CHAPTER 6 - BACTERIAL GENETICS

These lecture notes will provide an outline of information from the lectures. They are not complete. They should be used to help follow the lecture and as a guideline for information I think is important. You will need to fill in the gaps.

These notes were updated January 20, 2001, and are ready for printing by Spring 2001 Med Micro. students.

CHAPTER 6 - BACTERIAL GENETICS

I. DNA A. Macromolecule
1. Its basic building block is the nucleotide
2. Nucleotides are twisted together in pairs to form a double helix (spiral staircase)
3. The sides of the spiral are alternating sugar and phosphate groups (sugar-phosphate backbone)
4. The rungs of the ladder are the nitrogenous bases
5. Each nitrogenous base is attached to a sugar
6. The rungs are held together by hydrogen bonds between nitrogenous bases
7. Nitrogenous bases always pair up in a specific way
a) A to T
b) C to G
c) Thus the base sequence of one DNA strand determines the sequence of the other strand 8. The two strands of DNA are complementary to each other B. Structure
1. The two strands of DNA are called antiparallel
a) Antiparallel means that they run in opposite directions
b) In a strand of DNA, a phosphate group will join to deoxyribose units between the third (3' or " 3-prime") carbon atom of one molecule of deoxyribose and the fifth (5' or "5-prime") carbon atom of the other molecule of deoxyribose
c) Thus, each strand of DNA will have a free 5' carbon at one end and a free 3' carbon at the other end
d) The DNA strand is said to have either a 3' to 5' orientation or a 5' to 3' orientation 2. Because the two strands of DNA are never entwined with the same orientation, they are said to be antiparallel
a) That is, one strand runs in the 3' to 5' direction, and the other strand runs in the 5' to 3' direction
b) These two strands are as different as if they had a head at one end and a tail at the other II. DNA Processes and Roles A. Replication 1. Chromosome
2. Plasmids B. Protein Synthesis 1. Transcription
2. Translation C. Mutation
D. Genetic Recombination 1. Transformation
2. Conjugation
3. Transduction III. DNA replication A. The process where the "parent" double-stranded DNA molecule is converted into two identical "daughter" DNA molecules 1. This occurs just before binary fission begins
2. Those bacteria having a plasmid present, in addition to the chromosome, will undergo replication of the plasmid at the same time as replication of the chromosome occurs
3. Thus, daughter cells will each receive a copy of the parent cell's plasmid, in addition to its chromosome a) Recall that a plasmid is an extrachromosomal, circular, double-stranded DNA fragment that is associated with plasma membrane proteins
b) Plasmids are not essential to bacterial life
c) Plasmids may provide selective advantages to those bacteria that carry them (1) Some plasmids contain genes that confer antibiotic resistance to the bacterium (a) These are called R factors or resistance factors (2) Other plasmids carry genes that code for the production of bacteriocins (a) Bacteriocins are proteins that are toxic to other bacteria (3) Some plasmids carry genes that code for the production of EXOTOXINS (a) Do not confuse exotoxins with endotoxins
(b) Exotoxins are proteins that are toxic or poisonous to humans
(c) Examples of exotoxins are the toxins produced by Clostridium botulinum, Clostridium tetani, and E. coli O157:H7 B. During replication, the double strands of DNA separate, and one DNA strand acts as the template for the production of a new, complementary DNA strand a) Replication is initiated at a genetically specified point on the chromosome called the origin
b) At the origin, there are two replication forks that form that travel simultaneously in opposite directions around the circular chromosome
c) They will meet halfway around the chromosome at a point called the terminus
d) Once at the terminus, the two completed chromosomes will separate, ready to be distributed to daughter cells C. This combination of new and parent strands is called the SEMICONSERVATIVE METHOD OF REPLICATION a) DNA polymerase enzymes join the nucleotides to the growing DNA strand (1) Nucleotides that are present in free form in the cytoplasm of the bacterial cell align themselves to their corresponding base on the single-strand of parental DNA
(2) The DNA polymerase joins the nucleotides to the growing DNA strand (a) The leading strand is synthesized continuously in the same direction as the replication fork moves
