scholarly journals Altered Substrate Selection of the Melibiose Transporter (MelY) of Enterobacter cloacae Involving Point Mutations in Leu-88, Leu-91, and Ala-182 That Confer Enhanced Maltose Transport

2003 ◽  
Vol 185 (12) ◽  
pp. 3672-3677 ◽  
Author(s):  
Steven G. Shinnick ◽  
Stephanie A. Perez ◽  
Manuel F. Varela
Keyword(s):  
Escherichia Coli ◽  
Point Mutations ◽  
Enterobacter Cloacae ◽  
Substrate Selection ◽  
Coupled Transport ◽  
Wild Type ◽  
Transmembrane Segment ◽  
Maltose Transport ◽  
Site Classes ◽  
Selection Of

ABSTRACT We isolated mutants of Escherichia coli HS4006 containing the melibiose-H+ symporter (MelY) from Enterobacter cloacae that had enhanced fermentation on 1% maltose MacConkey plates. DNA sequencing revealed three site classes of mutations: L-88-P, L-91-P, and A-182-P. The mutants L-88-P and L-91-P had 3.6- and 5.1-fold greater maltose uptake than the wild type and enhanced apparent affinities for maltose. Energy-coupled transport was defective for melibiose accumulation, but detectable maltose accumulation for the mutants indicated that active transport is dependent upon the substrate transported through the carrier. We conclude that the residues Leu-88, Leu-91 (transmembrane segment 3 [TMS-3]), and Ala-182 (TMS-6) of MelY mediate sugar selection. These data represent the first MelY mutations that confer changes in sugar selection.

scholarly journals ampR Gene Mutations That Greatly Increase Class C β-Lactamase Activity in Enterobacter cloacae

2000 ◽  
Vol 44 (3) ◽  
pp. 561-567 ◽  
Author(s):  
Akio Kuga ◽  
Ryoichi Okamoto ◽  
Matsuhisa Inoue
Keyword(s):  
Escherichia Coli ◽  
Clinical Setting ◽  
Point Mutations ◽  
Gene Mutations ◽  
Enterobacter Cloacae ◽  
Class C ◽  
Selection Of

ABSTRACT The ampC and ampR genes ofEnterobacter cloacae GN7471 were cloned into pMW218 to yield pKU403. Four mutant plasmids derived from pKU403 (pKU404, pKU405, pKU406, and pKU407) were isolated in an AmpD mutant ofEscherichia coli ML4953 by selection with ceftazidime or aztreonam. The β-lactamase activities expressed by pKU404, pKU405, pKU406, and pKU407 were about 450, 75, 160, and 160 times higher, respectively, than that expressed by the original plasmid, pKU403. These mutant plasmids all carried point mutations in theampR gene. In pKU404 and pKU405, Asp-135 was changed to Asn and Val, respectively. In both pKU406 and pKU407, Arg-86 was changed to Cys. The ease of selection of AmpR mutations at a frequency of about 10−6 in this study strongly suggests that derepressed strains, such as AmpD or AmpR mutants, could frequently emerge in the clinical setting.

scholarly journals MUTATIONS IN radA ARE RESPONSIBLE FOR ACQUIRED RADIO-RESISTANCE IN AN Escherichia coli STRAIN

2021 ◽  
Author(s):  
David Alcántara Díaz ◽  
Jorge Humberto Serment Guerrero ◽  
Gerardo Aguirre Escalona ◽  
Jorge Tonatiuh Ayala Sumuano
Keyword(s):  
Escherichia Coli ◽  
Gamma Radiation ◽  
Uv Light ◽  
Point Mutations ◽  
Coli Strain ◽  
Normal Strain ◽  
Wild Type ◽  
Different Strains ◽  
Strain Ab1157 ◽  
Selection Of

When bacteria are exposed to chronic or cyclic irradiation with ultraviolet (UV) light, it is observed that their resistance to this agent is increased by the selection of advantageous mutations under those conditions. UV light produces different damages in DNA, the repair of which is necessary to maintain the integrity of the genome. However, some damages can lead to such mutations when they are not properly repaired. In an earlier work, five subcultures of a wild-type Escherichia coli strain (PQ30) were cyclically irradiated with UV and different strains resistant to UV light and gamma radiation were obtained. In a preliminary mapping, different genes involved in their resistance to radiation were identified. In one of these strains, designated as IN801, the radA gene, the product of which is involved in recombinational DNA repair, was identified. In this work, cells from another wild-type strain (AB1157) were transformed with a plasmid (pUC19) that carries the radA gene from either PQ30 or IN801, in order to establish whether the radio-resistant phenotype can be transferred to a normal strain. Only cells that received the IN801 radA gene showed increased resistance to UV and gamma radiation. Further radA sequencing showed that the gene of IN801 acquired two-point mutations that replace two amino acids in the RadA protein, which most likely changed its enzymatic activities. These results confirm that radA participates in the radiation resistance of IN801.

