Loss of phenotypic inheritance associated with ydcI mutation leads to increased frequency of small, slow persisters in Escherichia coli

Whenever a genetically homogenous population of bacterial cells is exposed to antibiotics, a tiny fraction of cells survives the treatment, the phenomenon known as bacterial persistence [G.L. Hobby et al., Exp. Biol. Med. 50, 281–285 (1942); J. Bigger, The Lancet 244, 497–500 (1944)]. Despite its biomedical relevance, the origin of the phenomenon is still unknown, and as a rare, phenotypically resistant subpopulation, persisters are notoriously hard to study and define. Using computerized tracking we show that persisters are small at birth and slowly replicating. We also determine that the high-persister mutant strain of Escherichia coli, HipQ, is associated with the phenotype of reduced phenotypic inheritance (RPI). We identify the gene responsible for RPI, ydcI, which encodes a transcription factor, and propose a mechanism whereby loss of phenotypic inheritance causes increased frequency of persisters. These results provide insight into the generation and maintenance of phenotypic variation and provide potential targets for the development of therapeutic strategies that tackle persistence in bacterial infections.

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Superhydrophobic Nanocoatings as Intervention against Biofilm-Associated Bacterial Infections

Nanomaterials ◽

10.3390/nano11041046 ◽

2021 ◽

Vol 11 (4) ◽

pp. 1046

Author(s):

Yinghan Chan ◽

Xun Hui Wu ◽

Buong Woei Chieng ◽

Nor Azowa Ibrahim ◽

Yoon Yee Then

Keyword(s):

Biofilm Formation ◽

Bacterial Infections ◽

Therapeutic Strategies ◽

Public Healthcare ◽

Persistent Infections ◽

Promising Strategy ◽

Antimicrobial Interventions ◽

Novel Therapeutic Strategies ◽

Insight Into ◽

Microbial Attachment

Biofilm formation represents a significant cause of concern as it has been associated with increased morbidity and mortality, thereby imposing a huge burden on public healthcare system throughout the world. As biofilms are usually resistant to various conventional antimicrobial interventions, they may result in severe and persistent infections, which necessitates the development of novel therapeutic strategies to combat biofilm-based infections. Physicochemical modification of the biomaterials utilized in medical devices to mitigate initial microbial attachment has been proposed as a promising strategy in combating polymicrobial infections, as the adhesion of microorganisms is typically the first step for the formation of biofilms. For instance, superhydrophobic surfaces have been shown to possess substantial anti-biofilm properties attributed to the presence of nanostructures. In this article, we provide an insight into the mechanisms underlying biofilm formation and their composition, as well as the applications of nanomaterials as superhydrophobic nanocoatings for the development of novel anti-biofilm therapies.

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Proteomic Profiling, Transcription Factor Modeling, and Genomics of Evolved Tolerant Strains Elucidate Mechanisms of Vanillin Toxicity in Escherichia coli

mSystems ◽

10.1128/msystems.00163-19 ◽

2019 ◽

Vol 4 (4) ◽

Cited By ~ 9

Author(s):

Calum A. Pattrick ◽

Joseph P. Webb ◽

Jeffrey Green ◽

Roy R. Chaudhuri ◽

Mark O. Collins ◽

...

Keyword(s):

