Bohomolets Microbiology Lecture #6


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By Ms. Kostiuk from Microbiology department

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  • The genome of procaryotes is quite small compared with the genomes of eucaryotes. Bacterial DNA consists of a few thousand genes in one circular chromosome. Eucaryotic genomes range from thousands to hundreds of thousands of genes. Their DNA is packaged in tightly wound spirals arranged in discrete chromosome.
  • DNA copies itself just before cellular division by the process of semiconservative replication. Semiconservative replication means that each “old” DNA strand is the template upon which each “new” strand is synthesized. The circular bacterial chromosome is replicated at two forks as directed by DNA polymerase III. At each fork, 2 new strands and synthesized – 1 continuously and 1 in short fragments, so called “Okazaki fragments”.
  • There are hypotheses that introns serve as a stock for extra bits of genetic material that could be available for splicing into existing genes, thus promoting genetic change and evolution. With losing of introns procaryotes have lost and grate potential to evolution, that have eucaryotes, but bacteria have acquired very rapid metabolism and reproduction. Eucaryotic cells and viruses have introns
  • Operon contain various control genes (regulators, promoters, and operators) that govern the operation of related structural genes. Such gene regulation responds to external stimuli and usually occurs at the level of transcription.
  • The lac operon controls the utilization of lactose. Tree structural genes under the control of the lac promoter (Plac) code for the synthesis of the enzymes needed for lactose utilization. These enzymes are made only when lactose is present.
  • In inductive systems like the lac operon , the operon is normally in an off mode and does not initiate enzyme synthesis when the appropriate substrate is absent. Structural gene–regular codes special protein – repressor. In the absence of lactose, this repressor binds with the operator lacus, thereby blocking the transcription of the structural genes lying downstream (a). If lactose is added to the cell’s environment, it triggers several events that turn the operon on . The binding of lactose to the repressor protein causes a conformational changes in the repressor that dislodges it from the operator segment (b). The control segment that was previously inactive is now unlocked, and RNA polymerase can now bind to the promoter. The structural genes are transcribed in a transcript coding for all 3 enzymes. After that 3 separate proteins for degradation of lactose are synthesized and digest lactose.. Lactose vanish from environment, repressor becomes free and lock operator.
  • Bacterial systems for synthesis of amino acids, purines, and pyrimidines work on a different principles – that of repression. Similar factors such as repressor proteins, operators, and a series of structural genes exist for this operon, but with some important differences. Unlike the lac operon, this operon is normally in the on mode and will be turned off only when this nutrient is no longer required. The nutrient plays an additional role as a corepressor needed to block the action of the operon. In cell all synthesized arginine are immediately used in metabolism. Under this conditions, the arg operon is set to on , and arginine is being actively synthesized. If cell has surplus of arginine, it accumulate. The free arginine as then available to act as a corepressor by attaching to the repressor. Arginine activates inactive repressor. Repressor locks operator and stops transcription and arginine synthesis.
  • Because adding or deleting a single base pair changes the reading frame of the transcribed mRNA. The deletion or addition of a single or double (but not 3) base pair can have as great an effect as a large deficiency.
  • Because the genetic code is degenerate, the substitution of one nucleotide base for another nay not change the amino acid specified by the codon. Because same amino acid may be coded by triplets with different base, for instance, ACU, ACC, ACG and ACA all code for threonine, so a mutation that changes only last base will not alter the sense of the message in any way. Changes in a single amino acid within a polypeptide often do not drastically reduce the activity of an enzyme and are rarely fatal to the microorganism.
  • An insertion sequence can move around bacterial chromosomes so that at different times it is found at different locations on the chromosome. The nucleotide bases in the IS regions often do not appear to code for structural proteins but may have a regulatory function. They code for transposase, an enzyme that is required for transposition. Transposons are transposable genetic elements that contain genetic information for the production if structural proteins, usually for antibiotic resistance. IS elements and transposons can not be situated free in cytoplasm, but only in integrative form in chromosome or plasmid. They can not self-dependent replicate as plasmid. Transposable elements as IS and transposon can move from sites on the chromosome of cells into plasmid, which are rapidly transferred by conjugation to other cells.
  • An insertion sequence can move around bacterial chromosomes so that at different times it is found at different locations on the chromosome. The nucleotide bases in the IS regions often do not appear to code for structural proteins but may have a regulatory function. They code for transposase, an enzyme that is required for transposition. Transposons are transposable genetic elements that contain genetic information for the production if structural proteins, usually for antibiotic resistance. Transposable elements as IS and transposon can move from sites on the chromosome of cells into plasmid, which are rapidly transferred by conjugation to other cells.
