scholarly journals Utilization of gluconate by Escherichia coli. Uptake of d-gluconate by a mutant impaired in gluconate kinase activity and by membrane vesicles derived therefrom*

1974 ◽  
Vol 140 (2) ◽  
pp. 193-203 ◽  
Author(s):  
J. M. Pouysségur ◽  
Pelin Faik ◽  
H. L. Kornberg
Keyword(s):  
Escherichia Coli ◽  
Kinase Activity ◽  
Membrane Vesicles ◽  
Dry Mass ◽  
Coli Strain ◽  
Whole Cells ◽  
E Coli ◽  
Phosphogluconate Dehydrogenase ◽  
Rate Limiting Step ◽  
Rate Limiting

1. From Escherichia coli strain K2.1.5c.8.9, which is devoid of 6-phosphogluconate dehydrogenase (gnd) and 6-phosphogluconate dehydratase (edd) activities, a mutant R6 was isolated that was tolerant to gluconate though still edd-, gnd-. 2. Measurements of the fate of labelled gluconate, of the conversion of gluconate into 6-phosphogluconate, and of the induction of gluconate kinase by the two organisms show that, although both inducibly form a gluconate-transport system, strain R6 is impaired in its ability to convert the gluconate thus taken up into 6-phosphogluconate; it was therefore used for study of the kinetics and energetics of gluconate uptake. 3. Suspensions of strain R6 induced for gluconate uptake took up this substrate via a ‘high affinity’ transport process, with Km about 10μm and Vmax. about 25nmol/min per mg dry mass; a ‘low affinity’ system demonstrated to occur in certain E. coli mutants was not induced under the conditions used in this work. 4. The uptake of gluconate was inhibited by lack of oxygen and by inhibitors of electron transport; such inhibitors also promoted the efflux of gluconate taken up. 5. Membrane vesicles prepared from strain R6 also manifested these properties when incubated with suitable electron donors, at rates similar to those observed with whole cells. 6. The results indicate that the active transport of gluconate into the cells is the rate-limiting step in gluconate utilization by E. coli, and that the mechanism of this process can be validly studied with membrane vesicles.

scholarly journals Quantitative analysis of proton-linked transport systems. The lactose permease of Escherichia coli

1979 ◽  
Vol 182 (3) ◽  
pp. 687-696 ◽  
Author(s):  
I R Booth ◽  
W J Mitchell ◽  
W A Hamilton
Keyword(s):  
Escherichia Coli ◽  
Membrane Vesicles ◽  
Transport Systems ◽  
Protonmotive Force ◽  
Lactose Permease ◽  
Whole Cells ◽  
E Coli ◽  
Ph Dependent ◽  
Ph Range ◽  
External Ph

Evidence is presented that lactose uptake into whole cells of Escherichia coli occurs by symport with a single proton over the range of external pH 6.5–7.7. The proton/lactose stoicheiometry has been measured directly over this pH range by comparison of the initial rates of proton and lactose uptake into anaerobic resting cell suspensions of E. coli ML308. Further, the relationship between the protonmotive force and lactose accumulation has been studied in E. coli ML308-225 over the range of external pH 5.9–8.7. At no point was the accumulation of the beta-galactoside in thermodynamic equilibrium with the protonmotive force. It is concluded that the concentration of lactose within the cell is governed by kinetic factors rather than pH-dependent changes in the proton/substrate stoicheiometry. The relevance of these findings to the model of pH-dependent proton/substrate stoicheiometries derived from studies with E. coli membrane vesicles is discussed.

A Zn(II)-translocating P-type ATPase from Proteus mirabilis

1998 ◽  
Vol 76 (5) ◽  
pp. 787-790 ◽  
Author(s):  
Christopher Rensing ◽  
Bharati Mitra ◽  
Barry P Rosen
Keyword(s):  
Escherichia Coli ◽  
Proteus Mirabilis ◽  
Membrane Vesicles ◽  
Coli Strain ◽  
E Coli ◽  
P Type ◽  
Zinc Resistance

A mutant of Proteus mirabilis had been previously isolated as defective in swarming. The mutation had been found to be in a gene related to the Escherichia coli zntA gene, which encodes the ZntA Zn(II)-translocating P-type ATPase. In this study the P. mirabilis genewas expressed in an E. coli strain in which the zntA gene had been disrupted. The P. mirabilis gene complemented the sensitivity to salts of zinc and cadmium. Everted membrane vesicles from the zntA-disrupted strain lost ATP-driven 65Zn(II) uptake. Membranes from the complemented strain had restored 65Zn(II) transport. These results demonstrate that the P. mirabilis homologue of ZntA is a Zn(II)-translocating P-type ATPase.Key words: zinc resistance, P-type ATPase, Proteus mirabilis.

