Genetics: From genes to genomes - Chapter 15: How genes are regulated

PowerPoint to accompanyGenetics: From Genes to GenomesFourth EditionLeland H. Hartwell, Leroy Hood, Michael L. Goldberg, Ann E. Reynolds, and Lee M. SilverPrepared by Mary A. BedellUniversity of Georgia*Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th editionHow Genes Are Regulated*PART VCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 15Gene Regulation in Prokaryotes15.1 Overview of Prokaryotic Gene Regulation15.2 The Regulation of Gene Transcription15.3 Attenuation of Gene Expression: Termination of Transcription15.4 Global Regulatory Mechanisms15.5 A Comprehensive Example: The Regulation of Virulence Genes in V. choleraeCHAPTER OUTLINECHAPTERRNA polymerase participates in all three phases of transcriptionInitiation – core RNA polymerase plus sigma (σ) factorCore has four subunits: two alpha (α), one beta (β), one beta prime (β')DNA is unwound and polymerization beginsElongation – core RNA polymerase without σ factor Continues until RNA polymerase recognizes termination signalTermination – two kinds in bacteriaRho-dependent – Rho (ρ) protein binds to RNA polymerase and removes it from RNARho-independent – 20 nt sequence in RNA forms stem-loop Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 15*Role of RNA polymerase in initiation and elongation phases of transcriptionCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 15*Fig. 15.2Two kinds of transcription termination in bacteriaCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 15*Fig. 15.2 (cont)Regulation of expression canoccur at many stepsTranscriptional controlBinding of RNA polymerase to promoterMost critical step in regulation of most prokaryotic genesShift from initiation to elongationRelease of mRNA at terminationPosttranscriptional controlStability of mRNAEfficiency of translation initiationStability of polypeptideCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 15*Utilization of lactose by E. coli providesa model system of gene regulationLactose utilization requires two enzymes (Fig. 15.3)Permease transports lactose into cellβ-Galactosidase (β-Gal) splits lactose into glucose and galactoseIn the absence of lactose, both enzymes are present at very low levelsLactose is the inducer of the genes encoding permease and β-Gal Induction – stimulation of synthesis of a specific proteinInducer – molecule responsible for inductionCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 15*Lactose utilization in an E. coli cellCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 15*Fig. 15.3Advantages of using lactose utilization by E. coli as a model for understanding gene regulationLac− mutants can be maintained on media with glucose and so lac genes are not essential for survivalIf both glucose and lactose are present, E. coli cells will use glucose firstSimple assays for lac expression - use of ONPG or X-gal as substrates for β-gal (color change)Lactose induces a 1000-fold increase in β-gal activityDetection and characterization of hundreds of lac− mutants defective in lactose utilization Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 15*Studies of lac− mutants revealed the operon theory of gene regulationJacques Monod and Francois Jacob – Pasteur InstituteNobel Prize in 1965 (with A. Lwoff) for their discoveries concerning genetic control of enzyme and virus synthesisCompared the effects of many different types of lac mutants on induction and repression of enzyme activity for lactose utilizationOperon theory - one signal can simultaneously regulate expression of several clustered genesHypothesized that lac genes are transcribed together as a single mRNA (polycistronic) from a single promoter Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 15*The lactose operon in E. coliThe playersThree structural genes - lacZ, lacY, and lacAPromoter - site to which RNA polymerase bindsCis-acting operator site – controls transcription initiationTrans-acting repressor - binds to the operator (encoded by lacI gene)Inducer - prevents repressor from binding to operatorCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 15*Fig. 15.5aRepression of lac gene expressionIn the absence of lactose, repressor binds to the operator and prevents transcriptionlac repressor is a negative regulatory elementCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 15*Fig. 15.2bInduction of the lac operon in E. coli When lactose (or IPTG) is present:Inducer binds to the lac repressorRepressor changes shape and cannot bind to operatorRNA polymerase binds to the promoter and initiates transcription of the polycistronic lac mRNACopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 15*Fig. 15.2cJacob and Monod defined the roles of the lac genes by genetic analysis of many lacI− mutantsComplementation analysis identified three genes in a tightly linked clusterlacZ encodes β-galactosidaselacY encodes permeaselacA encodes transacetylaseMost studies focused on lacZ and lacYConstitutive expression of β-galactosidase and permease was caused by mutations in the lacI geneConstitutive mutants (lacI−) express the enzymes in the absence and presence of inducer Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 15*The PaJaMo experiment provided evidence that lacI encodes a repressorlacI+ lacZ+ DNA transferred into lacI− lacZ− cellsβ-gal levels increased initially β-gal levels decreased as repressor accumulatedβ-gal accumulation resumed after addition of inducer Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 15*Fig. 15.7Jacob and Monod proposed that lacI encodes a repressor that binds to an operator site near the lac promoterHow the inducer acts to trigger synthesis of