scholarly journals Mutations that improve the efficiency of a weak-link enzyme are rare compared to adaptive mutations elsewhere in the genome

2019 ◽  
Author(s):  
Andrew B. Morgenthaler ◽  
Wallis R. Kinney ◽  
Christopher C. Ebmeier ◽  
Corinne M. Walsh ◽  
Daniel J. Snyder ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Weak Link ◽  
Experimental Studies ◽  
Gene Encoding ◽  
Evolutionary Trajectory ◽  
Adaptive Mutations ◽  
Parental Allele ◽  
Genes Encoding ◽  
Essential Function ◽  
Beneficial Allele

AbstractNew enzymes often evolve by amplification and divergence of genes encoding enzymes with a weak ability to provide a new function. Experimental studies to date have followed the evolutionary trajectory of an amplified gene, but have not addressed other mutations in the genome when fitness is limited by an evolving gene. We have adapted Escherichia coli in which an enzyme’s weak secondary activity has been recruited to serve an essential function. While the gene encoding the “weak-link” enzyme amplified in all eight populations, mutations improving the new activity occurred in only one. This beneficial allele quickly swept the amplified array, displacing the parental allele. Most adaptive mutations, however, occurred elsewhere in the genome. We have identified the mechanisms by which three of the classes of mutations increase fitness. These mutations may be detrimental once a new enzyme has evolved, and require reversion or compensation, leading to permanent changes in the genome.

scholarly journals Mutations that improve efficiency of a weak-link enzyme are rare compared to adaptive mutations elsewhere in the genome

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Andrew B Morgenthaler ◽  
Wallis R Kinney ◽  
Christopher C Ebmeier ◽  
Corinne M Walsh ◽  
Daniel J Snyder ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Metabolic Pathway ◽  
Weak Link ◽  
Experimental Studies ◽  
Wild Type ◽  
Gene Encoding ◽  
Evolutionary Trajectory ◽  
Adaptive Mutations ◽  
E Coli ◽  
Essential Function

New enzymes often evolve by gene amplification and divergence. Previous experimental studies have followed the evolutionary trajectory of an amplified gene, but have not considered mutations elsewhere in the genome when fitness is limited by an evolving gene. We have evolved a strain of Escherichia coli in which a secondary promiscuous activity has been recruited to serve an essential function. The gene encoding the ‘weak-link’ enzyme amplified in all eight populations, but mutations improving the newly needed activity occurred in only one. Most adaptive mutations occurred elsewhere in the genome. Some mutations increase expression of the enzyme upstream of the weak-link enzyme, pushing material through the dysfunctional metabolic pathway. Others enhance production of a co-substrate for a downstream enzyme, thereby pulling material through the pathway. Most of these latter mutations are detrimental in wild-type E. coli, and thus would require reversion or compensation once a sufficient new activity has evolved.

scholarly journals Essential and nonessential histone H2A variants in Tetrahymena thermophila.

1996 ◽  
Vol 16 (8) ◽  
pp. 4305-4311 ◽  
Author(s):  
X Liu ◽  
B Li ◽  
GorovskyMA
Keyword(s):  
Tetrahymena Thermophila ◽  
Essential Gene ◽  
Histone Variants ◽  
Gene Replacement ◽  
Histone H2a ◽  
Gene Encoding ◽  
Genes Encoding ◽  
Simple Mass ◽  
Essential Function ◽  
High Degree

