The Escherichia coli cellulose synthase subunit G (BcsG) is a Zn2+-dependent phosphoethanolamine transferase

Bacterial biofilms are cellular communities that produce an adherent matrix. Exopolysaccharides are key structural components of this matrix and are required for the assembly and architecture of biofilms produced by a wide variety of microorganisms. The human bacterial pathogens Escherichia coli and Salmonella enterica produce a biofilm matrix composed primarily of the exopolysaccharide phosphoethanolamine (pEtN) cellulose. Once thought to be composed of only underivatized cellulose, the pEtN modification present in these matrices has been implicated in the overall architecture and integrity of the biofilm. However, an understanding of the mechanism underlying pEtN derivatization of the cellulose exopolysaccharide remains elusive. The bacterial cellulose synthase subunit G (BcsG) is a predicted inner membrane–localized metalloenzyme that has been proposed to catalyze the transfer of the pEtN group from membrane phospholipids to cellulose. Here we present evidence that the C-terminal domain of BcsG from E. coli (EcBcsGΔN) functions as a phosphoethanolamine transferase in vitro with substrate preference for cellulosic materials. Structural characterization of EcBcsGΔN revealed that it belongs to the alkaline phosphatase superfamily, contains a Zn2+ ion at its active center, and is structurally similar to characterized enzymes that confer colistin resistance in Gram-negative bacteria. Informed by our structural studies, we present a functional complementation experiment in E. coli AR3110, indicating that the activity of the BcsG C-terminal domain is essential for integrity of the pellicular biofilm. Furthermore, our results established a similar but distinct active-site architecture and catalytic mechanism shared between BcsG and the colistin resistance enzymes.

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Characterization of the β-lactamase specified by the resistance factor R-1818 in Escherichia coli K12 and other Gram-negative bacteria

Biochemical Journal ◽

10.1042/bj1230501 ◽

1971 ◽

Vol 123 (4) ◽

pp. 501-505 ◽

Cited By ~ 13

Author(s):

J. W. Dale

Keyword(s):

Escherichia Coli ◽

Host Species ◽

Host Bacterium ◽

Gram Negative Bacteria ◽

Gram Negative ◽

E Coli ◽

R Factor ◽

Relationship Of ◽

The Relationship

1. The amino acid composition of the β-lactamase from E. coli (R-1818) was determined. 2. The R-1818 β-lactamase is inhibited by formaldehyde, hydroxylamine, sodium azide, iodoacetamide, iodine and sodium chloride. 3. The Km values for benzylpenicillin, ampicillin and oxacillin have been determined by using the R-factor enzyme from different host species. The same values were obtained, irrespective of the host bacterium. 4. The molecular weight of the enzyme was found to be 44600, and was the same for all host species. 5. The relationship of R-1818 and R-GN238 β-lactamases is discussed.

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Heterogeneous and Flexible Transmission ofmcr-1in Hospital-AssociatedEscherichia coli

mBio ◽

10.1128/mbio.00943-18 ◽

2018 ◽

Vol 9 (4) ◽

Cited By ~ 23

Author(s):

Yingbo Shen ◽

Zuowei Wu ◽

Yang Wang ◽

Rong Zhang ◽

Hong-Wei Zhou ◽

...

Keyword(s):

