STRESS IN BACTERIA

by Lena VOROBYOVA, Dr. Sc. (Biology), M.V. Lomonosov Moscow State University

Since long ago scientists have been showing much interest in stress, a phenomenon not studied well enough so far. After years and years of painstaking work they have found it to be proper to beings other than man, too. Even bacteria, the oldest inhabitants of the earth, are stress-affected. This very fact- along with the knowledge of survival opportunities under stress-is a major achievement toward fundamental problem solving in biology. This was discussed, among other things, at the International symposium "Food Microbiology and Hygiene" at Lillehammer, Norway in 2002.

Today it has been proved both in theory and in practice: that all forms of life are subjected to stress should their usual living conditions undergo a sudden change. The response is much alike both in bacteria (prokaryotes having no discrete cell nucleus) and in higher organisms (eukaryotes with a discrete cell nucleus).

For instance, an abrupt temperature rise draws a response from bacteria, fungi, plants, animals and man, and causes them to synthesize what we call heat-shock proteins (HSP). Some of the sites of these compounds preserve as much as 90 percent homology (correspondence of amino acid sequences), which shows that these proteins are evolutionally conserved. Consequently, the relatedness of the HSP structure and of molecular mechanisms responsible for the stress-induced reaction in organisms enables us to use bacteria as a simple model in studying responses to stress.

True, bacteria are all too small to control environmental conditions. Should these prove to be not favorable for proliferation, prokaryotic cells may lapse into a quiescent, dormant state and thus turn into a conserved form. Under lingering stress some of them have the only survival chance by turning into a spore, or a dehydrated cell clad in so many different coats. In this state bacteria can keep viable for thousands of years in most life-hostile conditions. Spore formation takes about 6 to 8 hours, a time when cell death is inevitable unless other stress-generated responses more common to living beings come to be implicated. In protozoa such responses are under very effective gene control.

Stress factors (stressors) are usually of chemical, physical or biological nature. A particular microorganism itself may be their source too. For example, under normal metabolism many bacteria produce FhCh (hydrogen peroxide) and active forms of oxygen touching off an oxidative stress responsible for denaturation of proteins (change of natural characteristics), breaks in nucleic acid chains, oxidation of macromolecules and other lesions.

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This phenomenon has been studied particularly well if a microorganism is acted upon by heat shock. The organism responds in a variety of ways: it switches on intracellular reparation mechanisms, and increases the number of HSP - the main function of these proteins is to see to the correct packing of newly synthesized polypeptides and in repairing the damages in polypeptide chains which then will be able to perform their regular function again. The partially denatured proteins have their hydrophobic (water-hostile) sites denuded and thus come into contact with other organic compounds, shaperons ^* in particular, that play a decisive part in the formation of the native quaternary (final) structure of protein molecules, the essential "building bricks" of animals and plants. Otherwise the polypeptide chains synthesized in rhibosomes would be unable to perform.

As a matter of fact, the stress-induced switchover of a cell is an extraordinary event. This can well be seen in the example of the lactic-acid bacterium Lactococcus lactis.

A temperature rise to 42 ^o C causes it to generate 12 to 17 proteins additionally; the amount of shaper - on GroEL soars 45 times over in the initial ten seconds of heating. And here's another example, relative to cold shock-when a suspension of cells that grow in norm at 20 - 30 ^o C finds itself placed in low- temperature conditions (+5 ^o C) for just one hour. In this case Bac. subtills will synthesize an extra 3, and

^* Shaperons - proteins that help newly formed polypeptides toward correct conformation, or shape. - Auth.

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E.coli - an extra 9 cold-shock proteins.

However, many complex organic compounds needed for sustaining normal life during the active synthesis of heat- or cold-shock proteins are produced in much smaller amounts and, as a result, corresponding metabolic reactions proceed at a low rate. Yet a timely switching of the stress-oriented program enables the organism to live on.

Different families of HSP are involved in the creation or recreation of protein conformations; some of these heat-shock proteins likewise belong in the shaperon group. They are small organic compounds with a molecular mass of 34 kDa or even less. One member of this family is the enzyme peptidyl-prolyl-cis-trans-isomerase (PP-isomerase). This large group of proteins is present in eukaryotes and bacteria, and has an important role to play. The point is that the packing of polypeptide chains, among other things, requires a rotation of peptydyl-prolyl bonds, a very slow process if it takes place spontaneously; PP-isomerase, however, speeds it up.

