Mechanisms of microorganism adaptation to stress factors
МІНІСТЕРСТВО
ОСВІТИ І НАУКИ УКРАЇНИ
Національний
авіаційний університет
Інститут
екологічної безпеки
Кафедра
біотехнології
КУРСОВА
РОБОТА
(пояснювальна
записка)
з
дисципліни «Мікробіологія біологічних агентів»
Тема:
«Механізми адаптації мікроорганізмів до стресових факторів»
Виконала студентка
304 групи
Літвін І.А.
Керівник:
Васильченко О.А.
Київ
2014
MINISTRY
OF EDUCATION AND SCIENCE OF UKRAINEAVIATION UNIVERSITYOF ECOLOGICAL SAFETYOF
BIOTECHNOLOGY
PAPER
(Explanatory
Note)the discipline: “Microbiology of biological agents”: “Mechanisms of
microorganism adaptation to stress factors”
Student:
Litvin IrinaIES 304: Vasylchenko O.A.
2014
CONTENT
ABSTRACT
INTRODUCTION
.
DEFINITIONS
.
MECHANISM OF STRESS ADAPTIVE RESPONSE
.
STRESS SENSING
.
REGULATION OF STRESS-RELATED PROTEIN SYNTHESIS
.
GENERAL STRESS RESPONSE
.
SPECIFIC STRESS RESPONSES
.1
Heat
.2
Cold
.3
Acid
.4
Osmotic Stress
.5
Oxidative Stress
.
MONITORING STRESS RESPONSE
.
INDUCTION OF STRESS ADAPTIVE RESPONSE: PRACTICAL
CONSIDERATIONS
.1
Heat
.2
Acid
.
DETECTING AND QUANTIFYING STRESS RESPONSE
.1
Detection of Stress Response Genes
.2
mRNA Analysis
.3
Detection of Stress Proteins
.4
Biosensors
.5
Measuring Increased Tolerance
.
PERSPECTIVES AND AREAS FOR FUTURE WORK
REFERENCES
ABSTRACT
microorganism adaptation
stress
Explanatory note to the semester
paper “Mechanisms of microorganism adaptation to stress factors” include 26
pages, 9 literary sources. of the study - industrially important
microorganisms.- to show different mechanisms of microorganism adaptation to
stress factors such as heat, cold, acid, osmotic pressure and so on.methods -
analysis, a systematic approach of observation., ADAPTATION, RESISTANCE, SIGMA
FACTOR, GENE EXPRESSION, REGULATORY PROTEINS
INTRODUCTION
To survive adverse and fluctuating
conditions, microorganisms possess mechanisms to recognize diverse
environmental changes and mount an appropriate response. Various mechanisms are
involved in its activation, depending on the type of stress factor and on the
metabolic characteristics of the microorganisms. Microorganisms frequently
react simultaneously to a wide variety of stresses and the various stress
response systems interact with each other by a complex of global regulatory
networks [1]. biological purpose of the stress response is to protect cell
components against potentially dangerous environmental factors and to repair
damage occurring in stress conditions. The stress response is manifested as a
change in the metabolic activity of the cell, resulting from the repression of
synthesis of most of the proteins formed in the cell under normal physiological
conditions, and induction of the synthesis of a specific group of proteins
enabling the cell to function in the new conditions. The biochemical changes
are accomplished by physiological changes, such as temporary slowing or
stoppage of the division cycle, morphological changes in the cell, or the
emergence of resistance to the same stress factor or other types of stress
factor [2].can survive under diverse environmental conditions and in order to
overcome these adverse and changing conditions, bacteria must sense the changes
and mount appropriate responses in gene expression and protein activity. The stress
response in bacteria involves a complex network of elements that acts against
the external stimulus. Bacteria can react simultaneously to a variety of
stresses and the various stress response systems interact (cross-talk) with
each other. A complex network of global regulatory systems leads to a
coordinated and effective response. These regulatory systems govern the
expression of more effectors that maintain stability of the cellular
equilibrium under the various conditions [3].bacteria some of the most
important stress response systems are:shock response, controlled by the sigma
factor sigma 32;stress response, controlled mainly by the sigma factor sigma E
and the Cpx two-component system;shock response, which governs expression of
RNA chaperones and ribosomal factors;stress response, which depends on the
sigma factor sigma S;
(p)ppGpp-dependent stringent
response which reduces the cellular protein synthesis capacity and controls
further global responses upon nutritional downshift.