lac enzymesBinding of inducer to repressor changes the shape of the repressor so that it can longer bind to DNAWhen no inducer is present, repressor is able to bind to DNARepressor is an allosteric protein – undergoes reversible changes in conformation when bound to another moleculeCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 15*lacl− mutants have a mutant repressor that cannot bind to operatorIn lacI− mutants, lac genes are expressed in the absence and the presence of inducer (constitutive expression) Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 15*Fig. 15.8lacls mutants have a superrepressor that binds to operator but cannot bind to the inducerIn lacIS mutants, lac genes are repressed in the absence and the presence of inducerCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 15*Fig. 15.9lac repressor has two separate domainsMutated sequences in many different lacI− mutants clustered in the DNA-binding domainMutated sequences in many different lacIS mutants clustered in the inducer-binding domainX-ray crystallography revealed the two separate domains Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 15*Fig. 15.10lacOc mutants have a mutant operator that cannot bind the repressorIn lacOc mutants, lac genes are expressed in the absence and the presence of inducer (constitutive expression) Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 15*Fig. 15.11Proteins act in trans, DNA sites act in cisJacob and Monod used partial diploids carrying different alleles of lac regulatory elements and structural genes to identify trans-acting and cis-acting elementsF' lac plasmids (Chapter 14) were used to generate partial diploidsTrans-acting elements: Can diffuse through the cytoplasm and act at target DNA sites on any DNA molecule in the cellCis-acting elements:Can only influence expression of adjacent genes on the same DNA moleculeCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 15*Lacl+ protein acts in transRepressor expressed from the plasmid can diffuse through the cytoplasm and bind to the operator on the chromosomeCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 15*Fig. 15.12Lacls protein acts in transSuperrepressor expressed from the plasmid can diffuse through the cytoplasm and bind to the operator on the chromosome, even in the presence of inducerCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 15*Fig. 15.13lacOc acts in cisThe lacOC mutation affects expression of genes only on the DNA that it is located onCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 15*Fig. 15.14The lac operon of E. coli is regulated by both lactose and glucoseWhen both glucose and lactose are present, only glucose is utilizedLactose induces lac mRNA expression, but only in the absence of glucoseLactose prevents repressor from binding to lacOlac repressor is a negative regulator of lac transcriptionlac mRNA expression cannot be induced if glucose is presentGlucose controls the levels of cAMP cAMP binds to cAMP receptor protein (CRP) CRP-cAMP is a positive regulator of lac transcriptionCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 15*Positive regulation by CRP–cAMPCatabolite repression – overall effect of glucose is to prevent lac gene expressionCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 15*Fig. 15.15Positive regulation of the araBADoperon by AraCThree structural genes required in the breakdown of the sugar arabinose - araB, araA, and araDArabinose genes are in an operon and are induced when arabinose is presentAraC is a positive regulator of the araBAD operonLoss of function of AraC results in no expression of the araBAD operon when arabinose was presentCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 15*AraC is a positive regulatorCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 15*Fig. 15.16Further studies revealed more about regulatory proteins and sitesBiochemical evidence for lac repressor binding to lacO (Fig. 15.17)X-ray crystallography revealed the structure of repressor proteinslac repressor has a helix-turn-helix (HTH) motif (Fig. 15.18)Evidence that specific amino acids in the α-helices of lac repressor are required for binding to lacO (Fig. 15.19)DNA sequences to which negative and positive regulators bind have a two-fold rotational symmetrye.g. CRP-binding site of the lac operonMost DNA-binding regulatory proteins are oligomeric, with two to four subunitsCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 15*The lac repressor binds to operator DNACopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 15*Fig. 15.17DNA recognition sequences by helix-turn-helix (HTH) motifA protein with an HTH motif has two α-helical regions separated by a turn in the proteinThe HTH motif fits into the major groove of DNAOne of the α-helices recognizes a specific DNA sequence Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 15*Fig. 15.18Changing amino acids in recognition sequence of a repressor protein 434 repressor binds to an operator in the DNA of the 434 virusP22 repressor binds to an operator in the DNA of the P22 virusAmino acid sequences in the α-helix of 434 repressor were modified to have amino acid sequence like that of P22 repressorHybrid 434-P22 functioned just like the P22 repressorCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 15*Fig. 15.19DNase footprint shows where proteins bindCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 15*Fig. 15.20Incubate radiolabeled DNA from lac+ operon with partially purified protein from lacI+ cellsPartial digest of DNA with DNase IGel electrophoresis and autoradiographyIf protein is bound to DNA, then specific fragments will be protected from DNase I digestionCRP–cAMP binds as a