PS05CIGB25 Quiz 2

Q1. Bolivar and Rodriguez constructed which vector?

a) Yip7
b) R6-5
c) pUC8
d) pBR322

Q2. Which of the following properties is not taken into account while looking for a suitable
vector?
a) Size
b) Parent organism
c) Restriction site
d) Origin of replication

Q3. Which antibiotic resistance is present in pBR322?

a) Ampicillin
b) Kanamycin
c) Lactase
d) Gentamycin

Q4. Size of pBR322 is _______

a) 100 kb
b) 10 kb
c) 4.3 kb
d) 1 kb

Q5. Who determined the sequence of Lambda phage?

a) Sanger
b) Maxam Gilbert
c) Ramachandran
d) John Williams

Sporulation

Definition: What is Sporulation?

Essentially, sporulation refers to the formation of spores from vegetative cells
during unfavorable environmental conditions. As such, it may be described as an
adaptive response that allows the organism to survive given adverse conditions
(radiation, extreme heat or cold, lack of nutrition etc).

Compared to vegetative cells, spores (formed during sporulation) are

multilayered structures that tend to be dormant (or relatively dormant).
These characteristics make it possible for some of the spores to preserve
the genetic content of the organism during harsh environmental
conditions.

During certain unfavorable conditions (depending on the organism), some

of the vegetative cells go through a series of morphological changes (and
some level of programmed gene expression) that ultimately produce
spores.

Apart from genetic material, spores also contain some cytoplasm, specific
acids, ribosome, and the appropriate enzymes among others that allow
the spore to germinate during favorable environmental conditions.

The term "spore" comes from the Greek word for Seed.

Some of the organisms that produce spores include:

 Plants
 Algae
 Protozoa
 Bacteria
 Fungi

Depending on the organism, spores vary significantly. 

There are also different types of spores including:

 Asexual spores (e.g. exogenous spores produced by Conidia oidia)

 Sexual spores such as Oospores and Zygote
 Vegetative spores (e.g. Chlamydospores)
 Megaspores of plants (female gametophyte)
 Microspores of plants (develop to formmale gametophyte)
 Endospores - Type of spores that are produced within the organism
(e.g. bacteria)
 Exospores - Exospores are a type of spores that are produced
outside the cell (e.g. in the genus Methylosinus)
Sporulation in Bacillus subtilis in stages

Sporulation in Bacteria

A majority of spore-forming bacteria are Gram-negative bacilli (rod-

shaped). These include aerobic Bacillus and anaerobic Clostridium
species. Although some Gram-negative bacteria have been shown to be
capable of producing spores, it's only a few species found in a few genera.

* Some filamentous cocci have also been shown to be capable of

sporulation (producing endospores)

Depending on the type of bacteria, four types of spores are produced.

These include:

Endospores: Endospores are the most common types of spores. They

are typically produced by Clostridium (e.g. Clostridium botulinum),
Bacillus (e.g. Bacillus anthracis) and Sporosarcina bacteria (e.g.
Sporosarcina ureae).

Cysts: Cysts are a group of cells that join together in order to survive
harsh environmental conditions. When conditions in the environment
become unfavorable, the cells come together in a manner that allows
them to survive. They are common in Azotobacter species
* Unlike endospores, cysts are non-reproductive cells. They are also less
resistant to tough environmental conditions and have a tough cell wall
that is waterproof in place of the peptidoglycan layer found on the cell
wall of bacterial endospores

Myxospores: A myxospore is a type of spore that is produced by

myxobacteria/ Myxobacterates (slimy molds bacteria). Referred to as
resting cells in some books, myxospores are capable of surviving such
harsh environmental conditions as UV light, high temperatures, and
desiccation.

They are formed within the fruiting body of the bacteria and in contrast to
other resting cells; they are formed from specialized spore-bearing
structures.

Exospores: Exospores are a type of spore that are produced by members

of the phylum Actinobacteria. These spores are produced through
the budding of mycelium and can survive in harsh environmental
conditions better than the vegetative cells.

* Although spores play an important role in the life cycle of bacteria, they
are not an obligatory stage. As such, they are typically common during
unfavorable environmental conditions.

* Sporulating bacteria are ubiquitous and can be found in soil, water, and
air among other environments in nature.

* Compared to the other types of spores, endospores are the most

defensive structures capable of surviving harsher environmental
conditions. This is largely due to the fact that beneath the thin and
delicate exosporium, they have a spore coat that is made up of layers of
proteins.

Some of the other characteristics associated with the high resistance

capacity of endospores include:
  High dipicolinic acid content
 High calcium concentration
 High amounts of SASP (small acid soluble DNA binding proteins)

Although unfavorable conditions such as diminishing sources of nutrition

play an important role in sporulation, in that they trigger the process,
sporulation does not necessarily start taking place right away. Rather, the
bacteria will start by trying to move to an area with favorable conditions.