Although variants have been identified for every class of histone, their functions remain unknown. We have been studying the histone H2A variant hv1 in the ciliated protozoan Tetrahymena thermophila. Sequence analysis indicates that hv1 belongs to the H2A.F/Z type of histone variants. On the basis of the high degree of evolutionary conservation of this class of histones, they are proposed to have one or more distinct and essential functions that cannot be performed by their major H2A counterparts. Considerable evidence supports the hypothesis that the hv1 protein in T. thermophila and hv1-like proteins in other eukaryotes are associated with active chromatin. In T. thermophila, simple mass transformation and gene replacement techniques have recently become available. In this report, we demonstrate that either the HTA1 gene or the HTA2 gene, encoding the major H2As, can be completely replaced by disrupted genes in the polyploid, transcriptionally active macronucleus, indicating that neither of the two genes is essential. However, only some of the HTA3 genes encoding hv1 can be replaced by disrupted genes, indicating that the H2A.F/Z type variants have an essential function that cannot be performed by the major H2A genes. Thus, an essential gene in T. thermophila can be defined by the fact that it can be partially, but not completely, eliminated from the polyploid macronucleus. To our knowledge, this study represents the first use of gene disruption technology to study core histone gene function in any organism other than yeast and the first demonstration of an essential gene in T. thermophila using these methods. When a rescuing plasmid carrying a wild-type HTA3 gene was introduced into the T. thermophila cells, the endogenous chromosomal HTA3 could be completely replaced, defining a gene replacement strategy that can be used to analyze the function of essential genes.

scholarly journals PhhB, a Pseudomonas aeruginosa Homolog of Mammalian Pterin 4a-Carbinolamine Dehydratase/DCoH, Does Not Regulate Expression of Phenylalanine Hydroxylase at the Transcriptional Level

1999 ◽  
Vol 181 (9) ◽  
pp. 2789-2796 ◽  
Author(s):  
Jian Song ◽  
Tianhui Xia ◽  
Roy A. Jensen
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Phenylalanine Hydroxylase ◽  
Transcriptional Level ◽  
Gene Encoding ◽  
Catalytic Function ◽  
Genes Encoding ◽  
Catalytic Role ◽  
Regulatory Effects

ABSTRACT Pterin 4a-carbinolamine dehydratase is bifunctional in mammals. In addition to playing a catalytic role in pterin recycling in the cytoplasm, it plays a regulatory role in the nucleus, where it acts as a dimerization-cofactor component (called DCoH) for the transcriptional activator HNF-1α. A thus far unique operon in Pseudomonas aeruginosa contains a gene encoding a homolog (PhhB) of the regulatory dehydratase, together with genes encoding phenylalanine hydroxylase (PhhA) and aromatic aminotransferase (PhhC). Using complementation of tyrosine auxotrophy in Escherichia colias a functional test, we have found that the in vivo function of PhhA requires PhhB. Strikingly, mammalian DCoH was an effective substitute for PhhB, and either one was effective in trans. Surprisingly, the required presence of PhhB for complementation did not reflect a critical positive regulatory effect of phhB onphhA expression. Rather, in the absence of PhhB, PhhA was found to be extremely toxic in E. coli, probably due to the nonenzymatic formation of 7-biopterin or a similar derivative. However, bacterial PhhB does appear to exert modest regulatory effects in addition to having a catalytic function. PhhB enhances the level of PhhA two- to threefold, as was demonstrated by gene inactivation ofphhB in P. aeruginosa and by comparison of the levels of expression of PhhA in the presence and absence of PhhB inEscherichia coli. Experiments using constructs having transcriptional and translational fusions with a lacZreporter indicated that PhhB activates PhhA at the posttranscriptional level. Regulation of PhhA and PhhB is semicoordinate; both PhhA and PhhB are induced coordinately in the presence of eitherl-tyrosine or l-phenylalanine, but PhhB exhibits a significant basal level of activity that is lacking for PhhA. Immunoprecipitation and affinity chromatography showed that PhhA and PhhB form a protein-protein complex.

scholarly journals GadY, a Small-RNA Regulator of Acid Response Genes in Escherichia coli

2004 ◽  
Vol 186 (20) ◽  
pp. 6698-6705 ◽  
Author(s):  
Jason A. Opdyke ◽  
Ju-Gyeong Kang ◽  
Gisela Storz
Keyword(s):  
Escherichia Coli ◽  
Stationary Phase ◽  
Small Rna ◽  
Acid Resistance ◽  
Base Pairs ◽  
Gene Encoding ◽  
Mrna Accumulation ◽  
Long Form ◽  
Genes Encoding ◽  
Opposite Strand

