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Summary Ecological & Evolutionary Genomics Research

Contents Genetic Variation ................................................................................................................................ 2

Mobilome (MGEs) ........................................................................................................................... 2

Horizontal Gene Transfer ................................................................................................................ 2

Vertical Genetic Change .................................................................................................................. 3

DNA transcription ............................................................................................................................... 4

Regulation ....................................................................................................................................... 4

Gene finding .................................................................................................................................... 6

Detecting HGT ................................................................................................................................. 6

Microbial processes ............................................................................................................................ 7

Environmental sensing .................................................................................................................... 7

Secretion systems ........................................................................................................................... 7

Membrane fluidity .......................................................................................................................... 8

Energy and Carbon. ......................................................................................................................... 9

Winogradsky ................................................................................................................................. 10

Agrobacterium tumefaciens .......................................................................................................... 11

Extremophiles ............................................................................................................................... 11

Reproductive success .................................................................................................................... 12

Mutability and starvation stress ................................................................................................... 12

Antibiotic resistance ...................................................................................................................... 13

Methods. ........................................................................................................................................... 14

Culture ........................................................................................................................................... 14

DGGE ............................................................................................................................................. 14

T-‐RFLP ............................................................................................................................................ 14

SIP .................................................................................................................................................. 14

Lezen:

-‐ Assembly olsen -‐ Brock

Print / e-‐reader -‐ Diversity of plants and micro-‐organisms -‐ biostatistiek

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Genetic Variation (EEGR evolutionary thinking(1)) Variation -‐> selection -‐> adaptation -‐> evolution (a change over time in the genetic composition of a population)

1. Mutation 2. Gene Flow 3. Nonrandom mating 4. Genetic Drift 5. Selection

(individual variation which is heritable, more individuals born than reproduce, some variants reproduce more than others) (Coll-‐EEGR2015-‐HGT) oriT Origing of Transfer start of replication for transfer mob mobilization mobilization function, relaxase: T4SS rep replication replicates DNA sequence tra transfer transfers DNA sequences vir virulent – transfer transfers DNA sequence int integration DNA integration / capture & expression rec recombination -‐ integration

Mobilome (MGEs) (Coll-‐EEGR2015-‐HGT) Plasmids – circular, self-‐replicating. oriT/mob, rep, tra pIPo2 = sampling plasmid. Mobilises other MGEs Transposons – can change their chromosomal localization. Insertion sequence (IS) elements – subset of transposons, small DNA sequence < 2,5 kb. Out to in: WXY short direct repeats, Inverted Repeats, transposase + integrase. Composite – transposase with extra gene Tn3-‐type / replicative – resolvase with transposase Transposable phage – replication and integration Conjugative transposons – Transposons able to transfer between cells via conjugation. oriT/mob, tra, int Bacteriophages – Virus that can integrate DNA (morons) in bacterial genome: prophages. Integrons – Integrase with extra gene without promoter region. Genomic Islands – Large chromosomal regions with cluster of functionally related genes.

Horizontal Gene Transfer Transformation Bacterium dies à transfer of free DNA through transformase à used for reparations and novel genes. Energy expensive.

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Transduction Transfer by viral delivery. Moron from bacteriophage à DNA integrated into bacteria = prophage. Defense against attack

-‐ Permissive, modifying, restrictive -‐ CRISPRs – clustered regularly interspaced short palindromic repeats. Adaptation, maturation,

interference. Cas + crDNA cleaves morons. Conjugation Type 4 (VIR) secretion. T-‐DNA from plasmid or conjugative transposon on relaxase à docks at virD4 à Relaxosome. Energy expensive.

