Our current research focuses on extreme pH stress in Escherichia coli
and other enteric bacteria. Much is made of the fact that "extremophiles," such
as thermophilic archeans, can grow under extreme conditions of temperature or
pH. But our work has shown that even enteric bacteria can survive for extended
periods under conditions considered extreme, such as pH 2 or pH 10, resuming
growth when favorable conditions return.
Recently our research has extended to look at the gram-positive model system,
Bacillus subtilis. Surprisingly little is known about pH regulation
in gram-positive bacteria. B. subtilis is a useful model for the pathogen
B. anthracis, causative agent of anthrax.
We study the physiology and genetic response of E. coli and Bacillus
subtilis to pH change. Current projects include:
In recent years great excitement has been generated about
bacterial genes which respond to environmental changes such as
heat shock, osmotic shock, and anaerobiosis. Since pathogens
experience major environmental changes as they enter the human
system, is not surprising that some of these genes play important
roles in pathogenesis.
The pH of the growth medium, or external pH, has been shown
to regulate gene expression in several enteric bacteria,
including virulence genes mediated by toxR in Vibrio cholerae.
Some of the acid-shock genes in Salmonella may turn out to assist
this organism to grow within macrophages, possibly by preventing
lysosomal acidification. Interestingly, acid also induces
virulence in the plant pathogen Agrobacterium tumefaciens.
Study of pH-regulated genes is at last answering questions
about pH homeostasis, an important capability of many enteric
bacteria. Furthermore, we increasingly recognize that pH
interacts in important ways with other environmental and
metabolic pathways involving anaerobiosis, Na+ and K+ levels, DNA
repair, and amino acid degradation. It may turn out that pH-
regulated systems will offer clues to the evolution of virulence
genes from multivalent environmental sensors.
The ability of bacteria to respond to pH change
metabolically has long been recognized. Fermentation pathways
show pH dependence in many species. In the forties Helen Epps
and Ernest Gale showed that both Escherichia coli and Micrococcus
luteus generate various amino acid decarboxylases at low external
pH and deaminases at high external pH -- in each case, acting to
neutralize the growth medium. The genetic basis for some of
these responses is now understood.
With respect to motility, several kinds of pH taxis have
been characterized in E. coli and Salmonella typhimurium. These
include repellent response to external acid and to membrane-
permeable weak acids, and attractant response to base and
membrane-permeable weak bases. In Robert Macnab's lab I showed
that signal transduction for acid and weak-acid taxis involves
methylation of the methyl-accepting chemotaxis protein MCP I.
This finding suggests a molecular basis for pH detection,
although it does not involve gene expresssion.
Until recently, few studies focused on pH regulation of gene
expression. For genetic screening, my group at Kenyon developed
plate and liquid media containing a range of sulfonic-acid
buffers specific for the range of pH to be fixed; this is an
important consideration, for even with 100 mM of an appropriate
buffer growth of Escherichia coli can change the plate pH by half
a unit. We used these media to screen pools of Mud lac operon
fusions for pH-regulated activity. The acid-inducible locus
exa(cadA) and the alkaline-inducible alx were both identified in
this manner.
In addition, we showed that a number of lac fusions
previously isolated by anaerobic induction were also dependent on
pH, for example hyd (hydrogenase) in Salmonella. Other well-
known loci in E. coli show induction at high pH: Etana Padan and
Shimon Schuldiner found that heat-shock genes are induced at high
external pH, and that components of the SOS response are induced
by internal alkalinization.
The acid-inducible locus exa turned out to be an allele of
cadA, encoding lysine decarboxylase. George Bennett's group
constructed similar fusions to cadA and to adi, arginine
decarboxylase. These fusions show several hundred-fold induction
in acidic medium. Anaerobic growth increases induction
several-fold across the pH range. This enormous output of
decarboxylases protects the cell by removing acid from a protein-
rich medium; the resulting alkalinization is the basis of the
well-known M eller Broth test.
The anaerobic enhancement of decarboxylase makes sense given
that fermentative growth can generate enough acid to retard
growth. David Clark's lab has evidence that lactate
dehydrogenase may also be induced anaerobically by acid, acting
to decrease the output of acidic endproducts.
The acidic induction of the cad operon has been dissected by
Eric Olson's group. Acid induction of cadAB requires
CadC, which is independently transcribed. The CadC-regulated
promoter is located upstream of cadB. Bennett has evidence that
CadB may act to exchange lysine and its alkaline product
cadaverine across the cell membrane.
The amino acid sequence of the regulator CadC shows striking
homology to known transcriptional regulators, in particular ToxR
of Vibrio cholerae. ToxR is a membrane-bound protein which binds
DNA to induce transcription of cholera toxin and other virulence
genes. Virginia Miller and John Mekalanos have shown that, like
cadA, ToxR-dependent genes are induced strongly by acid.
Moreover, ToxR is now known to induce at least one metabolic
enzyme, aldehyde dehydrogenase. It is tempting to speculate that
virulence genes may evolve from environmental regulators of
metabolism.