Growth of Escherichia coli on glucosamine 6-phosphate: selection of a constitutive hexose phosphate transport system mutant

1978 ◽  
Vol 24 (3) ◽  
pp. 203-208
Author(s):  
George W. Dietz Jr.
Keyword(s):  
Escherichia Coli ◽  
Transport System ◽  
Phosphate Transport ◽  
Wild Type ◽  
Hexose Phosphate ◽  
Selection Of

Glucosamine 6-phosphate was found to be a substrate but not an inducer for the hexose phosphate transport system of Escherichia coli. Wild-type cells grow very poorly on glucosamine 6-phosphate. A mutant was selected that will grow rapidly on glucosamine 6-phosphate because it contains a constitutive hexose phosphate transport system.

scholarly journals REGULATION OF NEWLY EVOLVED ENZYMES. IV. DIRECTED EVOLUTION OF THE EBG REPRESSOR

Genetics ◽  
1978 ◽  
Vol 90 (4) ◽  
pp. 673-681
Author(s):  
Barry G Hall
Keyword(s):  
Escherichia Coli ◽  
Directed Evolution ◽  
Enzyme Synthesis ◽  
Regulatory Function ◽  
Wild Type ◽  
Selection For ◽  
Selection Of

ABSTRACT In Escherichia coli, the wild-type repressor of ebg (evolved β-galactosidase) enzyme synthesis, specified by the ebgR  + gene, responds very weakly to lactulose (fructose-β-D-galactopyranoside). Selection for a functional repressor that responds strongly to lactulose as an inducer reveals the existence of ebgR+L mutants, which occur spontaneously at a frequency of about 2 x 10-10. ebgR+L mutants are pleiotropic in that they specify ebg repressor with a greatly increased response to lactulose, lactose, galactose-arabinoside and methyl-galactoside as inducers. Selection of ebgR+L mutants is discussed within the framework of directed evolution of a regulatory function.

scholarly journals Why Does Escherichia coli Grow More Slowly on Glucosamine than on N-Acetylglucosamine? Effects of Enzyme Levels and Allosteric Activation of GlcN6P Deaminase (NagB) on Growth Rates

2005 ◽  
Vol 187 (9) ◽  
pp. 2974-2982 ◽  
Author(s):  
Laura I. Álvarez-Añorve ◽  
Mario L. Calcagno ◽  
Jacqueline Plumbridge
Keyword(s):  
Escherichia Coli ◽  
Growth Rates ◽  
Sugar Transport ◽  
Point Mutations ◽  
Sole Source ◽  
Wild Type ◽  
Phosphotransferase System ◽  
Allosteric Activation

ABSTRACT Wild-type Escherichia coli grows more slowly on glucosamine (GlcN) than on N-acetylglucosamine (GlcNAc) as a sole source of carbon. Both sugars are transported by the phosphotransferase system, and their 6-phospho derivatives are produced. The subsequent catabolism of the sugars requires the allosteric enzyme glucosamine-6-phosphate (GlcN6P) deaminase, which is encoded by nagB, and degradation of GlcNAc also requires the nagA-encoded enzyme, N-acetylglucosamine-6-phosphate (GlcNAc6P) deacetylase. We investigated various factors which could affect growth on GlcN and GlcNAc, including the rate of GlcN uptake, the level of induction of the nag operon, and differential allosteric activation of GlcN6P deaminase. We found that for strains carrying a wild-type deaminase (nagB) gene, increasing the level of the NagB protein or the rate of GlcN uptake increased the growth rate, which showed that both enzyme induction and sugar transport were limiting. A set of point mutations in nagB that are known to affect the allosteric behavior of GlcN6P deaminase in vitro were transferred to the nagB gene on the Escherichia coli chromosome, and their effects on the growth rates were measured. Mutants in which the substrate-induced positive cooperativity of NagB was reduced or abolished grew even more slowly on GlcN than on GlcNAc or did not grow at all on GlcN. Increasing the amount of the deaminase by using a nagC or nagA mutation to derepress the nag operon improved growth. For some mutants, a nagA mutation, which caused the accumulation of the allosteric activator GlcNAc6P and permitted allosteric activation, had a stronger effect than nagC. The effects of the mutations on growth in vivo are discussed in light of their in vitro kinetics.

scholarly journals Effect of point mutations in the lac promoter on transient and severe catabolite repression of the lac operon of Escherichia coli

1971 ◽  
Vol 123 (4) ◽  
pp. 579-584 ◽  
Author(s):  
M. D. Yudkin
Keyword(s):  
Escherichia Coli ◽  
Catabolite Repression ◽  
Point Mutations ◽  
Severe Form ◽  
Lac Operon ◽  
Wild Type ◽  
Diploid Strain ◽  
Transient Repression ◽  
Lac Promoter

