Characterization of a Bifunctional Enzyme Fusion of Trehalose-6-Phosphate Synthetase and Trehalose-6-Phosphate Phosphatase of Escherichia coli

ABSTRACT To test the effect of the physical proximity of two enzymes catalyzing sequential reactions, a bifunctional fusion enzyme, TPSP, was constructed by fusing the Escherichia coli genes for trehalose-6-phosphate (T6P) synthetase (TPS) and trehalose-6-phosphate phosphatase (TPP). TPSP catalyzes the sequential reaction in which T6P is formed and then dephosphorylated, leading to the synthesis of trehalose. The fused chimeric gene was overexpressed in E. coli and purified to near homogeneity; its molecular weight was 88,300, as expected. The Km values of the TPSP fusion enzyme for the sequential overall reaction from UDP-glucose and glucose 6-phosphate to trehalose were smaller than those of an equimolar mixture of TPS and TPP (TPS/TPP). However, thek cat values of TPSP were similar to those of TPS/TPP, resulting in a 3.5- to 4.0-fold increase in the catalytic efficiency (k cat/Km ). The Km and k cat values of TPSP and TPP for the phosphatase reaction from T6P to trehalose were quite similar. This suggests that the increased catalytic efficiency results from the proximity of TPS and TPP in the TPSP fusion enzyme. The thermal stability of the TPSP fusion enzyme was quite similar to that of the TPS/TPP mixture, suggesting that the structure of each enzyme moiety in TPSP is unperturbed by intramolecular constraint. These results clearly demonstrate that the bifunctional fusion enzyme TPSP catalyzing sequential reactions has kinetic advantages over a mixture of both enzymes (TPS and TPP). These results are also supported by the in vivo accumulation of up to 0.48 mg of trehalose per g of cells after isopropyl-β-d-thiogalactopyranoside treatment of cells harboring the construct encoding TPSP.

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Dynamic regulation of extracellular ATP in Escherichia coli

Biochemical Journal ◽

10.1042/bcj20160879 ◽

2017 ◽

Vol 474 (8) ◽

pp. 1395-1416 ◽

Cited By ~ 7

Author(s):

Cora Lilia Alvarez ◽

Gerardo Corradi ◽

Natalia Lauri ◽

Irene Marginedas-Freixa ◽

María Florencia Leal Denis ◽

...

Keyword(s):

Escherichia Coli ◽

Atpase Activity ◽

Atp Release ◽

Membrane Vesicles ◽

Fold Increase ◽

Extracellular Atp ◽

Outer Membrane Vesicles ◽

Dynamic Regulation ◽

E Coli

We studied the kinetics of extracellular ATP (ATPe) in Escherichia coli and their outer membrane vesicles (OMVs) stimulated with amphipatic peptides melittin (MEL) and mastoparan 7 (MST7). Real-time luminometry was used to measure ATPe kinetics, ATP release, and ATPase activity. The latter was also determined by following [32P]Pi released from [γ-32P]ATP. E. coli was studied alone, co-incubated with Caco-2 cells, or in rat jejunum segments. In E. coli, the addition of [γ-32P]ATP led to the uptake and subsequent hydrolysis of ATPe. Exposure to peptides caused an acute 3-fold (MST7) and 7-fold (MEL) increase in [ATPe]. In OMVs, ATPase activity increased linearly with [ATPe] (0.1–1 µM). Exposure to MST7 and MEL enhanced ATP release by 3–7 fold, with similar kinetics to that of bacteria. In Caco-2 cells, the addition of ATP to the apical domain led to a steep [ATPe] increase to a maximum, with subsequent ATPase activity. The addition of bacterial suspensions led to a 6–7 fold increase in [ATPe], followed by an acute decrease. In perfused jejunum segments, exposure to E. coli increased luminal ATP 2 fold. ATPe regulation of E. coli depends on the balance between ATPase activity and ATP release. This balance can be altered by OMVs, which display their own capacity to regulate ATPe. E. coli can activate ATP release from Caco-2 cells and intestinal segments, a response which in vivo might lead to intestinal release of ATP from the gut lumen.