Further
examples include the secretion of protein domain, TauD
<#"867628.files/image001.jpg">
Figure 1. Interrelations among
physiological states of microbial cell subjected to different stresses
2. MECHANISM
OF STRESS ADAPTIVE RESPONSE
Response of microorganisms to stress
includes immediate emergency responses (e.g.,those produced in response to
shock) and longer-term adaptation. In some cases, the same proteins are
involved in both rapid and long-term responses. In addition to a general stress
response that helps protect cells from a variety of stresses, cells have
self-protective mechanisms against specific stresses. Overlap exists between
the proteins involved in the general stress response and some specific stress
responses.adaptation is a complex phenomenon that differs depending on the type
of stress and the bacterial species. Adaptation results from induction of
various stress-related proteins that protect the cell from stress. Many
stress-induced proteins have been identified.
3. STRESS
SENSING
For the cell’s metabolism to respond
to a stress, the stress must somehow be sensed. In general, bacterial sensing
of environmental changes is not well understood. Some stresses may affect
folding of mRNA or change a protein’s half-life, resulting in changes in gene expression
(Yura and Nakahigashi, 1999). Other stresses may affect protein structure. For
example, OxyR senses reactive oxygen species via cysteine residues that are
oxidized to form a disulphide bridge. The resulting oxidized protein positively
regulates oxidative stress response (Mongkolsuk and Helmann, 2002).of certain
cellular metabolites, such as guanosine phosphate, guanosine tetra-(ppGpp) and
pentaphosphates (pppGpp) and phosphate, may also trigger the synthesis of
stress-related proteins (Chatterji and Ojha, 2001; Rallu et al., 2000; Rao and
Kornberg, 1999). Ribosomes were suggested as sensors for temperature shocks
because of the sensitivity of these cellular components to heat (Duncan and
Hershey,1989). In addition, changes in the membrane structure or fluidity may
trigger a signal to synthesize proteins to counteract a
stress (Bremer and Krämer, 2000).
Two-component signal transduction
systems, consisting of a membrane-associated sensor kinase and an intracellular
response regulator, have been implicated in the sensing of and response to some
stresses. For example, in Bacillus subtilis, a two-component system is involved
in expression of cold-inducible genes. In this system, a membrane-bound
histidine kinase (DesK) that may sense changes in membrane fluidity transduces
the signal to a response regulator (DesR) that putatively activates the
transcription of fatty acid desaturase gene, des (Sakamoto and Murata, 2002).
4. REGULATION
OF STRESS-RELATED PROTEIN SYNTHESIS
Regulation of stress response is essential
for the synthesis of appropriate stress-related proteins only when necessary
for protection of the cell. Regulation of stress responses occurs at different
levels depending on the stress and the bacterium.may occur at the
transcriptional or translational levels or by adjusting the stability of the
mRNA or protein (Figure 2). Regulatory strategies vary considerably among
bacteria and stresses. To add to the complexity, one stress response factor may
be regulated at one or more levels.
Figure 2. A simplified
representation of general cellular processes involved in stress response,
molecular factors involved in sensing and controlling stress response, and
methods used to measure some of these responses.
The stress sensor is not depicted,
but this includes a lipid, protein, or nucleic acid component that senses the
stress and ultimately causes a change in transcription or translation. DSC:
differential scanning calorimetry; RT-PCR: reverse transcription-polymerase chain
reaction.control of stress-induced genes and operons is a frequently
encountered mechanism to control stress responses. One type of transcriptional
control employs alternative sigma factors. The sigma subunit of RNA polymerase
determines the specificity of promoter binding. Under non-stress conditions the
constitutive sigma factor (σ70
in E. coli and σA
in B. subtilis) directs expression of “housekeeping” genes. Binding of an
alternative sigma subunit to the RNA polymerase core enzyme changes its specificity,
directing it to transcribe a different group of genes and operons. Several
stress-related regulons (coordinately regulated operons) are positively
controlled by the synthesis of an alternative sigma factor. For example, the
presence of active σS
causes transcription of genes involved in the general stress response and
stationary phase in E. coli.strategy to negatively control transcription of
stress-related genes involves anti-sigma factors. Anti-sigma factors bind to a
specific sigma factor forming a complex that prevents the sigma factor from
binding to the RNA polymerase core enzyme (Hughes and Mathee, 1998). In E.
