Microbial Growth

Binary Fission: The most common means of cellular replication and reproduction in
prokaryotes, binary fission is a process in which a cell grows, replicates its DNA, and then
divides into two fully functional, genetically identical cells. SImilar to mitosis in eukaryotes.

FtsZ Protein: Directs cell division in binary fission by assembling into a Z ring on the
cytoplasmic membrane. Additional proteins are added to the ring, turning it into a divisome,
which produces a peptidoglycan cell wall septum, separating the two daughter cells. When the
septum is complete, the two cells are fully separated.

Generation TIme: In eukaryotes, the time between the same points of the life cycle in two
successive generations.

Doubling Time: A term for generation time for prokaryotes, equal to the amount of time it takes
for the population to double through one round of binary fission.

𝑛
Cell population can be determined using: 𝑁 = 𝑁02
𝑛

The Growth Curve

The Lag Phase: The beginning of the curve. Represents the original population, the inoculum.
The number of cells does not grow in the lag phase, but cells grow larger and more
metabolically active. If any cells are damaged or shocked during inoculation, the repair takes
place during the lag phase. Determined by species, genetic make-up, medium composition, and
original inoculum size.

The Log/Exponential Growth Phase: Cells divide through binary fission, increasing the
population size exponentially. Plotted as both on arithmetic scale (looks exponential) and
semilog scale (looks linear). Cell reproduction exceeds cell death rate.
● Intrinsic Growth Rate: The genetically determined generation time under specific
growth conditions.

Stationary Phase: Growth has stopped due to a build-up of waste and a lack of available
nutrients. Oxygen depletion also limits aerobic cell growth. Cells switch to survival instead of
growth metabolism. Cell reproduction equals cell death rate

Death Phase: Nutrients are fully exhausted an the medium is inundated with waste materials,
leading to an exponential decrease of cells. Death rate exceeds reproduction rate. Some cells
lyse (fall apart) and turn into nutrients.
● Persisters: The few cells that survive the death phase due to a slow metabolic rate.
Direct Microscopic Cell Count: A known volume of a culture is transferred to a calibrated
slide, and all cells are individually counted under a microscope.

Viable Plate Count: A count of live cells.

Indirect Cell Count: Calculating cell count based on indirect detection of cell density, often
through measuring turbidity. Light is shot through a sample, and density is estimated based on
the amount of light that escapes. More light escapes=Less cells.

Biofilms: Dynamic ecosystems that often form on, though aren’t limited to, solid surface
substrates. Any surface in a liquid environment with even the smallest amount of nutrients will
form a biofilm. This includes the human mouth.
● Steamers: Filamentous biofilms that form in rapidly flowing water. Anchored to the
substrate by a “head”. “Tail” floats downstream in the current.
Extracellular Polymeric Substances (EPS): A hydrated gel composed primarily of
polysaccharides and other macromolecules. Maintains integrity and function of the biofilm by
allowing nutrients, waste, and gases to flow freely. Secreted by organisms in the biofilm.

Biofilm Formation:
1. Reversible attachment of planktonic cells.
2. First colonizers become permanently attached.
3. Growth and cell division.
4. Production of EPS and formation of water channels.
5. Attachment of secondary colonizers and dispersion of microbes to new sites.

Oxygen Requirements for Microbial Growth

Obligate Aerobes: Bacteria that require oxygen to live.

Obligate Anaerobes: Bacteria that are killed by oxygen.

Facultative Anaerobes: Bacteria that thrive in the presence of oxygen, but can also survive
without it.

Aerotolerant anaerobes: Bacteria that is wholly unaffected by the presence or absence of

oxygen.

Microaerophiles: Bacteria that require a specific level of oxygen for growth, often 1-10% as
opposed to the 21% in our atmosphere.

Optimum Oxygen Concentration: Ideal oxygen concentration for a given organism.

Minimum Permissive Oxygen Concentration: The lowest survivable oxygen level.

Maximum Permissive Oxygen Concentration: Highest survivable oxygen level

Reactive Oxygen Species: Highly unstable ions and molecules derived from partial reduction
of oxygen.

pH Requirements for Growth

Neutrophiles: Bacteria that grow best at neutral pH close to 7.0, grow optimally at pH within
one or two pH units of the neutral pH 7.