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In vivo characterization of the activities of novel cyclodipeptide oxidases: new tools for increasing chemical diversity of bioproduced 2,5-diketopiperazines in Escherichia coli

Microbial Cell Factories ◽

10.1186/s12934-020-01432-y ◽

2020 ◽

Vol 19 (1) ◽

Author(s):

Fabien Le Chevalier ◽

Isabelle Correia ◽

Lucrèce Matheron ◽

Morgan Babin ◽

Mireille Moutiez ◽

...

Keyword(s):

Escherichia Coli ◽

Biological Activities ◽

Gene Clusters ◽

Chemical Diversity ◽

E Coli ◽

Chemical Structures ◽

In Vivo Characterization ◽

Culture Supernatants

Abstract Background Cyclodipeptide oxidases (CDOs) are enzymes involved in the biosynthesis of 2,5-diketopiperazines, a class of naturally occurring compounds with a large range of pharmaceutical activities. CDOs belong to cyclodipeptide synthase (CDPS)-dependent pathways, in which they play an early role in the chemical diversification of cyclodipeptides by introducing Cα-Cβ dehydrogenations. Although the activities of more than 100 CDPSs have been determined, the activities of only a few CDOs have been characterized. Furthermore, the assessment of the CDO activities on chemically-synthesized cyclodipeptides has shown these enzymes to be relatively promiscuous, making them interesting tools for cyclodipeptide chemical diversification. The purpose of this study is to provide the first completely microbial toolkit for the efficient bioproduction of a variety of dehydrogenated 2,5-diketopiperazines. Results We mined genomes for CDOs encoded in biosynthetic gene clusters of CDPS-dependent pathways and selected several for characterization. We co-expressed each with their associated CDPS in the pathway using Escherichia coli as a chassis and showed that the cyclodipeptides and the dehydrogenated derivatives were produced in the culture supernatants. We determined the biological activities of the six novel CDOs by solving the chemical structures of the biologically produced dehydrogenated cyclodipeptides. Then, we assessed the six novel CDOs plus two previously characterized CDOs in combinatorial engineering experiments in E. coli. We co-expressed each of the eight CDOs with each of 18 CDPSs selected for the diversity of cyclodipeptides they synthesize. We detected more than 50 dehydrogenated cyclodipeptides and determined the best CDPS/CDO combinations to optimize the production of 23. Conclusions Our study establishes the usefulness of CDPS and CDO for the bioproduction of dehydrogenated cyclodipeptides. It constitutes the first step toward the bioproduction of more complex and diverse 2,5-diketopiperazines.

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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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Increased Type 1 Fimbrial Expression among Commensal Escherichia coli Isolates in the Murine Cecum following Catabolic Stress

Infection and Immunity ◽

10.1128/iai.67.2.745-753.1999 ◽

1999 ◽

Vol 67 (2) ◽

pp. 745-753 ◽

Cited By ~ 29

Author(s):

Barbara A. Hendrickson ◽

Jun Guo ◽

Robert Laughlin ◽

Yimei Chen ◽

John C. Alverdy

Keyword(s):

Escherichia Coli ◽

Intestinal Epithelium ◽

Tissue Injury ◽

Fold Increase ◽

E Coli ◽

Type 1 Fimbriae ◽

Colon Cell ◽

Colon Cell Line

ABSTRACT Although indigenous bacteria intimately colonize the intestinal mucosa, under normal conditions the intestinal epithelial cell is free of adherent bacteria. Nonetheless, commensal bacteria such asEscherichia coli adhere to and translocate across the intestinal epithelium in association with a number of pathologic states including hemorrhagic shock, immunosuppression, traumatic tissue injury, and lack of enteral feedings. The adhesins involved in the adherence of indigenous E. coli to the intestinal epithelium in vivo following catabolic stress are unknown. We have developed a mouse model to study the bacterial adhesins which mediate the increased intestinal adherence of E. coliafter partial hepatectomy and short-term starvation. Our studies demonstrated that hepatectomy and starvation in the mouse were associated with a 7,500-fold increase in the numbers of E. coli bacteria adhering to the cecum. In addition, erythrocyte agglutination studies, as well as immunostaining of fimbrial preparations and electron micrographs of the bacteria, revealed that surface type 1 fimbriae were more abundant in the commensal E. coli harvested from the ceca of the stressed mice. These E. coli isolates adhered to a mouse colon cell line and injected cecal loops in a mannose-inhibitable manner, which suggests a role for type 1 fimbriae in the adherence of the E. coli isolates to the cecum in vivo following host catabolic stress.

