Osmoadaptation of haloalkaliphilic bacteria: role of osmoregulators and their possible practical application

The review discusses osmoadaptation of haloalkaliphilic bacteria from diverse taxonomic and physiological groups, inhabiting soda lakes. Our experimental research has confirmed the similarity of the osmoregulation strategies in neutrophilic and alkaliphilic halophiles, independent of their pH homeostasis mechanism. The external osmotic pressure is equilibrated either due to accumulation of ions from the environment, or by accumulation or synthesis of cytoplasmic osmoregulatory compounds. The alkaliphiles following the "compatible solutes" strategy contain low or moderate concentrations of salts in their cytoplasm; their proteins do not require adaptation to salts. Those that follow the "salt-in" strategy do not synthesize osmoregulators: they accumulate high levels of salts within the cell and thus equilibrate the osmotic pressures of the cell and the environment. The proteins of these bacteria contain more acidic amino acid residues compared to the proteins of neutrophiles. The functions of bacterial organic osmoregulatory compounds are discussed, as well as their characteristics of possible practical value. Applications for ectoine and betaine are discussed based on the published data.     

Compatible solute addition to biological systems treating waste/wastewater to counteract osmotic and other environmental stresses: a review

This study reviews the addition of compatible solutes to biological systems as a strategy to counteract osmolarity and other environmental stresses. At high osmolarity many microorganisms accumulate organic solutes called “compatible solutes” in order to balance osmotic pressure between the cytoplasm and the environment. These organic compounds are called compatible solutes because they can function inside the cell without the need for special adaptation of the intracellular enzymes, and also serve as protein stabilizers in the presence of high ionic strength. Moreover, the compatible solutes strategy is regularly being employed by the cell, not only under osmotic stress at high salinity, but also under other extreme environmental conditions such as low temperature, freezing, heat, starvation, dryness, recalcitrant compounds and solvent stresses. The accumulation of these solutes from the environment has energetically a lower cost than de novo synthesis. Based on this cell mechanism several studies in the field of environmental biotechnology (most of them on biological wastewater treatment) employed this strategy by exogenously adding compatible solutes to the wastewater or medium in order to alleviate environmental stress. This current paper critically reviews and evaluates these studies, and examines the future potential of this approach. In addition to this, a strategy for the successful implementation of compatible solutes in biological systems is proposed.     

Influence of osmoregulation processes on starvation survival of Escherichia coli in seawater.

The adaptation of enteric bacteria in seawater has previously been described in terms of nutrient starvation. In the present paper, we bring experimental arguments suggesting that survival of these microorganisms could also depend on their ability to overcome the effects of osmotic stress. We analyzed the influence of osmoregulatory mechanisms (potassium transport, transport and accumulation of organic osmolytes) on the survival of Escherichia coli in seawater microcosms by using mutants lacking components of the osmotic stress response. Long-term protection was afforded to cells by growth in a medium whose osmotic pressure was increased by either NaCl, LiCl, or saccharose. Achievement of the protection state depended at least partly on osmoregulatory mechanisms, but differed when these were activated or induced during prior growth or in resting cells suspended in phosphate buffer or in seawater. When achieved during growth, K+ transport, glycine-betaine (GBT) synthesis or transport, and trehalose synthesis helped increase the ability to survive in seawater. Protection by GBT was also obtained with resting cells in a phosphate buffer at high osmotic pressure. However, when added only to the seawater, GBT did not change the survival ability of cells no matter what their osmoregulation potential. These results showed that the survival of E. coli cells in seawater depends, at least partly, on whether they possess certain genes which enable them to regulate osmotic pressure and whether they can be stimulated to express those genes before or after their release into the environment. This expression requires nutrients as the substrates from which the corresponding gene products are made.     

中度嗜盐菌相容性溶质机制的研究进展

生活在高盐环境中的中度嗜盐菌不仅能抗衡外界的高渗透压胁迫,而且还能迅速适应短时间内的渗透冲击。为适应该环境,中度嗜盐菌依赖于一种被称为相容性溶质的物质,以执行渗透保护功能。这类物质属于极性的、易溶的和低分子量的有机化合物,其中包括糖类、氨基酸类、甜菜碱类和四氢嘧啶类等。中度嗜盐菌主要采用相容性溶质机制来适应盐环境。在此,就中度嗜盐菌的盐适应机理、相容性溶质的种类和特点,以及其作用的分子机制进行了阐述和讨论。     

Physiological and genetic responses of bacteria to osmotic stress.