Vertical Genetic Change (Coll-‐EEGR2015-‐Survfittest) (EEGR2015 genome & chromosome evolution) Point mutations -‐ random Replication Errors Repair Errors Asexual Recombination Duplications (multigene families) Deletions Unequal crossing over Replication slippage Slip-‐strand misrepair Transposition Polyploidisation Change in hierarchical position of genes Change in molecular functions of genes Change in transcriptional and/or translational regulation (maternal imprinting = silencing genes by methylation)

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DNA transcription

Regulation • nucleosomes • chromatin modifiers / remoddelers • insulators • cross-‐talk • combination of promoters • splicing • Enhancers

Nucleosomes DNA is wrapped around histone octamers: nucleosomes. Nucleosomes can block transcription sites (‘covered’) and therefore reduce transcription. Chromatin modifiers and remodellers are needed to alter the chromatin structure to allow access of transcription factors to the promoter region. This is how gene transcription can be regulated. Euchromatin = loosely packed, and can be transcribed Heterochromatin = more tightly packed

a. Constitutive = usually repetitive and forms structural domain such as telomeres and centromeres.

b. Facultative = silenced by eg. Histone de-‐acetylation. Genes with open promoters have a large nucleosome depleted region (NDR) upstream from the Transcription Start Site (TSS) with key cis-‐regulatory sequences. It also contains DNA sequences that resist bending (polydA:dT) to deter nucleosome formation and stability. This promotes gene expression. Regulated genes have covered promoters where nucleosomes cover the TSS, the regions flanking the TSS and most of the binding sites for transcriptional activators. These sites have nucleosome positioning sequences (NPS) which can bend more easily (AA/TT repeats) to cover the binding sites for transcription factor. However, at least one binding site is typically exposed in the linker DNA between nucleosomes, which allows a pioneer transcription factor access to the promoter. Before transcription can occur modifiers and remodelers have to be recruited (two-‐step model for activation). Chromatin modifiers: Histone acetylation = lysine in the N-‐terminal of the histone proteins = less affinity to DNA = gene activity Histone de-‐acetylation = gene silencing

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Acetylation controlled by HATs; Histone AceltylTransferases Phosphorylation Methylation Ubiquitination Modification by HMGN proteins Insulators:

• regulatory sequence which protects the DNA to become packed in nucleosomes. • keep the integrity of the region intact (insulate promoters of one region from activating a

genen in the next region). Cross-‐talk Loops of chromatin can communicate 'cross-‐talk' Combination of Promoters can also influence gene regulation (tissue specificity) Splicing -‐ exact mechanism not clear. Exon skipping. Cryptic Splice Site Selection. Spliceosome. Enhancers can be tissue specific. Enhancer can be put on or off by right location of insulator. Transcription factors (activators) bind to the promoter DNA (enhancer / cis-‐regulatory element) If the transcription factor is a repressor, it binds to a silencer. RNA polymerase is recruited; binds to the transcription factor complex Produces primary transcript RNA from template strand à pre-‐mRNA Splicing à mRNA

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Gene finding

Promoters + enhancers = cis-‐regulatory elements TATA-‐box (TATAA) TBP unwinds DNA TFII forms transcription factor complex Initiator element (Inr) (YYANWYY)1 TFII BRE (G/C G/C G/A CGCC) TFIIB GC-‐box CAAT-‐box (GGCCAATCT) RNA transcription factor Response elements: binds condition/stress specific transcription factors

DNA: Start protein transcription: ATG Start / end extron: no specific codon Start intron: GT End intron: AG End protein transcription: TAA, TAG, TGA PolyA: AATAAA

Detecting HGT 1) Phylogenetic incongruence 2) Unexpected similarity in unrelated species 3) Atypical G+C% in region/genomic island compared to genome (near tRNA, integrase) 4) Atypical codon usage (base at 3rd position)

1 In nucleic acid notation for DNA, Y (pYrimidine) stands for C/T (cytosine or thymine, which are both pyrimidines), N (Nucleobase) is any of the four bases, and W (Weak) stands for A/T (adenine or thymine, which both form only two hydrogen bonds)

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Microbial processes

Environmental sensing (Coll-‐EEGR-‐2015-‐I-‐final) Effector molecule à cytoplasmic protein à signal transduction à sensor molecule à changed physiology.

