Microbial Genetics Lecture 4 (BI 302) PDF

Lecture 4: Genetic Exchange Microbial Genetics BI 302 Instructor: Dr. Nalina Nadarajah Email: [email protected]  Agenda for This Week An introduction to genetic exchange between bacteria – Transformation – Conjugation...

Lecture 4: Genetic Exchange Microbial Genetics BI 302 Instructor: Dr. Nalina Nadarajah Email: [email protected]  Agenda for This Week An introduction to genetic exchange between bacteria – Transformation – Conjugation 2  Overview Bacteria reproduce by the process of binary fission – the chromosome is replicated and a copy is allocated to each of the daughter cells – two daughter cells are genetically identical If daughter cells are always identical to the parent, how are different strains created? 3 Events that Result in Heritable Changes in the Genome Recombination Genetic transfer Plasmids Transposons 4  Overview Mutations constantly arise due to errors made during replication If a particular mutation (e.g. AbR) provides any selective advantage, the mutant will quickly dominate the population Bacterial genes can be transferred from one bacteria to another bacteria – mutations as well 5  Gene Transfer in Bacteria Unidirectional – donor to recipient – donor: fragment of chromosome involved in a recombination event donor does not give the entire chromosome donates linear fragment – recipient: strain that carries intact circular chromosome and receives the extra DNA Recombination occurs in the recipient strain only Bacterial genes are usually transferred to members of the same species – can also occur between species 6 Gene Transfer Between Different Bacteria Image source: 7  Gene Transfer in Bacteria In bacteria, gene transfer is partial and unidirectional – donor: contributes DNA; exogenote – recipient: receives contributed DNA (has own complete genome); endogenote Merozygote – contains endogenote (recipient) and exogenote (donor) DNA – partial diploid – opportunity for recombination 8  Recombination Genetic recombination refers to the exchange between two DNA molecules – → new combinations of genes on the chromosome Remember the recombination event known as crossing over? – two homologous chromosomes break at corresponding points, switch fragments and rejoin – the result is two recombinant chromosomes 9 But, how does this happen in bacteria? They are haploid! how did the chromosome segment get in to the cell? Gene Transfer Mechanisms in Bacteria 1. Vertical – receives genetic material from its parent 2. Horizontal – any process in which an organism incorporates genetic material from another organism without being the offspring of that organism 11 Horizontal Gene Transfer Transposition 12  Horizontal Genetic Exchange Conjugation: direct contact between bacterial cells; DNA from donor to recipient Transformation: donor DNA is taken up from the environment and incorporated into the recipient genome Transduction: DNA goes from one bacteria to another via a phage Transposition: transfer of mobile DNA from one chromosome to another or plasmid to chromosome or chromosome to plasmid All are essentially one-way transfers from donor to recipient 13  Plasmids Extra-chromosomal DNA molecules that are capable of autonomous replication (replicon) Circular with different functional genes Genetic elements that can also provides a mechanism for genetic change – transmission of plasmid DNA from one bacteria to another can produce new strains Genetic exchange can occur between main chromosome and plasmid – episome: a plasmid that can integrate into the chromosome14 16 How do you Distinguish Plasmid from Chromosome? Shape? Size? Function? Plasmids do not carry essential genes and thus are ultimately dispensable In contrast, chromosomes are indispensable because they carry essential genes vital for survival 17  Genetic Exchange in Bacteria 18 Source: NATURE REVIEWS | GENETICS, VOLUME 2 | AUGUST 2001  Recombination The Rec proteins (RecA and RecB,C, D) have key roles in this process – mutations in Rec A, B, C, D disrupt recombination – the genes were given ‘Rec ’ names when first discovered since they were thought to function mainly in the ‘recombination pathways’ to promote genetic exchange – research later has indicated DNA replication and repair are the primary functions of these proteins Most bacterial recombination is ‘homologous’ – the 2 recombining segments have identical or near-identical sequences 19  Recombination Cont. Non-homologous or illegitimate recombination is less common – two unrelated sequences become connected by incorrect rejoining of broken ends by insertion of one DNA segment into another – the former is mediated by DNA ligase, which is essential for DNA replication and repair – the latter by transposase, which are encoded by the