Escherichia Coli ◽

Genomic Analysis ◽

Multidrug Resistant ◽

Fluoroquinolone Resistance ◽

Gram Negative Bacteria ◽

Colistin Resistance ◽

Gram Negative ◽

Content Type ◽

E Coli ◽

Genes Encoding

ABSTRACTThe recent emergence of a transferable colistin resistance mechanism, MCR-1, has gained global attention because of its threat to clinical treatment of infections caused by multidrug-resistant Gram-negative bacteria. However, the possible transmission route ofmcr-1amongEnterobacteriaceaespecies in clinical settings is largely unknown. Here, we present a comprehensive genomic analysis ofEscherichia coliisolates collected in a hospital in Hangzhou, China. We found thatmcr-1-carrying isolates from clinical infections and feces of inpatients and healthy volunteers were genetically diverse and were not closely related phylogenetically, suggesting that clonal expansion is not involved in the spread ofmcr-1. Themcr-1gene was found on either chromosomes or plasmids, but in most of theE. coliisolates,mcr-1was carried on plasmids. The genetic context of the plasmids showed considerable diversity as evidenced by the different functional insertion sequence (IS) elements, toxin-antitoxin (TA) systems, heavy metal resistance determinants, and Rep proteins of broad-host-range plasmids. Additionally, the genomic analysis revealed nosocomial transmission ofmcr-1and the coexistence ofmcr-1with other genes encoding β-lactamases and fluoroquinolone resistance in theE. coliisolates. These findings indicate thatmcr-1is heterogeneously disseminated in both commensal and pathogenic strains ofE. coli, suggest the high flexibility of this gene in its association with diverse genetic backgrounds of the hosts, and provide new insights into the genome epidemiology ofmcr-1among hospital-associatedE. colistrains.IMPORTANCEColistin represents one of the very few available drugs for treating infections caused by extensively multidrug-resistant Gram-negative bacteria. The recently emergentmcr-1colistin resistance gene threatens the clinical utility of colistin and has gained global attention. Howmcr-1spreads in hospital settings remains unknown and was investigated by whole-genome sequencing ofmcr-1-carryingEscherichia coliin this study. The findings revealed extraordinary flexibility ofmcr-1in its spread among genetically diverseE. colihosts and plasmids, nosocomial transmission ofmcr-1-carryingE. coli, and the continuous emergence of novel Inc types of plasmids carryingmcr-1and newmcr-1variants. Additionally,mcr-1was found to be frequently associated with other genes encoding β-lactams and fluoroquinolone resistance. These findings provide important information on the transmission and epidemiology ofmcr-1and are of significant public health importance as the information is expected to facilitate the control of this significant antibiotic resistance threat.

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Characterization of Two New Aminopeptidases in Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.187.11.3671-3677.2005 ◽

2005 ◽

Vol 187 (11) ◽

pp. 3671-3677 ◽

Cited By ~ 9

Author(s):

Yu Zheng ◽

Richard J. Roberts ◽

Simon Kasif ◽

Chudi Guan

Keyword(s):

Escherichia Coli ◽

Stop Codon ◽

Bacterial Species ◽

Start Codon ◽

Aminopeptidase Activity ◽

Peptide Substrates ◽

E Coli ◽

Terminal Domain ◽

Methionyl Aminopeptidase

ABSTRACT Two genes in the Escherichia coli genome, ypdE and ypdF, have been cloned and expressed, and their products have been purified. YpdF is shown to be a metalloenzyme with Xaa-Pro aminopeptidase activity and limited methionine aminopeptidase activity. Genes homologous to ypdF are widely distributed in bacterial species. The unique feature in the sequences of the products of these genes is a conserved C-terminal domain and a variable N-terminal domain. Full or partial deletion of the N terminus in YpdF leads to the loss of enzymatic activity. The conserved C-terminal domain is homologous to that of the methionyl aminopeptidase (encoded by map) in E. coli. However, YpdF and Map differ in their preference for the amino acid next to the initial methionine in the peptide substrates. The implication of this difference is discussed. ypdE is the immediate downstream gene of ypdF, and its start codon overlaps with the stop codon of ypdF by 1 base. YpdE is shown to be a metalloaminopeptidase and has a broad exoaminopeptidase activity.

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Quercetin Rejuvenates Sensitization of Colistin-Resistant Escherichia coli and Klebsiella Pneumoniae Clinical Isolates to Colistin

Frontiers in Chemistry ◽

10.3389/fchem.2021.795150 ◽

2021 ◽

Vol 9 ◽

Author(s):

Yishuai Lin ◽

Ying Zhang ◽

Shixing Liu ◽

Dandan Ye ◽

Liqiong Chen ◽

...