ABSTRACT A previous bioinformatics-based search for small RNAs in Escherichia coli identified a novel RNA named IS183. The gene encoding this small RNA is located between and on the opposite strand of genes encoding two transcriptional regulators of the acid response, gadX (yhiX) and gadW (yhiW). Given that IS183 is encoded in the gad gene cluster and because of its role in regulating acid response genes reported here, this RNA has been renamed GadY. We show that GadY exists in three forms, a long form consisting of 105 nucleotides and two processed forms, consisting of 90 and 59 nucleotides. The expression of this small RNA is highly induced during stationary phase in a manner that is dependent on the alternative sigma factor σS. Overexpression of the three GadY RNA forms resulted in increased levels of the mRNA encoding the GadX transcriptional activator, which in turn caused increased levels of the GadA and GadB glutamate decarboxylases. A promoter mutation which abolished gadY expression resulted in a reduction in the amount of gadX mRNA during stationary phase. The gadY gene was shown to overlap the 3′ end of the gadX gene, and this overlap region was found to be necessary for the GadY-dependent accumulation of gadX mRNA. We suggest that during stationary phase, GadY forms base pairs with the 3′-untranslated region of the gadX mRNA and confers increased stability, allowing for gadX mRNA accumulation and the increased expression of downstream acid resistance genes.

scholarly journals A Mutation in a New Gene, bglJ Activates the bgl Operon in Escherichia coli K-12

Genetics ◽  
1996 ◽  
Vol 143 (2) ◽  
pp. 627-635 ◽  
Author(s):  
Maryann Giel ◽  
Martine Desnoyer ◽  
Jane Lopilato
Keyword(s):  
Escherichia Coli ◽  
Dna Gyrase ◽  
New Mutation ◽  
Gene Encoding ◽  
Activating Mutations ◽  
Putative Protein ◽  
Genes Encoding ◽  
Chromosome Mutations ◽  
New Gene ◽  
K 12

Abstract A new mutation, bglJ4, has been characterized that results in the expression of the silent bgl operon. The bgl operon encodes proteins necessary for the transport and utilization of the aromatic β-glucosides arbutin and salicin. A variety of mutations activate the operon and result in a Bgl+ phenotype. Activating mutations are located upstream of the bgl promoter and in genes located elsewhere on the chromosome. Mutations outside of the bgl operon occur in the genes encoding DNA gyrase and in the gene encoding the nucleoid associated protein H-NS. The mutation described here, bglJ4, has been mapped to a new locus at min 99 on the Escherichia coli K-12 genetic map. The putative protein encoded by the bgygene has homolgy to a family of transcriptional activators. Evidence is presented that increased expression of the bglJ product is needed for activation of the bgl operon.

scholarly journals Optimization of (S)-3-Hydroxybutyric Acid Biosynthesis from Glucose through the Reversed Fatty Acid β-Oxidation Pathway by Recombinant Escherichia coli Strains

2021 ◽  
Vol 57 (2) ◽  
pp. 161-169
Author(s):  
A. Yu. Gulevich ◽  
A. Yu. Skorokhodova ◽  
V. G. Debabov
Keyword(s):  
Escherichia Coli ◽  
Fatty Acid ◽  
Product Yield ◽  
Target Product ◽  
Hydroxybutyric Acid ◽  
Acid Biosynthesis ◽  
Gene Encoding ◽  
Oxidation Pathway ◽  
Genes Encoding ◽  
Primary Evaluation

