Chapter 9. Microbial Genetics. Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

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1 Chapter 9 Microbial Genetics Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

2 9.1 Introduction to Genetics and Genes: Unlocking the Secrets of Heredity Genetics the study of heredity The science of genetics explores: 1. Transmission of biological traits from parent to offspring 2. Expression and variation of those traits 3. Structure and function of genetic material 4. How this material changes 2

3 The Levels of Structure and Function of the Genome Genome sum total of genetic material of a cell (chromosomes + mitochondria/chloroplasts and/or plasmids) Genome of cells: DN Genome of viruses: DN or RN DN complexed with protein constitutes the genetic material as chromosomes Figure 9.1 Levels of genetic study Organism level Cell level Chromosome level Molecular level T G C PhotoLink/Photodisc/Getty Images RF C C G T G C T G C C G T CG 3 T G C C G T

4 Figure 9.2 Microbial Genomes Bacterial chromosomes are a single circular loop Eukaryotic chromosomes are multiple and linear Figure 9.2 Forms of the genome Eukaryote (composite) Cells Chromosomes Prokaryote Nucleus Nucleolus Mitochondrion Chromosome Plasmids Plasmid (in some fungi and protozoa) Extrachromosomal DN Viruses Chloroplast DN RN 4

5 Genes Chromosome is subdivided into genes, the fundamental unit of heredity responsible for a given trait Site on the chromosome that provides information for a certain cell function Segment of DN that contains the necessary code to make a protein or RN molecule Three basic categories of genes: 1. Genes that code for proteins structural genes 2. Genes that code for RN 3. Genes that control gene expression regulatory genes 5

6 Genotypes and Phenotypes ll types of genes constitute the genetic makeup genotype The expression of the genotype creates observable traits phenotype 6

7 The Size and Packaging of Genomes Smallest virus 4-5 genes E. coli single chromosome containing 4,288 genes; 1 mm; 1,000X longer than cell Human cell 46 chromosomes containing 31,000 genes; 6 feet; 180,000X longer than cell Figure 9.3 a disrupted E. coli 7

8 The Structure of DN: Double Helix with Its Own Language Two strands twisted into a double helix Basic unit of DN structure is a nucleotide Each nucleotide consists of 3 parts: 5 carbon sugar: deoxyribose phosphate group nitrogenous base: adenine, guanine, thymine, cytosine Nucleotides covalently bond to form a sugar-phosphate backbone Each sugar attaches to two phosphates > 5 carbon and 3 carbon 8

9 Deoxyribose sugar N base D Backbone D P D C DN Hydrogen bonds T G D D P P Phosphate P P D G C D P Condensed metaphase chromosome DN DN double helix DN wrapped around histones with linker DN between them DN Histone Chemical tags attached to histoneproteins may increase the expression of nearby genes. Nucleosome Supercoiled condensed chromatin Condensed nucleosomes Loosely condensed chromosome Chromatin Uncondensed chromatin fiber 9

10 Figure 9.4 a Schematic DN Nitrogenous bases covalently bond to the 1 carbon of each sugar and span the center of the molecule to pair with an appropriate complementary base on the other strand denine binds to thymine with 2 hydrogen bonds Guanine binds to cytosine with 3 hydrogen bonds ntiparallel strands 3 to 5 and 5 to 3 H N Sugar Sugar phosphate G N-H N NH H-N Nitrogen base pair C N O Sugar phosphate P 3 5 OH 4 D P 5 P Phosphate P Deoxyribose 3 D C 2 with carbon number G C Cytosine 3 5 OH H N D Sugar N N H C H O N N H C H H N-H O CH 3 H-N T N O H 3 5 P H G T Guanine Thymin e denine Hydrogen bond Covalent bond 10 (a)

11 The Information in DN Each strand provides a template for the exact copying of a new strand semiconservative replication Order of bases constitutes the DN code 11

12 Figure 9.4 Three views of DN H H N O H-N H N G N-H C N N Sugar NH O H H Sugar phosphate Nitrogen base pair Sugar phosphate P 5 4 D P C G 3 OH 5 P Phosphate P 5 4 Deoxyribose 3 D 1 2 with carbon number C Cytosine 5 3 Base pairs Sugar phosphate backbone C G Guanine T Thymin e Minor groove a. Schematic non N Sugar N N H H denine Hydrogen bond Covalent bond 5 3 H helical model b. Simplified model c. space filling model H N N-H O CH 3 (a) 3 5 OH D C H-N T N O 3 5 P (b) Major groove (c) 12

