Applied Biochemistry and Microbiology, Vol. 40, No. 3, 2004, pp. 217224.

Translated from Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 40, No. 3, 2004, pp. 261269.
Original Russian Text Copyright 2004 by Vorobeva.

Stressors, Stress Reactions, and Survival of Bacteria: A Review

L. I. Vorobeva
Moscow State University, Moscow, 119899 Russia
e-mail: nina@NVorobjeva.home.bio.msu.ru
Received November 18, 2003

AbstractRecent data on the molecular mechanisms of the stress responses of bacteria are reviewed, with
emphasis on their reactions to a variety of stressors (heat, oxidation, cold, osmotic shock, etc.). The mechanisms
underlying the phenomenon of sensoring are discussed. It is shown that cross-resistance to stressors and cell-
to-cell communication, mediated by chemical metabolites, affect bacterial survival in food products. The stress-
antagonizing activity of bacteria is discussed in relation to food product biotechnology.

The concept of stress (a condition of strain; a term
borrowed from mechanics) was advanced by Hans
Selye, a Canadian physiologist, who wrote that stress
is life, and life is stress [1]. Humans frequently
encounter stressful situations, this being the reason for
the interest in stress and the nature of this phenomenon.
Stress can also be defined as a changein the
genome, the proteome, or the environmentproducing
a decrease in the growth rate or survival. Any form of
life may experience stress if the conditions that it is
used to undergo rapid changes. Stress responses are of
particular importance to microorganisms, because their
habitats are subject to continual changes (such parame-
ters as temperature, osmotic pressure, and substrate
availability are far from being constant). Of note, reac-
tions to stress in the most ancient inhabitants of Earth
(bacteria) are remarkably similar to those observed in
higher eukaryotes. All living organismsbacteria,
fungi, plants, animals, and humanssynthesize spe-
cific proteins in response to temperature elevation;
these molecules, known as heat shock proteins (HSPs),
are evolutionary conserved: certain regions in human
and bacterial HSPs retain homology (up to 90% of the
amino acid residues are common) [2], which is an indi-
cation of their extreme importance. Overproduction of
HSPs renders the cells resistant to high temperatures
and oxidative stress; this effect is observed in both
mammals and bacteria [3]. The Archeae (Sulfolobus
shibatae) contain chaperonin TF55, which has two sub-
units, and . Chaperonins are high-molecular-weight
biannular proteins with a subunit structure, exhibiting
ATPase activity; each subunit has a molecular weight of
60 kDa). The chaperonin of Sulfolobus, mentioned
above, accounts for 4% of the cellular protein in this
microorganism; its intracellular concentration exceeds
30 mg/ml, which is equivalent to 4600 molecules/cell.
Unlike their bacterial counterparts, chaperonins of
Archeae are only involved in the folding of several spe-
cific proteins under in vitro conditions [4]. Participation
in the formation of subcellular structures seems to be
their major function. In the presence of Mg2+ and nucle-
otides (i.e., under physiological conditions simulated in
vitro), chaperonins form filaments, which serve as
cytoskeleton-building blocks in vivo (in Sulfolobus and
other Archeae lacking rigid cell walls). TF55 is homol-
ogous to the eukaryotic protein TCP1 [5]. In eukary-
otes, TCP1 consist of 60 kDa subunits, the amino acid
sequence of which is more than 35% identical to chap-
erones of Archeae. In vitro, TCP1 is involved in actin
and tubulin assembly [6]; in vivo experiments involving
mutational analyses confirm their significant role in the
organization of the cytoskeleton. Thus, structurally
related chaperonins of Archeae and eukaryotes also
exhibit a functional similarity. The structural similarity
of the HSPs and the involvement of common molecular
mechanisms in triggering stress programs in diverse
organisms make it possible to use bacteria as a simple
model for studying stress responses. In view of the
above observations, Archeae, which exhibit a maxi-
mum similarity to eukaryotes in information processes,
offer the greatest promise as such model organisms.
In this work, we sought to review the current under-
standing of the mechanisms underlying (1) stress
responses in bacteria and (2) effects of stress-antago-
nizing activity on bacterial survival.
STRESSORS AND CONSEQUENCES
OF STRESSING CELLS
Stressors, or stress factors, may have a chemical,
physical, or biological nature. The organism itself may
be the source of stress factors. For example, many bac-
teria form 22, superoxide radicals ( O 2 ), and other
reactive oxygen species (ROSs). Stress factors cause
protein denaturing, induce breaks in nucleic acid
chains; other types of damage, including the oxidation
of macromolecules, are summarized in the table.
The existence of multiple stressors is matched by
the availability of diverse targets. An organism is alive