There is yet another essential condition for a correct packing of protein. Some anomalous, complex organic compounds produced in stress may aggregate into larger particles and, precipitating, become toxic for the cell and cause grave diseases. That is why all bacteria, evolutionally, possess protein-peptide degrading characteristics. To avoid such trouble, anomalous proteins should be either turned into native proteins or else eliminated altogether. Molecular shaperons, as said above, bring protein compounds to a normal functional state; and proteolytic proteins (otherwise known as shearonins) are responsible for the elimination of anomalous proteins.

Speaking of any stress responses in a cell, we must say we have but the most general notions about the mechanism of this process. Many regulatory systems by means of which cells react to external signals comprise two components with two different proteins-the sensor and the response (regulatory) protein. The former is localized in the cell membrane so that one part of it contacts the external medium, and the other part is in contact with the cell cytoplasm. The specific sensor protein possesses kinase activity (kinases are enzymes implicated in the phosphorylation of compounds). Detecting an external signal, the sensor protein catalyzes the phosphorylation of the specific sensor residue of histidine, a heterocyclic amino acid (by the autophosphorylation principle) within the cytoplasmic region. Next, the phosphate group is transferred to the other, regulatory (response) protein within the cell. This is a typical protein binding to DNA and regulating transcription. For example, the bacterium E.coli has at least as many as 50 different two-component systems at work.

Transcription initiation is the most important stage of gene regulation in bacteria. For this RNA polymerase should bind to the protein by the -factor to regulate gene expression in response to changes in the ambient medium. For instance, in L.lactis cells the induction of genes GroEL and Dnak occurs via formation of the gene product ³² responsible for HSP transcription.

DNA superhelices (supercoils), i.e. macromolecular structures characterized by a changed number of bonds between the two DNA strands, may also act as a sensor with respect to environmental conditions. Correspondingly, the number of helical coils is changed as well.

There is also a non-protein mechanism defending cells against the heat shock-for one, accumulation of trehalose ^* sugar.

In order to show the different nature of bacterial stress responses to stressors, we should also consider the cold shock. Unlike the heat shock, it causes no denaturation of proteins but changes molecules and biochemical reactions. The cold stress stabilizes the secondary structure of nucleic acids and thus inhibits (slows down) DNA replication ^** , gene transcription and translation. This suppresses the activity of many enzymes and general metabolism, and impedes the cell transport. Besides, the ice crystals formed in the cell damage many structures and lead to the death of the organism involved. Simultaneously, more of the cold-shock proteins are synthesized in bacteria, but their mechanism is obscure yet. What we know is that psychrophiles (bacteria living in a low -5 to +20C temperature range) have membranes whose fluidity ^*** is enhanced due to the presence of fatty acids; their enzymes are remarkable for the structural and functional plasticity (adaptiveness), and they synthesize anti-freeze proteins against the ice crystals. It might be that organisms with such characteristics can live on other planets colder than the earth.

Considering stress responses of cells, we can hardly bypass yet another important question related to the aftereffects of the oxidative shock which is caused by radiation, traumatic lesions, including insult and infarction. The state induced by such shock is thought to be the cause of ageing and apoptosis, or the induced suicide of cells. A process like that is responsible for change in the concentration of oxidants within the cell and may entail deformations of nucleic acids, breaks of protein molecules as well as their linkers, blocks of enzyme reaction centers and the overall inanition of the organism.

^* Trehalose - a disaccharide formed by two glucose residues; trehalose is found in yeast and in other fungi as well as in higher plants. - Ed.

^** Replication-process of duplication of each of the two strands of DNA, a new complementary strand being formed on each parent strand. Thereby genetic information is passed from generation to generation. - Ed.

^*** Fluidity of the cell membrane depends on the melting temperature of its components, the fatty acids, and on their length and saturation. Here fluidity is understood as a liquid-crystal state of the membrane in which it functions in an optimal mode. - Ed.

