1 Chapter 9: Gene Transfer, Mutations, and Genome Evolution

2 Chapter Overview ● The mosaic nature of genomes ● Gene transfer: Transformation; conjugation; and transduction ● Genetic recombination ● Mutations: Types and causes ● Mechanisms of DNA repair ● Mobile genetic elements - Insertion sequences and transposons ● How genomes evolve

3 Introduction DNA sequences change over generations through various mutations, rearrangements, and inter- and intraspecies gene transfer. But what are the consequences of DNA plasticity? This chapter explores long-standing evolutionary questions and shows how microbial genomes continually change.

4 A surprise arising from bioinformatic studies is the mosaic nature of all microbial genomes. - For example, E. coli’s genome is rife with genomic islands, inversions, deletions, and paralogs and orthologs - This is the result of heavy horizontal gene transfer, recombinations, and a variety of mutagenic and DNA repair strategies. The Mosaic Nature of Genomes

5 In bacteria recombination occurs in a number of ways: Transformation: Free DNA is transferred Transduction: DNA transfer via a virus Conjugation: Cell-to-cell contact and a plasmid is involved. Recombination: Mechanisms of Genetic Transfer

6 Gene Transfer by Transformation Transformation is the process of importing free DNA into bacterial cells. - the cells need to be competent. Many cells are capable of natural transformation and naturally competent. -others require artificial manipulations. - Perturbing the membrane by chemical (CaCl 2 ) or electrical (electroporation) methods

7 Not all bacteria can take up free or naked DNA (<1%). Some microbes become competent sometime during their growth cycle Gene Transfer by Transformation

8 Natural Transformation occus  Bacillus sp., Haemophilus sp., Neisseria sp., Acinetobacter sp., Streptococcus sp., Pseudomonas sp. Gene Transfer by Transformation

9 Gram-positive bacteria transform DNA using a transformasome complex.

10 Gram-negative bacteria transform DNA without the use of competence factors (CF). some Gram negative organisms are always competent or they become competent when starved. also, they do not use transformasomes. most Gram-negative species is sequence- specific. Thus limiting gene exchange between genera

11 Conjugation (mating) Conjugation involves a cell-to-cell contact mediated by a special plasmid, conjugative plasmid Gram Negative: The plasmid carries genes that code for a sex-pilus Gram Positive: Sticky molecules help bind two cells together. Gram Negative Bacteria with conjugative plasmids are males and without it are females

12 Gene Transfer by Conjugation Conjugation is the transfer of DNA from one bacterium to another, following cell-to- cell contact by pilus on the donor cell. - The pilus attaches to the receptor on the recipient cell - Two cell fuse and single- stranded DNA passes from donor to recipient cell.

13 Conjugation requires the presence of special transferable plasmids (conjugative plasmids). A well-studied example in E. coli is the fertility factor (F factor). Also called fertility plasmid Conjugation begins with contact between the donor cell, called the F + cell, and a recipient F – cell.

14 Conjugation Female cells become male cells and be able to transfer the plasmid

15 Relaxosome: many genes necessary for DNA transfer (halicase, endonuclease, etc.

16 Conjugation The F-factor plasmid can integrate into the chromosome. - The cell is now designated Hfr, or high- frequency recombination strain.

17 Conjugation between an Hfr and F-, the recipient gets some of the Hfr genes plus some of the donor’s genes. The recipient becomes a recombinant F-, since not all Hfr genes are transferes. The entire chromosome take about 100 min to transfer as opposed only 5 min for free plasmid Conjugation Hfr + F-  Hfr + F-

18

19 An integrated F-factor can excise from the chromosome. - Aberrant excision results in an F′ factor or F′ plasmid, which carries chromosomal genes. Figure 9.5

20 Some bacteria can actually transfer genes across biological domains. Transfer of Genes into Eukaryotes - Agrobacterium tumefaciens, which causes crown gall disease - Contains a tumor-inducing plasmid (Ti) that can be transferred via conjugation to plant cells Figure 9.6

21 Gene Transfer by Transduction Transduction is the process in which bacteriophages carry host DNA from one cell to another. There are two basic types: - Generalized transduction: Can transfer any gene from a donor to a recipient cell - Specialized transduction: Can transfer only a few closely linked genes between cells

22 Generalized Transduction Any gene from a donor chromosome is packaged into a bacteriophage and transferred to a new cell upon infection. Salmonella enterica

23 Bacteriophases with a foreign DNA are called transducing particles. The transducing particles transfer any part of the host DNA to a new host (recipient) cells. Recombination occurs at low frequency  P1 phage of E.Coli. and P22 phage of Samonella are examples of generalized transduction. Steps of generalized transduction

24 Specialized Transduction

25 Specialized Transduction

26 The Phage genome is integrated into the host DNA at a specific site. On induction (UV light), the viral DNA separates from the host genome. Under rare events, the phage DNA maybe excised incorrectly. Some of the adjacent bacterial genes are excised along with the viral genome. When the phage infects new crop of cells, it allows transduction to occur at high frequency Specialized Transduction

27 Bacteria have developed a kind of “safe sex” approach to gene exchange. This protection system, called restriction and modification, involves: - Enzymatic cleavage (restriction) of alien DNA, by restriction endonucleases - Protective methylation (modification) of host DNA DNA Restriction and Modification

28 Figure 9.9

29 Recombination Two different DNA molecules in a cell can recombine by one of several mechanisms: - Generalized recombination requires that the two recombining molecules have a considerable stretch of homologous DNA sequences (>50 bp). - Site-specific recombination requires very little sequence homology between the recombining DNA molecules. - But it does require a short sequence recognized by the recombination enzyme

30 Homologus DNA Recombinants Crossing over Recombination

31 RecA proteins or Synaptases play critical role in recombination -double stranded DNA becomes single- stranded DNA by creating a nick -DNA unwinds -single-stranded binding proteins bind to the ssDNA -RecA finds homology and mediated strand invasion

32 A mutation is a heritable change in the DNA. Mutations can come in several different forms: Types of Mutations - Point mutation: Change in a single base - Insertion (addition) and deletion (subtraction) of one or more bases - Inversion: DNA is flipped in orientation - Reversion: DNA mutates back to original sequence

33 Mutations can be categorized into several information classes: - Silent mutation: Does not change the amino acid sequence DNA template  TTT point mutation  T TC DNA coding  AAA  AAG m-RNA  UUU  UUC Amino acid  Phenylalanine  Phenylalanine Though DNA strand has changed, the protein sequence is the same Mutations

34 Missense mutation: Changes the amino acid sequence to another

35 Nonsense mutation: Changes the amino acid sequence to a stop codon

36 Frame-shift mutation: Changes the open- reading frame of the gene

37 Mutation due to inversion in DNA strands

38 Spontaneous mutations are rare because of the efficiency of DNA proofreading and repair pathways. However, they can arise for many reasons: 1)Tautomeric shifts in DNA bases that alter base- pairing properties [ G  T or A  C] 2) Oxidative deamination of bases Mutations Arise in Diverse Ways

39 3) Formation of apurinic sites [loss of purine]

40 Mutations can be caused by mutagens: Chemical agents - Base analogs - Base modifiers - Intercalators Electromagnetic radiation - X-rays and gamma rays: Break the DNA - Ultraviolet rays: Form pyrimidine dimers Mutations Arise in Diverse Ways

41 Mutagenic agents and their effects.

42 UV radiation can induce dimerization