Bacteria Genetics & Exchange of Genetic Information

Transcription

1 Bacteria Genetics & Exchange of Genetic Information Di Qu ( 瞿涤 ) MOH&MOE Key Lab of Medical Molecular Virology School of Basic Medical Sciences Shanghai Medical College of Fudan University 复旦大学上海医学院分子病毒学 教育部 / 卫生部重点实验室 Chapter 7

2 Bacterial Structures Cell Wall All bacteria -Lipopolysaccharides -Teichoic Acids Cell Membrane & Cytoplasm -Inclusions Ribosomes Nucleoid -Chromosome & Plasmids Capsule Flagella Pili some bacteria Spores Chapter 2

3 Key Words Chromsome Plasmid Transposable Genetic Elements Phage -Lysophage (temperate), virulent phage prophage, lysogen /lysogeny Gene transfer Transformation Transduction General transduction Lysogenic (specific)transduction/conversion Conjugation (transfer of genetic material between bacterial cells by direct cell-to-cell contact or by a bridge-like connection between two cells) F factor,hfr, R plasmid

4 All organisms have DNA and RNA as genetic material All organisms use the same nucleotides All organisms replicate, transcribe and translate DNA The central dogma of molecular biology

5 Chapter 7, p101 Genetics of Bacterial Chromsome: the prokaryotic genome is circular, haploid Plasmid: mobile Transposable Genetic Elements:mobile elements, integrate into chromsome or plasmid, -carried genes, drug resistant -insertion mutation Bacteriaphage (phage) RNA-mRNA, rrna -In prokaryotes, an mrna molecule carry information for several genes (eukaryotes an mrna for one gene) -The ribosomes are 70S in prokaryotes vs 80S in eukaryotes -Transcription: synthesis of RNA from a DNA template -Translation: formation of a protein (amino acid sequence) from RNA sequence

6 Some fundamentals of bacterial genetics Bacterial DNA can be altered by mutations Mutations can result in changes in proteins -diversity -acquisition of resistance New traits can be transmitted to other microbes

7 Mutations in Bacteria Mutations arise in bacterial populations Point mutation (synonymous/nonsynonymous substitution) Induced or spontaneous Genetic recombination Rare mutations are expressed Bacteria are haploid Rapid growth rate (bacteria generation time/doubling time? E.coli) Selective advantage enriches for mutants antibiotics, nutrients Horizontal gene transfer

8 Mutations are inheritable changes in the base sequence of nucleic acid -- the genetic material. An organism with these changes is called a mutant. Genetic recombination is the process where genes from two genomes are combined together. A mutant will be different from its parent, its genotype or genetic makeup has been altered. synonymous/nonsynonymou s substitution

9 Plasmids Plasmids are circular double strand DNA molecules Definition: Extrachromosomal genetic elements Replicate independently of the bacterial chromosome (replicon) encode a variety of genes usually not essential bacterial genes but may give bacterium new properties (antibiotic resistance, virulent, etc.), can lost during culture. Size vary widely, mobile and can be transferred between individuals and among species (host range) Plasmids are used in genetic engineering as gene transfer vectors Episome (virology) - a plasmid that can integrate into the chromosome 9

10 Table 7-2, p.105 Table 9-2, p.153

11 Table 7 2 p.105 Examples of Metabolic Activities Determined by Plasmids Organism Pseudomonas species Bacillus stearothermophilus Alcaligenes eutrophus Activity Degradation of camphor, toluene, octane, salicylic acid a-amylase Utilization of H 2 as oxidizable energy source Escherichia coli Klebsiella species Streptococcus (group N) Rhodospirillum rubrum Flavobacterium species Sucrose uptake and metabolism, citrate uptake Nitrogen fixation Lactose utilization, galactose phosphotransferase system, citrate metabolism Synthesis of photosynthetic pigment Nylon degradation

12 Classification of Plasmids Transfer properties Conjugative plasmid (containing tra genes, which perform the complex process of conjugation, the transfer of plasmids to another bacterium) Nonconjugative plasmid(incapable of initiating conjugation) Phenotypic effects Fertility (F plasmid) Resistance plasmid (R factors) Bacteriocinogenic plasmid - controls the synthesis of bacteriocin

13 Extrachromosomal Integration

14 Conjugative plasmid Bacterial conjugation

15 Transposable Genetic Elements Definition: Segments of DNA that are able to move from one location to another on the chromosome- jumping gene Bacteria contain a wide variety of transposable elements The smallest and simplest insertion sequences (IS elements) 1 3 kb in length and encode the transposase protein required for transposition and one or more additional proteins that regulate the rate of transposition Properties Random movement hot spot Not capable of self replication (not a replicon) Transposition mediated by site-specific recombination Transposase Transposition may be accompanied by duplication