replication of transposable genetic elements Both homologous and non-homologous recombination are carried out by proteins that have other important cellular functions 20  Transformation After death or lysis, some bacteria release their DNA into the environment Other bacteria that come into contact with these fragments may take them up and incorporate them into their chromosome by recombination This method of transfer is the process of transformation Any DNA that is not integrated into the chromosome will be degraded 21  Transformation Overview Cont. The genetically transformed cell is called a recombinant cell – it has a different genetic makeup than the donor and the recipient All of the descendants of the recombinant cell will be identical to it Recombination give rise to genetic diversity in the population 22  Griffith’s Famous Experiment Worked with Pneumococcus – S strain – smooth, shiny colonies, virulent virulence due to the polysaccharide coat (capsule) which gives the smooth shiny appearance – R strain – rough colonies, avirulent (harmless) R strain has a mutation that prevents it from forming the coat S strain R strain 23 Photo source: iGenetics – A Molecular Approach – 3rd edition  Griffith’s Expt. Cont. Several types of S strain – each with distinct chemical composition of polysaccharide coat (e.g. IIS and IIIS) S type can mutate to R type and vice versa – but type specific! – if a IIS cell mutates into R cell, it can only mutate back into a IIS cell NOT IIIS cell When Griffith injected mice with killed IIIS bacteria and living IIR bacteria → mouse died – viable IIIS bacteria were present in mouse blood – can not be due to mutation; Why? 24  25 Source: Concepts of Genetics, 8 th edition  Some living IIR bacteria has been somehow changed into IIIS bacteria by interaction with the dead IIIS bacteria How can a combination of these two strains cause pneumonia when either strand alone does not cause the disease? Transformation! – the living non-capsulated cells came in contact with DNA fragments of the dead capsulated cells – the genes that code for the capsule entered some of the living cells and a crossing over occurred – the recombinant cell now can form a capsule and cause pneumonia – all of the recombinant's offsprings have the same ability → mouse developed pneumonia and died Griffith called this agent transforming principle – he thought it was the proteins 26 Avery, MacLeod and McCarty Expt. 27  Factors Affecting Transformation DNA size – high MW double stranded DNA of at least 5 x 105 daltons – transformation is sensitive to nucleases in the environment Competence of the recipient – some bacteria are able to take up DNA naturally – they only take up DNA during a particular time in their growth cycle when they produce a specific protein called a competence factor → bacteria are said to be competent – the genes required are all chromosomal → benefit of DNA uptake is to the recipient 28  Natural Competence Natural Competence occurs in several genera of Gram positive and Gram negative bacteria – Bacillus subtilis (soil bacterium), Streptococcus pneumoniae (throat infections), Hemophilus influenzae (spinal meningitis), Neisseria gonorrhoeae (gonorrhea) and species of cyanobacteria from the genera Synechococcus and Synechocystis 29  Competence Cont. Mutations in competence genes do not affect viability ▪ mutants are unable to take up DNA, but usually grow well under standard culture conditions Competence can be artificially induced with chemicals ▪ heat shock with CaCl2 or by pulse electrical shock (electroporation) Artificial competence animation 30  Natural vs. Artificial Competence Naturally-competent cells take up only linear double- stranded DNA (incorporate only one of the two strands) Artificially-rendered competent cells can be transformed with double or single-stranded, linear or circular plasmid DNAs Artificially- competent cells are used in experiments involving transformation with recombinant DNA molecules produced in vitro (e.g. your pGLO experiment) 41  Why does Competence Exist? to favour genetic exchange homologous DNA can also be used as a template to repair otherwise-lethal DNA damage – repair could be more important to the cell than genetic exchange – if competence evolved to provide templates for DNA repair, DNA damage should induce competence – research results show no connection between DNA damage and inducing competence DNA repair may not be the main function of competence 31  Like other molecules taken up by bacterial cells, DNA can be used as a nutrient – bacteria may use as a source of C and N, but likely as a source of nucleotides for DNA and RNA synthesis – spares resources from a very ‘expensive’ cellular process Many competent bacteria live in very DNA-rich environments – e.g. H. influenzae, Streptococcus pneumoniae and Neisseria meningitidis live in respiratory tract mucus (~300 μg DNA per ml of mucus); Helicobacter pylori and Campylobacter jejuni live in gastrointestinal mucus (~200–400 μg DNA secreted into the gastric lumen every 10 min ; and B. subtilis lives in soil (>10 μg DNA per g of soil) 32 Source: NATURE REVIEWS | GENETICS, VOLUME 2 | AUGUST 2001  Why do Bacteria Take Up DNA Three non-mutually exclusive models can account for the evolution of DNA uptake systems: 1. DNA for genetic diversity – the acquisition of potential useful genetic information (e.g. novel metabolic functions, virulence traits or antibiotic resistance) 2. DNA repair – environmental DNA from closely related bacteria as template for repairing DNA damage 3. DNA as food – DNA can be used as a source of carbon, nitrogen and nucleotides 33 Transporting DNA from Extracellular Matrix to Cytoplasm Complex Process The incoming DNA must cross the cell wall and the cytoplasmic membrane – the outer membrane in Gram- negative bacteria → an extra barrier DNA uptake is defined as the conversion of exogenous, DNase-sensitive DNA into a DNase-protected state – in Gram-negative bacteria, this protection can be achieved by crossing the outer membrane – in Gram-positive bacteria, DNA uptake is synonymous with passage across the cytoplasmic membrane 35  Transporting DNA from Extracellular Matrix to Cytoplasm Only one strand of the DNA molecule is effectively transported into the cytoplasm – the other strand is degraded into nucleotides – released into the extracellular environment (in Gram positive bacteria) or presumably into the periplasmic space (in Gram-negative bacteria) 36 Structure of DNA Uptake Competence System of a Gram Positive Bacterium 37 DNA Binding and Uptake in Gram-Neg DNA uptake in most organisms is not sequence-specific Some Gram-negative microorganisms, such as Haemophilus influenzae and Neisseria species, efficient uptake occurs only if a specific sequence is present – called as DUS (DNA uptake sequences) – favour the uptake of DNA from the same or closely related species Haemophilus influenzae Neisseria gonorrhoeae & N. meningitidis 5’ –AAGTGCGGTCA- 3’ 5’ –GCCGTCTCAA- 3’ 3’ –TTCACGCCAGT- 5’ 3’ –CGGCAGAGTT- 5’ 38 Structure of DNA Uptake Competence System of a Gram Negative Bacterium 39 Competence in Gram Negative vs. Positive Outer membrane Peptidoglycan layer Periplasmic space Inner membrane Gram Negative Gram Positive Only one strand enters the cytoplasm; the other is degraded & degradation products are released – into the periplasmic space in Gram Negatives – into the extracellular matrix in Gram Positives 40  Conjugation Do bacteria possess any processes similar to sexual reproduction and recombination? – answered in 1946 by Joshua Lederberg and Edward Tatum – studied two strains of Escherichia coli with different nutritional requirements (auxotrophs) – strain A would grow on a minimal medium only if it was supplemented with methionine and biotin (met− bio− thr+ leu+ thi+) – strain B would grow on a minimal medium only if it was supplemented with threonine, leucine, and thiamine (met+ bio+ thr− leu− thi−) 42 Demonstration by Lederberg and Tatum of Genetic Recombination between Bacteria  What Happened? Cells of type A or type B cannot grow on minimal medium – both A and B are auxotrophs When A and B are mixed for a few hours and then plated → few colonies appear on the agar plate – these colonies derived from single cells in which an exchange of genetic material has occurred – they are capable of synthesizing all the required constituents of metabolism (prototrophs) – suggests some form of recombination of genes between the genomes of the two auxotrophs to produce prototrophs Can it be due to leakage of substances?  demonstrated by Bernard Davis pores of the filter were too small to allow bacteria to pass but large enough to allow easy Source: Fig. 8.11 from Genetics: A Conceptual Approach by B.A. Pierce passage of the fluid medium and any dissolved substances strains remained auxotrophic physical contact between the two strains was needed for wild- type prototrophs cells to form Tatum Lederberg  Conjugation in E.coli Conjugation is the mode of gene transfer between two live bacterial cells that are in direct contact Forms a temporary cytoplasmic bridge between two compatible cells – driven by plasmid called the fertility factor or sex factor (F) found in some but not all E. coli – F plasmids contain ~ 100 genes Cells carrying F plasmid are F+; those lacking it are F- Mating is only between F+ and F– Transfer of information is one-way from donor to recipient – not reciprocal – not true sexual reproduction 43  F plasmid Replicates inside host cell – this allows the plasmid to be