Bacterial Genetics PRINCIPLES OF BACTERIAL GENETICS • Study of heredity and variation seen in bacteria. • All hereditary characteristics of the bacteria are encoded in their DNA (deoxyribonucleic acid). • Bacterial DNA is present in chromosome as well in extrachromosomal genetic material as plasmid. BACTERIAL DNA • Bacteria possess a single haploid chromosome, comprising of super coiled circular double stranded DNA of 1 mm length. • Bacterial DNA - lacks basic proteins. • Bacteria do not have a true nucleus; but the genetic material is located in an irregularly shaped region called the nucleoid. • There is no nuclear membrane or nucleolus. Structure of DNA (Watson & Crick model) • • Bacterial DNA - is composed of two strands of complementary nucleotides that are coiled together in the form of a double helix as described first by Watson & Crick. Each strand is composed of three elements o Backbone of deoxyribose sugar and phosphate groups o Nitrogenous bases are attached to the sugar group o Nucleotide and nucleoside - components of the DNA strand.  Nucleoside = Sugar + nitrogenous base  Nucleotide = Sugar + nitrogenous base + phosphate Structure of DNA (Watson & Crick model) • • • • There are four nitrogenous bases, o Two purines: adenine (A) and guanine (G) o Two pyrimidines: thymine (T) and cytosine (C) Pairing- The two DNA strands are held together by hydrogen bonds occurring between the nitrogenous bases on the opposite strands. The pairing follows a specific ruleo Adenine of one strand binds with thymine (A-T) of other strand by double hydrogen bonds. o Guanine of one strand binds with cytosine (G-C) of other strand by triple hydrogen bonds. In a molecule of DNA, the number of A=T; G=C. The ratio of A + T to G + C is constant for each species but varies widely from one bacterial species to another. DNA replication • In eukaryotes, during DNA replication, the two strands of the double helix unwind from one another and separate. • Each strand acts as template for a new DNA strand which is synthesized through complementary base pairing- A with T, and G with C. Bidirectional replication • Replication begins at a single point, the origin. • DNA helix is unwound at a region called replication fork; the site at which the DNA synthesis occurs and individual strands are replicated. • Two replication forks move outwards from the origin - until they have copied the whole replicon and a structure shaped like the Greek letter theta (θ) is formed. • Since the forks meet on the other side and two chromosomes are separated. • e.g. in E.coli Rolling-circle mechanism • Occurs during bacterial conjugation and during the reproduction of viruses and bacteriophages. • Outer strand is nicked and the free 3′ end is extended by replication enzymes (growing point rolls around the circular inner strand). • 5′ end of the outer strand is displaced and forms a single-stranded tail which later may be converted to the dsDNA by complementary strand synthesis. Replication enzymes Enzyme Function Helicases DNA unwinding Topoisomerase (e.g. DNA gyrase in E.coli) relieves the tension generated by rapid unwinding by removing the super twists. DNA polymerase • Forms complementary strand synthesis by adding nucleotides to the growing end of the strand (5′ to 3′ direction). • DNA polymerase III plays the major role in replication, although it is probably assisted by polymerase I. • Polymerases I and II participate - repair of damaged DNA. DNA ligase- helps in joining of the fragments BACTERIAL RNA • RNA (ribonucleic acid) is structurally similar to DNA, except for o In sugar- ribose is present instead of deoxyribose o In nitrogenous base- uracil replaces thymine. • Three different types of RNA in a cell: o Messenger RNA (mRNA) o Ribosomal RNA (rRNA) o Transfer RNA (tRNA). • Main function of RNA - protein synthesis. Genetic Code • Gene - segment of DNA that stores information for a particular polypeptide synthesis. • Genetic information that is stored in DNA is transcribed into RNA and then translated to form the particular polypeptide. Genetic Code Terminology Explanation Codon (Nirenberg and Khorana – 1968) Sequence of three nucleotide bases present on mRNA that stores the information of an amino acid synthesis. Sense codons • There are 64 codons, out of which 61 are sense codons. • Each codon directs the production of a single amino acid. • There are only 20 amino acids, so more than one codon exist for the same amino acid. Non-sense codons • Three codons (UGA, UAG, and UAA) do not code for any amino acids. • Involved in the termination of translation. Genetic Code Terminology Explanation Start codon • First codon of an mRNA from which the translation begins. • Most common start codon - AUG (codes