REGULATION OF ACETOHYDROXY ACID SYNTHETASE IN STREPTOMYCIN-DEPENDENT ESCHERICHIA COLI

1966 ◽  
Vol 44 (5) ◽  
pp. 599-606 ◽  
Author(s):  
W. J. Polglase
Keyword(s):  
Escherichia Coli ◽  
Minimal Medium ◽  
Specific Activity ◽  
Product Inhibition ◽  
Biosynthetic Pathways ◽  
Regulatory Effect ◽  
Antibiotic Concentration ◽  
E Coli ◽  
End Products ◽  
Glutamic Dehydrogenase

The formation of acetolactate from pyruvate by extracts of streptomycin-dependent Escherichia coli was inhibited, competitively, by L-valine, thus establishing the potential in the dependent mutant for regulation of the activity of acetohydroxy acid synthetase through end-product inhibition. Furthermore, the level of acetohydroxy acid synthetase was decreased in streptomycin-dependent E. coli by growth in minimal medium to which had been added the end products of the biosynthetic pathways initiated by this enzyme, thus establishing the potential in dependent cells for regulation of acetohydroxy acid synthetase through repression. In contrast, the specific activity of acetohydroxy acid synthetase in extracts of streptomycin-dependent E. coli increased in proportion to the concentration of antibiotic (dihydrostreptomycin) present in the medium during growth, thereby indicating a positive regulatory effect of dihydrostreptomycin on the formation of this enzyme. The formation of two enzymes used as monitors, glucokinase, and glutamic dehydrogenase, was not affected by variation in antibiotic concentration.

scholarly journals Repression by glucose of acetohydroxy acid synthetase in Escherichia coli B

1969 ◽  
Vol 111 (3) ◽  
pp. 273-278 ◽  
Author(s):  
M. B. Coukell ◽  
W. J. Polglase
Keyword(s):  
Escherichia Coli ◽  
Catabolite Repression ◽  
Product Inhibition ◽  
Biosynthetic Pathways ◽  
Single System ◽  
Ph Optimum ◽  
Isoleucine Leucine ◽  
E Coli ◽  
End Products ◽  
Escherichia Coli B

Acetolactate formation in Escherichia coli B results from the activity of a single system, acetohydroxy acid synthetase, which has a pH optimum of 8·0 and is sensitive to end-product inhibition by l-valine. Acetohydroxy acid synthetase was found to be subject to catabolite repression, and the nature and concentration of the carbon source had a greater effect on the formation of the enzyme than had the known end products (valine, isoleucine, leucine and pantothenate) of the biosynthetic pathways of which this enzyme is a member. The results suggest that acetohydroxy acid synthetase may play an amphibolic role in E. coli B.

scholarly journals Identification and Function of the pdxY Gene, Which Encodes a Novel Pyridoxal Kinase Involved in the Salvage Pathway of Pyridoxal 5′-Phosphate Biosynthesis in Escherichia coli K-12

1998 ◽  
Vol 180 (7) ◽  
pp. 1814-1821 ◽  
Author(s):  
Yong Yang ◽  
Ho-Ching Tiffany Tsui ◽  
Tsz-Kwong Man ◽  
Malcolm E. Winkler
Keyword(s):  
Escherichia Coli ◽  
De Novo ◽  
Specific Activity ◽  
Salvage Pathway ◽  
Pyridoxal Kinase ◽  
Triple Mutant ◽  
Reading Frame ◽  
E Coli ◽  
Specific Activities ◽  
K 12

