This document summarizes microbial responses to acid stress. It discusses how bacteria like E. coli, Salmonella, and Helicobacter pylori have developed mechanisms to regulate internal pH and withstand acidic environments. These include proton pumps, amino acid decarboxylases, and the urease enzyme system. Regulatory systems like sigma factors and two-component systems help induce acid shock proteins and responses during acid exposure. Gram-positive bacteria like streptococci take a more flexible approach by allowing internal pH to decrease along with external pH.

1. Microbial response to acid stress
Presented by Nyasha Zenda

2. Introduction
• Ability to sense and respond to potentially lethal
changes in the environment is a trait crucial to the
survival of any microorganism
• Microorganisms have evolved elegant regulatory
networks designed to survive stress, e.g. acid stress
• What is acid stress?
– The combined biological effect of H+ ions (i.e. pH)
and weak acid concentrations.
• Microorganisms live in fluctuating pH conditions.
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3. • Permeability of membrane to protons is very is low,
however extremely low external pH (pH0) will cause
H+ to leak across the membrane and acidify the
internal pH (pHi).
• Decrease in pHi causes lethal effects on biochemical
reactions and macromolecular structures.
• Even moderate acidic environments can be lethal if
combined with weak acids (FA’s and OA’s)
• Bacteria have evolved strategies to minimize acid or
alkaline damage
• The study focuses on tactics used by neutrophiles to
combat biological effects of acid 3

4. • E.g. of neutrophiles are E. coli and Salmonella
enterica serovar Typhimurium.
• Gram negative and gram positive neutrophiles utilize
different as well as overlapping approaches to cope
up with acid stress.
• The systems involved in alkalization of internal
pH are,
– F0F1 ATPase
– Specific amino acid decarboxylases
– Complex global changes in proteome that protects
crucial, acid sensitive cellular components.
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5. • Regulatory mechanisms involved in these adaptations
to acid environments include;
– σ factors
– Specific signal transduction system.
• Adaptive strategies important for enteric pathogens
e.g. Salmonella, Shigella, E.coli and H. pylori.
 pH Homeostasis: Gram-Negative Approach
• Enteric pathogens try to keep internal pH 7.7 to 7.8
even as external pH changes during growth.
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6. • Differences between pHi and pH0 , known as ∆pH,
also changes.
• Stable pHi is maintained by pumps that either move
protons into the cell at alkaline pH0 or extrude them
at acidic pHi.
• Two principal systems recognized as potential
generators of pH gradients.
 Potassium–proton antiporters.
 Sodium-proton antiporters.
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7. • Shifts to acidic enviroments are handled by the K+/proton
antiporters resulting in alkalization of cytoplasm
• Whilst, shifts to alkaline pH conditions are handled by the
Na+/proton antiporters resulting in acidification of
cytoplasm.
• In E.coli system responsible for adaptation to Na+ and
alkaline conditions is a 349-amino acid membrane bound,
Na+ / H+ antiporter encoded by nhaA.
• Exchange reaction, 2H+ brought in for every Na+
extruded, resulting is the acidification of the cytoplasm.
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8. • NhaA antiporter activity
– is activated at the protein levels by shifts to
alkaline pH with a consistent role in pH
homeostasis.
• Control of NhaA activity by pH has been localized to
– Histine residue 226
– Glycine residue 338 e.g. G338S substitution
creates a pH-relaxed antiporter constitutively
active at pH6.9-pH9.
– nhaA gene is also subject to transcriptional control
by the NhaR regulator.
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9. – NhaR senses intracellular Na+ concentrations and
induces nhaA under Na+ or Li+ stress
– pH, also modulates NhaR regulation of nhaA with
alkaline pH causing elevated expression of nhaA
and a connected change in the NhaR footprint.
 pH homeostasis: The Gram-Positive Approach
• Gram positive microorganisms e.g. streptococci
regulate differently than do enteric pathogens.
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10. • While E.coli struggles to keep internal pH at 7.8 as
the external pH acidifies,
– Streptococci is more flexible, allowing internal pH
to decrease along with the environment.
