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The Protein Journal Jun 2019Cells in all domains of life must translocate newly synthesized proteins both across membranes and into membranes. In eukaryotes, proteins are translocated into the... (Review)
Review
Cells in all domains of life must translocate newly synthesized proteins both across membranes and into membranes. In eukaryotes, proteins are translocated into the lumen of the ER or the ER membrane. In prokaryotes, proteins are translocated into the cytoplasmic membrane or through the membrane into the periplasm for Gram-negative bacteria or the extracellular space for Gram-positive bacteria. Much of what we know about protein translocation was learned through genetic selections and screens utilizing lacZ gene fusions in Escherichia coli. This review covers the basic principles of protein translocation and how they were discovered and developed. In particular, we discuss how lacZ gene fusions and the phenotypes conferred were exploited to identify the genes involved in protein translocation and provide insights into their mechanisms of action. These approaches, which allowed the elucidation of processes that are conserved throughout the domains of life, illustrate the power of seemingly simple experiments.
Topics: Artificial Gene Fusion; Cell Membrane; Escherichia coli; Escherichia coli Proteins; Gene Fusion; Lac Operon; Protein Transport; Recombinant Fusion Proteins; SEC Translocation Channels; beta-Galactosidase
PubMed: 30684070
DOI: 10.1007/s10930-019-09813-y -
Research in Microbiology Jun 2014
Topics: Gene Expression Regulation, Bacterial; Genetics, Microbial; History, 20th Century; Molecular Biology; Operon
PubMed: 24859142
DOI: 10.1016/j.resmic.2014.05.029 -
Nucleic Acids Research Dec 2017Significant, otherwise-unavailable information about mechanisms and transition states (TS) of protein folding and binding is obtained from solute effects on rate...
Significant, otherwise-unavailable information about mechanisms and transition states (TS) of protein folding and binding is obtained from solute effects on rate constants. Here we characterize TS for lac repressor(R)-lac operator(O) binding by analyzing effects of RO-stabilizing and RO-destabilizing solutes on association (ka) and dissociation (kd) rate constants. RO-destabilizing solutes (urea, KCl) reduce ka comparably (urea) or more than (KCl) they increase kd, demonstrating that they destabilize TS relative to reactants and RO, and that TS exhibits most of the Coulombic interactions between R and O. Strikingly, three solutes which stabilize RO by favoring burial/dehydration of amide oxygens and anionic phosphate oxygens all reduce kd without affecting ka significantly. The lack of stabilization of TS by these solutes indicates that O phosphates remain hydrated in TS and that TS preferentially buries aromatic carbons and amide nitrogens while leaving amide oxygens exposed. In our proposed mechanism, DNA-binding-domains (DBD) of R insert in major grooves of O pre-TS, forming most Coulombic interactions of RO and burying aromatic carbons. Nucleation of hinge helices creates TS, burying sidechain amide nitrogens. Post-TS, hinge helices assemble and the DBD-hinge helix-O-DNA module docks on core repressor, partially dehydrating phosphate oxygens and tightening all interfaces to form RO.
Topics: Algorithms; Amides; DNA; Kinetics; Lac Operon; Lac Repressors; Models, Molecular; Nucleic Acid Conformation; Potassium Chloride; Protein Binding; Protein Domains; Protein Folding; Thermodynamics; Urea
PubMed: 29036376
DOI: 10.1093/nar/gkx862 -
Briefings in Bioinformatics Jul 2021Mechanistic computational models enable the study of regulatory mechanisms implicated in various biological processes. These models provide a means to analyze the... (Review)
Review
Mechanistic computational models enable the study of regulatory mechanisms implicated in various biological processes. These models provide a means to analyze the dynamics of the systems they describe, and to study and interrogate their properties, and provide insights about the emerging behavior of the system in the presence of single or combined perturbations. Aimed at those who are new to computational modeling, we present here a practical hands-on protocol breaking down the process of mechanistic modeling of biological systems in a succession of precise steps. The protocol provides a framework that includes defining the model scope, choosing validation criteria, selecting the appropriate modeling approach, constructing a model and simulating the model. To ensure broad accessibility of the protocol, we use a logical modeling framework, which presents a lower mathematical barrier of entry, and two easy-to-use and popular modeling software tools: Cell Collective and GINsim. The complete modeling workflow is applied to a well-studied and familiar biological process-the lac operon regulatory system. The protocol can be completed by users with little to no prior computational modeling experience approximately within 3 h.