Like ToxR, CadC has both a putative membrane-spanning domain
and a putative DNA-binding domain; thus it is believed to act
both as environmental sensor and as a transcription regulator.
The working model proposed for CadC action suggests that an
external sensory domain of the molecule is exposed to the
periplasmic space, where external proton concentration can be
detected directly or indirectly, while the amino-terminal DNA-
binding domain acts to turn on transcription at the cadAB
promoter. If this model is correct, CadC will provide a useful
system for mutagenesis to study the molecular mechanism by which
pH affects a sensory molecule.
While acidic induction of cadA requires the linked regulator
locus cadC, the induction of cadA by lysine involves an unlinked
regulator, cadR. A second unlinked regulator of the cad operon,
exaR, was recently identified by Olson as a Tn10 insertion which
partly deregulates cadA. My lab has preliminary evidence that
exaR is required for aerobic growth of E. coli below pH 6. In a
strain containing exaR::Tn10, the aerobic expression of cadA::lac
is increased to the anaerobic expression levels across the pH
range, while acid induction is unaffected.
Michael Leonardo in David Clark's group and Bradley Hersch
in my group have evidence that exaR regulates a number of
fermentation genes, including alcohol dehydrogenase, lactate
dehydrogenase, and fumarate reductase. The exaR::Tn10 is closely
linked to the nar operons, but Tn10 insertions in n
exaR may be the first known case of an anaerobic regulator
required for aerobic growth in E. coli at low pH. It might act
by repressing metabolic activities such as lactate dehydrogenase
which protect the cell from acidic fermentation at low external
pH, but retard cell growth under aerobic conditions.
Several other regulator genes are known to mediate pH-
dependent expression. earA, identified by John Foster, represses
acid-induced transcription of aniG in Salmonella. My group
identified inaR, which represses protonophore-dependent induction
of a weak acid-inducible locus in E. coli (see below.) Camille
LaCroix in my lab identified a regulator of alkaline-sensitive
expression, alxC, which represses alx.
An interesting consideration is that, in theory, bacterial
regulators could detect pH in two different compartments: the
cytoplasm (internal pH), and the periplasm, whose pH presumably
would equal that of the external medium. When I first began to
look at pH-regulated genes, I predicted that expression of
particular genes would be found to respond specifically to
internal or external pH. Since then, we and others have found
this to be the case: pH-dependent genes respond specifically to
internal or external pH change. The internal alkaline induction
of the SOS response is a particularly interesting case, since
stability of DNA could be affected by internal alkalinization.
In our lab Tania Gonzalez found a number of proteins in two-
dimensional SDS gels which were induced by benzoate, a membrane-
permeable weak acid which depresses internal pH. She also
identified the locus ina which shows pH-dependent expression only
in the presence of membrane-permeable weak acids or uncouplers.
Under certain conditions the expression of ina correlates with
internal acidification, although other effects of the weak acids
cannot be ruled out.
Do pH-regulated genes play a role in internal pH
homeostasis? For a long while pH homeostasis in E. coli was
generally believed to be constitutive, in contrast to the pH-
dependent ATPase in Streptococcus faecalis, for example.
Recently however two different kinds of inducible pH
homeostasis have been demonstrated: acid tolerance, and the
sodium-proton antiporter NhaA. Both cases are dauntingly
complex, involving several different stimuli and gene loci.
Foster's group discovered in Salmonella typhimurium that
growth in moderately acid medium (pH 5.5-6.0) induces genes whose
products enable cells to retain viability under more extreme acid
conditions (below pH 4) where growth is not possible. Close to
100% of acid-tolerant (or acid-adapted) cells can recover from
extreme-acid exposure and grow at neutral pH. The inducible
survival mechanism is called acid tolerance response. The
retention of viability by acid-tolerant cells correlates with
improved pH homeostasis at low external pH; thus, acid tolerance
may represent inducible pH homeostasis. Unlike constitutive pH
homeostasis, acid tolerance requires a functional proton-
translocating ATPase as well as the regulator fur, although its
iron uptake function is not involved.
Acid tolerance may be significant for pathogenesis because
Salmonella faces extreme acidification during uptake by
phagocytic cells, which the bacteria then colonize. Some of the
acid tolerance functions may protect the cell from effects of
acidification. Alternatively, Brett Finlay finds that Salmonella
may actually prevent or reverse acidification of its environment
within the phagocyte; perhaps some of the acid tolerance genes
function in this regard.
Extreme acid tolerance is not confined to Salmonella. Pam Small finds that clinical isolates of E. coli and Shigella exhibit acid tolerance below pH 2, either constitutively or under control of the stationary-phase regulator KatF.
At high external pH, Padan and Schuldiner have found in E.
coli an inducible system for internal pH homeostasis, the sodium-
proton antiporter NhaA. nhaA is induced at high external pH in
the presence of high sodium. In the presence of 10 mM
NaCl, the induction of nhaA increases with pH over precisely the
range in which external pH exceeds E. coli internal pH
(maintained at 7.4-7.9). The NhaA antiporter acts to acidify the
cytoplasm through electrogenic proton/sodium exchange.
