Microbial stress

responses

Microbial Stress
• Bacteria may alter cell morphology, cell
metabolism, gene transcription, and cell
behavior in response to environmental
fluctuations such as the availability of
respiratory electron acceptors, the
supply of carbon, nitrogen or phosphate,
changes in the osmolarity and
temperature of the medium.

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Microbial Stress Responses
• The ability of microbes to sense
and respond to alternations in the
environment is crucial to their
survival.

Adaptive and
Developmental changes
• Bacteria may undergo physiological
or developmental responses to
signaling molecules produced by
other cells and engage in
cooperative behavior.

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 Stress and Response
• Two Component signaling systems
• Aerobic to anaerobic transitions
• Nitrate response
• Osmotic stress and osmoregulation
• Response to attractants and
repellents

Two Component signaling

systems
• INFORMATION PROCESSING IN
CELLS
• Phosphate

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 The first component
functions as a sensor

The second is a
response regulator.

1. Histidine kinase
2. autophosphorylate

N-terminal

C-terminal

phosporelay

Asparate phosphorylated

N-terminal

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• The functional state of the regulatory protein
was thus modified by binding to the metabolic
intermediate.
• The consequence of ligand binding, an altered
state of the regulatory protein, was directed to
the appropriate gene(s) by the protein's DNA
binding activity.
• Initially, all such regulation was believed to occur
in a negative fashion - that is, the proteins
acted as repressors of gene expression.

• EnvZ/OmpR - osmoregulation in E. coli

• PhoR/PhoB - phosphate scavenging in E. coli
• NtrB/NtrC - nitrogen assimilation in a variety
of bacteria
• DctB/DctD - dicarboxylate transport in
Rhizobium leguminosarum
• VirA/VirG - virulence by Agrobacterium
tumefaciens
• Spo0A - sporulation
• Spo0F - sporulation
• CheY - chemotaxis
• CheB - chemotaxis

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 Components
• Histidine kinase (HK; sensor/kinase)
– Receives a signal and autophosphorylates at
a histidine residue
• Response regulator (RP)
– Phosphorylated at an aspartate residue by
HK-P and sends a signal to its target
• Phosphatase
– Inactives the RP-P
– Maybe the
• histidine kinase
• the response regulator
• a separate protein

1. Histidine kinase
2. autophosphorylate

N-terminal

C-terminal

phosporelay

Asparate phosphorylated

N-terminal

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 Two Component signaling
systems
• As of 11/07/97, there are more
than 160 two component signal
transduction systems that have
been identified by numerous
workers
• On 9/9/99 this had increased to
more than 500.

Two Component signaling

systems
• These signal transduction systems are found
in all kingdoms of life, ranging from

– Bacteria
– Archae
– single-celled eukaryotic organisms
– fungi
– higher plants

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• Stimulus: low external concentration of Pi
• Sensor: Pst transporter--PhoU
• Signal: Protein phosphorylation
• Transducer: PhoR
• Regulator: PhoB
• Output: Proteins such as PhoA
• Response: Improved Pi acquisition

Transmitter Domain

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 Receiving Doamin

Histidine kinase
Histidine residure

Span the membrane

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 Response-regulator protein
Interact with the sensor

Interact with
target

Phosphate regulation
Inorganic phosphate is a limiting nutrient
in many environments, and the ability to
scavenge Pi when it is only present at
low concentration is very important.

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 Phosphate regulation
• Alkaline phosphatase (PhoA) is an
important scavenging enzyme that is
inducible, secreted, and only activated
once it reaches the periplasmic space.
• It cleaves Pi from organic phosphates.
However, Pi starvation leads to the
elevated production of more than 100
proteins.

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• But, with genome sequences becoming
available, we now know of some
exceptions.
• For example, mycoplasma, which have
parred down their genome to ~500,000
base pairs, have done away with genes
encoding two-component regulation
systems, as well as removed all Helix-
turn-Helix DNA binding proteins except
for that found in their sigma factor

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Aerobic to anaerobic
transitions

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 Aerobic to anaerobic
transitions
• Facultative microorganisms
• The transition in metabolism is
accompanied by alterations in the rate,
route and efficiency of pathways of
electron flow.
• Metabolic changes accompanying shift to
anaerobiosis

Aerobic

NADH dehydrogenase

α-ketoglutarate
α-ketoglutarate
dehydrogenase

FUMA: fumarase A
SDH: succinate dehydrogenase

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 C4 C6

C5

Anaerobic 3 FUMB: fumarase B

FRD: fumarate reductase

menaquinone

α-ketoglutarate
1 α-ketoglutarate
dehydrogenase
inactive

2 Change electron
acceptors from oxygen
to fermentate

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 Gene regulation
• In aerobic
– repressed genes
• Nitrate reductase
• Fumarate reductase
• other reductase
• In anaerobic
– Induced genes
• Nitrate reductase : induced by nitrate
• Fumarate reductase : induced in no O2, no nitrate
• other reductase: case by case

Globally regulation in
anaerobiosis
• Arc system: activated by anaerobiosis
• FNR system: activated by anaerobiosis
• Nitrate response: activated by nitrate and nitrite
• FhlA/formate regulon:
– activated by anaerobiosis and formate
– and repressed by nitrate

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 Arc system
• Aerobic respiratory control system
• ArcB/ArcA
• ArcB monitors the level of oxygen indirectly
• when oxygen levels are sufficiently low, the
ArcB autophosphorylates using ATP as the
phosphoryl donor and the transfers the
phosphoryl group to ArcA.

