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ninth edition TORTORA ⏐ FUNKE ⏐ CASE MICROBIOLOGY an introduction Chapter 8 Microbial Genetics PowerPoint® Lecture Slide Presentation prepared by Christine L. Case! Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Structure and Function of the Genetic Material § Genetics – science of heredity § Study of what genes are, how they determine the characteristics of an organism, how they carry information, how the information is copied, how information is passed on to subsequent generations and between organisms § Genome – all the genetic information in a cell § Includes chromosomes and plasmids § Genomics – sequencing, characterization of genomes Structure and Function of the Genetic Material § Chromosomes – a structure that contains the DNA § Physically carries the hereditary information, genes § Bacteria typically have one circular chromosome § Attached to membrane at several points DNA is twisted and supercoiled to fit into cell Chromosome is 1000x longer than width of cell Chromosome erupting from one E. coli cell Structure and Function of the Genetic Material § Genes – segments of DNA that carry information to produce functional products, proteins § Genetic code – the set of rules that determines how a nucleotide sequence of a gene is converted into the amino acid sequence of a protein § Linear sequence of nucleotides, bases provides the information for making proteins § Much of anabolism is making proteins from DNA § When a product is made, the gene is expressed Structure and Function of the Genetic Material § Genotype – an organism’s genetic makeup § The information found in its DNA § Represents potential characteristics § Phenotype – an organism’s expressed properties § Eg, an ability to resist an antibiotic § Phenotype is the display of an organism’s genotype § Genotype is collection of DNA in cell § Phenotype is collection of proteins in cell The Flow of Genetic Information Genetic information can flow in a number of ways “Horizontal gene transfer” “Vertical gene transfer” “DNA replication” DNA Replication § One parental DNA molecule converted into 2 identical daughter molecule § Parental DNA strand acts as template for new strand § Structure of DNA § Complementary base pairing § DNA has direction § 3’ (3 prime) and 5’ (5 prime) § DNA strands are counter-parallel to each other DNA has direction Nucleotides added to 3’ OH 3’ OH required for nucleotide addition DNA Replication Structure of DNA 5’ end attached to PO4 DNA strands run anti-parallel 5’ à 3’ 3’ à 5’ 3’ end attached to OH DNA Replication Overview 1 – double stranded DNA unwound by helicase enzyme 2 – exposed bases matched up with bases in cytoplasm A to T C to G 3 - DNA polymerase joins new nucleotides to forming DNA molecule 4 – each new DNA molecule contains one parental strand, one daughter strand à semi-conservative replication DNA Replication The Flow of Genetic Information Genetic information can flow in a number of ways “Horizontal gene transfer” “Vertical gene transfer” “DNA replication” RNA and Protein Synthesis § Genetic information from DNA follows the “Central Dogma of Biology” § DNA is used to make RNA, which is used to make protein § DNA à RNA à Protein § DNA à RNA = Transcription, RNA synthesis § RNA à Protein = Translation, Protein synthesis RNA and Protein Synthesis Transcription – synthesis of RNA from DNA § Recall, RNA is single stranded, uses U instead of T § Three kinds of RNA § Ribosomal RNA, rRNA –integral part of ribosomes § Transfer RNA, tRNA – involved in protein synthesis § Messenger RNA, mRNA – carries information for making protein § mRNA is synthesized from a gene by enzyme called RNA polymerase RNA and Protein Synthesis § Transcription begins when RNA polymerase binds to the promoter sequence § RNA polymerase joins nucleotides into new mRNA strand using DNA as template § New mRNA complementary to DNA template § A à U § T à A § C à G § G à C RNA and Protein Synthesis Transcription 2 – mRNA synthesized by RNA polymerase 3 – RNA polymerase binds to DNA at the promoter 1 – double stranded DNA unwound by RNA polymerase enzyme RNA and Protein Synthesis RNA polymerase synthesizes mRNA using DNA as template DNA template is read 3’ à 5’ mRNA is made 5’ à 3’ 3’ 5’ 5’ 3’ RNA and Protein Synthesis § RNA is complementary to DNA § If DNA template is CGATAAG § What