Gene Regulation & Genetic Transfer in Bacteria Lecture 13 PDF

Gene regulation: gene expression Not all genes are needed all the time The ones that are, are called constitutive genes Constitutively active genes = continuously expressed. Are often important for homeostasis The ones that aren’t, are called inducible genes...

Gene regulation: gene expression Not all genes are needed all the time The ones that are, are called constitutive genes Constitutively active genes = continuously expressed. Are often important for homeostasis The ones that aren’t, are called inducible genes Genes can be induced (“turned on”) when specific conditions are met. *Gene regulation can occur at multiple levels. The book focuses on transcriptional control. Gene regulation: gene expression Inducible genes require an inducer (a substrate that turns on gene expression) Operon = a cluster of functionally related genes (structural and regulatory) that share a promoter. Case study: Induction of the lac operon Often found in prokaryotes, can occur in some eukaryotes Contains 3 structural genes: Z, Y, A Codes for 3 proteins: β-galactosidase, a permease, a transacetylase β-Galactosidase converts lactose into glucose and galactose. The permease transports lactose into the cell The transacetylase plays a role in lactose metabolism (function unknown) Genes share a promoter and terminator Also contains a regulatory gene (I) upstream of the promoter Codes for a regulator protein (a transcription factor) [is constitutively expressed] Gene regulation: gene expression If lactose is not present, the repressor protein binds the operator site An operator is a DNA sequence that a repressor binds to silence transcription The repressor protein can be inhibited by allolactose, a lactose intermediate There are only trace amounts of β-galactosidase when the operon is suppressed If lactose is present, transcription initiates: The structural proteins are produced until lactose is broken down. β-galactosidase concentration can increase 1000-fold *The lac operon can also be induced by isopropyl β- thiogalactoside (IPTG). E. coli cannot degrade IPTG. IPTG can be used as a lab tool to control LacZ expression Gene regulation: gene repression Gene repression detects an end-product to mediate repression Case study: Repression of the trp operon Contains 5 structural genes Proteins involved in tryptophan biosynthesis Also contains a repressor gene (I [R in figure]) upstream of the promoter Codes for a repressor protein (a transcription regulation factor) [constitutively expressed] If tryptophan is not present, transcription continues : The structural proteins are produced, and tryptophan is synthesized. Gene regulation: gene repression If tryptophan is present: Tryptophan binds to and activates the repressor protein Activation causes the repressor to bind to the operator Tryptophan therefore is a corepressor for the repressor protein The trp operon is turned off until tryptophan is used up The repressor inactivates  The Griffith Experiment (1928) Had two strains of Streptococcus pneumoniae: one virulent, one avirulent Virulent strain was protected from macrophages via a capsule Avirulent strain lacked a capsule Had 4 conditions: 1. Mice died after injection with virulent strain 2. Mice lived after injection with avirulent strain 3. Mice lived after injection with heat-killed virulent strain 4. Mice died after injection with a mix of heat-killed virulent strain and live avirulent strain How can this be? Genetic transfer in bacteria Genes are normally transferred vertically, i.e. from the previous generation to the next However, organisms can also transfer genes horizontally, i.e. between individual cells Bacteria cannot use meiosis to do this But there are still several mechanisms for horizontal gene transfer in bacteria Genetic transfer in bacteria: Transformation Many bacterial types can uptake naked DNA fragments released from dead cells Cells can only uptake the DNA when competent Competence = a physiological state that occurs in some bacteria, often during late log phase Depends on the expression of competence factors (proteins necessary for transformation) Mechanism varies by bacteria DNA must both enter the cell and integrate into the host genome Bacteria cannot be transformed with chromosomal DNA from unrelated species Unrelated species lack a homologous sequence for integration into the host’s chromosome Genetic transfer in bacteria: Transformation – Induced competence Transformation can also be artificially induced, for example: Transformation does not occur naturally in E. coli E. coli cells can be made to take up DNA DNA can come from an unrelated species Must be in a self-replicating vector DNA replication of vectors occur without needing to be integrated into the host genome There are established lab techniques for inducing artificial competence Genetic transfer in bacteria: Conjugation Most common mode of horizontal transfer in bacteria Genetic information can be directly transferred between living cells via conjugation Differs mechanistically from transformation Self-replicating fertility (F) plasmids contain dozens of genes required for its replication and sex pilus synthesis Cells that contain the F plasmid are called F+ cells Cells without the plasmid are F- cells The F plasmid DNA can be transferred to another cell via the sex pilus Plasmid is transferred into recipient cell as a single DNA strand The other strand remains in the donor cell DNA replication reproduces double stranded DNA Both the donor and receipt cells are now F+ cells Genetic transfer in bacteria: Hfr Conjugation Although F plasmids can be free in the cytosol, they can sometimes integrate into the host chromosome via recombination These cells are called high frequency recombination (Hfr) cells The integrated F plasmid replicates with the genome, rather than independently Plasmid genes are still active, including sex pilus genes During conjugation, starting at the origin of replication site of the F plasmid, DNA is transferred to the recipient cell, along with the chromosomal DNA The amount of chromosomal DNA transferred is time dependent Very slow process because of the size of the host chromosome (90 - 100 minutes) The rear portion of the F plasmid enters the recipient cell last If process if interrupted, the complete F plasmid DNA is not transferred, and the recipient cell will not be F+. Genetic transfer in bacteria: Generalized transduction DNA can be transferred via bacteriophages Phages package phage DNA into the virus coat fragmented host chromosomal DNA can be randomly packaged if it is of a similar size to phage DNA When phages infect another bacteria, some bacterial DNA is transferred This bacterial DNA can be incorporated into the host chromosome via homologous recombination Occurs at low frequencies Genetic transfer in bacteria: Specialized transduction When phage DNA is being cut out of the host chromosome, a few adjacent host genes may also be removed Sometimes host chromosomal DNA is accidentally packaged Results in a high frequency transfers Genetic transfer: Transposition Transposable elements are sections of DNA that can move from one location to another The simplest type of transposable elements are insertion sequences (IS) IS elements are short pieces of DNA that only code for transposase and are flanked by very short inverted sequences Transposase = enzyme that recognizes, cuts, and regulates the transposon. Recognizes and cuts at inverted sequences Transposable elements can cut out and relocated (conservative transposition) Transposable elements can also be duplicated and inserted elsewhere (replicative transposition Transposable elements that contain additional genes are called transposons