The capacity of organisms to respond to fluctuations in their osmotic environments is an important physiological process that determines their abilities to thrive in a variety of habitats. The primary response of bacteria to exposure to a high osmotic environment is the accumulation of certain solutes, K+, glutamate, trehalose, proline, and glycinebetaine, at concentrations that are proportional to the osmolarity of the medium. The supposed function of these solutes is to maintain the osmolarity of the cytoplasm at a value greater than the osmolarity of the medium and thus provide turgor pressure within the cells. Accumulation of these metabolites is accomplished by de novo synthesis or by uptake from the medium. Production of proteins that mediate accumulation or uptake of these metabolites is under osmotic control. This review is an account of the processes that mediate adaptation of bacteria to changes in their osmotic environment.     

Water transport parameters and regulatory processes inEremosphaera viridis

Summary The water relations parameters and the osmoregulatory response ofEremosphaera viridis were investigated both by using the pressure probe technique and by analyzing the intracellular pool of osmotically active agents. In the presence of various concentrations of different salts a biphasic osmoregulatory response was recorded, consisting of a rapid decrease in turgor pressure due to water loss followed by an increase in turgor pressure to the original turgor pressure value (depending on the salt). The values of turgor pressure, volumetric elastic modulus and hydraulic conductivity depended on the composition of the media. Nonelectrolytes did not cause a turgor recovery after the initial water efflux. The second phase of turgor regulation in the presence of salts was characterised by the intracellular accumulation of ions and sugars and required at least 24 hr. Analysis of the cell sap showed that the increase in the internal osmotic pressure was mainly achieved by accumulation of sucrose. Additionally, accumulation of glucose was observed in illuminated cells in the presence of Rb and K. Electron micrographs suggested that the sucrose was produced by degradation of starch granules. Turgor pressure recovery after salt stress seemed to be dependent on temperature and is well correlated with the according photosynthetic activity. The data suggest that a temperature-dependent enzyme which is activated by potassium or rubidium is involved in the regulatory response.     

Evolution of the term “cellular senescence” and its impact on the current cytogerontological research

The term “cellular/cell senescence” was first introduced by Leonard Hayflick to describe the “age-related” changes in normal eukaryotic cells during aging in vitro, i.e., over the exhaustion of their mitotic potential. In the “classic” variant, it was assumed that cells “grow old” with the help of some internal mechanism, which leads to accumulation of various macromolecular defects (DNA damage in the first place). Currently, as a rule, “cellular senescence” means accumulation/appearance of particular “biomarkers of aging” in cells (they are most often transformed cells that do not demonstrate any replicative senescence) under the influence of various external factors (oxidative stress, H₂O₂, mitomycin C, ethanol, ionizing radiation, doxorubicin, etc.) that cause DNA damage. This phenomenon has been called DDR (DNA Damage Response). Among the said biomarkers, there are senescence-associated beta-galactosidase activity, expression of p53 and p21 proteins as well as of proteins involved in the regulation of inflammation, such as IL-6 or IL-8, activation of oncogenes, etc. Thus, “aging/senescence” of cells does not occur simply by itself—it takes place because of the influence of DNA-damaging agents. This approach, in my opinion, despite being very important to define a strategy to fight cancer, distracts us, yet again, from the study of the real mechanisms of aging. It should be emphasized that the “stationary phase aging” model developed in my laboratory also allows registering the occurrence of certain biomarkers of aging in cultured cells, but in this case they arise due to the restriction of their proliferation by contact inhibition, i.e., due to a rather physiological impact, which does not cause any damage to cells by itself (the situation is similar to what we observe in a whole multicellular organism).     

Obtaining and characterization of DNA-containing micromummies of yeasts and gram-positive bacteria with enhanced cell wall permeability: Application in PCR

The procedure of obtaining DNA-containing cell envelopes (“micromummies”) of bacteria, yeasts, and fungi using chaotropic salts has been developed previously and the possibility of their direct application in PCR has been demonstrated. The fine structure of micromummies has been studied by electron microscopic methods. This work has demonstrated that additional treatment of micromummies of yeasts and gram-positive bacteria with proteinase K results in hydrolytic degradation of cell proteins and drastic enhancement of cell wall permeability for macromolecules (DNA). Thus, the efficiency of PCR ex situ using resultant micromummies after washing off the products of protein hydrolysis and proteinase K can be increased. The results of electron microscopic study of ultrathin sections of yeasts (Pichia pastoris, Saccharomyces cerevisiae) and gram-positive bacteria (Micrococcus luteus, Arthrobacter globiformis, Bacillus subtilis) support the biochemical data that treatment with chaotropic salts and proteinase K results in the loosening of microbial cell walls and in a decrease in the intracellular protein content. At the same time, cell walls generally maintain their integrity (continuity) and initial spherical or rodlike shape. The optimal modes of treatment of the cells of different microbial species with chaotropic salts and proteinase K have been selected to obtain permeabilized cell envelopes containing denatured or native DNA.     

