GENERAL MICROBIOLOGY - T.P. Pyrog - 2004

6. MICROBIAL GROWTH

6.2. MICROBIAL ADAPTIVE RESPONSES TO STRESSFUL CONDITIONS

6.2.2. Production of Protective Compounds

Under adverse conditions, protective compounds may include CARBOHYDRATES (including exopolysaccharides), carotenoids, Melanins, protective Proteins, low-molecular-weight compounds (such as osmoprotectants), and others. It should be noted that the synthesis of certain protective compounds in microorganisms is constitutive (EPS, pigments), but their synthesis can be upregulated under unfavorable conditions. A number of protective compounds, including protective proteins, are produced in response to stress factors.

Osmoprotectants. Each microorganism has an optimal Water activity ($a_w$) for growth. If the water activity in the presence of dissolved solutes drops below the optimal level for growth, the microorganism is forced to expend a portion of available energy on osmoregulation, powering transport systems, and synthesizing low-molecular-weight osmoprotectants. Consequently, under conditions of reduced water activity, one observes an elongation of the lag phase, a decrease in growth rate, and a lower biomass yield: the Organism experiences osmotic stress. Microorganisms possess quite complex and sophisticated systems for adapting to changes in environmental solute concentrations. Among these, the presence of osmosensors in the cytoplasmic membrane that respond to Changes in membrane tension is particularly noteworthy, as are alterations in The ratio of outer membrane porin proteins (which form hydrophilic channels for the passage of relatively small molecules into the periplasmic space) and the synthesis of osmoprotectants.

Osmoprotectants are low-molecular-weight compounds whose cytoplasmic concentration balances external osmotic pressure. In prokaryotes, such osmoprotectants include Glycine betaine, Proline, glycosylglycerol, glutamate, and dimethylglycine. Osmoprotectants are either synthesized de novo by The Cell or taken up from the surrounding environment, with The activity of the corresponding transport systems also being induced by high environmental osmolarity.

High osmophilicity in lower eukaryotes, particularly filamentous Fungi, is accompanied by the accumulation of trehalose and polyols within the Cells. It has been suggested that the different nature of osmoprotectants in eukaryotes and prokaryotes may be related to differences in the membrane Lipid Composition of these organisms; in other words, The Nature of the protector correlates with the composition of Membrane Lipids. This hypothesis is based on the premise that the primary biological function of protectants is to stabilize The Lipid Bilayer of the membrane. Perhaps the protection of bacterial membranes—which are more polymorphic in Structure and lipid composition compared to eukaryotic membranes—required more highly specialized protectants, such as glycine betaine and glycosylglycerol, which are absent in eukaryotes. It should be noted, however, that under certain conditions trehalose can serve as an osmoprotectant in some prokaryotes. Archaebacteria, including halophilic Bacteria, use only potassium as an osmoregulator. Recently, literature reports have emerged indicating that certain petroleum-degrading halophilic bacteria can utilize aspartic and glutamic acids, proline, and glycine betaine as protective compounds.

Pigments. The ability to synthesize pigments of various types (melanins, carotenoids, prodigiosins) is a characteristic feature of many microorganisms. These pigments perform various Functions within the microbial cell.

Melanins. The functions of melanins mainly involve protection against UV and ionizing radiation, desiccation, and the action of lytic Enzymes. It is also believed that The Role of melanins in protecting against UV and ionizing radiation may stem from the Flavonoids/14.html">Antioxidant Properties of these compounds. Literature reports indicate that melanins can act as cellular antioxidants. Specifically, irradiation causes the radiolysis of water, generating reactive free radicals (hydroxyl, peroxide, etc.) that cause damage to cellular Biomolecules. Antioxidants are capable of scavenging and neutralizing such reactive radicals.

Melanins apparently also play a certain role in microbial resistance to toxic heavy metals due to their binding capacity. For instance, an increase in melanin content in cells of Gaeumannomyces graminis var. graminis grown on a medium containing CuSO₄ is regarded by researchers as a response to the presence of Cu²⁺.

Carotenoids. In non-phototrophic microorganisms, carotenoids act as protectors against photosensitization, whereas in phototrophs, they additionally participate in The transfer of light energy to the reaction centers of Photosystems. Carotenoids in the Cells of the aerobic heterotrophic bacteria Micrococcus lysodeikticus (M. luteus) protect Quinones from degradation. The hypothesis that carotenoids stabilize the cell membranes of heterotrophic bacteria—Micrococcus lysodeikticus and Sarcina lutea—was not confirmed in subsequent studies.

