Bacterial Growth,

Metabolism and
Genetics

Questions:
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Bacterial Growth
Bacterial Growth
 As the previous video indicates:
 Bacteria replicate by binary fission
– The process by which one bacterium splits
into two.
 Bacterial growth occurs via geometric
progression.
– A population of bacteria doubles every
generation time. This is due to the nature
of replication, binary fission.
 Bacterial Growth:
Binary fission
 During DNA replication, each strand of DNA
attaches to proteins at the cell division plane. The
newly replicated DNA become separated as the
bacterium grows to its full size.
 Fts proteins interact, forming a ring at the cell
division plane.
 Fts proteins are the cell division apparatus known
as the divisome
 Proteins at the divisome synthesize new
peptidoglycan and plasma membrane that splits
the bacterium into 2 daughter cells.
Bacterial Growth:
Binary fission: Divisome
proteins
 4 major Fts proteins exist:
 FtsZ, similar to tubulin in eukaryotic
cells, forms a constricting ring at the
division site.
 FtsA, along with FtsZ are the driving
force behind membrane invagination
at the division site.
 FtsK helps in separating the replicated
bacterial chromosome.
 FtsI plays a role in the later stages of
peptidoglycan synthesis.
Bacterial Growth:
Generation Time
 Generation time refers to the time
required for a population to double.
 Most bacteria have a short generation
time
– Between 20 min, 1 hour (optimal
conditions)
– Pathogenic bacteria in humans usually
have a longer generation time, between 5-
10 hours
 As you have seen in the introductory
video bacteria can reach astronomical
numbers in a short period of time!
Bacterial Growth:
Generation Time
 The number of bacteria at a given time
can be estimated via the following
formula:
 Nt = No X 2 n
 Nt is the number at a given time
 No is the original number of bacteria
 n is the number of divisions the
bacteria have undergone during the
period of time
Bacterial Growth:
Generation Time
 Test yourself
 If we start with 5 E. coli cells
(which have a generation time of
20 minutes) and allowed them to
grow for 2 hours how many cells
would we have?
 Remember: Nt = No X 2 n
 Nt is the number at a given time
 No is the original number of bacteria
 n is the number of divisions the bacteria have undergone during the
period of time
Bacterial Growth:
Generation Time
 Answer

