1.

7 BACTERIAL GENETICS MICROBIOLOGY

Dr. Europa | July 9, 2013 1st 2013-2014

OUTLINE Promoter- site on DNA where RNA polymerase binds to initiate

I. Key terms transcription; promoter consensus sequences identifying strong
II. Introduction
III. Organization and expression of Genetic Information
promoters.
a. DNA Organization and Replication Regulation of gene expression- use by bacteria of DNA sequences and
b. Regulation and expression of Genetic information proteins that bind them (sigma factors, activators, repressors) to alter
IV. Transfer of Genetic Information the number of transcripts or proteins made from a particular gene.
a. Transformation Repressor- protein that binds close to operator and prevents
b. Transduction
c. Conjugation
transcription.
V. Genetic Basis of Bacterial Diversity Ribosomes- protein-rRNA complexes on which mRNA is translated
a. Mutation into ptotein; bind mRNA at ribosome binding site; leave mRNA at stop
b. Genetic Recombination codon.
VI. Additional Notes RNA polymerase- an enzyme that moves along selected regions of the
VII. Appendix
DNA, forming a complementary strand of mRNA; requires sigma factor
OBJECTIVES for initiation of transcription and rho factor for termination of some
At the end of the lecture, the student should be able to understand the transcripts.
following: Transcription-synthesis of an mRNA strand complementary to a single
 Steps in Bacterial DNA Replication strand of DNA, rsulting RNA can be mRNA(encodes protein) or
a. Opening up the supercoiled double helix
b. Copying both strands
ribosome associated RNA (rRNA, tRNA)
c. Restoration of supercoiling Transfer RNA (tRNA)- the form of RNA that carries an amino acid to
d. Bacterial cell division the ribosome; at the ribosome the anticodon region of the tRNA binds
e. Asymmetrical cell division during sporulation to a codon on the mRNA specific for the amino acid that the tRNA
 Correcting Mistakes and Healing Lesions carries and transfers the amino acid to the growing peptide chain.
a. Damage to DNA
Translation- synthesis of a protein by a ribosome as it moves along
b. Homologous recombination
 Plasmids and Plasmid Replication mRNA, converting mRNA sequence into amino acid sequence of the
a. Characteristics of plasmids protein.
b. Gene cloning: an application of plasmid characteristics Bacteriophage- viruses that attack bacteria; also called phages; may
c. The importance of plasmids for bacteria introduce new genes into bacteria by lysogeny (integration of viral
 Other Applications of Information about DNA Replication genome) or generalized transduction (accidental transfer of bacterial
a. DNA hybridization and Southern blotting
b. DNA sequencing
genes).
c. Polymerase chain reaction Conjugation- direct cell to cell transfer of bacterial DNA via
 Genetic Engineering and the Race for Human Survival multiprotein mating bridge.
Conjugative transposon- self transmissible chromosomal element that
References:
Dr. Europa’s lecture, powerpoint and notes. excises itself from the chromosome to form a circular intermediate,
2015B Trans which is transferred to a recipient then integrates into the recipient
genome.
Legend: Italicized – quoted from the lecturer; bold – emphasis, or from references
Gene disruption mutants- mutants generated by using single
I. KEY TERMS
crossover or double crossover homologous recombination to interrupt
Activator- protein that enhances binding of RNA polymerase to the
a gene so that the protein it encodes is no longer produced.
promoter and helps it to initiate transcription.
Horizontal gene transfer- transfer of DNA from one organism to
Gene- segment of DNA that encodes a protein or an RNA species such
another; in bacteria, mediated by transformation, transduction or
as rRNA or tRNA.
conjugation.
Gene expression- the process of converting DNA sequence (the gene
Insertion sequence(IS element)- DNA segment that integrates
or open reading frame) to RNA sequence (transcription) and from
randomly into DNA; contains gene encoding transposase; can activate
there to amino acid sequence of protein (translation); process can be
expression of genes or disrupt genes.
regulated at each level.
Integron- transposon that also contains an integrase gene, the protein
Constitutive expression- gene expressed under all conditions.
product of which faculiatates the integration of gene cassettes inro
Regulated expression- gene expressed only under some conditions.
special site in integron, creating gene clusters that are under the
Messenger RNA(mRNA)- RNA molecule transcribed from DNA that
control of a promoter provided by the integron.
contains the coding sequence for at least one protein.
Plasmid- DNA segments (often circular) that replicate autonomously.
Mutations- changes in DNA sequence of genes or regulatory regions
Self transmissible plasmid-Some carry gene sthat allow them to
can lead to altered expression of a gene, altered amino acid sequence,
transfer themselves by conjugation from a bacteria donor to a
or altered protein length; may result in ninfunctional protein.
bacterial recipient.
Open reading frame(orf)- region og the DNA sequence that starts with
Mobilizable plasmid-some carry genes that allow self-transmissible
a start codon and ends with a stop codon.
plasmids to mobilize them from a donor to recipient cell.
Operator site- region in DNA to which a repressor bind and prevents
Transformation- uptake of DNA from the extracellular fluid
mRNA synthesis.
Natural transformation system- consists of cytoplasmic membrane
Operon- a series of genes controlled by a single operator; single
complex that imports single-stranded DNA into the bacterial
transcript encodes multiple proteins.
cytoplasm.

