Bacterial Bacterial GeneticsGenetics

Xiao-Kui GUO PhDXiao-Kui GUO PhD

Bacterial GenomicsBacterial Genomics

Microbial GenomicsMicrobial Genomics

68%Deinococcus radiodurans

Microbial Genome Features

G+C content29%Borrelia

burgdorferi

Genome organization

single circular chromosome

large linear chromosome plus21 extrachromosomal elements

circular chromosome plus one or more

extrachromosomal elements

two circular chromosomes

PLASMIDS PLASMIDS Plasmids are extrachromosomal genetic elements capable of autonoPlasmids are extrachromosomal genetic elements capable of autonomous replication. An mous replication. An episomeepisome is a plasmid that can integrate into the bacterial chromo is a plasmid that can integrate into the bacterial chromosomesome

Classification of PlasmidsClassification of Plasmids Transfer properties Transfer properties Conjugative plasmidsConjugative plasmids Nonconjugative plasmidsNonconjugative plasmids Phenotypic effects Phenotypic effects Fertility plasmid (F factor)Fertility plasmid (F factor) Bacteriocinogenic plasmidsBacteriocinogenic plasmids.. Resistance plasmids 7 factors)Resistance plasmids 7 factors) . .

Insertion sequences (IS)- Insertion sequences (IS)- Insertion sequences are transposable genetic Insertion sequences are transposable genetic elements that carry no known genes except those that are required for transposition.elements that carry no known genes except those that are required for transposition.

• a. a. NomenclatureNomenclature - Insertion sequences are given the designation IS followed by a number. e.g. IS1 - Insertion sequences are given the designation IS followed by a number. e.g. IS1• b. b. StructureStructure Insertion sequences are small stretches of DNA that have at their ends repeated sequences, Insertion sequences are small stretches of DNA that have at their ends repeated sequences,

which are involved in transposition. In between the terminal repeated sequences there are genes involved iwhich are involved in transposition. In between the terminal repeated sequences there are genes involved in transposition and sequences that can control the expression of the genes but no other nonessential genen transposition and sequences that can control the expression of the genes but no other nonessential genes are present.s are present.

• c. c. ImportanceImportance• i) Mutation - The introduction of an insertion sequence into a bacterial gene will result in the inactivation of ti) Mutation - The introduction of an insertion sequence into a bacterial gene will result in the inactivation of t

he gene.he gene.• ii) Plasmid insertion into chromosomes - The sites at which plasmids insert into the bacterial chromosome aii) Plasmid insertion into chromosomes - The sites at which plasmids insert into the bacterial chromosome a

re at or near insertion sequence in the chromosome.re at or near insertion sequence in the chromosome.• iii) Phase Variation - The flagellar antigens are one of the main antigens to which the immune response is diiii) Phase Variation - The flagellar antigens are one of the main antigens to which the immune response is di

rected in our attempt to fight off a bacterial infection. In Salmonella there are two genes which code for two rected in our attempt to fight off a bacterial infection. In Salmonella there are two genes which code for two antigenically different flagellar antigens. The expression of these genes is regulated by an insertion sequenantigenically 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 actices. 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 ive. 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 s 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).mechanism of phase variation may differ in different species of bacteria (e.g. Neisseria; transformation).

Transposons (Tn) - Transposons (Tn) - Transposons are transposable genetic elements thTransposons are transposable genetic elements that carry one or more other genes in addition to those which are essential for tranat carry one or more other genes in addition to those which are essential for transposition.sposition.

• NomenclatureNomenclature - Transposons are given the designation Tn followed by a n - Transposons are given the designation Tn followed by a number.umber.

• StructureStructure - The structure of a transposon is similar to that of an insertion s - The structure of a transposon is similar to that of an insertion sequence. The extra genes are located between the terminal repeated sequenequence. The extra genes are located between the terminal repeated sequences. In some instances (composite transposons) the terminal repeated sequeces. In some instances (composite transposons) the terminal repeated sequences are actually insertion sequences. nces are actually insertion sequences.