ABSTRACT pdxK encodes a pyridoxine (PN)/pyridoxal (PL)/pyridoxamine (PM) kinase thought to function in the salvage pathway of pyridoxal 5′-phosphate (PLP) coenzyme biosynthesis. The observation that pdxK null mutants still contain PL kinase activity led to the hypothesis that Escherichia coli K-12 contains at least one other B6-vitamer kinase. Here we support this hypothesis by identifying the pdxY gene (formally, open reading frame f287b) at 36.92 min, which encodes a novel PL kinase. PdxY was first identified by its homology to PdxK in searches of the complete E. coli genome. Minimal clones of pdxY + overexpressed PL kinase specific activity about 10-fold. We inserted an omega cassette intopdxY and crossed the resultingpdxY::ΩKanr mutation into the bacterial chromosome of a pdxB mutant, in which de novo PLP biosynthesis is blocked. We then determined the growth characteristics and PL and PN kinase specific activities in extracts ofpdxK and pdxY single and double mutants. Significantly, the requirement of the pdxB pdxK pdxY triple mutant for PLP was not satisfied by PL and PN, and the triple mutant had negligible PL and PN kinase specific activities. Our combined results suggest that the PL kinase PdxY and the PN/PL/PM kinase PdxK are the only physiologically important B6vitamer kinases in E. coli and that their function is confined to the PLP salvage pathway. Last, we show thatpdxY is located downstream from pdxH (encoding PNP/PMP oxidase) and essential tyrS (encoding aminoacyl-tRNATyr synthetase) in a multifunctional operon.pdxY is completely cotranscribed with tyrS, but about 92% of tyrS transcripts terminate at a putative Rho-factor-dependent attenuator located in thetyrS-pdxY intercistronic region.

scholarly journals Expression of Active Human Tissue-Type Plasminogen Activator in Escherichia coli

1998 ◽  
Vol 64 (12) ◽  
pp. 4891-4896 ◽  
Author(s):  
Ji Qiu ◽  
James R. Swartz ◽  
George Georgiou
Keyword(s):  
Escherichia Coli ◽  
Plasminogen Activator ◽  
Disulfide Bonds ◽  
Specific Activity ◽  
Active Form ◽  
Tissue Type ◽  
Heterologous Proteins ◽  
E Coli ◽  
Disulfide Isomerases

ABSTRACT The formation of native disulfide bonds in complex eukaryotic proteins expressed in Escherichia coli is extremely inefficient. Tissue plasminogen activator (tPA) is a very important thrombolytic agent with 17 disulfides, and despite numerous attempts, its expression in an active form in bacteria has not been reported. To achieve the production of active tPA in E. coli, we have investigated the effect of cooverexpressing native (DsbA and DsbC) or heterologous (rat and yeast protein disulfide isomerases) cysteine oxidoreductases in the bacterial periplasm. Coexpression of DsbC, an enzyme which catalyzes disulfide bond isomerization in the periplasm, was found to dramatically increase the formation of active tPA both in shake flasks and in fermentors. The active protein was purified with an overall yield of 25% by using three affinity steps with, in sequence, lysine-Sepharose, immobilized Erythrina caffra inhibitor, and Zn-Sepharose resins. After purification, approximately 180 μg of tPA with a specific activity nearly identical to that of the authentic protein can be obtained per liter of culture in a high-cell-density fermentation. Thus, heterologous proteins as complex as tPA may be produced in an active form in bacteria in amounts suitable for structure-function studies. In addition, these results suggest the feasibility of commercial production of extremely complex proteins inE. coli without the need for in vitro refolding.

scholarly journals Modification by an R determinant for streptomycin of hissd phenotype in a mutant strain of Escherichia coli B

1968 ◽  
Vol 12 (2) ◽  
pp. 109-116 ◽  
Author(s):  
A. M. Molina ◽  
L. Calegari ◽  
G. Conte
Keyword(s):  
Escherichia Coli ◽  
Cell Membrane ◽  
Mutant Strain ◽  
Minimal Medium ◽  
Specific Requirement ◽  
Resistance Level ◽  
E Coli ◽  
Level Characteristic ◽  
Escherichia Coli B