Distribution in tissue homogenates,and the nature of the linkage of injected proteins to subcellular particles

Intravenously injected labeled proteins were recovered mostly in particulate fractions of rat liver homogenate. Distribution showed changes depending on the time elapsed from the injection. ¹³¹I-albumin undergoes an intraparticulate hydrolysis which shows the highest activity in the gradient fractions associated with the highest level of acid phosphatase. The labeled albumin-bearing particles separated at 27,000 g × 10 minutes released their radioactive protein at the same rate as acid phosphatase appeared in the medium, under the effect of such agents as distilled water, salts, homogenization, sonication and pH changes. The substitution of sucrose for distilled water or salts showed that the particles behave as an osmotic system as do lysosomes. These experiments prove that secondary lysosomes involved in the hydrolysis of foreign proteins, whose existence was shown by other authors only at the histochemical level, may survive the distrupting action of conventional homogenization and maintain many properties characteristic of primary lysosomes in addition to the ability of hydrolysing “in vitro” the engulfed material.     

Hatching of the Anostracan Branchiopod Chirocephalus diaphanus Prévost I. Osmotic processes and the possible role of glycerol

An osmotic hatching mechanism is proposed for the Anostracan Branchiopod Chirocephalus diaphanus, Prevost. In this mechanism, glycerol is thought to accumulate in the egg as a result of the metabolism of the embryo. The osmotic pressure exerted by the egg, largely due to this glycerol accumulation, causes water to enter from the environment. Eventually the hydrostatic pressure thus generated is sufficient to cause rupture of the eggshell. It is recognised that hatching in this Anostracan is a two-staged phenomenon, a process of “breaking”, in which glycerol is involved, preceding true “hatching”, in which glycerol is not involved. The egg is described and the physiology and ecological significance of the proposed hatching mechanism are discussed.     

Cardiolipin and the osmotic stress responses of bacteria

Cells control their own hydration by accumulating solutes when they are exposed to high osmolality media and releasing solutes in response to osmotic down-shocks. Osmosensory transporters mediate solute accumulation and mechanosensitive channels mediate solute release. Escherichia coli serves as a paradigm for studies of cellular osmoregulation. Growth in media of high salinity alters the phospholipid headgroup and fatty acid compositions of bacterial cytoplasmic membranes, in many cases increasing the ratio of anionic to zwitterionic lipid. In E. coli, the proportion of cardiolipin (CL) increases as the proportion of phosphatidylethanolamine (PE) decreases when osmotic stress is imposed with an electrolyte or a non-electrolyte. Osmotic induction of the gene encoding CL synthase (cls) contributes to these changes. The proportion of phosphatidylglycerol (PG) increases at the expense of PE in cls⁻ bacteria and, in Bacillus subtilis, the genes encoding CL and PG synthases (clsA and pgsA) are both osmotically regulated. CL is concentrated at the poles of diverse bacterial cells. A FlAsH-tagged variant of osmosensory transporter ProP is also concentrated at E. coli cell poles. Polar concentration of ProP is CL-dependent whereas polar concentration of its paralogue LacY, a H⁺-lactose symporter, is not. The proportion of anionic lipids (CL and PG) modulates the function of ProP in vivo and in vitro. These effects suggest that the osmotic induction of CL synthesis and co-localization of ProP with CL at the cell poles adjust the osmolality range over which ProP activity is controlled by placing it in a CL-rich membrane environment. In contrast, a GFP-tagged variant of mechanosensitive channel MscL is not concentrated at the cell poles but anionic lipids bind to a specific site on each subunit of MscL and influence its function in vitro. The sub-cellular locations and lipid dependencies of other osmosensory systems are not known. Varying CL content is a key element of osmotic adaptation by bacteria but much remains to be learned about its roles in the localization and function of osmoregulatory proteins.     