Gene Expression ◽

Escherichia Coli ◽

Transcription Factor ◽

Aromatic Aldehyde ◽

Bacterial Cells ◽

Data Set ◽

Flavor Compound ◽

Content Type ◽

Starting Point ◽

Factor Modeling

ABSTRACT Vanillin (4-hydroxy-3-methoxybenzaldehyde) is an economically important flavor compound that can be made in bacterial cell factories, but toxicity is a major problem for cells producing this aromatic aldehyde. Using (i) a global proteomic analysis supported by multiple physiological experiments, mutant analyses, and inferred transcription factor modeling and (ii) adaptive laboratory evolution (ALE) of vanillin tolerance combined with genome-wide analysis of the underlying mutations, mechanisms of vanillin toxicity in Escherichia coli have been elucidated. We identified 147 proteins that exhibited a significant change in abundance in response to vanillin, giving the first detailed insight into the cellular response to this aldehyde. Vanillin caused accumulation of reactive oxygen species invoking adaptations coordinated by a MarA, OxyR, and SoxS regulatory network and increased RpoS/DksA-dependent gene expression. Differential fumarase C upregulation was found to prevent oxidative damage to FumA and FumB during growth with vanillin. Surprisingly, vanillin-dependent reduction pf copper (II) to copper (I) led to upregulation of the copA gene and growth in the presence of vanillin was shown to be hypersensitive to inhibition by copper ions. AcrD and AaeAB were identified as potential vanillin efflux systems. Vanillin-tolerant strains isolated by ALE had distinct nonsynonymous single nucleotide polymorphisms (SNPs) in gltA that led to increased citrate synthase activity. Strain-specific mutations in cpdA, rob, and marC were also present. One strain had a large (∼10-kb) deletion that included the marRAB region. Our data provide new understanding of bacterial vanillin toxicity and identify novel gene targets for future engineering of vanillin-tolerant strains of E. coli. IMPORTANCE A particular problem for the biotechnological production of many of the valuable chemicals that we are now able to manufacture in bacterial cells is that these products often poison the cells producing them. Solutions to improve product yields or alleviate such toxicity using the techniques of modern molecular biology first require a detailed understanding of the mechanisms of product toxicity. Here we have studied the economically important flavor compound vanillin, an aromatic aldehyde that exerts significant toxic effects on bacterial cells. We used high-resolution protein abundance analysis as a starting point to determine which proteins are upregulated and which are downregulated by growth with vanillin, followed by gene expression and mutant studies to understand the mechanism of the response. In a second approach, we evolved bacterial strains with higher vanillin tolerance. Their genome sequences have yielded novel insights into vanillin tolerance that are complementary to the proteomics data set.

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Conserved Region 2.1 of Escherichia coli Heat Shock Transcription Factor σ32 Is Required for Modulating both Metabolic Stability and Transcriptional Activity

Journal of Bacteriology ◽

10.1128/jb.186.22.7474-7480.2004 ◽

2004 ◽

Vol 186 (22) ◽

pp. 7474-7480 ◽

Cited By ~ 21

Author(s):

Mina Horikoshi ◽

Takashi Yura ◽

Sachie Tsuchimoto ◽

Yoshihiro Fukumori ◽

Masaaki Kanemori

Keyword(s):

Escherichia Coli ◽

Transcription Factor ◽

Heat Shock ◽

Transcriptional Activity ◽

Heat Shock Transcription Factor ◽

Metabolic Stability ◽

Wild Type ◽

Shock Proteins ◽

Insight Into

ABSTRACT Escherichia coli heat shock transcription factor σ32 is rapidly degraded in vivo, with a half-life of about 1 min. A set of proteins that includes the DnaK chaperone team (DnaK, DnaJ, GrpE) and ATP-dependent proteases (FtsH, HslUV, etc.) are involved in degradation of σ32. To gain further insight into the regulation of σ32 stability, we isolated σ32 mutants that were markedly stabilized. Many of the mutants had amino acid substitutions in the N-terminal half (residues 47 to 55) of region 2.1, a region highly conserved among bacterial σ factors. The half-lives ranged from about 2-fold to more than 10-fold longer than that of the wild-type protein. Besides greater stability, the levels of heat shock proteins, such as DnaK and GroEL, increased in cells producing stable σ32. Detailed analysis showed that some stable σ32 mutants have higher transcriptional activity than the wild type. These results indicate that the N-terminal half of region 2.1 is required for modulating both metabolic stability and the activity of σ32. The evidence suggests that σ32 stabilization does not result from an elevated affinity for core RNA polymerase. Region 2.1 may, therefore, be involved in interactions with the proteolytic machinery, including molecular chaperones.