 Nt = No X 2 n
=5X26
= 5 X 64
= 320 cells after 2 hours
 Bacterial Growth:
Growth curves
 A single bactera can reach a
population of 5 billion in
about 12 hours in 2 mL of
media
 Unhindered growth requires
– Unlimited supply of nutrients
and
– Unchanging environment
 This is a theoretical example.
Sooner or later conditions
become unfavorable
 Bacterial Growth:
Growth curves
 In actuality conditions
change so we only
observe exponential
growth of bacteria for a
certain amount of time.
 We view 4 discrete
phases of bacterial
growth (Ie. in a test
tube)
 Bacterial Growth:
Growth curves
 Lag phase- Bacteria
require time to adapt
to their new
environment before
they begin dividing.
 Bacterial Growth:
Growth curves
 Log phase- Bacteria
begin dividing. Their
rate of division is
characteristic of the
strain of bacteria and
the growth
conditions.
 Bacterial Growth:
Growth curves
 Stationary Phase- The
bacteria eventually run
out of nutrients, and
toxic (waste) substances
build up in the media.
– This triggers the
production of alarmones
which cause DNA
synthesis to stop.
– During late stationary
phase some Gram +
strains produce spores
 Bacterial Growth:
Growth curves
 Death phase- The
buildup of toxic
compounds (Ie EtOH)
becomes so great
that the number of
microbes decrease.
Bacterial Growth:
Temperature
 Different strains of bacteria have
varying optimal growth temperatures.
These include:
 Psychrophiles - Optimum growth
temperature is between -5C and 15C.
Found near Arctic and Antarctic
regions.
 Mesophiles - Optimum growth
temperature is between 25C and 45C.
Most bacteria we examine will be
mesophilic (Ie bacteria associated with
humans)
 Bacterial Growth:
Temperature
 Thermophiles – Optimum
growth temperature between
45C and 70C. Commonly found
in hot springs and in compost
heaps.
 Hyperthermophiles - Optimum
growth temperature is between
70C and 110C. Found near
hydrothermal vents deep in the
ocean.
 Bacterial Growth:
Oxygen
 Different bacteria have
varying growth
requirements for oxygen.
 Obligate aerobes require
oxygen, thus grow on the
top of a flask
 For Obligate anaerobes,
oxygen is toxic, thus they
grow at the bottom of a
flask.
 Bacterial Growth:
Oxygen
 Facultative aerobes
grow under both
aerobic and anaerobic
conditions
 Microaerophilic bacteria
require a bit of oxygen
however too much is
toxic
Bacterial Growth:
pH
 Bacteria have varying pH
requirements.
 Acidophiles - Prefer a more acidic
media
 Neutrophiles – Prefer a near neutral pH
 Alkalinophiles – Prefer a more basic
media
Bacterial Growth:
Nutritional
Requirements
 Bacteria are often grouped according
to their Energy source and their source
of carbon
 Energy source
 1. Phototrophs use radiant energy
(light) as their primary energy source.
 2. Chemotrophs use the oxidation
and reduction of chemical compounds
as their primary energy source.
Bacterial Growth:
Nutritional
Requirements
 Carbon source
 1. Autotrophs: require only carbon
dioxide as a carbon source. An
autotroph can synthesize organic
molecules from inorganic nutrients.
 2. Heterotrophs: require organic
forms of carbon. A Heterotroph cannot
synthesize organic molecules from
inorganic nutrients.
Bacterial Growth:
Nutritional
Requirements
 All organisms in nature can be placed
into one of the following groups
 1. Photoautotrophs - use light as an
energy source and carbon dioxide as
their main carbon source.
 2. Photoheterotrophs - use light as
an energy source but cannot convert
carbon dioxide into energy. Instead
they use organic compounds as a
carbon source.
Bacterial Growth:
Nutritional
Requirements
 3. Chemolithoautotrophs - use
inorganic compounds such as
hydrogen sulfide, sulfur, ammonia,
nitrites, hydrogen gas, or iron as an
energy source and carbon dioxide as
their main carbon source.
 4. Chemooganoheterotrophs - use
organic compounds as both an
energy source and a carbon source.
Bacterial Growth:
Nutritional
Requirements
 Nitrogen source
 Nitrogen is needed for the
synthesis of such molecules as
amino acids, DNA, RNA and ATP.
Depending on the organism,
nitrogen, nitrates, ammonia, or
organic nitrogen compounds may
be used as a nitrogen source
Bacterial Growth:
Nutritional
Requirements
 Minerals
 Sulfur
 Sulfur is needed to synthesize
sulfur-containing amino acids and
certain vitamins. Depending on
the organism, sulfates, hydrogen
sulfide, or sulfur-containing amino
acids may be used as a sulfur
source.
Bacterial Growth:
Nutritional
Requirements
 Phosphorus
 Phosphorus is needed to synthesize
phospholipids, DNA, RNA, and ATP.
Phosphate ions are the primary source
of phosphorus.
 Potassium, magnesium, and
calcium
 These are required for certain
enzymes to function as well as
additional functions.
 Iron
 Iron is a part of certain enzymes
Bacterial Growth:
Nutritional
Requirements
 Trace elements
 Trace elements are elements required
in very minute amounts, and like
potassium, magnesium, calcium, and
iron, they usually function as
cofactors in enzyme reactions. They
include sodium, zinc, copper,
molybdenum, manganese, and cobalt
ions.
 Cofactors usually function as electron
donors or electron acceptors during
enzyme reactions.
Bacterial Growth:
Nutritional
Requirements
 Growth factors
 Growth factors are organic
compounds that a cell must have
for growth but cannot synthesize
itself.
 Organisms having complex
nutritional requirements and
needing many growth factors are
said to be fastidious.
Bacterial Metabolism
 Metabolism - All of the organized
chemical reactions in a cell.
 Catabolic reactions - Biochemical
reactions that generate energy from
the breakdown of chemical compounds
 Anabolic reactions - Require energy
in order to synthesize chemical
compounds
 All of these biochemical reactions are
catalyzed of enzymes
Bacterial Metabolism
ATP
 The energy currency of cells
 Phosphate groups are all
negatively charged, they repel
each other and stress the bond
holding them together, much like
a bent diving board
 Bacterial Metabolism
Generating ATP
 Substrate-level
phosphorylation

 The production of ATP

from ADP by a direct
transfer of a high-energy
phosphate group from a
phosphorylated
intermediate metabolic
compound in an
exergonic catabolic
pathway
Bacterial Metabolism
Generating ATP
 Oxidative
phosphorylation
 Electron transfer during
oxidation-reductions
permits pumping H+
across a membrane.
 As the H+ concentration
increases on one side of
the membrane, an
electrochemical
gradient develops.
Bacterial Metabolism
Glycolysis
Bacterial Metabolism
Glycolysis
 Starting with: glucose (6C) + 2 NAD+
2 ADP +2 inorganic phosphates (Pi)

