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Mutation Research Jan 2005Under stressful conditions mechanisms that increase genetic variation can bestow a selective advantage. Bacteria have several stress responses that provide ways in which... (Review)
Review
Under stressful conditions mechanisms that increase genetic variation can bestow a selective advantage. Bacteria have several stress responses that provide ways in which mutation rates can be increased. These include the SOS response, the general stress response, the heat-shock response, and the stringent response, all of which impact the regulation of error-prone polymerases. Adaptive mutation appears to be process by which cells can respond to selective pressure specifically by producing mutations. In Escherichia coli strain FC40 adaptive mutation involves the following inducible components: (i) a recombination pathway that generates mutations; (ii) a DNA polymerase that synthesizes error-containing DNA; and (iii) stress responses that regulate cellular processes. In addition, a subpopulation of cells enters into a state of hypermutation, giving rise to about 10% of the single mutants and virtually all of the mutants with multiple mutations. These bacterial responses have implications for the development of cancer and other genetic disorders in higher organisms.
Topics: Adaptation, Biological; Bacteria; Bacterial Proteins; DNA-Directed DNA Polymerase; Genes, Bacterial; Lac Operon; Mutation; Recombination, Genetic; SOS Response, Genetics
PubMed: 15603749
DOI: 10.1016/j.mrfmmm.2004.07.017 -
Genetics Mar 2018To test whether growth limitation induces mutations, Cairns and Foster constructed an strain whose mutant allele provides 1-2% of normal ability to use lactose. This...
To test whether growth limitation induces mutations, Cairns and Foster constructed an strain whose mutant allele provides 1-2% of normal ability to use lactose. This strain cannot grow on lactose, but produces ∼50 Lac revertant colonies per 10 plated cells over 5 days. About 80% of revertants carry a stable mutation made by the error-prone DinB polymerase, which may be induced during growth limitation; 10% of Lac revertants are stable but form without DinB; and the remaining 10% grow by amplifying their mutant allele and are unstably Lac Induced DinB mutagenesis has been explained in two ways: (1) upregulation of expression in nongrowing cells ("stress-induced mutagenesis") or (2) selected local overreplication of the and genes on lactose medium (selected amplification) in cells that are not dividing. Transcription of dinB is necessary but not sufficient for mutagenesis. Evidence is presented that DinB enhances reversion only when encoded somewhere on the F' plasmid that carries the mutant gene. A new model will propose that rare preexisting cells (1 in a 1000) have ∼10 copies of the F' plasmid, providing them with enough energy to divide, mate, and overreplicate their F' plasmid under selective conditions. In these clones, repeated replication of F' in nondividing cells directs opportunities for reversion and increases the copy number of the gene. Amplification of increases the error rate of replication and increases the number of revertants. Thus, reversion is enhanced in nondividing cells not by stress-induced mutagenesis, but by selected coamplification of the and genes, both of which happen to lie on the F' plasmid.
Topics: Alleles; Drug Resistance, Bacterial; Escherichia coli; Escherichia coli Proteins; Genome, Bacterial; Lac Operon; Models, Biological; Mutagenesis; Phenotype; Plasmids; Selection, Genetic
PubMed: 29301907
DOI: 10.1534/genetics.117.300409 -
Biophysical Journal Jun 2007Multistability is an emergent dynamic property that has been invoked to explain multiple coexisting biological states. In this work, we investigate the origin of...
Multistability is an emergent dynamic property that has been invoked to explain multiple coexisting biological states. In this work, we investigate the origin of bistability in the lac operon. To do this, we develop a mathematical model for the regulatory pathway in this system and compare the model predictions with other experimental results in which a nonmetabolizable inducer was employed. We investigate the effect of lactose metabolism using this model, and show that it greatly modifies the bistable region in the external lactose (Le) versus external glucose (Ge) parameter space. The model also predicts that lactose metabolism can cause bistability to disappear for very low Ge. We have also carried out stochastic numerical simulations of the model for several values of Ge and Le. Our results indicate that bistability can help guarantee that Escherichia coli consumes glucose and lactose in the most efficient possible way. Namely, the lac operon is induced only when there is almost no glucose in the growing medium, but if Le is high, the operon induction level increases abruptly when the levels of glucose in the environment decrease to very low values. We demonstrate that this behavior could not be obtained without bistability if the stability of the induced and uninduced states is to be preserved. Finally, we point out that the present methods and results may be useful to study the emergence of multistability in biological systems other than the lac operon.