is the RNA strand formed? § RNA synthesized is GCUAUUC RNA and Protein Synthesis § Transcription continues until RNA polymerase reaches site on DNA called the terminator mRNA, RNA polymerase “fall off” DNA 5’ mRNA strand Transcription RNA and Protein Synthesis Translation – protein synthesis from mRNA § “Translates” the “language” of nucleic acids into “language” of proteins § Codons – groups of three bases used to translate nucleic acids into amino acids § Each codon “codes” for an amino acid § Sequence of codons on mRNA determines sequence of amino acids § Codons to amino acids is the genetic code Translation: The Genetic Code The Genetic Code § Written as mRNA, 5’ to 3’ § Two types of codons § Sense codons – code for amino acids § 61 codons for 20 amino acids § Degeneracy of genetic code – amino acids coded for by multiple codons § Nonsense codons – code for stops in translation § Aka stop codons RNA and Protein Synthesis § Translation starts with AUG start codon § Codes for amino acid methionine § In Bacteria, proteins start with formylmethionine § tRNA carries amino acids to ribosomes tRNA carries amino acid on one end … … and has anticodon at other end Anticodon recognizes codon in mRNA What’s the codon this tRNA recognizes? RNA and Protein Synthesis RNA and Protein Synthesis 3 – tRNA binds to next codon 1 - ribosome binds at start codon 3’ 5’ 2 - ribosome moves along mRNA in 5’ to 3’ direction RNA and Protein Synthesis 4 – ribosome forms peptide bond between amino acids 3’ 5’ RNA and Protein Synthesis 5 – translation continues until ribosome reaches stop codon RNA and Protein Synthesis 6 – ribosome breaks up, mRNA, protein released Translation Which of the following is a product of transcription? 1. RNA polymerase 2. DNA polymerase 3. Proteins 4. mRNA RN A 0% m in s 0% P ro te po l ym er a se D NA er a ym po l NA R 0% se 0% Copies RNA from DNA 1. DNA polymerase 2. RNA polymerase 3. tRNA 4. DNA ligase 5. DNA helicase ic a se 0% D NA he l as e lig NA 0% D NA po l NA R 0% tR ym er a er a ym po l D NA 0% se se 0% Is required for protein synthesis 1. DNA polymerase 2. RNA polymerase 3. tRNA 4. DNA ligase 5. DNA helicase ic a se 0% D NA he l as e lig NA 0% D NA po l NA R 0% tR ym er a er a ym po l D NA 0% se se 0% Where does RNA polymerase bind at? 1. AUG start codon 2. The promoter 3. The initiator 4. Transcriptase ra n T T he in it i m pr o he T 0% sc rip at or ot e do n co ar t A UG st 0% ta se 0% r 0% Which of the following is a product of transcription? 1. RNA polymerase 2. DNA polymerase 3. Proteins 4. mRNA RN A 0% m in s 0% P ro te po l ym er a se D NA er a ym po l NA R 0% se 0% The template strand is the strand that the RNA polymerase uses to produce the mRNA product. What is the sequence of the start codon on the template strand? 1. AUG 2. ATG 3. UAC 4. TAC AC 0% T 0% U AC 0% A TG A UG 0% Transcription and Translation Consider the following "Template" strand of DNA AGA GGA TTA TAC TAT TCG TGG CGC AGT ATG TAA ACT TCT 1. Transcribe this DNA molecule 2. Translate the mRNA to find the "hidden message" in the protein. a. Hint #1 - Start at the first codon, not the first AUG b. Hint #2 - Use the first letter of each amino acid to spell out the message AGA GGA TTA TAC TAT TCG TGG CGC AGT ATG TAA ACT TCT AGA GGA TTA TAC TAT TCG TGG CGC AGT ATG TAA ACT TCT 1. Change 13th base from “T” to “G” 2. Change 15th base from “T” to “A” 3. Change 26th base from “G” to “T” AGA GGA TTA TAC TAT TCG TGG CGC AGT ATG TAA ACT TCT 1. Change 13th base from “T” to “G” 2. Change 15th base from “T” to “A” 3. Change 25th base from “G” to “T” Mutation: Change in the Genetic Material § Mutation – change in the nucleotide, or base, sequence of DNA § Change in nucleotide sequence can cause change in protein sequence § Change in protein sequence can cause change in protein function § Can become less active, more active, bind different substrate(s), etc… § The effect of a mutation depends on type of mutation Mutation: Change in the Genetic Material Type of mutations § Base substitution (point mutation) – one base is replaced by a different base § May cause change in one amino acid, stop codon § Frameshift mutation – one or a few bases are deleted of inserted (not in multiples of three) § Shifts “translational reading frame” of mRNA § Causes change in all amino acids after frameshift § Almost always results in nonfunctional