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ISOLATION AND CHARACTERIZATION OF dnaX AND dnaY TEMPERATURE-SENSITIVE MUTANTS OF ESCHERICHIA COLI

Genetics ◽

10.1093/genetics/92.4.1041 ◽

1979 ◽

Vol 92 (4) ◽

pp. 1041-1059

Author(s):

Joan M Henson ◽

Herman Chu ◽

Carleen A Irwin ◽

James R Walker

Keyword(s):

Escherichia Coli ◽

Dna Synthesis ◽

Fold Increase ◽

Temperature Sensitive ◽

E Coli ◽

Temperature Sensitive Mutants ◽

Isolation And Characterization ◽

Ts Mutations

ABSTRACT Escherichia coli mutants with temperature-sensitive (ts) mutations in dnaX and dnaY genes have been isolated. Based on transduction by phage PI, dnaX and Y have been mapped at minutes 10.4-10.5 and 12.1, respectively, in the sequence dnaX purE dnaY. Both dnaXts36 and YtslO are recessive to wild-type alleles present on episomes. F13 carries both dnaX + and Y +; the shorter F210 carries dnaY +, but not X +. Lambda transducing phages that carry dnaX + or Y + have been isolated, and hybrid plasmids of Col E1 and E. coli DNA from the CLARKE and CARBON (1976) collection also carry portions of the dnaX purE dnaY region. Results obtained with the λ transducing phages and the hybrid plasmids suggest that dnaX is a different gene from the previously characterized dnaZ gene, which is also near minute 10.5.—The dnaXts36 mutant, after a shift to 42°, stopped DNA synthesis gradually, and the total amount of DNA increased two-fold. When this mutant was shifted to M°, the rate of DNA synthesis dropped immediately and the final increment of DNA was only 10% of the initial amount. Replicative DNA synthesis in toluene-treated cells was completely inhibited at 42° and was partially in-hibited even at 30°.—When the dnaYtslO mutant was shifted to 42°, DNA synthesis gradually stopped, and the amount of DNA increased 3.6-fold. At 44°, residual DNA synthesis amounted to a two-fold increase. Replicative DNA synthesis in vitro in toluene-treated cells was inactivated after 20 minutes at 42° or by "preincubation" of cells at 42° before toluene treatment.— The dnaX and dnaY products probably function in polymerization of DNA, although participation also in initiation cannot be excluded.

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Assembly of the Escherichia coli FoF1 ATP synthase involves distinct subcomplex formation

Biochemical Society Transactions ◽

10.1042/bst20130096 ◽

2013 ◽

Vol 41 (5) ◽

pp. 1288-1293 ◽

Cited By ~ 11

Author(s):

Gabriele Deckers-Hebestreit

Keyword(s):

Escherichia Coli ◽

Atp Synthase ◽

Cytoplasmic Membrane ◽

Atp Synthesis ◽

E Coli ◽

Assembly Pathway ◽

Polypeptide Chains ◽

Fof1 Atp Synthase

The ATP synthase (FoF1) of Escherichia coli couples the translocation of protons across the cytoplasmic membrane by Fo to ATP synthesis or hydrolysis in F1. Whereas good knowledge of the nanostructure and the rotary mechanism of the ATP synthase is at hand, the assembly pathway of the 22 polypeptide chains present in a stoichiometry of ab2c10α3β3γδϵ has so far not received sufficient attention. In our studies, mutants that synthesize different sets of FoF1 subunits allowed the characterization of individually formed stable subcomplexes. Furthermore, the development of a time-delayed in vivo assembly system enabled the subsequent synthesis of particular missing subunits to allow the formation of functional ATP synthase complexes. These observations form the basis for a model that describes the assembly pathway of the E. coli ATP synthase from pre-formed subcomplexes, thereby avoiding membrane proton permeability by a concomitant assembly of the open H+-translocating unit within a coupled FoF1 complex.