Topics: Evolution, Molecular; Gene Regulatory Networks; Lac Operon; Models, Biological
PubMed: 17351004
DOI: 10.1529/biophysj.106.101717 -
Methods in Molecular Biology (Clifton,... 2013The nucleus is a complex organelle containing numerous highly dynamic, structurally stable domains and bodies, harboring functions that have only begun to be defined....
The nucleus is a complex organelle containing numerous highly dynamic, structurally stable domains and bodies, harboring functions that have only begun to be defined. However, the molecular mechanisms for their formation are still poorly understood. Recently it has been shown that a nuclear body can form de novo by self-organization. But little is known regarding what triggers the formation of a nuclear body and how subsequent assembly steps are orchestrated. Nuclear bodies are frequently associated with specific active gene loci that directly contribute to their formation. Both coding and noncoding RNAs can initiate the assembly of nuclear bodies with which they are physiologically associated. Thus, the formation of nuclear bodies occurs via recruitment and consequent accumulation of resident proteins in the nuclear bodies by nucleating RNA acting as a seeder. In this chapter I describe how to set up an experimental cell system to probe de novo biogenesis of a nuclear body by nucleating RNA and nuclear body components tethered on chromatin.
Topics: Cell Line, Tumor; Cell Nucleus; Cloning, Molecular; Coiled Bodies; Green Fluorescent Proteins; HeLa Cells; Histones; Humans; In Situ Hybridization, Fluorescence; Lac Operon; Lac Repressors; Nuclear Proteins; Transcription, Genetic
PubMed: 23980018
DOI: 10.1007/978-1-62703-526-2_23 -
Philosophical Transactions of the Royal... Nov 2016Chromatin immunoprecipitation, followed by quantification of immunoprecipitated DNA, can be used to measure RNA polymerase binding to any DNA segment in Escherichia coli...
Chromatin immunoprecipitation, followed by quantification of immunoprecipitated DNA, can be used to measure RNA polymerase binding to any DNA segment in Escherichia coli By calibrating measurements against the signal from a single RNA polymerase bound at a single promoter, we can calculate both promoter occupancy levels and the flux of transcribing RNA polymerase through transcription units. Here, we have applied the methodology to the E. coli lactose operon promoter. We confirm that promoter occupancy is limited by recruitment and that the supply of RNA polymerase to the lactose operon promoter depends on its location in the E. coli chromosome. Measurements of RNA polymerase binding to DNA segments within the lactose operon show that flux of RNA polymerase through the operon is low, with, on average, over 18 s elapsing between the passage of transcribing polymerases. Similar low levels of flux were found when semi-synthetic promoters were used to drive transcript initiation, even when the promoter elements were changed to ensure full occupancy of the promoter by RNA polymerase.This article is part of the themed issue 'The new bacteriology'.
Topics: DNA-Directed RNA Polymerases; Escherichia coli; Lac Operon; Promoter Regions, Genetic; Transcription, Genetic
PubMed: 27672157
DOI: 10.1098/rstb.2016.0080 -
Biophysical Journal Nov 2020Gene regulation by control of transcription initiation is a fundamental property of living cells. Much of our understanding of gene repression originated from studies of...
Gene regulation by control of transcription initiation is a fundamental property of living cells. Much of our understanding of gene repression originated from studies of the Escherichia coli lac operon switch, in which DNA looping plays an essential role. To validate and generalize principles from lac for practical applications, we previously described artificial DNA looping driven by designed transcription activator-like effector dimer (TALED) proteins. Because TALE monomers bind the idealized symmetrical lac operator sequence in two orientations, our prior studies detected repression due to multiple DNA loops. We now quantitatively characterize gene repression in living E. coli by a collection of individual TALED loops with systematic loop length variation. Fitting of a thermodynamic model allows unequivocal demonstration of looping and comparison of the engineered TALED repression system with the natural lac repressor system.