0003-6838/04/4003-0217 2004 MAIK Nauka /Interperiodica

218 VOROBEVA
Stresses and stress responses in Lactococcus lactis [15]
Stress factor (stressor) Effect
Acid Low pH
Heating Protein denaturing
Increasing salt Dehydration
concentration (causes drying)
Irradiation DNA damage
Effects of oxidants Oxidation of macromolecules
Cooling Slowing down translation,
inhibition of transcription, replication
as long as its cell membrane and nucleic acids retain
their integrity, and processes of protein folding are duly
maintained. Damage to other macromolecules may be
considerable, particularly in the case of mRNAs, but
their short life allows the cell to avoid any conse-
quences of the damage, which are self-eliminated in
turnover processes. According to a recent observation
[7], cells that either cease to grow, grow slowly, or enter
the steady state, acquire stress resistance. Irrespective
of its actual cause, any decrease in the growth rate is a
cellular signal initiating processes that result in stress
resistance. Rapid molecular reactions have developed,
which repair stress-induced damages and protect the
cells subsequently, in case of exposure to the same or
different stressor. The reaction involving the formation
of stress proteins is currently the best studied stress
response.
Heat shock and molecular chaperones. Protein
denaturing, which is the major event associated with
effects of heat on living organisms, induces a stress
response involving cellular mechanisms. The increase
in the amount of HSPsmolecules responsible for the
proper folding of newly synthesized polypeptides and
the refolding of polypeptides that were either damaged
or improperly foldedis one of such mechanisms.
Only properly folded polypeptide chains adopt the
native confirmation and are functional. The accumula-
tion of partially denatured proteins exposes their hydro-
phobic regions, to which chaperones adhere. Depletion
of the chaperone pool serves as a signal for activation
of their synthesis.
Chaperones prevent undesirable interactions
between potentially complementary surfaces of pro-
teins; they are found in all bacteria, as well as Archeae
and eukaryotes. Pursuant to conventional HSP nomen-
clature, the name of each species includes its molecular
weight expressed in kilodaltons (e.g., GroEL60,
DnaK70).
DnaK, GroEL, GroES, and a number of other chap-
erones are found in all bacteria. It is worth asking how
these wonderful proteins work. Figure 1 [8] shows the
route of the polypeptide chain released from the ribo-
some to the location where native folding is completed.
APPLIED BIOCHEMISTRY AND MICROBIOLOGY
Mechanism of resistance
Arginine deamination pathway resulting
in the formation of NH3 (neutralizes acids)
Chaperones facilitating proper protein folding
Osmolyte transportation system
DNA repair
Elimination of reactive oxygen species
Cold shock proteins, RNA chaperones,
stimulatory, Translation efficiency
The polypeptide released from the ribosome is first
bound by the chaperone DnaJ; the resulting protein
DnaJ complex becomes a target (substrate), to which
DnaK is bound. The tertiary complex thus formed has a
specific feature: it stabilizes DnaK in the ADP-binding
state, which increases the affinity of this chaperone to
unfolded proteins. DnaK exhibits weak ATPase activ-
ity, which is increased as a result of binding to the
ATPase region of the small chaperone GrpE. Following
ATP hydrolysis and ADP release, a new ATP molecule
is bound to DnaK. The release of the polypeptide chain
is coupled to changes in DnaK conformation, which
occur after ATP binding and hydrolysis. The partially
folded polypeptide released from the complex with
DnaK/DnaJ is passed to the complex GroEL/GroES.
The function of its constituent proteins, termed chaper-
onins, is ATP-dependent. The polypeptide is bound to
the inner surface of GroEL, which causes dissociation
of the complex GroEL/GroES and concomitant ATP
hydrolysis. Subsequent binding of ATP and its hydrol-
ysis makes possible the transfer of the protein inside
GroEL, where folding occurs. In the absence of ATP,
GroEL-bound protein is not folded. Thus, chaperones
play a critical role in the formation of the native struc-
ture of proteins. Short of the involvement of chaperones
the polypeptide chains synthesized in ribosomes would
be functionally inactive. When the conformation of a
denatured protein is restored, HSPs act in a similar way.
In this case, they act in concert with proteases, which
recognize improperly or incompletely folded proteins
as their substrates (Fig. 2). Low folding rate, chemical
or thermal stress, structural instability, and errors in the
course of biosynthesis are the factors contributing to
the appearance of such defective proteins [10]. The
major function of classic chaperonesDnaK, or Hsp70
(and its co-chaperones DnaJ and GrpE), and GroEL, or
Hsp60 (and it co-chaperone GroES)is to shift folding
along the pathways that prevent improper folding and
aggregation, facilitating refolding and the proper
assembly of proteins [11]. Considerable amounts of
HSP60 are detected in Archeae in the course of their
normal growth; this HSP likely functions as a chapero-
nin [12]. In light of the cellular folding model described
above, chaperonin activity in Archeae should be highly
Vol. 40 No. 3 2004
 STRESSORS, STRESS REACTIONS, AND SURVIVAL OF BACTERIA 219