for methionine in eukaryotes and modified methionine [NFormyl methionine (fMet)] in prokaryotes). Anticodon- • Set of three nucleotide bases present on tRNA that is complementary to the nucleotide bases of codon on mRNA. Transcription • Process in which a particular segment of DNA is copied into RNA by the enzyme RNA polymerase. • DNA acts as a template for synthesis of m-RNA, therefore, the bases in m-RNA are complementary to one strand of DNA. Translation • mRNA transcribed from DNA is decoded by a ribosome to produce a specific amino acid chain (cytoplasm). Translation phases Function Initiation First tRNA is attached at the start codon of mRNA. Elongation tRNA transfers an amino acid to the adjacent tRNA corresponding to the next codon. Translocation Ribosome then moves (translocates) to the next mRNA codon to continue the process, creating an amino acid chain Termination: When a stop codon is reached, the ribosome releases the polypeptide. PLASMID • Plasmid - extra chromosomal ds circular DNA molecules that exist in free state in the cytoplasm of bacteria and also found in some yeasts. • May be present singly or in multiple numbers- up to >40 plasmids per cell. • Plasmids are capable of replicating independently. PLASMID • Episome-Plasmid may integrate with chromosomal DNA of bacteria and such plasmids are called as episomes. They replicate along with bacterial chromosome. • Curing-The process of eliminating the plasmids from bacteria is known as curing. o Occur spontaneously or may be induced by treatment with acridine, radiations, thymine starvation, and growth at higher temperatures (does not destroy host cell). Classification of plasmids – Based on ability to perform conjugation o Conjugative plasmids- Possess ability to transfer themselves to other bacteria by means of conjugation (self transmissible or conjugative plasmids). o Non-conjugative plasmids-They cannot transfer themselves (nontransmissible plasmids). Classification of plasmids – Based on compatibility between the plasmids o Compatible plasmids- Different plasmids can exist in a single bacterial cell only if they are compatible to each other. o Incompatible plasmids-If two plasmids are not compatible, one or the other will be rapidly lost from the cell. They normally share the same replication or partition mechanisms, hence compete with each other. Classification of plasmids – Based on function • Fertility or F-plasmids • Resistance (R) plasmids • Col plasmids • Virulence plasmids • Metabolic plasmids Plasmid as vector • Ability to transfer DNA from one cell to another, plasmids have become important vectors in genetic engineering. • Plasmids contain certain sites where genes can be inserted artificially by recombinant DNA technology. • Uses - protein production, gene therapy. BACTERIAL VARIATION • Phenotypic variation • Genotypic variation MUTATION • Mutation - Random, undirected heritable variation caused by change in nucleotide sequence of the genome of the cell. • Frequency of mutation -10- 2 to 10-10 per bacterium per division. Mutations - Two ways • Spontaneous mutations- Occur naturally in any dividing cells that arise occasionally without adding any mutagen. • Induced mutations - As a result of exposure of the organism to a mutagen- an agent capable inducing mutagenesis. Examples of mutagens includeo Physical agent- e.g. ultraviolet radiations- cytosine and thymine are more vulnerable to UV rays. o Chemical agents-alkylating agents, 5-bromouracil and acridine dyes. Mutation • Most mutants go unrecognised as the mutation may be lethal or may involve some minor functions that may not be expressed. • Mutation is best appreciated when it involves a function, which can be readily observed by experimental methods. Mutation • Mutation can affect any gene and hence may modify any characteristic of the bacterium. o Sensitivity to bacteriophages o Loss of ability to produce capsule or flagella o Loss of virulence o Alteration in colony morphology o Alteration in pigment production o Drug susceptibility o Biochemical reactions o Antigenic structure Classification of mutation types • Small-scale mutations • Large-scale mutations Practical importance of bacterial mutation –Study of drug resistance and development of live vaccines. Type of mutations Forward mutations - Substitutions at single nucleotide base pair At DNA Level Point mutation that changes a purine nucleotide to another purine (A Transition G) or a pyrimidine nucleotide to another pyrimidine (C T). Transversion Substitution of a purine for a pyrimidine or vice versa -DNA, (C/T A/G). Type of mutations Forward mutations - Substitutions at single nucleotide base pair At codon level Silent New codon codes for the same amino acid: AGG→ CGG both