The impact of abiotic factors (temperature and glucose) on physicochemical properties of lipids from Yersinia pseudotuberculosis

The impact of the availability of glucose in nutrition medium and growth temperature on the composition and thermotropic behavior of lipids from Yersinia pseudotuberculosis (Enterobacteriaceae) was studied. Y. pseudotuberculosis was grown in nutrition broth (NB) with/without glucose at 8 and 37 degrees C, corresponding to the temperatures of saprophytic and parasitic phases of this bacterium life. The decrease of phosphatidylethanolamine, phosphatidylglycerol and unsaturated fatty acids and the parallel increase of lysophosphatidylethanolamine and diphosphatidylglycerol and saturated and cyclopropane acids were the most significant changes with temperature in bacterial phospholipid (PL) classes and fatty acids, respectively. Glucose did not effect the direction of temperature-induced changes in the contents of PLs, fatty acids, however it enhanced (for PLs) or diminished (for fatty acids) intensity of these changes. The thermally induced transitions of lipids were studied by differential scanning calorimetry (DSC). It was revealed that the addition of glucose to NB induced a sharp shift of DSC thermograms to lower temperatures in the "warm" variants of bacteria. The peak maximum temperature (Tmax) of thermal transitions dropped from 50 to 26 degrees C that is the optimal growth temperature of Y. pseudotuberculosis. Tmax of total lipids of the cells grown at 8 degrees C without glucose in NB was equal to growth temperature that corresponded to the classical mechanism of homeoviscous adaptation of bacteria. An addition of glucose to NB at this growth temperature caused the subsequent reduction of Tmax to -8 degrees C, while the temperature ranges of thermograms were not substantially changed. So, not only the temperature growth of bacteria, but also the presence of glucose in NB can modify the physical state of lipids from Y. pseudotuberculosis. In this case, both factors affect additively. It is suggested that glucose influences some membrane-associated proteins and then the fluidity of lipid matrix through temperature-inducible genes.     

Pathogenetic significance of the psychrophilia of Yersinia pseudotuberculosis

The work presents the data indicating that the temperature of Y. pseudotuberculosis cultivation is very important in regulating the activity of pathogenicity factors, necessary for the initiation of the pathogenic process in the cells of the macroorganism. Low temperature (8-10 degrees C), necessary for the growth of Y. pseudotuberculosis, facilitates the activation of invasive and toxic pathogenicity factors. At a growth temperature of 37 degrees C the inhibition of such necessary attributes of virulence as adhesion and invasion into epithelial cells occurs in Y. pseudotuberculosis, which decreases the capacity of these bacteria for inducing the infectious process. The virulence of Y. pseudotuberculosis population, lost as the result of its cultivation in synthetic culture media at a temperature of 37 degrees C, has been found to be restored at low temperature.     

Effect of cultivation temperature on nucleic acid level in bacteria Listeria monocytogenes and Yersinia pseudotuberculosis

The relationship between the multiplication of bacteria, the content of nucleic acid and the specific rate of their growth during their batch cultivation in nutrient broth and mineral medium at temperatures of 37 degrees C and 4-6 degrees C was studied in the causative agents of saprozoonotic infections with L. monocytogenes and Y. pseudotuberculosis used as typical representatives of such bacteria. The content of DNA was shown to remain practically unchanged after the alteration of cultivation temperature and the conditions of nutrition. The linear relationship between the content of RNA and specific growth rate was registered both at 37 degrees C and 4-6 degrees C. However a higher content of RNA at low temperatures was found to correspond to one and the same specific growth rate, which was linked with the additional synthesis of this nucleic acid.     

