Lecture 6: General Microbiology - MLAB 213 PDF

General microbiology MLAB 213 Microbial Genetics Mira El Chaar, PhD Medical laboratory sciences  Bacterial genetics Bacterial chromosome: Most bacteria possess one chromosome, which usually consists of a long, continuous (circular), double-stranded DNA mo...

General microbiology MLAB 213 Microbial Genetics Mira El Chaar, PhD Medical laboratory sciences  Bacterial genetics Bacterial chromosome: Most bacteria possess one chromosome, which usually consists of a long, continuous (circular), double-stranded DNA molecule, with no protein on the outside Bacterial plasmid: Plasmid is a DNA molecule that is separate from chromosomal DNA It can replicate independently They are double-stranded and circular It can integrate into the chromosome and is referred to as an episome  1origin of replication Replication of E. coli chromosome - E. coli has a circular chromosome with a single origin of replication (oriC). 5' to 3' -There are 2 replication forks on each chromosome, replicating in opposite directions topoisimerase  The point at which replication occurs is called the replication fork  As the replication fork moves along the parental DNA, each of the unwound single strands combines with new nucleotides  As the replication fork moves along the parental DNA, the two new strands must grow in different directions.  DNA replication requires a great deal of energy.  DNA replication by some bacteria, such as E. coli, goes bidirectionally around the chromosome  Two replication forks move in opposite directions away from the origin of replication.  After duplication, if each copy of the origin binds to the membrane at opposite poles, then each daughter cell receives one copy of the DNA molecule-that is, one complete chromosome. 1. The two strands of parental DNA are unwound by helicase (break the hydrogen bond) no mistake as opposed to RNA polymerase 2. DNA polymerase (proofreading Capability) add nucleotide to a free 3’ end of growing chain. 3. Synthesis of one strand of the DNA, called the leading strand proceed from 5’ to 3’  Bacterial protein synthesis 1-Transcription During transcription, a strand of mRNA is synthesized using a specific portion of the cell's DNA as a template The process of transcription requires both an enzyme called RNA polymerase and a supply of RNA nucleotides Transcription begins when RNA polymerase hinds to the DNA at a site called the promoter RNA is synthesized in the 5' - 3' direction  Transcription le RNA qui vient d'etre produit sort et les DNA strands initiaux se reassemblent Translation  Translation Translation is the process in which the information in the nucleotide base sequence of mRNA is used to dictate the amino acid sequence of a protein. Specific amino acids are attached to molecules of tRNA. Another portion of the tRNA has a base triplet called an anticodon. The ribosome moves along the mRNA strand as amino acids are joined to form a growing polypeptide; mRNA is read in the 5' - 3' direction There are 64 possible codons but only 20 amino acids  Eukaryotic Vs Prokaryotic replication Transcription takes place in nucleus mRNA completed prior to entry in cytoplasm Exons : Expressed DNA, code for protein Introns : intervening DNA, do not code for protein Intron derived RNA are removed by ribozymes  -virulent factors and resistance and Bacterial plasmids bacteriocins pas forcement circulaires genetic elements that replicate independently of the chromosome 1- 5% the size of the bacterial chromosome exist mostly as circular dsDNA (but many linear plasmids are known) they carry only unessential genes (but often helpful) Most of plasmid DNA isolated from cells is supercoiled plasmids copy number varies among cells (from 1 to > 100 copies)  Types of plasmids a- Resistance plasmids - they are among the most widespread and most studied plasmids - they confer resistance to antibiotics and other various growth inhibitors - the infectious nature of these R plasmids permitted their spread though populations by cell to cell contact. - They are a major problem in clinical medicine - several antibiotic resistance genes can be carried by R plasmids. These genes encode enzymes that inactivate the drug or inhibit its uptake by the cell Ex: the R100 plasmid  The R100 plasmid if someone has these genes, we cant give them tetracycline or sulfonamide or chloramphenicol because they have a resistance to them. complex insertion - oriT = origin of conjugative transfer sequence : CONTAINS MANY RESISTANT GENES - tra = transfer functions BETWEEN THEM - tet = tetracycline resistance coded by insertion sequences , can jump from plasmid to another or even from plasmid to chromosome - cat = chloramphenicol resistance SIMPLE IS : NO - sul = sulfonamide resistance GENE BETWEEN THEM insertion sequences stick on right and left of tet gene this is where a protein binds and can transfer it to another bacteria Can be transferred between enteric Gram-negative bacteria of the genera Escherichia, Klebsiella, Proteus, Shigella, Salmonella, b- Plasmids encoding toxins and other virulence factors Two major characteristics are involved in the virulence of pathogens: i- ability to attach and to colonize specific host tissues ii- formation of toxins, enzymes and other factors that cause damage to the host In many bacteria each of these virulence characteristics is carried on plasmids (Note: virulence factors can also be encoded in chromosomes as well as in transposons and bacteriophages) Ex: In E. coli  - the colonization factor antigen, a surface protein that allows cells to attach to epithelial cells of the midgut, is encoded on a plasmid watery diarrhea  - at least 2 toxins, hemolysin (which lyses blood cells) and enterotoxin (which induces extensive secretion of water and salts into the bowel) are encoded on a plasmid can be coded by a plasmid found in the bacteria c- Plasmids encoding bacteriocins can be found in only some bacterias proteins that have characteristics like antibiotics but with a narrower spectrum - Bacteriocins Inhibit or kill closely related bacterial species or even different strains of the same species - they have narrow spectrum of activity unlike antibiotics can destroy some species around them - they are encoded on plasmids or transposons - on peut voir les 3 types : bacteriocins, virulent factors et resistance dans le meme plasmid pas obliges qu'ils soient separes promoter : place where RNA polymerase sits operator (controlling site) : activator or repressor site like traffic lights : start or stop transcription operon = promoter + operator + coding genes  The Regulation of Bacterial Gene Expression  Operon: A set of operator and promoter sites and the structural genes they control (lac operon).  Operator: which is like a traffic light that acts as a go or stop signal for transcription of the structural genes (Controlling site)  The promoter: is the region of DNA where RNA polymerase initiates transcription.  Regulatory gene: encode a repressor protein that switches inducible and repressible operons on or off.  Many of the genes in E. coli are expressed constitutively  Other genes are expressed only when their products are needed by the cell, so their expression must be regulated.  When lactose enter the cell, a small number amount of it is permease protein : allows lactose to enterconverted to allolactose metabolizes lactose The Lac operon (Inducible operon)  Lactose Transport of Enzyme that transfers an catabolism lactose into the acetyl group from acetyl coA cell to a beta galactoside Most prokaryotic genomes are organized into clusters of more than one coding region that often collaborate on a single task such as metabolism of sugar (ex: Lactose) into by product that can be used for energy  Inducible operon (Lac operon) repressive proteins are coded by regulatory genes In the absence of Lactose: No transcription repressive protein active sits on promoter (acitve) = RNA polymerase can't sit = no transcription In the presence of Lactose: enzyme transcribed allolactose binds to repressive proteins (active -> inactive repressive protein) can't sit on operator because it is inactive so there is free space on operator for RNA polymerase and transcription, translation of the 3 enzymes can occur and metabolism is possible  Lactose as a Carbon Source for E. coli ALWAYS PRESENT 1. E. coli expresses genes for glucose metabolism constitutively, but the genes for metabolizing other sugars are regulated in a “sugar specific” sort of way. Presence of the sugar stimulates synthesis of the proteins needed 2. Lactose is a disaccharide (glucose 1 galactose). If lactose is E. coli’s sole carbon source, three genes are expressed: a. β-galactosidase has two functions: i. Breaking lactose into glucose and galactose. Galactose is converted to glucose, and glucose is metabolized by constitutively produced enzymes. ii. Converting lactose to allolactose (an isomerization). Allolactose is involved in regulation of the lac operon b. Lactose permease (M protein) is required for transport of lactose across the cytoplasmic membrane. c. Transacetylase (an enzyme that transfers an acetyl group from acetyl-CoA to β- galactosidesis ) is poorly understood. 