(b) The lagging strand is synthesized discontinuously in the opposite direction to the replication fork's movement (i) The result is a series of segments called OKAZAKI FRAGMENTS that are later joined by an enzyme, DNA ligase b) DNA polymerase also performs a proofreading function that prevents mismatched bases from attaching (1) This assures that each daughter DNA to be identical to its parent
(2) Although not 100% accurate, mistakes occur at the rate of about 1 for every 10¹⁰ bases
(3) This DNA polymerase function is also known as a MISMATCH REPAIR mechanism D. Another type of PARTIAL replication of the DNA strand occurs during conjugation 1. This is called the rolling circle mechanism and will be covered under conjugation E. Summary 1. Double strands of DNA separate
2. The leading strand is synthesized continuously in the same direction as the replication fork moves
3. The lagging strand is synthesized discontinuously in the opposite direction to the replication fork's movement
4. DNA polymerase is the enzyme responsible for proofreading and joining the nucleotides to form the daughter strands F. Once the bacterial chromosome is completely replicated, a membrane and cell wall material will grow between the two chromosomes 1. This growth divides the cell into two identical bacterial cells
2. This is called binary fission IV. Mutation
A. Mutations occur when there is a change in the nitrogen base sequence of DNA
B. Mutations are either spontaneous or the result of a mutagen
1. Spontaneous mutations occur in the absence of mutation-causing agents (mutagens)
2. Mutagens are agents that chemically or physically react with DNA to produce mutations a) Chemicals (nitrous acid and base analogs) (1) Base analogs are substances that are similar to nitrogenous bases that are incorporated into bacterial or viral DNA
(2) The clinical significance of a base analog is that certain antimicrobials contain them; bacteria incorporate base analogs into their DNA, thus rendering the replicating DNA functionless
(3) Base analogs are also important in anti- viral therapy b) Radiation (X-rays and ultraviolet light) 3. Not all mutations are harmful
a) Some produce no obvious effect
b) Others may prove to be beneficial, such as antibiotic resistance in bacteria C. Point mutation
1. Also called a base substitution
2. Replacement of one nitrogen base with a different nitrogen base
3. This causes a change in the sequence of nitrogen bases in the gene
a) The result may be silent (neutral)
(1) Because of the degeneracy of the genetic code, the resulting new codon may still code for the same amino acid b) OR, the amino acid may be changed, but the function of the protein itself may not change or may change so little that the protein is essentially very similar to the original in its function
c) OR the change may be very dramatic, resulting in conditions that are incompatible with life
(1) The protein may be very different in its function from the original
(2) The nitrogen base change may create a stop codon in the middle of the mRNA molecule, and only a fragment of the original protein is then produced 4. When the change results in an amino acid substitution in the synthesized protein, the change in the DNA is known as a missense mutation
5. When the change results in a nonsense codon that stops synthesis of the complete protein, the change in the DNA is known as a nonsense mutation D. Frameshift mutation
1. When one or a few nucleotide pairs are deleted or inserted in the DNA
2. This causes a shift in the "translational reading frame"
a) The three-by-three grouping of nucleotides has been regrouped because of the extra or missing nitrogen bases
b) Thus, rather than a "one for one" substitution that does not change the total number of nucleotides, the increase or decrease in nitrogen bases has shifted the codon groupings E. Transposable Elements (Jumping Genes or Transposins) 1. Special segments of DNA that can move spontaneously from one site to another a) This movement may occur in the same DNA molecule 2. This movement may occur between two different DNA molecules, such as plasmid to plasmid, plasmid to chromosome, or chromosome to plasmid
3. The gene into which the transposin is inserted generally will no longer encode the protein it encoded before the transposition
4. The transposin may encode for some other protein V. Genetic Transfer and Recombination A. Genetic recombination occurs when two DNA molecules exchange genes to form new combinations of genes 1. Although this may occur in the same cell or across different cells, in this class it will be used to mean the genetic process in which two organisms are involved, a DONOR cell and a RECIPIENT cell
2. The donor cell contributes chromosomal or plasmid DNA to the recipient cell, which is now called a RECOMBINANT cell a) When plasmid DNA is contributed, it exists independently in the recipient's cytoplasm and encodes proteins immediately