Arc system

O2 low

Anoxia

Sexual
state

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Low aeration
Stationary phase

High aeration
Log phase

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How does ArcB detect changes in
the levels of oxygen?

• The level of expression of the succinage

dehydrogenase
– Varied the concentration of electron
acceptors
– Accumulated flavoprotein, quinone,
cytochrome, or NADH

FNR system
• Fumarate nitrate reductase system
• Active FNR is a homodimer of an iron-sulfur
protein with an oxygen labile [4Fe-4S] cluster
in each monomer
• Upon exposure to oxygen, the [4Fe-4S] cluster
is oxidized and can even be lost from the
protein

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FNR system

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 FhlA regulon

• In anerobic
– Formate regulon
– FhlA trascriptional factor and formate
(coactivator)
– repressed by oxygen and nitrate
– induced by formate
– formate-hydrogen lyase
– Formate dehydrogenase H
– Hydrogenase 3
– Convert the formate to H2 and CO2

Mixed fermentation

3. pyruvate-
formate lyase

Positive
regulate by
5. formate-
activated
FNR hydrogen lyase

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 Nitrate response

Nitrate response
• Nitrate respiration
– E. coli can be grown anaerobically using
nitrate (NO3-) as the electron acceptor.
– In anaerobically, NO3- induces the synthesis
of a membrane bound nitrate reductase and
represses the synthesis of the other
reductase (e.g. fumarate reductase,
DMSO/TMAO reductase, et al.)

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 Pathway of nitrate
reduction
• Nitrate (NO3-) is first reduced to nitrite (NO2-)
– Membrane-bound Nitrate reductase
– In narG operon
• Nitrite (NO2-) is then reduced to ammonia (NH4+)
– Cytoplasmic NADH-linked nitrite reductase
• Ammonia is assimilated into cellular nitrogenous
compounds, and part is excreted into the medium.

nar system and regulation

• Regulated by two-component signaling system
• Sensor kinases:
– NarX and NarQ
• Cytoplasmic response regulator proteins:
– NarL and NarP

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NO3-
stimulate [NarL-P]

NO2-
stimulate [NarL-P] 

NO2-
Stimulate [NarP-P]

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 Response to Nitrogen
Supply
The Ntr Regulon

Response to Nitrogen Supply

The Ntr Regulon
• Inorganic nitrogen:
– Ammonia (NH4+)
– nitrate(NO3-)
– dinitrogen gas(N2)
Ammonia
• Organic nitrogen:
– Amino acid
– Urea
– other nitrogenous compounds Glutamine
synthetase

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 Ammonia regulation
• [NH4+]
:
– adequate
– ammonia as nitrogen source
– Represses genes required for the assimilation
of other nitrogenous compounds
• [NH4+]  :
– limited
– Induces genes required for the assimilation
of other nitrogenous compounds
– Glutamine synthetase (GS) is more active

Glutamate synthesis
• [ammonia] > 1mM
– Glutamate dehydrogenase
– Reductive amination of α-ketoglutarate

Glutamate
dehydrogenase
α-ketoglutarate Glutamate

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 Role of glutamate

Glutamate
synthesis
•[ammonia] < 1mM GS

•Glutamine synthetase (GS)

•glutamate synthase (GOGAT)
GOGAT:
Glutamine oxoglutarate
aminotransferase GOGAT

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 85%
Nitrogen
from Glu

15%
Nitrogen
from Gln

[NH4+] 

Ntr regulon
• The genes that are regulated by the ammonia supply
• NtrC
– response regulator protein sensor

• NtrB
Gln
– histidine kinase
• E. coli: glnALG
• Salmonella typhimurium: glnABC

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σ70 σ54 glnA glnL glnG
Ap1 Ap2 Lp

GS NtrB NtrC

PII (glnB)

ATP NtrC Pi
NtrB+
NtrB
PII
ADP NtrC-p H2O

[NH4+]
PII UMP

UTP
glutamine - glutamine
α-keto +
UT/UR +
glutarate PPi PII -UMP H2O

[NH4+]  UT/UR (glnD)

• When the cells are limited for

ammonia,
• glutamine is the immediate source
of nitrogen for glutamate

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 Nitrogen regulation
• Nitrogen excess (high ammonia)
– gln operon transcribe from glnAp1 and glnLp
by σ70 RNA polymerase
– Low level transcription is sufficient to
guarantee the synthesis of GS to meet the
cell’s need
– high glutamine enhance the production of PII
– NtrB bind to PII to become a phosphotase
– NtrC-P becomes to NtrC

Nitrogen regulation
• Nitrogen limited (low ammonia)
– gln operon transcribe from glnAp2 by
σ54 RNA polymerase
– high a-ketoglutarate enhance the production
of PII-UMP
– PII-UMP will activate GS to GS-AMP
(activate foam)
– Free NtrB could activate NtrC to NtrC–P
and then active the transcription of GS to
produce glutamine

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 NtrC-P

NtrC-P

NtrC-P

GS activity is regulated
by PII
• Adenylylation of GS regulates its activity

Low ammonia High ammonia

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