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Overproduction of the Bacillus subtilis glutamyl-tRNA synthetase in its host and its toxicity to Escherichia coli

Canadian Journal of Microbiology ◽

10.1139/w98-014 ◽

1998 ◽

Vol 44 (4) ◽

pp. 378-381 ◽

Cited By ~ 9

Author(s):

Martin Pelchat ◽

Lucille Lacoste ◽

Fu Yang ◽

Jacques Lapointe

Keyword(s):

Escherichia Coli ◽

Bacillus Subtilis ◽

Recombinant Plasmid ◽

Specific Activity ◽

Shuttle Vector ◽

Fold Increase ◽

Trna Synthetase ◽

E Coli ◽

Downstream Region

The Bacillus subtilis glutamyl-tRNA synthetase (GluRS), encoded by the gltX gene, aminoacylates its homologous tRNAGlu and tRNAGln with glutamate. This gene was cloned with its sigmaA promoter and a downstream region including a rho-independent terminator in the shuttle vector pRB394 for Escherichia coli and B. subtilis. Transformation of B. subtilis with this recombinant plasmid (pMP411) led to a 30-fold increase of glutamyl-tRNA synthetase specific activity in crude extracts. Transformation of E. coli with this plasmid gave no recombinants, but transformation with plasmids bearing an altered gltX was successful. These results indicate that the presence of B. subtilis glutamyl-tRNA synthetase is lethal for E. coli, probably because this enzyme glutamylates tRNA1Gln in vivo as it does in vitro.Key words: glutamyl-tRNA synthetase overproduction, Bacillus subtilis, toxicity, Escherichia coli.

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Characterization of GlnK1 from Methanosarcina mazei Strain Gö1: Complementation of an Escherichia coli glnK Mutant Strain by GlnK1

Journal of Bacteriology ◽

10.1128/jb.184.4.1028-1040.2002 ◽

2002 ◽

Vol 184 (4) ◽

pp. 1028-1040 ◽

Cited By ~ 21

Author(s):

Claudia Ehlers ◽

Roman Grabbe ◽

Katharina Veit ◽

Ruth A. Schmitz

Keyword(s):

Escherichia Coli ◽

Biochemical Characterization ◽

Ammonium Assimilation ◽

Regulatory Function ◽

Native Form ◽

E Coli ◽

Methanosarcina Mazei ◽

Apparent Homogeneity

ABSTRACT Trimeric PII-like signal proteins are known to be involved in bacterial regulation of ammonium assimilation and nitrogen fixation. We report here the first biochemical characterization of an archaeal GlnK protein from the diazotrophic methanogenic archaeon Methanosarcina mazei strain Gö1 and show that M. mazei GlnK1 is able to functionally complement an Escherichia coli glnK mutant for growth on arginine. This indicates that the archaeal GlnK protein substitutes for the regulatory function of E. coli GlnK. M. mazei GlnK1 is encoded in the glnK1 -amtB1 operon, which is transcriptionally regulated by the availability of combined nitrogen and is only transcribed in the absence of ammonium. The deduced amino acid sequence of the archaeal glnK1 shows 44% identity to the E. coli GlnK and contains the conserved tyrosine residue (Tyr-51) in the T-loop structure. M. mazei glnK1 was cloned and overexpressed in E. coli, and GlnK1 was purified to apparent homogeneity. A molecular mass of 42 kDa was observed under native conditions, indicating that its native form is a trimer. GlnK1-specific antibodies were raised and used to confirm the in vivo trimeric form by Western analysis. In vivo ammonium upshift experiments and analysis of purified GlnK1 indicated significant differences compared to E. coli GlnK. First, GlnK1 from M. mazei is not covalently modified by uridylylation under nitrogen limitation. Second, heterotrimers between M. mazei GlnK1 and Klebsiella pneumoniae GlnK are not formed. Because M. mazei GlnK1 was able to complement growth of an E. coli glnK mutant with arginine as the sole nitrogen source, it is likely that uridylylation is not required for its regulatory function.

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In VivoEvolution of CMY-2 to CMY-33 β-Lactamase in Escherichia coli Sequence Type 131: Characterization of an Acquired Extended-Spectrum AmpC Conferring Resistance to Cefepime

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01804-15 ◽

2015 ◽

Vol 59 (12) ◽

pp. 7483-7488 ◽

Cited By ~ 11

Author(s):

João Pires ◽

Magdalena Taracila ◽

Christopher R. Bethel ◽

Yohei Doi ◽

Sara Kasraian ◽

...

