De novo creation of new genes 1.Retrotransposition (+/- cooption of other sequences) AAAAA Pre-mRNA AAAAA Splicing to remove intron Reverse transcription by TE polymerases (in CYTOSOL) Integration into the genome (in NUCLEUS) Often see short flanking repeats due to mechanism of TE integration 1

De novo creation of new genes 1.Retrotransposition (+/- cooption of other sequences) AAAAA Pre-mRNA AAAAA Splicing to remove intron Reverse transcription by TE polymerases (in CYTOSOL) Integration into the genome (in NUCLEUS) Often see short flanking repeats due to mechanism of TE integration 2

De novo creation of new genes 1.Retrotransposition (+/- cooption of other sequences) 2.Gene duplication into other sequences = chimeric structure/regulation 3

De novo creation of new genes 1.Retrotransposition (+/- cooption of other sequences) 2.Gene duplication into other sequences = chimeric structure/regulation 3.Cooption of non-coding DNA (from introns, intergenic sequence) 4

De novo creation of new genes Challenge in distinguishing Novel Gene vs. missed orthology due to rapid evolution 1.Retrotransposition (+/- cooption of other sequences) 2.Gene duplication into other sequences = chimeric structure/regulation 3.Cooption of non-coding DNA (from introns, intergenic sequence) 4.Horizontal gene transfer (very prevalent in bacteria) - also observed from bacterial parasites to insect hosts 5

Horizontal (or Lateral) Gene Transfer 6 Vertical Transfer (e.g. along species tree) Horizontal Transfer

7 Mechanisms of HGT Steps 1-3: DNA Transfer Step 4: Persistence (replication) in Recipient Step 5: Selection to maintain sequence From Thomas & Nielsen. Nat Rev Microbiol. 2005

8 Mechanisms of HGT: DNA Transfer A. Transformation: direct uptake of naked DNA Donor and recipient do NOT need to co-exist in the same time/space Can occur across distantly related species Efficiency depends on ‘competency’ of recipient Some species readily take up DNA Other species have transient (e.g. stress/starvation) competency B. Transduction via bacteriophages Phage can package random or adjacent donor DNA DNA size limited by capsid packaging (but still can be 100 kb) Recipient must be able to take up phage (through specific receptors, etc)

9 Mechanisms of HGT: DNA Transfer C.Conjugation: direct connection between two bacteria Species need to co-exist in the same environment Need pairs of species that can conjugate DNA transferred as mobile element or plasmid

10 Mobile (Transposable) Elements & Bacteriophages are a major force of HGT Transposase Antibiotic resistance genes IR (inverted repeat) IR (inverted repeat) Some mobile elements excise and reintegrate, others are replicative. Some integrate at specific sites (“att” sites) & often adjacent to tRNAs. Many can excise or replicate neighboring DNA Many triggered to move upon environmental stress

11 Mechanisms of HGT: DNA Stabilization Transferred DNA needs to replicate & get passed on Episomal replication (e.g. plasmid) Integration along with phage genome or mobile element Homologous recombination Non-homologous (“illegitimate”) recombination Benefit of transferred DNA needs to outweigh its cost Burden of extra DNA and/or protein synthesis Famous cases of HGT involve antibiotic resistance or pathogenicity New DNA needs to be expressed to provide beneficial functions

12 Question: How does the prevalence of operons in bacteria influence evolution by Horizontal Gene Transfer? Having suites of functionally related genes linked and co-expressed = easy to transfer whole pathways

13 From Juhas et al FEMS Micro Genomic Islands: families of horizontally transferred genes Often near tRNA Often contain own mobility genes & sequences Evolve through gene acquisition & loss

14 From Juhas et al FEMS Micro Grey = sequence homology around 4 genomic islands (2 related to pathogenicity and 2 related to environmental responses); black = Genomic Islands

15 Detecting HGT sequences 1.Often have unusual sequence characteristics (GC content, codon usage, di-nt frequencies) compared to the rest of the genome Signatures of other genomes speckled in the host. 2.Often flanked by repeat elements (from phage or mobile element insertion) or tRNAs (since integration often near tRNAs) 3.Gene tree is very different from the species tree 1.These days, easily detected by sequencing many isolates of the same ‘species’ and detecting variable gene sequences

16 From Tenaillon et al. Nat Revs Micro 2010

17 From Keeling & Palmer Nat Rev Genetics 2008 Effects of HGT on Gene Trees

Best evidence for HGT: sequencing of many strains of the same ‘species’ … but What is a bacterial species?? No sex, lots of HGT across species … the idea of the Pan Genome: the total gene pool represented within a ‘species’ Core Genome: genes common to ALL isolates of a given species Accessory Genome: variable parts found in subsets of isolates

Bacterial Pan Genomes In study of 8 E. coli genomes: Only 40% of the Pan Genome was made up of the Core Genes But extrapolation suggests many more accessory genes in E. coli (but not all species … why?) From Mira et al Internat. Micro

Mobile elements more prominent for some species Some species more readily take up DNA; others do not do homologous recombination well Some species occupy very narrow niche – little exposure to other DNA, etc Bacterial Pan Genomes In study of 8 E. coli genomes: Only 40% of the Pan Genome was made up of the Core Genes But extrapolation suggests many more accessory genes in E. coli (but not all species … why?)

Different genes enriched in the Core vs. Accessory Genomes Core Genomes: ‘Housekeeping’ functions Accessory Genomes: * Environmental genes * Poorly characterized genes * Orphan genes (no homology to any known gene) * More mobile elements, phage sequences, repeats Orphan genes: Considerably shorter than normal genes Some are fragments of other genes Some may be non-functional May original from poorly sampled world of phage genes

Metagenomics: uncovering the world of new bacterial/phage genes Metagenomics: sequencing the entire pool of DNA found in environmental sample * Done without cloning or culturing (most bacteria cannot be cultured!) * Computational methods of linking sequence back to particular species * Work to try to assemble genomes * Most analysis to date done on pools of sequences, not genomes assembled from those sequences

23 Ed DeLong: 3:30 pm Thursday, February 12: Microbial Sciences Seminar Series