Lecture 5. GENETICS OF PROKARYOTES

1. 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. 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 transcriptionand translation(when the gene product is a protein; implies mRNA) Regulatory sequences 5’ 3’ Coding sequence (ORF: open reading frame) Transcription DNA RNA PROTEIN Translation GENETIC CODE Reverse transcription Replication Replication

3. 2. THE PROKARYOTIC GENOME Chromosome: carries genes essential for survival Plasmid/s: non essential* genes. Selectve advantages Chromosome Plasmids

4. 2. THE PROKARYOTIC GENOME 2.1. 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. Chromosome RNA coding sequences (0.8%) Non-coding sequences (0.7%) Regulatory non-coding sequences (11%) Proteincodingsequences (87%) Escherichia coli chromosome

6. 2. THE PROKARYOTIC GENOME 2.1. CHROMOSOME REGULON OPERON Only one promoter. Co-transcription of several genes. Polycistronic RNA One regulatory molecule Co-expression of several operons

7. 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) High/low copy number Incompatibility groups Curation: plasmid loss (induced or spontaneous) Plasmid Plasmid types: Cryptic Conjugative Resistance Metabolic Virulence Episome “Engineered” Chromosome PlasmidsR

8. 2. THE PROKARYOTIC GENOME 2.2. PLASMIDS ROLLING CIRCLE REPLICATION

9. 3. THE PAN-GENOME Core genome vs. Accessory genome (strain and environmental sequences)

10. 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)

11. 4. GENETIC INTERCHANGE AND RECOMBINATION These mechanisms transfer DNA to receptor cells. This DNA will “stay” if it recombines with the receptor genome

12. 4. GENETIC INTERCHANGE AND RECOMBINATION 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)

13. 4. GENETIC INTERCHANGE AND RECOMBINATION 4.1. RECOMBINATION Barrier to the recombination: restriction-modification systems Exogenous DNA Methylase CH3 Restriction enzyme Methylated Chromosomic DNA CH3

14. 4. GENETIC INTERCHANGE AND RECOMBINATION 4.1. RECOMBINATION RESTRICTION ENZYMES

15. 4. GENETIC INTERCHANGE AND RECOMBINATION 4.1. TRANSFORMATION

16. 4. GENETIC INTERCHANGE AND RECOMBINATION 4.1. TRANSFORMATION Definition? Competent cells Viral DNA: transfection DNA binding proteins Autolysines Nucleases DNA carrier proteins

17. Natural or recombinant plasmid (Genetic Engineering) 4. GENETIC INTERCHANGE AND RECOMBINATION 4.1. TRANSFORMATION

18. 4. GENETIC INTERCHANGE AND RECOMBINATION 4.1. CONJUGATION

19. 4. GENETIC INTERCHANGE AND RECOMBINATION 4.1. CONJUGATION DNasa

20. 4. GENETIC INTERCHANGE AND RECOMBINATION 4.1. CONJUGATION Conjugative plasmids [e.g.: plasmid F (factor F)] F+ F- F+ F+

21. 4. GENETIC INTERCHANGE AND RECOMBINATION 4.1. CONJUGATION Integrationintothegenome (episomes) Plasmid F Chromosome Plasmid F‘ INtegrated plasmid F bbe

22. 4. GENETIC INTERCHANGE AND RECOMBINATION 4.1. CONJUGATION

23. 4. GENETIC INTERCHANGE AND RECOMBINATION 4.1. CONJUGATION

24. 4. GENETIC INTERCHANGE AND RECOMBINATION 4.1. CONJUGATION From Hfr to F- From F ‘ to F- 2 cells with F ‘ 2 recombinat cells

25. 4. GENETIC INTERCHANGE AND RECOMBINATION 4.4. TRANDSUCTION DNase

26. 4. GENETIC INTERCHANGE AND RECOMBINATION 4.4. TRANDSUCTION LYTIC vs LYSOGENIC CYCLES Virulent phages Always lysis Temperate phages Integration/Lysis

27. 4. GENETIC INTERCHANGE AND RECOMBINATION 4.4. TRANSDUCTION Defective phage GENERALIZED TRANSDUCTION Transduced cell

28. SPECIALIZED TRANSDUCTION

29. 4. GENETIC INTERCHANGE AND RECOMBINATION 4.4. TRANSDUCTION Gene Transfer Agents (GTAs)

30. 5. TRANSPOSABLE ELEMENTS DNA fragments that can move and integrate in a new genomic region (transposition) Insertion sequences Transposons Replicative transposons

31. 5. TRANSPOSABLE ELEMENTS CUT AND PASTE Genomic region A (with a mobile element) Genomic region B (without a mobile element) Transposable element Transposable element Genomic region A (without a mobile element) Genomic region B (with a mobile element) COPY AND PASTE Genomic region A (with a mobile element) Genomic region B (without a mobile element) Transposable element Transposable element Genomic region A (with a mobile element) Genomic region B (with a mobile element)

32. 5. TRANSPOSABLE ELEMENTS 5’ 3’ Gene expression inactivation 5’ 3’ Over-expression 5’ 3’ P No effect 3’ 5’

33. 6. GENETIC MANIPULATION OF MICROORGANISMS 6.1. GENE CLONING Restriction enzymes / Taq polimerase Restriction enzymes DNA ligases bb Transformation

34. 6. GENETIC MANIPULATION OF MICROORGANISMS 6.2. CLONING VECTORS BBE

35. 6. GENETIC MANIPULATION OF MICROORGANISMS 6.2. CLONING VECTORS

36. 6. GENETIC MANIPULATION OF MICROORGANISMS 6.3. METAGENOMICS