scholarly journals Cloning and Heterologous Expression of an Enantioselective Amidase from Rhodococcus erythropolis Strain MP50

2002 ◽  
Vol 68 (7) ◽  
pp. 3279-3286 ◽  
Author(s):  
Sandra Trott ◽  
Sibylle Bürger ◽  
Carsten Calaminus ◽  
Andreas Stolz
Keyword(s):  
Nitrile Hydratase ◽  
Rhodococcus Erythropolis ◽  
Nitrogen Sources ◽  
Whole Cells ◽  
E Coli ◽  
Cell Extracts ◽  
Rate Limiting Step ◽  
Amide Hydrolysis ◽  
Rate Limiting ◽  
Amidase Gene

ABSTRACT The gene for an enantioselective amidase was cloned from Rhodococcus erythropolis MP50, which utilizes various aromatic nitriles via a nitrile hydratase/amidase system as nitrogen sources. The gene encoded a protein of 525 amino acids which corresponded to a protein with a molecular mass of 55.5 kDa. The deduced complete amino acid sequence showed homology to other enantioselective amidases from different bacterial genera. The nucleotide sequence approximately 2.5 kb upstream and downstream of the amidase gene was determined, but no indications for a structural coupling of the amidase gene with the genes for a nitrile hydratase were found. The amidase gene was carried by an approximately 40-kb circular plasmid in R. erythropolis MP50. The amidase was heterologously expressed in Escherichia coli and shown to hydrolyze 2-phenylpropionamide, α-chlorophenylacetamide, and α-methoxyphenylacetamide with high enantioselectivity; mandeloamide and 2-methyl-3-phenylpropionamide were also converted, but only with reduced enantioselectivity. The recombinant E. coli strain which synthesized the amidase gene was shown to grow with organic amides as nitrogen sources. A comparison of the amidase activities observed with whole cells or cell extracts of the recombinant E. coli strain suggested that the transport of the amides into the cells becomes the rate-limiting step for amide hydrolysis in recombinant E. coli strains.

scholarly journals Profiling of β-Lactam Selectivity for Penicillin-Binding Proteins in Escherichia coli Strain DC2

2015 ◽  
Vol 59 (5) ◽  
pp. 2785-2790 ◽  
Author(s):  
Ozden Kocaoglu ◽  
Erin E. Carlson
Keyword(s):  
Escherichia Coli ◽  
Binding Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Coli Strain ◽  
Bacterial Cell Division ◽  
Content Type ◽  
Penicillin Binding Proteins ◽  
Whole Cells ◽  
E Coli

ABSTRACTPenicillin-binding proteins (PBPs) are integral players in bacterial cell division, and their catalytic activities can be monitored with β-lactam-containing chemical probes. Compounds that target a single PBP could provide important information about the specific role(s) of each enzyme, making identification of such molecules important. We evaluated 22 commercially available β-lactams for inhibition of the PBPs in liveEscherichia colistrain DC2. Whole cells were titrated with β-lactam antibiotics and subsequently incubated with a fluorescent penicillin derivative, Bocillin-FL (Boc-FL), to label uninhibited PBPs. Protein visualization was accomplished by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation and fluorescent scanning. The examined β-lactams exhibited diverse PBP selectivities, with amdinocillin (mecillinam) showing selectivity for PBP2, aztreonam, piperacillin, cefuroxime, cefotaxime, and ceftriaxone for PBP3, and amoxicillin and cephalexin for PBP4. The remaining β-lactams did not block any PBPs in the DC2 strain ofE. colior inhibited more than one PBP at all examined concentrations in this Gram-negative organism.

scholarly journals A hybrid cyt c maturation system enhances the bioelectrical performance of engineered Escherichia coli by improving the rate-limiting step

2020 ◽  
Author(s):  
Lin Su ◽  
Tatsuya Fukushima ◽  
Caroline M. Ajo-Franklin
Keyword(s):  
Escherichia Coli ◽  
Electron Flux ◽  
Electrical Response ◽  
Shewanella Oneidensis ◽  
Electrical Performance ◽  
E Coli ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Cyt C ◽  
Rate Limiting