For instance, in the event that food sources diminish, the bacteria, using
such structures will attempt to move to a new location through such
processes as chemotaxis.

Other mechanisms to survive may involve an attempt to adapt to the new

conditions or changing their mode of reproduction among others.
Therefore, sporulation occurs as the last survival strategy. Before the cell
commits to sporulation, several conditions must be met.

These include:

 Chromosome integrity
 Functional Krebs cycle

The following is a basic mode of sporulation of Bacillus subtilis:

The end of vegetative growth

Phosphorelay system (consisting of a number of kinases (histidine

kinases)) transmit information regarding the environmental conditions
and conditions of the cell to the Spo0A (master transcriptional regulator)
thus activating it - This phase also plays an important role in determining
the state of phosphorylation of the intercellular pool of the master
transcriptional regulator

* Here, Spo0A with a phosphyl group play an important role in regulating

expression of about 121 genes some of which are involved in sporulation -
High levels of Spo0A~P promote the process while low levels result in the
formation of biofilm.

Stage 1: Axial filamentation and chromosome division - Activation of the

master transcriptional regulator is followed by chromosome replication
with each chromosome moving to the opposite poles of the cell – One of
the chromosomes remains in the mother cell while the other is contained
in the forespore
Stage II: Asymmetric septation - Origin-proximal regions of the cell are
tethered to the opposite poles of the cell

Chromosomal DNA stretch forming axial filaments

Stage III: Engulfment - As the cell divides, 30 percent of the origin-

proximal portion containing chromosome is covered. This is known as the
forespore – A section of the cell containing chromosome is engulfed by a
membrane as the peptidogycan is synthesized. 

Stage IV-V: Cortex and Coat assembly - Synthesis of the

peptidoglycan cortex followed by formation of proteinaceous spore coat:
In this stage, the spore (mature) is enclosed in two different layers that
include the outer coat consisting of different types of proteins and the
cortex that consists of a special type of peptidoglycan.

Lysis of the mother cell - The mother cell undergoes

lysis/disintegration. Some material from the mother cell may be used to
build on the spore coat.

Spore release - Mature spores are released into the environment and
are capable of surviving high temperatures, certain chemicals that can
destroy the vegetative cell and radiation among other extreme
environments. Once environmental conditions improve, the spore can
germinate as the organism returns to the original vegetative growth.

* Mutation has been shown to result in the production of twin endospores

(two viable spores). Some mutations may also produce more than two
endospores.

Sporulation of Fungi

Fungi are some of the most studied spore-producing organisms in the

world. They produce a wide variety of spores that significantly vary in
size, shape and other surface features that suit their environment (for
dispersal etc).

Whereas the spores produced sexually (through meiosis) remain dormant

for survival (e.g. ascospores), those that are produced asexually
(mitospores) are for dispersal.

Produced through mitosis, asexual spores are released in high numbers

and are genetically identical. This allows them to play an important role in
reproduction when they land on the appropriate substrate in the
environment following dispersal.
* Chlamydospore - This is a type of fungal spore that develops from the
hyphal structures during unfavorable conditions. Chlamydospores are
characterized by a thick, melanized wall that protects the contents of the
spore.

Different types of fungi may produce different types of spores.

The following are some of the main groups of fungi and the type of spores
they produce:

· Zygomycota - Members of Zygomycota are known as zygomeycetes.

They produce both sexual (zygospores) and asexual (sporangiospores)
spores.

· Ascomycota - Ascomycetes also produce both sexual (ascospores) and

asexual (conidia) spores.

· Basidiomycota - Compared to the other groups of

fungi, basidiomyecetes are largely known to produce sexual spores that
are known as basidiospores.

See more about Fungi here

Sporulation Process in Yeast (Saccharomyces cerevisiae)

Budding Yeast Life Cycle

* Saccharomyces cerevisiae is also known as Brewer's yeast and is used

for brewing, baking and winemaking.
As with bacteria (Gram-positive bacteria), fungi do not necessarily start
forming spores when environmental conditions change. Rather, they
attempt to adjust to the new conditions through a number of strategies.

When nitrogen sources diminish, Saccharomyces cerevisiae may respond

by going into a stationary phase or modifying their morphology. However,
in the event that unfavorable conditions persist, spore formation becomes
necessary. This involves a process known as meiosis.

Early Phase

During unfavorable conditions, the organism responds by entering the

early phase (first phase of sporulation) of sporulation. During this phase,
cell division processes shift from the mitotic cycle in G1 (An interphase
stage) and enter the S phase in meiosis.