() (b) (c)
ADP
GrpE
Pi DnaJ
DnaK
Ribosome Ribosome DnaK DnaK
DnaJ N DnaJ DnaJ
DnaK Nascent DnaK
mRNA protein
ATP
5' 3'
ATP

DnaK
DnaJ
GroEL

ADP
GroES

(d) (e) (f)

ADP ATP
+
Pi
Complete protein folding

ADP
+Pi
ATP

Fig. 1. Schematic representation of the involvement of molecular chaperones in the proper folding of polypeptide chains in bacteria
[8] (for details, see the text). (a) DnaK and DnaJ interact with the nascent polypeptide chain. (b) A tertiary complex is formed,
including DnaK, DnaJ, and the unfolded protein. (c) GrpE induces ADP formation, followed by ATP binding; the partially folded
protein is released and transferred to the GroEL/GroES complex. (d) Binding of the folded intermediate in the central cavity of
GroEL. Hydrolysis of ATP (bound to GroEL), the protein enters the cavity, where it is folded. (e) Chaperonin GroES moves between
the two ends of the GroEL cylinder. (f) The completely folded protein loses its affinity for the chaperonin and exits the cavity of
GroEL.

regulated: we have already seen that folding occurs
inside the central cavity, access to which may be
blocked by the filaments of chaperonins. It is believed
that cells are capable of isolating chaperonins from the
filaments or causing their dissociation [4]; this provides
a source of chaperonin proteins, which does not require
their de novo synthesis.
The HSPs of Archeae are structurally and function-
ally similar to bacterial chaperonins, in spite of consid-
erable differences in their amino acid sequences. In
fact, the HSPs of Archeae are homologous to a protein,
known as TCP1s, which is widespread in eukaryotes
[13, 14]. A switch to a stress program is an emergency
event in the life cycle of a cell. The majority of HSPs
are synthesized at low concentrations in the absence of
any stress, but their synthesis is rapidly induced if the
temperature is increased or another stressor appears.
For example, exposure of the lactic acid bacterium Lac-
tococcus lactis to a temperature of 42 results in the
synthesis of 12 to 17 new proteins [15]; the amount of
GroEL increases 45-fold within the first 10 s of heating.
In Bacillus subtilis, the synthesis of cold shock protein
increases 200-fold [13]. Exposure of Pyrodictium
occultum (a hyperthermophilic representative of
Archeae) to a temperature exceeding its optimum
(108) by 3C increases the synthesis of chaperonins
to a level where they account for 80% of cytosolic pro-
teins and form what is known as thermosomes [13]. In
the presence of a thermosome, the cells survive auto-
claving (121) for 1 h [13]. Certain representative of
the kingdom Crenarcheota survive temperature condi-
tions at which life is barely possible; by exposing these
organisms to a short heat shock (during which chaper-
onins are synthesized) extreme thermal stability is
attained. When heated at 60, halobacteria (meso-
philic representatives of Archeae) form 46 new pro-
teins (with molecular weights of 75104, 4445, and
2128 kDa) [16]. Because of the active HSP synthesis,
however, many other proteins required for normal life
are formed in lesser amounts, and this slows down the
corresponding metabolic reactions. At the expense of
this, stress programs are switched on, saving the organ-
isms life. HSP induction in Escherichia coli and Bacil-
lus subtilis involves alternative sigma () factors (32,
E, N, and B) altering the ability of RNA polymerase
to recognize its promoters; as a result, selective HSP
expression occurs [15].
Other representatives of the HSP family are also
involved in creating or restoring functional protein con-
formations; some of them belong to chaperonins. These
small proteins have molecular weights of 34 kDa or
less; one of them is the enzyme peptidyl-prolyl cis
trans-isomerase (PP isomerase; EC 5.2.1.8). This
group comprises many proteins found in eukaryotes,
bacteria, and Archeae. Their importance is underlain by
the fact that the folding of polypeptide chains requires,

APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 40 No. 3 2004

220
Denatured proteins
Protease Chaperone
+ATP +ATP
1 2
Degraded Active
protein protein
Fig. 2. Mechanism of HSP action [9, 10]. Inactive, dena-
tured, or unstable proteins are recognized by proteases
(1) or the chaperone complex (2); correspondingly, such
proteins are either cleaved into small peptides or recovered
by HSPs (resulting in active proteins with natural conforma-
tion and normal stability).
above all, that peptidyl-prolyl bonds be rotated. The
spontaneous rotation of these bonds has a very low rate,
and PP isomerase increases it [16].
Yet another important condition should be met in
order to maintain proper folding and produce a native
protein conformation. Anomalous proteins formed
under stress conditions may aggregate, form precipi-
tates, exert toxic effects in the cells, and cause serious
diseases. For this reason, organisms are equipped with
a defense system, which is based on their ability to
degrade proteins and peptides. In order to avoid prob-
lems, anomalous proteins should be either converted to
native forms or undergo elimination. As shown above,
restoration of normal function is performed by molecu-
lar chaperones; elimination is the specialty of pro-
teolytic enzymes known as chaperonins (Fig. 2) [9, 10].
The key proteolytic system in eukaryotes involves
ubiquitin and the proteosome. Proteins (or protein frag-
ments) to be cleaved are first labeled with ubiquitins
and then digested by the proteosome.
Oxidative stress. A review of the stress responses of
cells cannot bypass the problem of oxidative stress.
This phenomenon is believed to be involved in the
pathogenesis of radiation-induced diseases and dis-
eases arising from mass damage of cells (stroke and
myocardial infarction). Oxidative stress may be of cen-
APPLIED BIOCHEMISTRY AND MICROBIOLOGY
VOROBEVA
tral importance in the mechanisms of ageing and apop-
tosis (programmed cell death, or induced cell suicide).
The term oxidative stress is used herein to designate
changes in cellular levels of oxidizing agents.
Oxidative stress induces breaks in nucleic acid
chains, cleaves protein molecules (after which the frag-
ments may be cross-linked), and blocks the active sites
of enzymes; energy depletion is its consequence at the
organism level [17].
Bacteria routinely encounter ROSs (it is exactly
these forms of oxygen, rather than oxygen itself, that
cause the above damages).
ROSs attacking bacteria most frequently include
. .
both radicals ( O 2 , , O, N O ) and non-radicals
substances (22 and singlet oxygen, 12). The high
reactivity and short life of hydroxyl radicals and singlet
oxygen prevent cells from developing mechanisms of
their elimination. Conversely, O 2 and 22 are rela-
tively stable, and cells possess enzymes degrading
these ROSs. The antioxidative defense system of cells
includes superoxide dismutase (SOD; EC 1.15.1.1),
catalase (EC 1.11.1.61), and peroxidase (EC 1.11.1.7),
which perform the following reactions:
O 2 + 1e O2 ;
+ SOD
O 2 + O 2 + 2H H2 O2 + O2 ;
2H 2 O 2 catalase 2H 2 O + O 2 ;
peroxidase
H 2 O 2 + H 2 O 2 R 2H 2 O + O = R = O,
where R is an organic radical. In addition, cells contain
molecules with antioxidative activity. These include
fat-soluble vitamins, carotenoids, and polyamines, all
of which protect DNA from oxidation. New antistress
molecules have been discovered at the Bach Institute of
Biochemistry (methylerythritol cyclopyrophosphate,
natural nitroxyls, e.g., the trisaccharide lysodektose, a
trehalose derivative) [17, 18].
OxyR and SoxR are the two transcription factors
operating in E. coli under the conditions of oxidative
stress. The first factor activates antioxidative genes in
response to the appearance of hydrogen peroxide; the
second one induces antioxidative enzymes in the pres-
ence of O 2 . OxyR serves both as a receptor (sensing the
appearance of ROSs) and a transcription activator: the
oxidized and reduced forms of this factor have different
conformations, and a change in its conformation serves
as a cellular emergency signal [19]. OxyR contains six