16 Types of Transposable Genetic Elements Insertion sequences (IS) Definition: Elements that carry no other genes except those involved in transposition Nomenclature - IS1 (ISn) Structure (flanking inverted repeats- palindrome) Importance ABCDEFG Transposase GFEDCBA Insertional Mutation Plasmid insertion Phase variation Integration

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18 Phase Variation in Salmonella H Antigens H1 gene IS H2 gene H1 flagella H2 flagella

19 Types of Transposable Genetic Elements Transposons (Tn) Definition: Elements that carry other genes in addition to those involved in transposition, gene that moves from one DNA molecule to another within the same cell or from one site on a DNA molecule to another site on the same molecule Nomenclature - Tn10 Structure Composite Tns Importance Antibiotic resistance Integration

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22 BACTERIOPHAGES =bacterial virus infect host cell - bacteria

23 Phage Composition and Structure Composition Nucleic acid genome ds, ss DNA Head/Capsid - Protein head Capsid or Tail fibers etc. -Protection -Infection Contractile Sheath Tail Fibers Tail Base Plate

24 Types of Bacteriophage Lytic or virulent phage Phage that multiply within the host cell, lyse the cell and release progeny phage (e.g. T4) Lysogenic or temperate phage: Phage that can either multiply via the lytic cycle or genome integrating into chromosome of bacteria, entering a quiescent state in the bacterial cell. In lysogenic status: Expression of most phage genes repressed Prophage: Phage DNA integrated in chromosome of bacteria Lysogen: Bacteria harboring a prophage

25 TAIL HEAD CAPSOMER CORE SHEATH TAIL FIBER (6) PHAGE T4 lytic phage COLLAR BASE PLATE Adsorption Tail fibers Receptor is LPS for T4 Irreversible attachment Base plate Sheath Contraction Nucleic acid injection EXTERIOR SPIKES WALL - OUTER MEMBRANE CYTOPLASMIC MEMBRANE NOBEL (1969) Alfred Hershey discovery on the replication of viruses and genetic structure INJECTION - PENETRATION RECEPTOR PROTEIN CYTOPLASM

26 BACTERIOPHAGES - LYTIC GROWTH LYTIC PHAGE GROWTH (5 steps) Attachment (adsorption, specificity) Penetration (injection) Replication -Transcription, translation - Host provides: energy, ribosomes, RNA polymerase. etc. for macromolecular synthesis - Production of viral proteins and nucleic acids Assemble (maturation) (packaging) intact progeny viruses Release - cell Lysis - release of progeny

27 General Phage Life Cycle attach Host cell Inject DNA Lytic Cycle total time = ~15 mins replication cell lysis releasing ~200 phage assemble

28 TEMPERATE PHAGES AND LYSOGENY Lambda - Infection : Attachment, Penetration, genome integrated into chromosome Repression of lytic genes, Integration, Lysogeny Prophage, Lysogen (host cell) Prophage Induction (a high stress environment) Inducing agent Repression abolished, Lytic gene expression. Excision Lytic growth

29 Lysogenic Cycle All phage species can undergo a lytic cycle Phages capable of only the lytic cycle are called virulent lysogenic cycle: -no new phage produced -the infected bacterium survives -a phage DNA is transmitted to each bacterial progeny cell when the cell divides lysogenic cycle Those phages that are also capable of the lysogenic cycle are called temperate Integration 29

30 Lytic phase Lysogenic phase Lysogen- bacteria

31 Events Leading to Lysogeny Site-specific recombination Phage coded enzyme Repression of the phage genome Repressor protein Specific Immunity to superinfection Induction Adverse conditions Role of proteases reca protein Destruction of repressor Gene expression Excision Lytic growth Lysogenic phase Lytic phase

32 Exchange of Genetic Information -horizontal gene transfer

33 General Features of Gene Transfer in Bacteria Unidirectional Donor to recipient Donor does not give an entire chromosome Gene transfer can occur between species Transformation- uptake of naked DNA Transduction- by bacteriophages Lysogenic conversion Conjugation- bacterial cells come in direct contact with each other. Plasmid is often transferred (Hfr)

34 Transformation Definition: Gene transfer resulting from the uptake of DNA from a donor. Factors affecting transformation DNA size and state Sensitive to nucleases Competence of the recipient (Bacillus, Haemophilus, Neisseria, Streptococcus) Competence factors Induced competence

35 Transformation Steps - Donor DNA - Uptake of DNA Gram + Gram - Competence of the recipient - Recombination Legitimate, homologous or general reca, recb and recc genes Significance Phase variation in Neiseseria Recombinant DNA technology

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37 Transformant identified by selection Recombination

38 R form Griffith s transformation experiment (1928) S form Peter J. Russell, igenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.

39 Avery, MacLeod, McCarty Experiment (1944)

40 Experiment showed that DNA, not RNA, was the transforming principle Peter J. Russell, igenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.