protein Mutation: Change in the Genetic Material Results of mutations § Silent mutations - mutations that have no effect § Change in base, no change in amino acid § Due to degeneracy of genetic code § Missense mutations - mutations that result in an amino acid substitution in protein § Nonsense mutations - mutation that introduces premature stop codon Mutation: Change in the Genetic Material Missense mutation § Mutated DNA molecule, makes § Mutated mRNA with codons AUG, AAG, UUU, AGC, UAA, makes § Mutated protein Met-Lys-Phe-Ser § “Normal” DNA molecule, makes § “Normal” mRNA with codons AUG, AAG, UUU, GGC, UAA, makes § “Normal” protein Met-Lys-Phe-Gly Mutation: Change in the Genetic Material Nonsense mutation § Mutated DNA molecule, makes § Mutated mRNA with codons AUG, UAG, UUU, GGC, UAA, makes § Mutated short protein Met § “Normal” DNA molecule, makes § “Normal” mRNA with codons AUG, AAG, UUU, GGC, UAA, makes § “Normal” protein Met-Lys-Phe-Gly Mutation: Change in the Genetic Material Frameshift mutations Result in dramatic changes to protein § Mutated DNA molecule, makes § Mutated mRNA with codons AUG, AAG, UUG, GCU, AA…, makes § Mutated protein Met-Lys-Leu-Ser-… § “Normal” DNA molecule, makes § “Normal” mRNA with codons AUG, AAG, UUU, GGC, UAA, makes § “Normal” protein Met-Lys-Phe-Gly Mutations Mutagens § Spontaneous mutations – occur due to occasional errors in DNA replication § Mutagens – environmental agents that cause mutations § May be physical or chemical § Any agent that interacts with DNA is potential mutagen Mutagens § Chemical mutagens – interact with DNA to cause improper base pairing, deletions, insertions § Analogs of DNA look like bases § But don’t base pair properly § HNO2 alters A § “A” binds with C instead of T Mutagens Radiation § Ionizing radiation – X-rays, gamma rays § Ionize molecules § Cause structural damage to DNA, leads to errors in DNA replication Mutations A mutation is ____. a. .. m th e a in ge ch an 0% in o nc ... th e se q ue ot i.. 0% in ge ch an a a ch an ge in th e se q th e in ge ch an a 0% nu c le ue nc ... 0% a 1. a change in the sequence in DNA 2. a change in the nucleotide in mRNA 3. a change in the sequence in tRNA 4. a change in the amino acid in protein Which type of mutation is the most damaging? 1. Silent mutation 2. Missense mutation 3. Frameshift mutation 0% ut ... es hi f t am Fr m se iss en M m ut at .. . tio ut a nt m le Si 0% ... 0% Genetic Transfer and Recombination § Genetic recombination – exchange of genes between 2 DNA molecules § Contributes to genetic diversity of population § In eukaryotes, genetic recombination happens regularly as part of sexual cycle § Chromosomes are close together in nucleus § Recombination within one organism § In prokaryotes, transfer of genes happens by: § Vertical gene transfer § Horizontal gene transfer (recombination between 2 organisms) Genetic Transfer and Recombination Horizontal gene transfer – genes passed between cells of same generation Vertical gene transfer – transfer of genes from parent to offspring (daughter cells) Genetic Transfer and Recombination Horizontal gene transfer § Donor transfers part of its genome to recipient cell § Recipient can incorporate (recombine) part of donor DNA § Rest is degraded for use as building blocks § Recipient that incorporates DNA is called recombinant § Rare event, occurs in less than 1% of population § Three mechanisms of horizontal gene transfer § Transformation, conjugation, transduction Genetic Transfer and Recombination Transformation – transfer of “naked” DNA “Naked” DNA in environment Transformation – uptake of DNA Recipient cell Recombination – integration of DNA into genome Cell that recombines foreign DNA is a recombinant cell Genetic Transfer and Recombination § Griffiths experiment (1928) demonstrated transformation 2 - Nonencapsulated bacteria don’t cause disease, mice live when injected 3 – Dead encapsulated bacteria injected into mouse, mouse lives 4 – Dead encapsulated bacteria and live nonencapsulated bacteria injected, mouse dies 1 - Encapsulated bacteria cause disease, kill mice when injected 1 - Many encapsulated colonies isolated from dead mouse 2 – Few nonencapsulated 3 – No colonies colonies isolated, most isolated bacteria killed in mouse Only encapsulated bacteria cause disease 4 – Many encapsulated colonies isolated from mouse, “capsule” DNA