Abstract The microaerobic synthesis of 3-hydroxybutyric acid by the Escherichia coli strain BOX3.1 ∆4 PL-atoB PL-tesB (MG1655 lacIQ, ∆ackA-pta, ∆poxB, ∆ldhA, ∆adhE, ∆fadE, PL-SDphi10-atoB, Ptrc-ideal-4-SDphi10-fadB, PL-SDphi10-tesB), which was previously directly engineered for the biosynthesis of the target compound from glucose through the reversed fatty acid β-oxidation pathway, was studied. A target product yield of 0.12 mol/mol was achieved. Inactivation of the nonspecific YciA thioesterase gene in the strain led to an increase in the yield of 3-hydroxybutyric acid to 0.15 mol/mol. For the optimization of biosynthesis of target product the strain MG∆4 PL-tesB (MG1655 ∆ackA-pta, ∆poxB, ∆ldhA, ∆adhE, PL-SDphi10-tesB) was engineered, and the genes encoding key enzymes of fatty acid β-oxidation were overexpressed in the strain from the plasmid pMW118m-atoB-fadB. The level of microaerobic synthesis of 3-hydroxybutyric acid by the strain MG∆4 PL-tesB (pMW118m-atoB-fadB) achieved in primary evaluation conditions reached 0.35 mol/mol. Inactivation in the strain of the gene of nonspecific thioesterase YciA led to only minor decrease in acetate byproduction. Further inactivation in the strain of gene encoding nonspecific thioesterase YdiI had virtually no effect on the level of synthesis of side products. Cultivation of the constructed strain MG∆4 PL-tesB ∆yciA (pMW118m-atoB-fadB) in bioreactor under the controlled conditions ensured achievement of a yield of 3‑hydroxybutyric acid amounting to 0.75 mol/mol.

scholarly journals Hidden resources in the Escherichia coli genome restore PLP synthesis and robust growth after deletion of the essential gene pdxB

2019 ◽  
Vol 116 (48) ◽  
pp. 24164-24173 ◽  
Author(s):  
Juhan Kim ◽  
Jake J. Flood ◽  
Michael R. Kristofich ◽  
Cyrus Gidfar ◽  
Andrew B. Morgenthaler ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Carbon Source ◽  
Sole Carbon Source ◽  
Essential Gene ◽  
Alternative Pathway ◽  
Gene Encoding ◽  
Genes Encoding ◽  
Phosphoglycerate Dehydrogenase ◽  
Functional Pathway

PdxB (erythronate 4-phosphate dehydrogenase) is expected to be required for synthesis of the essential cofactor pyridoxal 5′-phosphate (PLP) in Escherichia coli. Surprisingly, incubation of the ∆pdxB strain in medium containing glucose as a sole carbon source for 10 d resulted in visible turbidity, suggesting that PLP is being produced by some alternative pathway. Continued evolution of parallel lineages for 110 to 150 generations produced several strains that grow robustly in glucose. We identified a 4-step bypass pathway patched together from promiscuous enzymes that restores PLP synthesis in strain JK1. None of the mutations in JK1 occurs in a gene encoding an enzyme in the new pathway. Two mutations indirectly enhance the ability of SerA (3-phosphoglycerate dehydrogenase) to perform a new function in the bypass pathway. Another disrupts a gene encoding a PLP phosphatase, thus preserving PLP levels. These results demonstrate that a functional pathway can be patched together from promiscuous enzymes in the proteome, even without mutations in the genes encoding those enzymes.

scholarly journals RegR Virulence Regulon of Rabbit-Specific Enteropathogenic Escherichia coli Strain E22

2013 ◽  
Vol 81 (4) ◽  
pp. 1078-1089 ◽  
Author(s):  
Yogitha N. Srikhanta ◽  
Dianna M. Hocking ◽  
Judyta Praszkier ◽  
Matthew J. Wakefield ◽  
Roy M. Robins-Browne ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Regulatory Protein ◽  
Bioinformatic Analysis ◽  
Coli Strain ◽  
Mobility Shift ◽  
Gene Encoding ◽  
Gene Target ◽  
Content Type ◽  
E Coli ◽  
Genes Encoding

