Transcript Document

Bacterial Genetics
• The science of genetics describes and analyze heredity of
physiologic functions that form the properties of organism.
• These properties are determined by the total of all the genetic
information named genome.
• The basic unit of genetics is gene, a segment of DNA that
carries in its nucleotide sequence information for a specific
biochemical or physiologic property.
• A gene is relatively stable but occasionally may undergo a
nucleotide change, such a change is called as mutation.
• Mutations may occur spontaneously or can be induced by a
number of physical or chemical agents.
Bacteria may have changes including:
Morphological and/or structural changes
(L form)
Variations of cultural characteristics and
biochemical reactions
Changes in virulence
Variation of antigenicity
Changes in drug resistance
These variations of bacteria can be divided into
two types:
a) Phenotypic variation: non-heritable
b) Genotypic variation: heritable (mutation)
Phenotypic variations
Normal physiologic responses of bacteria due to the
change of bacteria growth environment. The
changes are limited, non-hereditary, and revert back
to their original state when the conditions are
changed back.
Flagella of Salmonella spp. are absent due to the
presence of 0.1 % phenol in culture medium.
Genotypic variations (Mutation)
Stable, heritable changes of bacteria. The changes are
due to the mutations in bacterial genomic nucleotide
sequences.
In this lecture, emphases are given to the content about
genotypic variations containing bacterial genome,
mutation types and mechanisms.
What are the basic genetic
materials in bacteria?
Bacterial genome
Bacterial Genome
DNA/Genome:
the genetic materials relative to bacterial
heredity and mutation.
A. chromosome
B. out of chromosome:
a) plasmid
b) bacteriophage/phage
c) transposable genetic elements
Microbial Genome Features
29%
Borrelia
burgdorferi
G+C content
68%
Deinococcus
radiodurans
single circular chromosome
two circular
chromosomes
circular chromosome
plus one or more
extrachromosomal
elements
Genome organization
large linear chromosome plus
extrachromosomal elements
Bacterial Genomics
Chromosomal DNA
• Bacterial chromosome consists of a single, circle of
double-strand DNA.
– in average 2 mm long
– Usually < 5000 Kb)
• The chromosome carry many genes.
Bacterial Genome
DNA/Genome:
chromosome
out of chromosome:
plasmid
bacteriophage/phage
transposable genetic elements
a). Plasmids
• Are small，circular/line, extrachromosomal
DNA
that
double-stranded
are
capable
of
autonomous replication.
•Carry genes associated with
specialized functions
The characteristics of plasmids
•Self-replication
•Encoding some bacterial properties
-F/R/Col/Vi plasmid
•Not necessary for bacterial viability
•Transferability
Classification of plasmids
• Transfer properties
– Conjugative plasmids (mediate conjugation through sex
pilus)
– Non-conjugative plasmids (can not mediate conjugation
because of no gene for encoding sex pilus)
Classification of plasmids
• Phenotypic effects
– Fertility plasmid (F factor: carrying a gene that encoding
sex pilus protein)
– Bacteriocinogenic plasmid (carrying genes that
encoding bacteriocins that kill other bacteria)
– Resistance plasmid (R factors: carrying genes that
encoding enzymes to destroy antibiotics)
F plasmid
Structure of R Factors
• RTF (Resistance Transfer Factor)
RTF
– Conjugative plasmid
– Transfer genes
• R determinant
– Resistance genes
– Are often parts of
transposons (Tn)
R determinant
Mode of action of resistance genes
a) Modification (detoxification) of antibiotics
β-lactamase
b) Alteration of the target sites of antibiotics
Streptomycin resistance
c) Alteration of the uptake ability of antibiotics
Tetracycline resistance
d) Replacement of sensitive pathway
resistance to sulfa drugs
Bacterial Genome
DNA/Genome:
chromosome
out of chromosome:
plasmid
bacteriophage/phage
transposable genetic elements
b). Bacteriophages/phages
Phages
are
obligate
intracellular
parasites that multiply inside bacteria
by making use of some or all of the
host biosynthetic machinery.
They are viruses that specially infect
bacteria (“bacterial virus”).
