This document discusses various stress responses in microbes that allow them to survive in adverse environmental conditions. It focuses on bacteria and their mechanisms for responding to elevated oxygen levels, extreme pH, high osmotic pressure, heat shock, and other stressful conditions. It describes different types of microbes based on their tolerance ranges for these stresses, such as thermophiles, halophiles, and alkaliphiles. Key stress response systems discussed include antioxidant enzymes and molecular chaperones that help bacteria adapt to environmental changes.

1. STRESS PHYSIOLOGY
IN MICROBES:
Dr. Harinatha Reddy M.sc, Ph.D.
biohari14@gmail.com
Department of Microbiology
Sri Krishnadevaraya University
Anantapur, A.p. India

2. The bacterial stress response provide bacteria to survive in
adverse conditions.
Various bacterial mechanisms recognize different
environmental changes and mount an appropriate
response.
Bacteria can survive under diverse environmental
conditions bacteria must sense the changes and mount
appropriate responses in gene expression and protein
activity.
Stress response systems can play an important role in the
virulence of pathogenic organisms.

3. Effect of elevated oxygen concentration on bacteria:
• Most aerobic and micro-aerophilic organisms have
developed protective responses to tolerate environmental
oxygen concentrations.
• But when oxygen concentration exceed the air saturation
level, reactive oxygen species (ROS), such as superoxide
(O2-) and hydrogen peroxide (H2O2), accumulate as
byproducts of aerobic metabolism.
• These molecules are toxic to the organisms since they are
more reactive than molecular oxygen.
• It was reported that oxygen concentrations above 100% air
saturation cause inhibition of metabolism and respiration in
microorganisms.

4. • Accumulation of reactive oxygen species (ROS), effect of on
protein and lipid oxidation.
• When cells are exposed to high extracellular oxygen
concentration, oxygen diffuses through the membranes
and abstract electrons from reduced flavoenzymes to produce
partially reduced oxygen species such as superoxide (O2- )
and hydrogen peroxide (H2O2).
• At lower oxygen concentrations, catalases and glutathione
peroxidase systems minimize the accumulation of H2O2.
• But at higher oxygen concentrations, these antioxidant
defenses are supressed resulting in accumulation of H2O2
which can diffuse freely from the mitochondria reaching targets
that can be damaged the cellular organells and DNA.

5. • Oxygen accepts electrons and is readily reduced because its
two outer orbital electrons are unpaired.
• Flavo-proteins several other cell components, promote
oxygen reduction.
• The result is usually some combination of the reduction
products superoxide radical, hydrogen peroxide, and
hydroxyl radical.
O2 + e- → O2– (superoxide radical)
O2– + e- +2H → H2O2 (hydrogen peroxide)
H2O2 + e- + H+ → H2O +OH (hydroxyl radical)
• These products of oxygen reduction are extremely toxic
because they are powerful oxidizing agents and rapidly
destroy cellular organelles.

6. • Many microorganisms possess enzymes protect cells against O2
toxic products.
• Obligate aerobes and facultative anaerobes usually contain
the enzymes superoxide dismutase (SOD), glutathione
peroxidase and catalase, which catalyse the destruction of
superoxide radical and hydrogen peroxide, respectively.
2O2- +2H+ --- superoxide dismutase-- O2+H2O2
H2O2 ------Catalase----- 2H2O+ O2
• Aerotolerant microorganisms may lack catalase but almost
always have superoxide dismutase.
• All Obligate anaerobes lack both enzymes or have them in
very low concentrations and therefore cannot tolerate O2.

7. Oxygen Concentration:
Obligate aerobes: Completely dependent on atmospheric O2 for
growth. Micrococcus luteus, Pseudomonas, Mycobacterium; most
algae, fungi, and protozoa.
 Facultative anaerobes: Does not require O2 for growth, but grows
better in its presence. Escherichia, Enterococcus, Saccharomyces
cerevisiae.
 Aerotolerant anaerobe: Grows equally well in presence or absence
of O2 Streptococcus pyogenes.
 Obligate anaerobe: Does not tolerate O2 and dies in its presence.
Clostridium, Bacteroides, Methanobacterium.
 Microaerophile: Requires O2 levels below 2–10% for growth
Campylobacter, Spirillum volutans, Treponema pallidum.

