Lecture 3 Microbiology: Bacterial Heredity & Variation Lecture Notes PDF

Lecture 3 Microbiology: Bacterial Heredity &Variation 1  Content Genetic material of bacteria (Chromosome, plasmid, transposable element, integron and phage genome). Bacterial phages (an overview). Mechanisms of transfer an...

Lecture 3 Microbiology: Bacterial Heredity &Variation 1  Content Genetic material of bacteria (Chromosome, plasmid, transposable element, integron and phage genome). Bacterial phages (an overview). Mechanisms of transfer and recombination of bacterial genes (transformation, transduction, conjugation and lysogenic conversion); gene mutation. The significance of bacterial genetic variation (in drug resistance, pathogenesis or virulence and variation, diagnosis, and vaccination), and manipulation of cloned DNA. 2  Bacterial Genetics No feature is more central in bacterial diversity and power to produce disease than their genetic mechanisms. New media now deliver a constant stream of reports of new antibiotic resistance and emerging pathogens. Bacteria treated successfully with an antimicrobial for decades suddenly appear to be resistant; Diseases seemingly under control reappear; New diseases (at least new for us) emerge and spread. When traced to their origin most of these involve the spread and breadth of genetic mechanisms, such as mutation, recombination, transformation, transduction, conjugation and transposition. 3  Bacterial genome DNA molecules that replicate as discrete genetic units in bacteria are called replicons. In some bacterial strains chromosome is the only replicon present in the cell. Other bacterial strains have additional replicons such as plasmids and bacteriophages. 4  Bacterial genome Bacterial genome vary in size from about 4x109 to 8.6 x109 daltons (Da). NOTE: 1 dalton = 1.660530000001 x 10-24 gram 1 gram = 6.02217364335 x 10+23 dalton The smallest microbial genome is that of Mycoplasma genitalium at 580,070 base pairs encoding 525 genes. Some of the largest genomes are those of the Cyanobacteria. Scytonema hofmanni's genome is 12,073,012 base pairs in size and has around 12,356 putative protein coding genes. Most bacteria have haploid genome, a single chromosome – consisting of a circular double stranded DNA molecule. 5  Bacterial genome Bacterial genomes usually consist of a single circular chromosome, but species with more than one chromosome (eg. Deinococcus radiodurans), linear chromosomes (eg. some Bacillis subtilis strains). Linear chromosomes have been found in Gram-positive Borrelia (cause of Lyme disease) and Streptomyces spp. One linear and one circular chromosome is found in the Gram-negative bacteria Agrobacterium tumerfaciens. V.cholerae possesses 2 circular chromosoms. 6  How bacterial genome differs from higher forms of life? Every gene is composed of introns and exons: these are names given to (functionally) different parts of a gene. EXONS: coding portions of any gene - the exons of a gene contain the sequence that actually codify the GENE'S PRODUCT - THE PROTEIN. INTRONS: non-coding parts of any gene - the introns of a gene are sequences of DNA that don't codify proteins. Their function is not totally clear, but they can be transcripted into different RNAs, and these regions can also regulate genes expression. 7 Binary fission and generation time Most bacteria reproduce by a relatively simple asexual process called binary fission: each cell increases in size and divides into two cells. The time interval required for a bacterial cell to divide or for a population of cells to double is called the generation time. Generation times for bacterial species growing in nature may be as short as 15 minutes (20 min for E.coli) or as long as several days (e.g. for Mycobacteria). 8 9 Binary fusion 10 11  Mutation Mutations are heritable changes in the structure of genes. The normal, usually active type of a gene is called wild type allele. The mutated, usually inactive form is called mutant allele. 12  Mutation Spontaneous development of mutations is a major factor in the evolution of bacteria. Mutations occur in nature with a low frequency about 10-6 (one cell in a million). Because bacteria is haploid, the consequences of a mutation, even of a recessive one, are immediately evident in the mutated cell. Because the generation time of bacteria is short, it does not take many hours for a mutant cell that has arisen by chance to grow to the dominant cell type if the mutation gives it a survival advantage. 