 Yields: 2 pyruvate (3C) + 2 NADH +

2 H+ + 2 net ATP

Glycolysis does not require oxygen

and can occur under aerobic and
anaerobic conditions.
 Bacterial Metabolism
Transition Reaction
 As the two acetyl
groups become
oxidized to acetyl-
CoA, two molecules
of NAD+ are reduced
to 2NADH + 2H+.
 In bacteria this
occurs in the
cytoplasm
Bacterial Metabolism
Citric Acid Cycle
 STEP 1
 Coenzyme A
transfers its 2-
carbon acetyl group
to the 4-carbon
compound
oxaloacetate to
form the 6-carbon
molecule citrate.
Bacterial Metabolism
Citric Acid Cycle
 STEP 2
 Citrate is
rearranged to form
an isomer
Bacterial Metabolism
Citric Acid Cycle
 STEP 3
 The 6-carbon isocitrate
is oxidized and a
molecule of carbon
dioxide is removed
producing the 5-carbon
molecule alpha-
ketoglutarate. During
this oxidation, NAD+ is
reduced to NADH +
H+.
 Bacterial Metabolism
Citric Acid Cycle
 STEP 4
 Alpha-ketoglutarate is
oxidized, carbon
dioxide is removed,
and coenzyme A is
added to form the 4-
carbon compound
succinyl CoA. During
this oxidation, NAD+ is
reduced to NADH +
H+.
Bacterial Metabolism
Citric Acid Cycle
 STEP 5
 CoA is removed from
succinyl-CoA to produce
succinate. The energy
released is used to
make guanosine
triphosphate (GTP) from
guanosine diphosphate
(GDP) and Pi by
substrate-level
phosphorylation. GTP
can then be used to
make ATP.
Bacterial Metabolism
Citric Acid Cycle
 STEP 6
 Succinate is oxidized
to fumarate. During
this oxidation, FAD is
reduced to FADH2
Bacterial Metabolism
Citric Acid Cycle
 STEP 7
 Water is added to
fumarate to form
malate.
Bacterial Metabolism
Citric Acid Cycle
 STEP 8
 Malate is oxidized to
produce
oxaloacetate, the
starting compound
of the citric acid
cycle. During this
oxidation, NAD+ is
reduced to NADH +
H+.
Bacterial Metabolism
Citric Acid Cycle
 The NADH and FADH2 carry
protons and electrons to the
electron transport chain to
generate additional ATP by
oxidative phosphorylation.
 The intermediate molecules
pyruvate, oxaloacetate and
alpha-ketoglutarate are used in
the synthesis of amino acids and
Bacterial Metabolism
Electron Transport
Chain

Yield
3 ATP per NADH
2 ATP per FADH2
Bacterial Metabolism
Total Theoretical Yield
 38 ATP:
 4 from substrate-level
phosphorylation
 34 from oxidative
phosphorylation
Bacterial Metabolism
Anaerobic Respiration
 Some bacteria undergo anaerobic
respiration in which an inorganic
molecule other than oxygen is the
final electron acceptor.
 For example, (SO42-) reducing it to
H2S.
 (NO3-) reducing it to nitrite (NO2-)
 (NO) reducing it to nitrogen gas
(N2)
Bacterial Metabolism
Fermentation
 Occurs in anaerobic and
facultative anaerobic bacteria
 Terminal electron acceptor is an
organic molecule
 Involves only glycolytic pathway
– Thus only 2 ATP are generated per
molecule of glucose.
Bacterial Metabolism
Fermentation
 The two molecules of pyruvate are
converted into many different end
products depending on the species of
bacteria.

 Saccharomyces: ethyl alcohol and CO2

 Streptococcus and Lactobacillus: lactic
acid
 Escherichia coli: acetic acid, lactic
acid, succinic acid, ethyl alcohol, CO2,
 Bacterial Genetics
DNA Replication
 DNA replication is
initiated at the OriC
sequence
 It occurs in both
directions along the
bacterial
chromosome
Bacterial Genetics
DNA Replication
Bacterial Genetics
DNA Replication
Bacterial Genetics
DNA Replication
 Replication ends once the two
replication forks meet, 180o from
OriC
 Multiple growing replication forks
can be initiated in a cell before
complete septum formation and
cell division.
– The daughter cells are “born
pregnant”
Bacterial Genetics

