Gene Regulation in Bacteria PDF

Chapter 14 Regulation of Cellular Processes Notice: This mater...

Chapter 14 Regulation of Cellular Processes Notice: This material is subject to the U.S. Copyright Law; further reproduction in "Bobtail Squid" by Christian Gloor is licensed under CC BY 2.0. violation of the law is prohibited.  Sensing and Responding to Environmental Fluctuations Microorganisms constantly face a changing environment Ex: E. coli in mammalian intestinal tract Alternating periods of feast and famine I. Host eats, bacteria take up nutrients - Up regulate catabolic enzymes and transport proteins - Shut down biosynthetic pathways II. Between meals, nutrients are depleted - Bacterial biosynthetic pathways are activated to make their own nutrients III. Animal defecates, some E. coli excreted - Face an entirely different set of conditions Photo by Eric Erbe, colorized by Christopher Pooley, USDA Agricultural Research Service. Public Domain. Sensing and Responding to Environmental Fluctuations Cells do not express every gene in their genome under all conditions for several reasons. Physical space limitations Energy and resource conservation Contradictory functions Microbes use information of their internal and external environments and direct the synthesis of needed proteins. © McGraw Hill,  Bacterial Gene Regulation zymes/proteins can be grouped by type of regulation Constitutive enzymes produced constantly “Housekeeping” enzymes Ex: enzymes involved in glycolysis Inducible enzymes NOT produced routinely Synthesize only when needed -> Avoid waste of resources Enzymes often involved in catabolism Transport and breakdown of specific energy sources. Ex: β-galactosidase only made when lactose present Repressible enzymes produced routinely Turned off when not required Bacterial Gene Regulation Metabolic pathways are regulated in 2 general ways to conserve energy 1. Allosteric inhibition & activation of enzymes 2. Gene regulation via signal transduction a) Sigma factors b) Operons & DNA binding proteins ‣ Only make transcripts/proteins when needed Mcy jerry at English Wikipedia CC BY-SA 3.0 Sensing and Responding to Environmental Fluctuations How do cells monitor and react to external stimuli? Signal Transduction transmits information from outside cell to inside via signal transduction pathway Sun, Z.; Popp, P.F.; Loderer, C.; Revilla-Guarinos, A. Genetically Engineered Bacterial Biohybrid Microswimmers for Sensing Applications. Sensors 2020, 20, 180. Signal Transduction Ex: Quorum Sensing Some prokaryotic organisms can “sense” density of their population Allows cells to activate genes useful at a critical biomass Ex: Pathogens coordinate genes for infection Ex: biofilm consortia coordinate genes for competition 2019 Tanet, Tamburini, Baumas, Garel, Simon and Casalot. Frontiers in Signal Transduction Two-Component Regulatory Systems 1. Membrane-spanning sensor protein/kinase In response to external stimulus: Sensor catalyzes a reaction to add a phosphate to an Amino acid on internal region of the kinase (autophosphorylation) 2. Cytosol response regulator Phosphate transferred from sensor to regulator The regulator turns genes on or off Ex: Nitrate is the stimulus for E. coli that turns on genes so that cell can use nitrate as terminal electron acceptor © McGraw Hill, Signal Transduction Two-Component Regulatory Systems EnvZ/Ompr system is example of a two-component signal transduction system. Controls porin production in E. coli EnvZ is the sensor kinase and senses osmolarity changes in the periplasm During high periplasmic osmolarity conditions, the EnvZ catalytic domain phosphorylates a histidine residue on the cytoplasmic portion of the protein (autophosphorylation) The phosphate group is transferred to an aspartic acid of OmpR, the response regulator OmpR has a DNA-binding domain that then activates gene expression of ompC and represses ompF OmpF pores are larger and OmpC pores are © McGraw Hill, Bacterial Gene Regulation Signal transduction and Two-component regulatory systems affect gene expression through interaction with 1. Sigma factors 2. DNA Binding Proteins (repressors and activators) © McGraw Hill, Bacterial Gene Regulation Sigma Factors - Standard sigma factors recognize Response Regulator specific promoters for housekeeping genes - Alternative sigma factors immediately change the expression of many genes as they direct R NA polymerase to specific subsets of bacterial promoters. - Ex: many alternative sigma factors are produced at each sporulation stage in Bacillus subtilis - Anti-sigma factors are used to Methylobacterium extorquen inhibit alternative sigma factors “In Unstressed cells, PhyR is inactive, and “In response to a stress, PhyR is phosphorylated and interacts with the sigma factor NepR, thus releasing σEcfG1 and allowing - Anti-sigma factors can themselves be (σEcfG1) is sequestered by its anti-sigma factor σEcfG1 to associate with RNA polymerase to transcribe stress genes.” neutralized by anti-anti-sigma (NepR)” factors. Francez-Charlot et al. Proceedings of the National Academy of Sciences Bacterial Gene Regulation Two-component regulatory systems affect gene expression (transcription) through interaction with 1. Sigma factors 2. DNA Binding Proteins (repressors and activators) © McGraw Hill, Operon gene regulation expresses a protein acting as a repressor or activator In prokaryotes - regulated genes are found on an… Operon - Genes coding for functionally related enzymes are clustered together on the chromosome and co-expressed/regulated on one transcript (polycistronic mRNA) Several operons being controlled simultaneous using a single regulatory mechanism (repressor/activator) is called a regulon This process is called Global control Parker, N., et. al. (2019) Microbiology. Openst Transcriptional Control by Repressors and Activators The initiation of transcription in bacteria is controlled by regulatory proteins. Synthesized by upstream regulatory gene Bind DNA at or near gene promoters Stimulate or prevent binding of RNA polymerase to promoter 14 Parker, N., et. al. (2019) Microbiology. Openst  Bacterial Gene Regulation Operon gene regulation via DNA-binding proteins Two types: I. Repressor blocks transcription (negative regulation) Binds to operator immediately downstream of promoter Stops RNA polymerase from progressing/attaching II. Activator facilitates transcription (positive regulation) Binds to promoter (or activator binding site) to help RNA polymerase initiate transcription Repressor/Activator Activator Binding Parker, N., et. al. (2019) Microbiology. Openst  Gene Regulation a) Induce r Repressors prevent the ability of RNA polymerase to bind to the operator and are b) allosteric Two functions: 1. Induction: repressor synthesized in a form that binds to the operator (active form) and blocks c) transcription An Inducer binds to repressor -> conformational change -> repressor unable to bind to the operator mRNA © McGraw Hill,  Represso Corepress r a) or Gene Regulation Repressors prevent the ability of RNA polymerase to bind to the operator and are mRNA allosteric b) Two functions: 2. Repression: repressor synthesized as an inactive form that is unable to bind to the operator Corepressor attaches to c) repressor -> conformational change -> complex binds to the operator Blocks transcription © McGraw Hill, Bacterial Gene Regulation Activators facilitate transcription (positive regulation) Genes controlled by an activator have an ineffective promoter immediately preceded by activator-binding site Inducer Binding of the activator enhances the ability of RNA polymerase to initiate transcription at the promoter Activators are allosteric (always mRN produced in inactive form) and A when an inducer binds to an activator the protein binds to the activator-binding site © McGraw Hill, The lac Operon as a Model Lac Operon – Contains genes for proteins that transport & breakdown lactose. Enzymes normally not produced unless lactose present. β-galactosidase (LacZ) – cleaves lactose, a disaccharide, into glucose and galactose Lactose permease (LacY) – membrane protein that transports lactose β- galactoside transacetylase (LacA) – role is unclear: hypothesized to inactivate toxic compounds that can be transported via the lactose permease © McGraw Hill, The lac Operon as a Model Lac Operon – Contains genes for proteins that transport & breakdown lactose. Enzymes normally not produced unless lactose present. β-galactosidase (LacZ) – cleaves lactose, a disaccharide, into glucose and galactose Lactose permease (LacY) – membrane protein that transports lactose