The multiplication dynamics of pathogenic bacteria in relation to the trophic and temperature cultivation conditions

The comparative study of the dynamics of multiplication of Listeria monocytogenes and Yersinia pseudotuberculosis in organic and synthetic media and in distilled water at temperatures of 37 degrees C and 6 degrees C was carried out. This study revealed that in organic media the multiplication of bacteria was good at 37 degrees C and 6 degrees C. In mineral media and distilled water their multiplication was observed only at 6 degrees C. Moreover, conditions necessary for the multiplication of pathogenic bacteria in distilled water were shown; these conditions depended on the inoculated dose, the number of autolyzed microbial cells and the state of the culture. Proofs of the multiplication of the bacteria under the conditions of minimum nutrition and low temperature were obtained with the use of labeled 3H-thymidine.     

DNA, RNA, and protein biosynthesis in cells of Yersinia pseudotuberculosis at various cultivation temperatures

Y. pseudotuberculosis cells cultivated at temperatures of 37 degrees C and 8 degrees C were found to be capable of incorporating exogenic precursors into DNA, RNA and protein. The linear growth of thymidine incorporation occurred during 8 hours of cultivation at 37 degrees C, then the amount of the incorporated label decreased. At 8 degrees C the level of thymidine incorporation into DNA gradually increased for 80 hours and longer, but not reaching the level of incorporation observed at 37 degrees C. The incorporation of uridine into RNA of Y. pseudotuberculosis cells reached its maximum after 4 hours of cultivation at 37 degrees C, at a lower temperature of cultivation the incorporation of uridine into bacterial cells was almost linear, though slower, and lasted for 20 hours. The content of radioactive alanine in Y. pseudotuberculosis protein increased during 16 hours of cultivation at a high temperature, while at 8 degrees C the growth of the incorporation level lasted for at least 40 hours. For all precursors under study the incorporation rate into the cell biopolymers at the initial stages of cultivation was higher at 37 degrees C, than at a lower temperature.     

Thermotropic behavior of lipids and the morphology of Yersinia pseudotuberculosis cells with a high content of lysophosphatidylethanolamine

The content of lysophosphatidylethanolamine (LPE) in Y. pseudotuberculosis cells was found to increase during their growth at 8 degrees C under stationary conditions (without stirring the medium) and at 37 degrees C when the medium contained glucose. The maximum level of LPE (up to 45% of the total phospholipids) was observed in cells grown at 8 degrees C under stationary conditions. Such cells showed an enhanced growth rate, a reduced yield of biomass, an altered cell morphology, and an increased cell area. The cells contained unsaturated fatty acids, phosphatidylethanolamine (PE), and total phospholipids in small amounts, whereas neutral lipids and diphosphatidylglycerol were abundant. In addition, the cells contained an amount of methylated PE and phospholipids of unknown structure. Irrespective of whether the temperature for growth was low or high, the LPE-rich cells showed a high value (32-36 degrees C) of the maximum temperature of thermal transition of lipids (Tmax). This finding is indicative of a densification of the membrane lipid matrix of the LPE-rich cells. The suggestion is made that LPE is accumulated in glucose-fermenting bacterial cells in response to stress caused by oxygen deficiency and low pH values of the growth medium. The possible relationship between LPE accumulation and the virulence of Y. pseudotuberculosis cells grown at low temperatures is discussed.     

Chemotaxis of Yersinia pseudotuberculosis as a mechanism in its search for target tissues of the host organism

Y. pseudotuberculosis cells grown at biologically low temperature have been shown capable of chemotaxis with respect to carbohydrates and amino acids. During cultivation at 36-37 degrees C Y. pseudotuberculosis cells retain this property for 10-15 hours and then lose it. The mechanism of chemotaxis makes it possible for Y. pseudotuberculosis to "find" human and animal tissues and can facilitate the realization of the pathogenicity potential of these bacteria. When administered orally to mice motile bacteria, i. e. those grown at 6-8 degrees C, have been more virulent for the animals than nonmotile ones cultivated at 36-37 degrees C.     