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Bacteria are always the target of native active forms of oxygen (AFO). It is they, not oxygen as such, that may be the cause of the above lesions. AFO are in fact free radicals containing one or several uncoupled electrons (usually indicated by a dot). These include the superoxide radical (O ₂ ^- ), the singlet oxygen ('O ₂ ), the hydroxyl radicals ( OH), the monoxide radical (NO) as well the hydrogen peroxide (H ₂ O ₂ ), which is a nonradical compound. All of them are very aggressive, out to gain a lacking electron or give away an extra one. In either case the target molecule is modified.

Higher eukaryotic organisms warn their kindred species about the looming threat, each making use of special alarm signals. Exposed to stress, bacteria have been found to resort to special communication messages and mutual assistance. Thus, taking a definite brand of cheese, biologists have isolated microorganisms forming bright- orange colonies of Luteococcus casei cells (their generic name describing the color, and their specific name, the chief protein of cheese, casein). The sound cells, not subjected to any stress effects, produce a protein endowed with amazing characteristics. Such cells, if subjected to heat shock or UV radiation and then incubated for several minutes in a solution of extracellular protein taken at a concentration of ~ l mg/ml, have survival chances hundreds of times better than the damaged cells in the control group. The worse the damage inflicted on a population (that is the lower viability of bacteria), the more effective the antistress protein is. It is surpris-

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Bacteria responding to an unfavorable change in environmental conditions.

ing indeed that this protein helps reactivate even eukaryotes, the heat-shock inactivated yeast for one. Now the antistress protein with a molecular mass of ca. 10 kDa is undergoing purification at a Moscow University laboratory, whereupon it will be sequenced (that is, its amino-acid sequences will be determined) and identified together with other protein compounds or their fragments.

Not so long ago, our microbiolo-gists discovered something else as well. If the density of cells in one particular culture attains a definite level of density, they will be able to pass stress signals to the entire population. This has much in common with regular feelings. Communications are realized by means of small peptides, proteins, amino acids, homoserine ^* lactones (esters) and other molecules functioning at lower concentrations and resistant to external effects.

Yuri Nikolayev, Cand. Sc. (Biol.), of the RAS Institute of Microbiology, has shown that tetracycline-treated E.coli cells produce a compound protecting tetracycline-sensitive cells against this antibiotic as well as against the osmotic ^** and the heat shock.

There is yet another intriguing example of chemical communication. The following phenomenon is now under study at the Russian Medical Academy by Professor Maria Pshennikova and coworkers. In response to stress effects in man, glucocorticoids and catecholamines (hormones produced by the adrenal cortex for controlling the metabolism of carbohydrates, proteins and fats) are ejected into blood. These hormones, interacting with receptors localized in cell membranes, activate enzymes and give rise to secondary messengers (mediators) sending a signal into the cell and triggering processes instrumental in an increase of calcium ions (Ca ²⁺ ) and stimulating the activity of various organic compounds. The signal aids the transcription and synthesis of adequate proteins. Something essentially similar to stress responses in bacteria, isn't it?

Adaptation to one particular stress enhances resistance to other stresses as well. These findings underlie a new method of prevention and treatment of certain diseases (like allergies, neuroses, hypertension, etc.) by getting a patient used to hypoxia (oxygen deficiency) in an altitude chamber.

Now what concerns the microorganisms: a profound knowledge of these systems is going to be decisive for the further development of biotechnology where bacteria under stress are assigned definite tasks. Stress-induced proteins are clear molecular markers indicating the "sound health" of stock cultures. Such proteins show the effect of stress, which means that the cells of these cultures might not be able to realize fermentation optimally. On the other hand, these proteins may be employed as the positive indicators of a culture fully adjusted to resistance to definite negative effects.

Knowing that bacteria with a diminished stress response are less virulent (pathogenic), we can take a new vision of microbial pathogenesis, that is the origin and outcome of all the various diseases.

Dr. Vorobyova's studies are supported by a grant from the Russian Foundation of Basic Research (No. 02 - 04 - 49930).

Illustrations supplied by the author

^* Serine - an aliphatic amino acid within proteins and some conjugated lipids; it plays an important part in stimulating the catalytic activity of proteolytic enzymes. - Ed.

^** Osmotic shock-excessive pressure of solution preventing a solvent from traveling to a more concentrated solution from a less concentrated one. - Ed.