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The Effects of β-Lactam Antibiotics on Surface Modifications of Multidrug-Resistant Escherichia coli: A Multiscale Approach

Microscopy and Microanalysis ◽

10.1017/s1431927618015696 ◽

2019 ◽

Vol 25 (1) ◽

pp. 135-150 ◽

Cited By ~ 7

Author(s):

Samuel C. Uzoechi ◽

Nehal I. Abu-Lail

Keyword(s):

Escherichia Coli ◽

Biofilm Formation ◽

Bacterial Infections ◽

Treatment Strategies ◽

Multidrug Resistant ◽

Multiscale Approach ◽

Bacterial Cells ◽

Adhesion Forces ◽

E Coli ◽

Resistant Strains

AbstractPossible multidrug-resistant (MDR) mechanisms of four resistant strains of Escherichia coli to a model β-lactam, ampicillin, were investigated using contact angle measurements of wettability, crystal violet assays of permeability, biofilm formation, fluorescence imaging, and nanoscale analyses of dimensions, adherence, and roughness. Upon exposure to ampicillin, one of the resistant strains, E. coli A5, changed its phenotype from elliptical to spherical, maintained its roughness and biofilm formation abilities, decreased its length and surface area, maintained its cell wall integrity, increased its hydrophobicity, and decreased its nanoscale adhesion to a model surface of silicon nitride. Such modifications are suggested to allow these cells to conserve energy during metabolic dormancy. In comparison, resistant strains E. coli D4, A9, and H5 elongated their cells, increased their roughness, increased their nanoscale adhesion forces, became more hydrophilic, and increased their biofilm formation upon exposure to ampicillin. These results suggest that these strains resisted ampicillin through biofilm formation that possibly introduces diffusion limitations to antibiotics. Investigations of how MDR bacterial cells modify their surfaces in response to antibiotics can guide research efforts aimed at designing more effective antibiotics and new treatment strategies for MDR bacterial infections.

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A Critical, Nonlinear Threshold Dictates Bacterial Invasion and Initial Kinetics During Influenza

10.1101/052175 ◽

2016 ◽

Cited By ~ 1

Author(s):

Amber M. Smith ◽

Amanda P. Smith

Keyword(s):

Growth Rate ◽

Kinetic Model ◽

Influenza A Virus ◽

Influenza A ◽

Bacterial Infections ◽

Therapeutic Strategies ◽

Bacterial Invasion ◽

Dose Requirement ◽

Dose Threshold ◽

Insight Into

ABSTRACTSecondary bacterial infections increase morbidity and mortality of influenza A virus (IAV) infections. Bacteria are able to invade due to virus-induced depletion of alveolar macrophages (AMs), but this is not the only contributing factor. By analyzing a kinetic model, we uncovered a nonlinear initial dose threshold that is dependent on the amount of virus-induced AM depletion. The threshold separates the growth and clearance phenotypes such that bacteria decline for dose-AM depletion combinations below the threshold, stay constant near the threshold, and increase above the threshold. In addition, the distance from the threshold correlates to the growth rate. Because AM depletion changes throughout an IAV infection, the dose requirement for bacterial invasion also changes accordingly. Using the threshold, we found that the dose requirement drops dramatically during the first 7d of IAV infection. We then validated these analytical predictions by infecting mice with doses below or above the predicted threshold over the course of IAV infection. These results identify the nonlinear way in which two independent factors work together to support successful post-influenza bacterial invasion. They provide insight into coinfection timing, the heterogeneity in outcome, the probability of acquiring a coinfection, and the use of new therapeutic strategies to combat viral-bacterial coinfections.

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Complexation of the nickel and cobalt transcriptional regulator RcnR with DNA

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x19017084 ◽

2020 ◽

Vol 76 (1) ◽

pp. 25-30

Author(s):

Chao Li ◽

Joseph W. Vavra ◽

Carolyn E. Carr ◽

Hsin-Ting Huang ◽

Michael J. Maroney ◽

...

Keyword(s):

Escherichia Coli ◽

Transcription Factor ◽

Transcriptional Regulator ◽

Bacterial Cells ◽

X Ray Diffraction ◽

Unit Cell Parameters ◽

X Ray ◽

Cell Parameters ◽

Dna Binding Site ◽

Cobalt And Nickel

RcnR is a transcription factor that regulates the homeostasis of cobalt and nickel in bacterial cells. Escherichia coli RcnR was crystallized with DNA that encompasses the DNA-binding site. X-ray diffraction data were collected to 2.9 Å resolution. The crystal belonged to space group P6122 or P6522, with unit-cell parameters a = b = 73.59, c = 157.66 Å, α = β = 90, γ = 120°.