code for mutation arginine. Neutral mutation New codon forms different but functionally equivalent amino acid: AAA (lysine) AGA (arginine) Missense mutation The new codon codes for a different amino acid. Nonsense mutation The new codon is a stop codon which causes induces termination. E.g, CAG (Glutamine) →UAG (stop) Type of mutations Addition or Deletion at single or many nucleotide base pairs: Frameshift Any addition or deletion of base pairs that is not a multiple of three results in a mutation shift in the normal reading frame of the coded message forming new set of triplet codon. They are usually very deleterious and may lead to synthesis of nonfunctional proteins. Reverse mutations A second mutation which nullifies the effect of the first mutation that results in gaining back the function of the wild phenotype is called as reverse mutation. True reversion A true reverse mutation converts the mutant nucleotide sequence back to the wild-type sequence. AAA (Lysine) forward mutation GAA(Glutamine) reverse mutation AAA(Lysine) (wild type) (mutant) (wild type) Type of mutations Addition or Deletion at single or many nucleotide base pairs: Equivalent Second mutation produces a different codon which codes for the same aminoacid reversion of wild type sequence. UCC (Serine) forward mutation GAA (Cystine) reverse mutation AAA(Serine) (wild type) (mutant) (wild type) Suppressor It is a second mutation in a different gene, that reverts the phenotypic effects of mutation an already existing mutation. Detection and Isolation of Mutants – Fluctuation test • Demonstrates the spontaneous mutation in bacteria (Luria and Delbruck -1943). • When bacterial suspension is subjected to selective pressure by subculturing on to agar plate containing a growth limiting substance (e.g. streptomycin or bacteriophage etc.),they undergo spontaneous mutation. • Rate of mutation vary widely(some bacteria mutate early, some late) which leads to fluctuations. Detection and Isolation of Mutants – Fluctuation test • Fluctuations in mutation are wide when small volume sub cultures are made (more frequent mutations) as compared to large volume subcultures (less frequent mutations). • This experiment was not widely appreciated, probably due to the complicated statistical evaluation. Detection and Isolation of Mutants – Replica plating method • Detects auxotrophic mutants (Lederberg -1952). • Differentiates between the normal strains from auxotrophic mutants • E.g. Lysine auxotroph will grow on lysine-supplemented media but not on a medium lacking lysine. Detection and Isolation of Mutants – Replica plating method Ames test (carcinogenicity testing) • Used to identify the environmental carcinogens (Bruce Ames 1970). • Mutational reversion assay that uses the mutant strains (histidine auxotroph) of Salmonella which are subcultured on two agar plates containing small amount of histidine; one of the plate is added with the test mutagen. • The plates are incubated for 2 to 3 days at 37°C. • All of the histidine auxotrophs will grow for the first few hours until the histidine is depleted. Ames test (carcinogenicity testing) HORIZONTAL GENE TRANSFER IN BACTRIA • Gene transfer in bacteria can be broadly divided in too Vertical gene transfer (transmission of genes from parents to offspring) o Horizontal gene transfer (transmission of genes from one bacterium to another neighbour bacterium) • Horizontal gene transfer occurs in bacteria by several methods such aso Transformation (uptake of naked DNA) o Transduction (through bacteriophage) o Lysogenic conversion o Conjugation (plasmid-mediated) TRANSFORMATION • Definition - Process of random uptake of free or naked DNA fragment from the surrounding and incorporated into its bacterial chromosome in a heritable form. • Natural transformation studied in -Streptococcus, Bacillus, Haemophilus, Neisseria, Acinetobacter and Pseudomonas. Mechanism of transformation :When bacteria lyse, they release large amounts of dsDNA into the surrounding environment. Their uptake is usually mediated by the competent bacteria present in the surroundings TRANSFORMATION • Competency for transformation • Bacteria expressing competence factors (S. pneumoniae) can uptake any DNA fragment irrespective of source. • If competence factors not expressed (Haemophilus influenza) - uptake of DNA occurs only from the closely related species. STEPS IN TRANSFORMATION Frequency – In competent cells it is around 10-3 for most genera when an excess of DNA is used Griffith experiment • Griffith experiment (1928) on mice using pneumococci strains provided the direct evidence of existence of transformation. Griffith experiment • Stated that the live non-capsulated strains were transformed into the capsulated