ABSTRACTBioelectronic devices can use electron flux to enable communication between biotic components and abiotic electrodes. We have modified Escherichia coli to electrically interact with electrodes by expressing the cytochrome c from Shewanella oneidensis MR-1. However, we observe inefficient electrical performance, which we hypothesize is due to the limited compatibility of the E. coli cytochrome c maturation (Ccm) systems with MR-1 cytochrome c. Here we test whether the bioelectronic performance of E. coli can be improved by constructing hybrid Ccm systems containing protein domains from both E. coli and S. oneidensis MR-1. The hybrid CcmH increased cytochrome c expression by increasing the abundance of CymA 60%, while only slightly changing the abundance of the other cytochromes c. Electrochemical measurements showed that the overall current from the hybrid ccm strain increased 121% relative to the wildtype ccm strain, with an electron flux per cell of 12.3 ± 0.3 fA·cell-1. Additionally, the hybrid ccm strain doubled its electrical response with the addition of exogenous flavin, and quantitative analysis of this demonstrates CymA is the rate-limiting step in this electron conduit. These results demonstrate that this hybrid Ccm system can enhance the bioelectrical performance of the cyt c expressing E. coli, allowing the construction of more efficient bioelectronic devices.

scholarly journals Aberrant Membrane Structures in Hypervesiculating Escherichia coli Strain ΔmlaEΔnlpI Visualized by Electron Microscopy

2021 ◽  
Vol 12 ◽  
Author(s):  
Yoshihiro Ojima ◽  
Tomomi Sawabe ◽  
Mao Nakagawa ◽  
Yuhei O. Tahara ◽  
Makoto Miyata ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Escherichia Coli ◽  
Outer Membrane ◽  
Fluorescent Protein ◽  
Gene Knockout ◽  
Single Gene ◽  
Membrane Vesicles ◽  
Coli Strain ◽  
Outer Membrane Vesicles ◽  
E Coli

Escherichia coli produces extracellular vesicles called outer membrane vesicles (OMVs) by releasing a part of its outer membrane. We previously reported that the combined deletion of nlpI and mlaE, related to envelope structure and phospholipid accumulation in the outer leaflet of the outer membrane, respectively, resulted in the synergistic increase of OMV production. In this study, the analysis of ΔmlaEΔnlpI cells using quick-freeze, deep-etch electron microscopy (QFDE-EM) revealed that plasmolysis occurred at the tip of the long axis in cells and that OMVs formed from this tip. Plasmolysis was also observed in the single-gene knockout mutants ΔnlpI and ΔmlaE. This study has demonstrated that plasmolysis was induced in the hypervesiculating mutant E. coli cells. Furthermore, intracellular vesicles and multilamellar OMV were observed in the ΔmlaEΔnlpI cells. Meanwhile, the secretion of recombinant green fluorescent protein (GFP) expressed in the cytosol of the ΔmlaEΔnlpI cells was more than 100 times higher than that of WT and ΔnlpI, and about 50 times higher than that of ΔmlaE in the OMV fraction, suggesting that cytosolic components were incorporated into outer-inner membrane vesicles (OIMVs) and released into the extracellular space. Additionally, QFDE-EM analysis revealed that ΔmlaEΔnlpI sacculi contained many holes noticeably larger than the mean radius of the peptidoglycan (PG) pores in wild-type (WT) E. coli. These results suggest that in ΔmlaEΔnlpI cells, cytoplasmic membrane materials protrude into the periplasmic space through the peptidoglycan holes and are released as OIMVs.

scholarly journals The effect of replacing the conserved active-site residues His-264, Asp-312 and Arg-314 on the binding and catalytic properties of Escherichia coli citrate synthase

1994 ◽  
Vol 300 (3) ◽  
pp. 765-770 ◽  
Author(s):  
W J Man ◽  
Y Li ◽  
C D O'Connor ◽  
D C Wilton
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Citrate Synthase ◽  
Salt Bridge ◽  
Site Directed Mutagenesis ◽  
Active Site Residues ◽  
E Coli ◽  
Rate Limiting Step ◽  
Rate Limiting

