Bacterial GeneticsBacterial Genetics

Medical Medical MicrobiologyMicrobiology

You have known…You have known…Science of Science of geneticsgenetics describes and analyze describes and analyze heredityheredity of of physiologic functions that form the properties of organism. physiologic functions that form the properties of organism.

These properties are determined by the total of all the These properties are determined by the total of all the genetic information named genetic information named genomegenome..

Basic unit of genetics isBasic unit of genetics is genegene, a segment of DNA that , a segment of DNA that carries in its nucleotide sequence information for a specific carries in its nucleotide sequence information for a specific biochemical or physiologic property.biochemical or physiologic property.

A geneA gene is relatively stable but occasionally may undergo a is relatively stable but occasionally may undergo a nucleotide change, such a change is called as nucleotide change, such a change is called as mutationmutation. .

Mutations may occur Mutations may occur spontaneouslyspontaneously or can be or can be inducedinduced by a by a number of physical or chemical agents. number of physical or chemical agents.

Bacteria may have changes including:Bacteria may have changes including:Morphological and/or structural changesMorphological and/or structural changes

Variations of cultural characteristics and Variations of cultural characteristics and

biochemical reactionsbiochemical reactions

Changes in virulenceChanges in virulence

Variation in antigenicityVariation in antigenicity

Changes in drug resistanceChanges in drug resistance

These variations can be divided into two types:

▲ Phenotypic variation:Phenotypic variation: non-heritable▲ Genotypic variation:Genotypic variation: heritable (mutation)

Bacterial Bacterial GenomeGenome

DNA/Genome:

Genetic materials relative to bacterial mutation.

A. chromosome

B. out of chromosome: a) plasmid

b) phage

c) transposable genetic element

1. Chromosomal DNA1. Chromosomal DNA

Bacterial chromosome consists of a

single, circle of double-stranded DNA.

▲ 2 mm long in average

▲ Usually < 5 Mb (1 Mb = 1024 Kb)

Transfer property

▲ Conjugative plasmid ： mediate conjugation through sex

pilus

▲ Non-conjugative plasmid: can not mediate conjugation

because of no gene for encoding sex pilus

Phenotypic effect

▲ Fertility plasmid (F factor): has a sex pilus-encoding gene

▲ Resistance plasmid (R factors): contains genes that

encoding enzymes to destroy antibiotics

2. Plasmid2. Plasmid

Structure of R Factor

RTF (Resistance Transfer Factor)

▲ Conjugative plasmid

▲ Transfer genes

Tn 9

Tn

21

Tn 10

Tn 8RTF

R determinant

R determinant▲ Resistance genes

▲ Transposons (Tn, 转座子 )

3. Phage3. Phage

Phages are obligate

intracellular parasites that

specially propagate in bacteria

(bacterial virus).

▲Nucleic acid: DNA (dsDNA or

ssDNA) or RNA (dsRNA or ss

RNA).

▲ Protein: function in infection

and protect nucleic acid.

▲ Head: DNA or RNA+protein coat (capsid)

▲ Tail: is composed of a hollow core surrounded by a sheath with base plate and at the end.

Tail

Tail Fibers

Base Plate

Head

Contractile Sheath

CapsidDNA

Structure of T4 phage

Interaction between phages and bacteria

Phages are wide spread in nature but usually has a

specificity of host bacteria.

Infection with a virulent phage results in phage replication

with the production of new phage particles and their

subsequent release causes death of host bacteria.

adsorption penetration biosynthesis maturation /

release

Infection with a temperate phage does not necessarily

lead to bacterial death, but phage’s nucleic acid integrates

into bacterial chromosome without new phase production.

adsorption penetration integration

I. Adsorption

Recognition of host bacterial surface receptors by tail fibers

Phage’s nucleic acid is injected through hollow core into bacterial cytoplasm.

II. Penetration

Virulent phage

III. Biosynthesis IV. Maturation and Release

Virulent phage

Temperate Phage (Prophage (Prophage formation) formation)

I. adsorptionII. peneration

III. integrate of phage DNA into host genome

IV. prophage replicates along with chromosome of lysogenic bacterium

4. Transposable Genetic 4. Transposable Genetic ElementsElements

Types of Transposable Genetic Elements ▲ Insertion sequences (IS) ▲ Transposons (Tn)

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 the same one DNA molecule

(jumping gene / movable gene)

Insertion sequence (IS)

▲ Definition: a type of transposable Genetic Elements

only has a transposase-encoding gene alone.

▲ Structure: a small DNA has reverse repeated

sequences at the two ends that are involved in

transposition. In the middle there is a transposase

coding gene.

▲ Function: introduction of an insertion sequence into

a bacterial gene will result in inactivation of the

gene.TransposaseABCDEFG GFEDCBA

Transposon (Tn)

▲ Definition: a type of transposable Genetic Elements has both

insertion sequences (IS) and other genes.

▲ 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-resistant genes,

the transposons mediate drug resistance in bacteria.