When an R determinant for streptomycin is transferred into a conditionally streptomycin-dependent E. coli B mutant—which requires in minimal medium either histidine or streptomycin—the latter behaves like a histidineless strain. This phenotype modification shows that the repairing action of streptomycin is prevented. The specific requirement of the strain is not now replaced even by streptomycin concentrations up to 10000 µg/ml at which the conditionally streptomycin-dependent mutant could originally grow, and which are well beyond the resistance level characteristic of the R determinant itself. These data seem to suggest that a reduction in permeability of the cell membrane cannot be held responsible for the phenomenon observed.

scholarly journals Properties of human testis-specific lactate dehydrogenase expressed from Escherichia coli

1991 ◽  
Vol 273 (3) ◽  
pp. 587-592 ◽  
Author(s):  
K M LeVan ◽  
E Goldberg
Keyword(s):  
Escherichia Coli ◽  
Lactate Dehydrogenase ◽  
Prokaryotic Expression ◽  
Specific Activity ◽  
Human Testis ◽  
Reading Frame ◽  
E Coli ◽  
Heat Stable ◽  
Prokaryotic Expression Vector ◽  
Tac Promoter

The cDNA encoding the C4 isoenzyme of lactate dehydrogenase (LDH-C4) was engineered for expression in Escherichia coli. The Ldh-c open reading frame was constructed as a cassette for production of the native protein. The modified Ldh-c cDNA was subcloned into the prokaryotic expression vector pKK223-3. Transformed E. coli cells were grown to mid-exponential phase, and induced with isopropyl beta-D-thiogalactopyranoside for positive regulation of the tac promoter. Induced cells expressed the 35 kDa subunit, which spontaneously formed the enzymically active 140 kDa tetramer. Human LDH-C4 was purified over 200-fold from litre cultures of cells by AMP and oxamate affinity chromatography to a specific activity of 106 units/mg. The enzyme was inhibited by pyruvate concentrations above 0.3 mM, had a Km for pyruvate of 0.03 mM, a turnover number (nmol of NADH oxidized/mol of LDH-C4 per min at 25 degrees C) of 14,000 and was heat-stable.

scholarly journals The Putrescine Importer PuuP of Escherichia coli K-12

2009 ◽  
Vol 191 (8) ◽  
pp. 2776-2782 ◽  
Author(s):  
Shin Kurihara ◽  
Yuichi Tsuboi ◽  
Shinpei Oda ◽  
Hyeon Guk Kim ◽  
Hidehiko Kumagai ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Gene Cluster ◽  
Growth Phase ◽  
Exponential Growth ◽  
Minimal Medium ◽  
Exponential Growth Phase ◽  
E Coli ◽  
Genes Encoding ◽  
K 12 ◽  
Utilization Pathway

ABSTRACT The Puu pathway is a putrescine utilization pathway involving gamma-glutamyl intermediates. The genes encoding the enzymes of the Puu pathway form a gene cluster, the puu gene cluster, and puuP is one of the genes in this cluster. In Escherichia coli, three putrescine importers, PotFGHI, PotABCD, and PotE, were discovered in the 1990s and have been studied; however, PuuP had not been discovered previously. This paper shows that PuuP is a novel putrescine importer whose kinetic parameters are equivalent to those of the polyamine importers discovered previously. A puuP + strain absorbed up to 5 mM putrescine from the medium, but a ΔpuuP strain did not. E. coli strain MA261 has been used in previous studies of polyamine transporters, but PuuP had not been identified previously. It was revealed that the puuP gene of MA261 was inactivated by a point mutation. When E. coli was grown on minimal medium supplemented with putrescine as the sole carbon or nitrogen source, only PuuP among the polyamine importers was required. puuP was expressed strongly when putrescine was added to the medium or when the puuR gene, which encodes a putative repressor, was deleted. When E. coli was grown in M9-tryptone medium, PuuP was expressed mainly in the exponential growth phase, and PotFGHI was expressed independently of the growth phase.