coli, the RssB protein has anti-sigma factor properties; it inhibits the
expression of σS-dependent genes
in the presence of high σS
levels (Becker et al., 2000). A stress sensor may trigger release of the sigma
factor from the anti-sigma factor complex, resulting in transcription of
stressrelated genes. A sigma factor may be released from the anti-sigma factor
by an antianti-sigma factor that binds to the anti-sigma factor. For example, σB,
required for general stress response in B. subtilis, is bound by an anti-sigma
factor. An anti-antisigma factor is present in a phosphorylated form in the
absence of stress. Stress increases the level of non-phosphorylated
anti-anti-sigma factor, which is then able to bind to the anti-sigma factor,
releasing σB (Hecker and
Volker, 1998).transcriptional control mechanisms utilize repressor proteins
that bind to the promoter region of a specific gene or operon, preventing
transcription until conditions are appropriate, at which time the repressor
protein is released from the DNA allowing transcription to proceed. The heat
stress operons, dnaK and groE, are controlled in this manner in B. subtilis.
They are under the negative regulation by the HrcA repressor protein binding to
the CIRCE (controlling inverted repeat of chaperone expression) operator
(Narberhaus, 1999).of stress-related proteins can also be controlled at the
translational level. Messenger RNA secondary structure near the ribosome
binding site or translation start site can inhibit ribosome binding and
translation of mRNA until stress conditions are experienced (Takayama and
Kjelleberg, 2000). Translation of mRNA for the heat shock sigma factor (σ32)
is regulated in this manner. Heat disrupts the hydrogen bonds holding the mRNA
secondary structure together allowing the translation of the transcript under
hot conditions (Yura and Nakahigashi, 1999).in mRNA and protein stability
provide another method of controlling the activity of stress-related proteins.
The half-life of some molecules can be increased or decreased in response to
stress. For example, the CspA mRNA involved in cold tolerance is extremely
unstable at 37°C and dramatically stabilized at lower temperatures (Phadtare et
al., 1999). Proteolytic degradation of stress-related proteins is also observed
as a control mechanism. The ClpXP protease degrades σS
under non-stress conditions (Hengge-Aronis, 1999).
5. GENERAL
STRESS RESPONSE
A general stress response system can
be activated by several different stresses and protects against multiple
stresses. Activation of the general stress response usually results in reduced
growth rate or entry into stationary phase (Hengge-Aronis, 1999).best-characterized
general stress response systems are controlled by alternative sigma factors, σS,
in E. coli and other Gram-negative bacteria and σB
in B. subtilis and other Gram-positive bacteria.general stress response induces
multiple physiological changes in the cell including “multiple stress
resistance, the accumulation of storage compounds, changes in cell envelope
composition and altered overall morphology” (Hengge-Aronis, 1999).induced by σS
and σB
include those for catalase, DNA repair, and osmoprotectant importation,
suggesting that the cell is preparing for oxidative and osmotic stress (Hecker
and Volker, 1998; Petersohn et al., 2001).adaptive response in E. coli is
coordinated by σS.
Very little if any σS
is detectable in non-stressed E. coli cells. When cells are exposed to stress, σS
is
induced,
activating the σs-controlled
promoters. Expression of these genes is necessary for survival under stress
conditions. σS is regulated by
transcriptional and translational control as well as by proteolysis (by ClpXP
protease) in E. coli
(Hengge-Aronis,
1999). Different stresses differentially affect these various levelscontrol. In
B. subtilis, the activity of σB
is modulated by an anti-sigma factor and an anti-anti-sigma factor as described
in the previous section [4].
6. SPECIFIC
STRESS RESPONSES
6.1 Heat
Industrially important bacteria
commonly encounter heat stress during preservation and processing. Heat causes
damage to macromolecular cell components; thus the main function of
heat-induced stress proteins is to repair or destroy these damaged components
so they do not disrupt cellular metabolism. Many heat-induced stress proteins
are protein chaperones that assist in folding and assembly of heat-damaged
proteins (e.g., GroEL and DnaK) or are ATP-dependent proteases that degrade
damaged proteins [5].addition to these changes, some bacteria also alter their
cell membrane in response to heat by increasing the ratio of trans to cis fatty
acids in the membrane. This structural change is thought to decrease fluidity
caused by increasing temperatures (Cronan, 2002).E. coli, the major
heat-induced genes are controlled by the alternative sigma factor, σ32.
Approximately 50 genes are induced by σ32
when denatured proteins are detected in the cytoplasm (Yura and Nakahigashi,
1999). σ32
is present at low levels under non-heat-stress conditions. This low level is
governed by the short mRNA half-life and the low translation rate resulting
from secondary structure at the 5′ end of the mRNA. After a temperature
increase, the secondary structure is destabilized allowing translation to
proceed. The half-life of σ32
also increases dramatically upon exposure to heat
(Arsène et al., 2000; Yura and Nakahigashi, 1999).