β- galactoside transacetylase (LacA) – role is unclear: hypothesized to inactivate toxic compounds that are transported into the cell along with lactose © McGraw Hill, The lac Operon as a Model In E. coli, the genes that encode LacY and LacZ, LacA form an operon. The expression of all three genes is regulated by a single promoter (PlacZYA) The lactose repressor is encoded by a regulatory gene (LacI gene) that is situated immediately upstream of the operon. LacI is expressed constitutively from its own promoter (Placl). © McGraw Hill, The lac Operon as a Model Lac Operon When lactose is not present: LacI repressor binds as a tetramer to the operator region and represses transcription by preventing open complex formation with RNA polymerase. © McGraw Hill, The lac Operon as a Model Lac Operon When lactose is present Some lactose is converted to the inducer – allolactose - Binds to allosteric site on repressor - Repressor falls off operator and transcription progresses Only occurs when glucose is not present in environment (or at low levels) © McGraw Hill,  The lac Operon as a Model Glucose and the lac Operon When glucose and lactose are both present, bacteria use glucose first Global control system that represses the lac operon as well as other genes involved in catabolism of other compounds Two mechanisms: 1. Inducer exclusion Diauxic growth curve 2. Carbon catabolite Cells preferentially use glucose repression When glucose supply decreases cells start metabolizing lactose In between, growth stalls -> enzymes needed to break down lactose synthesized © McGraw Hill,  The lac Operon as a Model Unphosphorylated Inducer exclusion glucose transporter When glucose and component = glucose present lactose both present, bacteria use glucose first Glucose transport Glucose system acts as a sensor of glucose P availability HPr IIA Remember, glucose is transported into cell by group transport – Glucose LacY X Lactose transporter component phosphorylates glucose as it enters © McGraw Hill,  The lac Operon as a Model Inducer exclusion Unphosphorylated glucose transporter High glucose levels: component = glucose Unphosphorylated present Glucose transporter GlucoseP component binds to the lactose permease Preventing lactose from Glucose entering cell and acting as inducer on lac operon P This is called inducer HPr IIA exclusion LacY X Lactose © McGraw Hill,  The lac Operon as a Model Inducer exclusion Phospohrylated glucose transporter Low glucose levels component = glucose Phosphorylated glucose absent/low transporter component cannot block lactose permease Lactose transported -> allolactose -> repressor falls off lac operon operator HPr IIA P P P Lactose LacY © McGraw Hill,  The lac Operon as a Model Phospohrylated Lac operon is also regulated by carbon glucose transporter catabolite repression (CCR) so that component = glucose absent/low glucose is the 1st sugar metabolized In E. coli this involves a small molecule called cyclic AMP (cAMP). Increased cAMP levels signal glucose depletion. When a cell is starved for glucose, ATP levels decrease in the cell. Some ATP is converted to cAMP by adenylyl HPr IIA cyclase. P P Adenylyl cyclase is activated by phosphorylated EIIA (=low glucose) Cyclic AMP controls the expression of many genes, including those in the lactose operon, by combining with a regulator called cAMP receptor protein (CAP) and binding near the lacZYA promoter. © McGraw Hill,  The lac Operon as a Model Phospohrylated Carbon catabolite glucose transporter component = glucose repression absent/low Low glucose levels: Lactose LacY Phosphorylated glucose transporter component (EIIA) activates adenylyl cyclase converts ATP to Cyclic AMP Cyclic AMP concentration ⬆ HPr IIA P P © McGraw Hill,  The lac Operon as a Model Carbon catabolite repression Low glucose levels: Cyclic AMP acts as inducer and cAM binds to the lac operon activator P (cAMP receptor protein – CAP) CAP CAP changes shape allowing it to bind to activator binding site (upstream of lac operon) cAM P Now, RNA polymerase can bind CAP and transcribe the lac operon © McGraw Hill,  The lac Operon as a Model Carbon catabolite repression High glucose levels: Unphosphorylated glucose cAM XP transporter component (EIIA) -> X adenyl cyclase not activated -> cAMP not made – does not bind CAP to CAP and CAP