Acidophiles: Bacteria that grow optimally at pH near 3.0, microorganisms that grow optimally at
pH less than 5.55

Alkaliphiles: Organisms that grow optimally between pH of 8 and 10.5

Temperature Requirements for Growth

Psychrophiles grow best in the temperature range of 0-15C

Psychrotrophs thrive between 4 and 25C
Mesophiles grow best at moderate temperatures in the range of 20-45C. pathogens are usually
mesophiles
Thermophiles and hyperthermophiles are adapted to life at temperatures above 50C

Extreme temperature require changes in the composition of membrane lipids and proteins

Proteins in psychrophiles- rich in hydrophobic residues, display an increase in flexibility, and

have lower number of secondary stabilizing bonds when compared with homologous proteins
from mesophiles. Antifreeze proteins and solutes that decrease the freezing temperature of the
cytoplasm are common. Lipids in the membranes tend to be unsaturated to increase fluidity

Thermophiles and hyperthermophiles- ratio of saturated to polysaturated lipids increases to

limit the fluidity of the cell membranes. DNA sequences show a higher proportion of
guanine-cytosine nitrogen bases, which are held together by 3 H-bonds.

Other Environmental Conditions That Affect Growth

Halophile: Requires high salt concentration.

Halotolerant: Grows and multiplies in the presence of high salt, but does not require it.
Photosynthetic: Depends on visible light for energy
 Media Used for Bacterial Growth

Defined- contain only chemically known components

Selective- favor growth of some microorganisms while inhibiting others
Enriched- contain added essential nutrients a specific organism needs to grow
Differential- help distinguish bacteria by the colour of the colonies or the change in the medium

Using Microbiology to Discover the Secrets of Life

On the study of Acetabularia: In the 1930s and 1940s, German scientist Joachim Hammerling
removed the ‘foot’ of a set of single celled Acetabularia and found that, unlike other parts, it
would grow back, suggesting that hereditary information was stored in the foot. Later, he grafted
the foot of a Acetabularia mediterranea onto the footless stem of an Acetabularia crenulata and
found that the resulting regenerated cap was that of a Acetabularia mediterranea, confirming
that the foot contained genetic information, and that the stem did not. This implied the existence
of a specific in the cell that contained genetic information, as opposed to it being ubiquitous.

On Beadle and Edward: George Beadle and Edward Tatum irradiated the red mold
Neurospora crassa and noticed that the mold lost its ability to produce certain proteins and
nutrients. This implied that genetic material could be damaged and changed by radiation.
Subsequent tests led them to form the One Gene-One Polypeptide Hypothesis.

One Gene-One Polypeptide Hypothesis: Each gene is responsible for the production of a
single polypeptide.

On Griffith’s Transformation Experiments: Frederick Griffith discovered that hereditary

information could be transferred “horizontally” (between organisms in the same generation). He
injected mice with a HARMLESS strain of LIVE bacteria and a HARMFUL strain of DEAD
bacteria, and found that the mice died as if infected by the LIVE HARMFUL BACTERIA. This
implied that the live bacteria had somehow taken genetic information from the dead bacteria.

Proof of DNA as Genetic Material: Alfred Hershey and Martha Chase infected bacteria with
viruses filled either with irradiated DNA or irradiated proteins, and found that only the bacteria
infected by DNA filled viruses went on to produce more viruses.

Structure and Function of DNA

DNA Structure: Composed of a phosphate group, a deoxyribose sugar (oxygen atom bonded
to all but one carbon), and a nitrogenous base. The lowest unit of genetic material. DNA strands
match up in an antiparallel double helix stabilized by hydrogen bonds between parallel
nitrogenous bases:
● Pyrimidines: Nitrogenous bases that only have a single six carbon ring
○ Cytosine: Pairs with Guanine. Distinguished by its amine group.
 ○ Thymine: Pairs with Adenine. Distinguished by its methyl group. Present in DNA
but not RNA.
● Purines: Nitrogenous bases with a double ring structure.
○ Adenine: Pairs with Thymine. Distinguished by its LACK of an oxygen R group.
○ Guanine: Pairs with Cytosine. Distinguished by its oxygen R group.

Phosphodiester Bonds: Covalent bonds in which the phosphate group to the 5’ carbon one
DNA’s sugar connects to the 3’ carbon of another, forming a sugar-phosphate backbone.

C-G forms 3 hydrogen bonds

A-T forms 2 hydrogen bonds

Vertical Gene Transfer: DNA splits and is replicated, resulting in the formation of two
semiconservative DNA helixes in which one strand per helix is an original and the other a
copy. When the cell divides, one helix goes to each daughter cell.