IS ISResistance Gene(s)

IS ISResistance Gene(s)

Types of Mutation Self Mutations: Spontaneously occur with low frequency.

Gene transfer and recombination: high frequency.

One bacterium (recipient) uptake exogenous DNA

segment from another bacterium (donor) or phage (Gene transfer) and then the DNA segment is incorporated into DNA of itself (recombination). Major mechanisms of bacterial gene transfer

▲ Transformation ▲ Transduction

▲ Conjugation ▲ Lysogenic conversion

Transformation

Definition: a bacterial recipient uptake naked DNA segment offered by bacterial donor in environment and then the DNA segment recombinate with the recipient’s chromosomal DNA.

Significance for transformation

Transformation occurs in nature and it can lead to increased virulence ( e.g. S. pneumoniae) and drug resistance.

Transduction

Definition: a bacterial donor’s chromosomal DNA

segment transferred to a bacterial recipient by way

of a phage, and then the DNA segment recombinate

with the recipient’s chromosomal DNA . ▲ Generalized Transduction: incorrect assemblage (any

genes).

▲ Specialized transduction: Sometimes, during

activation of prophage, the excised phage’s DNA

contains host DNA segments at its ends.

Lysogenic conversion

Definition: a bacterial recipient is infected

with phage from a bacterial donor, and the

phage’s genes recombinate with 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.

Conjugation

Definition: Gene transfer from

a donor to a recipient by direct

contact between two bacterial

cells through sex pili.

▲ Donor: a bacterium with F factor

can produce sex pili.

▲ Recipient: a F factor-absent

bacterium.

Donor

Recipient

F+ factor-depecdent Conjugation

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.

F+ F+F+ F+

F+ F- F+ F-F+ F-

High-frequency recombination (Hfr)F factor is integrated into bacterial chromosome.

Bacterial DNA is transferred with a high frequency.

F factor is difficulty to be transferred.

F-Hfr Hfr F-

Hfr

Hfr F-

F-

F factor in bacterial DNA is excised but it carries

bacterial chromosomal DNA at its two sides (F’).

F’ factor-depecdent Conjugation

F’Hfr F’ F’F’ F-

SummarySummary

The most important contents in this lecture are

displayed as the followings:1) The Agents (plasmid, phage, bacterial chromosomal DNA)

associated with bacterial mutation.

2) Concepts of Transformation, Transduction, Conjugation

and Lysogenic conversion.

3) The Significance of bacterial mutation (e.g. bacterial

virulence, drug resistance and antigenicity).

Bacterial InfectionBacterial Infection

Medical Medical MicrobiologyMicrobiology

Bacterial PathogenicityTerms :Terms :

Pathogen: disease-causing microbe.

Opportunistic pathogen: A microbe does not

cause diseases in normal conditions, but can

cause diseases in some certain conditions.

Pathogenicity: ability of a microbe to cause

diseases.

Virulence: quantitative ability of a microbe to

cause disease (invasiveness and toxigenicity).

LD50: the number of pathogen required to cause

death in half of the exposed hosts.

Source of infectionSource of infection

Exogenous infection

Infectious agents come from environment or

hosts (patient, diseased animal or carrier).

▲ carrier: individuals (human and animal) infected with microbes but no clinical signs or symptoms.

Endogenous infection

Normal flora act as infections agents under

some certain conditions (opportunistic

infection).

Normal flora

Microbial populations inhabit on skin

and mucosa of healthy normal

persons. ▲ Gastrointestinal tract

▲ Urogenital tract

▲ Skin and Conjunctiva (结膜 )

Physiological role of normal flora

▲ Antagonism: a) biolfilm; b) antimicrobial

agents.

▲ Trophism: a) digestion of foods; b)

production of vitamins (K and B).

▲Immunoenhancement: promotes

development of mucosal immune system.

▲ Anti-tumorigenesis and anti-apolexis( 衰老 ):

e.g. eliminate nitrite and anti-oxidation

(SOD).

Opportunistic infectionsOpportunistic infections

▲ Translocation of normal

flora.

▲ Suppression of normal flora.

▲ Low immunity of human

body.

The conditions required by The conditions required by normal flora to cause normal flora to cause diseases.diseases.

Hospital acquired

infections

(Nosocomial

infections)New infectionsinfections after 48 hours of hospital admission.

Pathogenic process of bacteria

Generally, infection process caused by a

bacterial pathogen involves the four steps

as the following: ▲Adhesion (chemotaxis) Adhesion (chemotaxis)

▲▲ Survival / propagation Survival / propagation

▲▲ Penetration and sPenetration and spreadpread

▲▲ Tissue injuryTissue injury

Virulence: adhesins, invasive enzymes and

toxins.

adhesinadhesin

EPITHELIUMEPITHELIUM

receptorreceptor

BACTERIUMBACTERIUM

1. AdhesionA process of recognition and combination of microbial adhesins with receptors on the surface of host cells▲ lipoteichoic acid (G+ bacteria)

▲ ordinary pilus and outer membrane proteins (G- bacteria)

2. Survival / propagation2. Survival / propagation

•Anti-phagocytosis: e.g. capsules.