Keyword(s):

Escherichia Coli ◽

Klebsiella Pneumoniae ◽

Alternative Pathway ◽

Membrane Damage ◽

New Drugs ◽

Colistin Resistance ◽

Cell Membrane Damage ◽

E Coli

Colistin is being considered as “the last ditch” treatment in many infections caused by Gram-negative stains. However, colistin is becoming increasingly invalid in treating patients who are infected with colistin-resistant Escherichia coli (E. coli) and Klebsiella Pneumoniae (K. pneumoniae). To cope with the continuous emergence of colistin resistance, the development of new drugs and therapies is highly imminent. Herein, in this work, we surprisingly found that the combination of quercetin with colistin could efficiently and synergistically eradicate the colistin-resistant E. coli and K. pneumoniae, as confirmed by the synergy checkboard and time-kill assay. Mechanismly, the treatment of quercetin combined with colistin could significantly downregulate the expression of mcr-1 and mgrB that are responsible for colistin-resistance, synergistically enhancing the bacterial cell membrane damage efficacy of colistin. The colistin/quercetin combination was notably efficient in eradicating the colistin-resistant E. coli and K. pneumoniae both in vitro and in vivo. Therefore, our results may provide an efficient alternative pathway against colistin-resistant E. coli and K. pneumoniae infections.

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Activity of Tigecycline or Colistin in Combination with Zidovudine against Escherichia coli Harboring tet(X) and mcr-1

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01172-20 ◽

2020 ◽

Vol 65 (1) ◽

pp. e01172-20 ◽

Cited By ~ 1

Author(s):

Yu-Feng Zhou ◽

Ping Liu ◽

Shu-He Dai ◽

Jian Sun ◽

Ya-Hong Liu ◽

...

Keyword(s):

Escherichia Coli ◽

Multidrug Resistant ◽

Therapeutic Options ◽

Infection Model ◽

Synergistic Activity ◽

Colistin Resistance ◽

Content Type ◽

E Coli ◽

Antiretroviral Drug

ABSTRACTAlternative therapeutic options are urgently needed against multidrug-resistant Escherichia coli infections, especially in situations of preexisting tigecycline and colistin resistance. Here, we investigated synergistic activity of the antiretroviral drug zidovudine in combination with tigecycline or colistin against E. coli harboring tet(X) and mcr-1 in vitro and in a murine thigh infection model. Zidovudine and tigecycline/colistin combinations achieved synergistic killing and significantly decreased bacterial burdens by >2.5-log10 CFU/g in thigh tissues compared to each monotherapy.

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Helicobacter pylori DnaB helicase can bypass Escherichia coli DnaC function in vivo

Biochemical Journal ◽

10.1042/bj20050062 ◽

2005 ◽

Vol 389 (2) ◽

pp. 541-548 ◽

Cited By ~ 28

Author(s):

Rajesh K. Soni ◽

Parul Mehra ◽

Gauranga Mukhopadhyay ◽

Suman Kumar Dhar

Keyword(s):

Escherichia Coli ◽

Helicobacter Pylori ◽

Temperature Sensitive ◽

Chromosomal Dna ◽

E Coli ◽

Dnab Helicase ◽

H Pylori

In Escherichia coli, DnaC is essential for loading DnaB helicase at oriC (the origin of chromosomal DNA replication). The question arises as to whether this model can be generalized to other species, since many eubacterial species fail to possess dnaC in their genomes. Previously, we have reported the characterization of HpDnaB (Helicobacter pylori DnaB) both in vitro and in vivo. Interestingly, H. pylori does not have a DnaC homologue. Using two different E. coli dnaC (EcdnaC) temperature-sensitive mutant strains, we report here the complementation of EcDnaC function by HpDnaB in vivo. These observations strongly suggest that HpDnaB can bypass EcDnaC activity in vivo.

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Nonclonal Emergence of Colistin Resistance Associated with Mutations in the BasRS Two-Component System in Escherichia coli Bloodstream Isolates

mSphere ◽

10.1128/msphere.00143-20 ◽

2020 ◽

Vol 5 (2) ◽

Cited By ~ 2

Author(s):

Axel B. Janssen ◽

Toby L. Bartholomew ◽

Natalia P. Marciszewska ◽

Marc J. M. Bonten ◽

Rob J. L. Willems ◽

...