Keyword(s):

Escherichia Coli ◽

Bronchoalveolar Lavage Fluid ◽

Lavage Fluid ◽

Sequence Type ◽

Content Type ◽

E Coli ◽

Extended Spectrum ◽

Repetitive Extragenic Palindromic Pcr

ABSTRACTCefepime is frequently prescribed to treat infections caused by AmpC-producing Gram-negative bacteria. CMY-2 is the most common plasmid-mediated AmpC (pAmpC) β-lactamase. Unfortunately, CMY variants conferring enhanced cefepime resistance have been reported. Here, we describe the evolution of CMY-2 to an extended-spectrum AmpC (ESAC) in clonally identicalEscherichia coliisolates obtained from a patient. The CMY-2-producingE. coliisolate (CMY-2-Ec) was isolated from a wound. Thirty days later, one CMY-33-producingE. coliisolate (CMY-33-Ec) was detected in a bronchoalveolar lavage fluid sample. Two weeks before the isolation of CMY-33-Ec, the patient received cefepime. CMY-33-Ecand CMY-2-Ecwere identical by repetitive extragenic palindromic-PCR (rep-PCR), being of hyperepidemic sequence type 131 (ST131) but showing different β-lactam MICs (e.g., cefepime MIC, 16 and ≤0.5 μg/ml for CMY-33-Ecand CMY-2-Ec, respectively). Identical CMY-2-Ecisolates were also found in a rectal swab. CMY-33 differs from CMY-2 by a Leu293-Ala294 deletion. Expressed inE. colistrain DH10B, both CMYs conferred resistance to ceftazidime (≥256 μg/ml), but the cefepime MICs were higher for CMY-33 than CMY-2 (8 versus 0.25 μg/ml, respectively). Thekcat/Kmor inhibitor complex inactivation (kinact)/Kiapp(μM−1s−1) indicated that CMY-33 possesses an extended-spectrum β-lactamase (ESBL)-like spectrum compared to that of CMY-2 (e.g., cefoxitin, 0.2 versus 0.4; ceftazidime, 0.2 versus not measurable; cefepime, 0.2 versus not measurable; and tazobactam, 0.0018 versus 0.0009, respectively). Using molecular modeling, we show that a widened active site (∼4-Å shift) may play a significant role in enhancing cefepime hydrolysis. This is the firstin vivodemonstration of a pAmpC that under cephalosporin treatment expands its substrate spectrum, resembling an ESBL. The prevalence of CMY-2-Ecisolates is rapidly increasing worldwide; therefore, awareness that cefepime treatment may select for resistant isolates is critical.

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Improved production of the non-native cofactor F420 in Escherichia coli

Scientific Reports ◽

10.1038/s41598-021-01224-3 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Mihir V. Shah ◽

Hadi Nazem-Bokaee ◽

James Antoney ◽

Suk Woo Kang ◽

Colin J. Jackson ◽

...

Keyword(s):

Escherichia Coli ◽

Expression System ◽

Carbon Sources ◽

Fold Increase ◽

High Yield ◽

Antibiotic Biosynthesis ◽

Bacterial Strains ◽

E Coli ◽

A Genome

AbstractThe deazaflavin cofactor F420 is a low-potential, two-electron redox cofactor produced by some Archaea and Eubacteria that is involved in methanogenesis and methanotrophy, antibiotic biosynthesis, and xenobiotic metabolism. However, it is not produced by bacterial strains commonly used for industrial biocatalysis or recombinant protein production, such as Escherichia coli, limiting our ability to exploit it as an enzymatic cofactor and produce it in high yield. Here we have utilized a genome-scale metabolic model of E. coli and constraint-based metabolic modelling of cofactor F420 biosynthesis to optimize F420 production in E. coli. This analysis identified phospho-enol pyruvate (PEP) as a limiting precursor for F420 biosynthesis, explaining carbon source-dependent differences in productivity. PEP availability was improved by using gluconeogenic carbon sources and overexpression of PEP synthase. By improving PEP availability, we were able to achieve a ~ 40-fold increase in the space–time yield of F420 compared with the widely used recombinant Mycobacterium smegmatis expression system. This study establishes E. coli as an industrial F420-production system and will allow the recombinant in vivo use of F420-dependent enzymes for biocatalysis and protein engineering applications.

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