Microbiology

An Evolving Science
Third Edition

Joan L. Slonczewski and John W. Foster

Genomes and
Chromosomes
PowerPoint Lecture Outlines
Prepared by Johnny El-Rady, University of South Florida
Copyright 2014 W. W. Norton & Company, Inc. Permission required for reproduction or display

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
2

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
3

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

4

Bacterial Genomes
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.
5

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.
7

Functional Units of Genes

A gene can operate independently of others.
- Or, it may exist in tandem with other genes in a unit
called an operon.

Figure 7.3

DNA Structure
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.

 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 (50oC90oC), the hydrogen bonds
in DNA break and the duplex falls apart, or denatures,
into two single strands.
10

Figure 7.4A

11

Figure 7.5

12

Figure 7.6

The DNA double helix

has two grooves
- A wide major groove
and a narrow minor
groove

- These provide DNAbinding proteins

access to base
sequences
Figure 7.7

13

RNA Structure
RNA differs from DNA:
- Usually single-stranded
- Contains ribose sugar
- Uracil replaces thymine

Figure 7.4B

14

The Bacterial Nucleoid

Bacteria pack their DNA into a series of loops or
domains, collectively called the nucleoid.
- Loops are anchored by histone-like proteins.

Figure 7.8

15

DNA Supercoiling

But how does

DNA achieve
this supercoiled
state?

Figure 7.9

16

 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.
17

Topoisomerases Supercoil DNA

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
18

Figure 7.10

19

Figure 7.11

Figure 7.12

20

Topoisomerases
Animation: supercoiling and
topoisomerases

21

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.

22

Overview of Bacterial DNA Replication

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

Figure 7.13

23

Replication from a Single Origin

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.
24

Figure 7.14

25

 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
26

Initiation of Replication
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).
27

Figure 7.15

28

 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
29

Elongation of Replicating DNA

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.
30

 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.

31

Figure 7.18 (Part 1)

32

Figure 7.18 (Part 2)

33

Figure 7.18 (Part 3)

34

Figure 7.18 (Part 4)

35

DNA Replication
Animation: DNA replication

36

Elongation of Replicating DNA

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).
37

Figure 7.19

38

Terminating Replication
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 counterhelicase.
Ringed catenanes formed at the completion of
replication are separated by topoisomerase IV and
the proteins XerC and XerD.
39

Figure 7.20
B

40

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
41

Plasmids Replicate Autonomously

Plasmids are much smaller
than chromosomes.
- Found in archaea,
bacteria, and
eukaryotic microbes
- Usually circular
- Need host proteins
to replicate
Figure 7.22
42

 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.
43

Figure 7.23

44

Plasmids
Animation: rolling-circle mechanism of
plasmid replication

45

Plasmid Properties
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.

46

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
47

Figure 7.25

48

Archaeal Genomes
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.
49

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.
50

Restriction Endonuclease Digestion

Figure 7.26A

Restriction
endonucleases
cleave DNA at
specific
recognition sites,
which are usually
4 to 6 bp and
palindromes.
- May generate
blunt or
staggered ends
51

Restriction Endonuclease Digestion

Figure 7.26B

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

52

Cloning
Restriction endonucleasedigested 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
53

Figure 7.27c

54

PCR Amplifies Specific Genes

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

Figure 7.28

55

DNA Sequencing
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.

Figure 7.29

56

DNA Sequencing
Animation: DNA sequencing

57

Sequencing an Entire Genome

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.
58

Figure 7.30 (Part 1)

59

Figure 7.30 (Part 2)

60

 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
61

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.

62

Chapter Summary
DNA replication is divided into three phases:
1. Initiation: occurs at the origin (oriC)
2. Elongation: occurs at the replication forks
3. Termination: occurs at the terminus (ter)
Each phase requires a number of different proteins.
Plasmids are autonomously replicating, extrachromosomal DNA elements.
- They benefit the host under certain conditions.
Analysis of DNA involves restriction enzymes, gel
electrophoresis, PCR, and DNA sequencing.

63

Concept CheckSection 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
64

Concept CheckSection 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
65

Concept CheckSection 7.2

Supercoiling in bacteria is typically introduced
by an enzyme called DNA
a) Gyrase
b) Helicase
c) Ligase
d) Polymerase
e) Endonuclease
66

Concept CheckSection 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

67

Concept CheckSection 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

68

Concept CheckSection 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.
69

Concept CheckSection 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
70

Concept CheckSection 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
71

Concept CheckSection 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
72

Concept CheckSection 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
73