Composition of Bacteriophage
• nucleic acid: either DNA or RNA but not both
– dsDNA, ssRNA, ssDNA
– Contain unusual or modified bases
– Encode 3-5 gene products ~ approximate 100 gene
products
• protein: function in infection and protect the nucleic
acid
Structure of Bacteriophage
different sizes and shapes
icosahedral
like a tadpole
filamentous
Structure of T4 phage
Capsid
DNA
Contractile
Sheath
Head
Tail
Tail Fibers
Base Plate
– Head consists of DNA surrounded by a protein coat (capsid)
– Tail is composed of a hollow core surrounded by a contractile
sheath with base plate at the end through which are tail fibers.
Interaction between phages and bacteria
• Phages are wide spread throughout nature and can be
found infecting many different genera of bacteria.
• However, the host range of any specific phage is limit. A
given phage can usually infect only a single species of bacteria
or closely related species.
•Based on the pattern of interaction between a given phage
and it’s host, phages are divided into two major groups:
virulent/Lytic phages and temperate/Lysogenic phages.
Interaction between phages and bacteria
• Infection with a virulent phage results in phage replication
with the production of new phage particles and their
subsequent release that causing death and lysis of the host
bacteria.
• Infection with a temperate phage does not necessarily lead to
bacterial lysis and death, and the phage may integrate into
the bacterial genome without new phase production.
Virulent/Lytic Phages
• Virulent phages can multiply in bacteria and kill the bacterial
cell by lysis at the end of their life cycle.
• The life cycle of a virulent phage can be divided into four
phases:
I. adsorption / attachment
II. penetration
III. biosynthesis / intracellular development
IV. maturation and release
I. Adsorption
Recognition of host bacterial surface receptors by the tail fibers
II. Penetration
The tail sheath contracts, pushing the rigid tail core through the
bacterial cell envelope and the phage’s nucleic acid is injected
through the hollow core into the bacterial cytoplasm.
III. Biosynthesis
Protein synthesis and production of many new copies of new
phage DNA or RNA.
IV. Maturation and Release
Irreversible combination of phage nucleic acid with it’s
protein coat. Induce cell lysis and release of the newly
formed phages.
Temperate/Lysogenic Phages
• Temperate phages are capable to invade host bacteria and
inducing lysogenic state without necessarily producing a lethal
lytic infection.
• A temperate phage can either go through the lytic cycle or induce
lysogeny by integrating the host DNA in the form of a prophage.
• Prophage is only a genome of the phage that integrated in
genomic DNA of its host bacterium.
• The bacterial cell harboring a prophage is termed as lysogenic
bacterium and this state is called as lysogenization.
Prophage formation
I. adsorption
III. integrate of phage
DNA into host genome
II. peneration
IV. prophage replicates along
with host chromosome
Type
Life cycle
Virulent phage
Lytic
Temperate phage
Lysogenic
lytic
Prophages in
lysogenic bacteria
will spontaneously
proceed through
the lytic cycle.
Prophage
Genome of a temperate phage integrating
with bacterial genome
Lysogenic bacterium
A bacterium containing a prophage
The medical significance of phages
• Phage typing
• Genetic recombination in bacteria
Bacterial Genome
DNA/Genome:
chromosome
out of chromosome:
plasmid
bacteriophage/phage
transposable genetic elements
Transposable Genetic Elements
• Definition:
segments of DNA that have the capacity to move
from one bacterial DNA molecule (bacterial
chromosome or plasmid) to another or from one
location to another in one DNA molecule.
(jumping gene / movable gene)
Properties of transposable genetic elements
“Random” movement: move with no any regularity.
Transposase: coded by the transposable genetic elements and
mediates transposition.
Not capable of self replication: usually replicated as a part of
some other replicon (plasmid or chromosome).
Site-specific recombination: dependent on the inserted sites,
but does not require homology between the recombining
molecules.
Transposition may be accompanied by replication: In some cases,
one copy remains of the element at the original site and the other
is moves to a new site.
Types of Transposable Genetic Elements
I. Insertion sequences (IS)
II. Transposons (Tn)
Insertion sequences (IS)
– Definition: a type of transposable Genetic Elements that
carry no other genes except the genes involving in
transposition (transposase coding genes).
– Structure: a small DNA that has repeated sequences at its
ends, which are involved in transposition. In the middle
between the terminal repeated sequences there is a
transposase coding gene (usually one and occasionally more).
– Function: introduction of an insertion sequence into a
bacterial gene will result in the inactivation of the gene.
