Microbial Genetics
Structure and Function of the Genetic Material
1. Genetics is the study of what genes are, how they carry information, how
their information is expressed, and how they are replicated and passed to
subsequent generations or other organisms.
2. DNA in cells exists as a double-stranded helix; the two strands are held
together by hydrogen bonds between specific nitrogenous base pairs: A-T
and C-G.
3. A gene is a segment of DNA, a sequence of nucleotides that codes for a
functional product, usually a protein.
4. When a gene is expressed, DNA is transcribed to produce RNA, mRNA is
then translated into proteins.
5. The DNA in a cell is duplicated before the cell divides, so each daughter
cell receives the same genetic information.
Genotype and Phenotype
1. Genotype is the genetic composition of an organism-its entire DNA.
2. Phenotype is the expression of the genes-the proteins of the cell and the
properties they confer on the organism.
DNA and Chromosomes
1. The DNA in a chromosome exists as one long double helix associated
with various proteins that regulate genetic activity.
2. Bacterial DNA is circular; the chromosome of E. coli, for example, contains
about 4 million base pairs and is approximately 1000 times longer that the
cell.
3. Genomics is the molecular characterization of genomes.
4. Information contained in the DNA is transcribed into RNA and translated
into proteins.
DNA Replication
1. During DNA replication, the two strands of the double helix separate at the
replication fork, and each strand is used as a template by DNA
polymerase to synthesize two new strands of DNA according to the rules
of nitrogenous base pairing.
2. The result of DNA replication is two new strands of DNA, each having a
base sequence complementary to one of the original strands.
3. Because each double-stranded DNA molecule contains one original and
one new strand, the replication process is called semi-conservative.
4. DNA is synthesized in one chemical direction called 5’3’. At the
replication fork, the leading strand is synthesized continuously and the
lagging strand, discontinuously.
5. DNA polymerase proofreads new molecules of NDA and removes
mismatched bases before continuing DNA synthesis.
6. Each daughter bacterium receives a chromosome identical to the
partners.
RNA and Protein Synthesis
1. During transcription, the enzyme RNA polymerase synthesizes a strand of
RNA from one strand of double-stranded DNA, which serves as a
template.
2. RNA is synthesized from nucleotides containing the bases A, C, G, and U,
which pair with the bases of the DNA sense strand.
3. The starting point for transcription, where RNA polymerase binds to DNA,
is the promoter site; the region of DNA that is the endpoint of transcription
is the terminator site; mRNA is synthesized in the 5’3’ direction.
4. 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.
5. The mRNA associates with ribosomes, which consist of rRNA and protein.
6. Three-base segments of mRNA that specify amino acids are called
codons.
7. The genetic code refers to the relationship among the nucleotide base
sequence of DNA, the corresponding codons of mRNA, and the amino
acids for which the codons code.
8. The genetic code is degenerate; that is, most amino acids are coded for
by more than one codon.
9. Of the 64 codons, 61 are sense codons (which code for amino acids, and
3 are nonsense codons (which do not code for amino acids and are stop
signals for translation).
10. The start codon, AUG, codes for methionine.
11. Specific amino acids are attached to molecules of tRNA. Another portion
of the tRNA has a base triplet called an anticodon.
12. The base pairing of codon and anticodon at the ribosome results in
specific amino acids being brought to the site of protein synthesis.
13. The ribosome moves along the mRNA strand as amino acids are joined to
forma growing polypeptide; mRNA is read in the 5’ 3’ direction.
14. Translation ends when the ribosome reaches a stop codon on the mRNA.
15. In prokaryotes, translation can begin before transcription is complete.
16. In eukaryotes, coding regions of a gene (the expressed regions, or exons)
are often interrupted by noncoding regions (intervening sequences, or
introns). In the nucleus, RNA polymerase synthesizes an RNA transcript
containing exons and introns.
17. The introns must be removed from the RNA transcript before the resulting
mRNA can be translated – ribozymes remove the introns and splice the
exons together.
18. The mRNA is then moved through the nuclear membrane and into the
cytoplasm, where translation takes place.
