Mechanisms of microorganism adaptation to stress factors

Induction of stress adaptive response: practical considerations. Detecting and quantifying stress response. Perspectives and areas for future work. Mechanisms of microorganism adaptation to stress factors: heat, cold, acid, osmotic pressure and so on.

18.11.2014

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2014

MINISTRY OF EDUCATION AND SCIENCE OF UKRAINE

NATIONAL AVIATION UNIVERSITY

INSTITUTE OF ECOLOGICAL SAFETY

DEPARTMENT OF BIOTECHNOLOGY

SEMESTER PAPER

(Explanatory Note)

On the discipline: Microbiology of biological agents

Theme: Mechanisms of microorganism adaptation to stress factors

Student: Litvin Irina

Group IES 304

Leader: Vasylchenko O.A.

Kyiv 2014

CONTENT

ABSTRACT

INTRODUCTION

1. DEFINITIONS

2. MECHANISM OF STRESS ADAPTIVE RESPONSE

3. STRESS SENSING

4. REGULATION OF STRESS-RELATED PROTEIN SYNTHESIS

5. GENERAL STRESS RESPONSE

6. SPECIFIC STRESS RESPONSES

6.1 Heat

6.2 Cold

6.3 Acid

6.4 Osmotic Stress

6.5 Oxidative Stress

7. MONITORING STRESS RESPONSE

8. INDUCTION OF STRESS ADAPTIVE RESPONSE: PRACTICAL CONSIDERATIONS

8.1 Heat

8.2 Acid

9. DETECTING AND QUANTIFYING STRESS RESPONSE

9.1 Detection of Stress Response Genes

9.2 mRNA Analysis

9.3 Detection of Stress Proteins

9.4 Biosensors

9.5 Measuring Increased Tolerance

10. 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.

Object of the study - industrially important microorganisms.

Purpose - to show different mechanisms of microorganism adaptation to stress factors such as heat, cold, acid, osmotic pressure and so on.

Research methods - analysis, a systematic approach of observation.

STRESS, 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].

The 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].

Bacteria 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].

In bacteria some of the most important stress response systems are:

Heat shock response, controlled by the sigma factor sigma 32;

Envelope stress response, controlled mainly by the sigma factor sigma E and the Cpx two-component system;

Cold shock response, which governs expression of RNA chaperones and ribosomal factors;

General 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, to breakdown taurine into sulphur in times of need and YodA in toxic metal response.

1. DEFINITIONS

Stress

Stress has different meanings depending on the context of usage. When used in the field of biology, stress refers to the imposition of detrimental nutritional conditions, toxic chemicals and suboptimal physical conditions (Neidhardt and VanBogelen, 2000). Stress, as used in this term papper, refers to any deleterious factor or condition that adversely affects microbial growth or survival.

Stresses encountered by microorganisms vary in magnitude and outcome. We use the word mild to describe sublethal stress levels that do not result in viability loss, but reduce or arrest growth rate. Moderate stress not only arrests microbial growth but also causes some loss in cell viability. Extreme or severe describes a stress level that is normally lethal to the cells, resulting in death of the majority of the population. Stresses that food microbiota encounter include uncontrollable pre-harvest environmental factors (e.g., radiation and dry air) and the deliberate postharvest application of preservation factors. Stresses to the microorganisms during production and processing include:

1. Physical treatments such as heat, pressure, electric pulses, ultrasonic waves, light/radiation, and osmotic shock.

2. Addition of chemicals such as acids, salts, and oxidants

3. Biological stresses, e.g., competition, microbial metabolites and antagonism.

Stress Response

Once microorganisms sense a stress, the cells respond in various ways. Bacteria sense stresses that change membrane fluidity (e.g., cold shock), alter cell protein structure or disrupt ribosomes (e.g., heat), or affect nucleic acids (e.g., radiation). At the molecular level, stress response includes transcription leading to the synthesis of regulatory proteins.