Topics: DNA, Bacterial; Escherichia coli; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Lac Operon; Lac Repressors; Nucleic Acid Conformation; Transcription Activator-Like Effectors
PubMed: 33091377
DOI: 10.1016/j.bpj.2020.10.007 -
Cell Aug 2002Although genes in prokaryotes and eukaryotes are transcribed and translated by very different mechanisms, they may be organized in their respective chromosomes in... (Comparative Study)
Comparative Study Review
Although genes in prokaryotes and eukaryotes are transcribed and translated by very different mechanisms, they may be organized in their respective chromosomes in surprisingly similar ways. Here, I examine common modes of maintaining nonrandom gene organization in both prokaryotes and eukaryotes, the different ways these organizations have likely arisen, and classes of organization that may be unique to one group or the other.
Topics: Animals; Base Sequence; Eukaryotic Cells; Gene Expression Regulation; Genes; Genes, Regulator; Humans; Lac Operon; Multigene Family; Prokaryotic Cells; Protein Biosynthesis
PubMed: 12202031
DOI: 10.1016/s0092-8674(02)00900-5 -
Microbial Cell Factories Mar 2020The genome-integrated T7 expression system offers significant advantages, in terms of productivity and product quality, even when expressing the gene of interest (GOI)...
BACKGROUND
The genome-integrated T7 expression system offers significant advantages, in terms of productivity and product quality, even when expressing the gene of interest (GOI) from a single copy. Compared to plasmid-based expression systems, this system does not incur a plasmid-mediated metabolic load, and it does not vary the dosage of the GOI during the production process. However, long-term production with T7 expression system leads to a rapidly growing non-producing population, because the T7 RNA polymerase (RNAP) is prone to mutations. The present study aimed to investigate whether two σ promoters, which were recognized by the Escherichia coli host RNAP, might be suitable in genome-integrated expression systems. We applied a promoter engineering strategy that allowed control of expressing the model protein, GFP, by introducing lac operators (lacO) into the constitutive T5 and A1 promoter sequences.
RESULTS
We showed that, in genome-integrated E. coli expression systems that used σ promoters, the number of lacO sites must be well balanced. Promoters containing three and two lacO sites exhibited low basal expression, but resulted in a complete stop in recombinant protein production in partially induced cultures. In contrast, expression systems regulated by a single lacO site and the lac repressor element, lacI, on the same chromosome caused very low basal expression, were highly efficient in recombinant protein production, and enables fine-tuning of gene expression levels on a cellular level.
CONCLUSIONS
Based on our results, we hypothesized that this phenomenon was associated with the autoregulation of the lac repressor protein, LacI. We reasoned that the affinity of LacI for the lacO sites of the GOI must be lower than the affinity of LacI to the lacO sites of the endogenous lac operon; otherwise, LacI autoregulation could not take place, and the lack of LacI autoregulation would lead to a disturbance in lac repressor-mediated regulation of transcription. By exploiting the mechanism of LacI autoregulation, we created a novel E. coli expression system for use in recombinant protein production, synthetic biology, and metabolic engineering applications.
Topics: DNA-Directed RNA Polymerases; Escherichia coli; Gene Expression Regulation, Bacterial; Genome, Bacterial; Green Fluorescent Proteins; Lac Operon; Lac Repressors; Promoter Regions, Genetic; Recombinant Proteins; Viral Proteins
PubMed: 32138729
DOI: 10.1186/s12934-020-01311-6 -
PLoS Genetics Jun 2013Transmission of cellular identity relies on the faithful transfer of information from the mother to the daughter cell. This process includes accurate replication of the...