ABCDEFG
Transposase
GFEDCBA
– Importance
• i) Mutation - The introduction of an insertion sequence into a
bacterial gene will result in the inactivation of the gene.
• ii) Plasmid insertion into chromosomes - The sites at which
plasmids insert into the bacterial chromosome are at or near
insertion sequence in the chromosome.
– Importance
• iii) Phase Variation - In Salmonella there are two genes which code
for two antigenically different flagellar antigens. The expression of
these genes is regulated by an insertion sequences. In one
orientation one of the genes is active while in the other orientation
the other flagellar gene is active. Thus, Salmonella can change their
flagella in response to the immune systems' attack.
• Phase variation is not unique to Salmonella flagellar antigens. It is
also seen with other bacterial surface antigens. Also the mechanism
of phase variation may differ in different species of bacteria (e.g.
Neisseria; transformation).
Transposons (Tn)
Definition: a type of transposable Genetic Elements that carry
other genes and insertion sequences (IS).
Structure: the extra genes are located between the terminal
repeated sequences.
Function: Since transposons can jump from one DNA molecule
to another and frequently carry antibiotic resistance genes,
these transposons participate the development of drug
resistance in bacteria.
Importance: Many antibiotic resistance genes are located on transposons.
Since transposons can jump from one DNA molecule to another, these
antibiotic resistance transposons are a major factor in the development of
plasmids which can confer multiple drug resistance on a bacterium
harboring such a plasmid. These multiple drug resistance plasmids have
become a major medical problem because the indiscriminate use of
antibiotics have provided a selective advantage for bacteria harboring
these plasmids.
Mutation types
• Self Mutations: low frequency
– Spontaneous mutation: Mutations for a given gene spontaneously occur
with a certain frequency (from 10-8-10-6) in a population derived from a
single bacterium.
– Induced mutation: Some chemical agents and radiation can induce
bacterial mutation.
• Gene transfer and recombination: high frequency
– one bacterium uptake exogenous DNA segment from another bacterium
or phage (Gene transfer) and then the DNA segment is incorporated into
DNA of itself (recombination).
Terms about Bacterial Gene Transfer
• Donor: a bacterium to offer DNA segment (but not entire
chromosome) to other bacteria.
• Recipient: a bacterium to receive DNA segment offered by
other bacteria.
Bacterial genes are usually transferred among members of the same species
but occasionally transferred to other species.
Major mechanisms and modes
of Bacterial Gene Transfer
• Gene mutation
• Gene transfer and recombination
–
Transformation
–
Conjugation
–
Transduction
–
Lysogenic conversion
–
Protoplast fusion
Types of mutation
• Base substitution
• Frame shift
• Insertion sequences
Types of Mutations
Normal DNA
Base Substitution Mutation
C
Missense Mutation
Base Substitution Mutation
T
Nonsense Mutation
Frame Shift Mutation
• ATG CAT GCA TGC ATT TCC TGC TTA AAA
• 1. Addition Mutation
• AAT GCA TGC ATG CAT TTT CCT GCT TAA
•
Reading Frame is Shifted
• 2. Deletion Mutation
• (A)TGC ATG CAT GCA TTT CCT GCT TAA
•
Reading Frame is Shifted
What can cause mutation?
• Chemicals:
nitrous acid; alkylating agents
5-bromouracil
benzpyrene
• Radiation: X-rays and Ultraviolet light
• Viruses/ phage
Gene transfer and recombination
• Transfer:
a relatively small fragment of a donor genome to a
recipient cell
• Recombination:
Exogenous DNA integrated into the chromosome
Transformation
• Definition: a bacterial recipient uptake naked
chromosomal DNA segment offered by bacterial
donor in environment and then the DNA segment
recombined with the recipient’s chromosomal DNA .
Factors affecting transformation
DNA size: Double stranded DNA segment with at
least 500 kbp works best.
Competence of the recipient: Only the bacteria in a
particular time during their growth cycle called as
competent stage can take up DNA by transformation,
while the non-competent bacteria can not.
Steps in transformation
• Steps
– Uptake of DNA
– Recombination
Significance for transformation
• Transformation occurs in nature and it can lead to increased
virulence ( e.g. Streptococcus pneumoniae) and drug
resistance.
•
In addition transformation is widely used in recombinant
DNA technology.
Conjugation
• Definition: Gene transfer from a donor to
a recipient by direct physical contact
between two bacterial cells.