• ImportanceImportance - Many antibiotic resistance genes are located on transposons. - Many antibiotic resistance genes are located on transposons. Since transposons can jump from one DNA molecule to another, these antiSince transposons can jump from one DNA molecule to another, these antibiotic resistance transposons are a major factor in the development of plasbiotic resistance transposons are a major factor in the development of plasmids which can confer multiple drug resistance on a bacterium harboring smids which can confer multiple drug resistance on a bacterium harboring such a plasmid. These multiple drug resistance plasmids have become a majuch a plasmid. These multiple drug resistance plasmids have become a major medical problem because the indiscriminate use of antibiotics have provor medical problem because the indiscriminate use of antibiotics have prov

ided a selective advantage for bacteria harboring these plasmidsided a selective advantage for bacteria harboring these plasmids..

Mechanism of bacterial Mechanism of bacterial variationvariation

• Gene mutationGene mutation• Gene transfer and recombinaGene transfer and recombina

tiontion • TransformationTransformation• Conjugation Conjugation • TransductionTransduction• Lysogenic conversion Lysogenic conversion • Protoplast fusion Protoplast fusion

Types of Types of mutationmutation

• Base substitutionBase substitution

• Frame shefitFrame shefit

• Insertion sequencesInsertion sequences

What can cause mutation?What can cause mutation?

• Chemicals: Chemicals:

nitrous acid; alkylating agentsnitrous acid; alkylating agents

5-bromouracil5-bromouracil

benzpyrenebenzpyrene

• Radiation: X-rays and Ultraviolet Radiation: X-rays and Ultraviolet lightlight

• VirusesViruses

Bacterial Bacterial mutationmutation

• Mutation rateMutation rate• MutationMutation and selectivityand selectivity• Backward mutationBackward mutation

TransformationTransformation

• Transformation is gene transfer resulting Transformation is gene transfer resulting from the uptake by a recipient cell of nakfrom the uptake by a recipient cell of nakeded DNA from a donor cell. Certain bacteriDNA from a donor cell. Certain bacteria (a (e.g. e.g. Bacillus, Haemophilus, Neisseria, PBacillus, Haemophilus, Neisseria, Pneumococcus)neumococcus) can take up DNA from the can take up DNA from the environment and the DNA that is taken uenvironment and the DNA that is taken up can be incorporated into thep can be incorporated into the recipient's recipient's chromosome.chromosome.

ConjugationConjugation

• Transfer of DNA from a donor to a recipiTransfer of DNA from a donor to a recipient by direct physical contact between tent by direct physical contact between thehe cells. In bacteria there are two matincells. In bacteria there are two mating types a donor (male) and a recipient (fg types a donor (male) and a recipient (female) and the directionemale) and the direction of transfer of gof transfer of genetic material is one way; DNA is transfenetic material is one way; DNA is transferred from a donor to a recipient.erred from a donor to a recipient.

Physiological States of F Physiological States of F FactorFactor

• Autonomous (FAutonomous (F++))– Characteristics of FCharacteristics of F++ x F x F--

crossescrosses•FF-- becomes F becomes F++ while F while F++

remains Fremains F++

•Low transfer of donor Low transfer of donor chromosomal geneschromosomal genes

F+

PhysiologicalPhysiological StatesStates of F of F FactorFactor

• Integrated (Hfr)Integrated (Hfr)– Characteristics of Characteristics of

Hfr x FHfr x F-- crosses crosses•FF-- rarely becomes rarely becomes

Hfr while Hfr remHfr while Hfr remains Hfrains Hfr

•High transfer of cHigh transfer of certain donor chroertain donor chromosomal genesmosomal genes

F+ Hfr

Physiological States of F Physiological States of F FactorFactor

• Autonomous with Autonomous with donor genes (F’)donor genes (F’)– Characteristics of Characteristics of

F’ x FF’ x F-- crosses crosses

•FF-- becomes F’ becomes F’ while F’ remains while F’ remains F’F’

•High transfer of High transfer of donor genes on donor genes on F’ and low F’ and low transfer of other transfer of other donor donor chromosomal chromosomal genesgenes

Hfr F’