residues of the amino acid cysteine, of which two
(Cys199 and Cys208) are critical for building up a stress
response (Fig. 3) [19]. The reduced form of OxyR,
which is predominant in cells, is inactive (because its
redox potential, equaling 185 mV, is higher than that
of the cytosol, e.g. in the cells of E. coli). The oxidation
of OxyR is believed to induce enzymes reducing the
Vol. 40 No. 3 2004
 STRESSORS, STRESS REACTIONS, AND SURVIVAL OF BACTERIA
disulfide bonds bridging the two key cysteines; as a
result, the reduced form of OxyR is formed, which is
ready for an encounter with another molecule of hydro-
gen peroxide (Fig. 3).
Excess O 2 is sensed by the proteinaceous transcrip-
tion factor SoxR; the signal is transmitted to the appro-
priate genes, resulting in the induction of SoxR. The
active site of SoxR contains four cysteine residues and
a metal; a change in the redox state of the metal likely
activates SoxR, and the active form of the regulatory
protein transmits the emergency signal.
Cold shock. The temperature in most parts of the
biosphere is cold; in the case of humans, it is lower than
the living optimum of 2527. Nevertheless, animals
and humans maintain homeostasis (a state of internal
balance of the organism) using any possible way. The
question of how this problem is solved by bacteria,
which are exposed to all dangers (e.g., in polar regions,
at temperatures below the freezing of water), is worthy
of detailed consideration. Unlike heat shock, pH fluctu-
ations, and osmotic stress (to give just a few examples),
cold shock does not cause protein denaturing. What it
does change, however, is the state of molecules, and
this, in turn, affects biochemical reactions (if we com-
pare cellular metabolism under the conditions of cold
stress to processes occurring in mesophilic or hyper-
thermophilic organisms). Cold stress stabilizes the sec-
ondary conformation of nucleic acids, which inhibits
DNA replication, gene transcription, and translation.
The activity of enzymes and the rate of metabolic pro-
cesses decrease, as well as membrane fluidity; taken
together, these changes hinder the transmembrane
influx of compounds. The intracellular formation of ice
crystals damages subcellular structures, causing death
[16]. In response to low temperatures, microorganisms
synthesize cold shock proteins (CSPs), which are
involved in processes of protein synthesis and mRNA
folding [13].
Lysteria monocytogenes, Yersinia enterocolitica,
B. cereus, and Clostridium botulinum are psychrophilic
thermophiles capable of infecting food. Microbial
adaptation to low temperatures has received much
recent attention, because of the increasing use of prod-
uct freezing (for the purpose of long-term storage and
protection from microbial deterioration). In response to
cold shock, B. subtilis synthesizes three new proteins;
in E. coli, the number of these proteins is as high as nine
[16]; in either case, however, the molecular mechanism
of the action of these factors remains unknown. In
many bacteria, cold shock induces increased synthesis
of small proteins (7 kDa) highly homologous (by more
than 45%) to a region conserved between gram-positive
and gram-negative microorganisms (including those
routinely found in foods) [20, 21]. Psychrophilic
microorganisms living at temperatures from 5C to
+20 are characterized by (1) increased membrane
fluidity (which is underlain by the abundance of short-
chain polyunsaturated fatty acids), (2) structural and
APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 40
221
OxyR-C199-SH
C208-SH
H2O2
OxyR-C199-SOH
C208-SH
OxyR-C199-S
C208-S
Fig. 3. Schematic representation of the activation and deac-
tivation of transcription factor OxyR [19]. In the presence of
H2O2, Cys199 is first oxidized (to sulfenic acid). This reac-
tive intermediate reacts with Cys208 with the formation of a
disulfide bond, which locks OxyR in its active form. The
oxidized OxyR recovers the reduced state when the disul-
fide bond is cleaved (reduced) by the glutaredoxin system.
Since OxyR activates the transcription of two genes, i.e.,
grxA (encoding glutathione synthase) and gorA (glutathione