In light of modern concepts, carotene is considered one of the bioantioxidants that protect microbial cells from the action of toxic peroxide compounds, which can likewise accumulate in cells under METABOLISM/18.html">The Influence of UV and ionizing radiation. Thus, radioresistant bacteria (Representatives of the genera Deinococcus, Methylobacterium, Rubrobacter) contain carotenoids. Soil bacteria isolated from the 10-kilometer zone of the Chornobyl NPP have also proven to be pigmented (ranging in color from yellow to orange-red).

Carbohydrates. In recent years, researchers have focused on the fact that trehalose accumulates in significant quantities in organisms (particularly micromycetes) capable of surviving dehydration (anhydrobiosis). The correlation between high trehalose levels and cellular dehydration during the transition of microorganisms into an anabiotic state suggests that this disaccharide alters the Physical Properties of membrane lipids, conferring exceptional stability in the anhydrobiotic state; in other words, trehalose acts as a specific protective mechanism preventing membrane desiccation.

It is believed that one of the mechanisms of thermostability in microorganisms is the organism's ability to withstand dehydration, as it is precisely through this process that a number of cellular polymers—proteins in particular—are stabilized. Maintaining membrane integrity during dehydration is of no lesser importance. In bacterial spores, dipicolinic acid performs the function of protecting molecular structures from deformation, while an analogous role is attributed to trehalose in fungi. Accordingly, it is believed that an increase in trehalose levels leads to an increase in the organism's thermostability.

It has been demonstrated that Yeast cultures of Saccharomyces vini and Torulopsis dattila with a high content of intracellular carbohydrates (Glycogen, glucan, mannan, trehalose) are radioresistant. Radiosensitive cultures differ from radioresistant ones by having a lower carbohydrate reserve.

Exopolysaccharides. Among the diverse BIOLOGICAL FUNCTIONS OF EPS, the primary place is assigned to the protective function of these polymers. Microbial EPS protect producer cells from various unfavorable factors (desiccation, toxic heavy metals, Antibiotics, biocides, detergents, phages, ultraviolet radiation).

Due to their hydrophilicity, extracellular Polysaccharides retain water for prolonged periods, maintaining cell viability. For example, polysaccharides produced by soil and rhizosphere microorganisms prevent soil desiccation and reduce water stress. Mucoid strains of Escherichia coli, Acinetobacter calcoaceticus, and Erwinia stewartii are significantly more resistant to desiccation than corresponding mutants that do not synthesize EPS. EPS are capable of forming insoluble metal-EPS complexes with heavy metals.

The EPS synthesized by Beijerinckia derxii exhibits protective functions toward Nitrogenase. Extracellular polysaccharides from certain microorganisms are able to protect the producer's exoenzymes from proteolytic degradation. The survival of microbial cells under ultraviolet irradiation may also be attributed to the protective action of exopolysaccharides.

Protective proteins. Upon transition from the exponential to the stationary growth phase, E. coli synthesizes 20–30 new specific (so-called post-exponential) proteins that ensure cell survival in response to adverse factors. The processes occurring during the transition of cells to the stationary state can be regarded as normal under the natural conditions of microbial existence. Indeed, in nature, bacteria rarely encounter optimal growth conditions. Between brief bursts of growth, they spend long periods under starvation conditions, in unbalanced media, and so forth. Under such unfavorable conditions, cells do not completely halt their metabolic activity; rather, they transition to a new phase of their life cycle—the maintenance phase. They become metabolically less active and more resistant to environmental stressors. Thus, stationary-phase E. coli cells are more resistant to heat, peroxides, antibiotics, and osmotic stress than exponentially growing cells.

In response to prolonged starvation, bacteria synthesize protective proteins, such as protein D, which directly protects DNA. Under the influence of unfavorable factors, the synthesis of so-called stress proteins is observed, which are produced to protect essential proteins.

In many microorganisms, metal resistance is driven by the synthesis of metal-binding proteins. The Mechanism of Cd²⁺ detoxification involves The formation of a metal-protein complex, the synthesis of which is induced by Cd²⁺. Metal-binding proteins are produced by the thermoacidophilic bacteria Sulfolobus solfataricus and Bacillus caldarius when grown in the presence of Zn²⁺ and Cu²⁺. An extracellular Cu²⁺-binding protein has been found in cultures of Vibrio alginolyticus and Pseudomonas aeruginosa that survived Cu²⁺ stress. Heavy metal-resistant bacteria of the genus Citrobacter precipitate metals as a cell-associated MeHPO₄ complex mediated by a membrane-bound acid phosphatase. It was previously hypothesized that the overproduction of this enzyme is the cause of bacterial metal resistance; however, recent studies have revealed an inverse relationship between actual enzyme activity and bacterial copper resistance.