Effects of growth temperature and pVM82 plasmid on fatty acids of lipid A from Yersinia pseudotuberculosis

Effects of cultivation temperature (8 or 37 degrees C) and plasmid profile on the lipid A fatty acids of three isogenic Yersinia pseudotuberculosis strains (plasmidless (82-) and strains containing pVM82 (82+) or p57 (57+) plasmids) obtained by alkaline hydrolysis of the whole bacterial cells and differentiated from fatty acids of other membrane lipids were investigated. On the basis of the analysis, it is concluded that lipids A of all studied samples contain 3-hydroxytetradecanoic and dodecanoic acids, a part of which exists as the 3-dodecanoyloxytetradecanoic derivative. The effect of temperature appears in the higher contents of ester- and amide-linked 3-acyloxyalkanoic residues in lipid A from the "cold" variants of the bacteria and is determined by chromosomal genes. The plasmid effect is seen as various responses of the isogenic derivatives to change of growth temperature: in cells of strains 82+ and 82- grown in the cold, the share of lipid A fatty acids in the total population of cellular fatty acids is reduced, while in strains with plasmid p57 it is increased. The temperature variants of the 57+ strain differ by the low contents of amide-linked 3-acyloxyalkanoic acids. Finally, lack of plasmid pVM82 in the "warm" variants of the bacteria results in accumulation of glycolipid molecules deprived of dodecanoic acid. Correlation between growth temperature and plasmid profiles, on one hand, and lipid A fatty acid composition and potential pathogenic properties of the Y. pseudotuberculosis, on the other hand, and also possible mechanisms of thermal adaptation of this organism are discussed.     

Fatty-acid composition of cells and lipopolysaccharides in different Yersinia species under the conditions of growth at low temperature

Y. pestis, Y. pseudotuberculosis, Y. enterocolitica, Y. frederiksenii, Y. intermedia, Y. kristensenii and Y. ruckeri grown at 4 degrees C were characterized by fatty acid composition with a high content of C16:1 and C18:1, as well as the proportion of saturated to nonsaturated fatty acids equal to, on the average, 2.0. In Yersinia lipopolysaccharides a relatively high level of C16:1 and C12:0 was observed with the prevalence of 3-OH-C14:0. In the fatty-acid spectra of both cells and lipopolysaccharides no essential difference was noted. Thus, during growth at low temperature differences, earlier detected in the studied Yersinia species grown at 37 degrees C and making it possible to divide 7 Yersinia species into 2 groupes, were completely leveled. These results confirmed the close phylogenetic relationship between the Yersinia species under study and were indicative of more pronounced biological community of Yersinia under the conditions of growth at low temperature.     

The biochemical mechanisms of energy support in Yersinia pseudotuberculosis cells at a low cultivation temperature

The object of the study was the first stage of biological oxidation: the transfer of hydrogen electrons to the components of the respiratory chain of Y. pseudotuberculosis cells by NAD and NADF, coenzymes of pyridine-dependent dehydrogenases, having labile redox properties. The study revealed that in the low-temperature cultivation of Y. pseudotuberculosis an increase in the content of NAD and NADF was 1.5- to 2.0-fold greater than that observed at 37 degrees C, which was indicative of the fact that at low environmental temperature pyridine-dependent dehydrogenases played a more important role than at high temperature (37 degrees C). This, in combination with other mechanisms, made it possible for bacterial cells to maintain the level of energy metabolism, necessary for their survival, in the environment with low and constantly changing temperature.     

Fermentative mechanisms of the psychrophilicity of Yersinia tuberculosis

The specific activity of urease, nitrogenase, hialuronidase and neuraminidase in Y. pseudotuberculosis grown in different culture media and at different temperature has been studied. These enzymes have been found capable of functioning at both relatively low (2-8 degrees C) and high (37 degrees C) temperatures. The thermoadaptive properties of Y. pseudotuberculosis within a wide range of temperatures are ensured by the constant presence of isoenzymes, functioning only at low temperatures or only at high temperatures, in the microbial cells. Low temperature in combination with a definite culture medium triggers the activity of certain enzymatic systems, which explains, to some extent, the biochemical mechanisms of the psychrophilic properties of Y. pseudotuberculosis.     

