University of Guyana BIO 2107 Lecture 5 - Microbial Genetics PDF

UNIVERSITY OF GUYANA FACULTY OF NATURAL SCIENCES DEPARTMENT OF BIOLOGY BIO 2107 THE BIOLOGY OF MICROORGANISMS LECTURE 5: MICROBIAL GENETICS Dr. Sabrina Dookie 11th October, 2024  Microbial Genetics Bac...

UNIVERSITY OF GUYANA FACULTY OF NATURAL SCIENCES DEPARTMENT OF BIOLOGY BIO 2107 THE BIOLOGY OF MICROORGANISMS LECTURE 5: MICROBIAL GENETICS Dr. Sabrina Dookie 11th October, 2024  Microbial Genetics Bacteria possess two genetic structures: chromosomes and plasmids. These structures consist of a single circular DNA (plasmids) and double helix twisted counterclockwise about its helical axis (chromosomes). The plasmids are autonomous DNA molecules of varying sizes located in the cytoplasm. Many plasmids carry genes that code for certain phenotypic characteristics of the host cell.  Stages of molecular processes underlying genetic information flow Central Dogma Theory The central dogma theory of molecular biology is represented by a simple pathway: DNA →RNA → protein, which demonstrates the flow of genetic information in a living cell. The major processes involved in this pathway are replication, transcription, and translation.  Stages of molecular processes underlying genetic information flow In DNA replication, the DNA polymerase enzyme replicates all the DNA in the nuclear genome in a semi-conservative manner, meaning that the double-stranded DNA is separated into two and a template is made by DNA polymerase. This allows genomic material to be duplicated so it can be evenly partitioned between two somatic cells (daughter cells) upon division.  Stages of Molecular processes underlying genetic information flow The process in which DNA is copied into RNA by RNA polymerase is called transcription. Three forms of RNA are produced here: messenger RNA (mRNA), ribosomal RNA (rRNA), and transfer RNA (tRNA). The process by which mRNA directs protein synthesis with the assistance of tRNA is called translation.  Stages of molecular processes underlying genetic information flow Another process in this pathway is reverse transcription, which involves copying RNA information into DNA using reverse transcriptase. Recently, this process has been defined and may expand the central dogma. For example, retroviruses use the enzyme "reverse transcriptase" to transcribe DNA from an RNA template to make complementary DNA (cDNA). The viral DNA then integrates into the nucleus of the host cell. Then it is transcribed, and further translated into proteins. This biological process effectively adds another pathway to the central dogma of molecular biology.  DNA Replication in Bacteria ▪ Bacteria contain one chromosome while many contain plasmids. ▪ When bacterial chromosomes replicate both strands are duplicated. ▪ Each strand functions as a template. ▪ During replication, enzymes known as polymerases transport nucleotides from the cytoplasm that are complimentary to the template and fit them into place, resulting in two strands, one parental and one new one.  DNA Replication in Bacteria DNA replication begins at a specific spot on the DNA molecule called the origin of replication. At the origin, enzymes unwind the double helix making its components accessible for replication. The helix is unwound by helicase to form a pair of replication forks. The unwound helix is stabilized by SSB proteins and DNA topoisomerases. Primase forms RNA primers (10 bases), which serve to initiate the synthesis of both the leading and lagging strands. The leading strand is synthesised continuously in the 5′to 3′ direction by DNAP III. The lagging strand is synthesised discontinuously in the 5′to 3′ direction through the formation of Okazaki fragments. DNAP I remove the RNA primers and replace the existing gap with the appropriate deoxynucleotides. DNA ligase seals the breaks between the Okazaki fragments as well as around the primers to form continuous strands. Towards the fork Away from the fork  DNA Synthesis in Bacteria Transcription is the synthesis of RNA and involves the assembly of nucleotides by an enzyme, RNA polymerase. Transcription converts DNA to mRNA. 1. RNA polymerase binds to DNA at a promoter site near the gene to be transcribed. 2. RNA polymerase travels the length of the DNA using it as a template to duplicate. 3. The RNA polymerase continues until it reaches a termination site at which time the transcription is complete.  Protein Synthesis in Bacteria Protein synthesis is carried out in the cytoplasm. It begins with DNA duplication by mRNA (Transcription) mRNA then migrates to the ribosome where tRNA transfers information from mRNA to rRNA (Translation). Protein synthesis is continuous and takes place in three stages: 1. Initiation 2. Elongation 3. Termination ▪In prokaryotes, the processes of transcription and translation occur simultaneously in the cytoplasm, allowing for a rapid cellular response to an environmental cue.  