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Group 7: Bautista, Beltran, Bernardo C., Bernardo P., Bilas, Biong, Bompat
 Microbiology 1.7
Artificial transformation- employs conditions that force bacteria to B. Regulation and Expression of Genetic Information
take up DNA from the extracellular environment.  Bacteria use specific DNA sequences whereby the proteins can bind
Transposon- DNA segment flanked by two insertion sequence so you can regulate gene expression.
elements, which now move as a unit; may contain antibiotic resistance  Bacteria make sure they are in a balanced growth under different
genes or genes for a variety of other metabolic traits. nutritional condition.
Transposon mutagenesis- use of transposons to create collection of
mutants in which most mutants will have the transposon interrupting
a gene; employed to generate mutations that identify genes important
for a particular trait.
II. INTRODUCTION
 Bacterial genetics has proved to be an enormously effective tool
for the study of basic gene organization and function.
 It employs such techniques as fine-structure mapping and
complementation testing as well as transformation and
transduction.
 Genetically, the normal bacterial cell is haploid.
 During no stage of its life doe sthe cell undergo sexual
reproduction; therefore, meiotic segregation does not occur.
 Owing to the haploid nature of the organisms, the genotype and
phenotype almost always are the same, making genetic selection
and screening much easier and faster.
III. ORGANIZATION AND EXPRESSION OF GENETIC INFORMATION
A. DNA Organization and Replication
 The genetic material that defines bacteria is carried in a
chromosome.
Figure 1.Growth Curve of Bacteria
 Bacterial DNA is transcribed into both monocistronic and
Growth curve of bacteria in laboratory medium. When bacteria find themselves
polycistronic messenger RNA (mRNA) in the cytoplasm withot the in a new environment, they must first adapt those conditions before they start
need for any subsequent processing. dividing (lag phase). After they have adapted, bacteria divide rapidly by binary
 (Each eukaryotic mRNA contains information coding for only one fission which results in an exponential rise in the number of bacteria
protein,hence monocistronic, whereas Prokaryotic mRNAs may (exponential phase). After a period of starvation the bacteria may begin to lyse
encode more than one protein and are said to be polycistronic.) and the number of cell in the culture declines (death phase).
 This simple mechanism is possible because of the absence of Table 1. Growth phases of bacteria in laboratory medium
introns(intervening sequences) and the absence of nuclear Lag phase  Bacteria adapt to new conditions
membrane.  numbers remain constant
 Chromosomes Exponential phase Bacteria divide exponentially (N=N02n)
o Structure is single, continuous strands of DNA
9 N0= initial number of bacteria
 Average molecular weight is 3x10
n= number of divisions
 It is a closed circular structure N= number of bacteria after n divisions
 It is made up of DNA which is double-stranded; and has the Stationary phase  One or more nutrients become limiting
usual base pairing A-T and G-C (Adenine-Thymine and  Increase in number of bacteria ceases
Guanine-Cytosine) Death phase  Bacteria begins to lyse (probably as result of
o Replication – is precise and carefully controlled process activity of autolysins (proteins that break down
ensuring that each daughter cell receives an exact copy. the cell wall)
 Origin of Replication- a specific site where replication  Number of viable bacteria decreases
begins, where the two DNA strand are denatured.
 Semi-Conservative replication- means that a template 1. Processes Affecting Gene Expression
strand is copied and a new strand is synthesized. o Transcription- is the transfer of genectic information from DNA
 In bacteria, DNA replication is coordinated with cell division to mRNA.
and requires both protein and RNA synthesis.  Mediated by RNA polymerase
-Has 2 forms: Core enzyme & Holoenzyme
 Genes
-Sigma factor: is an activator protein which attaches
o DNA is organized in series of units called gene, one copy of
RNA polymerase enzyme to specific initiation site.
each gene is a haploid
 Initiation of Transcription
o Structure – chain of Nucleotides; transcript is read without
- enzyme + sigma factor → bind to promoter →
interruption.
polypeptide chain formation & elongation
 Triple nucleotide groups coding for one specific amino acid
-Promoter: Specific sequence of DNA that bind RNA
 Contiguous DNA sequences
polymerase
- A Prokaryote does not have a nucleus, no nuclear
 Termination of Transcription
membrane, and no intervening sequences called
-Goes to a G-C rich region where stability of DNA
introns which are found in higher forms of organism.
structure is altered
o Phenotype – observable characteristics such as physiological,