13 Development of antibiotic resistance 14  Kinds of mutations There are several kinds of mutations based on the nature of the change in nucleotide sequence of the affected gene(s). A. Spontaneous or B. Induced. Also mutations may be typed as: Replacement or Substitution (Point mutation), – involves Missense mutation substitution of one base with another. Missense mutation – when changes in sequence of genes changes mRNA transcript to a different amino-acid (e.g. an AAG – Lysine; GAG – Glutamate) 15  Kinds of mutation Insertion – involves addition of many base pairs of nucleotides at a single site. Nonsense mutation –when replacement changes a codon specifying one amino-acid to one specifying none (e.g.UAG - Tyrosine to UAA - STOP) Deletion – remove a contiguous segments of many base pairs. Microdeletion and microinsertion – involves removal of addition of a single nucleotide. Frame- shift mutation is caused by microdeletion and microinsertion, which results in changes in reading frame by which the ribosomes translate the mRNA from the mutated gene. Duplication – produce a redundant segment of DNA, usually adjuscent to (tandem) to original one. Transversion – when purine nucleotide is changed with a pyrimidine or vice versa Suppressor mutation (reversal of mutant phenotype), Lethal mutation Conditional lethal mutant (Temperature sensitive mutant or ts mutant; permissive temperature 37⁰C, Non-permissive temperature 39⁰C). 16 Dr SOMESHWARAN, ASSISTANT PROFESSOR & CLINICAL MICROBIOLOGIST at KARPAGAM FACULTY OF MEDICAL SCIENCES AND RESEARCH 17  Recombination Recombination is the process in which nucleic acid from different sources are combined or re-arranged to produce a new nucleic sequence. The source of recombinant chromosome may be another part of the same chromosome (endogenote) or from outside the cell (exogenote) from genetic mechanisms such as conjugation, transformation or transduction. Homologous (generalized) recombination is a type of genetic recombination in which nucleotide sequences are exchanged between two similar or identical molecules of DNA. Generalized recombination requires participation of the RecA enzyme, however other enzymes are also necessary. Site-specific recombination, also known as conservative site-specific recombination, is a type of genetic recombination Site specific recombination operates only in the unique sequences with participation of RecBCD enzymes that are encoded by exogenote genes. Rec A enzyme is not essential for the specific recombination. 18 19  Plasmids The term plasmid was first introduced by the American molecular biologist Joshua Lederberg in 1952. Plasmids are considered as transferable genetic Joshua Lederberg elements - “replicons”, capable of autonomous replication within the suitable host. Plasmid may be found in all three major kingdoms: Bacteria, Arhea and Eukaria. 20  Plasmid A plasmid is an extra-chromosomal DNA molecule separate from the chromosomal DNA, capable to replicate independently of the chromosomal DNA. In many cases it is circular and double stranded. Plasmids occur naturally in bacteria, but sometimes they are found in Eukaria as well. 21  Plasmids Plasmid size varies from 1 to 200 kilobase pairs (kbp). The number of identical plasmids within the single cell can range anywhere from one to even thousands. Plasmids do not carry genes essential for survival, but info for selective advantage. 22  Types of plasmids There are two types of plasmid integration into the host bacteria: a) Non-integrating plasmids replicate independently from the host chromosome b) Integrated plasmid is called episome. Episomes can insert into bacterial chromosome, where they become permanent part of the genome. 23  Grouping of plasmids One way of grouping plasmids is by their ability to transfer to other bacteria. Conjugative plasmids carry so called tra genes that mediate process of conjugation and are responsible for transfer of plasmids into other bacteria. Non-conjugative plasmids are incapable of initiating conjugation however they can be transferred only with assistance of conjugative plasmid. 24  Plasmids according to compatibility groups It is possible for plasmids of different types to co-exist in a single cell. Seven different plasmids have been found in E.coli. But related plasmids are often incompatable, in the sense that only one of them survives in the cell line due to regulation of the vital plasmid functions. Therefore, plasmids can be assigned into compatibility groups. 