Genetic Variability of Bacteria Changes in the bacterial DNA are the result of spontaneous mutations in individual genes as well as recombination processes resulting in new genes or new genetic information. Mutations: A heritable change in the nucleotide sequence of a gene is called a mutation. Mutations are usually detrimental, but they can also lead to beneficial changes. A mutant is called an auxotroph if the mutation leads to a new nutrient requirement, while the wild-type strain is known as a prototroph.  Mutagens Chemical and physical agents that cause mutations. Two types – spontaneous and induced. – Spontaneous mutation occurs naturally, about one in every million to one in every billion divisions, and is probably due to low level natural mutagens present in the environment. – Induced mutation is caused by mutagens that cause a much higher rate of mutation; induced by chemicals or radiations. Other mutations are caused by a transposable genetic element (DNA sequences capable of entering a cell’s genome, altering its genotype).  Mutagens ▪UV light is absorbed by pyrimidines (cytosine and thymine). UV causes adjacent thymines in the same strand to react and bond with each other. ▪Thymine dimers are replication errors in transcription; if not correct, they can lead to cellular death. ▪Nitrous acid alters the chemical structures of adenine, cytosine, and guanine so that they change the base pairing which introduces mutation during DNA replication. ▪Ionization radiation ionizes water and other molecules to form free radicals that can break DNA strands and alter purine and pyrimidine bases. ▪Most mutations are harmful but some are beneficial because they introduce variability into the progeny which promotes survival.  Types of Mutations Point mutation (affects DNA) ▪substitution of one base for another during DNA replication; ▪most common mechanism of mutation; ▪substitution of one nucleotide for another may be a result of tautomeric shift, a process by which the hydrogen atoms of a base shift in a way that changes the properties of its hydrogen bonding. ▪For example, a shift in the hydrogen atom of adenine enables it to form hydrogen bonds with cytosine rather than thymine.  Types of Mutations A sense/ ‘silent’ mutation (affects DNA) A single substitution mutation which results in a new codon still coding for the same amino acid.  Types of Mutations Frame shift mutation (affects DNA) ▪a genetic mutation caused by a deletion or insertion in a DNA sequence that shifts the way the sequence is read. ▪Causes a shift in open reading frame and all of the codons and amino acids after that mutation are usually wrong. ▪Frequently one of the wrong codons turns out to be a stop or nonsense codon and the protein is terminated at that point.  Types of mutations A mis-sense mutation (affects protein) A single substitution mutation which results in one wrong codon and, therefore, one wrong amino acid.  Types of mutation A non-sense mutation (affects protein) A single substitution mutation occurs in DNA when a sequence change gives rise to a stop codon rather than a codon specifying an amino acid. The presence of the new stop codon results in the production of a shortened protein that is likely non-functional.  Genetic Recombination in Bacteria ▪Genetic recombination is the transfer of DNA from one organism to another. ▪The donor’s DNA may then be integrated into the recipient’s DNA by various mechanisms - homologous (identical) or heterologous (non- identical). ▪Unidirectional - Donor to recipient. ▪Donor does not give an entire chromosome – Merozygotes (partially diploid temporarily). ▪Gene transfer can occur between the same or different species.  Homologous recombination of bacterial DNA Deoxyribonuclease (Dnase) degrades DNA and inserts a nick in one strand of the donor DNA. Then single-stranded binding protein (yellow) bind to nicked strand to stabilise. Rec A protein (recombinant) then binds to the single-strand fragment. Strand exchange between donor and recipient DNA followed by annealing (joining of strands).  Other Mechanisms of Genetic Recombination in Bacteria Transformation is a method of genetic recombination in which a naked DNA from a donor bacteria is transferred to a competent recipient bacteria and incorporated into chromosome of the latter, e.g. in Bacillus, Haemophilus, Neisseria, Pneumococcus. ▪Transformation occurs in nature. ▪It is widely used in recombinant DNA technology. ▪In Gram +ve bacteria, the DNA is taken up as a single stranded molecule and the complementary strand is synthesized in the recipient. ▪In Gram -ve bacteria, double stranded DNA is transformed.  