1. Experiments were devised to show whether the point mutations L8 and L29 in the lac promoter alleviate transient repression. 2. Several recombinants were picked from matings between a single F−p+strain and Hfr strains carrying mutations L8 and L29. All of the 19 p−recombinants tested proved to suffer no transient repression, whereas all of the eight p+recombinants tested suffered prolonged transient repression. 3. A diploid strain was constructed in which more than 90% of the thiogalactoside transacetylase is synthesized from the episome with a wild-type lac promoter, whereas 100% of the β-galactosidase is synthesized from the chromosome with a promoter carrying mutation L8. In this diploid the synthesis of thiogalactoside transacetylase suffered transient repression but the synthesis of β-galactosidase did not. 4. Exactly similar results were obtained with a diploid strain in which the chromosomal promoter carried mutation L29. 5. The same diploid strains were used in experiments to show whether mutations L8 and L29 alleviate the severe catabolite repression caused by growth in glucose plus gluconate. In both strains glucose+gluconate repressed the synthesis of β-galactosidase much less than the synthesis of thiogalactoside transacetylase. 6. These and previously reported results can be explained by assuming (a) that both mutations L8 and L29 render the lac promoter partially, but not completely, insensitive to catabolite repression, and (b) that transient repression is an exceptionally severe form of catabolite repression.

scholarly journals Precise Excision of IS5 from the Intergenic Region between the fucPIK and the fucAO Operons and Mutational Control of fucPIK Operon Expression in Escherichia coli

2010 ◽  
Vol 192 (7) ◽  
pp. 2013-2019 ◽  
Author(s):  
Zhongge Zhang ◽  
Ming Ren Yen ◽  
Milton H. Saier
Keyword(s):  
Escherichia Coli ◽  
Insertion Site ◽  
Point Mutations ◽  
Wild Type ◽  
Sequence Element ◽  
Low Frequencies ◽  
Precise Excision ◽  
Wild Type Phenotype ◽  
Unique System ◽  
Operon Expression

ABSTRACT Excision of transposable genetic elements from host DNA occurs at low frequencies and is usually imprecise. A common insertion sequence element in Escherichia coli, IS5, has been shown to provide various benefits to its host by inserting into specific sites. Precise excision of this element had not previously been demonstrated. Using a unique system, the fucose (fuc) regulon, in which IS5 insertion and excision result in two distinct selectable phenotypes, we have demonstrated that IS5 can precisely excise from its insertion site, restoring the wild-type phenotype. In addition to precise excision, several “suppressor” insertion, deletion, and point mutations restore the wild-type Fuc+ phenotype to various degrees without IS5 excision. The possible bases for these observations are discussed.

scholarly journals Twelve-Transmembrane-Segment (TMS) Version (ΔTMS VII-VIII) of the 14-TMS Tet(L) Antibiotic Resistance Protein Retains Monovalent Cation Transport Modes but Lacks Tetracycline Efflux Capacity

2001 ◽  
Vol 183 (8) ◽  
pp. 2667-2671 ◽  
Author(s):  
Jie Jin ◽  
Arthur A. Guffanti ◽  
Catherine Beck ◽  
Terry A. Krulwich
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Monovalent Cation ◽  
Cation Transport ◽  
Wild Type ◽  
Transmembrane Segment ◽  
Transmembrane Segments ◽  
Resistance Protein ◽  
Transport Modes ◽  
Better Than

ABSTRACT A “Tet(L)-12” version of Tet(L), a tetracycline efflux protein with 14 transmembrane segments (TMS), was constructed by deletion of two central TMS. Tet(L)-12 catalyzed Na+/H+antiport and antiport with K+ as a coupling ion as well as or better than wild-type Tet(L) but exhibited no tetracycline-Me2+/H+ antiport inEscherichia coli vesicles.

scholarly journals NEGATIVE DOMINANT MUTATIONS OF THE uidR GENE IN ESCHERICHIA COLI: GENETIC PROOF FOR A COOPERATIVE REGULATION OF uidA EXPRESSION

Genetics ◽  
1986 ◽  
Vol 112 (2) ◽  
pp. 173-182
Author(s):  
Carlos Blanco ◽  
Paul Ritzenthaler ◽  
Mireille Mata-Gilsinger
Keyword(s):  
Escherichia Coli ◽  
Gene Dosage ◽  
Regulatory Gene ◽  
Active Form ◽  
Point Mutations ◽  
Multicopy Plasmid ◽  
Wild Type ◽  
Cooperative Process ◽  
Mutant Phenotypes ◽  
K 12

ABSTRACT The uidA gene is the first gene involved in the hexuronide-hexuronate pathway in Escherichia coli K-12 and is under the dual control of the uidR and uxuR encoded repressors. Point mutations affecting the uidR regulatory gene were sought to investigate the regulation of uidA. When the uidR mutant allele was on a multicopy plasmid and the wild-type allele was on the chromosome, some of the mutant phenotypes were dominant to the wild-type phenotype, indicating that the active form of the UidR repressor is multimeric. We have demonstrated that expression of the mutant phenotype is dependent on gene dosage. The dominance of the uidR allele was also sensitive to the presence of the wild-type uxuR allele in the cell. This behavior probably results from UidR-UxuR repressor interactions. A mechanism is proposed: we suggest that the UidR and UxuR repressors interact after their binding to the operator site of uidA; the binding of one regulatory molecule may facilitate the binding of the other one in a cooperative process.

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