MicrobiologyAn Evolving Science

Third Edition

Joan L. Slonczewski and John W. Foster

Copyright © 2014 W. W. Norton & Company, Inc. Permission required for reproduction or display

PowerPoint® Lecture Outlines Prepared by Johnny El-Rady, University of South Florida

Genomes and Chromosomes

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Chapter Overview DNA 101 The organization of prokaryotic and eukaryotic

genomes The mechanism of DNA replication Plasmids ’R Us The features of eukaryotic chromosomes DNA analysis

- Restriction enzymes, gel electrophoresis, the polymerase chain reaction (PCR), and DNA sequencing

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Introduction

A genome is all the genetic information that defines an organism.

Microbial genomes consist of one (usually) or more DNA chromosomes.

This chapter explores the structure of genomes and their replication.

Figure 7.1

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7.1 DNA: The Genetic Material

Two types of gene transfer are known:

1. Vertical transmission: from parent to child

2. Horizontal transmission: transfer of small pieces of DNA from one cell to another

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

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In the 1950s, conjugation was discovered.

- A horizontal gene transfer mechanism requiring cell-to-cell contact, which could transfer large segments of some bacterial chromosomes.

This process allowed genes to be mapped relative to one another according to time of transfer.

- The results suggested that bacterial chromosomes were circular.

We now know that there is tremendous diversity in prokaryotic genomes.

Bacterial Genomes

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7.2 Genome Organization Bacterial and archaeal chromosomes range in size from

490 to 9,400 kilobase pairs (kb).

- For comparison, eukaryotic chromosomes range from 2,900 kb (Microsporidia) to over 100 million kb (flowering plants).

- The human genome is over 3 million kb.

Another distinction between genomes is the presence of noncoding DNA.

- It is typically > 90% of eukaryotic genomes, but < 15% of prokaryotic genomes.

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A gene can operate independently of others.

- Or, it may exist in tandem with other genes in a unit called an operon.

Functional Units of Genes

Figure 7.3

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DNA is a polymer of nucleotides.

Each nucleotide consists of three parts:

1. Nitrogenous base

- Purine: adenine (A) and guanine (G)

- Pyrimidine: cytosine (C) and thymine (T)

2. Deoxyribose sugar

3. Phosphate

Nucleotides are connected to each other by 5′-3′ phosphodiester bonds.

DNA Structure

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Hydrogen bonding allows complementary base interactions.

- A pairs only with T (via two H bonds).

- G pairs only with C (via three H bonds).

These interactions allow the two phosphodiester backbones to come together in an antiparallel fashion.

- Thus forming the double helix

At high temperatures (50oC–90oC), the hydrogen bonds in DNA break and the duplex falls apart, or denatures, into two single strands.

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Figure 7.4A

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Figure 7.5

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The DNA double helix has two grooves

- A wide major groove and a narrow minor groove

- These provide DNA-binding proteins access to base sequences

Figure 7.6

Figure 7.7

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RNA differs from DNA:

- Usually single-stranded

- Contains ribose sugar

- Uracil replaces thymine

RNA Structure

Figure 7.4B

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Bacteria pack their DNA into a series of loops or domains, collectively called the nucleoid.

- Loops are anchored by histone-like proteins.

The Bacterial Nucleoid

Figure 7.8

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But how does DNA achieve this supercoiled state?

Figure 7.9

DNA Supercoiling

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There are two types of supercoils:

- Positive supercoils: DNA is overwound

- Negative supercoils: DNA is underwound

Eukaryotes, bacteria, and most archaea possess negatively supercoiled DNA.

Archaea living in acid at high temperature possess positively supercoiled DNA.

Enzymes that change DNA supercoiling are called topoisomerases.

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A cell has two types of topoisomerases:

- Type I topoisomerases

- Usually single proteins

- Cleave one strand of DNA

- Type II topoisomerases

- Have multiple subunits

- Cleave both strands of DNA

- Example: DNA gyrase

- Targeted by quinolone antibiotics

Topoisomerases Supercoil DNA

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Figure 7.10

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Figure 7.11 Figure 7.12

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Animation: supercoiling and topoisomerases

Topoisomerases

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7.3 DNA Replication

Microbial DNA needs to replicate itself as accurately and as quickly as possible so that the organism can grow and compete with other species.

The process of bacterial replication involves an amazing number of proteins and genes coming together in a complex machine.

- A list of 20 DNA replication components can be found in eTopic 7.1.

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Replication of cellular DNA in most cases is semiconservative.- Each daughter cell receives one parental and one

newly synthesized strand.

Overview of Bacterial DNA Replication

Figure 7.13

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Replication in bacteria begins at a single origin (oriC).

After initiation, a replication bubble forms.

- Contains two replication forks that move in opposite directions around the chromosome

Replication ends at defined termination (ter) sites located opposite to the origin.

Note: A partially replicated chromosome can start new rounds of replication at the two daughter origins even before the first round is complete.