The Regulation of Gene Expression in Bacteria
1. Regulating protein synthesis at the gene level is energy-efficient because
proteins are synthesized only as they are needed.
2. Constitutive enzymes are always present in a cell. Examples are genes for
most of the enzymes in glycolysis.
3. For these gene regulatory mechanisms, the control is aimed at mRNA
synthesis.
Repression and Induction
1. Repression controls the synthesis of one or several (repressible)
enzymes.
2. When cells are exposed to the end product of a metabolic pathway, the
synthesis of enzymes related to that product decreases (enzyme
synthesis is repressed).
3. In some instances, induction of enzymes to metabolize a less desirable
substrate also requires the absence of the preferable substrate. The
preferred substrate, when present, represses the metabolism of
alternative substrates. This is termed catabolite repression.
4. In the presence of certain chemicals (inducers), usually a substrate, cells
synthesize enzymes to metabolize that substrate. These enzymes are
inducible enzymes and this process is called enzyme induction.
5. An example of induction is the production of B-galactosidase by E. coli in
the presence of lactose, so lactose can be metabolized.
The Operon Model of Gene Expression
1. The formation of enzymes is determined by structural genes.
2. In bacteria, a group of coordinately regulated structural genes with related
metabolic functions and the promoter and operator sites that control their
transcription care called an operon.
3. In the operon model for an inducible system, a regulatory gene codes for
the repressor protein.
4. When the inducer is absent, the repressor binds to the operator and no
mRNA is synthesized.
5. When the inducer is present, it binds to the repressor so that it cannot bind
to the operator; thus, mRNA is made and enzyme synthesis is induced.
6. In the lac operon the inducer is allolactose, which is synthesized when
lactose enters the cell.
7. In repressible systems, the repressor requires a corepressor in order to
bind to the operator site; thus, the corepressor controls enzyme synthesis.
8. The corepressor is a product of the anabolic pathways catalyzed by the
enzymes coded for by the genes in the operon (i.e. tryptophan).
9. The metabolic product builds up and exerts negative feedback on the
transcription of the operon by binding to the inactive repressor protein and
forming an active repressor which binds to the operator and inhibits RNA
polymerase activity.
10. Transcription of structural genes for catabolic enzymes (such as Bgalactosidase) is induced by the absence of glucose.
11. Glucose is preferentially catabolized by cells, so enzymes that catabolize
other sugars are not transcribed in the presence of glucose.
12. When glucose levels drop cAMP is synthesized, which functions as a
cellular alarm signal or alarmone.
13. cAMP binds to its receptor protein, CRP, and the complex then binds to
the lac promoter to enhance transcription.
14. The inhibition of metabolism of alternative carbon sources by glucose is
catabolic repression (glucose effect).
Mutation: Change in the Genetic Material
1. A mutation is a change in the nitrogenous-base sequence of DNA; that
change causes a change in the product coded for by the mutated gene.
2. Many mutations are neutral, some are disadvantageous, and others are
beneficial.
Types of Mutations
1. A base substitution occurs when one base pair in DNA is replaced with a
difference base pair.
2. Alterations in DNA can result in missense mutations (which cause amino
acid substitutions) or nonsense mutations (which create stop codons).
3. In a frameshift mutation, one or a few base pairs are deleted or added to
DNA.
4. Mutagens are agents in the environment that cause permanent changes in
DNA.
5. Spontaneous mutations occur without the presence of a mutagen.
Mutagens
1. Chemical mutagens include base-pair mutagens (for example, nitrous
acid), nucleoside analogs (for example, 2-aminopurine and 5bromouracil), and frameshift mutagens (for example, benzpyrene).
2. Ionizing radiation causes the formation of ions and free radicals that react
with DNA; base substitutions or breakage of the sugar-phosphate
backbone result.
3. Ultraviolet radiation is nonionizing; it causes bonding between adjacent
thymines.
4. Damage to DNA caused by ultraviolet radiation can be repaired by
enzymes that cut out and replace the damaged portion of DNA.
5. Photoreactivation enzymes repair thymine dimers in the presence of
visible light.
Frequency of Mutation
1. Mutation rate is the probability that a gene will mutate when a cell divides;
the rate is expressed as 10 to a negative power.