b) If chromosomal DNA is contributed, it replaces a homologous region in the recipient's DNA
c) Thus, there is no change in the quantity of the recipient's chromosomal DNA B. There are three methods for bacterial recombination 1. Transformation
2. Conjugation
3. Transduction C. Genetic recombination contributes to a population's genetic diversity
D. Genetic diversity is the source of variation in evolution
E. In organisms that have evolved a great deal, recombination is more likely to be beneficial than mutation
F. Transformation 1. Bacteria acquire DNA directly from the environment, soon after it is released from another, non-living cell (the DNA is called "naked DNA") a) This is most likely to occur during the stationary and death phase of bacterial growth 2. Not all bacteria are capable of transformation a) The bacteria must demonstrate COMPETENCE, or the ability of the recipient bacterium to take up DNA from the environment (1) The competent cell is usually at the end of its log phase of growth
(2) Although double-stranded DNA molecules bind to the competent cells, only a single-strand of DNA enters, because nucleases at the cell surface degrade the other strand
(3) Once inside the recipient cell, the single-stranded donor DNA is positioned next to the homologous region of the recipient DNA that would be complementary to the donor strand
(4) The recipient cell's homologous DNA strand is cleaved by an enzyme in two places, releasing a fragment of DNA into the cytoplasm that is degraded
(5) The donor DNA then replaces the recipient DNA precisely
(6) This is called breakage and reunion (this is also similar to what happens during conjugation and transduction) b) Competence is related to membrane permeability and surface receptors such that the passage of DNA strands is encouraged
c) Competence can also be induced within the laboratory (example is E. coli when it is used for genetic engineering) 3. Generally speaking, transformation is more likely to take place in organisms whose DNA is very similar (same species) a) Best examples are Bacillus, Haemophilus, Neisseria, Azotobacter, and certain strains of Streptococcus and Staphylococcus
b) A famous example is the pneumococcal bacterium, Streptococcus pneumoniae, which has two strains, a pathogenic and a non-pathogenic strain 4. One of the effects of transformation is to increase an organism's pathogenicity a) Transformed bacteria may also display enhanced drug resistance if they acquire R factors from plasmids G. Conjugation 1. Involves the transfer of DNA from one living bacterium to another through direct contact
2. Requires the presence of a conjugative plasmid (the "F plasmid" is one example and is found in E. coli) that contains the genes necessary for conjugation
3. Involves cell-to-cell contact as DNA crosses a sex pilus from donor to recipient
4. Requires that conjugating cells are of opposite mating type a) We call these F+, or male cells, and F-, or female cells
b) This means the donor cell MUST carry the plasmid with the genes for conjugation (F+ E. coli mating with F- bacterium)
c) In Gram negative cells, this plasmid carries genes coding for the synthesis of sex pili (1) Sex pili are projections from the donor's cell surface
(2) The sex pili contact the recipient and help bring the two cells into direct contact
(a) In E. coli, the the F+ plasmid DNA replicates and enters the F- cell
(b) Within the F- cell, the plasmid DNA forms a circle and duplicates
(c) Now the F- cell is F+ and is capable of transferring a copy of its F plasmid to another F- cell
(d) The original F+ cell still retains its copy of the conjugative plasmid
(e) The F plasmid is only transferred between strains of E. coli and closely related species d) In Gram positive cells, sticky surface molecules are produced that cause the cells to come into direct contact with each other 5. Once cells are attached, the conjugative plasmid begins replicating by the rolling circle mechanism a) One strand of the donor DNA is separated from the ring of DNA, serving as a template for the opposite strand
b) These strands join to form a double helix, which is then transferred to the recipient cell
c) The original DNA ring will then use the complementary strand that was joined to the separated strand serve as a template for rebuilding the missing strand 6. The conjugative plasmid then transfers chromosomal and plasmid genes to the recipient cell a) Recall that plasmids can carry genes that code for antibiotic resistance, bacteriocins, or exotoxins, all of which provide a survival advantage to the bacteria (but are not essential to life)