maintained in a dividing cell population Contains genes encoding synthesis of pili which allow F+ cells to attach to F- cells to conjugate F+ F- 46  F+ cells conjugate with F– – F+ donates single-stranded (ss) copy of F plasmid to F– cell (via rolling circle mechanism) – donor F+ retains copy of plasmid – in the recipient cell, ss F is converted to ds F+ – rapidly F- population are converted to F+ F+ cells are inhibited from contacting other F + Animation 47 Source: Fig. 8.13 from Genetics: A Conceptual Approach by B.A. Pierce  Insertion Sequence (IS) F carries one or more insertion sequence (IS) elements – repeated segments of DNA that can move from place to place within the host chromosome or between chromosome & plasmid – permit homologous recombination with same IS in genome – leads to integration of F plasmid into the bacterial chromosome (episome) 48 – cells with F factor integrated into chromosome → high frequency of recombination (Hfr) strains 49 Source: Fig. 8.14 from Genetics: A Conceptual Approach by B.A. Pierce High Frequency of Recombination (Hfr) – integration of F into genome forms Hfr strain – can be isolated and grown into a pure culture every cell has the F factor in chromosome donates chromosomal DNA during conjugation Source: Fig. 8.15 from Genetics: A Conceptual Approach by B.A. Pierce frequency of recombinants is much higher Animation 50  Hfr Strain In Hfr x F- mating, the recipient (F -) almost never becomes F+. Why? – F Factor is nicked in the middle (oriT) in initiation of strand transfer – part of F factor at the beginning and rest at the end of strand to be transferred – to become F+ or Hfr, the recipient must receive the entire F factor (i.e. entire bacterial chromosome needs to be transferred) – rarely happens as conjugating cells break apart before entire chromosome transfer happens 49  F’ Cells » When an F factor excises from the integrated chromosome, it may remove some bacterial genes with it » These genes will be carried with the F plasmid » Cells containing an F plasmid with bacterial genes are known as F prime (F’) Source: Fig. 8.16 from Genetics: A Conceptual Approach by B.A. Pierce » During conjugation between F’ lac cell and F- cell, all genes on F plasmid are transferred (incl. lac) => sexduction 52  Different Forms F factor 3 Different Forms 1. F+ - cells containing an autonomously replicating F plasmid they efficiently transfer the F plasmid to a suitable recipient (F-) 2. Hfr (high frequency of recombination) - F factor is able to integrate into the cell chromosome can efficiently transfer donor cell chromosomal DNA to a recipient cell by conjugation 3. F’ (F-prime) - F factor carrying some chromosomal DNA with it arises when F factor excises out of the Hfr chromosome and returns to the autonomously replicating state 51 F Plasmids have 3 Distinct Properties 1. Ability to synthesize the F pilus 2. Mobilization of DNA for transfer to another cell 3. Alteration of surface receptors so the cell is no longer able to behave as a recipient in conjugation 52  55 Source: Table 8.2 from Genetics: A Conceptual Approach by B.A. Pierce  56 Source: Table 8.3 from Genetics: A Conceptual Approach by B.A. Pierce Mapping by Interrupted Conjugation Hfr strains transmit host chromosome to F – in linear manner, about 1% of chromosome per minute After timed intervals, mating is interrupted and cells are plated on selective medium to recover recombinants Genes are mapped according to time of appearance of recombinants Circular, low resolution map is made by combining maps from different Hfr donors Higher resolution map is made by RF (recombinant frequencies) analysis 55  Interrupted-mating conjugation experiments with E. coli F− cells that are strr are crossed with Hfr cells that are strs F− cells have a # of mutations → can’t carry out specific metabolic steps Hfr cells are capable of carrying out all metabolic steps After cells are mixed, at different times, samples are disrupted to break conjugation and plated on media containing streptomycin F- tested for their ability to carry out the four metabolic steps 60 59 60 61 62  References Text Book – Chapter 6 iGenetics – A Molecular Approach – 3rd edition Ch. 15 University of South Carolina School of Medicine Do bacteria have sex? by Rosemary J. Redfield in NATURE REVIEWS, Genetics, VOLUME 2, AUGUST 2001 DNA uptake during bacterial transformation by Ines Chen and David Dubnau in NATURE REVIEWS, MICROBIOLOGY, VOLUME 2, MARCH 2004 Dubnau, D. 1999. DNA uptake in bacteria, Annu. Rev. Microbiol. 53:217–44 An Introduction to Genetic Analysis. 7th edition. Griffiths AJF, Miller JH, Suzuki DT, et al. New York: W. H. Freeman; 2000 Chapter 8 from Genetics: A Conceptual Approach, 3rd edition © 2008 by B.A. Pierce, New York: W. H. Freeman 61