– Only requirement is to maintain pHi 0.5 to 1 unit
higher than pH0.
• Primary mechanism for extruding protons in
Streptococci is the F0F1 ATPase rather than the
antiporters.
• Decrease in cytoplasmic causes an increase in the
amount of this complex and enhanced acid tolerance
by proton extrusion.
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11. • Regulation of ATPase
– occurs at the assembly stage
– when pH is acceptable unassembled F1 and F0
subunits are degraded ,thus not assembled into an
active membrane-associated complex.
– In contrast assembled complexes are stable.
– Cytoplasm acidification prevents degradation and
stimulates subunit assembly into an active
complex, therefore increase in ATPase in response
to external acidification allows the cell to
compensate for concomitant increases in proton
influx.
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12. • Medical significance of this membrane complex as an
acid tolerance mechanism;
– is best explained in tooth decay microorganisms
– tolerance to acid enviroments a major importance
to the ecology of dental plaque microorganisms
involved in tooth mineral dissolution.
– They grow and metabolize CHO’s at low pH
values, they acidify their environment via
carbohydrate fermentation.
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13.  THE ACID TOLERANCE RESPONSE OF
SALMONELLA ENTERICA SEROVAR
TYPHIMURIUM
• During its travel within the host this microorganism,
encounters extreme low pH in the stomach, volatile
FA’s in the intestines and feces, and also the
macrophage phagosome and phagolysosome
environments.
• Possesses both Log-phase and stationary-phase acid
tolerance response (ATR) systems that will protect
the organism at pH 3 for several hours.
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14. – Lowering adaptive pH to 4.5 for exponential-phase
cells results in the induction of 50 acid shock
proteins(ASP’s) responsible for acid survival.
• Some ASP’s induced by pH0 and others by internal
pHi.
• Induction of acid tolerance also provides cross
protection against
– high temperature
– Oxidative damage
– High osmolarity
• Several genes play a role in the log-phase ATR,
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15. – The alternate σs factor encoded by rpoS; Fur and
PhoPQ a two-component signal transduction
system.
– Each regulator controls expression of a subset of
ASP’s
• 10 ASP’s for σs, 5 for Fur, and 4 for PhoPQ.
• Following acid shock, σs level increases because a
response regulator, MViA which decreases proteolytic
turnover(a control of the σs by ClpXP protease)
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16. • σs-dependent ATR system is required to survive acid
stress imposed by volatile FA’s and contributes to
inorganic acid tolerance.
• The iron regulator Fur normally acts as a repressor of
gene expression when bound to excess intracellular
Fe2+, but controls a subset of ASPs in an iron-
independent manner
• PhoPQ important in acid tolerance response and
nonessential for log-phase in the presence of σs. This
system important in Samonella virulence.
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17. • This PhoPQ activated in macrophage phagolysosome
where there is an acidic pH and low Mg2+ levels.
• Stationary-phase acid tolerance in this
microorganism, also involves σs-dependent and –
independent systems.
• σs-dependent SP does not require acid induction, as
opposing to LP σs-dependent system.
– Entry into SP is enough to induce σs levels and
acid tolerance
• σs-independent system is regulated by OmpR
response regulators
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18.  Acid tolerance, acid resistance and acid
habituation of Shigella flexneri and E. coli
• S. enterica and E.coli, Posses both LP and SP acid
survival mechanisms.
• Three systems named in E. coli
– Acid tolerance response
– Acid habituation
– Acid resistance
• ATR in E coli similar to serovar Typhimurium
• Whilst Shigella do not appear to have distinguishable
ATR.
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19. • Cyclopropane FA’s formation in E. coli membrane is
important for the organism to survive acid stress.
• This is regulated by cfa (CFA synthase) which is
dependent on RpoS and induced by acid conditions.
• cfa is responsible for the CFA synthase that carries
out post-synthetic modification of the lipid bilayer.
• CFAs decrease proton permeability of membranes
and also interact with membrane proteins that
influence proton traffic
• Phosphate and cAMP also influence induction of acid
habituation
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20. • Acid habituation significantly enhances the survival
of exponential phase cultures exposed to a lethal acid
challenge.