ABSTRACTAraC-like regulators play a key role in the expression of virulence factors in enteric pathogens, such as enteropathogenicEscherichia coli(EPEC), enterotoxigenicE. coli, enteroaggregativeE. coli, andCitrobacter rodentium. Bioinformatic analysis of the genome of rabbit-specific EPEC (REPEC) strain E22 (O103:H2) revealed the presence of a gene encoding an AraC-like regulatory protein, RegR, which shares 71% identity to the global virulence regulator, RegA, ofC. rodentium. Microarray analysis demonstrated that RegR exerts 25- to 400-fold activation on transcription of several genes encoding putative virulence-associated factors, including a fimbrial operon (SEF14), a serine protease, and an autotransporter adhesin. These observations were confirmed by proteomic analysis of secreted and heat-extracted surface-associated proteins. The mechanism of RegR-mediated activation was investigated by using its most highly upregulated gene target,sefA. Transcriptional analyses and electrophoretic mobility shift assays showed that RegR activates the expression ofsefAby binding to a region upstream of thesefApromoter, thereby relieving gene silencing by the global regulatory protein H-NS. Moreover, RegR was found to contribute significantly to virulence in a rabbit infection experiment. Taken together, our findings indicate that RegR controls the expression of a series of accessory adhesins that significantly enhance the virulence of REPEC strain E22.

scholarly journals Identification, Cloning, and Characterization of l-Phenylserine Dehydrogenase from Pseudomonas syringae NK-15

2010 ◽  
Vol 2010 ◽  
pp. 1-10 ◽  
Author(s):  
Sakuko Ueshima ◽  
Hisashi Muramatsu ◽  
Takanori Nakajima ◽  
Hiroaki Yamamoto ◽  
Shin-ichiro Kato ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Syringae ◽  
Homology Search ◽  
Hydroxyl Group ◽  
Enzymatic Reactions ◽  
Reaction Products ◽  
Gene Encoding ◽  
Genes Encoding ◽  
Single Operon

The gene encoding d-phenylserine dehydrogenase from Pseudomonas syringae NK-15 was identified, and a 9,246-bp nucleotide sequence containing the gene was sequenced. Six ORFs were confirmed in the sequenced region, four of which were predicted to form an operon. A homology search of each ORF predicted that orf3 encoded l-phenylserine dehydrogenase. Hence, orf3 was cloned and overexpressed in Escherichia coli cells and recombinant ORF3 was purified to homogeneity and characterized. The purified ORF3 enzyme showed l-phenylserine dehydrogenase activity. The enzymological properties and primary structure of l-phenylserine dehydrogenase (ORF3) were quite different from those of d-phenylserine dehydrogenase previously reported. l-Phenylserine dehydrogenase catalyzed the NAD+-dependent oxidation of the β-hydroxyl group of l-β-phenylserine. l-Phenylserine and l-threo-(2-thienyl)serine were good substrates for l-phenylserine dehydrogenase. The genes encoding l-phenylserine dehydrogenase and d-phenylserine dehydrogenase, which is induced by phenylserine, are located in a single operon. The reaction products of both enzymatic reactions were 2-aminoacetophenone and CO2.

scholarly journals Translational Paradigms in Cerebrovascular Gene Transfer

2003 ◽  
Vol 23 (11) ◽  
pp. 1251-1262 ◽  
Author(s):  
Vini G Khurana ◽  
Fredric B Meyer
Keyword(s):  
Gene Transfer ◽  
Blood Vessels ◽  
Ex Vivo ◽  
Present Report ◽  
Cerebral Arteries ◽  
Experimental Studies ◽  
Gene Encoding ◽  
Expression Of Genes ◽  
Genes Encoding

Gene transfer involves the use of an engineered biologic vehicle known as a vector to introduce a gene encoding a protein of interest into a particular tissue. In diseases with known defects at a genetic level, gene transfer offers a potential means of restoring a normal molecular environment via vector-mediated entry (transduction) and expression of genes encoding potentially therapeutic proteins selectively in diseased tissues. The technology of gene transfer therefore underlies the concept of gene therapy and falls under the umbrella of the current genomics revolution. Particularly since 1995, numerous attempts have been made to introduce genes into intracranial blood vessels to demonstrate and characterize viable transduction. More recently, in attempting to translate cerebrovascular gene transfer technology closer to the clinical arena, successful transductions of normal human cerebral arteries ex vivo and diseased animal cerebral arteries in vivo have been reported using vasomodulatory vectors. Considering the emerging importance of gene-based strategies for the treatment of the spectrum of human disease, the goals of the present report are to overview the fundamentals of gene transfer and review experimental studies germane to the clinical translation of a technology that can facilitate genetic modification of cerebral blood vessels.

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