyt bd -II shows no NO release at a pH above 8. • D58B (bd -I) and E58B (bd -I) are critical for NO release. [ABSTRACT FROM AUTHOR]

The small molecule alarmone (p)ppGpp mediates bacterial adaptation to nutrient deprivation by altering the initiation properties of RNA polymerase (RNAP). ppGpp is generated in Escherichia coli by two related enzymes, RelA and SpoT.We show that ppGpp is robustly, but transiently, induced in response to DNA damage and is required for efficient nucleotide excision DNA repair (NER). This explains why relA-spoT-deficient cells are sensitive to diverse genotoxic agents and ultraviolet radiation, whereas ppGpp induction renders them more resistant to such challenges. The mechanism of DNA protection by ppGpp involves promotion of UvrD-mediated RNAP backtracking. By rendering RNAP backtracking-prone, ppGpp couples transcription to DNA repair and prompts transitions between repair and recovery states. [ABSTRACT FROM AUTHOR]

Background: The impact of historical contingency, i.e. the past evolutionary history of a population, on further adaptation is mostly unknown at both the phenotypic and genomic levels. We addressed this question using a two-step evolution experiment. First, replicate populations of Escherichia coli were propagated in four different environmental conditions for 1000 generations. Then, all replicate populations were transferred and propagated for further 1000 generations to a single new environment. Results: Using this two-step experimental evolution strategy, we investigated, at both the phenotypic and genomic levels, whether and how adaptation in the initial historical environments impacted evolutionary trajectories in a new environment. We showed that both the growth rate and fitness of the evolved populations obtained after the second step of evolution were contingent upon past evolutionary history. In contrast however, the genes that were modified during the second step of evolution were independent from the previous history of the populations. Conclusions: Our work suggests that historical contingency affects phenotypic adaptation to a new environment. This was however not reflected at the genomic level implying complex relationships between environmental factors and the genotype-to-phenotype map. [ABSTRACT FROM AUTHOR]

Bacteria navigate within inhomogeneous environments by temporally comparing concentrations of chemoeffectors over the course of a few seconds and biasing their rate of reorientations accordingly, thereby drifting towards more favorable conditions. This navigation requires a short-term memory achieved through the sequential methylations and demethylations of several specific glutamate residues on the chemotaxis receptors, which progressively adjusts the receptors’ activity to track the levels of stimulation encountered by the cell with a delay. Such adaptation also tunes the receptors’ sensitivity according to the background ligand concentration, enabling the cells to respond to fractional rather than absolute concentration changes, i.e. to perform logarithmic sensing. Despite the adaptation system being principally well understood, the need for a specific number of methylation sites remains relatively unclear. Here we systematically substituted the four glutamate residues of the Tar receptor of Escherichia coli by non-methylated alanine, creating a set of 16 modified receptors with a varying number of available methylation sites and explored the effect of these substitutions on the performance of the chemotaxis system. Alanine substitutions were found to desensitize the receptors, similarly but to a lesser extent than glutamate methylation, and to affect the methylation and demethylation rates of the remaining sites in a site-specific manner. Each substitution reduces the dynamic range of chemotaxis, by one order of magnitude on average. The substitution of up to two sites could be partly compensated by the adaptation system, but the full set of methylation sites was necessary to achieve efficient logarithmic sensing. [ABSTRACT FROM AUTHOR]

The authors comment on the article "The icmF3 Locus is Involved in Multiple Adaptation- and Virulence-related Characteristics in Pseudomonas aeruginosa PAO1" by J. Lin and colleagues. He says that Lin and colleagues reported multiple interesting yet moderate phenotypes associated with the IcmF3 of the H3-T6SS. He also says that Lin and colleagues tested Escherichia coli survival after co-incubation with PAO1 or the icmF3 mutant.

RelA and SpoT synthesize ppGpp, a key effector molecule that facilitates the adaptation of bacteria to nutrient starvation and other stresses, known as the stringent response. To investigate the role of Rsh, a putative RelA/SpoT homolog (encoded by BAS4302) in Bacillus anthracis, we examined the alteration of the secretome profiles after the overexpression of a functional His-Rsh protein in the B. anthracis strain Sterne at the stationary growth phase. In the ppGpp-deficient E. coli mutant strain CF1693, overexpression of Rsh restored a ppGpp-dependent growth defect on minimal glucose media. The secretome profiles obtained using a two-dimensional electrophoresis (2-DE) analysis were altered by overexpression of Rsh in B. anthracis. Among the 66 protein spots differentially expressed >1.5-fold, the 29 proteins were abundant for further identification using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Functional categorization of those proteins implicated their involvement in various biological activities. Taken together, our results imply that overexpression of a functional His-Rsh can lead to the increased levels of intracellular ppGpp in B. anthracis, resulting in the significant changes in its secretome profiling. The stringent response-controlled proteins identified are likely useful as potential targets for serodiagnostic applications. [ABSTRACT FROM AUTHOR]

We explored whether bacteria could respond adaptively to the presence of carbon nanotubes (CNTs) by investigating the influence of CNTs on the viability, composition of fatty acids, and cytoplasmic membrane fluidity of bacteria in aqueous medium for 24 h exposure. The CNTs included long single-walled carbon nanotubes (L-SWCNTs), short single-walled carbon nanotubes (S-SWCNTs), short carboxyl single-walled carbon nanotubes (S-SWCNT-COOH), and aligned multiwalled carbon nanotubes (A-MWCNTs). The bacteria included three common model bacteria, Staphyloccocus aureus (Gram-positive), Bacillus subtilis (Gram-positive), and Escherichia coli (Gram-negative), and one polybrominated diphenyl ether degrading strain, Ochrobactrum sp. (Gram-negative). Generally, L-SWCNTs were the most toxic to bacteria, whereas S-SWCNT-COOH showed the mildest bacterial toxicity. Ochrobactrum sp. was more susceptible to the toxic effect of CNTs than E. coli Compared to the control in the absence of CNTs, the viability of Ochrobactrum sp. decreased from 71.6-81.4% to 41.8-70.2%, and E. coli from 93.7-104.0% to 67.7-91.0% when CNT concentration increased from 10 to 50 mg L-1. The cytoplasmic membrane fluidity of bacteria increased with CNT concentration, and a significant negative correlation existed between the bacterial viabilities and membrane fluidity for E. coli and Ochrobactrum sp. (p < 0.05), indicating that the increase in membrane fluidity induced by CNTs was an important factor causing the inactivation of bacteria. In the presence of CNTs, E. colt and Ochrobactrum sp. showed elevation in the level of saturated fatty acids accompanied with reduction in unsaturated fatty acids, compensating for the fluidizing effect of CNTs. This demonstrated that bacteria could modify their composition of fatty acids to adapt to the toxicity of CNTs. In contrast, S. aureus and B. subtilis exposed to CNTs increased the proportion of branched-cliain fatty acids and decreased the level of straight-chain fatty acids, which was also favorable to counteract the toxic effect of CNTs. This study suggests that the bacterial tolerances to CNTs are associated with both the adaptive modification of fatty acids in the membrane and the physicochemical properties of CNTs. This is the first report about the physiologically adaptive response of bacteria to CNTs, and may help to further understand the ecotoxicological effects of CNTs. [ABSTRACT FROM AUTHOR]