Cloning and expression of the glycerol-3-phosphate transport genes of Escherichia coli

1978 ◽  
Vol 56 (6) ◽  
pp. 611-617 ◽  
Author(s):  
Joel H. Weiner ◽  
Elke Lohmeier ◽  
Anthony Schryvers
Keyword(s):  
Escherichia Coli ◽  
Membrane Vesicles ◽  
Phosphate Transport ◽  
Cloning And Expression ◽  
In Vitro Synthesis ◽  
Whole Cells ◽  
Glycerol 3 Phosphate Dehydrogenase ◽  
Plasmid Size ◽  
And Control

The two thousand Escherichia coli: Col E1 hybrid plasmid strains of the Clarke and Carbon colony bank (Clarke, L. &Carbon, J. (1976) Cell 9, 91–96) were screened by conjugation for those that correct the deficiency of a mutant unable to transport glycerol-3-phosphate. Six strains harbouring recombinant plasmids carrying the glpT region were identified and characterized with respect to plasmid size and transport properties. The initial rate of glycerol-3-phosphate transport in both whole cells and membrane vesicles prepared from such strains was elevated 3- to 10-fold over strains carrying random DNA inserts, whereas the Km of glycerol-3-phosphate transport was near 12 μM in both experimental and control strains. Four of the six glpT carrying plasmid strains demonstrated elevated levels of the anaerobic glycerol-3-phosphate dehydrogenase coded for by the neighbouring glpA gene.We have transferred the glpT hybrid plasmids into a minicell-producing strain of E. coli X1197 and have used the minicells for specific in vitro synthesis of plasmid-coded proteins. The glpT plasmids code for a 40 000 polypeptide which is localized in the periplasmic space. In addition, they code for a membrane-associated protein of 26 000 which may be the carrier polypeptide.

The anaerobic sn-glycerol-3-phosphate dehydrogenase: cloning and expression of the glpA gene of Escherichia coli and identification of the glpA products

1982 ◽  
Vol 60 (3) ◽  
pp. 224-231 ◽  
Author(s):  
Anthony Schryvers ◽  
Joel H. Weiner
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Phosphate Transport ◽  
Cloning And Expression ◽  
Relative Mass ◽  
Recombinant Plasmids ◽  
Sds Page ◽  
Glycerol 3 Phosphate Dehydrogenase ◽  
Definition Of ◽  
Different Strains

The expression of recombinant plasmids carrying the glpA gene (anaerobic glycerol-3-phosphate dehydrogenase) and the closely linked glpT gene (glycerol 3-phosphate transport) were studied under aerobic and anaerobic conditions. When the pattern of expression of enzymatic activity in different strains was compared with sodium dodecyl sulphate – polyacrylamide gel electrophoresis (SDS–PAGE) analysis from the same cells the glpA products were identified. Two polypeptides of 62 000 and 43 000 relative mass correlated with enzymatic activity.Five recombinant plasmids that contained one or both of the glpT or glpA genes were isolated and subjected to restriction endonuclease cleavage analysis. The regions of overlap from the inserts in these plasmids allowed definition of the regions of DNA containing the glpT and glpA genes. SDS–PAGE analysis revealed a partial product of the glpA locus from one plasmid, pLC42-17, which allowed more precise definition of the glpA locus on the physical DNA map and prediction of the direction of transcription.

Effect of thyroxine administration on phosphate transport across renal cortical brush border membrane

1984 ◽  
Vol 246 (2) ◽  
pp. F133-F139 ◽  
Author(s):  
R. E. Espinosa ◽  
M. J. Keller ◽  
A. N. Yusufi ◽  
T. P. Dousa
Keyword(s):  
Brush Border ◽  
Brush Border Membrane ◽  
Renal Cortex ◽  
Membrane Vesicles ◽  
Phosphate Transport ◽  
Treated Group ◽  
Renal Tubular Reabsorption ◽  
Renal Tubular ◽  
And Control ◽  
Cortical Slices

Previous studies indicate that in hyperthyroid states the renal tubular reabsorption of phosphate is enhanced. To determine the cellular basis of this phenomenon, we investigated the effect of L-thyroxine (T4) administration on Pi transport across brush border membrane vesicles (BBMV) from rat renal cortex. Rats were thyroparathyroidectomized, fed a diet containing 1.2% phosphate, and treated intraperitoneally for 6 days with 200 micrograms T4 X 100 g body wt-1 X day-1. At the end of the treatment period, the rats had a significantly (+ delta 25%) elevated plasma Pi and a slightly decreased plasma Ca compared with controls. The renal clearance of Pi was not different between the two groups. Na+ gradient-dependent uptake of 32Pi by BBMV from renal cortex was significantly enhanced in T4-treated rats. BBMV uptake of 32Pi in the absence of Na+ -gradient as well as Na+ gradient-dependent uptake of D-[3H]glucose and L-[3H]proline did not differ between BBMV from T4-treated and control rats. Kinetic analysis showed that the Na+ gradient-dependent 32Pi transport system in BBMV from T4-treated rats had a significantly increased Vmax compared with controls (5.2 +/- 0.4 vs. 4.1 +/- 0.4 nmol Pi X 30 s-1 X mg protein-1) and also a slightly higher affinity for Pi (apparent Km in controls, 95 +/- 9; in T4-treated, 78 +/- 8 microM). Gluconeogenesis in cortical slices was not significantly different between T4-treated rats and controls. Specific activities of alkaline phosphatase and gamma-glutamyltransferase were significantly lower in BBMV from the T4-treated group compared with controls.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Repression of the Inner Membrane Lipoprotein NlpA by Rns in Enterotoxigenic Escherichia coli