Osmoregulatory function of large vacuoles found in notochordal cells of the intervertebral disc running title: an osmoregulatory vacuole

The nucleus pulposi of many species contain residual cells from the embryonic notochord, which exhibit a very unusual appearance (large vacuoles occupying approximately 80% of the cell volume, surrounded by an actin cytoskeleton). While the vacuoles have been qualitatively described, their composition and function has remained elusive. Given that these cells are believed to generate and experience significant osmotic pressures in both the notochord and intervertebral disc, we hypothesized that the vacuoles may serve as osmoregulatory organelles. Using both experimental and theoretical means, we demonstrated that the vacuoles contain a low-osmolality solution, generated via ion pumps on the vacuolar membrane. During hypotonic stress the vacuoles release their contents into the cytoplasm, diluting the cytoplasm and restoring the osmotic balance across the cell membrane. Thus the vacuoles function to regulate the cell volume and tonicity during rapid osmotic stress, protecting the cells from potentially damaging swelling pressures.     

Factors reducing and promoting the effectiveness of proline as an osmoprotectant in Escherichia coli K12

Proline accumulation in Escherichia coli is mediated by three proline porters. Proline catabolism is effected by proline porter I (PPI) and proline/delta 1-pyrroline carboxylate dehydrogenase. Proline did not accumulate cytoplasmically when E. coli was subjected to osmotic stress in minimal salts medium. Although PPI is induced when proline is provided as carbon or nitrogen source, its activity decreased following growth of the bacteria in minimal salts medium of high osmotic strength. Proline dehydrogenase was induced by proline in low or high osmotic strength media. Proline porter II (PPII) was both activated and induced in osmotically stressed bacteria, though the dependencies of the two responses on medium osmolarity differed. Osmotic downshift during the transport measurement decreased the uptake of proline, serine and glutamine by bacteria cultured in media of high osmotic strength. Thus, while osmotic upshift caused specific activation of PPII, osmotic downshift caused a non-specific reduction in amino acid uptake. Glycine betaine inhibited the uptake of [14C]proline via PPII and PPIII but not via PPI. The dependence of that inhibition on glycine betaine concentration was similar when PPII was uninduced, induced or activated by osmotic stress, or induced by amino acid limited growth. Thus PPII and PPIII, not PPI, contribute to the mechanism of osmoprotection by proline and glycine betaine. The tendency for exogenous proline to accumulate in the cytoplasm of bacteria exposed to osmotic stress would, however, be countered by increased proline catabolism.     

Metabolism of chloride in halophilic prokaryotes

While much understanding has been achieved on the intracellular sodium and potassium concentrations of halophilic and halotolerant microorganisms and on their regulation, we know little on the metabolism of anions. Archaea of the family Halobacteriaceae contain molar concentrations of chloride, which is pumped into the cells by cotransport with sodium ions and/or using the light-driven primary chloride pump halorhodopsin. Most halophilic and halotolerant representatives of the bacterial domain contain low intracellular ion concentrations, with organic osmotic solutes providing osmotic balance. However, some species show a specific requirement for chloride. In Halobacillus halophilus certain functions, such as growth, endospore germination, motility and flagellar synthesis, and glycine betaine transport are chloride dependent. In this organism the expression of a large number of proteins is chloride regulated. Other moderately halophilic Bacteria such as Halomonas elongata do not show a specific demand for chloride. A very high requirement for chloride was demonstrated in two groups of Bacteria that accumulate inorganic salts intracellularly rather than using organic osmotic solutes: the anaerobic Halanaerobiales and the aerobic extremely halophilic Salinibacter ruber. It is thus becoming increasingly clear that chloride has specific functions in haloadaptation in different groups of halophilic microorganisms.     

The distance between bacterial species in sequence space

Despite the revolution caused by information from macromolecular sequences, the basis of bacterial classification remains the genus and the species. How do these terms relate to the variety of bacteria that exist on earth? In this paper, the inter- and intraspecies differences in amino acid sequence of several bacterial electron transport proteins, cytochromesc, and blue copper proteins are compared. For the soil and water organisms studied, bacterial species can be classed as “tight” when there is little intraspecies variation, or “loose” when this variation is large. For this set of proteins and organisms, interspecies variation is much larger than that within a species. Examples of “tight” species arePseudomonas aeruginosa andRhodobacter sphaeroides, whilePseudomonas stutzeri andRhodopseudomonas palustris are loose species. The results are discussed in the context of the origin and age of bacterial species, and the distribution of genomes in “sequence space.” The situation is probably different for commensal or pathogenic bacteria, whose population structure and evolution are linked to the properties of another organism.     