scholarly journals Cloning, Nucleotide Sequence, and Overexpression of the l-Rhamnose Isomerase Gene from Pseudomonas stutzeri in Escherichia coli

2004 ◽  
Vol 70 (6) ◽  
pp. 3298-3304 ◽  
Author(s):  
Khim Leang ◽  
Goro Takada ◽  
Akihiro Ishimura ◽  
Masashi Okita ◽  
Ken Izumori
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Specific Activity ◽  
Sodium Dodecyl ◽  
Pseudomonas Stutzeri ◽  
Fold Increase ◽  
E Coli ◽  
Apparent Homogeneity ◽  
Volumetric Yield

ABSTRACT The gene encoding l-rhamnose isomerase (l-RhI) from Pseudomonas stutzeri was cloned into Escherichia coli and sequenced. A sequence analysis of the DNA responsible for the l-RhI gene revealed an open reading frame of 1,290 bp coding for a protein of 430 amino acid residues with a predicted molecular mass of 46,946 Da. A comparison of the deduced amino acid sequence with sequences in relevant databases indicated that no significant homology has previously been identified. An amino acid sequence alignment, however, suggested that the residues involved in the active site of l-RhI from E. coli are conserved in that from P. stutzeri. The l-RhI gene was then overexpressed in E. coli cells under the control of the T5 promoter. The recombinant clone, E. coli JM109, produced significant levels of l-RhI activity, with a specific activity of 140 U/mg and a volumetric yield of 20,000 U of soluble enzyme per liter of medium. This reflected a 20-fold increase in the volumetric yield compared to the value for the intrinsic yield. The recombinant l-RhI protein was purified to apparent homogeneity on the basis of three-step chromatography. The purified recombinant enzyme showed a single band with an estimated molecular weight of 42,000 in a sodium dodecyl sulfate-polyacrylamide gel. The overall enzymatic properties of the purified recombinant l-RhI protein were the same as those of the authentic one, as the optimal activity was measured at 60�C within a broad pH range from 5.0 to 11.0, with an optimum at pH 9.0.

scholarly journals Pyruvate Formate Lyase and Acetate Kinase Are Essential for Anaerobic Growth of Escherichia coli on Xylose

2004 ◽  
Vol 186 (22) ◽  
pp. 7593-7600 ◽  
Author(s):  
Adnan Hasona ◽  
Youngnyun Kim ◽  
F. G. Healy ◽  
L. O. Ingram ◽  
K. T. Shanmugam
Keyword(s):  
Escherichia Coli ◽  
Minimal Medium ◽  
High Capacity ◽  
Redox Balance ◽  
Anaerobic Growth ◽  
Acetate Kinase ◽  
Anaerobic Conditions ◽  
Pyruvate Formate Lyase ◽  
E Coli ◽  
Xylose Transport

ABSTRACT During anaerobic growth of bacteria, organic intermediates of metabolism, such as pyruvate or its derivatives, serve as electron acceptors to maintain the overall redox balance. Under these conditions, the ATP needed for cell growth is derived from substrate-level phosphorylation. In Escherichia coli, conversion of glucose to pyruvate yields 2 net ATPs, while metabolism of a pentose, such as xylose, to pyruvate only yields 0.67 net ATP per xylose due to the need for one (each) ATP for xylose transport and xylulose phosphorylation. During fermentative growth, E. coli produces equimolar amounts of acetate and ethanol from two pyruvates, and these reactions generate one additional ATP from two pyruvates (one hexose equivalent) while still maintaining the overall redox balance. Conversion of xylose to acetate and ethanol increases the net ATP yield from 0.67 to 1.5 per xylose. An E. coli pfl mutant lacking pyruvate formate lyase cannot convert pyruvate to acetyl coenzyme A, the required precursor for acetate and ethanol production, and could not produce this additional ATP. E. coli pfl mutants failed to grow under anaerobic conditions in xylose minimal medium without any negative effect on their survival or aerobic growth. An ackA mutant, lacking the ability to generate ATP from acetyl phosphate, also failed to grow in xylose minimal medium under anaerobic conditions, confirming the need for the ATP produced by acetate kinase for anaerobic growth on xylose. Since arabinose transport by AraE, the low-affinity, high-capacity, arabinose/H+ symport, conserves the ATP expended in pentose transport by the ABC transporter, both pfl and ackA mutants grew anaerobically with arabinose. AraE-based xylose transport, achieved after constitutively expressing araE, also supported the growth of the pfl mutant in xylose minimal medium. These results suggest that a net ATP yield of 0.67 per pentose is only enough to provide for maintenance energy but not enough to support growth of E. coli in minimal medium. Thus, pyruvate formate lyase and acetate kinase are essential for anaerobic growth of E. coli on xylose due to energetic constraints.