Transmission of cellular identity relies on the faithful transfer of information from the mother to the daughter cell. This process includes accurate replication of the DNA, but also the correct propagation of regulatory programs responsible for cellular identity. Errors in DNA replication (mutations) and protein conformation (prions) can trigger stable phenotypic changes and cause human disease, yet the ability of transient transcriptional errors to produce heritable phenotypic change ('epimutations') remains an open question. Here, we demonstrate that transcriptional errors made specifically in the mRNA encoding a transcription factor can promote heritable phenotypic change by reprogramming a transcriptional network, without altering DNA. We have harnessed the classical bistable switch in the lac operon, a memory-module, to capture the consequences of transient transcription errors in living Escherichia coli cells. We engineered an error-prone transcription sequence (A9 run) in the gene encoding the lac repressor and show that this 'slippery' sequence directly increases epigenetic switching, not mutation in the cell population. Therefore, one altered transcript within a multi-generational series of many error-free transcripts can cause long-term phenotypic consequences. Thus, like DNA mutations, transcriptional epimutations can instigate heritable changes that increase phenotypic diversity, which drives both evolution and disease.
Topics: DNA Replication; Epigenesis, Genetic; Escherichia coli; Evolution, Molecular; Genetic Variation; Green Fluorescent Proteins; Humans; Lac Operon; Lac Repressors; Mutation; Phenotype; Protein Conformation; RNA, Messenger; Transcription, Genetic
PubMed: 23825966
DOI: 10.1371/journal.pgen.1003595 -
Journal of Bacteriology Oct 1992The complete nucleotide sequences of lacRABCDF and partial nucleotide sequence of lacE from the lactose operon of Streptococcus mutans are presented. Comparison of the... (Comparative Study)
Comparative Study
Nucleotide and deduced amino acid sequences of the lacR, lacABCD, and lacFE genes encoding the repressor, tagatose 6-phosphate gene cluster, and sugar-specific phosphotransferase system components of the lactose operon of Streptococcus mutans.
The complete nucleotide sequences of lacRABCDF and partial nucleotide sequence of lacE from the lactose operon of Streptococcus mutans are presented. Comparison of the streptococcal lac determinants with those of Staphylococcus aureus and Lactococcus lactis indicate exceptional protein and nucleotide identity. The deduced polypeptides also demonstrate significant, but lower, sequence similarity with the corresponding lactose proteins of Lactobacillus casei. Additionally, LacR has sequence homology with the repressor (DeoR) of the Escherichia coli deoxyribonucleotide operon, while LacC is similar to phosphokinases (FruK and PfkB) from E. coli. The primary translation products of the lacRABCDFE genes are polypeptides of 251 (M(r) 28,713), 142 (M(r) 15,610), 171 (M(r) 18,950), 310 (M(r) 33,368), 325 (M(r) 36,495), 104 (M(r) 11,401), and 123 (NH2-terminal) amino acids, respectively. As inferred from their direct homology to the staphylococcal lac genes, these determinants would encode the repressor of the streptococcal lactose operon (LacR), galactose-6-phosphate isomerase (LacA and LacB), tagatose-6-phosphate kinase (LacC), tagatose-1,6-bisphosphate aldolase (LacD), and the sugar-specific components enzyme III-lactose (LacF) and enzyme II-lactose (LacE) of the S. mutans phosphoenolpyruvate-dependent phosphotransferase system. The nucleotide sequence encompassing the S. mutans lac promoter appears to contain repeat elements analogous to those of S. aureus, suggesting that repression and catabolite repression of the lactose operons may be similar in these organisms.
Topics: Amino Acid Sequence; Bacterial Proteins; Base Sequence; DNA-Binding Proteins; Escherichia coli Proteins; Genes, Bacterial; Hexosephosphates; Lac Operon; Lactose; Membrane Transport Proteins; Molecular Sequence Data; Monosaccharide Transport Proteins; Nucleic Acid Hybridization; Promoter Regions, Genetic; Protein Biosynthesis; Protein Conformation; Repressor Proteins; Sequence Homology; Streptococcus mutans; Symporters
PubMed: 1400164
DOI: 10.1128/jb.174.19.6159-6170.1992