8. The Effects of pH on Microbial Growth:
Extreme pH affects the structure of all macromolecules.
The hydrogen bonds holding together strands of DNA break up at high pH.
Lipids are hydrolyzed by an extremely basic pH.
The proton motive force responsible for production of ATP in cellular
respiration depends on the concentration gradient of H+ across the plasma
membrane.
If the pH increases H+ ions are neutralized by hydroxide ions(OH-), the
concentration gradient collapses and impairs energy production.
Moderate changes in pH modify the ionization of amino-acid functional
groups and disrupt hydrogen bonding, which, in turn, promotes changes in
the folding of the molecule, promoting denaturation and destroying activity.

9. • Most bacteria are neutrophiles, meaning they grow optimally
at neutral pH of 7.
• Most familiar bacteria, like Escherichia coli, Staphylococci, and
Salmonella spp. are neutrophiles and but grow well at acidic
pH of the stomach.
• However, there are pathogenic strains of E. coli, S. typhi, and
other species of intestinal pathogens that are much more
resistant to stomach acid.

10. • The real cause of most peptic ulcers was discovered to be corkscrew-
shaped bacterium, Helicobacter pylori.
• The ability of Helicobacter pylori to survive the low pH of the stomach, but
it is not acidophile.
• In fact, H. pylori is a neutrophile. So, how does it survive in the
stomach?.
• H. pylori creates a microenvironment in which the pH is nearly neutral.
• It achieves this by producing large amounts of the urease enzyme, which
breaks down urea to form NH4+ and CO2.
• These react with the strong acids in the stomach and produce a neutralized
area around H. pylori.
• H. pylori is able to sense the pH gradient in the mucus and move towards
the less acidic region (chemotaxis).

11. • Some Microorganisms frequently change the pH of their own habitat
by producing acidic or basic metabolic waste products.
•
• Fermentative microorganisms form organic acids from carbohydrates.
• Whereas chemolithotrophs like Thiobacillus bacteria reduced sulfur
components to sulfuric acid.
• Nitrobacteria species make their environment more alkaline by
generating ammonia through amino acid degradation.
• Buffers often are included in media to prevent growth inhibition by
large pH changes.
• Phosphate buffer is a commonly used buffer and a good example of
buffering by a weak acid (H2PO4–) and its conjugate base (HPO4 2–
).

12. Effect of osmotic pressure on bacteria:
• Having the correct osmotic pressure in the culture medium is essential.
• If the medium is hypertonic — a concentrated solution with a lower water
concentration than the cell — the cell will lose water by osmosis.
• If the medium is isotonic — a solution with exactly the same water
concentration as the cell — there will be no net movement of water across the
cell membrane.
• If the medium is hypotonic — a diluted solution with a higher water
concentration than the cell — the cell will gain water through osmosis .
•
•

13. • Cells respond to variations in external osmotic pressure by
accumulating or releasing solutes.
• Those solutes include inorganic ions (often K+), and organic
molecules denoted as “osmolytes”.
• In the extreme halophiles accumulate KCl to molar
concentrations, and their proteins function only in high salt
environments.
• Osmolytes Or Osmo-regulatory solutes accumulate via active
transport or synthesis.
• Osmolytes such as Trehalose, Glutamate, Proline, Carnitine,
accumulate in E. coli in high osmotic pressure media.

14. • For example, bacteria inhabiting seawater face a higher
osmotic pressure than those inhabiting most freshwater
environments.
• Salts predominate in seawater, and marine organisms
simultaneously face both a high osmotic pressure and a
high Na+ concentration.
• Solute accumulation powerfully stimulates bacterial growth
at high osmotic pressure, and solute release allows cells to
survive osmotic downshocks.