13 Significance of DN Structure 1. Maintenance of code during reproduction - Constancy of base pairing guarantees that the code will be retained 2. Providing variety - order of bases responsible for unique qualities of each organism 13

14 DN Replication: Preserving the Code and Passing It On Replication occurs on both strands simultaneously G C 5 3 G C T T T G C T Parental helix Figure 9.5 Simplified DN replication Creates complementary strands T G C C C T G C G T G Replication fork G G Semiconservative replication process T T T C G C T T T T T C G C T T 3 G C 5 3 G C 5 Parental New New Parental Replicas

15 Figure 9.6 Overall Bacterial DN Replication 4.Before synthesis of the lagging strand can start, a primase first constructs a short RN primer to direct the DN polymerase III. Synthesis can proceed only in short sections and produces segments of RN primer and new DN called Okazaki fragments. 3.The template for the lagging strand runs the opposite direction (3 to 5 ) and must be replicated backwards away from the replication fork so the DN polymerase can add the nucleotides in the necessary 5 to 3 arrangement. 5. second polymerase (DN polymerase I) acts on the Okazaki fragments by removing the primers Open spaces in the lagging strand are filled in by a ligase that adds the correct nucleotides (a) Forks Lagging strand synthesis Nick 1. The chromosome tobe replicated is continuously unwound by a helicase, forming a replication fork with two template strands The template for the leading strand (bottom) is correctly oriented for the DN polymerase III to add nucleotides in the 5 to 3 direction towards the replication fork, so it can be synthesized as a continuous strand. Note that direction of synthesis refers to the order of the new strand (red). Lagging strand synthesis See Table 9.1 for summary of enzyme function Key: Template strand New strand RN primer Helicase Primase DN polymerase III DN polymerase I Ligase Daughter cell Daughter cell (b)

16 9.2 pplications of the DN code: Transcription and Translation (a) Figure 9.8 (b) Information stored on the DN molecule is conveyed to RN molecules through the process of transcription Transcription of DN DN DSRN SSRN Regulatory RNs trn mrn rrn The information contained in the RN molecule is then used to produce proteins in the process of translation Ribosome (rrn+protein Translation of RN Protein trn mrn Micro RN, interfering RN, antisense RN, and riboswitches regulate transcription and translation Expression of DN for structures and functions of cell 16

17 Gene-Protein Connection 1. Each triplet of nucleotides on the RN specifies a particular amino acid 2. protein s primary structure determines its shape and function 3. Proteins determine phenotype. Living things are what their proteins make them. 4. DN is mainly a blueprint that tells the cell which kinds of proteins to make and how to make them DN mrn (copy of one strand) mino acids Figure 9.9 DN- protein relationship Triplets 1 Codon Single nucleotide Variations in the order and types will dictate the shape 17 and function of the protein

18 The Major Participants in Transcription and Translation Single-stranded molecule made of nucleotides 5 carbon sugar is ribose 4 nitrogen bases: adenine, uracil, guanine, cytosine Phosphate 18

19 Messenger RN: Carrying DN s Message 3 types of RN: Messenger RN (mrn): carries DN message through complementary copy; message is in triplets called codons (a) Messenger RN (mrn) short piece of messenger RN (mrn illustrates the general structure of RN: single strandedness, repeating phosphate-ribose sugar backbone attached to single nitrogen bases; use of uracil instead of thymine. Figure 9.10 a U G C U G C U P P P P P P P P Codon 1 Codon 2 Codon 3 P = Phosphate R = Ribose U = Uracil Transfer RN (trn) Ribosomal RN (rrn) 19