25  Classification of plasmids by their functions Another way to classify plasmids is by their functions. There are 5 main classes: Fertility – F-plasmids which contain tra genes. They are capable of conjugation (transfer of genes between the bacteria being in physical contact) Resistance – R-plasmids that can build resistance against antibiotics or poisons. Historically known as R-factors before the nature of plasmids was understood. 26  Classification of plasmids by their functions Col plasmids that code i.e. determine production of proteins called bacteriocines since they able to kill other bacteria. Bacteriocines are antibacterial agent that are active only against similar or closely related bacterial strains. These are growth inhibiting proteins which can inhibit the growth of some closely related organisms, proteins produced by bacteria to kill other microbes Degradative plasmids that are capable for digestion of unusual substances, e.g. toluene or salicylic acid. Virulence plasmid which turn the bacteria into a pathogen (one that causes a disease). 27  Use of plasmids Plasmids are used in molecular biology for production of large amounts of proteins. In this case the researchers grow the bacteria containing plasmids harboring the genes of interest. The plasmid carrier bacteria is producing the protein in large amounts. This is a cheap and easy way for mass-production of certain proteins e.g. insulin or even antibiotics. Plasmids are now used to manipulate DNA and may possibly be a tool for curing many diseases. 28 29  Mobile genetic elements – “jumping genes” Transposons are the segments of DNA that can move from one site of DNA molecule to other target sites of the same or a different DNA molecule e.g. plasmid. The process is called transposition and occurs by a mechanism that is independent from generalized recombination. Transposones are not self replicating elements. They must integrate into other replicons to be stably maintained in bacterial genomes. The transposition relies of their ability to Barbara McClintock – synthesize their own specific recombination Nobel Prize 1983 enzymes – transposases. 30  Transposons were first discovered in corn, where they cause varied expression of color 31 The major kind of transposable elements are insertion sequence (IS) elements and transposons. IS elements encode only proteins for their own transposition. Insertion of IS elements into a gene causes mutation. 32 IS elements are flanked by inverted repeats IR elements.  Transposons IS –insertion sequences are the simplest structures that encode only the functions needed for transposition. Complex transposons vary in length from about 2,000 to more than 40,000 nucleotide pairs and contain insertion sequences (or closely related sequences) at each end usually as inverted repeats. 33  Role of transposons Most transposons share a number of common features. Each transposon encodes the functions necessary for transposition, including production of transposase – an enzyme that integrates with specific sequences present at the ends of the transposon. Transposons encode functions (e.g. antibiotic resistance, substrate metabolism, etc.) beyond those needed for their own transposition Insertion of transposons often interrupts the linear sequence of a gene and inactivates it. Transposons have a major role in causing deletions, duplications and inversions of DNA segments as well as fusions between the replicons. 34 Transposons sequences can hop to another region of the genome and integrate 35  Mechanisms of transposition A. Direct transposition moves the transposon to a new site –”cut and paste mechanism”. B. Replicative transposition leaves a copy behind – “copy and paste mechanism” 36  Mechanisms of transposition During transposition a short sequence of target DNA is duplicated. The duplication is presumed to involve an assymetric cleavage of DNA at the target site. If the transposon at the donor site is replicated and a copy is inserted into the target site the process is called integrative transposition. 37 38  Regulation of gene transfer Species of bacteria differ by their ability to transfer DNA, but all the three mechanisms are distributed among both Gram + and Gram- species, however: Only transformation is governed by bacterial chromosome genes; Transduction is totally mediated by bacteriophage genes; Conjugation is regulated by plasmid genes. 