15. • Osmotolerant: Able to grow over wide ranges of water
activity or osmotic concentration. Ex: Staphylococcus aureus.
• Halophile: Requires high levels of sodium chloride, usually
above about 0.2 M, to grow. Ex: Halobacterium sp.

16. Heat shock response on Bacteria:
• Heat shock response is the cellular response to heat shock includes the
transcriptional up-regulation of genes encoding heat shock proteins
(HSPs) as part of the cell's internal repair mechanism.
• When bacteria cells are exposed to higher temperature, a set of heat-
shock proteins (HSPs) is induced rapidly.
• The HSPs include chaperones and proteases that are essential for
overcoming changes that involve protein denaturation.
• In E. coli the heat-shock response is controlled by a specific sigma factor-
32 (σ32), this factor coded by the rpoH gene and binds to heat-shock
promoters located upstream of heat-shock genes AND regulate
expression of genes.
• Many molecular chaperones have developed to control protein folding
and protection of the cell from high temperature.
•

17. Chaperones:
• Chaperones are proteins that assist the covalent folding or
unfolding and the assembly or disassembly of other
macromolecular structures.
• Many chaperones are heat shock proteins, that is, proteins
expressed in response to elevated temperatures or other
cellular stresses.
• The protein folding is severely affected by heat and, therefore,
some chaperones act to prevent damage and correct damage
caused by misfolding.
• Other chaperones are involved in folding newly made proteins
as they are extruded from the ribosome.

18. • Some chaperone systems work as foldases: they support the
folding of proteins in an ATP-dependent manner (for example
HSP60 and HSP70).
• Other chaperones work as holdases: prevent their aggregation of
proteins. for example Hsp33.
• There are many different families of chaperones (HSP60, 70,
90,100) each family acts to aid protein folding in a different
way.
• In bacteria like E. coli, many of these proteins are highly expressed
under conditions of high stress and temperatures. For this reason,
they named as "heat shock protein“.
• .

19. HSP90:
• The 90-kDa HSP are member of molecular chaperones
family that are wide spread in prokaryote and eukaryote cell.
• HSP90 is a dimer conserved from bacteria to eukaryotic cell.
• HSP90 proteins have a major role in cellular functions, such
as protein folding, receptor maturation and signal
transduction.
• HSP90 proteins contain three conserved domains:
• N-terminal domain that binds and hydrolyze ATP.
• C-terminal and middle domain that is important for
dimerization.

20. HSP60:
Heat shock protein 60 found in both eukaryotic and prokaryotic
cell with high sequences similarity.
 HSP60 of Helicobacter pylari and Mycobacterium leprae can
interact with immune cell and produce antibody response in the
body.
One of major heat shock protein an H.pylori is HSP60 that
antibody's found against in most of patients.
This HSP60 can induce cytokine such a IL-8, IL-1 and TNF-α.

21. Thermophiles:
• Thermophile: Can grow at 55°C; optimum temperature often
between 55 and 65°C.
Example: Bacillus stearothermophilus, Thermus aquaticus.
• Hyperthermophile: Has an optimum temperature between
80°C and abouve113°C.
Example: Sulfolobus, Pyrococcus, Pyrodictium.

22. • Thermophiles are found in various heated regions of the
Earth, such as hot springs like those in Yellowstone National
Park and deep sea hydrothermal vents.
• Thermophiles can survive at high temperatures, whereas
other bacteria would be damaged and sometimes killed if
exposed to the same temperatures.
• The enzymes in thermophiles necessarily function at high
temperatures.
• Taq polymerase is a thermostable DNA polymerase, optimum
temperature for activity is 75–80°C, Identified in Thermus
aquaticus.