•propagation: probably due to

stress.

3. Penetration and Spread3. Penetration and Spread

▲ Collagenase: hydrolyze collagens.

▲ Hyaluronidas: hydrolyze hyaluronic acid.

▲ Coagulase: agglutinate fibrinogen.

▲ Streptokinase/fibrinolysin: the former activates fibrinogenase

to thrombin, and the latter directly hydrolyze fibrin.

▲ Cytolysins: 1) hemolysin (to lyse erythrocyte or tissue cells )

2) Leukocisin (to kill leukocyte or tissue cells )

4. Tissue injury

▲ Exotoxin

▲ Endotoxin

▲ Immunopathological

reaction

4.1. Exotoxin

▲ Cytotoxin: e.g. diphtheria toxin

▲ Nuerotoxin: e.g. tetanospasmin

▲ Enterotoxin: e.g. cholera enterotoxin

diphtheria toxin

4.1. Exotoxin

tetanospasmin

cholera

enterotoxin

ToxicToxicBindingBinding

AA

Cell surfaceCell surface

BB

A-B type exotoxinsA-B type exotoxins

▲ Subunit A is toxic, while subunit B is the ligand of cell’s receptor to mediate A subunit into host cells.

4.2. Endotoxin (LPS)4.2. Endotoxin (LPS)

LPS

▲Fever: a typical pyrogen.

▲ Leukocytoreaction: leukopenia/leukocytosis.

▲ DIC: disseminated intravascular coagulation.

▲ Shock: dilatation of small blood vessels

▲ Inflammation: IL-1β , TNF-αand IL-6.

Major difference between endotoxin and Major difference between endotoxin and exotoxinexotoxin

Property Endotoxin Exotoxin

Chemical nature Lipopolysaccharide( MW = 800-1, 000 kDa )

Protein or peptide( MW = 50-1, 000 kDa )

Relationship to bacteria Part of outer membrane Extracellular

Denatured by boiling No Usually

Antigenicity Yes Yes

Form toxoid No Yes

Toxicity Relatively low( > 100 µg )

Relatively high( approximate 1 µg )

Pyrogenicity Yes Occasionally

Specificity to host cells Low degree High degree

Enzymatic activity No Usually

4.3. Immunopathological reaction

Human immune responses to bacteria may cause tissue injury by:

1. Numerous cytokines and complement activation.

2. Continuously generated bacterial antigens will

subsequently causet humoral antibodies and cell

mediated immunity, which resulting in chronic

immunopathological injury.

3. Some of bacterial antigens (e.g. M protein of

Streptococcus) react with host tissue antigens to cause

autoimmunity.

Number and route▲ Invaded mumber of bacteria:

Different pathogens need different bacterial number to cause diseases.

▲ Suitable invading route of bacteria:

Most of bacteria require suitablec invading routes to cause diseases (e.g. Clostridum tetani causes disease through wounds and Mycobacterium tuberculosis has multiple invading routes).

Types of infection

According to infectious state:According to infectious state:

▲ Inapparent or subclinical infection: The

infection with no manifesting clinical

signs and symptoms.

▲ Latent infection: The infection is inactive

but maintain potential to cause diseases.

▲ Apparent infection: clinical signs and

symptoms.According to infectious site:According to infectious site:

▲ Local infection; ▲ Systemic infection

Systemic infection

▲ Bacteremia: Bacteria enter bloodstream without propagation in bloodstream.

▲ Toxemia: Exotoxin or endotoxin rather than bacteria enters bloodstream.

▲ Septicemia: Bacteria enter bloodstream with propagation and release virulent factors.

▲ Pyemia:Pyogenic bacteria enter bloodstream with propagation and release virulent factors, and then spread through bloodstream into the target organs to form pyogenic foci.

SummarySummaryThe most important contents in this lecture are displayed as the

followings:

1) Concepts of virulence, normal flora, hospital acquired

infection, latent infection, toxemia, septicemia, endotoxemia

and pyemia.

2) The physiologic role of normal flora.

3) The conditions for generation of opportunistic infection.

4) The difference between exotoxin and endotoxin.

5) The pathogenic effects of endotoxin.

Anti-infection Immunity

Medical Medical MicrobiologyMicrobiology

Innate ImmunityPhagocytosis: mononuclear-

macrophages, neutrophils.

Complements: lyse bacteria

Others: lysozyme, antibacterial peptide.

Acquired Immunity

Antibody: antibacterial antibody and

antitoxin (IgM, IgG and SIgA).

T lymphocyte: cytotoxicity (CD4Th1, CTL).

Medical Medical MicrobiologyMicrobiology

Laboratory Diagnosis

Diagnostic procedure

▲ Sample: collect fresh sample from focus of infection using aseptic technique.

▲ Isolated culture: “gold standard”, strains.

▲ Etiological agent detection: Bacterial

antigens (ELISA, etc.) and DNA (PCR).

▲ Serological examination:early dignosis

(IgM).