  • Note the relative sizes of the 2 kinds of DNA.
  • Colicin is protein similar to antibiotic but it toxic only to closely related bacteria. Activity of colicinigenic plasmids acts to eliminate competitors.
  • Four natural process lead to movement of DNA from a donor to a recipient cell. Plasmid transfer, transformation (transfer of naked DNA), transduction (transfer of DNA via a phage), and conjugation (transfer by direct mating contact) can lead to recombination of DNA.
  • Depending upon the mode of transmission, the means of genetic recombination is called C., T., Tr.
  • The physical contact between mating cells is established by the F pilus.
  • Some bacterial cells contain F plasmids that contain the genes that code for the F pilus and the transfer of DNA from a donor cell to a recipient.
  • The F plasmid in the donor cell carries the genetic information for the synthesis of the sex pilus, the organelle that attaches the donor to the recipient cell. If the donor cell F+ loses the F plasmid, it becomes F – because it can no longer synthesize the sex pilus and attacn to the recipient cell. Within minutes after contact, the F plasmid from the donor cell enters the recipient cell. Because F – cell receive the F plasmid, it quickly becomes F+. Cell donor do not loss F plasmid, because only one strand is transferred to donor cell. And the complementary strand is synthesized in both cells. In addition, any other plasmids the donor cell contains, such R plasmid, may also be transferred, but not cell chromosome.
  • In the Hfr cell, the F plasmid replicates as part of the chromosome. Thus, the progeny of an Hfr cell are also Hfr.
  • The F plasmid must be incorporated into the chromosome for the donor chromosome to be transferred into the recipient cell. When the Hfr and F – cells contact each other, the circular donor chromosome breaks at the site at which the F plasmid is integrated. The part of chromosome is transported into the recipient cell and integrate into the recipient chromosome. Donor cell synthesizes complementary strand.
  • DNA from lysed dead cell get into solution and recipient bacterium takes up the DNA. To take up DNA, a recipient call must be competent, that is, it must have a site for binding the donor DNA at the cell surface and its plasma membrane must be in a state so that free DNA can pass across it. When dead cells which can form capsule are mixed with live noncapsuled bacteria, transformation occurs, and DNA containing genes for capsule production is taken up by the living bacteria that normally lack the genetic information for capsule production.
  • Griffith experiment. The Streptococcus pneumoniae exists in 2 major strains based on the presence of the capsule. Encapsulated strains bears a smooth S colonies and are virulent, strains lacking a capsule have a rough R colonies and are nonvirulent. When a mouse was infected with a live, virulent strain, it soon died.
  • When another mouse was infected with a live nonvirulent strain, the mouse remained alive and healthy.
  • Next, Griffith heat-killed an virulent S strain and injected it into a mouse, which remained healthy.
  • Then Griffith injected both dead S virulent encapsulated streptococci and live R cells without capsule into a mouse. At result, mouse have dead from pneumococcal infection. And live encapsulated virulent Streptococcus pneumoniae was isolated from the mouse. Recombination occurs and the progeny of the transformed bacteria become capable of producing capsules and were transformed into virulent.
  • In generalized transduction, random fragments of disintegrating host DNA are taken up by the phage during assembly. Virtually any gene from the bacterium can be transmitted through this means. After that, phage with fragment of donor DNA infect other bacterium, and the DNA incorporate into its chromosome.
  • The DNA of a temperate phage enters into the bacterial host cell The phage DNA may become integrated with host cell DNA as a prophage When the prophage is induced to leave the bacterial chromosome, it may carry along a piece of bacterial DNA in place of phage DNA. The phage that are replicated are defective because they lack viral genes that have been replaced by bacterial DNA The defective phage DNA enters new host cell but cannot cause the production of new phage particles Bacterial genes introduced into the new host cell are integrated into the DNA, become a part of the bacterial chromosome, and are replicated along with the rest of the bacterial DNA. Several cases of specialized transduction have medical importance. The virulent strains of Corinebacterium diphtheriae produce toxin, whereas nonvirulent strains do not produce toxin. It turn out that virulence is due entirely to lysogenic conversion, in which a bacteriophage introduces genes that code for toxin. Only those bacteria infected with a temperate phage are toxin formed.