dimer to a regulatory regionCRP-binding sites have a two-fold rotational symmetryCRP protein binds as a dimerCRP-binding site consists of two recognition sequences, one for each subunit of the CRP dimerCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 15*5'TGTGAGTTAGCTCACA 3'3'ACACTCAATCGAGTGT 5'Fig. 15.21lac repressor tetramer binds to two siteslac repressor is a tetramer, with each subunit containing a DNA-binding HTH motiflac operon has three operators (O1, O2, and O3) each of which contains two recognition sequences for lac repressorO1 has the strongest binding affinity for lac repressorMaximal repression occurs when all four repressor subunits are boundCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 15*Fig. 15.2Two repressor subunits bind to O1Two repressor subunits bind to either O2 or O3AraC acts as both a repressor and an activator AraC can bind to three sites (araO, araI1, and araI2) with different affinitiesCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 15*Fig. 15.23(a) No arabinose present: When AraC is bound to araO and to araI1, looping of DNA occurs and prevents transcription(b) Arabinose present: Arabinose causes allosteric change in AraC so that it cannot bind to araOAraC interacts with RNA polymerase only when both araI1 and araI2 are occupiedInteraction of regulatory proteins with RNA polymeraseMany negative regulators (e.g. lac repressor) prevent transcription initiation by blocking the functional binding of RNA polymerase (Fig. 15.24)Many positive regulators (e.g. CRP-cAMP) establish contact with RNA polymerase that enhances transcription initiation (Fig. 15.25)Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 15*Overlapping binding sites for RNA polymerase and lac repressorWhen lac repressor is bound to lac operator, functional binding of RNA polymerase to the promoter is blockedCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 15*Fig. 15.24CRP-cAMP complex makes direct contact with RNA polymeraseWithout interaction with CRP-cAMP, RNA polymerase can bind to the promoter but is less likely to unwind DNA and initiate transcriptionCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 15*Fig. 15.25Using the lacZ gene as a reporter of gene expressionReporter gene – protein-encoding gene whose expression in the cell is quantifiable by sensitive and reliable techniquesMeasuring gene expressionFuse coding region of lacZ to cis-acting regulatory regions from other genes (Fig. 15.26)Identifying sets of genes regulated by the same stimulusCreate library of cells with promoter-less lacZ inserted by transposition into random sites in the genome (Fig. 15.27)Controlling gene expressionFuse the lac regulatory sequences to the coding region of a foreign gene (Fig. 15.28)Inducible expression of the foreign gene controlled by IPTGCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 15*lacZ fusion used to perform genetic studies of the regulatory region of gene XConditions that regulate expression of the test regions from gene X will alter the levels of β-galactosidaseSpecific regulatory sites can be identified by constructing and testing mutations in the test regions of gene XCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 15*Fig. 15.26Using lacZ to identify sets of genes regulated by the same stimulusCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 15*Fig. 15.27Transposition of promoter-less lacZ coding region Library of clones containing lacZ insertions at random sitesScreen library to identify all the genes that express lacZ in response to a signalUse of fusions to overproduce a geneproductCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 15*Fig. 15.28bFig. 15.28aExpression of human growth hormone in E. coli controlled by lac control regionExpression of gene X under control of the lac regulatory systemRegulation of the tryptophan (trp) operon in E. coliStructural genes for tryptophan (Trp) biosynthesis are expressed only in the absence of Trp Two mechanisms for trp operon regulationTrpR gene encodes the trp repressor that can bind to the Trp operator (TrpO)When Trp is present, TrpR repressor binds to TrpOWhen Trp is absent, TrpR repressor cannot bind to TrpOAttenuation controls termination of transcription in the trp leader (TrpL)When Trp is present, transcription terminates in TrpLWhen Trp is absent, transcription doesn't terminate in TrpL Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 15*Tryptophan acts as a corepressorBinding of tryptophan to TrpR repressor allows TrpR to bind to TrpO and inhibit transcription of the five structural genesIn the absence of tryptophan, TrpR repressor cannot bind to TrpOCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 15*Fig. 15.29aEvidence that TrpR repressor is not the only regulator of the trp operonConstitutive expression of Trp biosynthesis doesn't occur in TrpR− mutantsIf TrpR were the sole regulator, maximal expression of trp genes would occur in the absence or presence of tryptophanSecond regulatory mechanism is attenuation – control of gene expression by premature termination of transcriptionCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 15*Table15.1Attenuation controls termination of transcription in the trp leader (TrpL)Truncated mRNA - terminates in TrpL, only 140 basesFull-length mRNA - continues through TrpL and encodes all five structural genesTranscription from the trp promoter produces two alternative mRNAsCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 15*Fig. 15.29bAlternate stem-loop structures in trpL RNADifferent regions of trpL have complementary base-pairingFormation of the 1-2 stem-loop allows formation of the 3-4 stem-loopFormation of the 2-3 stem-loop prevents