CHAPTER 11 MICROBIAL GENETICS AND INFECTIOUS DISEASE WHY IS THIS IMPORTANT? Understanding genetic mechanisms lets us study how microorganisms can mutate and change in ways that allow them to defeat host defenses These changes are one of the most important topics in health care today To understand pathogenesis and virulence, we must be familiar with microbial genetics WHY IS THIS IMPORTANT? One of the most difficult problems in medicine today is antibiotic resistance Organisms can become resistant to antibiotics through mutations Mutations can be transferred to other bacteria Mutations can make a harmless bacterium pathogenic and a pathogenic bacterium more virulent and even lethal 1
OVERVIEW Microbial Genetics and Infectious Disease THE STRUCTURE GENETIC CODE MUTATION AND TRANSFER OF GENETIC OF DNA AND RNA REPAIR OF DNA INFORMATION DNA REPLICATION GENE EXPRESSION GENETICS AND PATHOGENICITY REGULATION OF GENE EXPRESSION DNA DNA stands for deoxyribonucleic acid DNA is a blueprint for all components of the cell The blueprint can be faithfully passed on from one generation to the next The structure of DNA maximizes the ease of replication and makes gene expression, defined as the process of transcription (DNA to RNA) and translation (RNA to protein), efficient and almost error-free DNA STRUCTURE DNA is a double-stranded helical structure It is composed of nucleotides Each nucleotide is a combination of a phosphate, a sugar (which in DNA is deoxyribose), and a nucleotide base 2
DNA STRUCTURE The two strands are complementary and wind around each other to form the double helix The bases project inward The components of DNA bind together in a very specific way This permits a correct and precise orientation of the nucleotide DNA STRUCTURE DNA STRUCTURE 3
DNA STRUCTURE DNA STRUCTURE Nucleotides join to each other to form a chain The 3 hydroxyl group of a sugar joins to the 5 hydroxyl of another nucleotide This makes the linkage inherently polarized And gives structural orientation to the growing chain DNA STRUCTURE 4
DNA STRUCTURE DNA has two types of bases Purines adenine and guanine Purines are large double-ring structures Pyrimidines thymine and cytosine Pyrimidines have smaller single ring structures DNA STRUCTURE DNA STRUCTURE DNA has a helical geometry governed by how the bases pair up Adenine always pairs with thymine Cytosine always pairs with guanine 5
DNA STRUCTURE The strands are anti-parallel One of the strands is oriented upside down relative to the other The bases are stacked on top of each other DNA is a chemically stable molecule Any mismatched pairing is chemically unstable RNA RNA stands for ribonucleic acid RNA differs from DNA in several ways It contains the sugar ribose (rather than deoxyribose) It contains uracil instead of thymine Uracil pairs up with adenine It is usually found in single-stranded form RNA There are three forms of RNA: Messenger RNA contains information derived from DNA Transfer RNA carries amino acids to ribosomes Ribosomal RNA helps maintain the proper shape of ribosomes 6
RNA DNA REPLICATION This is the process by which DNA is copied DNA replication involves specific components and mechanisms It is a critical cellular procedure accomplished with remarkable accuracy and at astounding speed DNA REPLICATION: Separation and Supercoiling Supercoiling is a characteristic of helical structures Strands must be uncoiled, unwound, and separated before replication This is accomplished by two enzymes: Topoisomerase unwinds the supercoils Helicase separates and unwinds the strands 7
DNA REPLICATION: Requirements There are two requirements for replication: An ample supply of each of the four nucleotides A primer:template junction Each single strand of DNA is a template A portion of the DNA is paired with a short piece of RNA called a primer DNA REPLICATION: Requirements DNA REPLICATION: Direction DNA replication proceeds in only one direction The primer at the primer:template junction gives the DNA polymerase a place to add the next base Binding is between the 3 end of one base and the 5 end of the next base 8
DNA REPLICATION: Direction Elongation of the bases is from the 3 end This is required for chemical stability The binding of a new base uses energy released from pyrophosphate DNA REPLICATION: Direction DNA POLYMERASE DNA replication is performed by an enzyme called DNA polymerase DNA polymerase forms new strands of DNA using the primer:template junction as a guide 9
DNA POLYMERASE It works incredibly quickly The addition of nucleotides is in the millisecond range There are several types of DNA polymerase They perform specific functions and work at different speeds DNA POLYMERASE: Proofreading DNA replication is extraordinarily accurate There are always some mistakes mutations Evolution relies on mutations During replication, an error occurs approximately once in 10 10 pairings DNA POLYMERASE: Proofreading Proofreading takes place at the newly synthesized strand DNA polymerase contains an exonuclease component to remove improperly paired bases 10
