Objectives

Chapter 8 Microbial Genetics

Define genetics, genome, chromosome, gene, genetic code, genotype, phenotype, and genomics. Describe the process of DNA replication. Describe protein synthesis, including transcription, RNA processing, and translation. Classify mutations by type, and describe how mutations are prevented and repaired. Define mutagen. Describe two ways mutations can be repaired. Outline methods of direct and indirect selection of mutants. Identify the purpose and outline the procedure for the Ames test. Compare the mechanisms of genetic recombination in bacteria. Differentiate between horizontal and vertical gene transfer. Describe the functions of plasmids and transposons.
2004 by Jones and Bartlett Publishers

Terminology
Genetics
Complementary but antiparallel

Genome
Gene Chromosome Base pairs Genetic code

Genomics
Genotype

Phenotype

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DNA

The Bacterial DNA

Mostly single circular chromosome

Attached to plasma membrane

DNA is supercoiled

Number of genes in E. coli

Extra-chromosomal bacterial DNA: _________(1-5% of chromosome size)
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E. coli

Fig 8.1
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Figure 8.1a

Chromosome Map of E. coli Chromosome length: 1mm Cell length ?

Figure 8.1b

Flow of Genetic Information

Fig 8.2 Foundation Figure

DNA Replication
DNA polymerase initiated by RNA primer

bidirectional
origin of replication leading strand: continuous DNA synthesis lagging strand: discontinuous DNA synthesis Okazaki fragments semiconservative
2

Replication fork
Replication in 5' 3' direction Fig 5.8

Replication 1; 2; 3 of circular bacterial Chromosome

Fig 8.6

Protein Synthesis
Genetic code: universal and degenerate (or redundant)
Fig 8.8

Transcription

produces 3 types of RNA (?) Enzyme necessary ? Promoters and terminators

Fig 8.7

Translation

produces the protein Sense codons vs. nonsense codons anticodons

Fig 8.9

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Compare to Fig 8.8

Transcription
RNA polymerase binds to promotor sequence proceeds in 5' 3' direction stops when it reaches terminator sequence

Fig 8.7
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More Details on Translation Nucleotide sequence of mRNA is translated into amino acid sequence of protein using three letter words = codons Translation of mRNA begins at the start codon: AUG Translation ends at a stop codon: UAA, UAG, UGA Requires various accessory molecules and 3 major components: ? In Prokaryotes: Simultaneous transcription and translation Polyribosomes
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The Translation Process in Protein Synthesis

Compare to Fig 8.9

Continuous Transcription and Translation Simultaneous Transcription and Translation in Prokaryotes

Compare to Fig 8.10

Mutations
Change in genetic material. 1. Point mutations = base pair substitution (silent, missense, nonsense) 2. Frameshift mutations = Insertion or deletion of one or more nucleotide pairs

Review Fig 8.17

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Various Point Mutations

Missense
Nonsense

Silent

Fig 8.17

Mutations cont.
May be neutral (silent), beneficial, or harmful. Spontaneous mutation rate 10-6 1 mutation per million replicated genes Mutagens increase mutation rate 10 1000x

Chemical mutagens
Nucleoside (base) analogs have altered basepairing properties. They can be

randomly incorporated into growing cells (cancer drugs) only used by viral enzymes (e.g. AZT)

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Frameshift mutagens such as intercalating agents (e.g.:, aflatoxin, ethidium bromide)

Fig 8.19a

Distortion due to intercalating agent will lead to one or more base-pairs inserted or deleted during replication.
Potent carcinogens!

Radiation as a Mutagen
1. Ionizing radiation (x-rays and -rays) lead to deletion mutations (ds breaks) 2. UV rays lead to thymine dimers (intrastrand bonding) Photolyases = light repair enzymes
(use energy from visible light to fix UV light damage)

Nucleotide excision repair for repair of all mutations

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Repair
Photolyases separate thymine dimers
Nucleotide excision repair

Fig 8.20

ANIMATION Mutations: Repair

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Mutagen Identification: Ames

Wild type vs. mutant Auxotroph vs. prototroph
Many mutagens are carcinogens

Test

Combine animal liver cell extracts with Salmonella auxotroph Expose mixture to test substance
Examine for signs of mutation in Salmonella, i.e. Look for cells (colonies) that have reverted from his to his+
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Ames Reverse Gene Mutation Test

Fig. 8.22

Professor Richard A. Muller of UC Berkeley on the Ames Test and Natural Foods

Positive or negative Ames test? Explain what happened

Genetic Transfer and Recombination

Vertical gene transfer: Occurs during
reproduction between generations of cells. Horizontal (lateral) gene transfer: Transfer of genes between cells of the same generation. Leads to genetic recombination

Three mechanisms of horizontal gene transfer: Transformation Conjugation Transduction

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Genetic Recombination
Vertical gene transfer: Occurs during reproduction between generations of cells. Horizontal gene transfer: The transfer of genes between cells of the same generation. Leads to genetic recombination.

Three mechanisms of horizontal gene transfer:

1. Transformation 2. Conjugation 3. Transduction Copyright 2006 Pearson Education, Inc., publishing as Benjamin Cummings ANIMATION Horizontal Gene Transfer: Overview

Genetic Recombination
Exchange of genes between two DNA molecules Crossing over occurs when two chromosomes break and rejoin

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Figure 8.23

1) Transformation
Naked DNA transfer

Recipient cells have to be competent

Occurs naturally among very few genera (G+ and G) Simple laboratory treatment will make E. coli competent workhorse for genetic engineering
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Griffiths historical experiment in 1928

Griffiths Experiment to Demonstrate Genetic Transformation

Fig 8.24

ANIMATION Transformation

Transformation and Recombination

Fig 8.25

2) Conjugation
Plasmid and chromosomal DNA transfer via direct cell to cell contact High efficiency F+ = donor cell. Contains F plasmid (factor) and produces conjugation (F) pilus (aka sex pilus) Recipient cell (F ) becomes F+ In some cells F factor integrates into chromosome Hfr cell R plasmids (R factors) are also transferred via conjugation
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Fig 8.26

Fig 8.27

ANIMATIONs

3) Transduction
DNA Transfer from donor to recipient cell with help of bacteriophage (= transducing phage) 2 types of phage-bacteria interaction: 1. Generalized transduction happens via lytic cycle caused by virulent phages
Fig 8.27

2. Specialized transduction will be covered in Ch 13

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Transduction by a Bacteriophage

ANIMATION Generalized Transduction

ANIMATION Specialized Transduction

Fig 8.28