Topics: Algorithms; Gene Regulatory Networks; Models, Genetic; Software; Systems Biology
PubMed: 33064138
DOI: 10.1093/bib/bbaa236 -
MBio Feb 2022Persisters represent a small subpopulation of cells that are tolerant of killing by antibiotics and are implicated in the recalcitrance of chronic infections to...
Persisters represent a small subpopulation of cells that are tolerant of killing by antibiotics and are implicated in the recalcitrance of chronic infections to antibiotic therapy. One general theme has emerged regarding persisters formed by different bacterial species, namely, a state of relative dormancy characterized by diminished activity of antibiotic targets. Within this framework, a number of studies have linked persister formation to stochastic decreases in energy-generating components, leading to low ATP and target activity. In this study, we screen knockouts in the main global regulators of Escherichia coli for their effect on persisters. A knockout in integration host factor (IHF) had elevated ATP and a diminished level of persisters. This was accompanied by an overexpression of isocitrate dehydrogenase (Icd) and a downregulation of isocitrate lyase (AceA), two genes located at the bifurcation between the tricarboxylic acid (TCA) cycle and the glyoxylate bypass. Using a translational fusion, we sort out rare bright cells, and this subpopulation is enriched in persisters. Our results suggest that noise in the expression of produces rare cells with low Icd/high AceA, diverting substrates into the glyoxylate bypass, which decreases ATP, leading to antibiotic-tolerant persisters. We further examine noise in a simple model, the operon, and show that a knockout of the repressor increases expression of the operon and decreases persister formation. Our results suggest that noise quenching by overexpression serves as a general approach to determine the nature of persister genes in a variety of bacterial species and conditions. Persisters are phenotypic variants that survive exposure to antibiotics through temporary dormancy. Mutants with increased levels of persisters have been identified in clinical isolates, and evidence suggests these cells contribute to chronic infections and antibiotic treatment failure. Understanding the underlying mechanism of persister formation and tolerance is important for developing therapeutic approaches to treat chronic infections. In this study, we examine a global regulator, IHF, that plays a role in persister formation. We find that noise in expression of IHF contributes to persister formation, likely by regulating the switch between the TCA cycle that efficiently produces energy and the glyoxylate bypass. We extend this study to a simple model operon and show that when grown on lactose as the sole carbon source, noise in its expression influences ATP levels and determines persister formation. This noise is quenched by overexpression of the operon, providing a simple approach to test the involvement of a gene in persister formation.
Topics: Humans; Adenosine Triphosphate; Anti-Bacterial Agents; Bacteria; Escherichia coli; Glyoxylates; Integration Host Factors; Persistent Infection
PubMed: 34982597
DOI: 10.1128/mbio.03420-21 -
Journal of Microbiology & Biology... Apr 2024Many undergraduates struggle to interpret abstract concepts in molecular biology. Modeling can facilitate learning by making these abstract concepts tangible. Here, we...
Many undergraduates struggle to interpret abstract concepts in molecular biology. Modeling can facilitate learning by making these abstract concepts tangible. Here, we present an exercise based on the operon designed for undergraduate students using LEGO bricks. The operon is a classic example of transcriptional regulation taught in a variety of undergraduate biology courses and is fundamental to understanding the regulation of gene expression. This easy-to-implement active learning exercise demonstrates how the various components of the operon are oriented under a variety of nutritional conditions to control gene expression. In addition, higher-order concepts, such as the effect of mutation on operon expression, can be readily modeled. Overall, students not only found this exercise to be enjoyable but also helpful as a tool to engage with this course material.
PubMed: 38661411
DOI: 10.1128/jmbe.00034-24 -
Frontiers in Genetics 2021Zebrafish are a foundational model organism for studying the spatio-temporal activity of genes and their regulatory sequences. A variety of approaches are currently...