o Translation
functional, structural characteristics of an expressed genes.
 mRNA + tRNA + ribosomes
-boundaries of your gene are identified genetically by
 transfer RNA (tRNA)
observing the phenotype.
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 Microbiology 1.7
2. Regulation of Gene Expression
A. Metabolic Stage
1. End-product inhibition / Feedback inhibition
 when you have a sufficient amount of an end product in
a pathway this end product would inhibit the first
enzyme of the pathway.
a)Direct and intermediate
b)Competitive inhibition (between product and
substrate of an enzyme)
2. Enzyme Activation
 Positive effector molecules can stimulate the first
enzyme.
 Contribute to the translation of message expressed by
structural genes.
B. Initiation of transcription
o There is a positive and negative regulation at this level.
o Negative Control Systems = active repressor molecules
1. Repressor molecules
Figure 2. The Operon Theory
 Recognize specific DNA sequences in a region which
A. An operon consist of one or more structural genes, an operator and a
you call the operator. promoter. A regulatory gene is found at the site separate from operon.
 Ex:The lactose repressor – M.W. 150,000 B. The regulatory gene encodes a repressor protein that binds to the operator,
 Lac repressor will bind to an Operator then disallows thereby preventing the transcription of the structural genes by the RNA
the binding of RNA polymerase to the Promoter region. polymerase.
 The operator is a rigidity defined DNA sequence that C. If the repressor protein is blocked somehow from binding to the operator, the
involved genetic regulation of bacteria. RNA polymerase is free to transcribe the structural genes.
 Repressor molecules recognize and bind to the Table 2. Summary of signals that control transcription and translation of a gene
operator, leading to reduction of transcription and Signals Determine Mutation can affect
expression of the structural genes (ie.,negative control). Promoter, Length of transcript
Length of transcript
terminator Strength of promoter
2. End – product repression Whether transcript is
 In the presence of the end product there is decrease Operator,
made and how many are Regulation of
activator binding
formation of the enzyme, the inactive repressor is made (level of gene expression
site
activated and binds to the end product. expression)
 End-product repression reduces the formation of the Ribosome
Where ribosome binds Whether protein is
enzymes in the own biosynthetic pathway rather than Sequence affects amount made and how much is
binding site (rbs)
through alteration of their enzymatic activity. of protein made made
Where protein starts,
 Although most commonly found in biosynthetic Start codon, stop Whether protein starts,
stops
pathway, it is not limited to these pathways. codon stops in right places
Length of protein
Premature
o Positive control system- initiation of transcription in response
termination(nonsense)
to binding of an activator protein Change of amino
o Operongroup of gene controlled by an operator(regulatory DNA sequence Amino acid sequence of acid(missense)
gene) which bind to repressor molecules to stop the of orf protein Different amino acid
transcription of mRNA, it means that RNA polymerase cannot sequence after a
bind to the Promoter region. certain point
 Operon begins with a promoter and are followed by an (frameshift)
operator, whichis followed by the structural genes.
 Mutations in operator means that there are alterations Table 3. Mechanism for Gene regulation
in DNA sequensences so there is failure to bind Type of regulation Level at which regulation occurs
repressor which lead to the non-repressible phenotype Transcriptional control Ability of RNA polymerase to bind a
Sigma factor certain set of promoters.
referred to as the operator constitutive.
Repressor, activator Ability of RNA polymerase to bind a
C. Transcription, Termination and Translation Two-component promoter and start transcribing gene
o Two mechanisms control gene expression at the level of Quorum sensing under different conditions
transcription termination: Translational control Ability of ribosome to bind mRNA and
 Polarity in Operons that are transcribed as a single Ribosome binding site start translating
mRNA species → weak terminator sites within the Post-translational control
operon → higher levels of mRNA, proximal to distal Feedback inhibition Activity of protein affected
 In some operons, there is variable, premature Covalent modification
termination of transcription in the early mRNA called
attenuation. IV. TRANSFER OF GENETIC INFORMATION
o Regulation of Translation = minor significance.  Bacteria use a series of mechanism for gene transfer; these
o Transcriptional control of gene regulation in bacteria is what’s mechanisms produce partial diploids as a prelude to a
important. recombinant event.