2006 ◽  
Vol 189 (5) ◽  
pp. 1627-1632 ◽  
Author(s):  
Maria D. Bodero ◽  
M. Carolina Pilonieta ◽  
George P. Munson
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Enterotoxigenic Escherichia Coli ◽  
Start Codon ◽  
Inner Membrane ◽  
E Coli ◽  
Inner Membrane Protein

ABSTRACT The expression of the inner membrane protein NlpA is repressed by the enterotoxigenic Escherichia coli (ETEC) virulence regulator Rns, a member of the AraC/XylS family. The Rns homologs CfaD from ETEC and AggR from enteroaggregative E. coli also repress expression of nlpA. In vitro DNase I and potassium permanganate footprinting revealed that Rns binds to a site overlapping the start codon of nlpA, preventing RNA polymerase from forming an open complex at nlpAp. A second Rns binding site between positions −152 and −195 relative to the nlpA transcription start site is not required for repression. NlpA is not essential for growth of E. coli under laboratory conditions, but it does contribute to the biogenesis of outer membrane vesicles. As outer membrane vesicles have been shown to contain ETEC heat-labile toxin, the repression of nlpA may be an indirect mechanism through which the virulence regulators Rns and CfaD limit the release of toxin.

In vitro synthesis of β-galactosidase by membrane fractions of Escherichia coli

1970 ◽  
Vol 48 (8) ◽  
pp. 893-907 ◽  
Author(s):  
W. Chefurka ◽  
A. Yapo ◽  
B. Nisman
Keyword(s):  
Escherichia Coli ◽  
Actinomycin D ◽  
Heavy Fraction ◽  
Synthetic Activity ◽  
In Vitro Synthesis ◽  
Absolute Requirement ◽  
Discontinuous Sucrose Gradient ◽  
Reconstituted System ◽  
High Concentrations

The induction of β-galactosidase by a membranous fraction P1, prepared by digitonin lysis of spheroplasts of Escherichia coli, was studied in vitro. Electron micrographs of P1 show it to be a heterogeneous mixture of smooth vesicles, rough vesicles, rough vesicles attached to DNA, and ribosomes attached to DNA. P1 was subfractionated by differential centrifugation into an active heavy fraction, P4, and a relatively inactive light fraction, Pm. The P4 fraction consisted mainly of rough vesicles while the Pm fraction consisted mainly of smooth vesicles, but also of some rough vesicles. These vesicles of the Pm fraction were further separated by discontinuous sucrose gradient centrifugation.The induction of β-galactosidase by P1, P4, and Pm fractions was not related to mild contamination by unbroken viable spheroplasts. It was only partially sensitive to DNase and RNase. High concentrations of actinomycin D were required for complete inhibition of activity. This suggests that the transcription and translation components were shielded by the membranes. The synthetic activity of Pm was enhanced by fortification with DNA and/or S30. Only lac-containing DNA was active. The induction of β-galactosidase by this reconstituted system showed an absolute requirement for Pm membranes and for the inducer but only a partial requirement for nucleoside triphosphates. It was completely inhibited by puromycin and chloramphenicol.

scholarly journals Identification of a Two-Partner Secretion Locus of Enterotoxigenic Escherichia coli

2006 ◽  
Vol 74 (4) ◽  
pp. 2245-2258 ◽  
Author(s):  
James M. Fleckenstein ◽  
Koushik Roy ◽  
Julia F. Fischer ◽  
Michael Burkitt
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
Epithelial Cells ◽  
High Molecular Weight ◽  
Cloning And Expression ◽  
Peptide Sequence ◽  
Enterotoxigenic Escherichia Coli ◽  
Virulence Plasmid ◽  
E Coli

ABSTRACT Enterotoxigenic Escherichia coli (ETEC) remains a formidable cause of diarrheal illness worldwide. At present, there is no vaccine that provides broad-based protection against ETEC. A ′phoA-based self-cloning mutagenesis system, TnphoA.ts, employed to identify novel ETEC surface antigens, led to identification of an ETEC two-partner secretion locus (etpBAC) on the pCS1 virulence plasmid of prototype strain H10407. Cloning and expression of etpBAC in recombinant E. coli LMG194(pJY019) resulted in secretion of a high-molecular-weight (HMW) glycosylated exoprotein. This glycoprotein, EtpA, exhibits linear peptide sequence and predicted structural homologies with known HMW adhesins produced by other two-partner secretion loci. Antibodies directed against recombinant EtpA (anti-rEtpA.6H) recognized an HMW protein in culture supernatants of ETEC strains H10407 and LMG194(pJY019) but not in culture supernatant of strain H10407-P, which lacks the 92-kb pCS1 plasmid, or an isogenic etpA mutant. etpA mutants were deficient in adherence to intestinal epithelial cells in vitro, and anti-rEtpA.6H antibodies inhibited association of H10407 with target epithelial cells. Cloning and expression of etpB in recombinant E. coli were sufficient to confer adherence. Screening of multiple ETEC isolates for the etpBAC locus by colony hybridization and by EtpA immunoblotting suggested that EtpA is one of the most common antigens secreted by these pathogens. Together, these results indicate that the newly identified ETEC two-partner secretion locus directs the secretion of a high-molecular-weight glycosylated protein, EtpA, that in concert with the putative EtpB transporter participates in adherence of H10407 to epithelial cells, thereby expanding the repertoire of potential ETEC virulence proteins and vaccine candidates.

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