Two other alternative sigma factors,
σE
and σ54,
control other regulons induced by heat. σE,
an extracytoplasmic function (ECF) sigma factor, responds to the appearance of
non-native proteins within the periplasm by means of an inner membrane-bound
anti-sigma factor (Raivio and Silhavy, 2001). Release of σ
E from the anti-sigma factor activates transcription of about 10 genes involved
in proper assembly of outer membrane proteins (Raivio and Silhavy, 2001). How
non-native proteins are sensed resulting in release of σE
is not understood. σ54
controls one operon and is activated by disturbances in the cytoplasmic membrane
by an unknown mechanism (Kuczynska-Wisnik et al., 2001).positive bacteria
differ markedly in their regulation of heat shock response. In B. subtilis,
several classes of heat shock genes have been identified. Class I consists of
the chaperone-encoding dnaK and groE operons. These operons have σA-dependent
promoters that are under the negative regulation of the HrcA repressor protein
binding to the CIRCE operator. This regulatory system is widespread and
conserved within the bacterial kingdom and has been described in more than 40
different species (Hecker et al., 1996). The σB
regulon constitutes the Class II genes, the largest group of heat-induced genes
in B. subtilis. These genes are not only induced by heat, but also by other
stresses, as discussed above (Hecker and Volker 1998). Class III heat-induced
genes are negatively controlled at the transcriptional level by a repressor
protein, CtsR. CtsR binds to a specific sequence in the promoter region
upstream of clp genes, clpP, clpE and clpC. These three genes are components of
the Clp protease system which degrades damaged proteins (Derre et al., 1999).
It is not clear how CtsR activity is changed after an increase in temperature.
Other heat-induced genes, not controlled by the above mechanisms, are yet to be
classified.
6.2
Cold
secondary
structures resulting in reduced efficiency of translation, transcription and
DNA replication. These deleterious effects are overcome by induction of
cold-shock proteins that serve as nucleic acid chaperones [7]. CspA, the major
cold-shock protein of E. coli, is proposed to regulate gene expression by
functioning as an RNA chaperone at low temperatures. CspA-like proteins contain
two conserved RNA binding sequences. CspA is regulated at the transcriptional
and translational levels and by increased mRNA stability at low temperatures
(Phadtare et al., 1999).
In E. coli, Csps have been grouped
into two classes. Class I proteins consist of RNA/DNA chaperones (including
CspA), ribosome-associated proteins, a ribonuclease, and a protein involved in
termination of transcription. Class I genes are barely expressed at 37°C, but
dramatically increase after a shift to lower temperatures. Class II genes are
involved in DNA stability and structure and include the DNA binding protein,
H-NS, and a subunit of DNA gyrase. Class II proteins are present at 37°C; after
shift to colder temperatures, their transcription is only slightly higher
(<10-fold) (Phadtare et al., 1999).or synthesis of compatible solutes (see
osmotic stress section) was
reported
to confer cold shock tolerance. In E. coli, the σS-dependent
synthesis of trehalose by the otsAB gene products is cold-inducible. An
additional level of regulation is provided by the instability of otsAB mRNA at
higher temperatures (Kandror et al., 2002). Listeria monocytogenes transports
the compatible solutes,(Ko et al., 1994) and carnitine (Angelidis et al.,
2002), in response to cold temperatures. Regulation of this system has not been
reported.
6.3 Acid
Industrial important bacteria
encounter organic and inorganic acids in foods or in the gastrointestinal tract
and cells of the host. Bacteria respond to acid stress in many ways including
changes in membrane composition, increase in proton efflux, increase in amino
acid catabolism, and induction of DNA repair enzymes.in most bacteria, the acid
tolerance response (ATR) is a phenomenon whereby exposure to moderately low pH
induces the synthesis of proteins that promote survival at extremely low pHs.
ATR differs in exponential and stationary phase cells. This response also
differs dramatically among different bacterial species. signal for induction of
acid shock or adaptation proteins may be intracellular or extracellular pH.