23. • Thermophiles differ from mesophiles in having much more
heat-stable enzymes and protein synthesis systems able
to function at high temperatures.
• Their membrane lipids are also more saturated than those
of mesophiles and have higher melting points; therefore
thermophile membranes remain intact at higher
temperatures.
• Many of the hyperthermophiles Archea require elemental
sulfur for growth that oxidize sulfur to sulfuric acid as an
energy source.

24. • Thermophiles can be classified in various ways according to their
optimal growth temperatures:
1. Simply thermophiles: 50–64 °C
2. Extreme thermophiles 65–79 °C
3. Hyperthermophiles 80 °C but not < 50 °C.
• In a related classification, thermophiles are sorted as follows:
1. Obligate thermophiles (also called extreme thermophiles) require
such high temperatures for growth.
2. Facultative thermophiles (also called moderate thermophiles)
can thrive at high temperatures, but also at lower temperatures
(below 50 °C (122 °F)).
3. Hyperthermophiles are particularly extreme thermophiles for
which the optimal temperatures are above 80 °C (176 °F).

25. Halophiles:
• The genus Halobacterium (Domain: Archaea) ("salt" or
"ocean bacterium") consists of an aerobic metabolism which
requires high concentration environment of salt; many of their
proteins will not function in low-salt environments.
• They may be either rods or cocci, and in color, either red or
purple. They reproduce using binary fission (by constriction),
and are motile.
• Halobacterium species can be found in the Great Salt Lake,
the Dead Sea, and any other waters with high salt
concentration.

26. • Halophiles have adapted so completely to hypertonic, saline
conditions.
• That they require high levels of sodium chloride to grow,
concentrations between about 2.8 M for Halophiles and about
6.2 M for extreme halophilic bacteria.
• Halobacterium and other extremely halophilic bacteria have
significantly modified the structure of their proteins and
membranes rather than simply increasing the intracellular
concentrations of solutes, the approach used by most
osmotolerant microorganisms.

27. • These extreme halophiles accumulate enormous quantities of
potassium ions in order to remain hypertonic to their environment;
the internal potassium concentration may reach 4 to 7 M.
• The enzymes, ribosomes, and transport proteins of these bacteria
require high levels of potassium for stability and activity.
• In addition, the plasma membrane and cell wall of Halobacterium
are stabilized by high concentrations of sodium ion.
• If the sodium concentration decreases too much, the wall and
plasma membrane disintegrate.

28. • Example: Halobacterium salinarum and Halomonas
titanicae.
• Halomonas titanicae is a gram-negative, halophilic species
which was discovered on wreck of the RMS Titanic in 2010.
• The researchers, estimated that the action of microbes like
Halomonas titanicae may bring about the total decompose of
the Titanic by 2030.

29. Alkaliphiles:
• Alkaliphiles are a class of extremophilic microbes capable of
survival in alkaline pH roughly 8.5–11, growing optimally
around a pH of 10.
These bacteria can be further categorized as:
• Obligate alkaliphiles: (those that require high pH to survive),
• Facultative alkaliphiles: (those able to survive in high pH, but
also grow under normal conditions).

30. • The cell walls contain acidic polymers composed of
residues such as galacturonic acid, gluconic acid,
glutamic acid, aspartic acid, and phosphoric acid.
• In addition, the peptidoglycan in alkaliphilic B. subtilis
has been observed to contain higher levels of hexos-
amines and amino acids in the cell walls compared to that
of the neutrophilic B. subtilis.

31. • α-Amylases of alkaliphilic Bacillus strains had a pH optimum of
8.0 to 8.5 and displayed maximum activity at 55°C.
• Thermoactinomyces sp. And Streptomyces pactum produce
proteinase was optimally active in the pH range from 7 to 10 and
at temperatures from 40 to 75°C.
• Alkaliphiles have made a great impact in industrial applications.
Biological detergents contain alkaline enzymes, such as alkaline
cellulases and/or alkaline proteases that have been produced
from alkaliphiles.