 For homework: Please review

 Lac Operon
 Transcription
 Translation
Bacterial Genetics
Types of Mutations
 SPONTANEOUS MUTATION: A
mutation that occurs naturally
about one in every million to one
in every billion divisions
 Bacterial Genetics
Types of Mutations
 During DNA replication:
 Substitution,
 Deletion,
 Addition mutations
can occur
Bacterial Genetics
Results of Mutations
 Missense Mutation
 Results in one wrong codon, one
wrong amino acid.
Bacterial Genetics
Results of Mutations
 Nonsense Mutation
 Results in a "stop" codon and
premature termination of the
protein.
Bacterial Genetics
Results of Mutations
 Sense Mutation
 Results in a new codon which still
codes for the same amino acid.
Bacterial Genetics
Results of Mutations
 Frameshift Mutation
 Results in a reading frame shift.
All codons and all amino acid
after the shift are usually wrong.
Bacterial Genetics
Types of Mutations

 INDUCED MUTATION: A mutation

that occurs as a result of exposure to a
mutagen.
– Chemical mutagens
– Ultraviolet Radiation: 260 nm - 270 nm
range
– Ionizing Radiation: X-rays and gamma rays
Bacterial Genetics:
Genetic transfer between
cells.
 Transformation
 The Rec A protein promotes
genetic exchange between a
fragment of the donor's DNA and
the recipient's DNA.
Bacterial Genetics:
Genetic transfer between
cells.
 Transduction
 Occasionally a bacteriophage
head or capsid assembles around
a fragment of donor DNA or
plasmid instead of a phage
genome by mistake.
Bacterial Genetics:
Genetic transfer between
cells.
 F+ conjugation: codes for a
sex pilus
Bacterial Genetics:
Genetic transfer between
cells.
 Resistance plasmid (R-
plasmid): codes for antibiotic
resistance and sex pilus
Bacterial Genetics
Recombinant DNA
Bacterial Genetics
Recombinant DNA
 Restriction endonuclease
 Naturally occurring enzymes in
bacteria
 Help protect bacteria from viral attack
by cutting up the foreign viral DNA
while not harming the bacterium's own
DNA.
 Recognize specific palandromic
deoxyribonucleotide base sequences
and then split each DNA strand at a
specific site within that sequence.
Bacterial Genetics
Recombinant DNA
 EcoRI Escherichia Coli GAATTC
Sticky
 PvuI Proteus vulgaris CGATCG
Sticky
 PvuII Proteus vulgaris CAGCTG
Blunt
Bacterial Genetics
Recombinant DNA
 In order to be useful, the recombinant
DNA molecules have to be made to
replicate and function genetically
within a cell.
 One method for doing this is to use
plasmid DNA from bacteria. Small DNA
fragments can be inserted into the
plasmids, which are then introduced
into bacterial cells.
 As the bacteria reproduce, so do the
recombinant plasmids. The result is a
bacterial colony in which the foreign
gene has been cloned.
Bacterial Genetics
Recombinant DNA:
STEPS
 The first step in transformation is to select a
piece of DNA to be inserted into a vector.
 The second step is to cut that piece of DNA
with a restriction enzyme and then ligate the
DNA insert into the vector with DNA Ligase.
 The insert contains a selectable marker
which allows for identification of recombinant
molecules.
 An antibiotic marker is often used so a host
cell without a vector dies when exposed to a
certain antibiotic, and the host with the
vector will live because it is resistant.
Bacterial Genetics
Recombinant DNA:
STEPS
 The vector is inserted into a host
cell, in a process called
transformation. One example of a
possible host cell is E. Coli. The
host cells must be specially
prepared to take up the foreign
DNA. (Salt treatment, electric
shock)
Bacterial Genetics
Recombinant DNA:
STEPS
 Recombinant DNA works when
the host cell expresses protein
from the recombinant genes. A
significant amount of
recombinant protein will not be
produced by the host unless
expression factors are added.
Bacterial Genetics
Recombinant DNA:
STEPS
 Protein expression depends upon
the gene being surrounded by a
collection of signals which provide
instructions for the transcription
and translation of the gene by the
cell. These signals include the
promoter, the ribosome binding
site, and the terminator.
Bacterial Genetics
Recombinant DNA:
STEPS
 Expression vectors, in which the
foreign DNA is inserted, contain
these signals. Signals are species
specific. In the case of E. Coli,
these signals must be E. Coli
signals as E. Coli is unlikely to
understand the signals of human
promoters and terminators.
Bacterial Genetics
Recombinant DNA:
STEPS
 Another possible cloning vector is
a virus. The isolated gene is
inserted into the DNA of a virus
and "glued" using DNA ligase. The
virus then injects the gene into
the cell’s main DNA. The cell then
begins producing the desired
protein.