BACKGROUND
Zebrafish are a foundational model organism for studying the spatio-temporal activity of genes and their regulatory sequences. A variety of approaches are currently available for editing genes and modifying gene expression in zebrafish, including RNAi, Cre/lox, and CRISPR-Cas9. However, the operator-repressor system, an operon component which has been adapted for use in many other species and is a valuable, flexible tool for inducible modulation of gene expression studies, has not been previously tested in zebrafish.
RESULTS
Here we demonstrate that the operator-repressor system robustly decreases expression of firefly luciferase in cultured zebrafish fibroblast cells. Our work establishes the operator-repressor system as a promising tool for the manipulation of gene expression in whole zebrafish.
CONCLUSION
Our results lay the groundwork for the development of based reporter assays in zebrafish, and adds to the tools available for investigating dynamic gene expression in embryogenesis. We believe this work will catalyze the development of new reporter assay systems to investigate uncharacterized regulatory elements and their cell-type specific activities.
PubMed: 34220959
DOI: 10.3389/fgene.2021.683394 -
Frontiers in Microbiology 2021The operon is one of the best known gene regulatory circuits and constitutes a landmark example of how bacteria tune their metabolism to nutritional conditions. It is...
The operon is one of the best known gene regulatory circuits and constitutes a landmark example of how bacteria tune their metabolism to nutritional conditions. It is nearly ubiquitous in strains justifying the use of its phenotype, the ability to consume lactose, for species identification. Lactose is the primary sugar found in milk, which is abundant in mammals during the first weeks of life. However, lactose is virtually non-existent after the weaning period, with humans being an exception as many consume dairy products throughout their lives. The absence of lactose during adulthood in most mammals and the rarity of lactose in the environment, means that the selective pressure for maintaining the operon could be weak for long periods of time. Despite the ability to metabolize lactose being a hallmark of 's success when colonizing its primary habitat, the mammalian intestine, the selective value of this trait remains unknown in this ecosystem during adulthood. Here we determine the competitive advantage conferred by the operon to a commensal strain of when colonizing the mouse gut. We find that its benefit, which can be as high as 11%, is contingent on the presence of lactose in the diet and on the presence of other microbiota members in the gut, but the operon is never deleterious. These results help explaining the pervasiveness of the operon in , but also its polymorphism, as -negative strains albeit rare can naturally occur in the gut.
PubMed: 34367115
DOI: 10.3389/fmicb.2021.709259 -
Molecular Microbiology Jan 2021When Streptococcus mutans is transferred from a preferred carbohydrate (glucose or fructose) to lactose, initiation of growth can take several hours, and substantial...
When Streptococcus mutans is transferred from a preferred carbohydrate (glucose or fructose) to lactose, initiation of growth can take several hours, and substantial amounts of glucose are released during growth. Here, S. mutans strains UA159 and GS-5 were examined for stochastic behaviors in transcription of the lac operon. Using a gfp reporter fusion, we demonstrated that induction of the lac operon occurs in only a fraction of the population, with prior exposure to carbohydrate source and strain influencing the magniture of the sub-population response. Lower glucokinase activity in GS-5 was associated with release of substantially more glucose than UA159 and significantly lower lac expression. Mutants unable to use lactose grew on lactose as the sole carbohydrate when strains with an intact lac operon were also present in the cultures, indicative of the potential for population cheating. Utilizing a set of engineered obligate cheating and non-cheating strains, we confirmed that cheating can sustain a heterogeneous population. Futher, obligate cheaters of GS-5 competed well with the non-cheaters and showed a high degree of competitive fitness in a human-derived consortium biofilm model. The results show that bet-hedging behaviors in carbohydrate metabolism may substantially influence the composition and pathogenic potential of oral biofilms.
Topics: Biofilms; Carbohydrate Metabolism; Fructose; Gene Expression; Gene Expression Regulation, Bacterial; Glucose; Lac Operon; Lactose; Operon; Streptococcus mutans
PubMed: 32881164
DOI: 10.1111/mmi.14596