External or periplasmic pH may be sensed by membrane bound proteins (Foster,
1999). Internal pH may affect gene expression directly or may alter a cellular
component involved in gene expression.phase ATR in Salmonella typhimurium involves
several regulatory proteins that each control a subset of acid-induced proteins
[8]. These regulatory proteins include σS,
the two-component signaling system PhoPQ, and the iron regulator, Fur (Foster,
1999, 2000). The σS-dependent
ATR genes that have been identified consist of several proteins of unknown
function and a superoxide dismutase. Most of the PhoPQ-controlled genes are of
unknown function, though Adams et al. (2001) reported decreased flagellin
expression and cell motility upon activation of the PhoPQ pathway by acid. The
authors suggest that “flagellar repression at low pH conserves ATP for survival
processes and helps to limit the influx of protons into the cytosol.” The
Fur-controlled acid-induced genes in Salmonella have not been identified
(Foster, 2000), but Fur modulates urease expression in enterohemorrhagic E.
coli, and thus, may be involved in acid tolerance of this organism (Heimer et
al. 2002). Urease hydrolyzes urea into ammonia and carbon dioxide. The
resulting ammonium ions may accumulate and modify internal and/or external
pH.phase ATR in Salmonella involves stationary phase induction of σS
resulting in a general stress tolerance and induction of acid stress proteins
by OmpA (Foster, 2000). A deletion in the gene encoding σB
in L. mono Cyclopropane fatty acid (CFA) synthase catalyzes the synthesis of
CFAs from unsaturated fatty acids in the bacterial membrane. In E. coli, CFA
synthase gene
expression
increases with a decrease in pH to 5. Transcriptional activation is σSdependent.
The increase in cfa gene expression
results in increased survival to the lethal challenge of pH 3 (Chang and
Cronan, 1999). The investigators suggest that the resulting changes may affect
proton permeability through the membrane or the activity of a membrane-bound
protein involved in acid stress.information is available about the association
of extracellular cell-tocell signaling and stress adaptation. Acid adapted E.
coli is believed to secrete an
extracellular
protein that causes unadapted cells to become acid tolerant without acid
adaptation (Rowbury and Goodson, 1999; Chapter 8 of this book).
Gram-positive bacteria, which
regulate internal pH with an F0F1 ATPase, can increase synthesis or activity of
the ATPase upon pH decrease, providing the cell with a higher capacity for
proton efflux (Foster, 2000). The F0F1ATPase is acidinducible at the
transcriptional level in Lactobacillus acidophilus (Kullen and Klaenhammer,
1999), whereas in Streptococcus spp. or Enterococcus spp., enzyme activity is
controlled at the subunit assembly stage (Foster, 2000).cytoplasmic pH can
cause DNA damage. An acid-inducible DNA repair enzyme was identified in
Streptococcus mutans (Hahn et al., 1999). The importance of DNA repair in acid
stressed cells is supported by data revealing that mutations in the ada gene,
involved in DNA repair, cause acid sensitivity in Salmonella (Foster,
2000).acid catabolism can also help cells to fight a proton influx. Some
Grampositive bacteria use the arginine deiminase system to alkalinize the cytoplasm
(Foster, 1999). Arginine is broken down into ornithine, carbon dioxide and
ammonia. The glutamate decarboxylase/GadC antiporter system (E. coli, Shigella,
Lactococcus, [Foster, 2000], and Listeria [Gahan and Hill, 1999]) requires
extracelluar glutamate which is imported via the GadC antiporter and
decarboxylated within the cell, a reaction that consumes a proton. The
resulting gamma amino butyric acid is exported via GadC. This system is induced
by stationary phase or by acid in the exponential phase. A similar system
involving arginine decarboxylase also protects E. coli from pH (Foster, 2000).
6.4 Osmotic Stress
Bacteria may encounter osmotic
stresses in foods that are high in salt or sugar or in a dried state. Under
such conditions, it is essential for the cell to maintain turgor pressure and
hydration. The mechanisms described refer to bacteria that reside in environments
with moderate or occasional hyperosmotic conditions.best-characterized
mechanism by which bacterial cells respond to hyperosmotic conditions involves
intracellular accumulation of compatible solutes. This accumulation can be
accomplished by synthesis or import from the environment [9].solutes are polar,
highly soluble compounds that counteract osmotic pressure without affecting
normal cellular functions, even at very high concentrations. Glycine betaine,
proline, ectoine, carnitine, choline, and trehalose, among others, are common
compatible solutes. Accumulation of these compounds is regulated at the gene
transcription level or by modifying enzyme activity directly (Bremer and Krämer,
2000). σS
(E. coli) and σB (B. subtilis)
control synthesis of some proteins required for osmoprotectant synthesis or
transport. changes in cell metabolism in response to osmotic stress involve the
cell membrane. An increase in the ratio of trans to cis unsaturated fatty acids
is
observed
in cells exposed to high salt concentrations (Cronan, 2002). In
addition,proportion of anionic phospholipid and/or glycolipids is increased in
saltstressed, compared with unstressed, cells (Russell et al., 1995). In
addition to σS, the σ32
and σE
regulons are activated when E. coli experiences hyperosmotic conditions.