41 Avery, MacLeod, McCarty experiment

42 Recipient bacteria must be competent to take up and incorporate DNA Few strains of bacteria are naturally competent Bacteria can be made artificially competent - calcium solutions - electric current

43 Transduction Definition: Gene transfer from a donor to a recipient by a bacteriophage Resistant to environmental nucleases Bacteriophage (phage): a virus that infects bacteria can incorporate genetic material into chromosomal DNA. Bacterial cell can change characteristics and pathogenic factors: Diphtheria toxin Botulinum neurotoxin Staphylococcal enterotoxin Cholera toxin Table 9-2 P. 153

44 Transduction Types of transduction Generalized Transduction in which potentially any dornor bacterial gene can be transferred. Specialized Transduction in which only certain donor genes can be transferred Significance Common in Gram+ bacteria Lysogenic (phage) conversion e.g. Corynebacterium diptheriae toxin Toxin derived from lysogenic phage

45 Generalized Transduction Infection of Donor (phage) Phage replication and degradation of host DNA Assembly of phages particles Release of phage Infection of recipient (cell-bacterium) Homologous recombination Potentially any donor gene can be transferred

46 Transduction Recombination

47 General Transduction A bacteriophage transfers the DNA from one bacterial cell to another During a LYTIC infection, a transducing phage, such as P1 infecting E. coli, accidentally packages a piece of the bacterial chromosome into a virus particle instead of its own viral DNA. The phage carrying the bacterial DNA then delivers it to the recipient cell when it tried to infect again. The injected bacterial DNA may then be integrated into recipient chromosome by homologous recombination 47

48 Specialized Transduction Lysogenic Phage Excision of the prophage (carrying diphtheria toxin gene etc.) Replication and release of phage Infection of the recipient Lysogenization of the recipient Homologous recombination also possible

49 Lysogenic Cycle Lytic Cycle Integration Lysogen- bacteria prophage

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51 Conjugation Definition: Gene transfer from a donor to a recipient by direct physical contact between cells with F pili Mating types in bacteria Donor F factor (Fertility factor) F (sex) pilus Encoded by a plasmid F+ Recipient Lacks an F factor -F- Donor Recipient F+ F-

52 F factor and Conjugation F (fertility) factor is a conjugative plasmid transferred from cell to cell by conjugation F factor is an episome, genetic element that can insert into chromosome or replicate as circular plasmid ~100 kb in length A low-copy-number plasmid, 1 2 copies per cell Replicates once per cell cycle and segregates to both daughter cells in cell division 52

53 F-Pilus for Conjugation

54 Conjugation Direct contact between donor and recipient must occur Sex pilus is encoded by fertility (F) plasmid

55 Physiological States of F Factor Characteristics of F + x F - crosses: F - becomes F +, F + remains F + Low transfer of donor chromosomal genes F+

56 Mechanism of F + x F - Crosses Pair formation Conjugation bridge DNA transfer Origin of transfer Rolling circle replication F + F - F + F - F + F + F + F +

57 Physiological States of F Factor Integrated into chromosome (Hfr) (High Frequency of Recombination) Characteristics of Hfr x F - crosses: F - rarely becomes Hfr, while Hfr remains Hfr High transfer of certain donor chromosomal genes F + Hfr

58 Mechanism of Hfr x F - Crosses Pair formation Conjugation bridge DNA transfer Origin of transfer Rolling circle replication Homologous recombination Hfr F - Hfr F - Hfr F - Hfr F -

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60 Physiological States of F Factor Autonomous with donor genes (F ) Characteristics of F x F - crosses F - becomes F, while F remains F High transfer of donor genes on F, low transfer of other donor chromosomal genes ( Hfr ) Hfr F

61 Mechanism of F x F - Crosses Pair formation Conjugation bridge DNA transfer Origin of transfer Rolling circle replication F F - F F - F F F F

62 Structure of R Factors RTF(resistance transfer factor) Conjugative plasmid Transfer genes RTF R determinant Resistance genes Transposons R determinant

63 R plasmid R: drug resistance RTF: transfer of R plasmid

64 Bacteria do not reproduce sexually but can acquire new DNA through transformation, transduction or conjugation R plasmids - resistance to antibiotics, metals -Virulence factors (that make bacteria pathogenic) Transposons can insert themselves into genome or plasmid (and out of it) Thus bacteria have many ways of obtaining new genes horzontally to enhance survival -These natural processes have been modified so that DNA can be deliberately incorporated into host microbeseven genes that would normally never be transferred this way

65 Review questions 1. In p118: question 1, 2, 5 2. Question 3, 4, and give the explains, if possible. 3. Does the phenotype of an organism automatically change when a change in genotype occurs? Why or why not? 4.Can phenotype change without a change in genotype? In both cases, give some examples to support your answer. 5. List the biological significances of gene transfer in bacteria.

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67 Ribosome Protein synthesis; Targets of antibiotics 70S : 30S (16S rrna) 50S (5S & 23S rrna) Streptomycin Erythromycin

68 Procaryotic ribosome 68