scholarly journals Feedback Inhibition of Chorismate Mutase/Prephenate Dehydrogenase (TyrA) of Escherichia coli: Generation and Characterization of Tyrosine-Insensitive Mutants

2005 ◽  
Vol 71 (11) ◽  
pp. 7224-7228 ◽  
Author(s):  
Tina Lütke-Eversloh ◽  
Gregory Stephanopoulos
Keyword(s):  
Escherichia Coli ◽  
Dna Sequences ◽  
Molecular Level ◽  
Feedback Inhibition ◽  
Feedback Regulation ◽  
The Other ◽  
Chorismate Mutase ◽  
Amino Acid Substitutions ◽  
Prephenate Dehydrogenase

ABSTRACT In order to get insights into the feedback regulation by tyrosine of the Escherichia coli chorismate mutase/prephenate dehydrogenase (CM/PDH), which is encoded by the tyrA gene, feedback-inhibition-resistant (fbr) mutants were generated by error-prone PCR. The tyrA fbr mutants were selected by virtue of their resistance toward m-fluoro-d,l-tyrosine, and seven representatives were characterized on the biochemical as well as on the molecular level. The PDH activities of the purified His6-tagged TyrA proteins exhibited up to 35% of the enzyme activity of TyrAWT, but tyrosine did not inhibit the mutant PDH activities. On the other hand, CM activities of the TyrAfbr mutants were similar to those of the TyrAWT protein. Analyses of the DNA sequences of the tyrA genes revealed that tyrA fbr contained amino acid substitutions either at Tyr263 or at residues 354 to 357, indicating that these two sites are involved in the feedback inhibition by tyrosine.

scholarly journals Metabolic Engineering of Escherichia coli for l-Tyrosine Production by Expression of Genes Coding for the Chorismate Mutase Domain of the Native Chorismate Mutase-Prephenate Dehydratase and a Cyclohexadienyl Dehydrogenase from Zymomonas mobilis

2008 ◽  
Vol 74 (10) ◽  
pp. 3284-3290 ◽  
Author(s):  
María I. Chávez-Béjar ◽  
Alvaro R. Lara ◽  
Hezraí López ◽  
Georgina Hernández-Chávez ◽  
Alfredo Martinez ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Metabolic Engineering ◽  
Zymomonas Mobilis ◽  
Feedback Inhibition ◽  
Chorismate Mutase ◽  
Prephenate Dehydratase ◽  
E Coli ◽  
Prephenate Dehydrogenase ◽  
Expression Of Genes ◽  
Engineering Strategy

ABSTRACT The expression of the feedback inhibition-insensitive enzyme cyclohexadienyl dehydrogenase (TyrC) from Zymomonas mobilis and the chorismate mutase domain from native chorismate mutase-prephenate dehydratase (PheACM) from Escherichia coli was compared to the expression of native feedback inhibition-sensitive chorismate mutase-prephenate dehydrogenase (CM-TyrAp) with regard to the capacity to produce l-tyrosine in E. coli strains modified to increase the carbon flow to chorismate. Shake flask experiments showed that TyrC increased the yield of l-tyrosine from glucose (Y l-Tyr/Glc ) by 6.8-fold compared to the yield obtained with CM-TyrAp. In bioreactor experiments, a strain expressing both TyrC and PheACM produced 3 g/liter of l-tyrosine with a Y l-Tyr/Glc of 66 mg/g. These values are 46 and 48% higher than the values for a strain expressing only TyrC. The results show that the feedback inhibition-insensitive enzymes can be employed for strain development as part of a metabolic engineering strategy for l-tyrosine production.

scholarly journals Characterization of the Two Mycobacterium tuberculosis recA Promoters

2003 ◽  
Vol 185 (20) ◽  
pp. 6005-6015 ◽  
Author(s):  
Krishna K. Gopaul ◽  
Patricia C. Brooks ◽  
Jean-François Prost ◽  
Elaine O. Davis
Keyword(s):  
Escherichia Coli ◽  
Dna Damage ◽  
Mycobacterium Tuberculosis ◽  
Promoter Activity ◽  
The Other ◽  
Expression Level ◽  
Promoter Elements ◽  
Kinetics Of