A. Two-‐component regulatory systems. Effector molecule enters sensor kinase protein in membrane àphosphorylation at his-‐residue of sensor kinase prot by histidine kinase àP group transferred to response regulator (RR) à phosphorylated RR affects physiology, either repressor or activator.

a. Histidine kinase is highly conserved and thus easily identified in whole genome sequences.

B. Quorum sensing. Cells produce auto-‐inducers (AIs) à concentration above threshold à mass induction of cellular genes

a. Most important AI: acyl homoserine lactones (AHLs) b. Vibrio fischeri (bobtail squid) AI: 3-‐oxo-‐C6-‐HSL à binds to LuxR protein à induced

transcription of lux genes. c. Biofilms: increased concentration easily (polysaccharides).

Secretion systems Type 3 Secretion System (TTSS): excretion of effector proteins, energetically costly: ‘injectisome’ Type 4 Secretion System (VIR): excretion of effector proteins ánd DNA, secretion pump; energetically costly; secretion of virulence factors. (Pilus: conjugation). Conjugation: Relaxase / relaxosome, virD4

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Membrane fluidity Maintenance of membrane fluidity = homoviscous adaptation.

Fluid Not tightly packed

Stiff Tightly packed

Higher temperature Lower temperature Unsaturated Fatty Acids Saturated Fatty Acids Cis – unsaturated FAs Trans – unsaturated FAs Shorter chains (16 = palmitic) Longer chains (18 = cis-‐vaccenic acid)

Cold: Hik33 & DesK sense decreased fluidity à two-‐component regulatory system à Rer1 and DesR are response regulators and regulate expression of fatty acid desaturases.

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Energy and Carbon. (Coll-‐EEGR2015-‐II-‐III) CO2 Organic Carbon Photo Photosynthesis Aerobic Bacterial photosynthesis Photoheterotroof Anaerobic Chemo Lithotroof

Consumer Aerobic

Organotroof Anaerobic

Methanogen CO2 à CH4 Homoacetogens CO2 à acetaat Sulfate reducers SO42-‐ à H2S Sulfur reducers S0 à H2S Denitrifiers NO3-‐ à N2 Ferric iron reducers à Fe3+ à Fe2+ Fermenters

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Winogradsky Column with lake water, mud, cellulose, sodium sulfate, calcium carbonate. Light from above.

-‐ Sheathed bacteria -‐ organotrophs -‐ Cyanobacteria -‐ Purple non-‐S bacteria -‐ Purple S bacteria -‐ Green S bacteria -‐ Desulfovibrio (S. reducer) -‐ Clostridium (Fermenter)

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Agrobacterium tumefaciens Causes Crown Gall. The bacterium transfers part of its DNA to the plant (VIR secretion system), and it integrates into the plant’s genome. This causes the production of tumors and changes in metabolism. There is effective biological control for this disease. Can be used to artificially insert desirable genes into a plant. Ti (tumor inducing) plasmid. Plant wound: chemotaxis to sugars and phenolic compounds like acetosyringone. Recognized with the two-‐component regulatory system (VirA/VirG). This last compound also activated the Vir genes which coordinate the infection process. Permeases are produced and put into the cell membrane for opine uptake (will be produces by the plant tumor). Endonucleases (restriction enzymes) are produced to cut T-‐DNA (transferred DNA). T-‐DNA enters the plant through the wound (mediated by cytokinins? = plant hormone) and integrates with its chromosomes. Now the plant will produce cytokinins, indoleacetic acid and new plant metabolites (opines and agrocinopines). Opines (amino acid derivatives) and agrocinopines (phosphorylated sugar derivatives) are a unique food source and provide both carbon and energy. Radiobacter strain K84 prevents disease caused by Agrobacter which produce nopaline. It produces agrocin 84 (a bacteriocin) which is coded for by pAgK84 on the plasmid. It is a fraudulent adenine-‐type nucleotide with two sugar derivatives attached to it.The agrocinopine permease recognizes the agrocin 84 for uptake. It then blocks DNA synthesis.