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Evidence for Escherichia coli Diguanylate Cyclase DgcZ Interlinking Surface Sensing and Adhesion via Multiple Regulatory Routes

Journal of Bacteriology ◽

10.1128/jb.00320-16 ◽

2016 ◽

Vol 198 (18) ◽

pp. 2524-2535 ◽

Cited By ~ 13

Author(s):

Egidio Lacanna ◽

Colette Bigosch ◽

Volkhard Kaever ◽

Alex Boehm ◽

Anke Becker

Keyword(s):

Escherichia Coli ◽

Biofilm Formation ◽

Bacterial Infections ◽

Primary Role ◽

Bacterial Cells ◽

Diguanylate Cyclase ◽

Surface Attachment ◽

Content Type ◽

Surface Sensing

ABSTRACTDgcZ is the main cyclic dimeric GMP (c-di-GMP)-producing diguanylate cyclase (DGC) controlling biosynthesis of the exopolysaccharide poly-β-1,6-N-acetylglucosamine (poly-GlcNAc or PGA), which is essential for surface attachment ofEscherichia coli. Although the complex regulation of DgcZ has previously been investigated, its primary role and the physiological conditions under which the protein is active are not fully understood. Transcription ofdgcZis regulated by the two-component system CpxAR activated by the lipoprotein NlpE in response to surface sensing. Here, we show that the negative effect of acpxRmutation and the positive effect ofnlpEoverexpression on biofilm formation both depend on DgcZ. Coimmunoprecipitation data suggest several potential interaction partners of DgcZ. Interaction with FrdB, a subunit of the fumarate reductase complex (FRD) involved in anaerobic respiration and in control of flagellum assembly, was further supported by a bacterial-two-hybrid assay. Furthermore, the FRD complex was required for the increase in DgcZ-mediated biofilm formation upon induction of oxidative stress by addition of paraquat. A DgcZ-mVENUS fusion protein was found to localize at one bacterial cell pole in response to alkaline pH and carbon starvation. Based on our data and previous knowledge, an integrative role of DgcZ in regulation of surface attachment is proposed. We speculate that both DgcZ-stimulated PGA biosynthesis and interaction of DgcZ with the FRD complex contribute to impeding bacterial escape from the surface.IMPORTANCEBacterial cells can grow by clonal expansion to surface-associated biofilms that are ubiquitous in the environment but also constitute a pervasive problem related to bacterial infections. Cyclic dimeric GMP (c-di-GMP) is a widespread bacterial second messenger involved in regulation of motility and biofilm formation, and plays a primary role in bacterial surface attachment.E. colipossesses a plethora of c-di-GMP-producing diguanylate cyclases, including DgcZ. Our study expands the knowledge on the role of DgcZ in regulation of surface attachment and suggests that it interconnects surface sensing and adhesion via multiple routes.

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In Vitro Evaluation of a Phage Cocktail Controlling Infections with Escherichia coli

Viruses ◽

10.3390/v12121470 ◽

2020 ◽

Vol 12 (12) ◽

pp. 1470

Author(s):

Imke H. E. Korf ◽

Sophie Kittler ◽

Anna Bierbrodt ◽

Ruth Mengden ◽

Christine Rohde ◽

...

Keyword(s):

Escherichia Coli ◽

Bacterial Infections ◽

Animal Health ◽

High Specificity ◽

Resistant Mutant ◽

Resistant Bacteria ◽

Bacterial Cells ◽

E Coli ◽

Phage Cocktail

Worldwide, poultry industry suffers from infections caused by avian pathogenic Escherichia coli. Therapeutic failure due to resistant bacteria is of increasing concern and poses a threat to human and animal health. This causes a high demand to find alternatives to fight bacterial infections in animal farming. Bacteriophages are being especially considered for the control of multi-drug resistant bacteria due to their high specificity and lack of serious side effects. Therefore, the study aimed on characterizing phages and composing a phage cocktail suitable for the prevention of infections with E. coli. Six phages were isolated or selected from our collections and characterized individually and in combination with regard to host range, stability, reproduction, and efficacy in vitro. The cocktail consisting of six phages was able to inhibit formation of biofilms by some E. coli strains but not by all. Phage-resistant variants arose when bacterial cells were challenged with a single phage but not when challenged by a combination of four or six phages. Resistant variants arising showed changes in carbon metabolism and/or motility. Genomic comparison of wild type and phage-resistant mutant E28.G28R3 revealed a deletion of several genes putatively involved in phage adsorption and infection.