Both regulons encode protein
chaperones and proteases that assure proper
assembly
of proteins in the stressed cell (Bianchi and Baneyx, 1999). Hyperosmotic
stress not only activates the σB
regulon in B. subtilis, but also induces the extracytoplasmic function (ECF)
sigma factor σW (Petersohn et
al., 2001). This sigma factor controls expression of >30 genes, many
encoding membrane proteins of unknown function (Huang et al., 1999).
6.5 Oxidative Stress
Bacteria may be exposed to increased
levels of reactive oxygen species
such
as hydrogen peroxide, hydroxyl radicals and superoxide. Such oxidants cause
damage to cellular proteins, lipids and nucleic acids. Many of the known
proteinsby oxidative stress have antioxidant roles. Others are involved in
repair ofdamage, particularly damage to nucleic acids.
In E. coli, most oxidative
stress-induced genes are part of the oxyR and soxRS regulons induced by
hydrogen peroxide and superoxide, respectively (Storz and Zheng, 2000). OxyR
senses oxidative damage via cysteine residues that are oxidized to form a
disulphide bridge, altering the protein structure into the active form
(Mongkolsuk and Helmann, 2002). There is significant overlap between the
oxidative stress-induced proteins and those induced by σS,
suggesting that oxidative damage is significant in stationary phase or stressed
cells.
. MONITORING
STRESS RESPONSE
Microorganisms in food or
environment are often exposed to stresses and some of these evoke measurable
responses (Figure 2). The response varies mainly with the type and magnitude of
stress and the microorganism’s physiological state. Under some stress
conditions, microbial response is a protective effect, i.e., an adaptive
response. Food microbiologists and processors are interested in the stress
adaptive response since it alters the microorganism’s resistance to processing
and preservation factors. Higher levels of stress may injure the cells. Injured
cells probably become energy-exhausted by multiple responses which decrease
their capacity to react to additional insults. Additional stress usually kills
injured cells Injury is evident by the sensitization of treated cells to
selective agents, antibiotics and other deleterious factors, or the impairment of
cells’ ability to multiply.and measuring stress response have many beneficial
applications. Food processors may learn about the consequences of mild
treatments and the causes of resistance of pathogens to processes that are
presumed lethal to these microorganisms.the contrary, stresses that sensitize
pathogens to processing may have
beneficial
applications in food preservation. Using stress response to sense undesirable
agents (stressors) in the food processing environment is another area of
potential interest to food processors.
To determine the conditions likely
to lead to adaptive responses, researchers
may
vary stress level and apply stress at various physiological states of the
targeted. Based on experience and a large amount of published literature,adaptive
response is most apparent at sublethal levels of stress and whenmicroorganism
is in an active metabolic state, i.e., the exponential phase of. Many
researchers, however, have demonstrated appreciable stationary-phase inducible
adaptive responses (e.g., Buchanan and Edelson, 1999). Similarly, lethal doses
of stress may trigger considerable adaptive responses in the fraction of the
population that survives the treatment. After applying the stress under
investigation, procedures to detect or quantify the response should be
followed. Stress responses measured include changes in gene expression products
(RNA and proteins) and stress tolerance.
Although detection of stress
adaptive response is generally laborious, distinction of injury is relatively
simple. Stress-sensitized cells (i.e., injured) demonstrate reduced growth rate
(e.g., reduced colony size on agar media), impaired growth in the presence of
selective agents such as NaCl and bile salts, increased sensitivity to
antibiotics, and loss of aerotolerance. Details about adaptive responses are
included in this contribution, but sensitization by stress will not be
addressed.
8. INDUCTION
OF STRESS ADAPTIVE RESPONSE: PRACTICAL CONSIDERATIONS
The following are examples of the
most commonly investigated stresses, heat and acid. Included is a brief
description of methods of applying theses stresses for inducing adaptive
responses. Once the stress response is developed, cells should be handled in a
way to preserve the response. Active metabolism and multiplication of
stress-adapted cells deteriorate the adaptation and thus it becomes difficult
to detect.
8.1 Heat
Heat induces a universal protective
response that is relatively easy to detect. Temperatures conducive to growth
normally do not constitute stress to cells and thus are not used commonly in
developing a stress response. Severe thermal stress may eliminate sizable
proportion of the cell population and the adaptive response in the small
fraction of the population that survives the treatment may not be measurable.