strains due to transfer of the capsular genes released from the lysis of the killed capsulated strains which was confirmed later by Avery, Macleod and McCarty in 1944. TRANSDUCTION • Definition- Transmission of a portion of DNA from one bacterium to another by a bacteriophage. • Mechanism of transduction: o Part of the host DNA may accidentally be incorporated into the bacteriophage o Gets transferred to the recipient bacterium so that it leads to acquisition of new characters by the recipient bacterium coded by donor DNA. Bacteriophages -Life cycle • • Lytic or virulent cycle o Bacteriophage multiplies in host cytoplasm, produces large number of progeny phages. o Subsequently, released causing death and lysis of the host cell. Lysogenic or Temperate cycle o Host bacterium is unharmed. o Phage DNA remains integrated with the bacterial chromosome as the prophage, which multiplies synchronously with bacterial DNA. o When the phage DNA tries to come out, it is disintegrated from host chromosome, comes out into the cytoplasm, and behaves as a lytic phage. o It replicates to produce daughter phages which are subsequently released by host cell lysis. Types of TransductionGeneralised transduction • Involves transfer of any part of the donor bacterial genome into the recipient bacteria. • Occurs as a result of defective assembly during the lytic cycle of virulent and some temperate phages. Types of TransductionGeneralised transduction Types of TransductionRestricted or Specialized transduction Role of Transduction • Method of transfer of episomes and plasmids. • Drug resistance • Treatment- Transduction has also been proposed as a method of genetic engineering in the treatment of some inborn metabolic defects. LYSOGENIC CONVERSION • During the temperate or lysogenic life cycle, the phage DNA remains integrated with the bacterial chromosome as prophage, which multiplies synchronously with the bacterial DNA. • The prophage acts as an additional chromosomal element which encodes for new characters and is transferred to the daughter cells. This process is known as lysogeny or lysogenic conversion. LYSOGENIC CONVERSION • Bacterial toxins that are coded by lysogenic phages includeo Diphtheria toxin o Cholera toxin o Verocytotoxin of E.coli o Streptococcus pyrogenic exotoxin(SPE)- A & C o Botulinum toxin C and D CONJUGATION F + X F - mating CONJUGATION HFR X F - mating CONJUGATION F’ X F - mating Resistance Transfer Factor (RTF) • R factor (or the resistance factor) is a plasmid which has two components. (R factor = RTF+ r determinants). o Resistance transfer factor (RTF)- is the plasmid responsible for conjugational transfer (similar to F factor) o Resistance determinant (r) - An R factor can have several r determinants and each r determinant coding for resistance to one drug. FATE OF THE DONOR DNA FOLLOWING HORIZONTAL TRANSFER Recombination • Recombination takes place between the donor DNA and recipient chromosome in two ways: o General recombination o Site specific recombination General or Homologous Recombination • Most common form, can occur at any place on the recipient’s chromosome in general, and occurs between DNA of similar sequences Site Specific Recombination • In restricted transduction, the integration of bacteriophage DNA into bacterial chromosome is sitespecific. TRANSPOSITION • Transposons or transposable elements are the bacterial genes that are capable of intracellular transfer between: o Chromosome to chromosome o Plasmid to plasmid o Chromosome to plasmid or vice versa • and the process of such intracellular transfer Transposition. Transposons. A. Insertion sequence transposon; B. Hairpin loop structure of transposon; C. Composite transposon Recombinant DNA Technology • The procedure of recombinant DNA technology involves the following steps: o Treatment with restriction enzyme o Southern blot o Recombination with a vector o Introduction of the vector into bacteria o Cloning Applications of Genetic Engineering • • • • • Production of vaccines Production of antigens used in diagnostic kits Production of proteins used in therapy Transgenic animals Gene therapy NUCLEIC ACID PROBE • Nucleic acid probes are used to detect the specific nucleic acid either— o Directly in the clinical sample or o Following amplification of small quantity nucleic acid present in the clinical sample o Following enzymatic digestion of the extracted nucleic acid BLOTTING TECHNIQUES • • • • Southern blot - to detect DNA Northern blot - to detect RNA Western blot - to detect proteins Eastern blot: Modification of Western blot, used to analyze proteins for post-translational modifications using probes that may detect lipids, carbohydrate, phosphorylation or any other protein modification.