Extremophiles (Coll-‐EEGR2015-‐Extremorph) Thermophiles.

A) Protein folding – high temp denatures proteins. a. Compact folding to exclude H2O b. Less glycine to increase stability c. Chaperonin to stabilize d. Refolding of denatured proteins by thermosome

B) DNA a. Reverse DNA gyrase – catalyses more compact folding of DNA (double helix,

supercoiling) b. DNA binding proteins – stabilize folding c. High G+C (higher melting temp)

C) Membrane a. Tetraether lipids in Archaea – four ether bonds. Stability and limited permeability for

protons to maintain a viable proton motive force. Halophiles. Pigments: rhodopsins / bacterioruberins. Beta-‐carotenes.

A) Compatible solutes (osmolytes) to counter external osmotic pressure. Costs E. B) Salt-‐in strategy.

a. 10x more E efficient than osmolytes b. Proteins have to be adapted to higher salt concentration. à more Aspartic acid (E)

and Glutamic acid (D) which have lower isoelectric points.

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Reproductive success (Coll-‐EEGR2015-‐Survfittest) Fitness = efficiency & rate of metabolism = flux Sout à enzyme 1 à Sin à enzyme 2 à product V = k1 [E] [S] k1 = kcat / Km Increase fitness by

1) Increase [E] Protein production/degradation a. Eg porin pores (passive) b. Lac permease (active) c. Beta-‐Galactosidase (active)

2) Increase [S] uptake 3) Increase k1 amino acid sequence of the enzyme

Fitness w = 1 + selection coefficient

Mutability and starvation stress (CollEEGR-‐2015-‐survfittest / Starv) Starvation in stationary phase. Newest populations have mutants that can survive better under the starvation conditions than the older populations (selective sweeps). These are called GASP mutants (Growth Advantage in Stationary Phase). Under starvation they get mutations in rpoS (sigma factor for gene expression under stress) or other regulatory genes. This is called the stress or SOS reponse. GASP

• Stress induced hypermutation o starvation causes DNA strands to break: poly(P) o starvation causes release of ppGpp (alarmone) which activates poly(P)

• Single stranded DNA / poly(P) activates RecA o Activated RecA starts recombination o Activated RecA represses LexA.

§ LexA unblocks SOS / stress response § umuDC is transcribed § umuDC causes mutagenesis: allows replication without repairing damage but

in stead the insertion of random bases. § Mutability is enhanced.

• Mutation in rpoS / poly(P) activates rpoS o Decreased sigmaS activity o Enhanced uptake of amino acids o Decreased MutHLS activity -‐-‐> decreased mismatch repair = increased mutation rate o Other controlled genes: OsmB, osmotic stress; katE, oxidative stress; otsBA,

thermotolerance.

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Antibiotic resistance (Coll-‐EEGR2015-‐Antibiotres)

• Target site modification • Reduced cell permeability / uptake • Metabolic bypass • Multidrug efflux pumps • Antibiotic modification, phosphorylation, adenylation, acetylation, ring cleavage

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Methods. Determining microbial community diversity, DNA sequences, RNA, proteins, etc.

Culture (Coll-‐EEGR2015-‐II-‐III) Great Plate Count Anomaly.

DGGE (Coll-‐EEGR2015-‐II-‐III) Denaturing Gradient Gel Electrophoresis. 16S RNA. Increasing concentration of denaturant or increasing temperature. Determine % GC / AT AT – not strong – low melting temp – melts quickly GC – strong bond – high melting temp – melts slowly Richness, Evenness, Community Composition. Only numerically abundant populations. No information about function. No distinguishing between active and non-‐active cells.