The resulting regulation may lead to the synthesis of other proteins that cope

with the imposed stress. Microbial response to stress may produce these outcomes:

1. Production of proteins that repair damage, maintain the cell, or eliminate the stress agent.

2. Transient increase in resistance or tolerance to deleterious factors.

3. Cell transformation to a dormant state, i.e., spore formation or passage to the viable-but-not-culturable state.

4. Evasion of host organism defenses.

5. Adaptive mutations.

Adaptation

When microorganisms are stressed, an adaptive or protective response may follow. Response to stress, in this case, increases the organism's tolerance to the same or to a different type of stress. This phenomenon is occasionally described as adaptive response, induced tolerance, habituation, acclimatization or stress hardening.

Tolerance

Tolerance to a deleterious factor (e.g., low pH) refers to a microorganism's ability to survive a stress. Each microorganism has an inherent tolerance level to a particular stress, but a transient or adaptive tolerance may also be induced. For example, lactic acid bacteria are inherently more acid tolerant than many other bacteria, yet they can become even more acid tolerant after acid adaptation.

Injury

Damage to cellular components by stresses may impair the ability of microorganisms to multiply or may sensitize the cells to mildly deleterious factors. These changes are commonly described as injury. Injury is most noticeable when stress-exposed cells become sensitive to selective agents that healthy cells readily survive. The relationship between cell injury and stress adaptation has not been well characterized, but injury may result from a cell's inability to respond to stress or a delayed or inadequate adaptive response. Injured cells may recover or die. Leistner (2000) indicated that simultaneous exposure of bacteria to different stress factors requires increased energy consumption and leads bacteria to cellular death through metabolic exhaustion and disturbed homeostasis. Interrelations among physiological states of microbial cell subjected to different stresses are depicted on Figure 1.

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.

Stress 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).

Levels 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 Krmer, 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.

Control 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.

Transcriptional 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 (70 in E. coli and A 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 S causes transcription of genes involved in the general stress response and stationary phase in E. coli.

A 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 S-dependent genes in the presence of high S 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, B, 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 B (Hecker and Volker, 1998).

Other 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).

Synthesis 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 (32) 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).

Changes 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 37C 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 S 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).

The best-characterized general stress response systems are controlled by alternative sigma factors, S, in E. coli and other Gram-negative bacteria and B in B. subtilis and other Gram-positive bacteria.

The 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).

Genes induced by S and B 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).

Stress adaptive response in E. coli is coordinated by S. Very little if any S is detectable in non-stressed E. coli cells. When cells are exposed to stress, S is

induced, activating the s-controlled promoters. Expression of these genes is necessary for survival under stress conditions. S 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 levels

of control. In B. subtilis, the activity of B 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].

In 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).

In E. coli, the major heat-induced genes are controlled by the alternative sigma factor, 32. Approximately 50 genes are induced by 32 when denatured proteins are detected in the cytoplasm (Yura and Nakahigashi, 1999). 32 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 32 also increases dramatically upon exposure to heat (Arsne et al., 2000; Yura and Nakahigashi, 1999).

Two other alternative sigma factors, E and 54, control other regulons induced by heat. E, 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 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 E is not understood. 54 controls one operon and is activated by disturbances in the cytoplasmic membrane by an unknown mechanism (Kuczynska-Wisnik et al., 2001).

Gram-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 A-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 B 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

Physiological changes in response to cold include changes in the membrane fatty acid composition to promote optimum membrane fluidity (Russell et al., 1995), synthesis of DNA- and RNA-binding proteins that counteract the stabilizing effect of cold temperatures on nucleic acid secondary structures (Phadtare et al., 1999), and importation of compatible solutes (Ko et al., 1994; Angelidis et al., 2002).

Proteins synthesized in response to cold can be classified as Csps (cold shock proteins) or Caps (cold-shock acclimation proteins). Csps are rapidly, but transiently overexpressed in response to cold. Caps are synthesized during continuous growth at cold temperatures; they are rapidly induced, but remain overexpressed several hours after the temperature downshift. A slow temperature downshift results in synthesis of some Csps and Caps (Phadtare et al., 1999).

Upon decrease in temperature, the phospholipid bilayer membranes of all cells

decrease in fluidity. To maintain optimum fluidity, cells increase the unsaturation or decrease the chain length of the membrane fatty acids, resulting in increased fluidity at lower temperatures (Russell et al., 1995). After cold shock in B. subtilis and cyanobacteria, synthesis and stability of a fatty acid desaturase increase as controlled by a two-component signaling system [6].