Structure and Function of RNA

RNA Structure: Typically single stranded and made of ribonucleotides linked together by
phosphodiester bonds. Much like DNA, except ribose sugar has oxygen bonded to all of its
carbons and the nitrogenous base, thymine, is replaced with uracil. RNA strands can fold in on
themselves, forming hydrogen bonds between small sets of their own nitrogenous bases. THis
allows for the formation of loops and hairpins. As a whole less stable than DNA.

Uracil: Nitrogenous base present only in RNA that takes the place of thymine. Lacks thymine’s
methyl group.

Messenger RNA (mRNA): Made through transcription. Acts as a photocopy of specific

instructions for protein synthesis (translation). Carries said instructions from the DNA to
ribosomes and other organelles which use it to produce proteins. Unstable and short lived,
especially in prokaryotes.

Ribosomal RNA (rRNA): Encoded in the DNA then copied into long RNA molecules that are
cut into smaller mature RNA pieces. Becomes a structural part of a ribosome (60% of its mass)
and ensures proper alignment of mRNA, tRNA, and ribosome during protein synthesis.
Catalyzes peptide bond formation between amino acids

Transfer RNA (tRNA): Small, usually only 70-90 nucleotides long. Carries the correct amino
acid to the site of protein synthesis in the ribosome. Stable due to intramolecular base pairing

Structure and Function of Cellular Genomes

Gene - Segments of DNA molecules. Individual genes contain the instructional code necessary
for synthesizing various proteins, enzymes, or stable rna molecules.
Genome - The entire genetic content.
Phenotype - The expression (cell activity and observable characteristics) of the genes.
Noncoding DNA: DNA that does not encode proteins or stable RNA products.

Extrachromosomal DNA: DNA contained OUTSIDE the chromosomes, that are also part of
the genome. Includes mitochondrial DNA.
● Plasmids: Smaller loops of DNA found in prokaryotes that contain genes that are not
essential for growth. Can be exchanged with other bacteria (HGT) and occasionally
codes for something useful.
Viral Genomes: Can be single or double stranded DNA, or sometimes even single or double
stranded RNA. Typically smaller than bacterial genomes and rarely encodes more than a few
genes.

The Functions of Genetic Material

Genomes store and express the genetic information that gives rise to a cell’s function

The central dogma states that DNA organized into genes specifies the sequences of
messenger RNA (mRNA), which, in turn, specifies the amino acid sequence of proteins.

The genotype of a cell is the full collection of genes a cell contains. Not all genes are
used to make proteins simultaneously.

The phenotype is a cell’s observable characteristics resulting from the proteins it is

producing at a given time under specific environmental conditions.

DNA Replication

DNA IS ADDED IN THE 5’->3’ DIRECTION

FROM 5’ TO 3’

-For bacterial DNA replication to begin, the supercoiled chromosome is relaxed by

topoisomerase II/DNA gyrase.

-An enzyme called helicase then separates the DNA strands by breaking the H bonds between
the nitrogenous base pairs. (Recall that AT sequences have fewer hydrogen bonds, so they
have weaker interactions than guanine-cytosine (GC) sequences). These enzymes require ATP
hydrolysis.

-As the DNA opens up, Y-shaped structures called replication forks are formed. 2 replication
forks are formed at the origin of replication, allowing for bidirectional replication and formation of
a structure that looks like a bubble when viewed with a transmission electron microscope. This
structure is called a replication bubble.

-The DNA near each replication fork is coated with single-stranded binding proteins to
prevent the single-stranded DNA from rewinding into a double helix.

-Primase synthesizes a short RNA primer, providing a free 3’-OH group to which DNA
polymerase III can add DNA nucleotides

-Eukaryotic genomes are much more complex and larger than prokaryotic genomes and are
typically composed of multiple linear chromosomes.

-The rate of replication is approximately 100 nucleotides per second—10 times slower than
prokaryotic replication. (prokaryotic is faster)

(But for the actual replication process, eukaryotes and prokaryotes are quite similar).

RNA Transcription

Rolling circle replication is a type of rapid unidirectional DNA synthesis of a circular DNA
molecule used for the replication of some plasmids.

-During transcription, the information encoded in DNA is used to make RNA.

-RNA polymerase synthesizes RNA, using the antisense strand of the DNA as a
template by adding complementary RNA nucleotides to the 3’ end of the growing strand.