Keyword(s):

Escherichia Coli ◽

Lipid A ◽

Gram Negative Bacteria ◽

Colistin Resistance ◽

Anionic Lipid ◽

Gram Negative ◽

Content Type ◽

E Coli ◽

Two Component ◽

Resistant Strains

ABSTRACT Infections by multidrug-resistant Gram-negative bacteria are increasingly common, prompting the renewed interest in the use of colistin. Colistin specifically targets Gram-negative bacteria by interacting with the anionic lipid A moieties of lipopolysaccharides, leading to membrane destabilization and cell death. Here, we aimed to uncover the mechanisms of colistin resistance in nine colistin-resistant Escherichia coli strains and one Escherichia albertii strain. These were the only colistin-resistant strains of 1,140 bloodstream Escherichia isolates collected in a tertiary hospital over a 10-year period (2006 to 2015). Core-genome phylogenetic analysis showed that each patient was colonized by a unique strain, suggesting that colistin resistance was acquired independently in each strain. All colistin-resistant strains had lipid A that was modified with phosphoethanolamine. In addition, two E. coli strains had hepta-acylated lipid A species, containing an additional palmitate compared to the canonical hexa-acylated E. coli lipid A. One E. coli strain carried the mobile colistin resistance (mcr) gene mcr-1.1 on an IncX4-type plasmid. Through construction of chromosomal transgene integration mutants, we experimentally determined that mutations in basRS, encoding a two-component signal transduction system, contributed to colistin resistance in four strains. We confirmed these observations by reversing the mutations in basRS to the sequences found in reference strains, resulting in loss of colistin resistance. While the mcr genes have become a widely studied mechanism of colistin resistance in E. coli, sequence variation in basRS is another, potentially more prevalent but relatively underexplored, cause of colistin resistance in this important nosocomial pathogen. IMPORTANCE Multidrug resistance among Gram-negative bacteria has led to the use of colistin as a last-resort drug. The cationic colistin kills Gram-negative bacteria through electrostatic interaction with the anionic lipid A moiety of lipopolysaccharides. Due to increased use in clinical and agricultural settings, colistin resistance has recently started to emerge. In this study, we used a combination of whole-genome sequence analysis and experimental validation to characterize the mechanisms through which Escherichia coli strains from bloodstream infections can develop colistin resistance. We found no evidence of direct transfer of colistin-resistant isolates between patients. The lipid A of all isolates was modified by the addition of phosphoethanolamine. In four isolates, colistin resistance was experimentally verified to be caused by mutations in the basRS genes, encoding a two-component regulatory system. Our data show that chromosomal mutations are an important cause of colistin resistance among clinical E. coli isolates.

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Exogenous polyunsaturated fatty acids (PUFAs) promote changes in growth, phospholipid composition, membrane permeability and virulence phenotypes in Escherichia coli

BMC Microbiology ◽

10.1186/s12866-020-01988-0 ◽

2020 ◽

Vol 20 (1) ◽

Author(s):

Joshua L. Herndon ◽

Rachel E. Peters ◽

Rachel N. Hofer ◽

Timothy B. Simmons ◽

Steven J. Symes ◽

...

Keyword(s):

Escherichia Coli ◽

Fatty Acids ◽

Fatty Acid ◽

Membrane Permeability ◽

Phospholipid Composition ◽

Acid Oxidation ◽

Gram Negative Bacteria ◽

Membrane Phospholipids ◽

Gram Negative ◽

E Coli

Abstract Background The utilization of exogenous fatty acids by Gram-negative bacteria has been linked to many cellular processes, including fatty acid oxidation for metabolic gain, assimilation into membrane phospholipids, and control of phenotypes associated with virulence. The expanded fatty acid handling capabilities have been demonstrated in several bacteria of medical importance; however, a survey of the polyunsaturated fatty acid responses in the model organism Escherichia coli has not been performed. The current study examined the impacts of exogenous fatty acids on E. coli. Results All PUFAs elicited higher overall growth, with several fatty acids supporting growth as sole carbon sources. Most PUFAs were incorporated into membrane phospholipids as determined by Ultra performance liquid chromatography-mass spectrometry, whereas membrane permeability was variably affected as measured by two separate dye uptake assays. Biofilm formation, swimming motility and antimicrobial peptide resistance were altered in the presence of PUFAs, with arachidonic and docosahexaenoic acids eliciting strong alteration to these phenotypes. Conclusions The findings herein add E. coli to the growing list of Gram-negative bacteria with broader capabilities for utilizing and responding to exogenous fatty acids. Understanding bacterial responses to PUFAs may lead to microbial behavioral control regimens for disease prevention.