ABSTRACT The recA gene of Mycobacterium tuberculosis is unusual in that it is expressed from two promoters, one of which, P1, is DNA damage inducible independently of LexA and RecA, while the other, P2, is regulated by LexA in the classical way (E. O. Davis, B. Springer, K. K. Gopaul, K. G. Papavinasasundaram, P. Sander, and E. C. Böttger, Mol. Microbiol. 46:791-800, 2002). In this study we characterized these two promoters in more detail. Firstly, we localized the promoter elements for each of the promoters, and in so doing we identified a mutation in each promoter which eliminates promoter activity. Interestingly, a motif with similarity to Escherichia coli σ70 −35 elements but located much closer to the −10 element is important for optimal expression of P1, whereas the sequence at the −35 location is not. Secondly, we found that the sequences flanking the promoters can have a profound effect on the expression level directed by each of the promoters. Finally, we examined the contribution of each of the promoters to recA expression and compared their kinetics of induction following DNA damage.

scholarly journals Mutations in the uncE gene affecting assembly of the c-subunit of the adenosine triphosphatase of Escherichia coli

1983 ◽  
Vol 211 (3) ◽  
pp. 717-726 ◽  
Author(s):  
D A Jans ◽  
A L Fimmel ◽  
L Langman ◽  
L B James ◽  
J A Downie ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Cell Membrane ◽  
Dna Sequences ◽  
Adenosine Triphosphatase ◽  
Gene Dosage ◽  
Base Change ◽  
Amino Acid Substitutions ◽  
Helical Hairpin ◽  
C Subunit

The amino acid substitutions in the mutant c-subunits of Escherichia coli F1F0-ATPase coded for by the uncE429, uncE408 and uncE463 alleles affect the incorporation of these proteins into the cell membrane. The DNA sequence of the uncE429 allele differed from normal in that a G leads to A base change occurred at nucleotide 68 of the uncE gene, resulting in glycine being replaced by aspartic acid at position 23 in the c-subunit. The uncE408 and uncE463 mutant DNA sequences were identical and differed from normal in that a C leads to T base change occurred at nucleotide 91 of the uncE gene, resulting in leucine being replaced by phenylalanine at position 31 in the c-subunit. An increased gene dosage of the uncE408 or uncE463 alleles resulted in the incorporation into the membranes of the mutant c-subunits. The results are discussed in terms of the ‘Helical Hairpin Hypothesis’ of Engelman & Steitz [(1981) Cell 23,411-422].

scholarly journals TcsL Is an Essential Virulence Factor inClostridium sordelliiATCC 9714

2011 ◽  
Vol 79 (3) ◽  
pp. 1025-1032 ◽  
Author(s):  
Glen P. Carter ◽  
Milena M. Awad ◽  
Yibai Hao ◽  
Tennille Thelen ◽  
Ingrid L. Bergin ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Virulence Factor ◽  
Reference Strain ◽  
Molecular Level ◽  
Mortality Rates ◽  
The Other ◽  
Strain Atcc ◽  
Human Pathogen ◽  
Uterine Infection ◽  
Major Virulence Factor

ABSTRACTClostridium sordelliiis an important pathogen of humans and animals, causing a range of diseases, including myonecrosis, sepsis, and shock. Although relatively rare in humans, the incidence of disease is increasing, and it is associated with high mortality rates, approaching 70%. Currently, very little is known about the pathogenesis ofC. sordelliiinfections or disease. Previous work suggested that the lethal large clostridial glucosylating toxin TcsL is the major virulence factor, but a lack of genetic tools has hindered our ability to conclusively assign a role for TcsL or, indeed, any of the other putative virulence factors produced by this organism. In this study, we have developed methods for the introduction of plasmids intoC. sordelliiusing RP4-mediated conjugation fromEscherichia coliand have successfully used these techniques to insertionally inactivate thetcsLgene in the reference strain ATCC 9714, using targetron technology. Virulence testing revealed that the production of TcsL is essential for the development of lethal infections byC. sordelliiATCC 9714 and also contributes significantly to edema seen during uterine infection. This study represents the first definitive identification of a virulence factor inC. sordelliiand opens the way for in-depth studies of this important human pathogen at the molecular level.

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