• Acid habituation, a reversible adaptive tolerance to
low pH,
• Compounds such as glucose, glutamate, aspartate,
can induce habituation at neutral pH0.
• Habituation induced at pH 5 is CysB dependent
• Most dramatic resistance occurs in SP of the two
organisms and these systems are called acid
resistance systems, will protect the cells at pH 2 for
hours.
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21. • AR 1 and 2 present in both the organisms, and
dependent on the stress response alternative sigma
Rpos, cAMP receptor protein (CRP), and glucose
repressed.
• AR 1 requires activation by glutamate prior to
challenge at pH 2. echanism unknown.
• E.coli AR 2 also present in Shigella requires
extracellular glutamate during pH 2 acid challenge
• This system requires glutamate decarboxylase that
converts intracellular glutamate to GABA and the
glutamate/GABA antiporter gadC.
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22. • GadC antiporter provide the means to transport
substrate rapidly into the cell and expel
decarboxylation products. This ensures efficient
proton consumption
• This results in intracellular alkalization.
• AR 3, not present in Shigella, requires arginine
during pH 2 and functions much like GAD system,
– it is arginine(adiA)-dependent system that function
like the GAD system
– It coverts arginine to argmatine, consuming proton
in the process
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23. 23
Figure shows the AR resistance systems in E. coli

24.  Helicobacter pylori: urease-dependent and –
independent acid tolerance.
• H.pylori is a gut pathogen.
• Encounters acidic environment in the stomach,
resulting in the development of exceptional systems
of acid tolerance.
• Major mechanism involves urease produced by the
organism, which converts urea to CO2 and NH3 this
alkalizes the internal pH.
• Urease-independent ATR encoded by genes cagA and
wbcJ and dnaK also help in acid stress
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25. • How Wbcj contributes to acid resistance is unknown,
but it is involved in the O-antigen synthesis
– LPS proves a barrier, reducing proton influx
– O-antigen alters membrane permeability by
changing surface charge.
• The chaperone Hsp70(dnaK) is induced by low pH,
and is believed to be responsible for altered
glycolipid binding.
• It is believed that the altered binding facilitates the
association of organisms with the stomach l
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26.  Rhizobium: calcium, acid tolerance and dealing
with acid underground
• Low soil pH restricts legume productivity and thus
understanding the effects of low pH on growth and
survival of root nodule bacteria is important.
• Genes responsible for acid resistance in R.
leguminosarum are located on the second largest of
four mega plasmids, important in LPS structure and
interfere with pH homeostasis at low pH
• S. meliloti grows at low pH in the presence of
calcium
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27.  ATPase-independent acid tolerance mechanisms in
gram positive bacteria
• In L. monocytogenes, sigma factor σB is involved in
stationary-phase induced acid tolerance
• S. mutans decreased membrane permeability and
increased ability to expel protons, improves internal
pH and enhances resistance to acid killing.
• S. rattus increases ATPase levels and alteration of its
fermentation end products in response to
acidification, which effectively lowers the lipophilic
undissociated weak acid available to permeate the
membrane causing toxic effects.
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28. • In L lactis F0F1 H+–translocating ATPase is used to
aid in acid tolerance.
• Some bacteria genera utilize the arginine deiminase
pathway to survive acidic environments.
• The system converts arginine to ornithine, ammonia
and carbon dioxide. The generation of ammonia
alkalizes the environment.
• The system is repressed by carbohydrates and
induced by arginine
• Glutamate decarboxylase also plays an important role
in acid resistance.
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29. • Genes, gadCB encode for proteins, a glutamate:
GABA antiporter and a glutamate decarboxylase.
• Protect cells in the presence of glutamate and Cl- ion.
• Maximum induction of gadCB occurs in SP in the
presence of Cl- ion, low pH and glutamate.
 Conclusion
• The mechanisms used by microbes in response to
acid stress is important in food microbiology,
pathogenesis and agriculture.
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