Response to a mild heat shock is readily detectable when cells are treated at
sublethal or minimally lethal temperatures. According to our experience, heat
shock response is demonstrated best when L. monocytogenes exponential-phase
culture is heated at 45°C for 1 h (Lou and Yousef, 1997). By comparison, injury
of L. monocytogenes is most apparent at 55 to 60°C (El-Shenawy et al., 1989)
and neither stress response nor injury can be reliably detected at 70°C. Heat
shocking E. coli O157:H7 at 45 to 46°C for 15 to 30 min produces appreciable
thermal adaptation (Juneja et al., 1998; Lucore et al., 2002). Heat may be
applied rapidly, i.e., as a heat shock (Lou and Yousef, 1997) or gradually
(Stephens et al., 1994), since both procedures produce significant adaptive
response.
8.2 Acid
Acid Shock during Exponential Phasegrowing
microbial cells, in their mid-exponential phase, are treated with sublethal
levels of an acid, i.e., cells are acid shocked. Incubation is continued to
allow one to two doublings under the acid stress. During this additional
incubation period, cells normally develop an acid adaptive response. Since the
adaptive response is a transient phenomenon, further processing of these cells
(e.g., centrifugation and washing) should be done promptly and under
refrigeration conditions in order to preserve the developed response. This
technique produces a strikingly different response from that observed in the
non-treated culture and thus the adaptation is relatively easy to track.
Response of these cells, however, is transient and the adaptation may degrade
quickly before it can be measured, particularly if treated cells are
mishandled. Additionally, collecting cells from mid-exponential phase can be
tricky since cell density at this stage is normally low. Phase of growth should
be determined in advance by plating the culture after different incubation
periods and constructing a growth curve. Correlation of microbial counts with
culture turbidity (measured spectrophotometrically) allows estimation of growth
phase prior to the experiment. Researchers who successfully applied acid stress
to mid-exponential phase cultures include Foster and Hall (1990), Leyer and
Johnson (1992), and Lou and Yousef (1997).Acid Stressthat produce acid as a
byproduct of carbohydrate metabolism experience a gradual decrease in pH during
culturing. This gradual acidification induces a stationary-phase acid
resistance response (Buchanan and Edelson, 1999). Gradual acid exposure is a
simple and practical method of producing acid-adapted cells.of the adaptation,
however, occurs during the stationary phase when cells generally develop
resistance to various deleterious factors (Watson, 1990). Consequently, the
intrinsic stationary phase acid resistance may overshadow induction of acid
resistance by carbohydrate fermentation. The non-acid adapted cells (control
culture) are grown in the absence of a fermentable carbohydrate and thus
produce energy through alternative metabolic ways. Unfortunately, these control
cells may inadvertently be sensitized to acid or develop a starvation response
during growth in the carbohydrate-free medium. Gradual application of acid
stress may also be accomplished by manual incremental addition of acid to a
growing culture. Alternatively, a chemostat may be used to gradually apply acid
stress to a growing culture in a controlled manner. This latter procedure is
most useful when the test microorganism does not produce acid during growth.
9. DETECTING
AND QUANTIFYING STRESS RESPONSE
to detect and measure stress
response vary depending on the response measured. Evidence of stress response
includes presence of genes involved in stress response mechanisms, elevated
level of gene products such as
mRNA,
de novo protein synthesis in response to stress, and increased tolerance to
lethal levels of the stress.
9.1 Detection of Stress Response
Genes
Presence of genes encoding stress
response proteins may indicate that the microorganism is capable of responding
to a stress in a predictable fashion. Comparing the genomes of resistant and
sensitive strains may reveal these genes involved in stress response (Koonin et
al., 2000). Researchers have developed probes for detecting genes that
contribute to stress response; these are useful tools to determine potential
response to stress by an isolate.
9.2 mRNA Analysis
While presence of the gene is a
prerequisite for a response, expression of this gene is needed for the ultimate
manifestation of the response. Therefore, interest in detecting stress response
at the transcriptional level is increasing. Synthesis of proteins that protect
cells against stress is sometimes preceded by increased transcription of the
relevant mRNA. Measuring these mRNAs demonstrates, or even quantifies, the
stress response. Methods to measure mRNA include Northern analysis,
microarray-genome-wide expression monitoring (also known as microarray
analysis) and reverse transcription polymerase chain reaction (RT-PCR).
9.3 Detection of Stress Proteins
Synthesis of stress proteins
provides yet more direct evidence of the microorganism’s response to stress.
