MONITORING STRESS RESPONSE

Microorganisms in food or environment are often exposed to stresses and some of these evoke measurable
responses (see Figure 1.5). 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 (see Figure 1.1). 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, but this has not been
explored.

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 (see Figure 1.5). 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.
DETECTING AND QUANTIFYING STRESS RESPONSE
Methods to detect and measure stress response vary depending on the response measured (see Figure 1.5).
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.

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.

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).

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).

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. 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.

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.