b) Some plasmids are termed promiscuous, because cells that carry them mate with just about any bacterial cell, not just closely related cells 7. When donated conjugated plasmids share regions of homology with the recipient chromosome, genetic recombination occurs within the chromosome a) This means that the plasmid actually becomes integrated into the chromosome
b) The cell is then called an Hfr (high frequency of recombination) cell
c) Even when integrated into the chromosome, the plasmid can still bring about conjugation H. Transduction
1. Bacterial DNA is transferred from donor to recipient cell inside a virus that infects bacteria
2. Viruses that infect bacteria are called BACTERIOPHAGES or PHAGES for short
3. Phages are either virulent (always kill their host) or temperate (may be carried within bacterium without harming it)
4. Virulent phages bring about generalized transduction
5. Temperate phages bring about generalized transduction (during their "lytic cycle"), specialized transduction (during their "lysogenic cycle"), or both
6. Generalized transduction occurs when
a) Virulent phage OR temperate phage during its lytic cycle, attaches to surface of victim bacterial cell
b) Phage injects its DNA into bacterium
c) Phage DNA now directs bacterial cell to make phage components (DNA and protein)
d) Components are assembled into mature phage particles
e) Bacterium lyses
f) Phage particles are released, looking for other cells to infect
g) Rarely (once in 100,000 times), a mistake occurs, and instead of phage DNA, bacterial DNA is inserted into the new phage
(1) This abnormal phage particle is called a transducing particle, and it can attach to another bacterium and inject bacterial DNA into it
(2) The result is a genetic exchange: DNA from the bacterial cell where the transducing particle was formed is introduced into another cell
(3) The DNA fragment that the phage injects persists in the host cell ONLY if it becomes integrated by recombination into the host's genome 7. Specialized transduction occurs when
a) A temperate phage, during its lysogenic cycle, attaches to surface of victim bacterial cell
b) Temperate phage injects its DNA into bacterium
c) Phage DNA becomes quiescent (inactive or dormant)
(1) The quiescent phage DNA is called a PROPHAGE
(2) Some prophages exist in the bacteria as plasmids
(3) Other prophages become incorporated into the host cell's chromosome d) When a prophage becomes incorporated into the host cell's chromosome, specialized transduction occurs
e) These prophages may become reactivated and enter a lytic cycle
f) The lytic cycle produces phage particles
g) Specialized transducing particles are formed when a mistake is made during reactifvation and a few bacterial genes, along with the prophage, leave the chromosome
h) When a specialized transducing particle attaches to another bacterial host and injects its DNA, it is also injecting the bacterial genes it picked up by accident
i) Thus genetic recombination occurs between the original bacterial host cell and the bacterium infected by the specialized transducing particle from that host cell
j) Because prophages are inserted only at a specific site on the bacterial chromosome, this process is called specialized transduction, and ONLY those bacterial genes near to this site will be transferred by specialized transduction
k) Specialized transduction is important because this is the mechanism that transfers toxin-making genes into bacteria VI. Summary A. When genetic recombination occurs, bacterial genotypes change and are transferred to daughter cells through binary fission
B. Genetic recombination occurs through transformation, conjugation, and transduction
C. Phenotypes may or may not change as a result of genetic recombination
D. Genetic change in a few cells can change an entire population of bacteria
1. Using the example of antibiotic resistance
a) Certain genes encode antibiotic resistance, allowing bacteria to survive in the presence of otherwise lethal antibiotics
b) These genes are often carried on conjugative plasmids and are called R factors
c) Specifically, they are often found in transposons
(1) A transposon is a small segment of DNA that can move (be transposed) from one region of a DNA molecule to another d) The transposon can move to the chromosome OR to another plasmid that the cell contains
e) Either way, the stage is set for antibiotic resistance spreading through a bacterial population through conjugation or through transduction or transformation 2. This example is the same for toxin production

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