Proteins synthesized in response to stress include regulatory proteins (e.g., σ32
in E. coli and σB
in L. monocytogenes), chaperones (e.g., GroEL), ATP-dependent proteases (e.g.,
Lon), and DNA repair proteins (e.g., UspA) (Duncan et al., 2000; Diez et al.,
2000; Rosen et al., 2002). Many of these proteins have been successfully
detected using a two-dimensional electrophoresis (e.g., Rince et al., 2002).
Antibodies specific to some of the well-characterized stress proteins are
commercially available to detect a stress response by immunodetection methods
such as Western blotting (Duncan et al., 2000). If the corresponding antibodies
are not commercially available, the gene of a specific stress protein can be
cloned. The recombinant protein is then amplified, purified and used to
generate the corresponding specific antibodies (Jayaraman and Burne, 1995).
9.4 Biosensors
Microorganisms have been genetically
engineered for easy detection of stress response (LaRossa and Van Dyk, 2000).
Reporter genes (e.g., lacZ which encodes for β-galactosidase)
were fused to promoters of genes involved in adaptive response.useful reporter
genes include luxAB, which encodes bacterial luciferase, luc, encoding insect
luciferase, and gfp, for green fluorescence protein. When these fusion strains
respond to stress, the reporter gene is expressed and fluorescent or
luminescent products are produced. Gene fusion strains (biosensors) for
detecting DNA damage, heat shock, oxidative stress, and starvation have been
developed for basic research and are potentially useful in the field of food
microbiology.
9.5 Measuring Increased Tolerance
Adaptive responses may be measured
by comparing stress tolerance of cells that have been pre-exposed to sublethal
stress to those that have not. Measurement of inactivation by stress uses
simple plating techniques. A greater degree of survivability of the cells
exposed to sublethal stress may indicate that the stress induced an adaptive
response. Quantifying the stress by the cultural technique may require
measuring changes in death rates as a result of pre-exposure to stress.
Determining D-value (time required to decrease the population under stress by
one log CFU unit) is a useful quantitative measure of resistance. Culture
techniques provide direct evidence of stress adaptive response and the results
of the analysis have great practical value to food processors. These
techniques, however, are time-consuming and the results may be compromised by
experimental artifacts.
10. PERSPECTIVES
AND AREAS FOR FUTURE WORK
researchers question the relevance
of stress adaptation to food safety. This argument is based on these
observations:
• Stress adaptation is best
demonstrated at the exponential, rather than at the stationary, phase of
growth. Since pathogens in food are rarely in the exponential phase,
significant adaptation to stress under most processing and production practices
may be unlikely.
• Direct determination of the degree
of adaptation of microbiota in food is not currently feasible. Therefore, there
is no knowledge on how much of processing resistance that these microorganisms
experience is attributed to stress adaptive response.
• Although the number of reports
linking stress adaptation and virulence is rising, there is no evidence that
directly links stress adaptation of pathogens to foodborne disease
outbreaks.these arguments have some merits, we believe that the stress
adaptation phenomenon has a profound effect on the safety of food:
• Although stress adaptation is
remarkable in actively metabolizing cultures,at all phases of growth do adapt
to stress. Induction of stress adaptive response in stationary-phase cultures
is well documented. Nevertheless, demonstration and quantification of these
adaptive responses, under real processing conditions, need to be carefully
investigated.
• Lack of direct evidence is not a
proof of the absence of the relationship
between
stress adaptation and food safety. With the continuous improvements in
analytical tools and protocols, researchers may soon be able to verify these
associations. Rapid methods to differentiate between transient and inherent
resistance, and to quantify these traits in the food microbiota, are urgently
needed. Availability of these methods will not only reveal the risks associated
with stress adaptation, but processors may also use these techniques to gauge
processing severity with the anticipated tolerance of the microbiota in food.
Many researchers agree that there is
a considerable potential risk of disease as a result of stress adaptation,
particularly in food produced by minimal-processing or novel, alternative
processing technologies (Abee and Wouters, 1999; Archer,1996; Rowan, 1999;
Yousef, 2000). Interest in these technologies has increased appreciably in the
past decade. These technologies promise to maintain the critical balance
between safety and marketability of a new generation of foods. It is of concern
that processing conditions may be conducive to stress adaptive response in
foodborne pathogens. Currently, stress adaptive responses of microorganisms in
food processed by these technologies are poorly understood. As these novel food
processing technologies become commercialized or used more widely, it is
essential that researchers understand the adaptive responses that are induced
by these treatments.
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