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 Microbiology 1.7
A. Transformation Plasmids- are extrachromosomal genetic elements which carry
 Exchange of genetic information that involves uptake of free DNA genes for important traits. (e.g. Antimicrobial resistance)
by competent cells. o Classification:
o Competence  Conjugative plasmids- encode genes for conjugal
 means that the bacteria should have a well defined transfer; can integrate at multiple sites within a
metabolic state that is hospitable to the entry of the free bacterial chromosome but is limited to only a very few
DNA. species and to only chromosomal DNA.
 capability of bacteria to take up DNA from their a) Structure: Large (60-120)
environment through their cell membrane. b) Function: Encoded genes for autonomous
o Double stranded DNA will be converted to single stranded replication. Other genes are responsible for
DNA;some bacteria leave DNA in double stranded form until bacteriocin or toxin production.
the recombination. Ex: F factors – transfer of plasmid DNA
o Observed in cultures of both gram positive positive/negative R factors – mediate bacterial resistance to
organisms antimicrobials
o Useful genetic tool for gene mapping  Nonconjugative plasmid , except that
o Bacteriophage is not involved in transformation. carryingtransposons, do not untegrate into other DNA.
o Since the transforming DNA is in an unprotected (free/naked)
state, it is sensitive to DNase in the medium.

B. Transduction
 Excahnge of genetic information between bacteria that is
mediated by a bacteriophage carrier.
 2 Ways of Transduction:
o Generalized Transduction
 host DNA package transfer of any gene
 Only a fragment of the bacterial chromosome and no
bacteriophage DNA is packaged.
o Specialized transduction – phage DNA integrated into host
chromosomes; incorporates a few specific genes upon
excision and therefore the transfer of gene is limited.
 Specialized transducing phage –from small proportion of
the progeny from a lysogenic culture; containing both
phage and bacterial genes. Figure 3. Isolation of Plasmid DNA
 Lysogenic bacteria-any bacterial cell harbouring in its Bacteria is harvested from a culture and treated with a detergent to promote
genome the genetic material of a prophage and thus lysis. This allows the release of materials such as proteins, chromosomal DNA
reproducing the bacteriophage in cell division; and plasmid DNA. Increasing salt concentration will cause the much larger
occasionally the prophage develops into the mature molecules of chromosomal DNA to precipitate; Plasmid DNA and proteins will
remain soluble. The soluble portion will be treated to remove the proteins, and
form, replicates, lyses the bacterial cell and is free to
isolate the plasmid DNA.
infect other cells.
 Prophage- a bacteriophage whose genome incorporates
V. GENETIC BASIS OF BACTERIAL DIVERSITY
with and replicates with that of the host bacterium.
A. Mutations
 Has LOW frequency transduction & HIGH frequency transduction.
 Occurs in both both gram positive positive/negative organisms.  Heritable alterations in the DNA nucleotide sequence and the
 Does not require involvement of complement bacterial cells. changes would be on morphology, enzyme activity, nutritional
requirements and susceptibility on antibiotics.
C. Conjugation  Types of sequence changes:
 One – way transfer of Genes o Nucleotide deletions – single bases are involved.
 Donor conjugates to transfer a gene to a Recipient cell, there 1. Microdeletions of a single nucleotide shift the triplet
should be a physical contact between the Donor and the code reading frame. They totally change the amino acid
Recipient. sequence of the protein
o Fertility (F) Factor 2. Large deletions – result to loss of the coding capacity for
1. Codes for series of genes responsible for the conjugal part of the gene
transfer o Nucleotide insertions – involve single bases pair.
F⁺ - Donor cells o Nucleotide replacements
F⁻- Recipient cells 1. Transitions – replacement of a purine base with another
2. F factor genes – encoded by a plasmid purine or replacement of a pyrimidine with another
3. F pilus – establishes contact between donor and pyrimidine. (eg.Adenine replaced with Guanine, or