formation of the 3-4 stem-loopThe 3-4 stem loop is a transcription terminatorCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 15*Fig. 15.30aWhen tryptophan is present, transcription terminates in trpL because of translationThe trpL mRNA is translated and includes two trp codonsMovement of ribosomes through trpL mRNA depends on the availability of tRNATrpWhen Trp is present, tRNATrp is available and rapid ribosome movement allows the formation of 3-4 stem-loopCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 15*Fig. 15.30bWhen tryptophan isn't present, transcription doesn't terminate in trpL Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 15*Fig. 15.30cGlobal regulatory mechanismsDramatic shifts in environmental conditions can trigger expression of sets of genes or operonsRegulon – a group of genes whose expression is controlled by the same regulatory proteinTwo examples in E. coli:CRP-cAMP controls several catabolic operonsExpression of several genes induced by heat shockHighly conserved stress responseInduced proteins include those that recognize and degrade aberrant proteins and chaperones, which assist in preventing protein aggregationCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 15*Sigma factor (σ) recognition sequencesNormal, housekeeping sigma factor is σ70Active under normal physiological conditions, but is inactivated by heat shockrpoH genes encodes σ32, an alternative sigma factorHeat shock inducible genes have promoters that are recognized by σ32σ32 is resistant to inactivation by heat shockCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 15*Fig. 15.31Factors influencing increase in σ32 activity after heat-shock treatmentIncreased transcription of rpoH geneIncreased translation of σ32 mRNA because of increased stability of rpoH mRNAIncreased stability and activity of σ32 proteinNo longer inhibited by chaperones DnaJ/KNo competition from σ70 because it is removed by degradationCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 15*At normal temperatures, promoter for rpoH gene is recognized by σ70After heat shock, σ70 is degraded and transcription of the gene for σ24 is increasedσ24 recognizes a different promoter sequence at rpoH Increased expression of σ32 causes transcription of several genes Alternate sigma factor in the heat-shock responseCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 15*Fig. 15.32Translational control of another sigma factor encoded by the rpoS geneUnder normal conditions, rpoS gene is transcribed but rpoS mRNA is not translatedAfter stress response, a small RNA (dsrA) binds to rpoS mRNA and allows translationCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 15*Fig. 15.33Tools for studying genes regulated in a global responseMicroarrays of expression in different growth conditions, e.g. E. coli grown on glucose, glycerol, succinate, or alanineSwitch from glucose to glycerol or succinate caused increased expression of 40 genesSwitch from glucose to alanine caused increased expression of 188 genesMutants in specific genes, e.g. NtrC is a master control gene activated by lack of ammoniaComputer analysis to identify regulatory proteins, e.g. searches for HTH DNA binding motifCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 15*A comprehensive example: The regulation of virulence genes in V. choleraeV. cholerae is the bacterial species that causes cholera, a life-threatening diarrheal disease Bacteria are ingested in contaminated drinking waterRespond to changes in environment by increasing or decreasing transcription and/or translation of specific genesIn intestine, V. cholerae express proteins to make flagella and to degrade mucous so that they can reach epithelial cellsOnce the bacteria reach the epithelial cells, they secrete toxins that result in diarrhea Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 15*Identification of regulators of toxin production in V. choleraeTwo genes, ctxA and ctxB, identified that encode subunits of cholera toxinGene fusions of ctxA promoter and lacZ coding region created and transformed into E. coliTransformation of E. coli with ctxA-lacZ reporter gene with fragments of V. cholerae genomic DNAtoxR gene from V. cholerae identified in genomic DNA that caused increased expression of ctxA-lacZ reporter geneFurther experiments showed that mutation of toxR gene in V. cholerae abolished its virulenceCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 15*Identification of other V. cholerae genes regulated by toxRLibrary of V. cholerae cells created that had random insertions of lacZ coding regionGene fusions of a constitutive promoter and toxR coding region created and transformed into the lacZ libraryColonies with high β-gal expression had lacZ sequences inserted adjacent to promoters regulated by toxRIn E. coli, toxR could not affect expression of the same genesToxT was then identified as a positive regulator of many V. cholera virulence genesTcpP and ToxR both bind to ToxT promoter and are both required for ToxT transcriptionCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 15*Model for how V. cholerae regulatesgenes for virulenceCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 15*Fig. 15.34Unanswered questions about expression of virulence genes in V. cholera What is the signal that makes cholera bacteria stop swimming and start colonizing the intestinal epithelial cells?What molecular events differentiate swimming versus adherence?Why is there a cascade of regulatory factors (ToxR and ToxT)?A better understanding of V. cholerae pathogenesis will lead to more effective treatments and preventatives for choleraCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 15*