39 Transformation – Griffith’s experiment 40  Transformation Transformation – a genetic recombination in which a DNA fragment from a dead, degraded bacterium enters a competent recipient bacterium and it is exchanged for a piece of the recipient's DNA. Involves 4 steps: 1. A donor bacterium dies and is degraded 2. A fragment of DNA from the dead donor bacterium binds to DNA binding proteins on the cell wall of a competent, living recipient bacterium 3. The Rec A protein promotes genetic exchange between a fragment of the donor's DNA and the recipient's DNA 4. Exchange is complete 41  Transformation The ability to take up DNA from the environment is called competence. To be active in transformation DNA molecules must be at least 500 nucleotide pairs in length. Any DNA present in environment is bound indiscriminately, however its further fate depends on whether it shares homology with the portion of DNA of the recipient cell. In this case transformation can occur, but heterologous DNA is degraded. Other species do not naturally enter the competent state , can be made permeable to DNA by treatment with agents that damage the cell envelope, making an artificial transformation 42 possible.  Conjugation The process called conjugation is the transfer of genetic information from the donor (F+) to a recipient (F-) bacterial cell in the process that requires intimate cell contact. Conjugation is plasmid encoded process. Conjugative plasmids contain the genes responsible for transfer. Nonconjugative plasmids are The 3 conjugative processes are known: transferred by plasmid mobilization I. F + conjugation by conjugative plasmid. II. Hfr conjugation Conjugation may cross species lines III. Resistance plasmid conjugation 43  Conjugation Plasmids responsible for conjugation are known as Fertility factors (F) or Sex or Transfer factor. F factor (is an plasmid coding for synthesis of Sex pilus) Cells carrying F factor = F+ Cells; Cells lacking F factor = F- Cells; Hfr cells: High frequency cells – the F factor is the episome state i.e. in integrated with host chromosome. Conversion of F+ cell to Hfr cell is reversible; F factor to F Prime (F’) factor 44  F+ Conjugation Genetic recombination in which there is a transfer of an F+ plasmid (coding only for a sex pilus) but not chromosomal DNA from a male donor bacterium to a female recipient bacterium. Involves a sex (conjugation) pilus. Other plasmids present in the cytoplasm of the bacterium, such as those coding for antibiotic resistance, may also be transferred during this process. 45 Conjugation 46 Conjugation 47  The 4 stepped F+ Conjugation 1. The F+ male has an F+ plasmid coding for a sex pilus and can serve as a genetic donor 2. The sex pilus adheres to an F- female (recipient). One strand of the F+ plasmid breaks 3. The sex pilus retracts and a bridge is created between the two bacteria. One strand of the F+ plasmid enters the recipient bacterium 4. Both bacteria make a complementary strand of the F+ plasmid and both are now F+ males capable of producing a sex pilus. There was no transfer of donor chromosomal DNA although other plasmids the donor bacterium carries may also be transferred during F+ conjugation 48 49  II. Hfr Conjugation Genetic recombination in which fragments of chromosomal DNA from a male donor bacterium are transferred to a female recipient bacterium following insertion of an F+ plasmid into the nucleoid of the donor bacterium. Involves a sex (conjugation)pilus. 50  5 stepped Hfr Conjugation 1. An F+ plasmid inserts into the donor bacterium's nucleoid to form an Hfr male. 2. The sex pilus adheres to an F- female (recipient). One donor DNA strand breaks in the middle of the inserted F+ plasmid. 3. The sex pilus retracts and a bridge forms between the two bacteria. One donor DNA strand begins to enter the recipient bacterium. The two cells break apart easily so the only a portion of the donor's DNA strand is usually transferred to the recipient bacterium. 4. The donor bacterium makes a complementary copy of the remaining DNA strand and remains an Hfr male. The recipient bacterium makes a complementary strand of the transferred donor DNA. 5. The donor DNA fragment undergoes genetic exchange with the recipient bacterium's DNA. Since there was transfer of some donor chromosomal DNA but usually not a complete F+ plasmid, the recipient 51 bacterium usually remains F- 52  Composition of RTF Plasmid consists of two components: A transfer factor RT, helps conjugational transfer, and resistant determinant (r) to each of the several drugs. RTF + r are known as R factor R factor may contain determinants as many as 8 or > 8 drugs 53  III. Resistant Plasmid Conjugation Genetic recombination in which there is a transfer of an R plasmid (a plasmid coding for multiple antibiotic resistance and often a sex pilus) from a male donor bacterium to a female recipient bacterium. Involves a sex (conjugation) pilus. 