The cultural properties and virulence of Yersinia]

Study of the cultivation properties of 82 enterobacterial strains has revealed that the colonies of virulent Y. enterocolitica (serovars O3, O9) and Y. pseudotuberculosis (serovar I) are temperature-sensitive. This sign, closely connected with the presence and expression of the virulence plasmid with a molecular weight of 44-48 MD, is not characteristic of other strains. Virulent Yersinia grown in nutrient agar for 48 hours at 37 degrees C form colonies which are smaller in diameter than those formed during cultivation at 26 degrees C (with the significance of differences t greater than or equal to 4), their diameter at 37 degrees C not exceeding 1.0 mm. The test for the determination of the temperature-sensitive morphology of Yersinia colonies, along with the tests for other virulence markers, is probably suitable for the detection of the causative agents of yersiniosis or pseudotuberculosis.     

Virulence plasmid-associated autoagglutination in Yersinia spp.

The autoagglutination of Yersinia enterocolitica was dependent on the presence of the virulence plasmid and on the active growth of bacteria in tissue culture media at 37 degrees C. Cultures with a high initial concentration of bacteria failed to autoagglutinate , indicating that synthesis of new virulence plasmid-associated surface factors was essential for autoagglutination. The synthesis of a plasmid-encoded polypeptide (molecular weight, 240,000), designated P1, that could be dissociated under strongly reducing conditions into subunits of 52,500 daltons was found to be correlated with autoagglutination. Further, a strain of Yersinia pseudotuberculosis [ YPIII ( PIB102 )], which has Tn5 inserted within the structural gene of P1 that prevents the synthesis of P1, failed to autoagglutinate , in contrast to the wild-type strain, strongly indicating that P1 is involved in this phenomenon. It was also found by immunoblotting that in addition to the common response to temperature, the P1 proteins of Y. enterocolitica and Y. pseudotuberculosis were immunologically related.     

Metabolic control of the membrane fluidity in Bacillus subtilis during cold adaptation

Membrane fluidity adaptation to the low growth temperature in Bacillus subtilis involves two distinct mechanisms: (1) long-term adaptation accomplished by increasing the ratio of anteiso- to iso-branched fatty acids and (2) rapid desaturation of fatty acid chains in existing phospholipids by induction of fatty acid desaturase after cold shock. In this work we studied the effect of medium composition on cold adaptation of membrane fluidity. Bacillus subtilis was cultivated at optimum (40 degrees C) and low (20 degrees C) temperatures in complex medium with glucose or in mineral medium with either glucose or glycerol. Cold adaptation was characterized by fatty acid analysis and by measuring the midpoint of phospholipid phase transition T(m) (differential scanning calorimetry) and membrane fluidity (DPH fluorescence polarization). Cells cultured and measured at 40 degrees C displayed the same membrane fluidity in all three media despite a markedly different fatty acid composition. The T(m) was surprisingly the highest in the case of a culture grown in complex medium. On the contrary, cultivation at 20 degrees C in the complex medium gave rise to the highest membrane fluidity with concomitant decrease of T(m) by 10.5 degrees C. In mineral media at 20 degrees C the corresponding changes of T(m) were almost negligible. After a temperature shift from 40 to 20 degrees C, the cultures from all three media displayed the same adaptive induction of fatty acid desaturase despite their different membrane fluidity values immediately after cold shock.     

The Effect of Temperature on the Lipid Composition of the Green Alga Botryococcus

The lipid composition of the green alga Botryococcus was studied at three different cultivation temperatures: suboptimal (18°C), optimal (25°C), and supraoptimal (32°C). Cultivation at the supraoptimal temperature was found to considerably inhibit the synthesis of nearly all intracellular lipids, except for triacylglycerides, and to influence their fatty acid composition. In particular, the content of trienoic fatty acids was significantly lower at the supraoptimal than at the optimal cultivation temperature. At the same time, the fatty acid composition of the extracellular lipids of the alga virtually did not depend on cultivation temperature.     