3-The lac operon shows coordinate induction: a. In glucose medium, E. coli normally has very low levels of the lac gene products. b. When lactose is the sole carbon source, levels of the three enzymes increase coordinately (simultaneously) about 1,000-fold. i. Allolactose is the inducer molecule ii. The mRNA for the enzymes has a short half-life. When lactose is gone, lac transcription stops, and enzyme levels drop rapidly.  The regulatory mechanism that inhibits gene expression and decreases the synthesis of enzymes is called repression. Repression is mediated by regulatory proteins called repressors, which block the ability of RNA polymerase to initiate transcript ion from the repressed genes. The process that turns on the transcription of a gene or genes is induction A substance that acts to induce transcription of a gene is called an inducer (allolactose), and enzymes that are synthesized in the presence of inducers are inducible enzymes (beta galactosidase) What would the transcription be regulated if there is a lot of Glucose and/ or Lactose?  Positive regulation Lac Operon regulation Catabolic activator protein Negative regulation The concentration of cyclic AMP in E. coli is inversely proportional to the concentration of glucose: as the concentration of glucose decreases, the concentration of cyclic AMP increases LOW GLUCOSE = high cAMP (binds to inactive CAP and makes it active) binds to promoter before RNA polymerase and orders to produce RNA polymerase of the 3 enzymes High GLUCOSE = low cAMP (inactive CAP)  Positive Control of the lac Operon 1. Repressor exerts negative control by preventing transcription. Positive control of this operon also occurs when lactose is E. coli’s sole carbon source a.Catabolite activator protein (CAP) binds cyclic AMP (cAMP) b. CAP-cAMP complex is a positive regulator of the lac operon. It binds the CAP-site, a DNA sequence upstream of the operon’s promoter. c. Binding of CAP-cAMP complex causes the DNA to bend, facilitating protein- protein interactions between CAP and RNA polymerase, and leading to transcription. When both glucose and lactose are in the medium, E. coli preferentially uses glucose, due to catabolite repression. a. Glucose metabolism greatly reduces cAMP levels in the cell. b. The CAP-cAMP level drops, and is insufficient to maintain high transcription of the lac genes. c. Even when allolactose has removed the repressor protein from the operator, lac gene transcription is at very low levels without CAP-cAMP complex bound to the CAP-site. d. Experimental evidence supports this model. Adding cAMP to cells restored transcription of the lac operon, even when glucose was present. Inducible versus Repressible operon ????  Repressible operon (tryptophan operon) In repressible operons, the structural genes are transcribed until they are turned off, or repressed When low concentration of tryptophan: Enzyme are transcribed In the presence of tryptophan: No transcription if trp present : repressive proteins passes from inactive to active = blockage of promoter = RNA polymerase can't sit = inhibition of enzymes pas besoin de retenir ces enzymes  allolactose trp conserves energy production of enzymes : loses energy Two genetic control mechanisms known as repression and induction regulate the transcription of mRNA  Mutation, Repair, and Recombination during replication mutation can occur A mutation is any change in the base sequence of the DNA. A silent mutation is a change at the DNA level that does not result in any change of amino acid in the encoded protein. A missense mutation results in a different amino acid being inserted in the protein, but this may be a conservative mutation if the new amino acid has similar properties A nonsense mutation changes a codon encoding an amino acid to a stop codon (e.g., TAG [thymidine- adenine-guanine]), which will cause the ribosome to fall off the mRNA and end the protein prematurely immature protein And may lead to a non functional protein Frameshift mutation: A small deletion or insertion that is not in multiples of three. This results in a change in the reading frame, usually leading to a useless peptide and premature truncation of the protein. Null mutations, which completely destroy gene function, arise when there is an extensive insertion, deletion, or gross rearrangement of the chromosome structure. Spontaneous