Mechanism of FMechanism of F++ x F x F-- Crosses Crosses

• DNA transferDNA transfer– Origin of Origin of

transfertransfer– Rolling circle Rolling circle

replicationreplication

• Pair formation

– Conjugation bridge

F+ F- F+ F-

F+ F+F+ F+

Mechanism of Hfr x FMechanism of Hfr x F-- Crosses Crosses

• DNA transferDNA transfer– Origin of Origin of

transfertransfer– Rolling circle Rolling circle

replicationreplication

• Homologous Homologous recombinationrecombination

• Pair formation

– Conjugation bridge

Hfr F- Hfr F-

Hfr F-Hfr F-

Mechanism of F’ x FMechanism of F’ x F-- Crosses Crosses

• DNA transferDNA transfer– Origin of Origin of

transfertransfer– Rolling circle Rolling circle

replicationreplication

• Pair formation

– Conjugation bridge

F’ F’F’ F’

F’ F- F’ F-

R PlasmidR Plasmid

Transduction:Transduction:

• Transduction is defined as the transfer oTransduction is defined as the transfer of genetic information between cells throf genetic information between cells through the mediation of a virus (phage) parugh the mediation of a virus (phage) particle. It therefore does not require cell to ticle. It therefore does not require cell to cell contact and is DNase resistant.cell contact and is DNase resistant.

Generalized Generalized Transduction Transduction

• Generalized transduction is transductioGeneralized transduction is transduction in which potentially anyn in which potentially any bacterial gene bacterial gene from the donor can be transferred to the from the donor can be transferred to the recipient.recipient.

The mechanism of The mechanism of generalizedgeneralizedtransductiontransduction

Generalized Generalized transductiontransduction

1.1. It is relatively easy. It is relatively easy.

2.2. It is rather efficient (10It is rather efficient (10-3-3 per recipient per recipient with P22HT, 10with P22HT, 10-6-6 with P22 or P1), using with P22 or P1), using the correct phage. the correct phage.

3.3. It moves only a small part of the It moves only a small part of the chromosome which allows you to chromosome which allows you to change part of a strain's genotype change part of a strain's genotype without affecting the rest of the without affecting the rest of the chromosome. chromosome.

4.4. The high frequency of transfer and the The high frequency of transfer and the small region transferred allows fine-small region transferred allows fine-structure mappingstructure mapping

Specialized transductionSpecialized transduction

• Specialized transductionSpecialized transduction is transduction in whic is transduction in which only h only certain donorcertain donor genesgenes can be transferred to can be transferred to the recipient. the recipient.

• Different phages may transfer different genes but Different phages may transfer different genes but anan individual phage can only transfer certain geneindividual phage can only transfer certain geness

• Specialized transduction is mediated by lysogenicSpecialized transduction is mediated by lysogenic or temperate phage and the genes that get transfor temperate phage and the genes that get transferred will depend on where the prophage haserred will depend on where the prophage has insinserted in the chromosome.erted in the chromosome.

The mechanism of specialized The mechanism of specialized transductiontransduction

Specialized transduction Specialized transduction

1.1. Very efficient transfer of a small region--can be uVery efficient transfer of a small region--can be useful for fine-structure mapping seful for fine-structure mapping

2.2. Excellent source of DNA for the chromosomal regExcellent source of DNA for the chromosomal region carried by the phage, since every phage carriion carried by the phage, since every phage carries the same DNA. es the same DNA.

3.3. Can often be used to select for deletions of some Can often be used to select for deletions of some of the chromosomal genes carried on the phage. of the chromosomal genes carried on the phage.

4.4. Merodiploids generated using specialized phage Merodiploids generated using specialized phage can be quite useful in complementation analyses.can be quite useful in complementation analyses.

Lysogenic conversionLysogenic conversion

•The prophage DNA as a gene rThe prophage DNA as a gene recombined with chromosomecombined with chromosome of host cell.e of host cell.

Protoplast FusionProtoplast Fusion

• Fusion of two protoplasts treated with lyFusion of two protoplasts treated with lysozyme and penicillin. sozyme and penicillin.

Application of Bacterial Application of Bacterial VariationVariation

• UUse in medical clinic: Diagnosis, se in medical clinic: Diagnosis, Treatment, Prophylaxis.Treatment, Prophylaxis.

• UUse in Genetic Engineering se in Genetic Engineering