Cold shock also causes stabilization of the hydrogen bonds in nucleic acid

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 37C, 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 37C; after shift to colder temperatures, their transcription is only slightly higher (<10-fold) (Phadtare et al., 1999).

Transport or synthesis of compatible solutes (see osmotic stress section) was

reported to confer cold shock tolerance. In E. coli, the S-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,

betaine (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.

Observed 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.

The 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.

Exponential phase ATR in Salmonella typhimurium involves several regulatory proteins that each control a subset of acid-induced proteins [8]. These regulatory proteins include S, the two-component signaling system PhoPQ, and the iron regulator, Fur (Foster, 1999, 2000). The S-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.

Stationary phase ATR in Salmonella involves stationary phase induction of S resulting in a general stress tolerance and induction of acid stress proteins by OmpA (Foster, 2000). A deletion in the gene encoding B 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 Sdependent.

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.

Limited 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).

Low 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).

Amino 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.

The 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].

Compatible 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 Krmer, 2000). S (E. coli) and B (B. subtilis) control synthesis of some proteins required for osmoprotectant synthesis or transport.

Additional 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,

the proportion of anionic phospholipid and/or glycolipids is increased in saltstressed, compared with unstressed, cells (Russell et al., 1995). In addition to S, the 32 and E 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 B regulon in B. subtilis, but also induces the extracytoplasmic function (ECF) sigma factor W (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 proteins

induced by oxidative stress have antioxidant roles. Others are involved in repair of

oxidative damage, 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 S, suggesting that oxidative damage is significant in stationary phase or stressed cells.

7. 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.

Detecting 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.

On 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

microorganism. Based on experience and a large amount of published literature,

microbial adaptive response is most apparent at sublethal levels of stress and when

the microorganism is in an active metabolic state, i.e., the exponential phase of

growth. 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 45C for 1 h (Lou and Yousef, 1997). By comparison, injury of L. monocytogenes is most apparent at 55 to 60C (El-Shenawy et al., 1989) and neither stress response nor injury can be reliably detected at 70C. Heat shocking E. coli O157:H7 at 45 to 46C 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 Phase

Actively growing 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).

Gradual Acid Stress

Microorganisms that 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.

Most 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

Methods 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., 32 in E. coli and B 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 -galactosidase) were fused to promoters of genes involved in adaptive response.

Other 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

Some 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.

While 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,

microorganisms 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.

REFERENCES

Requena J.M. Stress Response in Microbiology. Caister Academic Press, 2009. - p.34.

Agata Swiecilo, Iwona Zych-Wezyk. Bacterial Stress Response as an Adaptation to Life in a Soil Environment. Pol.J.Environ.Vol.22, 6 (2013). - pp. 1577-1578.

Filloux. Bacterial Regulatory Networks. Caister Academic Press,1997 - p.18.

Rince, A., M. Uguen, Y. Le Breton, J.C. Giard, S. Flahaut, A. Dufour, and Y. Auffray. 2002. The Enterococcus faecalis gene encoding the novel general stress protein Gsp62,Microbiology. - p.148.

Yura, T. and K. Nakahigashi. 1999. Regulation of the heat-shock response, Curr. Opin. Microbiol. - p.2.

Aguilar, P.S., J.E. Cronan, and D. de Mendoza. A Bacillus subtilis gene induced by cold shock encodes a membrane phospholipid desaturase, J. Bacteriol., 1998. - p.180.

Kim, W.S. and N.W. Dunn. Identification of a cold shock gene in lactic acid bacteria and the effect of cold shock on cryotolerance, Curr. Microbiol., 1997. - p.35.

Leyer, G.J. and E.A. Johnson. Acid adaptation promotes survival of Salmonella spp. in cheese. - Appl. Environ. Microbiol., 1992. - p.58.

Timson, W.J. and A.J. Short. Resistance of microorganisms to osmotic pressure. - Biotechnol. Bioeng., 1965. - p.7.




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