Replication from a Single Origin

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Figure 7.14

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The major proteins involved in DNA replication include:

- DnaA: initiator protein

- DnaB: helicase

- DNA primase: synthesis of RNA primer

- DNA Pol III: major replication enzyme

- DNA Pol I: replaces RNA primer with DNA

- DNA gyrase: relieves DNA supercoiling

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The start of DNA replication is precisely timed and linked to the ratio of DNA to cell mass.

In Escherichia coli, DnaA accumulates during growth, and then triggers the initiation of replication.

- DnaA-ATP complexes bind to 9-bp repeats upstream of the origin.

- This binding causes DNA to loop in preparation for being melted open by the helicase (DnaB).

Initiation of Replication

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Figure 7.15

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A sliding clamp protein (the beta subunit) tethers DNA polymerase to the DNA.

- Without it, DNA pol would frequently “fall off” the DNA molecule.

What happens to new replication origins after replication begins?

- The origin starts in the center of the cell, and the newly replicated origins move toward opposite cell poles.

Figure 7.16

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After initiation, each replication fork contains two strands:

- A leading strand, which is replicated continuously in the 5′-to-3′ direction

- A lagging strand, which is replicated discontinuously in stages, each producing an Okazaki fragment

- These are progressively stitched together to make a continuous unbroken strand.

Elongation of Replicating DNA

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The cell coordinates the activity of two DNA Pol III enzymes in one complex.

- These two enzymes, together with DNA primase and helicase, form the replisome.

The replisome ensures that the leading and lagging strands are synthesized simultaneously in the 5′-to-3′ direction.

- This is possible because the problem (lagging) strand loops out after passing through its polymerase.

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Figure 7.18 (Part 1)

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Figure 7.18 (Part 2)

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Figure 7.18 (Part 3)

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Figure 7.18 (Part 4)

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Animation: DNA replication

DNA Replication

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To remove RNA primers, cells use RNase H.

A DNA Pol I enzyme then synthesizes a DNA patch using the 3′ OH end of the preexisting DNA fragment as a priming site.

Finally, DNA ligase repairs the phosphodiester nick using energy from NAD (in bacteria) or ATP (in eukaryotes).

Elongation of Replicating DNA

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Figure 7.19

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There are as many as ten terminator sequences (ter) on the Escherichia coli chromosome.

A protein called Tus (terminus utilization substance) binds to these sequences and acts as a counter-helicase.

Ringed catenanes formed at the completion of replication are separated by topoisomerase IV and the proteins XerC and XerD.

Terminating Replication

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Figure 7.20

A

B

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7.4 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

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Plasmids are much smaller than chromosomes.

- Found in archaea, bacteria, and eukaryotic microbes

- Usually circular

- Need host proteins to replicate

Plasmids Replicate Autonomously

Figure 7.22

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Plasmids can replicate in two different ways:

1. Bidirectional replication

- Starts at a single origin and occurs in two directions simultaneously

2. Rolling-circle replication

- Starts at a single origin and moves in only one direction

While most known plasmids use only one of these two replication strategies, a few can use either one, depending on the circumstance of the cell.

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Figure 7.23

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Animation: rolling-circle mechanism of plasmid replication

Plasmids

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Plasmids have tricks to ensure their inheritance:

- Low-copy-number plasmids segregate equally to daughter cells.

- High-copy-number plasmids segregate randomly to daughter cells.

Plasmids are advantageous under certain conditions:

- Resistance to antibiotics and toxic metals

- Pathogenesis

- Symbiosis

Plasmids can also be transferred between cells.

Plasmid Properties

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7.5 Eukaryotic Chromosomes In general, eukaryotic genomes are larger than those of

bacteria.

Because their chromosomes are linear, eukaryotes require a reverse transcriptase called telomerase to replicate their ends.

Eukaryotic cells pack their DNA within the nucleus using proteins called histones.

A large portion of eukaryotic chromosomes are composed of noncoding DNA:

- Introns and pseudogenes

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Figure 7.25

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Archaeal genomes combine features of bacteria and eukaryotes.

- Like bacteria, archaea have:

- Polygenic operons

- Asexual reproduction

- Cells lacking a nuclear membrane

- A single circular chromosome

- In most species of archaea, however, the processes of DNA replication, transcription, and translation more closely resemble those of eukaryotes.

Archaeal Genomes

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7.6 DNA Sequence Analysis

What are the basic techniques used to manipulate DNA?

- These include:

- Isolating genomic DNA from cells

- Snipping out DNA fragments with surgical precision

- Splicing them into plasmid vehicles, and reading their nucleotide sequences

- These are the techniques that drove the genomic revolution.

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Restriction endonucleases cleave DNA at specific recognition sites, which are usually 4 to 6 bp and palindromes.

- May generate blunt or staggered ends

Figure 7.26A

Restriction Endonuclease Digestion

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Agarose gel electrophoresis can be used to analyze the DNA fragments obtained by treatment with restriction enzymes.

Figure 7.26B

Restriction Endonuclease Digestion

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Restriction endonuclease–digested DNA molecules were first cloned into plasmids in the early 1970s.

