1. 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”)

2. 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

3. 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

4. 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

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

6. 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

7. 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

8. L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014 2. THE PROKARYOTIC GENOME 2.2. PLASMIDS ROLLING CIRCLE REPLICATION

9. L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014 Core genome vs. Accessory genome (strain and environmental sequences) 3. THE PAN-GENOME

10. L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014 4. GENETIC INTERCHANGE AND RECOMBINATION 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)

11. 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

12. 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

13. L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014 Barrier to the recombination: restriction-modification systems CH 3 Methylated Chromosomic DNA Methylase Restriction enzyme Exogenous DNA 4.1. RECOMBINATION 4. GENETIC INTERCHANGE AND RECOMBINATION

14. L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014 RESTRICTION ENZYMES 4.1. RECOMBINATION 4. GENETIC INTERCHANGE AND RECOMBINATION

15. L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014 4.1. TRANSFORMATION 4. GENETIC INTERCHANGE AND RECOMBINATION

16. 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

17. L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014 Natural or recombinant plasmid (Genetic Engineering) 4.1. TRANSFORMATION 4. GENETIC INTERCHANGE AND RECOMBINATION

18. L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014 4.1. CONJUGATION 4. GENETIC INTERCHANGE AND RECOMBINATION

19. L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014 DNasa 4.1. CONJUGATION 4. GENETIC INTERCHANGE AND RECOMBINATION

20. L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014 Conjugative plasmids [e.g.: plasmid F (factor F)] F+ F- F+ 4.1. CONJUGATION 4. GENETIC INTERCHANGE AND RECOMBINATION

21. 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

22. L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014 4.1. CONJUGATION 4. GENETIC INTERCHANGE AND RECOMBINATION

23. L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014 4.1. CONJUGATION 4. GENETIC INTERCHANGE AND RECOMBINATION

24. 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

25. L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014 DNase 4. GENETIC INTERCHANGE AND RECOMBINATION 4.4. TRANDSUCTION

26. 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

27. L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014 GENERALIZED TRANSDUCTION Defective phage Transduced cell 4. GENETIC INTERCHANGE AND RECOMBINATION 4.4. TRANSDUCTION

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

29. L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014 Gene Transfer Agents (GTAs) 4. GENETIC INTERCHANGE AND RECOMBINATION 4.4. TRANSDUCTION

30. 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

31. 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 B (with a mobile element) Transposable element

32. L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014 5. TRANSPOSABLE ELEMENTS 5’3’ 5’ P Gene expression inactivation Over-expression 5’ 3’ No effect 5’3’

33. 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

34. L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014 6. GENETIC MANIPULATION OF MICROORGANISMS 6.2. CLONING VECTORS BBE

35. L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014 6. GENETIC MANIPULATION OF MICROORGANISMS 6.2. CLONING VECTORS

36. L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014 6. GENETIC MANIPULATION OF MICROORGANISMS 6.3. METAGENOMICS