mutation: occasional mistakes made during DNA replication. These spontaneous mutations apparently occur in the absence of any mutation-causing agents (chemical, radiation) Conditional mutations, such as temperature-sensitive mutations, may result from a conservative mutation which changes the structure or function of an important protein at elevated temperatures Amino acid mutation spontaneous  Gene Exchange in Prokaryotic Cells Bacterial plasmid: Plasmid is a DNA molecule that is separate from chromosomal DNA It can replicate independently They are double-stranded and circular It can integrate into the chromosome and is referred to as an episome Genetic Transfer and Recombination There are four ways that the genetic composition of bacteria can be changed: Transduction Transformation Conjugation  Mechanisms of Genetic Transfer between Cells when a cell dies = lysis fragments of DNA released and some Bacteriophage of them have receptors on another cell = integration of DNA viral genome integrated LYTIC LYSOGENIC pilli F+ have pilli F- does not so F+ gives to F- F- becomes F+ jumping genes (insertion sequence for example) plasmid or whole chromosome can be transferred  Transformation A bacterial cell becomes genetically transformed following the uptake of DNA fragments (“naked DNA”) from the environment. not whole DNA!! It has been demonstrated to occur in several genera including Bacillus, Escherichia, Haemophilus, Pseudomonas, and Neisseria. oublie ce point Extracellular pieces of DNA molecules can only penetrate the cell wall and cell membrane of certain bacteria The ability to absorb “naked DNA” into the cell is referred to as competence, and bacteria capable of taking up “naked DNA” molecules are said to be competent bacteria. able to take free DNA Transformation was first demonstrated in Griffith’s experiment (Frederick Griffith), using Streptococcus pneumoniae, and is a process that occurs naturally among a few genera of bacteria DNA that codes for capsid transformed into DNA of nonencapsulated bacteria  Griffith’s experiment (a) Inject living encapsulated bacteria into mice, mice die, encapsulated bacteria isolated from dead mice. (b) Inject living nonencapsulated bacteria into mice, mice remain healthy, a few non-encapsulated bacteria can be isolated from the living mice – most phagocytized by leukocytes. (c) Inject heat-killed encapsulated bacteria into mice, mice remain healthy, no bacteria isolated from the living mice. (d) Inject living non-encapsulated and heat-killed encapsulated bacteria into mice, mice die, isolated encapsulated bacteria from dead mice The mechanism of genetic transformation in bacteria aquiring new characteristics due to recombination  Recombination RecA important to recombinate  Conjugation A bacterial cell (called the donor cell) possessing a sex pilus attaches by means of the sex pilus to another bacterial cell (called the recipient cell). Some genetic material (usually in the form of a plasmid) is then transferred through the hollow sex pilus from the donor cell to the recipient cell. This type of genetic recombination occurs mostly among species of enteric, Gram-negative bacilli, but has been reported within species of Pseudomonas and Streptococcus as well Although many different genes may be transferred via conjugation, the ones most frequently noted include those coding for antibiotic resistance and fertility factors (F)  Sex pilus produced by donor cells during conjugation - allows specific pairing between the donor and recipient cells - makes contact with a receptor on bacteria and then retracts pulling the 2 cells together - the contact between the cells is then stabilized probably by fusion of the outer membranes  Cell to cell contact induces a nick in one strand of plasmid DNA circle, and nicked strand is transferred to recipient cell Nick formation Unwinding of nicked strand by helicase DNA synthesis by the rolling circle mechanism in the donor replaces the transferred strand complementary DNA strand synthesis in the recipient  Transduction Some bacterial genetic material may be “carried across” from one bacterial cell to another by a bacterial virus (temperate bacteriophage) If a stimulating chemical, heat, or ultraviolet light activates the prophage, it begins to produce new viruses via the production of phage DNA and proteins As the chromosome disintegrates, small pieces of bacterial DNA may remain attached to the maturing phage DNA During the assembly of the virus particles, one or more bacterial genes may be incorporated into some of the mature bacteriophages When all the phages are released by cell lysis, they proceed to infect other cells, some injecting bacterial genes as well as viral genes  Role of bacteriophage in genetic transfer  There are two categories of bacteriophages (phages): virulent phages and temperate (lysogenic) phages.  