reductase), the overall response is self-regulated.
functional plasticity (adaptability) of enzyme systems
and (3) the ability to synthesize antifreeze proteins pre-
venting the growth of ice crystals.
Modification of DNA supercoiling may play a cer-
tain role in cold shock. It has been shown that cold
shock increases negative supercoiling of plasmid DNA
in E. coli [22]. Cold shock changes the physiology of
bacteria in such a way that their survival rates in frozen
foods considerably increases [10]. Moreover, bacteria
adapted to low temperatures are characterized by a
shorter lag phase and a higher growth rate; the extent to
which these features are pronounced depends on the
quality of the product and measures taken to its protec-
tion from infection.
High hydrostatic pressure. At pressures in the range
of 20130 MPa, the growth of microorganisms is inhib-
ited, and increasing the pressure further (130
800 MPa) may result in cell death. Ribosomes and
membranes are particularly sensitive to high pressure
[23]. At 55304 kPa, the growth of E. coli is inhibited,
but the synthesis of certain proteins involved in tran-
scription and translation is not affected. This unique
stress response to increased pressure (manifesting itself
as uninterrupted synthesis of strictly controlled pro-
teins) was also described in cold shock [24]. Exposure
of E. coli to high pressures increases the level of CSPs,
HSPs, and other proteins [24]; their total number in
No. 3 2004
222 VOROBEVA
E. coli is as high as 55. However, the induction of stress
protein synthesis is a slow process building up within
6090 min (compare to HSP induction on increasing
the temperature, which is complete in seconds). Resis-
tance to high hydrostatic pressure creates considerable
problems, because the technology of pressurization is
widely used as a means of preventing microbial deteri-
oration of foods. Using high pressure treatment of
foods in combination with moderate heating made it
possible to increase microbial inactivation [23].
Osmotic shock. To sustain extreme saltiness, various
microorganisms use a common twofold mechanism:
the cells (1) discharge excess salt and (2) accumulate
osmolytes, substances that are not damaging to intrac-
ellular structures. Osmolytes are small organic mole-
cules retaining water solubility at high concentrations;
high levels of osmolytes are detected in the cytosol of
cells subjected to osmotic stress. Carnitine, trehalose,
glycerol, sucrose, proline, mannitol, glycinebetaine,
ectoine, glucitin, and small peptides belong to this
group of substances.
Betaine (N,N,N-trimethylglycine) accumulated
intracellularly by bacteria is used as an osmoregulator.
E. coli, B. subtilis, and Staphylococcus aureus are capa-
ble of synthesizing betaine (in a reaction catalyzed by
choline dehydrogenase; EC 1.1.99.1) or effecting its
transmembrane transportation from the medium. Sugar
beet (Beta vulgaris L. var. saccharifera (Alef) Krass)
and other plant products are rich in betaine; its transpor-
tation has been characterized in E. coli and
S. typhimurium [25]. In E. coli, betaine is pumped into
the cells by ProP, a constitutively active Na+-dependent
transporter. Another system, which is ATP- and protein-
dependent, is induced under the conditions of osmotic
stress, on changing osmotic pressure.
The gene betA of E. coli was introduced into the
plasmid Ti of Agrobacterium tumefaciens, which trans-
formed the cells of dicotyledonous plants in the course
of its life cycle. Tobacco plants (Nicotiana tabacum L.)
expressing the gene turned out to be 80% more resistant
to high salinity than their wild counterparts [26]. Stress
proteins are also involved in osmotic stress, by facilitat-
ing the proper folding of partially unfolded polypep-
tides. Accumulation of betaine is also detected in
Archeae. Halophilic Archeae accumulate potassium
ions. Under high salinity conditions [27], the yeast Sac-
charomyces cerevisiae accumulates glycerol (a harm-
less osmolyte) and activates NaCl excretion (discharg-
ing the stressor into the medium). Trehalose is another
osmolyte accumulated by yeasts, but its amount is