20 Transfer RN: The Key to Translation 3 types of RN: Messenger RN (mrn) Transfer RN (trn) made from DN; secondary structure creates loops; bottom loop exposes a triplet of nucleotides called anticodon which designates specificity and complements mrn; carries specific amino acids to ribosomes (b) Transfer RN (trn) Left : The trn stand loops back on itself to form intrachain hydrogen bonds. The result is a cloverleaf structure, shown here in simplified form. t its bottom is an anticodon that specifies the attachment of a particular amino acid at the 39 end right three-dimensional view of trn structure. G H bonds G G G G G 5 3 mino acid attachment site G C C G G C G G G G G G G G G Hairpin loops nticodon nticodon mino acid attachment site 5 3 Ribosomal RN (rrn) 20

21 The Ribosome: Mobile Molecular Factory for Translation 3 types of RN: Messenger RN (mrn) Transfer RN (trn) Ribosomal RN (rrn): component of ribosomes where protein synthesis occurs mino acids Large subunit Exit site P E Small transcript 5 trns mrn transcript 21

22 Transcription: The First Stage of Gene Expression 1. RN polymerase binds to promoter region upstream of the gene 2. RN polymerase adds nucleotides complementary to the template strand of a segment of DN in the 5 to 3 direction 3. Uracil is placed as adenine s complement 4. t termination, RN polymerase recognizes signals and releases the transcript 100-1,200 bases long 22

23 Figure 9.11 Major Events in Transcription Each gene contains a specific promoter region and a leader sequence for guiding the beginning of transcription. Next is the region of the gene that codes for a polypeptide and ends with a series of terminal sequences that stop translation. DN is unwound at the promoter by RN polymerase. Only one strand of DN, called the template strand, is copied by the RN polymerase. This strand runs in the 3' to 59 direction. The RN polymerase moves along the strand, adding complementary nucleotides as dictated by the DN template. The mrn strand reads in the 5' to 39 direction. RN polymerase 5' Promoter region T Initiation codon T C G T emplate strand 5 Nontemplate strand Direction of transcription RN polymerase binding site Leader sequence 3' 5' 3 3' G C T C G T Unwinding of DN Nucleotide pool Termination sequences ( ) G T G C C T C G ( ) Intervening sequence of variable size T ermination sequence 4 The polymerase continues transcribing until it reaches a termination site, and the mrn transcript is released to be translated. Note that the section of the transcribed DN is rewound into its original configuration. 5' Early mrn transcript Late mrn transcript Elongation 23

24 Translation: The Second Stage of Gene Expression ll the elements needed to synthesize protein are brought together on the ribosomes Exit site Figure 9.12 Players in translation mino acids E P Large subunit The process occurs in five stages: initiation, elongation, termination, and protein folding and processing 5 trns Small transcript mrn transcript 24

25 First Base Position Third Base Position The Master Genetic Code: The Message in mrn Represented by the mrn codons and the amino acids they specify Code is universal among organisms Code is redundant U C G } Second Base Position U C G UUU UCU UU UGU U Phenylalanine T yrosine Cysteine UUC UCC UC UGC C Serine UU UC U UG STOP** Leucine } STOP** UUG } UCG UG UGG Tryptophan G CUU CCU CU Histidine CGU U CUC CCC CC CGC C Leucine Proline rginine CU CC C CG Glutamine } CUG CCG CG CGG G UU Isoleucine CU U sparagine GU Serine U UC CC C GC C Threonine U C G UG* Methionine CG G } Lysine rginine STRT GG } G GUU GCU GU GGU U spartic acid GUC GCC GC GGC C Valine lanine Glycine GU GC GC GG Glutamic acid GUG GCG GG GGG G *This codon initiates translation. **For these codons, which give the orders to stop translation, there are no corresponding trns with amino acids. } } } } } Figure 9.13 } } 25

26 Figure 9.14 Interpreting the DN Code Transcription produces an mrn complementary to the DN gene, the template strand DN triplets mrn codons Nontemplate strand Template strand trn anticodons UC GC UG UGC During translation, trns use their anticodon to interpret the mrn codons and bring in the amino acids Protein (amino acid specified) F-Methionine Leucine Threonine Threonine Same amino acid; has a different codon and anticodon 26

27 Figure 9.15 Translation Steps 1. Ribosomes assemble on the 5 end of an mrn transcript 2. Ribosome scans the mrn until it reaches the start codon, usually UG Exit site mino acids E P Large subunit 3. trn molecule with the complementary anticodon and methionine amino acid enters the P site of the ribosome and binds to the mrn 5 trns Small transcript mrn transcript 27