Mechanism of Transformation ▪A bacterial cell dies or is degraded releasing its dsDNA molecule in the environment. ▪Nuclease enzymes cut the released DNA into fragments of usually about 20 genes long. ▪The fragments bind to DNA binding proteins present on the surface of a competent recipient bacterium and subsequently translocated in the cytoplasm of recipient bacteria ▪The DNA fragment from the donor is then exchanged for a piece of the recipient's DNA by means of Rec A proteins (aids in recombination of DNA in bacteria). dsDNA – double-stranded DNA  STEPS IN TRANSFORMATION STEP 1: A donor bacterium dies and is STEP 2: A fragment of DNA from the dead donor bacterium degraded. binds to DNA binding proteins on cell wall of a competent live recipient bacterium. STEP 3: The Rec A protein promotes genetic exchange between a fragment of the donor's DNA STEP 4: Exchange is complete. and the recipient's DNA.  Factors affecting Transformation ▪DNA size and state. ▪Sensitive to nucleases (at least 5 X 105 daltons ). ▪Competence of the recipient (Bacillus, Haemophilus, Neisseria, Streptococcus). ▪The ability to take up DNA from the environment is known as competence. ▪ Only DNA from closely related bacteria (competent cells) would be successfully transformed. ▪Competence factor (a specific protein produced at a particular time in the growth cycle of competent bacteria and enables it to take up DNA naturally). ▪Induced competence (chemical manipulation under lab conditions).  Transduction Gene transfer from a donor to a recipient bacteria through a bacteriophage. Bacteriophage (phage): A virus that infects bacteria. Types of transduction: Generalized and Specialized. Phage Composition – Nucleic acid (DNA/RNA) Genome size Modified bases (protect from host nucleases) – Protein: Protection and Infection Structure (T4), Size (80 X 100 nm), Head or capsid – Tail (contractile sheath, base plate, tail fibres)  Types of Bacteriophages ▪Virulent phage: a phage that multiplies within the host cell, lyses the cell and releases progeny phage (e.g. T4) – lytic cycle ▪Temperate phage: a phage that can either multiply via the lytic cycle or enter a quiescent integrated state in the bacterial cell - lysogenic cycle. – Expression of most phage genes repressed – Prophage: Phage DNA in the quiescent integrated state – Lysogen – Bacteria harbouring a prophage  Lysogeny ▪Following the injection of a phage genome, it is integrated into the chromosome by means of region – specific recombination employing an integrase. ▪The phage genome thus integrated is called a prophage. The prophage is capable of changing to the vegetative state, either spontaneously or in response to induction by physical and chemical noxae (UV light etc.) ▪The process begins with the excision of the phage genome out of the DNA of the host cell, continues replication of the phage DNA and synthesis of phage structure proteins, and finally ends with host cell lysis (breakdown of cell membrane). ▪Cells carrying a prophage are called lysogenic because they contain the genetic information for lysis. ▪Lysogeny has advantages for both sides. It prevents immediate host cell lysis, but also ensures that the phage genome replicates concurrently with host cell reproduction.  Importance of Bacteriophages ▪Biological research: DNA replication, gene expression, gene regulation, viral morphogenesis etc. ▪Genetic engineering: vectors for gene cloning, adjuvants in sequencing. ▪Therapy and prevention: gastrointestinal infections, animal husbandry (EHEC infections). ▪Epidemiology: bacterial typing and DNA typing. This has been established for Salmonella typhi, Salmonella paratyphi B., Staphylococcus aureus, Pseudomonas aeruginosa.  Types of Transduction Generalized transduction can transfer any gene of donor bacteria to recipient bacteria. During the replication of a lytic phage the capsid sometimes encloses a small fragment of lysed bacterial DNA, instead of phage DNA, by a "head-full" mechanism. This is a defective phage. Such a phage cannot lyse another bacterium because the DNA in the phage head does not have the genetic information to produce a phage genome and proteins. On infection of another bacterium the defective phage injects the fragment of donor bacterial DNA into the recipient bacteria, where it can be exchanged for a piece of the recipient's DNA, if their sequences are homologous.  