recipient Cytosine replacted with thymine)
o F+ strains 2. Transversions - replacement of a purine with a
1. F factor must be incorporated into the bacterial pyrimidine or vice versa (eg.Adenine replaced with
chromosome Cytosine, or Thymine replaced Guanine)
2. High frequency recombination (Hfr) strains
3. Bacterial cultures in which the F factor is stabilized in the
chromosome
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 Microbiology 1.7
 Point mutations – change in single base
1. Nonsense mutations – single nucleotide change which
allows introduction of a termination codon. Premature
arrest or discontinuation of the synthesis of polypeptide
chain.
2. Missense mutations – single nucleotide change leading
to substitution of one amino acid for another.
 Mutagens
o Radiation – ex. X-ray (non-ionizing radiation), Gamma rays
(ionizing radiation)
o Chemical Mutagens
1. Base analogues are incorporated in DNA during normal
replication; copied incorrectly in the next round
2. Deaminating agents
3. Alkylating agents
4. Acridine derrivatives
 Reversions – mutant’s original enzymatic activity is restored
o True reversion
1. Phenotypic reversion - regaining an activity lost as a
consequence of a mutation
2. Genotypic mutation - regaining an activity lost as a result
of restoration of the original DNA sequence
o Suppression – allow the presence of mutation but in a certain
area, it will compensate or overcome the effect of a certain
mutation
B. Genetic Recombination Figure 4. Homologous Recombination
 Combination of a DNA from a donor and a DNA from a recipient (A) By the process of homologous recombination, two segments of DNA with
giving rise to a new genome containing genetic information identical sequences can exchange regions. This exchange is initiated when part
from both sources. of one strand of a DNA molecule invades a second DNA strand and vice versa.
o Symmetric assimilation – single DNA strands get together This realignment of strands forms crossover. The Rec proteins catalyze the
annealing and exchange of portions of the two strands of DNA. The final result
forming a DNA heteroduplex.
is each of the two original DNA segments now has new segments that it
o Assymetric – single DNA strand from only one parent is obtained from the other DNA segments. If the segments are completely
transferred. identical, there will be no change in the two DNA strands. But if the two DNA
VI. ADDITIONAL NOTES segments contain regions with sequence differences flanked by regions of
 The source of DNA thet may be cloned in a bacterial cell is not identity where the crossover occur, the two resulting DNA molecules will have
restricted, and that DNA need not share any to its regulatory exchanged the variable region.
sequences with te bacterial host. (B) The double stranded break can be repaired by homologous recombination.
After the recombination event occurs, each double stranded DNA molecule will
o The only requirement is that the foreign DNA be attached to a
have a single-stranded DNA molecule will have a single-stranded gap that can
suitable vector that contains the appropriate origin of be sealed by DNA polymerase.
replication for the host organism.
o Without the appropriate promoters and ribosome-binding
sequences, the clones DNA cannot be properly expressed as
functional protein in the cell, but DNA will be replicated as long
as the origin of replicatin is held intact.
o It is possible to have more than one origin of replication on a
given vector so that the vector may be used for more than one
host.
 One source of variability within bacterial species is the widespread
movement of genes, especially antibiotic resistance genes,
between different species.
o Transposons are highly mobile genetic elements capable of
inserting into a variety of DNA elements, including prophages,
plasmids and bacterial chromosomes.
o Many transposons encode genes for antibiotic resistance as
well as genes to promote their own recombination.
o Transposons appear to have a little species specificity; the
same transposons can be found in a avariety of different
microorganisms.
In contrast, bacteriophages are highly specific in both the species they
infect and the site at which they integrate to other DNA.