Die Mundhöhle als Ökosystem

Biomimetic approaches for the dental plaque control Tooth and gum diseases are widespread and are primarily based on the presence of bacterial biofilms. The characterization of biofilms can be carried out by means of state‐of‐the‐art microbiome analysis that can provide information on bacterial composition and diversity. Modern oral care products mostly contain different antimicrobial agents for biofilm control. These include chlorhexidine, metal salts, and quaternary ammonium compounds, which, however, often kill harmful (dysbiotic) and useful bacteria (homeostatic) (unspecific antimicrobial effect). Recent developments show that innovative concepts shift the ecological balance of plaque in the oral cavity to “physiological commensal bacteria” (homeostasis) or minimize the bacterial colonization on enamel surfaces without having pronounced antimicrobial properties. Examples are biomimetic approaches, i.e. based on selected salivary enzymes or hydroxyapatite.     

Osmotic relations during elongation growth in hypocotyls of Helianthus annum L.

The relationship between growth, change in cell osmotic pressure and accumulation of osmotic solutes was investigated in hypocotyls of sunflower (Helianthus annum L.) seedlings. During growth in darkness the osmotic pressure decreased by 50% between days 2 and 6 after sowing. After irradiation of dark-grown seedlings with continuous white light (WL) an inhibition of hypocotyl growth was measured, but the osmotic pressure of the growing cells was not lower than in the dark-grown control. Growth in darkness and after WL irradiation was accompanied by an increase in the amount of osmotic substances (soluble sugars) which was proportional to the increase in length of the organ. During growth in continuous WL the cell osmotic pressure decreased by 45 % between days 2 and 6 after sowing. The transfer of WL-grown seedlings to darkness (“re-etiolation”) resulted in a rapid acceleration of hypocotyl growth, but the cell osmotic pressure was the same as that of the WL grown control. Growth in continuous WL was accompanied by a corresponding accumulation of osmotic substances (soluble sugars). The transition from WL to darkness resulted in an enhanced accumulation of osmotica and an increase in cell-wall extensibility. The results indicate that the relative maintenance of cell osmotic pressure during rapid hypocotyl growth in darkness is caused by an enhanced accumulation of soluble sugars into the growing cells of the organ.     

The cell pole: the site of cross talk between the DNA uptake and genetic recombination machinery

Natural transformation is a programmed mechanism characterized by binding of free double-stranded (ds) DNA from the environment to the cell pole in rod-shaped bacteria. In Bacillus subtilis some competence proteins, which process the dsDNA and translocate single-stranded (ss) DNA into the cytosol, recruit a set of recombination proteins mainly to one of the cell poles. A subset of single-stranded binding proteins, working as “guardians”, protects ssDNA from degradation and limit the RecA recombinase loading. Then, the “mediators” overcome the inhibitory role of guardians, and recruit RecA onto ssDNA. A RecA·ssDNA filament searches for homology on the chromosome and, in a process that is controlled by “modulators”, catalyzes strand invasion with the generation of a displacement loop (D-loop). A D-loop resolvase or “resolver” cleaves this intermediate, limited DNA replication restores missing information and a DNA ligase seals the DNA ends. However, if any step fails, the “rescuers” will repair the broken end to rescue chromosomal transformation. If the ssDNA does not share homology with resident DNA, but it contains information for autonomous replication, guardian and mediator proteins catalyze plasmid establishment after inhibition of RecA. DNA replication and ligation reconstitute the molecule (plasmid transformation). In this review, the interacting network that leads to a cross talk between proteins of the uptake and genetic recombination machinery will be placed into prospective.     

Induction of synthesis of tetrahydropyrimidine derivatives in Streptomyces strains and their effect on Escherichia coli in response to osmotic and heat stress.

The metabolic responses of a number of Streptomyces strains to osmotic and heat stress were studied by 13C nuclear magnetic resonance spectroscopy. During cell growth in a chemically defined medium supplemented with 0.5 M NaCl, tetrahydropyrimidine derivatives (THPs), 2-methyl-4-carboxy-5-hydroxy-3,4,5,6-tetrahydropyrimidine [THP(A)] and, to a lesser extent, 2-methyl-4-carboxy-3,4,5,6-tetrahydropyrimidine [THP(B)], were found to accumulate in a significant amount in all bacteria examined. In addition, when the growth temperature was shifted from 30 to 39 degrees C, the intracellular concentration of THP(A) increased significantly. Moreover, exogenously provided THP(A) or THP(B) or both reversed inhibition of Escherichia coli growth caused by osmotic stress and increased temperature. Although the ability of Streptomyces strains to tolerate high concentrations of NaCl is well known, very little is known about the osmoregulatory strategy in Streptomyces strains. Similarly, the mechanism by which compatible solutes accumulate in a variety of microorganisms is not understood. Our findings suggest the possibility of a novel mechanism of protection of DNA against salt and heat stresses involving the THPs.