Prokaryotic Genetics Lecture Notes PDF

3-2: Prokaryotic Genetics Lecture Overview: • Overview of some basics of prokaryotic genetics: key genetics terms, mutations/mutants, genotypes & phenotypes, isolating mutants in the lab, types of mutations. • Horizontal gene transfer – recombination & transposition. Transformation, transduction & c...

3-2: Prokaryotic Genetics Lecture Overview: • Overview of some basics of prokaryotic genetics: key genetics terms, mutations/mutants, genotypes & phenotypes, isolating mutants in the lab, types of mutations. • Horizontal gene transfer – recombination & transposition. Transformation, transduction & conjugation • Textbook: Chapter 9 How do asexual microbes evolve/adapt efficiently? o As humans, we generate new genetic diversity every generation using sexual replication o Prokaryotes do not reproduce sexually. Simple binary fission produces genetically identical offspring o Yet prokaryotes are the masters of adaptation and rapid evolution…how is this possible? o Prokaryotes are not genetically stagnant! They mutate, exchange genes, etc. Tie (father) & Max (son) Domi. It’s hard to tell, but sexual reproduction did result in a son who is different than his father. Gene names and protein names aa ~ somethingt o Gene names, by convention are 4 letters. First 3 letters describe function – 4th letter designates a specific gene. ↳ numbering System o Example: btuC = B Twelve (B12) Uptake, gene C. There are also btuB, btuD, btuF genes o Gene names are italicized – first three letters lower case, end with upper case letter (btuC) I o Protein names are the same, but start with an upper-case letter and are NOT italicized (BtuC). 2 numbers generally use o Eukaryotic naming conventions vary, sometimes from species to species. frequently tested ~ ↳ We Will not - go into Example: “…which allows RpoS to bind to the promoter region to stimulate purF expression.” -- Talking about the RpoS protein, but the purF gene. Some key genetics terms Mutation - A heritable change in the DNA sequence of a genome. Includes substitution mutations, insertions, deletions – any change. Mutant (mutant strain): An organism whose genome carries a mutation Wild-type strain: – Strain isolated from nature and/or one being used as the parental strain in a genetic study. The term “wild-type” can also be applied to a single gene Genotype: The complete genetic makeup of an organism Phenotype: An observable characteristic of an organism. There can be many different sorts of phenotypes: metabolic, virulence, morphology, etc. In prokaryotic genetics, we often seek to make mutations. The resulting mutant strains have a different genotype compared to the wild-type (WT) strain. It is then common to test to see if the wildtype and mutant strains have different phenotypes. Wild-type and mutant strains - example Genomic locus (plural = loci): a specific position on the chromosome o Shown below are two genomic loci encoding genes involved in maltose fermentation (malK, lamB, malM, malQ) in E. coli o Two mutant strains shown: M1 features deletion of lamB, M2 features mutation in malQ. Both mutations result in loss of maltose fermentation o Indicator plates that detect maltose fermentation (purple) show this loss-of-function phenotype in these mutant strains Textbook - Fig 9.2 Mutant and phenotype names o In classic bacterial genetics, mutations named by adding numbers to gene name: hisC1, hisC2 – first and second hisC mutations isolated. o This is done far less now, since mutations are generally sequenced and so naming using the specific mutation is more common. o Mapped mutations can be described using nucleotide or amino numbers. Convention: WT base or amino acid, then number, then mutant base or amino acid. E.g. HisC (A77K) – residue 77 mutated from an weta alanine (A) to a lysine (K). What itMGaG - Un to Undescribe natureof that mutant o Deletion mutations shown using the delta (Δ) symbol (e.g. ΔbtuC) o Phenotype names have three letter (first letter = capital) designations and strains are shown with a plus (+) or a minus (–) for that phenotype: o E.g. His+ strain can make histidine. His- strain is a histidine auxotroph – can’t make histidine. ↳ need something CANNOT yourself BUT make it we Auxotroph Types of point mutations Mutations can be spontaneous (naturally-occurring mistakes) or induced (E.g. using mutagenic chemicals or UV to damage DNA) Point mutations (mutations to a single base pair) within a protein-coding sequence can be either: ~Tyrasine ↳ Some for are more coded than 1 sequence 1) Silent mutations: do not change amino acid sequence, different codon, same amino acid 2) Missense mutations (most common): lead to a change in that amino acid to a specific different amino acid gets ~ which a4 enuded 3) Nonsense mutations: lead to a change in that amino acid to a stop codon, leading to a premature end to the protein sequence (truncation) ↳very damagins : I disrupts stops function Textbook - Fig 9.5 Insertion or deletion mutations o Other mutations are not simple substitutions from one base pair to another, but instead result in DNA being added or lost. o Deletion mutations (DNA lost) and insertion mutations (DNA added to a specific location) can be as small as a single bp or can be as large as thousands of bp. o Deletions/insertions within protein coding regions often result in a frameshift mutation Frameshift mutations – Textbook Fig 9.5 Because proteins are encoded by 3 nucleotide codons, adding/losing 1 bp (for