T-‐RFLP (Coll-‐EEGR2015-‐II-‐III) Terminal Restriction Fragment Length Polymorphisms. 16S RNA. Analysis of fluorescently labeled terminal restriction fragments. Determine fragment abundance. Only numerically abundant populations. No information about function. No distinguishing between active and non-‐active cells. Less sensitive than DGGE due to shared terminal restriction sites. Sequencing

SIP (Coll-‐EEGR2015-‐II-‐III) Stable Isotopes (Biomarker). Natural vs. added. (12CO2 : 13CO2 = 19:1). C-‐fixing enzyme prefers 12CO2. Delta13C small = biological origin, big = heavy, geological origin. Antibodies

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Definitions. ABC DEFG HIJ KLMNOP QRST UVW XYZ 2D-‐gel electrophoresis proteomics; rotate gel; two different separation factors. Adapter ‘handvat’ known sequence ligated to fragmented DNA Allele Amensalism A is limited by B (eg toxic product, pH) Analogous genes Antagonistic co-‐evolution genes that increase fitness of males decrease fitness of females and vice versa. Antibodies Assembly assembling sequenced DNA fragments to obtain a de novo genome Associates See Partners. Base-‐pair BRE B recognition element, promoter sequence, upstream of TATA-‐box, 7 nucleotides. Bruijn graph all possible ways of ordering k-‐mers CAAT box Core promoter, upstream from the initial transcription site (27-‐100 bp), essential for good quality of transcription. Chromatin = nucleosome (DNA + histone) cDNA complementary? CREs cis-‐Regulatory elements; regions of non-‐coding DNA which regulate the transcription of nearby genes CRISPRs acquired immune system of bacterial cells Cloning growing DNA in bacterial cell Co-‐activator Co-‐repressor Coding strand ‘primary strand’, same as RNA (except T = U) Comparative genomics examines whole genome structure, gene arrangement and rearrangement Compensatory mutation resistance decreases fitness à mutation in other aspect to increase fitness à fitness same again, and resistance is still present. Conformation folding of proteins Constitutive heterochromatin Usually repetitive and forms structural domain such as telomeres and centromeres. Contigs overlapping DNA fragments added up (with gaps) Conserved regions Core promoter includes TSS, binding site for RNA polymerase and general transcription factor binding sites. Coverage Crystalography determine protein structure Deletions Domain Dominant population Central role in the functioning of the community Ecotypes Microbes of the same species but adapted for a specific environment

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EEG Ecological & Evolutionary Genomics; responses at genomic level to environmental variation through time and space in order to understand the structure and function of biodiversity at the individual, population and community level. Enhancer short DNA sequence (50-‐1500bp) that can bind with activators to activate the transcription of genes (usually cis-‐acting). Epistasis an effect of the interaction between two or more gene loci on the phenotype of fitness, whereby the joint effect differs from the sum of loci taken together. eQTL pinpoint genomic regions that control the expression of genes of interest. Euchromatin Loosely packed chromatin, and can be transcribed Exon Exon shuffling Facultative heterochromatin Silenced by eg. Histone deacetylation. Functional genomics the development of genome-‐wide or gene-‐related experimental approaches to assess the biological and biochemical roles of open reading frames of unknown function; to provide insight into multi-‐ gene, complex regulation. GASP mutant Growth Advantage in Stationary Phase Gene Gene fusions Genetic mutants method in functional genomics; manipulation of expression of individual genes in vivo using knock out, RNAi. Genetics analysis of one gene Genetic Network how genes are related: co-‐expression Genome complete set of genetic material of an organism Genomic conflict different genes affecting the same trait experience opposite selection pressures Genomics large-‐scale analysis of genomic architecture by mapping and sequencing Guild Microbes that are metabolically similar ‘functionele groep’ GWAS Genome Wide Association Studies; associate sequence variations to phenotypic variations. Helicase Unwind DNA Heterochromatin More tightly packed chromatin. Constitutive and Facultative. Homologous genes Homoviscous adaptation Maintenance of membrane fluidity Horizontal Gene Transfer Hygroscopy is the ability of a substance to attract and hold water molecules from the surrounding environment Incidentals Organisms without significant ecological interactions within the community.