A strain deleted for nhaA fails to grow at high pH in the
presence of 100 mM NaCl. In this deletion background, Padan and
Schuldiner identified a second antiporter locus nhaB. This
antiporter is constitutive and electroneutral. An nhaA nhaB
deletion strain is extremely sensitive to high pH. This suggests
that E. coli has both a constitutive mechanism for internal
acidification (over moderate pH values) and an inducible
electrogenic mechanism (at high pH, in the presence of Na+).
Interestingly, this confirms the observation of Anna Castle and
Robert Macnab that sodium transport is electroneutral at acid-to-
neutral pH and becomes electrogenic above pH 7.5.
How does the cell detect external alkalinization? Schuldiner and Padan have recently identified a positive regulator NhaR which shows homology to OxyR and LysR. This protein may be part of the alkaline signal transduction system.
Sodium-proton antiporters function in pH homeostasis in a
number of species, especially alkalophiles. In a particularly
intriguing experiment, Padan transferred an antiporter gene from
the alkalophile Bacillus alkalophilus into the nhaA nhaB deletion
strain, where it restored growth at high pH. Terry Krulwich
believes that B. alkalophilus may have as many as three sodium-
proton antiporters, and that the number of antiporters may relate
to the alkalophilicity of a species.
K+ transport at Low External pH
The work of Ian Booth and others has long suggested that
potassium transport may play a role in pH homeostasis in E. coli
under acid conditions, possibly a K+/H+ antiporter analogous to
the Na+/H+ antiporter at high pH. The study is complicated
however by the existence of multiple potassium transport systems,
both inducible and constitutive. It may well be that both
constitutive and inducible pH homeostasis mechanisms exist in the
acid range, as they do in the alkaline range.
Donald Dosch and Wolfgang Epstein constructed a strain in
which the three major K+ uptake systems are deleted. In my lab
we find that this strain requires 100 mM K+ for good growth at
pH 6, but only 10 mM K+ at pH 8.5. Furthermore, the internal pH
of the K+ uptake-deficient strain is depressed in low K+ medium.
At present we are using this strain background to test the
effects of internal acidification on gene expression, while
avoiding the side effects of membrane-permeable weak acids. We
also hope to identify additional loci involved in the
relationship between K+ and pH, possibly as mutations which
compensate for the K+ defect.
Clearly, pH interacts with a bewildering number of other
environmental effects on gene expression. The antiporter nhaA
requires both sodium and high pH for induction. For the cad
operon, a specific protein CadC mediates pH regulation, while
different regulators mediate induction by anaerobiosis or by
lysine. ToxR mediates response to osmolarity, temperature, and
amino acids, as well as pH; it induces a growing list of
virulence factors and metabolic enzymes. Fur regulates ferric
siderophores, Shiga-like toxin, and acid tolerance -- all of
which are known or potential virulence factors.
In addition, the mechanisms of pH homeostasis now appear
considerably more complex than was once thought, involving both
inducible and constitutive systems, which studies of pH-regulated
gene expression are at last helping to identify.
Some references (see also Publications):
Aliabadi, Z., Y. K. Park, J. L. Slonczewski, and J. W. Foster.
1988. Novel regulatory loci controlling oxygen- and pH-
regulated gene expression in Salmonella typhimurium. J.
Bacteriol. 170:842-851.
Auger, E. A., K. E. Redding, T. Plumb, L. C. Childs, S.-Y. Meng,
and G. N. Bennett. 1989. Construction of lac fusions to
the inducible arginine and lysine decarboxylase genes of
Escherichia coli K12. Mol. Microbiol. 3:609-620.
Bingham, R. J., K. S. Hall, and J. L. Slonczewski. 1990. Alkaline induction of a novel gene locus, alx, in
Escherichia coli. J. Bacteriol. 172:2184-2186.
Foster, J. W., and H. K. Hall. 1991. Inducible pH homeostasis and the acid tolerance response of Salmonella
typhimurium. J.
Bacteriol.
173:5130-5135.
Karpel, R. T. Alon, G. Glaser, S. Schuldiner, and E. Padan. In
press. Expression of a sodium proton antiporter (NhaA) in
Escherichia coli is induced by Na+ and Li+ ions. J. Biol.
Chem.
Parsot, C., and J. J. Mekalanos. 1991. Expression of the Vibrio
cholerae gene encoding aldehyde dehydrogenase is under
control of ToxR, the cholera toxin transcriptional
activator. J. Bacteriol. 173:2842-2851.
Slonczewski, J. L., T. N. Gonzalez, F. M. Bartholomew, and N. J.
Holt. 1987. Mu d-directed lacZ fusions regulated by low pH
in Escherichia coli. J. Bacteriol. 169:3001-3006.
Watson, N., D. S. Dunyak, E. L. Rosey, J. L. Slonczewski, and E.
R. Olson. In press. The Escherichia coli cad operon:
Identification of elements involved in its transcriptional
regulation by external pH. J. Bacteriol.