Some of the events that take place during early phase include:

 DNA replication
 Homolog recombination and pairing (prophase)

* This phase is therefore largely characterized by changes in the

machinery of the cell cycle as well as altered RNA processing.

Middle Phase 

The middle phase is characterized by various cytological events. Here, the

meiotic process results in the production of haploid nuclei (four) that form
four new daughter cells. This involves the modification of the spindle pole
bodies to contribute to the formation of prospores (membrane
compartments).

Some of the other events that take place during this phase include:

 Post-Golgi secretory vesicles are redirected to promote the regeneration

and expansion of the new compartments
 The prospores grow and engulf the nuclei
  Other organelles move to the cytoplasmic space that lies between the
nuclear membrane and the prospores
 The nucleus is completely separated from the cytoplasm of the mother cell

Late Phase

This is the last phase of sporulation and involves the following events:

 The prospore membrane closes completely

 A thick wall starts forming around the spores- This also contributes to the
maturation of the spores
 Chromatic compaction in the nucleus of the spores
 Some of the organelles start regenerating

* While given environments may contain various nutrients, the lack of

nitrogen and other appropriate nutrient sources may trigger sporulation of
species like A. nidulans and result in the growth of hyphae and
consequently conidiation.

* Before spores start forming, special reproductive hyphae known as

sporangiophores are produced.

* Compared to sexual reproduction, asexual reproduction is more

common in fungi given that it allows for the rapid spread of the
organisms.

* Some spores are produced in the sporangium (sac) through mitotic

division (e.g. conidia).

* Fungi spore vary in size and structure. The differences allow them to be
effectively dispersed by a variety of agents including wind, animals, and
water.

Learn more about Yeast here

Sporulation in Amoeba

Encystment

For the most part, amoeba reproduces asexually through such processes
as regeneration, binary fission, multiple fission, and
sporulation/encystment. For the purposes of this article, we will focus on
encystment and sporulation.

When unfavorable environmental conditions (extreme cold, heat, starvation etc)

such protozoa as amoeba ultimately respond by forming a protective cyst wall.

This type of response is known as encystment and is characterized by the following

events:

 Withdrawal of the pseudopodia

 Formation of a hard protective layer
 Dormancy

* Such bacteria as E. coli, S. enterica and L. monocytogenes among a few others

have been shown to survive extreme conditions when inside the cyst of
such amoeba as A. castellanii. The cyst protects these organisms from various
extreme conditions like high acidity, antibiotics, and insufficient nutrition among
others.

In the laboratory, encystation can be stimulated by using the non-nutrient medium.

For instance, by using a non-nutrient agar that contains about 80 mm of sodium
chloride, 15mM of magnesium chloride and 20mM of taurine, researchers have been
able to stimulate encystment of A. culbertsoni.

As the trophozoites transform into cysts, some of the morphological changes

observed include reduced rates of motility, changing into a spherical shape, general
cell shrinkage as well as gradual withdrawal of the pseudopodia (temporary
cytoplasm-filled projection ).

Various studies have shown cyclic AMP to play an important role in amoeba
encystation. Here, amines of taurine or other biogenic amines bind to specific
receptors on the organisms' membrane. This, in turn, activates adenylate cyclase on
the membrane which triggers the encystation process.

In addition to the morphological changes observed during encystation, a number of

chemical and molecular changes are also evident.
These include:

 A change from aerobic metabolism to anaerobic metabolism and complete

dormancy in the end
 Rapid turnover of phosphorylated compounds
 Significant loss of cellular RNA, DNA, and other proteins. However, some
species have been shown to gradually synthesis RNA and proteins
 Incorporation of glucose compounds into a polyssacharide

Sporulation in amoeba has been observed in social species like

Dictyostelium discoideum. This process is unique and results in a mass of spores
that are held up on a tapering stalk.

During starvation, the amoeba cells come together to form aggregates that in turn
elongate for form a migratory slug composed of over 100,000 cells. The slug then
becomes upright as prestalk cells located at its tip form a cellulose tube.

As cells enter the tube, they vacuolize in and contribute to its strength. From the
desired substrate at the bottom of the stalk, prestalk cells climb and enter the stalk
followed by the prespore. Once the stalk is complete, the prespore encapsulate and
turn to dormant spores that are protected by a protein coat.

* Here, it is worth noting that in the course of this process, the cells differentiate
into pre-stalk and pre-spores involved in sporulation.

* Cells that converge in large numbers are known as myxamoebae.

* Once the slug is formed, it has to move to an area with favorable conditions
(illuminated) where it transforms into a fruiting body. This is the stalk that is
composed of the spore cells.

* Vacuoles are created as prestalk cells form stalk cells.

* Spores at the top of the stalk are released into the environment and dispersed to
other environments. When environmental conditions are favorable in the new
environment, the spores germinate to produce active amoeba.