  • Bohomolets Microbiology Lecture #6

    1. 1. Genetic of microorganisms
    2. 2. Advantages of bacteria and viruses as objects for genetic researches <ul><li>Simplicity of genome structure </li></ul><ul><li>Universality of gene code </li></ul><ul><li>Lack of diploid set of chromosomes and dominant genes Відносна легкість культивування </li></ul><ul><li>Grate rapid of reproduction and plurality of population </li></ul><ul><li>Genetic heterogeneity of population </li></ul><ul><li>Accessibility modern methods of genetic analysis as sequencing of DNA, polymerase chain reaction (PCR) </li></ul>
    3. 3. Competitive structure genome of E.coli and human cell Hours Minutes Time existence of mRNA Histones Polyamines DNA combined in cell with Absent Present Plasmid Diploid Haploid Genome type Absent Present Introns Linear Supercoiled DNA shape 183 см 0,1 см DNA length 2х10 12 2х10 9 Molecular weight of DNA 100000 4000 Quantity of genes 46 1 Quantity of chromosomes Nucleus Nucleoid Place in cell Human cell E.coli Properties
    4. 4. The genome is the some total of genetic material of a cell
    5. 5. Semiconservative replication of DNA . Simplified steps <ul><li>A helicase unwinds the double helix into 2 parent strands (P1 and P2). </li></ul><ul><li>The replication of new complementary strands proceeds though the action of an enzyme that attaches nucleotides, using the exposed strands as templates. </li></ul><ul><li>Each completed daughter molecule contains one strand that is newly synthesized and one of the original parent strands. </li></ul>
    6. 6. Replication of bacterial DNA <ul><li>The theta stage of replication, in which one strand loops down as it grown in length. </li></ul><ul><li>Nicking, separation, repair, and release of two completed molecules that will be separated into daughter cells during binary fission. </li></ul>
    7. 7. Definitions <ul><li>Together the structural and regulatory genes constitute the genotype </li></ul><ul><li>Phenotype is determined by genotype and is the actual appearance and activities of the organism. </li></ul><ul><li>Exons are coding regions that will be translated into product – proteins, enzymes </li></ul><ul><li>Introns are sequences of bases that do not code for product </li></ul>
    8. 8. Structure of bacterial genome <ul><li>Structural genes are genes that code for proteins </li></ul><ul><li>Regulatory genes are genes that have regulatory function and act to control the expression of the structural genes </li></ul><ul><li>Operon is a specific unit of DNA that regulates genetic function in procaryotes </li></ul>
    9. 9. The lac operon <ul><li>Regulator – composed of the gene that codes for a protein capable of repressing the operon ( a repressor ) </li></ul><ul><li>Control lorus - composed of two genes, the promoter (recognized by RNA polymerase) and the operator , a sequence where transcription of the structural genes is initiated </li></ul><ul><li>Structural locus – made up of 3 genes, each coding for a different enzyme (b-galactosidase, permease, and transacetylase) needed to catabolize lactose </li></ul>Regulator Control locus Structural locus
    10. 10. Two possible ways work of operon <ul><li>Inducible. The operon can be turned on ( induced ) by the substrate of the enzyme for which the structural genes code </li></ul><ul><li>Repressible. The operon can be turned off ( repressed ) by the product its enzymes synthesize </li></ul>
    11. 11. A model of inducible regulation. Lac operon
    12. 12. A model of repressible regulation. Arg operon
    13. 13. Types of changes in the genetic code <ul><li>Mutation is a change in the nucleotide sequence of DNA that result is a recognizable change in the organism </li></ul><ul><li>Recombination means the addition of genes from an outside source, such as a virus or another cell </li></ul>
    14. 14. Categories based on causes of mutations <ul><li>A spontaneous mutation is a random change in the DNA arising from mistakes in replication of the detrimental effects of natural background radiation on DNA </li></ul><ul><li>An induced mutations result from exposure of the cell to exogenous DNA modifiers such as radiation or chemical substances </li></ul>
    15. 15. Categories of mutation based in alteration of base sequence in DNA <ul><li>A base substitution mutation occurs when one pair of nucleotide bases in the DNA is replaced by another that results in change in codon and synthesis different protein. </li></ul><ul><li>Transitions involves the raplacement of a purine by a different purine or pyrimidine by a different pyrimidine </li></ul><ul><li>Transversions occurs when purines replace pyrimidines and pyrimidines replace purines. </li></ul><ul><li>A deletion mutation involves the removal of one or more nucleotide base pairs from the DNA </li></ul><ul><li>An insertion mutation involves the addition of one or more pairs to the DNA </li></ul><ul><li>A frameshift mutation are addition or loss of one or two bases in a gene. It can result in the misreading of large numbers of codons </li></ul>
