Bacterial Genetics. The science of genetics describes and analyze heredity of physiologic functions...

Bacterial GeneticsBacterial 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: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 Bacterial GenomeGenomeDNA/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

68%Deinococcus radiodurans

Microbial Genome Features

G+C content29%Borrelia

burgdorferi

Genome organization

single circular chromosome

large linear chromosome plusextrachromosomal elements

circular chromosome plus one or more

extrachromosomal elements

two circular chromosomes

Bacterial Genomics

Chromosomal DNAChromosomal 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 Bacterial GenomeGenome

DNA/Genome:

chromosome

out of chromosome:

plasmid

bacteriophage/phage

transposable genetic elements

a). Plasmidsa). Plasmids

• Are small ， circular/line,

extra-chromosomal double-

stranded DNA that 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) – Conjugative plasmid– Transfer genes

Tn 9

Tn

21

Tn 10Tn 8

RTF

R determinant

• R determinant– Resistance genes– Are often parts of

transposons (Tn)

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 Bacterial GenomeGenome

DNA/Genome:

chromosome

out of chromosome:

plasmid

bacteriophage/phage

transposable genetic elements

b). Bacteriophages/phagesb). 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

filamentous

like a tadpole

– 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.

Tail

Tail Fibers

Base Plate

Head

Contractile Sheath

CapsidDNA

Structure of T4 phage

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. adsorptionII. peneration

III. integrate of phage DNA into host genome

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.

ProphageProphage

Lysogenic bacteriumLysogenic bacterium

Genome of a temperate phage integrating

with bacterial genome

A bacterium containing a prophage

The medical significance of phages

• Phage typing

• Genetic recombination in bacteria

Bacterial Bacterial GenomeGenome

DNA/Genome:

chromosome

out of chromosome:

plasmid

bacteriophage/phage

transposable genetic elements

Transposable Genetic ElementsTransposable 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.

TransposaseABCDEFG 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 TransferTerms 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 modesMajor mechanisms and modesof Bacterial Gene Transferof 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

T

Nonsense Mutation

Base Substitution 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

– Recombination

• Steps– Uptake of DNA

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

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.

Donor

Recipient

only one strand of DNA is transferred

Physiological States of F Factor (I)

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+.

According to the different patterns and characteristics of gene transfer, conjugation can divided into three types.

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

F-Hfr Hfr F-

Hfr

Hfr F-

F-

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'.

Physiological States of F Factor (III)

Model of autonomous Conjugation by F’

F’Hfr 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

Specialized transduction: is the transduction that only certain

bacterial genes can be transferred to the recipient.

Generalized Transduction: is the transduction in which

potentially any genes of the bacterial donor 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 and drug

resistance of recipient bacteria.

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.

SummarySummary

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).