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Inducer Infectious speciation reproductive isolation resulting from infection with microorganisms (Wolbachia) Initiator element Inr, does not occur together with the TATA-‐box usually. Core promoter. Insertions Insulator genetic boundary DNA element that blocks the interaction between enhancers and promoters. It is thought that an insulator must reside between the enhancer and promoter to inhibit their subsequent interactions. Insulators therefore determine the set of genes an enhancer can influence Intron K-‐mer all possible length sequences of a read L-‐50 Lateral Gene Transfer Horizontal Gene Transfer Ligation ‘Plakken’ Meiotic Drive an allele is represented in more than 50% of the gametes of a heterozygote Metabolome All low-‐molecular-‐weight (LMW) molecules (end products) Metagenomics treats the entire ecosystem as a single living organism with its own unique DNA. It examines community gene expression in order to understand ecosystem function. MGE Mobile Genetic Element Micro-‐array describe mRNAs in biological sample Mobilome Addition of all mobile genetic elements. Moron Genes uniquely carried by bacteriophages. N-‐50 Niche A position in n-‐dimensional space formed by different environmental factors and resources which are needed for a species to maintain viability. Non-‐coding DNA Northern Blot Transcriptome, RNA Nucleotide Nucleosomes DNA wrapped around histone octamers. Operon operon is a functioning unit of genomic DNA containing a cluster of genes under the control of a single promoter. ORF Open Reading Frame Partners Fixed components of a community, which do not have a great effect on it. Same as associates. PCR PE Paired End Phylogenomics reconstruct evolutionary history at the full or partial genome level; to identify differences between genomes of species; also for gene families and domains. Pleiotropy

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Pre-‐mRNA Promoter initiates DNA transcription, 100-‐1000 bp long Proteome all proteins, including post-‐translational modifications qPCR quantitative PCR, each cycle records amount of product (specific gene transcript) – use the graph to determine RNA abundance. Read sequenced DNA fragment Resolvase together with transposase makes a replicative transposon (Tn3 type) Response elements short sequences of DNA within a gene promoter region that are able to bind specific transcription factors and regulate transcription of genes. Under conditions of stress, a transcription activator protein binds to the response element and stimulates transcription rDNA repeated DNA sequences / ribosomal DNA?? Regulatory elements Restriction enzyme ‘Knipt’ RILs Recombinant Inbred Lines, to maximize homozygosity RNAi RNA polymerase produces the primary transcript RNA RNAseq sequencing RNA Scaffold added contigs with known gap length Selfish genetic element gene that enhances its own transmissive relative to the host genome, and is either neutral or detrimental to the host Sex ratio distorter segregation distorter that effects the sex of an individual -‐> driving X or Y chromosome Silencer DNA sequence that binds to repressor transcription factors: RNA polymerase can’t bind to the promoter region. SNP Single Nucleotide Polymorphism Southern Blot DNA Structural genomics see ‘Comparative Genomics’ TATA-‐box core promoter region, usually 25 bp upstream from the transcription start site TBP TATA Binding Protein; unwinds DNA and binds it through 80o. It binds on the minor groove with a beta sheet. Transcription factor Template strand RNA polymerase adds complementary RNA nucleotides to the template strand. Terminator TFIID transcription factor Transcription a segment of DNA is copied into RNA by the enzyme RNA polymerase Transcription factor protein that binds to the DNA and recruits (activator) or blocks (repressor) RNA polymerase. General definition for proteins involved in DNA transcription and bind to DNA Transcriptome mRNA, transcription level of all genes Translation Transposon TSS Transcription Start Site UTR untranslated regions

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Western Blot Proteins

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