32. Acidophiles:
• Acidophiles or acidophilic organisms are those that thrive under highly
acidic conditions (usually at pH 2.0 or below).
• Most acidophile organisms have evolved extremely efficient mechanisms
to pump protons out of the intracellular space in order to keep the
cytoplasm at or near neutral pH.
• Acidophilic enzymes have optimal structure and stability in acidic
environments and have been shown to be catalytically active at pH as low
as 1.
• Endo-𝛽-glucanase isolated from acidophilic Sulfolobus sp. This enzyme
has an optimum pH of 1.8 and consists of excess of glutamic and
aspartic acid residues.
• Another example is 𝛼-glucosidase from Ferroplasma acidiphilum, This
enzyme has an optimum pH of 3.0.

33. • Another example is 𝛼-glucosidase from Ferroplasma
acidiphilum.
• 𝛼-Glucosidase optimum pH is 3, which is the internal average
cytoplasmic pH of F. acidiphilum.
• Similarly carboxylesterase in F. acidiphilum was also shown to
have a pH optimum of approximately 2.
• All of these enzymes showed significantly lower activity after
the pH was higher than 5.

34. • Many of these acidophilic enzymes also fall into the thermophilic category
and have potential for biotechnological and industrial applications.
• One such example is in biofuels production where currently high sugar
compounds (e.g., corn) are used for ethanol production.
• Cellulases and xylanolytic enzymes of acidophiles could be used in a hot
acidic environment to hydrolyze lignocellulose materials.
• Further applications of thermal/ acid stable enzymes could be in mining
industries.
• Another potential application could be in the food industry where
glucoamylases could be used to break down complex polysaccharides into
basic dextrose and fructose sugars, this could improve the efficiency of
monosaccharide production.
• The technique known as bioleaching utilizes microorganisms and their
enzymes to harvest metals such as copper, nickel, cobalt, zinc, and
uranium

35. Psychrophiles:
• Psychrophiles or cryophiles are extremophilic organisms that
are capable of growth and reproduction in cold temperatures,
ranging from −20 °C to +10 °C.
• Bacteria that can tolerate extreme cold are Arthrobacter sp.,
Psychrobacter sp.
• The ability of psychrophiles to survive and proliferate at low
temperatures by reduced enzyme activity; decreased
membrane fluidity; altered transport of nutrients and waste
products; decreased rates of transcription, translation and
intracellular ice formation.

36. • In psychrophiles, enzymes are optimally active at low temperatures.
For example, a ribosomal extract.
• RNA polymerase, elongation factor and RNA helicases have all
been shown to retain activity near 0 °C in several psychrophilic
microorganisms.
• Decreasing temperatures have an adverse effect on the physical
properties and functions of membranes, typically leading to a
reduction in membrane fluidity.
• In general, lower growth temperatures produce a higher content of
unsaturated, polyunsaturated and methyl-branched fatty acids,
• This altered composition have a key role in increasing membrane
fluidity by reducing the number of interactions in the membrane.

37. • Cold-shock proteins(Csps) that are involved in various
cellular processes such as transcription, translation,
protein folding and the regulation of membrane fluidity.
• In particular, increased levels of nucleic-acid-binding
proteins (for example, CspA-related proteins and
chaperones, such as GroEL and DnaK have been
frequently reported.
• Antifreeze proteins (AFPs) have the ability to bind to ice
crystals and lower the temperature at which an organism
can grow.

38. • The high activity of psychrophilic enzymes at low and moderate
temperatures offers potential economic benefits.
• A typical example is the industrial 'peeling' of leather by proteases,
which can be done at the temperature of tap water by cold-active
enzymes.
• Psychrophilic enzymes can also be useful in domestic processes,
for washing clothes at low temperatures can protect the colours of
fabrics.
• Cell membranes of psychrophiles contain surfactants bearing
unique stability at low temperatures and that can be used in
pharmaceutical formulation.

39. THANK YOU……..