DNA POLYMERASE: Proofreading THE REPLICATION FORK In the replication fork, the double helix is unwound and the strands separate DNA replication occurs at the replication fork The separated strands at the replication fork are anti-parallel and are identified as: Leading strand Lagging strand THE REPLICATION FORK 11
THE REPLICATION FORK The leading strand is in a perfect position for the addition of nucleotides to its 3ʹ end Replication moves towards the replication fork THE REPLICATION FORK The lagging strand is anti-parallel It moves away from the replication fork For nucleotides to be added to a growing DNA strand, there has to be a free 3ʹ end that the polymerase can use The lagging strand is replicated in pieces called Okazaki fragments THE REPLICATION FORK Each Okazaki fragment has its own short RNA primer It is created by an RNA polymerase called primase When the fragment is finished, the enzyme RNAase H removes the primer The gap is filled in by DNA polymerase Fragments are linked together by DNA ligase 12
INITIATION AND TERMINATION OF REPLICATION Initiation begins at a specific site on the chromosome The origin of replication Termination occurs when the entire chromosome has been copied Replicated chromosomes are separated by topoisomerase INITIATION AND TERMINATION OF REPLICATION THE GENETIC CODE Information in DNA is based on a four letter alphabet (A, T, C, G) The genetic code employs three letter combinations called codons 13
THE GENETIC CODE There are 64 possible three letter combinations Only 20 amino acids are used to make proteins The genetic code is degenerate THE GENETIC CODE THE GENETIC CODE Three rules govern the arrangement and use of codons: Codons are always read in one direction The message is translated in a fixed reading frame There is no overlap or gap in the code 14
GENE EXPRESSION A gene is a segment of DNA that codes for a functional product Gene expression is the production of the functional product Gene expression has two features: It involves specific interactions between DNA and RNA It is highly regulated GENE EXPRESSION There are two parts to gene expression: Transcription construction of RNA from a DNA template Translation construction of the protein using RNA instructions TRANSCRIPTION The process by which RNA is made from a DNA template It does not require a primer:template junction RNA does not remain base-paired to DNA It is not as accurate as DNA synthesis RNA polymerase is a poor proofreader 15
TRANSCRIPTION Transcription has three steps: Initiation RNA polymerase binds to a DNA sequence called the promoter: This produces a bubble in the DNA Elongation RNA polymerase unwinds strands of DNA and synthesizes the RNA: It also re-anneals the strands Termination a sequence of DNA signals the end of transcription: RNA polymerase detaches from DNA TRANSCRIPTION TRANSLATION This is the process by which proteins are made The sequence of nucleotides in messenger RNA is translated into a sequence of amino acids It is directly affected by any errors in either DNA or RNA 16
TRANSLATION It is a highly conserved function seen in all cells It requires high levels of energy Translation requires all three types of RNA messenger, transfer, and ribosomal MESSENGER RNA (mrna) IN TRANSLATION An open reading frame (ORF) indicates the start of an amino acid sequence An ORF begins with a start codon Translation moves from the 5 end to the 3 end An ORF ends with a stop codon mrna IN TRANSLATION mrna contains a segment that recruits the ribosomal subunits Ribosome and mrna bind here through complementary base pairing 17
TRANSFER RNA (trna) IN TRANSLATION Each trna attaches to a specific amino acid at the acceptor arm It brings amino acids to the ribosome It binds to the ribosome at the anti-codon region using complementary base pairing trna IN TRANSLATION THE RIBOSOME IN TRANSLATION The ribosome is composed of three molecules of rrna and over 50 proteins It adds amino acids at a rate of 2-20 amino acids per second More than one ribosome can move along the same messenger RNA This is called a polyribosome or polysome 18
THE RIBOSOME IN TRANSLATION FORMATION OF PEPTIDE BONDS IN TRANSLATION Peptide bonds form between amino acids while on the ribosome The ribosome has three sites: A site trna brings in new amino acid P site trna holds the growing amino acid chain E site trna exits the ribosome FORMATION OF PEPTIDE BONDS IN TRANSLATION 19
FORMATION OF PEPTIDE BONDS IN TRANSLATION The ribosome is a honeycombed structure with tunnels The components of protein synthesis enter these tunnels and move through them mrna trna Growing polypeptide chain STAGES OF TRANSLATION There are three stages of transcription: Initiation Elongation Termination INITIATION Initiation requires: Recruitment of the ribosome to the mrna Placement of a methionine trna complex at the P site Precise positioning of the ribosome over the start codon of mrna 20
