Chapter 9 Microbial Genetics

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1 Chapter 9 Microbial Genetics

2 You are expected to know details of 1) DNA replication 2) RNA synthesis (transcription) 3) Protein synthesis (translation)

3 Genome & Genes A genome is all the genetic information that defines an organism. A gene is basic unit of heredity. It contains information for amking one RNA and one polypeptide. A structural gene produces a functional RNA, which usually encodes a protein. A DNA control sequence regulates the expression of a structural gene. Does not encode an RNA Two types of gene transfer are known: Vertical transmission: from parent to child Horizontal transmission: transfer of small pieces of DNA from one cell to another

4 Genetics 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 (phenotype) 3. Structure and function of genetic material (genotype) 4. How this material changes

5 Regulating gene expression A bacterial genome encodes thousands of different proteins needed to handle many types of environmental contingencies. To compete successfully with others, the microbe will not waste energy making unneeded proteins. It uses elegant mechanisms to control gene expression at transcription and translation steps.

6 Protein synthesis regulation and metabolism Genes are regulated to be active only when their products are required In prokaryotes this regulation is coordinated by operons Operons are a set of genes all of which are regulated as a single unit

7 Transcriptional control by regulatory proteins Regulatory proteins Repressors Bind regulatory sequences in the DNA and prevent transcription of target genes Some must first bind a small ligand. Activators Bind regulatory sequences in the DNA and stimulate transcription of target genes Most must first bind a small ligand.

8 In 1961, Jacques Monod and François Jacob proposed the revolutionary idea that genes could be regulated. They noticed that, in Escherichia coli, enzymes used to metabolize lactose were inducible, while those for glucose were constitutive.

9 Operons Inducible operon is turned ON by substrate catabolic operons made up of enzymes that metabolize a nutrient when needed Induced by presence of nutrient Repressible operon is turned OFF by the product synthesized anabolic operon made up of enzymes that stops amino acid synthesis when they are not needed Repressed by presence of amino acid

10 Lactose Operon: Inducible Operon Regulator Control locus Structural locus gene codes for repressor promoter & operator coding for enzymes needed to catabolize lactose β-galactosidase hydrolyzes lactose permease brings lactose across cell membrane β-galactosidase transacetylase uncertain function

11 Lactose Operon: Inducible operon

12 Normally off Lactose Operon: Inducible Operon In the absence of lactose, the repressor binds with the operator locus and blocks transcription of downstream structural genes

13 Lactose Operon: Inducible Operon Lactose turns the operon on by acting as the inducer

14 Arginine Operon: Repressible Normally on and will be turned off when the product of the pathway is no longer required

15 Arginine Operon: Repressible When excess arginine is present, it binds to the repressor and changes it. Then the repressor binds to the operator and blocks arginine synthesis. Arginine is the co-repressor.

16 Difference between operon types

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

18 Isolating mutants Replica plating technique allows identification of mutants (a) Treatment of culture with a mutagen. Replica block (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) All strains grow Mutant colonies do not grow No colony present Mutant colony can be located and isolated

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

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21 Categories of mutations 1) Point mutation addition, deletion, or substitution of a few bases 2) Missense mutation causes change in a single amino acid 3) Nonsense mutation changes a normal codon into a stop codon 4) Silent mutation alters a base but does not change the amino acid 5) Back-mutation when a mutated gene reverses to its original base composition 6) Frameshift mutation when the reading frame of the mrna is altered by insertion or deletion of nucleotides

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23 Effect of mutations

24 Repair of mutations Since mutations can be potentially fatal, the cell has several enzymatic repair mechanisms in place to find and repair damaged DNA 1) DNA polymerase proofreads nucleotides during DNA replication 2) Mismatch repair locates and repairs mismatched nitrogen bases that were not repaired by DNA polymerase 3) Light repair for UV light damage 4) Excision repair locates and repairs incorrect sequence by removing a segment of the DNA and then adding the correct nucleotides

25 The Ames test The Ames test is a biological assay to assess the mutagenic potential of chemical compounds Culture of Salmonella bacteria, histidine (-) Any chemical capable of mutating bacterial DNA can potentially mutate mammalian DNA In the control setup, bacteria are plated on a histidine-free medium containing liver enzymes but lacking the test agent. The experimental plate is prepared the same way except that it contains the test agent. Agricultural, industrial, and medicinal compounds are screened using the Ames test (a) Control Plate Minimal medium lacking histidine and test chemical (b) Test Plate Minimal medium with test chemical and no histidine Indicator organism is a mutant strain of Salmonella typhimurium that has lost the ability to synthesize histidine This mutation is highly susceptible to backmutation his( + ) colonies arising from spontaneous back-mutation Incubation (12 h) Any colonies that form have back-mutated to his( + ) his( + ) colonies in presence of the chemical (c) The degree of mutagenicity of the chemical agent can be calculated by comparing the number of colonies growing on the control plate with the number on the test plate. Chemicals that induce an increased incidence of backmutation (right side) are considered carcinogens.