REGULATION OF ACETOHYDROXY ACID SYNTHETASE IN STREPTOMYCIN-DEPENDENT ESCHERICHIA COLI K-12

1966 ◽  
Vol 44 (5) ◽  
pp. 607-612 ◽  
Author(s):  
W. J. Polglase
Keyword(s):  
Escherichia Coli ◽  
Optical Density ◽  
Density Increase ◽  
Regulatory Effect ◽  
Michaelis Constant ◽  
E Coli ◽  
K 12 ◽  
Positive Effect

The formation of acetohydroxy acid (AHA) synthetase in streptomycin-dependent Escherichia coli K-12 as a function of antibiotic (dihydrostreptomycin) concentration can be expressed by a Michaelis constant (KM = 2 × 10−4M). Valine exerts end-product repression (partial) of AHA synthetase in competition with the positive regulatory effect of dihydrostreptomycin. In dependent E. coli K-12, growth (as measured by optical density increase) correlated closely with the formation of AHA synthetase. The observed positive effect of dihydrostreptomycin on AHA synthetase formation suggests that derepression of this enzyme may be obligatory in antibiotic-dependent cells.

scholarly journals A fourth Escherichia coli gene system with the potential to evolve beta-glucoside utilization.

Genetics ◽  
1988 ◽  
Vol 119 (3) ◽  
pp. 485-490
Author(s):  
L L Parker ◽  
B G Hall
Keyword(s):  
Escherichia Coli ◽  
Liquid Medium ◽  
Adaptive Evolution ◽  
Minimal Medium ◽  
Artificial Substrate ◽  
Second Step ◽  
Wild Type ◽  
E Coli ◽  
Gene System ◽  
Single Organism

Abstract Escherichia coli K12 is being used to study the potential for adaptive evolution that is present in the genome of a single organism. Wild-type E. coli K12 do not utilize any of the beta-glucoside sugars arbutin, salicin or cellobiose. It has been shown that mutations at three cryptic loci allow utilization of these sugars. Mutations in the bgl operon allow inducible growth on arbutin and salicin while cel mutations allow constitutive utilization of cellobiose as well as arbutin and salicin. Mutations in a third cryptic locus, arbT, allow the transport of arbutin. A salicin+ arbutin+ cellobiose+ mutant has been isolated from a strain which is deleted for the both the bgl and cel operons. Because the mutant utilized salicin and cellobiose as well as arbutin, it is unlikely it is the result of a mutation in arbT. A second step mutant exhibited enhanced growth on salicin and a third step mutant showed better growth on cellobiose. A fourfold level of induction in response to arbutin and a twofold level of induction in response to salicin was observed when these mutants were assayed on the artificial substrate p-nitrophenyl-beta-D-glucoside. Although growth on cellobiose minimal medium can be detected after prolonged periods of time, these strains are severely inhibited by cellobiose in liquid medium. This system has been cloned and does not hybridize to either bgl or cel specific probes. We have designated this gene system the sac locus. The sac locus is a fourth set of genes with the potential for evolving to provide beta-glucoside utilization.

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