- By Stanley Cohen (Stanford University) and Herbert Boyer (UC San Francisco)

Genome libraries (also called clone libraries or clone pools) containing all the genes in an organism are routinely made today

Shuttle vector allows the study of eukaryotic proteins in prokaryotic cells

Cloning

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Figure 7.27c

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The polymerase chain reaction (PCR) can produce over a million-fold amplification of target DNA within a few hours.

PCR Amplifies Specific Genes

Figure 7.28

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The most commonly used DNA sequencing method relies on the Sanger dideoxy strategy.

- Incorporation of a 2′,3′-dideoxynucleotide into a growing chain prevents further elongation.

DNA Sequencing

Figure 7.29

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Animation: DNA sequencing

DNA Sequencing

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New sequencing technologies, called “next-generation sequencing,” have combined the power of robotics, computers, and fluidics such that an entire bacterial genome can be sequenced within a few days.

- One of these techniques, pyrosequencing, is described in Special Topic 7.1.

- Another popular technique is called sequencing by synthesis (a technology developed by Solexa, a company now part of Illumina, Inc.)

- The process is outlined in Figure 7.30.

Sequencing an Entire Genome

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Figure 7.30 (Part 1)

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Figure 7.30 (Part 2)

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Whole-genome shotgun (WGS) sequencing methods

- Genome is broken into thousands of pieces, which are all sequenced

- Computer determines sequence overlap to recreate entire genome sequence

Metagenomics uses modern genomic techniques to study microbial communities directly in nature

- Bypassing the need for isolating and cultivating individual species in the laboratory

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Chapter Summary A genome is all the genetic information that defines

an organism.

The prokaryotic genome is typically a single, circular chromosome, whereas the eukaryotic genome consists of multiple, linear chromosomes.

The DNA structure consists of a double helix, composed of four different nucleotides.

The bacterial chromosome is packed in a series of protein-bound loops collectively called the nucleoid.

Topoisomerases are enzymes that supercoil DNA.

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Chapter Summary DNA replication is divided into three phases:

1. Initiation: occurs at the origin (oriC)2. Elongation: occurs at the replication forks3. Termination: occurs at the terminus (ter)

Each phase requires a number of different proteins.

Plasmids are autonomously replicating, extra-chromosomal DNA elements.

- They benefit the host under certain conditions.

Analysis of DNA involves restriction enzymes, gel electrophoresis, PCR, and DNA sequencing.

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Concept Check—Section 7.1

The transfer of genetic information from one cell to another is termed _______ transfer.

a) vertical

b) horizontal

c) linear

d) oblique

e) inverse

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Concept Check—Section 7.2

If the sequence of one strand of DNA is 5’ TCGATC 3’, what is the sequence of the complementary strand?

a) 5’ CTAGCT 3’

b) 5’ GCTAGC 3’

c) 5’ AGCTAG 3’

d) 5’ GATCGA 3’

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Concept Check—Section 7.2 Supercoiling in bacteria is typically introduced

by an enzyme called DNA

a) Gyrase

b) Helicase

c) Ligase

d) Polymerase

e) Endonuclease

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Concept Check—Section 7.3

Which of these represents a correct order of proteins involved in bacterial DNA replication?

a) DnaA → DNA pol III → primase → ligase

b) Primase → DNA pol III → DnaA → ligase

c) DnaA → primase → DNA pol III → ligase

d) Primase → DNA pol III → ligase → DnaA

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Concept Check—Section 7.3 The primer in DNA replication is _______

starter sequence with a free _______ group.

a) a DNA; 3' OH

b) a DNA; 5' OH

c) an RNA; 3' OH

d) an RNA; 5' OH

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Concept Check—Section 7.4 How does rolling-circle replication differ from

chromosomal replication?

a) Polymerization starts at a nick.

b) Helicase moves around the DNA to melt the double-stranded DNA.

c) Single-strand binding proteins coat melted DNA.

d) It requires DNA polymerase.

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Concept Check—Section 7.4 Plasmids can be transferred from one bacterium

to another via the process of

a) Transformation

b) Transduction

c) Transfection

d) Coinfection

e) Conjugation

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Concept Check—Section 7.5 Noncoding sequences make up a large portion

of eukaryotic chromosomes. These include

a) Exons and introns

b) Bacteriophages and plasmids

c) Plasmids and introns

d) Introns and pseudogenes

e) Pseudogenes and bacteriophages

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Concept Check—Section 7.5 Archaea resemble eukaryotes in all of the

following EXCEPT

a) Nuclear membrane

b) DNA-packing proteins

c) RNA polymerase

d) Ribosomal components

e) DNA polymerase

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Concept Check—Section 7.6 PCR consists of three steps, whose order is

a) Denaturation, annealing, polymerization

b) Denaturation, polymerization, annealing

c) Annealing, denaturation, polymerization

d) Annealing, polymerization, denaturation

e) Polymerization, denaturation, annealing