example), shifts all downstream codons and scrambles the downstream sequence. Frameshift mutations are typically highly disruptive. Reversions & suppressors o Genetic reversion: Mutant strain has a specific a mutation. This strain then acquires a mutation that changes it back to the original wild-type sequence, so strain is now “WT” again. o Phenotypic reversion: Mutant strain has a certain phenotype. It then acquires another mutation to “revert” back to the wild-type phenotype. Might be a genetic reversion, might be some other mutation. o Suppressor mutations: Cause phenotypic reversions. Mutations that compensate for the effects of a prior mutation – return the phenotype of the strain partially or fully back to WT. o NOT genetic reversions – different genetic change “suppresses” phenotype of original mutation. Mutation often (but not always) to a different gene – “fixes problem” that was created by initial mutation. Isolating mutants: Selection o Identifying mutants at the heart of our ability to study microbial cells/processes. How does process X work? Find mutants that can’t do process X – study the genes/proteins containing those mutations. o Can induce mutations in different ways (e.g. mutagens, transposons) o In some instances, mutants can be isolated by selection – mutant grows, parent doesn’t (or grows significantly worse). E.g. antibiotic resistance. o Selection is highly efficient – can identify single mutant with a desired phenotype out of millions (or more) of cells Textbook Fig 9.3 White arrow shows a mutant colony that grows within the inhibition zone on an antibiotic disc assay. Appears to represent an antibiotic-resistant mutant strain. Screening for absence of growth o It is easier to identify mutants that grow better than parent by selection o Using replica plating (plating the same colony on two different plates – under two different conditions), you can identify mutants that grow worse than parent (or not at all) o E.g. – auxotrophs (see fig below) or antibiotic sensitive strains Textbook - Fig 9.4 Screening: Indicator plates Signe - o In some instances, you can link a phenotype to a change in colony appearance or to a detectable signal (colour, fluorescence, luminescence) o Mutants with a desired phenotype can then be isolated by detecting single colonies the do (or do not) produce that signal o The enzyme β-galactosidase (lacZ gene) is commonly used as a tool to identify colonies with altered gene expression in bacteria. o β-galactosidase substrates such as X-gal can be added to plates that produce a signal (blue) when modified by this enzyme o By placing lacZ under the control of a promoter you want to study, you can seek mutants that can “turn on” or “turn off ” this promoter Whatfactur e er - “Blue-white screening” How do bacterial genomes change (evolve over time) o Natural mutation rate ~ 10-6 – 10-7 per 1000 bp per round of replication for prokaryotes. DNA polymerase errors when copying genomic DNA o Average gene ~1000 bp, so each round of replication will introduce a mutation to a given gene in one out of every ~1-10 million cells. o If you have large enough numbers of bacteria in a population, you will likely have some genetic diversity in each gene (even though most genomes are identical or almost identical). o Acquiring mutations plays a major role in prokaryotic evolution o Horizontal gene transfer – acquiring new genetic material from (foreign DNA) from the environment plays an even bigger role Gene transfer in Bacteria (& Archaea) Foreign DNA can enter a prokaryotic cell in 3 major ways: 1) Transformation 2) Transduction 3) Conjugation Once inside the cell, this DNA can: 1) Be degraded/lost 2) Replicate as a separate entity (plasmids, phage) 3) Be integrated into the chromosome (recombination, transposition) How foreign DNA can be integrated into host genome • Genetic recombination (we’ll focus on homologous recombination, but there are other types) • Transposition Genetic recombination o Genetic recombination: Physical exchange of DNA between genetic elements. One important type is homologous recombination (HR) o HR is an important DNA repair mechanism used to repair double strand breaks. HR also important for horizontal gene transfer. o HR mechanism is beyond scope of this course. In a nutshell, DNA with similar sequences can get “shuffled around” using this machinery. o Foreign DNA with homology to a region of host chromosome can be inserted into genome in place of - or in addition to - the native DNA sequence o Also important for genome rearrangements – deletions, duplications, inversions of segments of genomic DNA Outcomes -> inserted replacing DNA chopped out , duplicated ↳ once , it gets in can change genetic , makeup ofcell Transposable elements o Transposable elements are mobile genetic elements found in almost all species. Contain transposase gene flanked by inverted repeats. o Transposase enzymes are able to (see image on next slide): o o o o o o Recognize inverted repeats of DNA sequences Cleave that DNA to free “transposable element” Cleave another DNA (e.g. chromosomal DNA) Insert the transposable element into that DNA Wow! That’s one impressive enzyme!!! This process called “transposition”. o “Insertion sequence elements” have only transposition machinery. o “Transposons” contain extra genes as well, such as antibioticresistance genes Transposable elements Often short duplication at insertion site because: - Host DNA gets cut unevenly - E.g. After D, before