Lysogenic conversion  lysogenic phages inject their DNA into the bacterial cell. The phage DNA integrates into the bacterial chromosome but does not cause the lytic cycle to occur (lysogeny)  During lysogeny, all that remains of the phage is its DNA; in this form, the phage is referred to as a prophage.  The bacterial cell containing the prophage is referred to as a lysogenic cell  Each time a lysogenic cell undergoes binary fission, the phage DNA is replicated along with the bacterial DNA and is passed on to each of the daughter cells.  Daughters cells are also lysogenic cells.  the bacterial cell can produce gene products that are coded for by the prophage genes.  The bacterial cell will exhibit new properties: lysogenic conversion  Virulent phage 1. Phage attaches to a specific host bacterium 2. Injection of its DNA 3. Disruption the bacterial genome and killing the bacterium 4. Biosynthesis: Overtaking the bacterial DNA and protein synthesis machinery to make phage parts. 5. The process culminates with the assembly of new phage 6. The lysis of the bacterial cell wall to release a hundred new copies Virulent phages always cause the lytic cycle to of the input phage into the occur, ending with the destruction (lysis) of the environment bacterial cell.  TRANSPOSONS Mobile genetic elements can transfer DNA within a cell, from one position to another in the genome, or between different molecules of DNA The simplest transposons are called insertion sequences and range in length from 150 to 1500 base pairs Complex transposons carry other genes, such as genes that provide resistance against antibiotics Transposons may insert into genes and inactivate those genes A, The insertion sequences code only for a transposase (tnp) and possess inverted repeats (15 to 40 base pairs) at each end. B, The composite transposons contain a central region coding for antibiotic resistances or toxins flanked by two insertion sequences (IS) C, Tn3, a member of the TnA transposon family. The central region encodes three genes—a transposase (tnpA), a resolvase (tnpR), and a β-lactamase—conferring resistance to ampicillin. Usage of bacteria for genetic engineering usage Genetic engineering or recombinant DNA technology: Techniques developed to transfer eukaryotic genes, particularly human genes, into other easily cultured cells to facilitate the large-scale production of important gene products (proteins, in most cases). Genetically engineered microorganisms can also be used to clean up the environment (e.g., to get rid of toxic wastes). Genetically engineered microorganisms is used to engineer antibodies, antibiotics , drugs and vaccines Gene therapy Gene therapy of human diseases involves the insertion of a normal gene into cells to correct a specific genetic or acquired disorder that is being caused by a defective gene  Gene cloning using bacterial plasmids Plasmids and steps in gene cloning: 1- isolation and fragmentation of source DNA (using shearing of genomic DNA, restriction enzymes or use PCR generate fragments) 2- Joining DNA fragments to a vector with DNA ligase 3- Introduction and maintenance of cloned DNA in a host organism This is how gene libraries are produced  Restriction enzymes - recognize and cleave DNA within particular sequences of 4 to 8 nucleotides which have a 2 fold axis of rotational symmetry often called palindromes -The bacterial DNA is protected from digestion because the cell methylates (adds methyl groups to) some of the cytosines in its DNA - they allow bacteria to monitor the origin of incoming DNA and to destroy it if it is recognized as foreign (ex: bacteriophage) 5'- GAA TTC -3' 3'- CTT AAG -5' 5'- G/AA TTC -3' Cleavage site for EcoRI 3'- CTT AA/G -5' 5'- G 5'- AATTC -3' Cohesive ends 3'- CTTAA-5' G -5' produced  The Frequency of Mutation MUTATION IN VIRUSES MORE THAN BACTERIA Spontaneous mistakes in DNA replication occur at a very low rate Mutations usually occur more or less randomly along a chromosome Most mutations either are harmful and likely to be removed from the gene pool when the individual cell dies or are neutral The bacteria have a repair system for the mutation Mutation may be beneficial (antibiotic resistance)