The first step in the overall catalytic mechanism of citrate synthase is the binding and polarization of oxaloacetate. Active-site residues Arg-314, Asp-312 and His-264 in Escherichia coli citrate synthase, which are involved in oxaloacetate binding, were converted by site-directed mutagenesis to Gln-314, Asn-312 and Asn-264 respectively. The R314Q and D312N mutants expressed negligible overall catalytic activity at pH 8.0, the normal assay pH, but substantial activities for the partial reactions that reflect the cleavage and hydrolysis of the substrate intermediate citryl-CoA. However, when the pH was lowered to 7.0, the overall reaction of the mutants became significant, in contrast to the wild-type enzyme, whereas the two mutants exhibited reduced activities for the partial reactions. This result is consistent with the existence of a rate-limiting step between the two partial reactions for these mutants that is pH-dependent. The Km for oxaloacetate for the two mutants was increased 10-fold and was paralleled by an increase in the Km for citryl-CoA, whereas the Km for acetyl-CoA was increased only 2-fold. Overall, there was a striking parallel between the results obtained for these two mutants, which suggests that they are functionally linked in the E. coli enzyme. The equivalent of these two residues form a salt bridge in the pig heart citrate synthase crystal structure. The H264N mutant, in which the amide nitrogen of asparagine should mimic the delta-nitrogen of histidine, showed negligible activity in terms of both overall and partial catalysis, which may result from a hindrance of conformational change upon oxaloacetate binding. The affinity of this mutant for oxaloacetate appeared to be greatly reduced when investigated using indirect fluorescence and chemical modification techniques.

scholarly journals Regulation of Expression of the TIR-Containing Protein C Gene of the Uropathogenic Escherichia coli Strain CFT073

Pathogens ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 549
Author(s):  
Julia Ittensohn ◽  
Jacqueline Hemberger ◽  
Hannah Griffiths ◽  
Maren Keller ◽  
Simone Albrecht ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protein C ◽  
Interleukin 1 ◽  
Coli Strain ◽  
Uropathogenic Escherichia Coli ◽  
Reporter Construct ◽  
Regulation Of Expression ◽  
E Coli ◽  
Strain Cft073 ◽  
Myeloid Differentiation Factor

The uropathogenic Escherichia coli strain CFT073 causes kidney abscesses in mice Toll/interleukin-1 receptor domain-containing protein C (TcpC) dependently and the corresponding gene is present in around 40% of E. coli isolates of pyelonephritis patients. It impairs the Toll-like receptor (TLR) signaling chain and the NACHT leucin-rich repeat PYD protein 3 inflammasome (NLRP3) by binding to TLR4 and myeloid differentiation factor 88 as well as to NLRP3 and caspase-1, respectively. Overexpression of the tcpC gene stopped replication of CFT073. Overexpression of several tcpC-truncation constructs revealed a transmembrane region, while its TIR domain induced filamentous bacteria. Based on these observations, we hypothesized that tcpC expression is presumably tightly controlled. We tested two putative promoters designated P1 and P2 located at 5′ of the gene c2397 and 5′ of the tcpC gene (c2398), respectively, which may form an operon. High pH and increasing glucose concentrations stimulated a P2 reporter construct that was considerably stronger than a P1 reporter construct, while increasing FeSO4 concentrations suppressed their activity. Human urine activated P2, demonstrating that tcpC might be induced in the urinary tract of infected patients. We conclude that P2, consisting of a 240 bp region 5′ of the tcpC gene, represents the major regulator of tcpC expression.

scholarly journals Pilot-Scale Production of Fatty Acid Ethyl Esters by an Engineered Escherichia coli Strain Harboring the p(Microdiesel) Plasmid

2010 ◽  
Vol 76 (13) ◽  
pp. 4560-4565 ◽  
Author(s):  
Yasser Elbahloul ◽  
Alexander Steinbüchel
Keyword(s):  
Escherichia Coli ◽  
Fatty Acid ◽  
Carbon Sources ◽  
Dry Mass ◽  
Pilot Scale ◽  
Coli Strain ◽  
Fatty Acid Ethyl Esters ◽  
Ethyl Esters ◽  
Scale Production ◽  
A Cell

ABSTRACT Fatty acid ethyl esters (FAEEs) were produced in this study by the use of an engineered Escherichia coli p(Microdiesel) strain. Four fed-batch pilot scale cultivations were carried out by first using glycerol as sole carbon source for biomass production before glucose and oleic acid were added as carbon sources. Cultivations yielded a cell density of up to 61 ± 3.1 g of cell dry mass (CDM) per liter and a maximal FAEE content of 25.4% ± 1.1% (wt/wt) of CDM.

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