4 stepped Resistant Plasmid Conjugation 1. The bacterium with an R-plasmid is multiple antibiotic resistant and can produce a sex pilus (serve as a genetic donor). 2. The sex pilus adheres to an F- female (recipient). One strand of the R-plasmid breaks 3. The sex pilus retracts and a bridge is created between the two bacteria. One strand of the R-plasmid enters the recipient bacterium. 4. Both bacteria make a complementary strand of the R-plasmid and both are now multiple antibiotic resistant and capable of producing a sex pilus 54  Phage T4 Phage structure Head Collar Tail Fibers Basal plate Phage genetic map 55 Phage Reproduction on the Bacterial Cell Bacteriophage in action - to find the target and neutralize 56 57 Lytic and Lysogenic cycles 58 Lytic cycle 59 Lysogenic cycle 60 61 62  Transduction Transduction is the transfer of DNA fragments from one bacterium to another bacterium by a bacteriophage. Transduction was discovered by Joshua Lederberg and Norton Zinder in 1952. 63 U tube experiment Transduction was discovered by Zinder and Lederberg while searching for conjugation in S. typhimurium. Lederberg-Zinder experiment - After placing two auxotrophic strain of Salmonella on opposite side of the U-tube, the researches recovered prototrophs from the side with the LA-22 strain, but not from the containing the LA-2 strain. These initial observations led to the discovery of the phenomenon called transduction. ---------------------------------- Phe=phenilalanin Trp=tryptophan Met=metionin 64 His=histidine 65 66  Generalized transduction 1 2 67 3 4 Generalized transduction 68  Seven steps in Generalized Transduction 1. A lytic bacteriophage adsorbs to a susceptible bacterium. 2. The bacteriophage genome enters the bacterium. The genome directs the bacterium's metabolic machinery to manufacture bacteriophage components and enzymes 3. Occasionally, a bacteriophage head or capsid assembles around a fragment of donor bacterium's nucleoid or around a plasmid instead of a phage genome by mistake. 4. The bacteriophages are released. 5. The bacteriophage carrying the donor bacterium's DNA adsorbs to a recipient bacterium 6. The bacteriophage inserts the donor bacterium's DNA it is carrying into the recipient bacterium. 7. The donor bacterium's DNA is exchanged for some of the recipient's DNA. 69 70  Six steps in Specialized transduction 1. A temperate bacteriophage adsorbs to a susceptible bacterium and injects its genome. 2. The bacteriophage inserts its genome into the bacterium's nucleoid to become a prophage 3. Occasionally during spontaneous induction, a small piece of the donor bacterium's DNA is picked up as part of the phage's genome in place of some of the phage DNA which remains in the bacterium's nucleoid. 4. As the bacteriophage replicates, the segment of bacterial DNA replicates as part of the phage's genome. Every phage now carries that segment of bacterial DNA. 5. The bacteriophage adsorbs to a recipient bacterium and injects its genome. 6. The bacteriophage genome carrying the donor bacterial DNA inserts into the recipient bacterium's nucleoid. 71 Genetic mechanism of drug-resistance Bacteria acquire drug resistance through several Mechanisms Mutations Genetic transfer Transformation Transduction Conjugation Several Biochemical Mechanisms Decreasing permeability of drugs Attaining alternative pathways Produce enzymes and inactivate drugs 72 73  Genetic manipulations of DNA DNA cloning and other techniques can be used to manipulate and analyze DNA and to produce useful new products and organisms. DNA cloning permits production of multiple copies of a specific gene or other DNA segment. A recombinant plasmid is made by inserting restriction fragments from DNA containing a gene of interest into a plasmid vector that has been cut open by the same enzyme. Gene cloning results when the recombinant plasmid is introduced into a host bacterial cell and the foreign genes are replicated along with the bacterial chromosome as the host cell reproduces. DNA technology is reshaping medicine and the pharmaceutical industry. Medical applications include diagnostic tests for genetic and other diseases, safer, more effective vaccines, and the prospect of treating or even curing certain genetic disorders. Pharmaceutical applications include the large-scale production of many new, and some previously scarce, drugs and other medicines 74 Your manual Chapter 7. Microbial genetics 75 Thank you 76