A’ (see fig) - Via process of single-stranded DNA being “filled in”, A-D gets duplicated Textbook - Fig 9.33 Transposable elements o Many transposable elements are conservative (cut and paste) mechanisms – move from one place to another o Others work via a replicative mechanism – transposon remains at its locus and a copy is produced & inserted elsewhere o Transposons are used extensively in the lab to generate mutant strains o Can insert randomly into genome, inactivating genes o Mutant with an interesting property/phenotype? Sequencing can identify transposon-inactivated gene Textbook - Fig 9.33 How foreign DNA can make its way into a prokaryotic cell • Transformation • Transduction • Conjugation Transformation o Process by which free DNA is incorporated into a recipient cell and brings about genetic change o Can come from a variety of sources – often lysed cells within their environment o DNA does not freely cross cell membrane – a cell capable of taking up free DNA is said to be competent. Some bacteria/archaea are naturally competent, others are not. o In naturally competent organisms, competence is often tightly regulated. o Many bacteria that are not naturally competent can be made competent artificially in the lab – a common way to transfer DNA into cells Textbook Fig 9.11 Transformation In many competent organisms, DNA from the environment is captured by pili, which retract, bringing DNA through outer membrane/cell wall Textbook Fig 9.15 One strand of DNA typically degraded & other strand passed through cytoplasmic membrane & into cell via a multi-protein competence system Textbook Fig 9.16 Bacteriophage infections o A bacteriophage (or phage) is a virus that infects a bacterium. ~1031 phages on the planet!!!!!! o Virus’ DNA packaged into virions, which feature protein coats that protect the DNA. Virions bind cells, inject DNA. o In lytic pathway, phage DNA replicated & new particles produced using host resources. Viruses then lyse host cell, released to infect new cell. o In lysogenic pathway, viral DNA integrated into host DNA – prophage. Can be induced, triggering the lytic cycle. o Some phages purely lytic (only operate via lytic pathway). Others are temperate and can operate via the lytic or lysogenic pathway Textbook Fig 5.18 Transduction Process in which a virus (phage) transfers DNA from one cell to another. Two types: Generalized transduction: o During the lytic cycle some host cell DNA is accidentally packaged into a viral particle. o This DNA injected into new cell in place of phage DNA. See image on next slide. Specialized Transduction: o When a prophage is induced, its DNA is excised from genome & packaged into phage particles. o Sometimes some neighboring DNA is also packaged by mistake o This DNA can then be injected into a new cell by that phage particle Generalized transduction Textbook Fig 9.18 Specialized transduction Textbook Fig 9.20 Conjugation (mating) o Horizontal gene transfer that requires cell-cell contact o Typically conjugation is mediated by plasmids called conjugative plasmids – the F plasmid (originally identified in E. coli) has served as a model. o Many similar systems (with unique aspects) have also be identified o F (fertility) plasmid is large (~100 kbp). Strains with an F plasmid are called F+ and are donor cells o F plasmid can be transferred to cells that lack the plasmid (F-), recipient cells o DNA transfer only from donor to recipient (unidirectional). Only between F+ and F- cells (two F+ cells won’t mate) Textbook Fig 9.11 Conjugation – F plasmids o F plasmid encodes many tra (transfer) genes that are involved in the conjugative transfer process o Some tra genes encode a conjugative pilus – produced by F+ cells, attach to F- cells only (F plasmid encodes genes that prevent attachment) o Pilus attaches, brings two cells together. Conjugative bridge forms. o Beginning at oriT, DNA is nicked and single strand is copied o Copied strand passed to type IV secretion system, which transfers F plasmid DNA from F+ cell to F- cell through the bridge o F- cell now F+, can act as donor. F+ cell remains F+ F+ and F- cells (mating pair) attached via a conjugative pilus Textbook Fig 9.23 Conjugative transfer of F plasmids Textbook Fig 9.24 Textbook Fig 9.11 Conjugative transfer: Hfr strains o F plasmid has insertion sequences & can integrate into chromosome producing an Hfr cell (high frequency recombination). o Conjugative transfer machinery still intact and active – oriT now on chromosome o Transfer to F- cell via a synonymous mechanism, but can also transfer part of donor’s chromosomal DNA o Transferred DNA can be incorporated into recipient strain’s genome (e.g. transposition or recombination) o Doesn’t transfer full F plasmid, so recipient strain remains F- Textbook Fig 9.27 Evolution via horizontal gene transfer o Much acquired DNA will not be evolutionarily useful and will ultimately be lost. For example: o Transposon or recombination-mediated processes o Random processes/errors during DNA replication or DNA repair o Genes that provide a selective advantage will be maintained and can outcompete parental strains that lack this new DNA o Microbial genomes contain a great deal of horizontally-acquired DNA (can tell by %GC content different from rest of genome, absence of these loci in related lineages) o Horizontal gene transfer has huge impacts in all aspects of microbiology. Most notoriously, infectious disease – new virulence mechanisms, antibiotic resistance