– Donor: the bacterium
(F+)
has fertility
Donor
plasmid called as F factor. The F factor
offers the bacterium an ability to produce a
sex pilus.
– Recipient: the bacterium (F- ) that lack of F
factor.
Recipient
only one strand of DNA is transferred
Physiological States of F Factor (I)
According to the different patterns and characteristics of gene
transfer, conjugation can divided into three types.
I. Autonomous (F+):
• the F factor is autonomous and carries only those genes
necessary for its replication and for DNA transfer (no
chromosomal genes of bacterial donor).
• So in this type of conjugation, there is low transfer level of
bacterial donor’s genes.
• In crosses of the F+ and F- bacteria, the F- bacterium becomes F+
and the F+ bacterium remains F+.
Model of Autonomous Conjugation by F+
F+
F+
F-
F+
F+
F-
F+
F+
F-
F+
The F+ bacterium transfers extra chain of F+ factor and then the
completed F+ factors in the two bacteria is synthesized by rolling
circle replication.
Physiological States of F Factor (II)
II. Integrated (Hfr):
• The F factor has integrated on bacterial chromosome, and
only bacterial DNA is transferred with a high frequency
(usually the F factor is not transferred).
• In crosses of the Hfr and F- bacteria, the F- bacterium rarely
becomes Hfr but obtaining DNA segment from donor
bacterium, and the Hfr bacterium remains Hfr.
Model of Integrated Conjugation
Hfr
Hfr
F-
Hfr
F-
F-
Hfr
F-
Physiological States of F Factor (III)
III. Autonomous (F’):
• In this pattern, the F factor is autonomous but it now
carries some of bacterial chromosomal genes (F’), because
this F factor is a excised integrated F factor with host’s
chromosomal sequences at its two sides.
• In crosses of the F' and F- bacteria, the F- bacterium
becomes F' and the F' bacterium remains F'.
Model of autonomous Conjugation by F’
Hfr
F’
F’
F-
F’
F’
Significance for conjugation
– In most of Gram negative bacteria, conjugation is the major
way of bacterial gene transfer, which frequently result in
multiple antibiotic resistance.
– In some of Gram positive bacteria, conjugation is also an
active way of bacterial gene transfer. Multiple antibiotic
resistance genes in a Gram positive bacterium can be obtained
by conjugation or by transduction.
Transduction
• Definition:
a chromosomal DNA segment of
bacterial donor transferred to a bacterial
recipient by way of a bacteriophage, and then the
DNA segment recombinate with the recipient’s
chromosomal DNA .
Types of Transduction
Generalized Transduction: is the transduction in which
potentially any genes of the bacterial donor can be transferred
to the recipient.
Specialized transduction: is the transduction that only certain
bacterial genes can be transferred to the recipient.
Generalized Transduction
• Virulent phages that mediate generalized transduction generally
breakdown host DNA into smaller pieces.
• Occasionally, one of the host DNA pieces is randomly packaged into the
phage particle. Thus, any genes of bacteria can be potentially transferred.
• When the bacterial DNA contained phage infect a bacterial recipient,
donor DNA enters the recipient.
• In the recipient, the event of a recombination of donor DNA and
recipient DNA can occur.
Specialized Transduction
•As the introduction above, sometimes prophage, like virulent phage, can
spontaneously entry the lytic cycle.
•During the excision of prophage, occasionally some of the host DNA segments at
either sides of prophage gene sequence is excised with the phage DNA.
•After a bacterial recipient is infected with this phage and then forms its
lysogenization, the recipient’s genomic DNA contains the donor DNA.
Significance for Transduction
Transduction occurs in nature and it can lead to
increased
virulence
recipient bacteria.
and
drug
resistance
of
Lysogenic conversion
• Definition: a bacterial recipient is infected with bacteriophage from a
bacterial donor, and the genes of phage itself, but not genes of the
bacterial donor, recombined with the recipient’s chromosomal DNA.
• As an example, Corynebacterium diphtheriae will produces diphtheria toxin
after it is infected by the β- phage, because the gene encoding the toxin is
carried by the phage.
Summary
1) The genetic materials of bacteria.
2) Concepts of Transformation, Transduction, Conjugation
and Lysogenic conversion
3) Four forms of genetic recombination in bacteria
4) The Significance of bacterial mutation (changes of
bacterial virulence, drug resistance, antigenicity and so on).