incorporated by nonencapsulated bacteria Genetic Transfer and Recombination § In nature, some bacteria release DNA into environment § After cell death, cell lysis § Some bacteria can take up this DNA via transformation § Occurs naturally in Bacillus, Haemophilus, Neisseria, Acinetobacter § Competence – physiological state in which recipient cell can take up DNA via transformation § ie, Haemophilus can take up DNA only when competent Transformation Genetic Transfer and Recombination Transduction – phage mediated DNA transfer § DNA transferred as a part of bacteriophage infection § Two types of transduction § Generalized transduction - phage mediated transfer of random segments of DNA § Specialized transduction - phage mediated transfer of specific genes § ie, some phages transfer only toxin genes Genetic Transfer and Recombination Generalized transduction 1 - Phage attaches to donor cell, injects DNA 3 – Phage particles assembled. Some phages mistakenly package bacterial DNA 2 – Phage DNA and proteins made, bacterial chromosome broken up 4 – Improperly packaged phages transfer bacterial DNA, not phage DNA 5 – Transferred DNA can recombine into recipient cell Transduction Plasmids § Plasmids are self-replicating circular molecules of DNA § Small, about 1-5% of genome § Often carry genes that are not essential for survival § Conjugative plasmids - carry genes for plasmid transfer § Conjugation Genetic Transfer and Recombination Conjugation – plasmid dependent DNA transfer § Requires cell to cell contact § Conjugating cells must be opposite “mating types” § Donor cell must carry plasmid § Recipient cell must not carry plasmid § “Bacterial sex” Conjugation Pilus Genetic Transfer and Recombination The F factor (fertility factor) § Conjugation requires contact between donor (F+) cell and recipient (F-) cell § Genes for conjugation on F plasmid Transfer of plasmid is through a “sex pilus” F+ F- F+ F factor Plasmid replicates during transfer F+ Recipient cell now F+ Genetic Transfer and Recombination § Sometimes F factor integrates into chromosome § F+ cell becomes Hfr (high frequency of recombination) F+ F+ Hfr Genetic Transfer and Recombination § Conjugation between Hfr and F- transfers part of donor chromosome § Recipient is recombinant, but still F§ Why? Hfr F- Only a part of F plasmid is transferred Part of donor chromosome Hfr Recombinant F- Conjugation Plasmids § Conjugative plasmids - carry genes for plasmid transfer § Dissimilation plasmids - carry genes crucial for survival § Catabolism of unusual compounds § Toxins, bacteriocins (toxins that kill bacteria) § Enhance pathogenicity § R factors - provide antibiotic resistance Which method of DNA transfer involves a plasmid? 1. Transformation 2. Conjugation 3. Transduction T 0% T ra n sd uc tio n io n 0% C on ju ga t ra n sfo rm at io n 0% Which of the following is not true of a bacterium that is R+? lso It is a It is re sis t ... ra n e t 0% F+ 0% an . .. 0% an b + c R + r ef er s t o t. .. 0% R 1. R+ refers to the possession of a plasmid. 2. R+ can be transferred to a recipient cell. 3. It is resistant to certain drugs. 4. It is also F+ Transduction is _____. 0% ca rr ie d ou t b y. .. .. . 0% e of "n ak u pt ak o f g e .. . 0% an sf er tr an sf er o f g e .. . 0% tr 1. transfer of genes from parent to offspring 2. transfer of genes due to a bacteriophage infection 3. uptake of "naked" DNA from environment 4. carried out by DNA polymerase When conjugation occurs between a Hfr donor and a F- recipient, which of the following describes the most likely outcome of the recipient cell? ns F. .. 0% C el l r em ai ns F. .. 0% l r em ai C el C el l r em ai .. . n to 0% ns F. .. 0% C on ve rs io 1. Conversion to an F + cell. 2. Cell remains F- with some chromosomal DNA from donor. 3. Cell remains F- with no change in genotype. 4. Cell remains F- with gain of F plasmid. In transduction, the recipient is _____. 1. a dead cell 2. a cell infected by a virus 3. a phage 4. a cell without a plasmid 0% 0% it h ou ll w ce a a p ha g t.. . e .. 0% nf ec te . ll i ce a a d e ad ce ll 0% In transformation, what is the donor cell? 1. A virus infected cell 2. A dead cell 3. A competent cell 4. A cell that contains a plasmid A ce 0% it h ou t.. . ll w co ... 0% ha t A ce pe A co m ll t ce l ea d A d 0% e. .. 0% l ... s i nf ec t iru A v plasmid 0% te nt c 5. A cell without a