insufficient for increasing the osmotic properties of the
cells; and protection against drying and thermal inacti-
vation is achieved by stabilizing proteins and maintain-
ing the integrity of the cell membrane.
APPLIED BIOCHEMISTRY AND MICROBIOLOGY
MECHANISMS OF SENSORING
Current understanding of how the cell determines
which proteins and molecular systems should be
switched in response to the effects of a stress factor is
rather poor. Many regulatory systems used by bacteria
for sensing the signal and responding to it have two
components: (1) sensory elements exposed at the sur-
face of the cells and (2) a specific sensor protein. The
transmembrane position of the sensor ensures its con-
tact with both the medium and the cytosol; the protein
exhibits a kinase activity (i.e., it catalyzes phosphoryla-
tion reactions). The external portion of the protein may
bind diverse signal molecules. The binding of such
molecules results in autophosphorylation of the protein
(at hystidine residues), followed by the transfer of
phosphate to the aspartate residue of another protein,
the responder. The responder protein may act as a tran-
scription activator or repressor. The initiation of tran-
scription is the most important step of gene regulation
in bacteria. The binding of RNA polymerase to a fac-
tor (a small protein recognizing various DNA
sequences and initiating the operation of silent genes) is
a prerequisite to the initiation. Fourteen classes of
factors have been described, which regulate gene
expression in response to changes in the environment.
For example, the induction of the genes encoding
GroEL and DnaK in L. lactis involves 32, which is
responsible for HSP transcription [28]. Supercoiled
DNA may serve as a sensor [29], because the number
of bonds between the two chains determines the num-
ber of coils. Changes in the extent of coiling are, in turn
determined by the activity of certain enzymes (gyrase
and topoisomerase).
CROSS-RESPONSES TO STRESS FACTORS
Many genes in E. coli are organized into functional
units (regulons), in which separate genes having indi-
vidual control mechanisms may be controlled in a coor-
dinated fashion, by a common regulatory gene. The
existence of two levels of control corresponds to the
concepts of common and specific stress responses.
More than 17 common stress proteins have been
detected thus far in lactic acid bacteria [30]. It is not
surprising, therefore, that pre-adaptation to one stressor
renders the cells resistant to effects of other stress fac-
tors (cross-resistance). For example, moderate UV irra-
diation renders the cells of L. lactis resistant to heating
and the effects of acids, alcohols, and 22 [30]. The
cells of E. coli, adapted to chemical stress (by pre-expo-
sure to 4-nitroquinoline 1-oxide) acquire resistance to
UV irradiation and treatment with nalidixic acid [31].
Cross-resistance creates added problems in preventing
the microbial deterioration of food products.
Vol. 40 No. 3 2004
 STRESSORS, STRESS REACTIONS, AND SURVIVAL OF BACTERIA
CELL COMMUNICATIONS AND MUTUAL AID
IN BACTERIA SUBJECTED TO STRESS
Unlike animals, microorganisms (and plants) lack
the ability to defend themselves from unfavorable con-
ditions of the environment by physical action. Never-
theless, bacteria are endowed with specific means of
transmitting alarm (and or emergency) signals. Such
signaling involves chemical communication, and is
species-specific. In addition to inducing stress
responses in the neighboring cells, components
released by the bacterium act as extracellular alarmones
(alarm signals) alerting other organisms potentially
amenable to effects of the stress factor.
Luteococcus japonicus subsp. casei is a microor-
ganism forming bright-orange colonies, which was iso-
lated by us from cheese of the brand Sovetskii. Healthy
cells of this microorganism, not subjected to stress,
release into the medium a small protein with remark-
able properties. The survival rate of L. casei cells
exposed to increased temperature or UV irradiation
increased several hundred times if the cells were incu-