28 Figure 9.15 Translation 4. second trn with the complementary anticodon fills the site f Met Leucine nticodion m RN Start codon Entrance of trns 1 and 2 CCG 28

29 Figure 9.15 Translation 5. peptide bond is formed is formed between the amino acids on the neighboring trns Peptide bond 1 CCG Fermationof peptide bond 29

30 Figure 9.15 Translation 6. The first trn is released and the ribosome slides down to the next codon Empty trn UC CCG P site Discharge of trn 1 at E site 30

31 Figure 9.15 Translation 7. nother trn fills the site and a peptide bond is formed Proline Peptide bond 2 2 First translocation: trn 2 shifts into p site ; trn 3 enters ribosome at UG Formation of peptide bond 31 UG

32 Figure 9.15 Translation 8. This process continues until a stop codon is reached lanine Peptide bond 3 3 G G C UC UC Discharge of trn 2; second translocation; trn 4 enters ribosome Formation of peptide bond Stop codon

33 Translation Termination 9. Termination codons U, UG, and UG are codons for which there is no corresponding trn 10. When this codon is reached, the ribosome falls off and the last trn is removed from the polypeptide 33

34 Polyribosomal Complex Polyribosomal complex allows for the synthesis of many protein molecules simultaneously from the same mrn molecule. mrn RN polymerase Figure 9.16 Transcription Start of translation mrn (a) Ribosomes Growing polypeptides 1 Polypeptide Polyribosomal complex Start (c) Steven McKnight and Oscar L Mille, Department of Biolog, University of virginia 34 (b)

35 Eukaryotic Transcription and Translation: Similar yet Different 1. Do not occur simultaneously transcription occurs in the nucleus and translation occurs in the cytoplasm 2. Eukaryotic start codon is UG, but it does not use formyl-methionine 3. Eukaryotic mrn encodes a single protein, unlike bacterial mrn which encodes many 4. Eukaryotic DN contains introns intervening sequences of noncoding DN which have to be spliced out of the final mrn transcript 35

36 Figure 9.17 Splicing of Eukaryotic pre-mrn Removal of introns and connection of exons DN template Primary mrn transcript E II E E I E Exon Intron E II E E I E Does not occur in Prokaryotes Occurs In nucleus Transcript processed by special enzymes Larat forming Spliceosomes E E E E Lariat excised Spliceosomes released Exons spliced together E E E E Occurs in cytoplasm mrn transcript can now be translated 36

37 9.4 Mutations: Changes in the Genetic Code change in phenotype due to a change in genotype (nitrogen base sequence of DN) is called a mutation natural, nonmutated characteristic is known as a wild type (wild strain) n organism that has a mutation is a mutant strain, showing variance in morphology, nutritional characteristics, genetic control mechanisms, resistance to chemicals, etc. 37

38 Isolating Mutants Figure 9.20 (a) Treatment of culture with a mutagen. Replica block Replica Plating technique allows identification of mutants (b) Inoculate a plate containing complete growth medium and incubate. Both wild-type and mutants form colonies. (c) Velvet surface (sterilized) Master plate (complete medium) (c) Replica plate (complete medium) Replica plate (medium minus nutrient) Incubation (d) ll strains grow No colony Mutant present colonies do not grow Mutant colony can be located and isolated 38

39 Causes of Mutations Spontaneous mutations random change in the DN due to errors in replication that occur without known cause Induced mutations result from exposure to known mutagens, physical (primarily radiation) or chemical agents that interact with DN in a disruptive manner 39

40 Categories of Mutations Point mutation addition, deletion, or substitution of a few bases Missense mutation causes change in a single amino acid Nonsense mutation changes a normal codon into a stop codon Silent mutation alters a base but does not change the amino acid Back-mutation when a mutated gene reverses to its original base composition Frameshift mutation when the reading frame of the mrn is altered 40

41 Effect of Mutations 41

42 Repair of Mutations Since mutations can be potentially fatal, the cell has several enzymatic repair mechanisms in place to find and repair damaged DN DN polymerase: proofreads nucleotides during DN replication Mismatch repair: locates and repairs mismatched nitrogen bases that were not repaired by DN polymerase Light repair: for UV light damage Excision repair: locates and repairs incorrect sequence by removing a segment of the DN and then adding the correct nucleotides 42