INITIATION ELONGATION After initiation, three things must occur in order for amino acids to be added to methionine A trna carrying the next amino acid is loaded into the A site A peptide bond forms between the amino acids Each trna moves the one at the A site to the P site, the one at the P site to the E site The ribosome moves along the messenger RNA TERMINATION Translation continues until a stop codon enters the A site Stop codons are recognized by specialized proteins These specialized proteins cause the translation complex to break down The peptide chain is released from the ribosome and begins to form secondary and tertiary structures 21
REGULATION OF GENE EXPRESSION Protein synthesis is energetically expensive and highly regulated Some genes are always turned on constitutive genes Some genes are on and can be turned off repressible genes Some genes are off and can be turned on inducible genes REGULATION OF GENE EXPRESSION Gene expression is controlled by regulatory proteins: Activators involved in positive regulation Repressors involved in negative regulation Both types are DNA binding proteins REGULATION OF GENE EXPRESSION Regulatory proteins recognize two sites on DNA near the genes they control The promoter where RNA polymerase binds The operator where regulatory proteins bind The two sites are adjacent 22
INDUCTION Induction turns on genes that are off (repressed) The best example is the lac operon: An operon is a set of genes that are co-regulated There are many operons in the chromosome lac Operon lac Proteins The lac system has two regulatory proteins The lac repressor protein The lac activator - CAP (catabolite activator protein). Both proteins bind at the operator site on DNA 23
lac Repressor The lac repressor is always produced It binds at the operator site and overlaps part of the promoter site This blocks the RNA polymerase from attaching This prevents transcription OPERATION OF THE lac OPERON lac ACTIVATOR CAP also binds at the operator site It recruits RNA polymerase to the site It then interacts with the polymerase so it binds properly 24
EXPRESSION OF lac OPERON For the genes of the lac operon to be turned on, the repressor must first be inhibited This occurs through an allosteric control mechanism EXPRESSION OF lac OPERON The expression of lac genes is leaky A few transcripts are made and there is always a low level of β-galactosidase This allows small amounts of lactose into the cell. Lactose is converted to allolactose Allolactose binds the lac repressor This changes the shape of the lac repressor and it can no longer bind the operator site EXPRESSION OF lac OPERON CAP acts in a similar fashion to allolactose Its activity is based on levels of cyclic AMP (camp) 25
EXPRESSION OF lac OPERON When camp levels rise, camp binds to CAP This causes a change in the three-dimensional shape of CAP The CAP-cAMP complex binds to DNA This helps the RNA polymerase bind to the promoter site The lac genes are expressed EXPRESSION OF lac OPERON When camp levels fall, no complex is formed RNA polymerase does not bind to the promoter site The lac genes are not expressed REPRESSION There are also cellular mechanisms that turn off (repress) genes This is very important for the conservation of energy Repression has similar mechanisms to feedback inhibition 26
REPRESSION A good example of repression is the synthesis of tryptophan The tryptophan repressor is always produced but cannot bind DNA in its normal form Excess tryptophan binds the repressor and changes its shape so it can bind DNA and prevent gene expression Tryptophan is a co-repressor of its own synthesis TRYPTOPHAN OPERON TRYPTOPHAN OPERON 27
TRYPTOPHAN OPERON MUTATION & REPAIR OF DNA Mutations are changes in the DNA sequence Change in DNA sequence can cause changes in proteins Mutations must be kept to a minimum MUTATION & REPAIR OF DNA The simplest type of mutation is classified as a point mutation In this instance, one nucleotide is switched for another More drastic mutations are classified as frameshift mutations This is caused by insertion or deletion of nucleotides 28
MUTATION & REPAIR OF DNA MUTATION & REPAIR OF DNA Spontaneous mutation rates are low Certain sections of the chromosome have a higher rate of spontaneous mutation These are called hot spots There are also suppressor mutations Suppressor mutations can reverse the primary mutation HOW DNA DAMAGE OCCURS DNA can be damaged by: Hydrolysis Deamination Alkylation Oxidation Radiation 29
HOW DNA DAMAGE OCCURS Gamma radiation and ionizing radiation cause double-strand breaks in DNA Ultraviolet radiation causes DNA damage through the formation of thymine dimers Radiation damage impairs replication HOW DNA DAMAGE OCCURS Base analogs look like DNA bases but are not They can be mistakenly used in replication This impairs further replication HOW DNA DAMAGE OCCURS 30