Effect of blue-green alga (Cyanobacteria) and their exometabolites on formation of resting forms and variability of Yersinia pseudotuberculosis

Research, carried out with the use of bacteriological methods and polymerase chain reaction, revealed that the transformation of Y. pseudotuberculosis, associated with blue-green algae Anabaena variabilis, into resting (noncultivable) forms took shorter time than in soil extract containing no algae. The exometabolites of "old" cultures of these algae sharply accelerated the formation of resting Y. pseudotuberculosis forms. The influence of the algae and the products of their metabolism was manifested far more intensively at 22 degrees C than at 4 degrees C. After passage through infusoria resting Y. pseudotuberculosis forms, preserved in the mucous covering of cyanobacteria, partially reverted into vegetative forms, capable of growing on solid culture media. The revertants essentially differed from the initial vegetative forms by having lower enzymatic activity, agglutinability and cytopathogenicity, as well as by the loss of plasmid p45. The probable role of blue-green algae, widely spread in soils and water reservoirs, in the processes of reversible transformation of Y. pseudotuberculosis vegetative and resting forms, closely connected with seasonal changes of temperature conditions.     

Enzymatic inactivation of levomycetin and penicillin by cells of the plague and pseudotuberculosis microbes that contain R plasmid depending on cultivation conditions]

The activity of enzymes, inactivating levomycetin and penicillin in the cells of plague and pseudotuberculosis microbes bearing extrachromosomal determinants resistant to a number of antibiotics was studied as dependent on some cultivation parameters: population age, aeration rate and temperature. It was shown that the highest capacity for levomycetin acetylation was characteristic of the cells in the late logarithmic and early stationary growth phages. Accumulation of levomycetin O-acetothers in the incubation medium markedly increased, when the cells were grown under the conditions of intensive aeration. An increase in the cultivation temperature up to 37 degrees C was accompanied by a reliable decrease in the activity of levomycetin acetylase in the transconjugant plague and pseudotuberculosis microbes though no correlation with the resistance levels in the same strains to the above antibiotics was observed. Optimal conditions for penicillinase production were determined. The maximum levels of penicillinase were found in the cells of Y. pestis 556/106 Rn with the episotic resistance type in the early exponential developmental phase under the aeration conditions and the temperature of 28 degrees C.     

Critical role of anteiso-C15:0 fatty acid in the growth of Listeria monocytogenes at low temperatures.

Listeria monocytogenes is a food-borne pathogen capable of growth at refrigeration temperatures. Membrane lipid fatty acids are major determinants of a sufficiently fluid membrane state to allow growth at low temperatures. L. monocytogenes was characterized by a fatty acid profile dominated to an unusual extent (> 95%) by branched-chain fatty acids, with the major fatty acids being anteiso-C15:0, anteiso-C17:0, and iso-C15:0 in cultures grown in complex or defined media at 37 degrees C. Determination of the fatty acid composition of L. monocytogenes 10403S and SLCC 53 grown over the temperature range 45 to 5 degrees C revealed two modes of adaptation of fatty acid composition to lower growth temperatures: (i) shortening of fatty acid chain length and (ii) alteration of branching from iso to anteiso. Two transposon Tn917-induced cold-sensitive mutants incapable of growth at low temperatures had dramatically altered fatty acid compositions with low levels of i-C15:0, a-C15:0, and a-C17:0 and high levels of i-C14:0, C14:0, i-C16:0, and C16:0. The levels of a-C15:0 and a-C17:0 and the ability to grow at low temperatures were restored by supplementing media with 2-methylbutyric acid, presumably because it acted as a precursor of methylbutyryl coenzyme A, the primer for synthesis of anteiso odd-numbered fatty acids. When mid-exponential-phase 10403S cells grown at 37 degrees C were temperature down-shocked to 5 degrees C they were able, for the most part, to reinitiate growth before the membrane fatty acid composition had reset to a composition more typical for low-temperature growth. No obvious evidence was found for a role for fatty acid unsaturation in adaptation of L. monocytogenes to cold temperature. The switch to a fatty acid profile dominated by a-C15:0 at low temperatures and the association of cold sensitivity with deficiency of a-C15:0 focus attention on the critical role of this fatty acid in growth of L. monocytogenes in the cold, presumably through its physical properties and their effects, in maintaining a fluid, liquid-crystalline state of the membrane lipids.     