cannot bind to activator binding site CAP X © McGraw Hill, Regulation of the lac Operon by the lac Repressor and CAP Lactose LacY H P IIA Pr P © McGraw Hill, The AraC Family of Transcriptional Regulators Many proteins act only as repressors (like LacI) or solely as activators (like CAP). AraC is a regulatory protein that has dual regulatory functions. AraC controls the expression of proteins that allow microbes to metabolize the sugar arabinose. AraC can assume one of two conformations depending on whether arabinose is available. When arabinose is absent, AraC represses gene expression, but when it is present, AraC activates these same genes. © McGraw Hill, Quorum Sensing Cell-to-cell communication mediated by small signaling molecules. Coordinates gene expression. Plays an essential role in the regulation of genes whose products are needed for virulence, symbiosis, biofilm production, and morphological differentiation in a wide range of bacteria. An autoinducer is a membrane-permeable molecule that allows bacteria to sense the density of cells in the environment and control gene expression accordingly. This phenomenon is called quorum sensing © McGraw Hill, Quorum Sensing and Cell-Cell Communication Quorum sensing was first was discovered in the aquatic Bioluminescenc e luminescent bacterium Aliivibrio fischeri Biomass Threshold A. fischeri colonizes the light organ Biomass of the Hawaiian bobtail squid. A. fischeri uses quorum sensing to produce light when their cell numbers are above a specific Time (h) critical biomass threshold. Quorum Sensing in A. fischeri (a) Chris Frazee/UW-Madison; i (b) ©Dr. Margaret Jean McFall- Nga "Bobtail squid light organ silhouette" by Pen-Yuan Hsing is licensed under CC BY-SA 4.0. © McGraw Hill, Quorum Sensing in A. fischeri LuxI gene synthesizes AHL, which diffuses out of the cell. When threshold levels of AHL are reached, autoinducer reenters cells and binds to LuxR (activator). LuxR activates transcription of the genes for AHL synthase (luxl) and luciferase proteins needed for light production. © McGraw Hill, Quorum Sensing and Pathogenesis Many pathogens use quorum sensing to control the expression of virulence genes for optimal effect on the host. Examples: Pseudomonas aeruginosa—forms a biofilm in the lungs of cystic fibrosis patients and secretes virulence factors like proteases only when it can overwhelm its host and avoid immune responses with high cell densities. Staphylococcus, Yersinia pestis, Vibrio cholerae, and many others Eukaryotic gene regulation Much more complex and includes multiple mechanisms: Regulating initiation of transcription (transcription factors) Regulating mRNA processing and modification RNA interference (RNAi) Short RNAs that decrease transcription or translation Transcription – directs enzymes to methylate DNA Translation - direct enzymes to degrade messenger RNA Eukaryotic gene regulation RNA interference (RNAi) - degrade messenger Cell produces RNA siRNA transcript specific short interfering RNAs (siRNAs) produced by cell siRNAs sequence complementary to target mRNA transcript siRNAs joins a protein complex (RNA- RNA-induced induced silencing complex – RISC) silencing complex Together: assembles the siRNA acts as probe to locate and bind to complementary mRNA transcripts RISC catalyzes the destruction of the siRNA in RISC binds to transcript complementary mRNA and enzymes cut Mocellin, S., Provenzano, M. RNA interference: learning gene knock-down from cell physiology. J Transl Med 2, 39 (2004). https://doi.org/10.1186/1479-5876-2-39 CC BY 2.0  The Gospel in Creation The gospel is the manifestation of God's power put forth to save men. It is in creation, therefore, that the power of God is to be seen by everybody… Therefore, the works of creation teach the gospel. -E.J. Waggoner The gospel demonstrates the power, love, creativity, grace of God. Psalm 19: The heavens declare the glory of God; the These same characters of skies proclaim the work of his hands. Day after day they pour forth speech; night after night they reveal knowledge. They God are have no speech; they use no words; no sound is heard from revealed/displayed in his them. Yet their voice goes out into all the earth, their words to the ends of the world. creation. "Bobtail Squid" by Christian Gloor is licensed under