    16. 16. Categories based in overall effect of mutation <ul><li>A silent mutation alters a base but not change the amino acid and has no effect </li></ul><ul><li>A missense mutation is a change in the code that leads to placement of a different amino acid. It can do one of the following: </li></ul><ul><li>create a faulty, nonfunctional protein </li></ul><ul><li>produce a different but functional protein </li></ul><ul><li>cause no significant alteration in protein function </li></ul><ul><li>A nonsense mutation changes a normal codon into a stop codon that does not code for an amino acid and stops the production of the protein wherever it occurs. It almost always results in a nonfunctional protein </li></ul><ul><li>A back-mutation (reversion mutation) occurs when a gene that has undergone mutation reverse (mutates back) to its original base composition </li></ul>
    17. 17. Selected mutagenic agents and their effects Cause frameshifts due to insertion between base pairs Acridine dyes Compete with natural bases for sites on replicating DNA Notrogen base analogs Radiation Causes cross-links between adjacent pyrimidines Ultraviolet Form free radicals that cause single or double breaks in DNA Ionizing (gamma rays) Causes cross-linkage of DNA strands Mustard gas Removes an amino group from some bases Nitrous acid, disulfite Chemical Effect Agent
    18. 18. Transposable genetic elements <ul><li>Insertion sequences (IS elements) </li></ul><ul><li>Transposons </li></ul><ul><li>Plasmids </li></ul>
    19. 19. Competitive characteristic IS, transposons, and plasmids 40-50 different genes Only few genes for transposase and resistance to antibiotics Only genes for transposase Quantity of genes No 800-1400 pair nucleic bases IS Yes 3000-5000 pair nucleic bases Plasmid No 2000-2500 pair nucleic bases Transposon Self-dependent replication DNA size Genetic element
    20. 20. Transposable genetic elements
    21. 21. Plasmid Chromosomal and plasmid DNA leaking out of a cell.
    22. 22. Cell functions coded for by some plasmids Utilization of camphor Formation of spores in streptomycetes Metabolic plasmids Enterotoxin production Fimbriae production Virulence factor plasmids Bacteriocin production Col plasmids Resistance to various antibiotics Resistance to cadmium and mercury Resistance to ultraviolet radiation Resistance plasmids (R) Transfer of DNA from one cell to another via conjugation (F-pili) Fertility plasmids (F) Function Group
    23. 23. Intermicrobial DNA transfer and recombination Transformation Transduction Conjugation Plasmid transfer
    24. 24. Types of intermicrobial exchange Toxins; enzymes for fermentation; drug resistance Indirect Donor is lysed bacterium Defective bacteriophage is carrier of donor DNA Live, competent recipient cell of came species as donor Transduc-tion Polysaccharide capsule Indirect Free donor DNA (fragment) Live, competent recipient cell Transfor-mation Drug resistance, resistance to metal; enzymes; degradation of toxic substrate Direct Sex pilus on donor Fertility plasmid in donor Both donor and recipient Gram-negative cells Conjuga-tion Genes transferred Direct of indirect Requirement Mode
    25. 25. Conjugation Conjugation is mode of sexual process mating in which a plasmid or other genetic material is transferred by a donor to a recipient cell via a specialized appendage
    26. 26. Conjugation process Sex, or F, pilus holding together donor and recipient cell of E.coli during DNA transfer
    27. 27. Participants of conjugation process Gram-negative bacterium that produce F pili act as donors during conjugation. Donor strains are designated F + if the F plasmid is independent Bacteria lacking the F plasmid are recipients and designated F - If the F plasmid DNA incorporated into the bacterial chromosome the donor cell designated Hfr ( high frequency recombination )
    28. 28. Cojugation – transfer of the F plasmid
    29. 29. Transformation F + to Hfr cell The F plasmid integrates at specific locations into the chromosome and F + cell is transformed to Hfr cell. The process is reversible.
    30. 30. Conjugation – transfer of chromosomal DNA
    31. 31. Transformation In transformation a free DNA molecule is transferred from a donor to a recipient bacterium
    32. 32. Infecting mouse with virulent encapsulated streptococci
    33. 33. Infecting mouse with nonvirulent streptococci without capsule
    34. 34. Infecting mouse with killed virulent streptococci
    35. 35. Infecting mouse with mix killed virulent streptococci and alive nonvirulent
    36. 36. Transduction is a process by which a bacteriophage serve a the carrier of DNA from a donor cell to a recipient cell. <ul><li>There are 2 version of transduction : </li></ul><ul><li>Generalized </li></ul><ul><li>Specialized </li></ul>
    37. 37. Generalized transduction
    38. 38. Specialized transduction In specialized transduction, a highly specific part of the host genome is regularly incorporated into the virus. It occurs only during infection caused by temperate phage a) b) c) d) e) f)