26 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 Any change that confers an advantage during selection pressure will be retained by the population

27 Plasmids Two kinds of extragenomic DNA molecules can interact with bacterial genomes: Horizontally transferred plasmids The genomes of bacteriophages (viruses that infect bacterial cells) Plasmid-encoded functions can contribute to the physiology of the cell. For example, antibiotic resistance

28 Plasmids Replicate Autonomously Plasmids are much smaller than chromosomes. -Found in archaea, bacteria, and eukaryotic microbes -Usually circular -Need host proteins to replicate

29 Plasmid Properties Plasmids are advantageous under certain conditions: Resistance to antibiotics and toxic metals Pathogenesis Symbiosis Plasmids can also be transferred between cells.

30 DNA recombination events Genetic recombination occurs when an organism acquires and expresses genes that originated in another organism Genetic recombination in bacteria Conjugation Transformation Transduction

31 Conjugation Conjugation transfer of a plasmid or chromosomal fragment from a donor cell to a recipient cell via a direct connection Gram-negative cell donor has a fertility plasmid (F plasmid, F factor) that allows the synthesis of a conjugative pilus (F + ) Recipient cell is a related species or genus without a fertility plasmid (F - ) Donor transfers fertility plasmid through pilus

32 Conjugation F+ F+ F (a) During conjugation, the pilus pulls two bacteria together. (b) Next, a bridge (essentially a pore) forms between the two cells. (c) Then one strand of plasmid DNA passes into the recipient bacterium, and each single strand becomes double stranded again. F- An Introduction to Genetic Analysis. 7th edition. Griffiths AJF, Miller JH, Suzuki DT, et al.new York: W. H. Freeman; 2000.

33 High-frequency recombination (Hfr)- Conjugation type (a) Occasionally, the independent F factor combines with the E. coli chromosome. (b) When the integrated F transfers to another E. coli cell during conjugation, it carries along any E. coli DNA that is attached, thus transferring host chromosomal markers to a new cell. An Introduction to Genetic Analysis. 7th edition. Griffiths AJF, Miller JH, Suzuki DT, et al.new York: W. H. Freeman; 2000.

34 Fertility plasmid Type of cell F + Donor cell F - Recipient cell Hfr High frequency of recombination

35 Summary events taking place in the conjugational cycle of E. coli. An Introduction to Genetic Analysis. 7th edition. Griffiths AJF, Miller JH, Suzuki DT, et al.new York: W. H. Freeman; 2000.

36 R factor/r plasmid R factor = RTF + r determinant R factor is plasmid that can be transferred by conjugation R factor consists of transfer factor (RTF) and a resistance determinant (r determinant) RTF responsible for its own replication and conjugal transfer r determinant contains genes that code for antibiotic resistance

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

38 Griffith s Work on transformation Strain of Colony Cell Type Effect Capsule (a) Smooth (S) Live S strain Dies (c) Heat-killed S strain Survives No capsule Live R strain Rough (R) Live R strain Survives (b) (d) Heat-killed S strain Live S and R strains isolated from dead mouse Dies

39 Streptococcus pneumoniae Pneumococcus Pneumococcus colony Photo courtesy: The Rockefeller University

40

41 Oswald T. Avery ( ) Maclyn McCarty ( ) Colin M. MacLeod ( ) Photo courtesy: Avery & McCarty: The Rockefeller University, Macleod: profiles.nlm.nih.gov

42 Transduction Transduction bacteriophage serves as a carrier of DNA from a donor cell to a recipient cell Generalized transduction Random fragments of disintegrating host DNA are picked up by the phage during assembly Specialized transduction A highly specific part of the host genome is regularly incorporated into the virus Any gene can be transmitted this way

43 Generalized transduction E.g. P22 phage in Salmonella An Introduction to Genetic Analysis. 7th edition. Griffiths AJF, Miller JH, Suzuki DT, et al.new York: W. H. Freeman; 2000.

44 Prophage within the bacterial chromosome Specialized transduction Excised phage DNA contains some bacterial DNA New viral particles are synthesized Infection of recipient cell transfers bacterial DNA to a new cell

45

46 James Wilson In: Molecular paradigms of infectious disease- a bacterial perspective Nickerson,C.A. & Schurr,M. (Eds). 2006

47 Intermicrobial DNA exchange

48 Transposons Special DNA segments that have the capability of moving from one location in the genome to another jumping genes

49 Jumping genes

50 Transposons Special DNA segments that have the capability of moving from one location in the genome to another jumping genes Cause rearrangement of the genetic material (1) 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 Transcription is prevented by, and stimulated by. A. activators; repressors B. inducers; corepressors C. repressors; inducers D. regulators; repressors E. repressors; activators

52 If an operon s repressor is in its active form that means A. Transcription from the operon is occurring B. Transcription from the operon is not occurring

53 An F+ Cell A. Has undergone Conjugation B. Can undergo Conjugation C. Has undergone Transformation D. Can undergo Transformation E. Has undergone Transduction F. Can undergo Transduction

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