bated in a 1 g/ml solution of the protein for minutes
(shocked cells not treated with the protein served as a
reference). Moreover, the greater the damage to the
microbial population, the more efficient the extracellu-
lar antistress protein. In fact, the protein switched the
response to stress in those cells that balanced between
life and death, lacking the requisite resource for repair-
ing the damage [32, 33]. Of greater importance, the
same protein resuscitated temperature-inactivated yeast
cells. Experiments on purifying the protein are in
progress (its molecular weight is 9 kDa), and we
expected to have it sequenced (which would in turn
allow identification with other proteins or their frag-
ments).
The recent term quorum sensing processes desig-
nates phenomena [34] manifesting themselves after the
cell density attains a certain value, at which the stress
signal is accessible to the whole population. Cell-to-
cell communications are mediated by short peptides
and proteins, amino acids, homoserine lactones, and
other small molecules released by the cells; these medi-
ators are active at low concentrations and resistant to
environmental factors. Researchers of the Institute of
Microbiology of the Russian Academy of Sciences [35]
demonstrated that tetracycline-treated E. coli cells
release into the medium an unidentified compound that
exerts protective effects on tetracycline-sensitive cells
grown in the presence of this antibiotic or subjected to
oxidative, osmotic, or heat shock. Another example of
chemical communication comes from experiments with
the culture E. coli K-12. When grown at pH 7.09.0, the
cells release into the medium a proteinaceous exometab-
olite; exposure of this compound to low pH, increased
temperature, or UV radiation converts it to an extracellu-
lar inducer rendering the cells acid resistant [36].
Many gram-positive bacteria (some of which are
known to cause food spoilage) frequently release into
APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 40
223
the medium N-acylhomoserine lactones (AHLs), which
serve as sensory molecules. AHLs directly affect
eukaryotic cells by suppressing their immune reactions
[37]. Experiments identifying inhibitors of AHL-com-
munication have provided some important results. One
such inhibitor is the halogenated furanone (QSI)
formed by Australian red algae (Delisea pulchra) [38].
Other furanones structurally similar to this product
offer promise as agents for meat preservation [38]. QSI
suppresses several key processes (bacterial coloniza-
tion of the meat surface, the formation of toxins, and,
possibly, reproduction of bacteria) by inhibiting the
sensory compounds.
Antistress activity of bacteria and their ability to
synthesize exometabolites with pheromone-like or
reactivating properties may have both positive and neg-
ative implications. Knowledge of these features of bac-
teria is important for the development of biotechnology,
because this applied science uses stressed bacteria as
operators fulfilling defined tasks. Stress-induced pro-
teins serve as molecular markers of the health of the
starting cultures of such bacteria. For example, the
appearance of these markers may show that the culture
has been subjected to stress and that fermentation under
optimum conditions is no longer possible. On the other
hand, these same markers may indicate that the culture
has been adapted to certain stressors. Fighting hazard-
ous bacteria, such those causing food spoilage, requires
that their antistress activity be accounted for. In order to
assess the antistress potential of microorganisms, they
are exposed to extremely high pressure in combination
with pulsed electric field [23]. Thereafter, qualitative
parameters of the population are studied using flow
cytometry [23]. This powerful analytical tool makes it
possible to measure the chemical parameters of individ-
ual cells in the course of their flow (1000 cells/s), using
optical and electronic sensors.
ACKNOWLEDGMENTS
This work was supported by the Russian Foundation
for Basic Research, project no. 02-04-49930.
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