Generalized Transduction Steps in Generalized Transduction Release of phage Phage replication and degradation of host DNA Assembly of phages particles and encapsidation of host DNA Infection of recipient Homologous recombination  Specialised Transduction ▪A transduction in which only certain donor genes can be transferred to the recipient ▪Occur during the lysogenic life cycle of a temperate phage ▪During spontaneous induction of lysogeny, a small piece of bacterial DNA may sometimes be exchanged for a piece of phage genome. ▪This piece of bacterial DNA replicates as a part of the phage genome and is incorporated into capsid of each phage progeny ▪On infection of a recipient bacteria, the phage DNA containing donor bacterium genes are injected into the recipient bacterium where donor ▪DNA fragments can be exchanged for a piece of the recipient's DNA, if their sequences are homologous. ▪Different phages may transfer different genes but an individual phage can only transfer certain genes ▪Lysogenic (phage) conversion occurs in nature and is the source of virulent strains of bacteria, e.g. toxin production in Cl. botulinum, C. diptheriae, STEC, etc.  Conjugation ▪Conjugation is the transfer of DNA from a donor to a receptor in a conjugal process involving cell–to–cell contact. Conjugation is made possible by two genetic elements: conjugative plasmids and conjugative transposons. ▪ In the conjugative process, the conjugative elements themselves are primarily transferred. ▪However, these elements can also mobilise chromosomal genes or otherwise non – transferrable plasmids. ▪Conjugation is seen commonly in gram – negative rods, and enterococci.  Conjugative plasmids Conjugative plasmids are extra-chromosomal DNA elements that are capable of horizontal transmission and are found in many natural isolated bacteria. ▪Although plasmids may carry beneficial genes to their bacterial host, they may also cause a fitness cost. ▪Three characteristics of conjugative plasmids promote a highly efficient horizontal spread of these determinant factors among different bacteria: ▪High frequency of gene transfer – due to the ‘transfer replication’ mechanism, each receptor cell that has received a conjugative plasmid automatically becomes a donor cell. ▪Wide range of hosts – can be transferred to different taxonomic species, genera, and even families. ▪Multiple determinants – many conjugative plasmids carry several genes determining the phenotype of the carrier cell.  Conjugative Transposons ▪Conjugative transposons are integrated DNA elements that excise themselves to form a covalently closed circular intermediate. ▪Occur mainly in Gram-positive cocci but have also been found in gram–negative bacteria (Bacteroides). ▪Carry many determinants for antibiotic resistance and contribute to horizontal gene transfer. ▪In the transfer process, the transposon is first excised from the chromosome and circularized. ▪A single strand of double helix is cut and the linearized single strand is transferred to the receptor cell. ▪Are also capable of mobilizing non – conjugative plasmids.  Restriction, Modification and Gene Cloning ▪A number of control mechanisms limit genetic exchange processes in bacterial species – restriction and modification. ▪Restriction endonucleases can destroy foreign DNA that bears no ‘fingerprint’ (modification) signifying ‘self’. ▪Bacterial restriction endonucleases are valuable tools in modern gene cloning techniques. ▪Cloning – involves replication of DNA that has been manipulated in vitro in a suitable host cell so as to produce identical copies of this DNA: OR GENE CLONES. This simplifies DNA replication, making experimental manipulations easier. ▪Bacteria can also be used to synthesize gene products of foreign genes – recombinant proteins. ▪Bacterial plasmids often function in the role of vectors into which the sequences to be cloned are inserted.  Genetic Engineering ▪Artificial means, when a gene of one species is transferred to another organism, it is called recombinant DNA technology. ▪Allows the introduction of new DNA into cells. ▪Tools of genetic engineering: ▪Restriction endonucleases – bacterial enzymes that can cut/ split DNA at specific sites. ▪Recognition sites – where DNA is cut by a restriction endonuclease (can recognise DNA at a particular sequence). ▪Cleavage patterns – cut DNA fragments by restriction endonucleases that may have blunt/sticky ends. ▪DNA ligases – cut DNA fragments that are covalently joined together.  Genetic Engineering ▪Host cells – living cells which the carrier of rDNA molecule can be propagated. Microorganisms are preferred as host cells due to high rates of multiplication (eg. E. coli) ▪Vectors – DNA molecules which carry DNA fragments to be cloned (usually self replicating). Examples: Plasmids (E. Coli) Bacteriophages Artificial chromosome vectors. END OF LECTURE 5