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 Microbiology 1.7

VI. APPENDIX
Each strand of DNA has a deoxyribose phosphate backbone to
which different bases (adenine, thymine, cytosine, and guanine
are attached. The two strands interact with each other through
hydrogen bonds that form between the bases on different
strands. The specificity of the interaction is determined by two
hydrogen bonds that restrict binding of A to T and three hydrogen
bonds that restrict binding of C to G. A and G are purines; C and T
are pyrimidines. Additional stability comes from interactions
between the adjacent bases in the stack of bases that forms when
the DNA is in its helical form. The structure of RNA is similar,
except that the nucleotides in the backbone contain ribose
instead of deoxyribose. RNA contains the base uracil (U) instead
of thymine.

Figure 5. DNAStructure
The original version of DNA replication process. For simplicity, only
one direction of replication is shown here. In reality the same
process is occurring in the other direction as well so that the
replication region resembles a bubble instead of a single fork.
Enzymes nick one strand of the DNA to open up the super coiled
structure. Then helicase causes the DNA strands to uncoil and
separate. On the leading strand, DNA polymerase begins at a
double stranded region and continuously synthesizes a new copy
of the single strand in a 5 to 3 direction. On the lagging strand,
complementary RNA primers are laid down and serve as a double
stranded region from which the DNA polymerase can synthesize
short segments in a 5 to 3 direction. RNA primers are removed and
the gaps between the segments are sealed by DNA ligase. After the
new DNA is formed, DNA gyrase rewinds it into the super coiled
form.

Figure 6. DNA Replication

The newer- but still controversial –understanding of DNA

replication. The process is the same except that both the
leading and lagging strands are copied in segments.

Figure 7. Newer DNA Replication

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 Microbiology 1.7
Damage to DNA by pyrimidine dimer formation.
Ultraviolet light may cause adjoining pyrimidine residues,
shown here as thymine residues, to become cross-linked
(thymine dimmers), thus making mistakes in copying the
DNA (mutations) likely. Bacterial remove thymine dimmers
and leave a gap in one strand. If DNA polymerase moves
quickly enough, it will fill the gap, and the chromosome
will remain intact. If the polymerase does not act quickly
enough occur, preventing replication and causing the
bacterium to die.

- S. pneumoniae  can become multiresistant by acquiring

mutant penicillin-binding protein genes.
- Different resistant strains of S. pneumoniae have genes
that differ slightly form susceptible strains
A.) Homologous recombination between DNA of
susceptible strain and resistant strain can introduce
mutations in the gene of susceptible strain, but may not
result in making the susceptible strain resistant. It may
require several recombinations to achieve the resistance
of the susceptible strain.
B.) Analysis of resistant strains show that variety of
homologous recombination events have occurred to
produce new proteins that resist binding to penicillin.

The chromosomal DNA contains gene X- the gene to be

cloned.

-The plasmid cloning factor and chromosomal DNA are

both digested by the same restriction enzyme. The
cloning vector then becomes linear, and the -
chromosomal DNA is cleaved.
-Restriction enzymes are then deactivated. Linear
vector and chromosomal fragments are mixed.
Figure 9. Acquisition of a new penicillin resistance -If restriction enzymes used produce sticky ends, the
gene by homologous recombination in Streptococcus pneumonia sticky ends of the chromosomal fragments will
hybridize with the sticky ends of the linear vector.
Addition of DNA ligase seals the gaps.
-Result: recombinant plasmid containing gene X that -
can be used to transform bacterial DNA.
-Selection for the antibiotic resistant gene by including
antibiotic in the medium yields only cells that contain
plasmid.
-Though in reality, large percentage of linear cloning
vectors re-anneal to form circular plasmid that does
not contain the gene of interest. That is why it is
necessary to select screen transformants for clones
that have the desired gene.

