L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014

Lecture 5. GENETICS OF PROKARYOTES

1. Basic concepts

2. The prokaryotic genome

3. The pan-genome

4. Genetic interchange and recombination

4.1. Recombination

4.2. Transformation

4.3. Conjugation

4.4. Transduction

5. Transposable elements

6. Genetic manipulation of microorganisms

(“Genetic engineering”)

L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014

1. BASIC CONCEPTS

Genome: molecule/s storing the genetic information (DNA in all cells; DNA and/or RNA in viruses) Gene: the basic unit of genetic information. A fragment of DNA or RNA, including regulatory sequences, coding for a protein or RNA.

Expression: transcription (when the gene product is a rRNA or tRNA) or transcription and translation (when the gene product is a protein; implies mRNA)

Coding sequence (ORF: open reading frame)

5’ 3’

Regulatory sequences

DNA RNA PROTEIN Translation

GENETIC CODE

Transcription

Reverse transcription

Replication

Replication

L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014

2. THE PROKARYOTIC GENOME

Chromosome: carries genes essential for survival Plasmid/s: non essential* genes. Selectve advantages

Chromosome

Plasmids

L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014

2. THE PROKARYOTIC GENOME 2.1. CHROMOSOME

Chromosome

Plasmid Number: normally, only one Copy number: 1 – 100 Size: 0.5 Mb – 10 Mb Structure: cccDNA (with some exceptions) supercoiled Packaging: basic proteins, cations, etc.

Protein coding sequences (87%)

RNA coding sequences (0.8%) Regulatory non-coding

sequences (11%)

Non-coding sequences (0.7%)

Escherichia coli chromosome

L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014

L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014

2. THE PROKARYOTIC GENOME 2.1. CHROMOSOME

OPERON REGULON

Only one promoter. Co-transcription of several genes. Polycistronic RNA

One regulatory molecule Co-expression of several operons

L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014

2. THE PROKARYOTIC GENOME 2.2. PLASMIDS

Circular (normally) DNA molecules Chromosome-independent replication

Genes non-essential* for growths Size range from 1 Kb to 1 Mb (megaplasmids)

Chromosome

Plasmid High/low copy number Incompatibility groups Curation: plasmid loss (induced or spontaneous)

Plasmid types:

Cryptic

Conjugative

Resistance

Metabolic

Virulence

Episome

“Engineered”

Plasmids R

L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014

2. THE PROKARYOTIC GENOME 2.2. PLASMIDS

ROLLING CIRCLE REPLICATION

L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014

Core genome

vs.

Accessory genome

(strain and environmental

sequences)

3. THE PAN-GENOME

L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014

4. GENETIC INTERCHANGE AND RECOMBINATION

GENETIC VARIABILITY

GENETIC VARIABILITY Eukaryotes: Individual level (mutation and recombination) Population level (sexual reproduction) Prokaryotes: Individual level (mutation and recombination) Population level (HGT: horizontal gene transfer or LGT: lateral GT)

L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014

These mechanisms transfer DNA to receptor cells. This DNA will “stay” if it recombines with the receptor genome

4. GENETIC INTERCHANGE AND RECOMBINATION

L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014

4.1. RECOMBINATION

Together with point mutations, this is a mechanisms of generating genetic diversity

Transfer of DNA between different molecules. Homologous recombination requieres large straches of homologous sequences (>100pb)

4. GENETIC INTERCHANGE AND RECOMBINATION

L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014

Barrier to the recombination: restriction-modification systems

CH3

CH3

Methylated Chromosomic DNA

Methylase

Restriction enzyme

Exogenous DNA

4.1. RECOMBINATION 4. GENETIC INTERCHANGE AND RECOMBINATION

L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014

RESTRICTION ENZYMES

4.1. RECOMBINATION 4. GENETIC INTERCHANGE AND RECOMBINATION

L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014

4.1. TRANSFORMATION 4. GENETIC INTERCHANGE AND RECOMBINATION

L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014

Definition? Competent cells

Viral DNA: transfection

DNA binding proteins Autolysines Nucleases

DNA carrier proteins

4.1. TRANSFORMATION 4. GENETIC INTERCHANGE AND RECOMBINATION

L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014

Natural or recombinant plasmid

(Genetic Engineering) 4.1. TRANSFORMATION 4. GENETIC INTERCHANGE AND RECOMBINATION

L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014

4.1. CONJUGATION 4. GENETIC INTERCHANGE AND RECOMBINATION

L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014

DNasa

4.1. CONJUGATION 4. GENETIC INTERCHANGE AND RECOMBINATION

L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014

Conjugative plasmids [e.g.: plasmid F (factor F)]

F+

F-

F+

F+

4.1. CONJUGATION 4. GENETIC INTERCHANGE AND RECOMBINATION

L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014

Integration into the genome (episomes)

bbe

Plasmid F

Chromosome

INtegrated plasmid F

Plasmid F‘

4.1. CONJUGATION 4. GENETIC INTERCHANGE AND RECOMBINATION

L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014

4.1. CONJUGATION 4. GENETIC INTERCHANGE AND RECOMBINATION

L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014

4.1. CONJUGATION 4. GENETIC INTERCHANGE AND RECOMBINATION

L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014

From Hfr to F- From F ‘ to F-

2 cells with F ‘ 2 recombinat cells

4.1. CONJUGATION 4. GENETIC INTERCHANGE AND RECOMBINATION

L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014

DNase

4. GENETIC INTERCHANGE AND RECOMBINATION 4.4. TRANDSUCTION

L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014

LYTIC vs LYSOGENIC CYCLES

Virulent phages Always lysis

Temperate phages Integration/Lysis

4. GENETIC INTERCHANGE AND RECOMBINATION 4.4. TRANDSUCTION

L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014

GENERALIZED TRANSDUCTION

Defective phage

Transduced cell

4. GENETIC INTERCHANGE AND RECOMBINATION 4.4. TRANSDUCTION

L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014

SPECIALIZED TRANSDUCTION

L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014

Gene Transfer Agents (GTAs)

4. GENETIC INTERCHANGE AND RECOMBINATION 4.4. TRANSDUCTION

L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014

5. TRANSPOSABLE ELEMENTS

DNA fragments that can move and integrate in a new genomic region (transposition)

Insertion sequences

Transposons

Replicative transposons

L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014

5. TRANSPOSABLE ELEMENTS

CUT AND PASTE

Genomic region B (without a mobile element) Genomic region A (with a mobile element)

Genomic region B (with a mobile element) Genomic region A (without a mobile element)

COPY AND PASTE

Genomic region B (without a mobile element) Genomic region A (with a mobile element)

Genomic region A (with a mobile element) Genomic region B (with a mobile element)

Transposable element Transposable element

Transposable element

Transposable element

L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014

5. TRANSPOSABLE ELEMENTS

5’ 3’

3’ 5’ P

Gene expression inactivation

Over-expression

5’ 3’ No effect

5’ 3’

L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014

6. GENETIC MANIPULATION OF MICROORGANISMS 6.1. GENE CLONING

Restriction enzymes / Taq polimerase

Restriction enzymes

DNA ligases

Transformation

bb

L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014

6. GENETIC MANIPULATION OF MICROORGANISMS 6.2. CLONING VECTORS

BBE

L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014

6. GENETIC MANIPULATION OF MICROORGANISMS 6.2. CLONING VECTORS

L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014

6. GENETIC MANIPULATION OF MICROORGANISMS 6.3. METAGENOMICS