Membrane lipid metabolism in Acholeplasma laidlawii A EF 22. Influence of cholesterol and temperature shift-down on incorporation of fatty acids and synthesis of membrane lipid species

1. Membrane lipid metabolism in Acholeplasma laidlowii A EF 22 has been studied under different conditions by applying three different techniques for changing membrane viscosity: fatty acid and cholesterol supplementation and temperature changes. 2. The molar relationship between the two dominating membrane lipids, monoglucosyldiglyceride and diglucosyldiglyceride, is to a large extent determined by membrane viscosity properties. This is shown by the varying metabolic responses occurring during incorporation of different fatty acids with and without cholesterol and by temperature shift-down experiments. Higher viscosity in membranes stimulates synthesis of monoglucosyldiglyceride at the expense of diglucosyldiglyceride. Synthesis of phospho and phosphoglucolipids is affected as well. 3. Temperature shift-down from 37 degrees C to 17 degrees C results in an immediate synthesis of monoglucosyldiglyceride accompanied by an increased incorporation of unsaturated fatty acids into this lipid. Synthesis of the other membrane lipid species (containing more unsaturated fatty acids) lags behind temporarily. 4. Incorporation from an equimolar mixture of palmitic and oleic acids together with cholesterol yields greater amounts of oleic acid in membrane lipids than incorporation in the absence of cholesterol, indicating that incorporation is viscosity dependent. 5. Studies of precursor relationships reveal that all main lipids have an active turnover which differs depending on membrane composition and conditions. Furthermore, this turnover proceeds with different intra-lipid pools. 6. Isolated membranes contain no detectable lipolytic enzymes capable of hydrolyzing membrane phospho or glycolipids. It is suggested that lipid turnover is partly mediated by enzymatic interlipid conversions, thus not allowing intermediates to accumulate.     

A hierarchical quorum-sensing system in Yersinia pseudotuberculosis is involved in the regulation of motility and clumping

In cell-free Yersinia pseudotuberculosis culture supernatants, we have chemically characterized three N-acyl homoserine lactone (AHL) molecules, N-octanoyl homoserine lactone (C8-HSL), N-(3-oxohexanoyl)homoserine lactone (3-oxo-C6-HSL) and N-hexanoyl homoserine lactone (C6-HSL). We have identified, cloned and sequenced two pairs of LuxR/I homologues termed YpsR/I and YtbR/I. In Escherichia coli at 37 degrees C, YpsI and YtbI both synthesize C6-HSL, although YpsI is responsible for 3-oxo-C6-HSL and YtbI for C8-HSL synthesis respectively. However, in a Y. pseudotuberculosis ypsI-negative background, YtbI appears capable of adjusting the AHL profile from all three AHLs at 37 degrees C and 22 degrees C to the absence of 3-oxo-C6-HSL at 28 degrees C. Insertion deletion mutagenesis of ypsR leads to the loss of C8-HSL at 22 degrees C, which suggests that at this temperature the YpsR protein is involved in the hierarchical regulation of the ytbR/I locus. When compared with the parent strain, the ypsR and ypsI mutants exhibit a number of phenotypes, including clumping (ypsR mutant), overexpression of a major flagellin subunit (ypsR mutant) and increased motility (both ypsR and ypsI mutants). The clumping and motility phenotypes are both temperature dependent. These data are consistent with a hierarchical quorum-sensing cascade in Y. pseudotuberculosis that is involved in the regulation of clumping and motility.