Figure 10. The Cloning Process

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 Microbiology 1.7

Figure 11. Control of transcription by repressor and activators

Figure 10. The Cloning Process
(A) The repressor binds to a site on the DNA called the operator, which is located between the promoter and the transcription start site of
the gene. If the repressor is not bound to the operator, the RNA polymerase is able to transcribe the gene. If the repressor binds to the
operator, the RNA polymerase does not begin transcription. Whether a repressor binds or not depends on some signal it receives from
the environment.
(B) Activators help RNA polymerase bind to weak promoters and to begin transcribing. If the activator does not bind, the RNA polymerase
will not bind well and the transcription will not occur. Some genes are controlled by both repressors and activators.

A substrate is converted to an end product in a series of reactions

catalyzed by different enzymes. Arrows show the flow of substrate
through the pathway.
(A) If bacterium has produces a sufficient amount of end
product, it is not energy efficient to continue to produce more of
that end product. In this case the end product will bind to one of
the enzymes in the pathway and alter it so that it is no longer
active. Once the end product is used, the inhibition of the enzyme
will be reversed and the process of converting the substrate to
product will continue.
(B) In industrial applications in which large amounts of end
product are desired, feedback inhibition creates a problem. One
way to avoid this is to select for mutant enzymes that will no
longer bind the end product, and thus, the process can continue
uninterrupted.

Figure 12. Example of Feedback inhibition

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 Microbiology 1.7
Table 4. DNA Structure and DNA Synthesis and their applications
Structure of DNA/step in DNA synthesis Application
Structure of DNA: complementary 2 strands DNA hybridization; Southern blot
Opening up double helix (topoisomerases relieve supercoiling,
helicases unwind the DNA)
DNA polymerase copies each DNA strand, starting from a Primers used for DNA sequencing and polymerase
double-stranded region created by an RNA primer chain reaction (PCR)
RNA primers are removed and gaps filled
Used in cloning to seal cloned segment into the
DNA ligase seals nicks in in newly synthesized strand
cloning vector
DNA gyrase restores supercoiling Target of fluoroquinolone antibiotics

Table 5. Comparison of different molecular techniques

Primers/DNA Size fractionation by Restriction
Technique Hybridization
polymerase gel electrophoresis enzymes
Spot blot No No Yes (probe) No
Southern blot No Yes Yes (probe) Yes
DNA Sequencing, Yes Yes Yes (primer) No
PCR
Cloning No Sometimes Yes (sticky Yes
ends anneal)

Table 6. Comparison of DNA replication by vegetative cells and DNA replication during sporulation
Steps in division by vegetative cells Differences in division associated with sporogenesis
Copies of chromosomes are separated as cell elongates Division is asymmetric. No elongation.
prior to division (Symmetric division)
Both daughter cells are the same size One daughter cell is smaller than the other.
Both viable. Only one destined to be a spore will survive.
Both daughter cells contain all the molecules necessary The spore contains all the molecules necessary for
for viability. viability; can later become a vegetative cell
Both daughter cells have normal peptidoglycan cell walls. Spore has a thick covering (spore coat) that includes
layers of protein as well as peptidoglycan

Table 7. Chemical Signals

Origin and Type of
Effect of signal
type of Signal regulation
Determine whether special sigma factor
is produced.
Sigma Factor
Binding of signal to repressor or activator
Repressors,
changes conformation, determines if it
activators
Environment will bind DNA and affect transcription.
Sensor responds to signal by
Two component
phosphorylating activator or repressor,
system
enabling it to aid RNA polymerase to
transcribe gene
Bacteria produce autoinducer molecule,
use it to assess their concentration,
autoinducer binds repressor or activator,
Internal Quorum sensing
changing its conformation so that the
repressor or activator binds to or ceases
to bind to DNA
Feedback Amino acid or other product of pathway
inhibition (exogenous or endogenous) inhibits an
Internal or
enzyme in the pathway.
external
Covalent Modification activates or inactivates
modification enzyme

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