43 Positive and Negative Effects of Mutations Mutations leading to nonfunctional proteins are harmful, possibly fatal Organisms with mutations that are beneficial in their environment can readily adapt, survive, and reproduce these mutations are the basis of change in populations ny change that confers an advantage during selection pressure will be retained by the population 43

44 9.5 DN Recombination Events Genetic recombination occurs when an organism acquires and expresses genes that originated in another organism 3 means for genetic recombination in bacteria: 1. Conjugation 2. Transformation 3. Transduction 44

45 Conjugation: Genetic Transmission through Direct Contact Conjugation transfer of a plasmid or chromosomal fragment from a donor cell to a recipient cell via a direct connection Figure 9.23 (1) F factor Physical Conjugation Bacterial chromosome F + Pilus Gram-negative cell donor has a fertility plasmid (F plasmid, F factor) that allows the synthesis of a conjugative pilus Recipient cell is a related species or genus without a fertility plasmid Donor transfers fertility plasmid through pilus (1 and 2) Sex pilus makes contact with F recipient cell F + Sex pilus contracts, bringing cells together. F + F 1 The pilus of donor cell (top) attaches to receptor on recipient cell and retracts to draw the two cells together. This is the mechanism for gram negative bacteria. 45

46 Figure 9.23 Conjugation (2) (3) High-frequency recombination (3) donor s fertility plasmid has been integrated into the bacterial chromosome F factor (plasmid) F Factor Transfer Bridge made with pilus Donor Recipient F + F Chromosome F + F Hfr cell Donor Hfr Transfer Recipient F factor Integration of F facter into choromosome Pilus Chromosome Partial copy of donor chromosome When conjugation occurs, a portion of the chromosome and a portion of the fertility plasmid are transferred to the recipient F factor being copied F + F + F factor 2 Transfer of the F facter, or 3 conjugative plasmid Bridge broken Donated genes High-frequency (Hfr) transfer involves transmission of chromosomal genes from a donor cell to a recipient cell. The donor chromosome is duplicated and transmitted in part to a recipient cell, where it is integrated into the chromosome.

47 Transformation: Free DN in Solution Transformation chromosome fragments from a lysed cell are accepted by a recipient cell; the genetic code of the DN fragment is acquired by the recipient Donor and recipient cells can be unrelated DN transport system Receptor Cap + Cap + Cap + Figure 9.25 ds DN fragment (blue) with new gene (red) binds to a surface receptor on a competent recipient cell. DN is converted to one strand and transported into the cell, by the DN transport system. The DN strand aligns itself with a compatible region on the recipient chromosome. Useful tool in recombinant DN technology The DN strand is incorporated into the recipient chromosome Transformed cell Cap + Recipient is now transformed with gene for synthesizing a capsule. 47

48 Transduction: Viruses as Vectors Transduction bacteriophage serves as a carrier of DN from a donor cell to a recipient cell Two types: Generalized transduction: random fragments of disintegrating host DN are picked up by the phage during assembly; any gene can be transmitted this way Specialized transduction: a highly specific part of the host genome is regularly incorporated into the virus 48

49 Transduction Generalized transduction (1) Phage DN Cell (donor) Donor (host) chromosome Parts of phage Excised phage DN Separated piece (2) contains some of host DN bacterial DN Figure 9.26 (2) Figure 9.27 (1) Prophage within the bacterial chromosome Specialized transduction (3) Newly assembled phage incorporating piece of host DN (3) New viral particles are synthesized Lysis (4) (4) DN from donor (5) Infection of recipient cell transfers bacterial DN to a new cell Cell B (ecipient) (6) Incorporated into chromosome (5) Cell survives and utilizes transduced DN Recombination results in two possible outcomes. 49

50 Intermicrobial DN Exchange 50

51 Transposons Special DN segments that have the capability of moving from one location in the genome to another jumping genes Cause rearrangement of the genetic material (1) Figure 9.28 Can move from one chromosome site to another, from a chromosome to a plasmid, or from a plasmid to a chromosome (2) (3) (4) May be beneficial or harmful 51