REPAIR OF DNA DAMAGE Three principle mechanisms of DNA repair Base excision Nucleotide excision Photoreactivation REPAIR OF DNA DAMAGE During base excision: Repair enzymes look for damaged bases The damaged base is removed (excised) from the double helix A DNA polymerase fills in the gap A DNA ligase repairs the break in the strand REPAIR OF DNA DAMAGE During nucleotide excision repair: Repair enzymes look for distortions in the helix A short section of DNA surrounding the distortion is removed DNA polymerase fills in removed sections DNA ligase repairs the break in the strand 31
REPAIR OF DNA DAMAGE REPAIR OF DNA DAMAGE Photoreactivation repairs thymine dimers It is accomplished by an enzyme called photolyase Photolyase binds to the dimer in the dark When the DNA is then exposed to light, the photolyase becomes activated and breaks the thymine thymine bond REPAIR OF DNA DAMAGE 32
TRANSFER OF GENETIC INFORMATION Bacteria can shuffle genes This is called genetic recombination There are four ways in which genetic recombination can occur: Transposition within the same cell Transformation between cells Conjugation between cells Transduction between cells TRANSPOSITION Transposition is caused by transposons Transposons move from one place on the chromosome to another They can move into or out of the chromosome They use cleavage and rejoining mechanisms TRANSPOSITION Transposition causes random rearrangements The results can be beneficial or detrimental Beneficial changes will be selected for and maintained They may be the reason for several human diseases 33
TRANSFORMATION Transformation involves the transfer of genetic material between cells It involves naked DNA This DNA is taken up by a bacterial cell and recombines with genes of that cell TRANSFORMATION The recipient cell must be competent Must be able to take up large molecules such as pieces of DNA Some bacteria are naturally competent, whereas others can become competent after chemical treatment Only a small amount of DNA is actually taken up TRANSFORMATION 34
TRANSDUCTION Transduction involves the transfer of genetic material between cells It is a common event in both Gram-positive and Gram-negative bacteria It uses a bacterial virus (phage) for transfer TRANSDUCTION There are two forms of transduction: Generalized random Specialized specific Transduction-related development of pathogenicity or increase in virulence is called phage conversion TRANSDUCTION There are three phases to generalized transduction The original infected cell chromosome is cleaved into pieces Some of this bacterial DNA is incorporated into a newly made phage When these phages infect the next cell, original DNA recombines with host chromosome 35
TRANSDUCTION TRANSDUCTION TRANSDUCTION During specialized transduction: Phage DNA incorporates into the host chromosome Phage DNA excises itself from the host chromosome Part of the host DNA is taken along Previous host DNA is incorporated into the next host chromosome 36
CONJUGATION Conjugation involves the transfer of material between cells Conjugation requires direct contact between the donor and recipient cells Gram-positive cells stick to each other Gram-negative cells use pili as a conduit for DNA transfer DNA moves from the donor to recipient cell CONJUGATION Dennis Kunkel CONJUGATION There are several steps in conjugation: The sex pilus of the donor cell recognizes specific receptors on the cell wall of recipient cell An enzyme in the donor cell causes the plasmid DNA to unwind One of the two single strands of plasmid DNA stays in the donor cell 37
CONJUGATION There are several steps in conjugation: The other moves across the plasmid into the recipient cell Both single strands are replicated After replication, the donor and the recipient contain identical plasmids At the completion of this transfer, the recipient cell becomes a donor and can conjugate with another recipient cell CONJUGATION CONJUGATION 38
CONJUGATION Conjugation can have several outcomes for the recipient cell: The plasmid can remain as a plasmid The plasmid can become incorporated into the recipient cell chromosome When this happens, the recipient cell is then referred to as Hfr DNA from Hfr can be moved into a new recipient This replaces sections of the host chromosome CONJUGATION CONJUGATION 39
GENETICS AND PATHOGENICITY Mutations that occur during DNA replication and the various processes of gene expression can lead to: a harmless bacterium becoming pathogenic increased virulence in a pathogen antibiotic resistance GENETICS AND PATHOGENICITY Genes found on plasmids can code for: toxins involved in infection and pathogenesis antibiotic resistance disinfectant resistance better adaptation to otherwise destructive environments Plasmids are easily transported through conjugation 40