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Antibacterial, Antibiofilm and Anti-adhesion Activities of Piper Betle Leaf Extract Against Avian Pathogenic Escherichia Coli

10.21203/rs.3.rs-620785/v1 ◽

2021 ◽

Author(s):

Pawinee Kulnanan ◽

Julalak Chuprom ◽

Thotsapol Thomrongsuwannakij ◽

Chonticha Romyasamit ◽

Suthinee Sangkanu ◽

...

Keyword(s):

Escherichia Coli ◽

Bacterial Infections ◽

Ethanol Extract ◽

Bacterial Biofilm ◽

Piper Betle ◽

Bacterial Cells ◽

Biofilm Inhibition ◽

E Coli ◽

Viable Cells ◽

Pathogenic Escherichia Coli

Abstract Piper betle leaves have traditionally been used to treat many diseases, including bacterial infections. The present study aimed to investigate the antibacterial, antibiofilm, and anti-adhesion activities of P. betle extract against Avian pathogenic Escherichia coli (APEC). The ethanol extract of P. betle leaves demonstrated strong antibacterial activity against clinical isolates of APEC with MIC and MBC values ranging from 0.5-1.0 mg/mL. Disruption and breakdown of the bacterial cells were detected when the cells were challenged with the extract at 2×MIC. Bacterial cells treated with the extract demonstrated longer cells without a septum, compared to the control. The extract at 1/8, 1/4, and 1/2×MIC significantly inhibited the formation of bacterial biofilm of the isolates (P<0.05) without inhibiting growth. At 1/2×MIC, 55% of the biofilm inhibition was detected in APEC CH09, a strong biofilm producer. At 32×MIC, 88% of the inhibition of viable cells embedded in the mature biofilm was detected in APEC CH09. Reduction in the bacterial adhesion to surfaces was shown when APEC were treated with sub-MICs of the extract as observed by SEM. The results suggested potential medicinal benefits of P. betle extract for the treatment of the infection caused by Avian pathogenic E. coli.

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Disruption of Membrane by Colistin Kills Uropathogenic Escherichia coli Persisters and Enhances Killing of Other Antibiotics

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01481-16 ◽

2016 ◽

Vol 60 (11) ◽

pp. 6867-6871 ◽

Cited By ~ 28

Author(s):

Peng Cui ◽

Hongxia Niu ◽

Wanliang Shi ◽

Shuo Zhang ◽

Hao Zhang ◽

...

Keyword(s):

Escherichia Coli ◽

Bacterial Infections ◽

Enhancement Effect ◽

Bacterial Cells ◽

Sulfa Drugs ◽

Content Type ◽

Bacterial Membranes ◽

Highly Active ◽

High Concentrations

ABSTRACTPersisters are small populations of quiescent bacterial cells that survive exposure to bactericidal antibiotics and are responsible for many persistent infections and posttreatment relapses. However, little is known about how to effectively kill persister bacteria. In the work presented here, we found that colistin, a membrane-active antibiotic, was highly active againstEscherichia colipersisters at high concentrations (25 or 50 μg/ml). At a clinically relevant lower concentration (10 μg/ml), colistin alone had no apparent effect onE. colipersisters. In combination with other drugs, this concentration of colistin enhanced the antipersister activity of gentamicin and ofloxacin but not that of ampicillin, nitrofurans, and sulfa drugsin vitro. The colistin enhancement effect was most likely due to increased uptake of the other antibiotics, as demonstrated by increased accumulation of fluorescence-labeled gentamicin. Interestingly, colistin significantly enhanced the activity of ofloxacin and nitrofurantoin but not that of gentamicin or sulfa drugs in the murine model of urinary tract infection. Our findings suggest that targeting bacterial membranes is a valuable approach to eradicating persisters and should have implications for more effective treatment of persistent bacterial infections.

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