-RNA polymerase binds to DNA at a sequence called a promoter during the initiation
of transcription.

Prokaryotes: Continuous: both transcription and translation happen in cytoplasm, mRNA is

unstable, translation is fast (20 a.a/sec)

Eukaryotes: Discontinuous: Transcription happens in nucleus and translation happens in

cytoplasm, mRNA is stable, translation is slow (1a.a/sec)

Protein Synthesis (Translation)

The relationship between an mRNA codon and its corresponding amino acid is called the
genetic code. Each amino acid is defined within the mRNA by a triplet of nucleotides
called a codon.
With the three-nucleotide code, there are 64 possible combinations in total (with four diff
nucleotides possible at each of the three positions within the codon).

This number is greater than the number of amino acids, and a given amino acid can be encoded
by more than one codon. The genetic code is degenerate, because several mRNA codons
code for the same amino acids (Ex: UCU and UCC both mean serine). So the genetic code is
almost universal among living organisms.

Initiation:

- the small ribosomal subunit binds with initiation factors and an initiator
tRNA at the start codon of an mRNA

Elongation:

- A charged tRNA binds to mRNA in the A site of the ribosome. A peptide

bond is catalyzed between the two adjacent amino acids, breaking the
bond between the first amino acid and its tRNA.
- The ribosome moves one codon along the mRNA; and the first tRNA is
moved from the P site of the ribosome to the E site and leaves the
ribosomal complex.

Termination:

- the ribosome encounters a stop codon, which does not code for a tRNA.
Release factors cause the polypeptide to be released, and the ribosomal
complex dissociates.

Mutations

A mutation is a heritable change in the DNA sequence.

Point mutation affects a single base. It most commonly occurs when one base is substituted or
replaced by another.
Insertion is the addition of one or more bases.
Deletion is the removal of one or more bases.
Silent mutation is when a change has no effect on the protein structure.
Missense mutation is when a different amino acid is incorporated into the resulting
polypeptide.
Conditional mutation happens because sometimes the effects of missense mutations may be
only apparent under certain environmental conditions.
Nonsense mutation is when a codon encoding an amino acid is converted into a stop codon.
Frameshift mutation is caused by insertions or deletions of a number of nucleotides (that are
not a multiple of three). It leads to a shift in the reading frame.

Chemical mutagens such as intercalating agents slide between the stacked nitrogenous base
of the DNA double helix, distorting the molecule and creating atypical spacing between
nucleotide base pairs. Therefore during DNA replication, DNA polymerase may either skip
replicating several nucleotides, or insert extra nucleotides.

The Ames test is a method that uses bacteria for rapid, inexpensive screening of the
carcinogenic potential of new chemical compounds. It measures the mutation rate associated
with exposure to the compound.

How Asexual Prokaryotes Achieve Genetic Diversity

o Transformation allows for competent cells to take up naked DNA, released from other cells on
their death, into their cytoplasm, where it may recombine with the host genome
o In generalized transduction, any piece of chromosomal DNA may be transferred by
accidental packaging of the degraded host chromosome into a phage head. In
specialized transduction, only chromosomal DNA adjacent to the integration site of a
lysogenic phage may be transferred as a result of imprecise excision of the prophage.
o Conjugation- use of a hollow tube called a conjugation pilus to transfer genes between
cells

o Transposons are molecules of DNA with inverted repeats at their ends that also
encode the enzyme transposase, allowing for their movement from one location in DNA
to another. Although found in both prokaryotes and eukaryotes, transposons are
clinically relevant in bacterial pathogens for the movement of virulence factors, including
antibiotic resistance genes.
o Process whereby DNA independently excises from one location in a DNA molecule
and integrates elsewhere.

Gene Regulation: Operon Theory

o Inducible- bacterial operon, typically containing genes encoding enzymes in a

degradative pathway, whose expression is induced by the substrate to be degraded
when the substrate is available for the cell to use, but that is otherwise prepressed in the
absence of the substrate. Always off until needed
o Repressible- bacterial operon, typically containing genes encoding enzymes required
for biosynthetic pathway and that is expressed when the product of the pathway
continues to be required but it repressed when the product of the pathway accumulates,
removing the need for continued expression. – always on until shut off.
o Constitutively expression- always on
o Prokaryotic structural genes of related function are often organized into operons, all
controlled by transcription from a single promoter. The regulatory region of an operon
includes the promoter itself and the region surrounding the promoter to which
transcription factors can bind to influence transcription
o Gene expression