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ucFabV Requires Functional Reductase Activity to Confer Reduced Triclosan Susceptibility in Escherichia coli

Journal of Molecular Microbiology and Biotechnology ◽

10.1159/000441640 ◽

2015 ◽

Vol 25 (6) ◽

pp. 394-402 ◽

Cited By ~ 1

Author(s):

Taylor L. Fischer ◽

Robert J. White ◽

Katherine F.K. Mares ◽

Devin E. Molnau ◽

Justin J. Donato

Keyword(s):

Escherichia Coli ◽

Reductase Activity ◽

Acid Synthesis ◽

Temperature Sensitive ◽

Wild Type ◽

E Coli ◽

Enoyl Acp Reductase ◽

Selection For

Background/Aims: We previously identified the Triclo1 fosmid in a functional metagenomic selection for clones that increased triclosan tolerance in Escherichia coli. The active enzyme encoded by Triclo1 is ucFabV. Although ucFabV is homologous to FabV from other organisms, ucFabV contains substitutions at key positions that would predict differences in substrate binding. Therefore, a detailed characterization of ucFabV was conducted to link its biochemical activity to its ability to confer reduced triclosan sensitivity. Methods: ucFabV and a catalytic mutant were purified and used to reduce crotonoyl-CoA in vitro. The mutant and wild-type enzymes were introduced into E. coli, and their ability to confer triclosan tolerance as well as suppress a temperature-sensitive mutant of FabI were measured. Results: Purified ucFabV, but not the mutant, reduced crotonoyl-CoA in vitro. The wild-type enzyme confers increased triclosan tolerance when introduced into E. coli, whereas the mutant remained susceptible to triclosan. Additionally, wild-type ucFabV, but not the mutant, functionally replaced FabI within living cells. Conclusion: ucFabV confers increased tolerance through its function as an enoyl-ACP reductase. Furthermore, ucFabV is capable of restoring viability in the presence of compromised FabI, suggesting ucFabV is likely facilitating an alternate step within fatty acid synthesis, bypassing FabI inhibition.

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Genomic Insights into a Colistin-Resistant Uropathogenic Escherichia coli Strain of O23:H4-ST641 Lineage Harboring mcr-1.1 on a Conjugative IncHI2 Plasmid from Egypt

Microorganisms ◽

10.3390/microorganisms9040799 ◽

2021 ◽

Vol 9 (4) ◽

pp. 799

Author(s):

Azza S. Zakaria ◽

Eva A. Edward ◽

Nelly M. Mohamed

Keyword(s):

Escherichia Coli ◽

Multidrug Resistant ◽

Coli Strain ◽

Colistin Resistance ◽

E Coli ◽

Agriculture Sector ◽

Potential Reservoir ◽

Clinical Source ◽

Tract Infections

The reintroduction of colistin, a last-resort antibiotic for multidrug-resistant pathogens, resulted in the global spread of plasmid-mediated mobile colistin resistance (mcr) genes. Our study investigated the occurrence of colistin resistance among Escherichia coli isolated from patients with urinary tract infections admitted to a teaching hospital in Egypt. Out of 67 isolates, three isolates were colistin-resistant, having a minimum inhibitory concentration of 4 µg/mL and possessing the mcr-1 gene. A double mechanism of colistin resistance was detected; production of mcr-1 along with amino acid substitution in PmrB (E123D and Y358N) and PmrA (G144S). Broth mating experiments inferred that mcr-1 was positioned on conjugative plasmids. Whole-genome sequencing of EC13049 indicated that the isolate belonged to O23:H4-ST641 lineage and to phylogroup D. The mcr-1-bearing plasmid corresponded to IncHI2 type with a notable similarity to other E. coli plasmids previously recovered from Egypt. The unbanned use of colistin in the Egyptian agriculture sector might have created a potential reservoir for the mcr-1 gene in food-producing animals that spread to humans. More proactive regulations must be implemented to prevent further dissemination of this resistance. This is the first characterization of mcr-1-carrying IncHI2:ST4 plasmid recovered from E. coli of a clinical source in Egypt.

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