2. Mutations usually occur randomly along a chromosome.
3. A low rate of spontaneous mutations is beneficial in providing the genetic
diversity needed for evolution.
Identifying Mutants
1. Mutants can be detected by selecting or testing for an altered phenotype.
2. Positive selection involves the selection of mutant cells and rejection of
nonmutated cells.
3. Replica plating is used for negative selection - to detect, for example,
auxotrophs that have nutritional requirements not possessed by the parent
(nonmutated) cell.
Identifying Chemical Carcinogens
1. The Ames test is a relatively inexpensive and rapid test for identifying
possible chemical carcinogens.
2. The test assumes that a mutant cell can revert to a normal cell in the
presence of a mutagen and that many mutagens are carcinogens.
3. Histidine auxotrophs (require histidine in the media) of Salmonella are
exposed to an enzymatically treated potential carcinogen, and reversions
to the nonmutant state are selected.
Genetic Transfer and Recombination
1. Genetic recombination, the rearrangement of genes from separate groups
of genes, usually involves DNA from different organisms; it contributes to
genetic diversity.
2. In crossing over, genes from two chromosomes are recombined into one
chromosome containing some genes from each original chromosome.
3. Vertical gene transfer occurs during reproduction when genes are passed
from an organism to its offspring.
4. Horizontal gene transfer in bacteria involves a portion of the cell’s DNA
being transferred from donor to recipient.
a. Transformation
b. Conjugation
c. Transduction
5. When some of the donor’s DNA has been integrated into the recipient’s
DNA, the resultant cell is called a recombinant.
Transformation in Bacteria
1. During this process, genes are transferred from one bacterium to another
as “naked” DNA in solution.
2. This process was first demonstrated in Streptococcus pneumoniae, and
occurs naturally among a few genera of bacteria.
3. 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 nonencapsulated and heat-killed encapsulated bacteria
into mice, mice die, isolated encapsulated bacteria from dead mice.
e. Nonencapsulated bacteria took up pieces of DNA from dead
encapsulated bacteria, some of the pieces contained genes for
capsule, avirulent bacteria were transformed by these genes into
the encapsulated virulent strain.
Conjugation in Bacteria
1. This process requires contact between living cells.
2. One type of genetic donor cell is an F+; recipient cells are F-.
3. F+ cells contain plasmids called F factors; these are transferred to the Fcells during conjugation.
4. When the plasmid becomes incorporated into the chromosome, the cell is
called an Hfr (high-frequency recombinant).
5. During conjugation, an Hfr can transfer chromosomal DNA to an F-,
Usually, the Hfr chromosome breaks before it is fully transferred.
Transduction in Bacteria
1. In this process, DNA is passed from one bacterium to another in a
bacteriophage and is then incorporated into the recipient’s DNA.
2. In generalized transduction, any bacterial genes can be transferred.
Plasmids and Transposons
1. Plasmids are self-replicating circular molecules of DNA carrying genes
that are not usually essential for survival of the cell.
2. There are several types of plasmids:
a. Conjugative plasmids – genes for sex pili and conjugation
b. Dissimulation plasmids – genes for enzymes that catabolize
unusual organic molecules (Pseudomonas species – toluene,
camphor, petroleum products)
c. Plasmids carrying genes for toxins or bacteriocins
d. Plasmids carrying genes for resistance factors
i.
Consist of two sets of genes – RTF (resistance transfer factor)
and specific resistance genes (r-determinant)
3. Transposons are small segments of DNA that can move from one region
of a chromosome to another region of the same chromosome or to a
different chromosome or a plasmid.
4. Transposons are found in the main chromosomes of organisms, in
plasmids, and in the genetic material of viruses. They vary from simple
(insertion sequences) to complex.
5. Complex transposons can carry any type of gene, including antibioticresistance genes, and are thus a natural mechanism for moving genes
from one chromosome to another.
Genes and Evolution
1. Diversity is the precondition of evolution.
2. Genetic mutation and recombination provide a diversity of organisms, and
the process of natural selection allows the growth of those best adapted
for a given environment.