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The chemical structure of the extracellular reactivating factor (RF) from Luteococcus japonicus subsp. casei was determined; this factor promotes survival of a small subpopulation of the producer cells under lethal stress impact. For the isolation and purification of this RF, the previously developed method for RF isolation from Saccharomyces cerevisiae was optimized. A total of 15 fractions were obtained from the culture liquid of Luteococcus casei, two of which (I and IV) exhibited reactivation activity against the cells subjected to a lethal stress impact (UV irradiation). The method included solid-phase extraction of the peptides on a hydrophobic sorbent with the C8 phase and subsequent multistage separation using RP-HPLC. Mass spectral analysis (MALDI-TOF) was used to determine the molecular characteristics of fraction IV. Efficient ionization was not achieved for fraction I. Mass charges for fraction IV were 773.394 and 788.102 Da. Edman automatic sequencing was used to identify these components as peptides: Ala-Pro-Asn-Glu-Asn-Gln-Gly and Ala-Pro-Asn-Glu-Glu-Gln-Gly. No similarity to any known full-size functional peptide molecules in the databases on polypeptide primary structures was revealed. Formation of biologically active peptides by L. casei may be associated with non-template synthesis and probably involves proteolysis of a large protein.

Protective effect of Saccharomyces cerevisiae exometabolites (RF1 and RF2) on seven strains of propionic acid bacteria under exposure to bile salts and acid stress was studied. RF2 had a stronger protective effect than RF1. Comparison of the analytical profiles of RF1 and RF2, separated by RP-HPLC, showed that nearly 80% of RF1 components were present in RF2. Separation of S. cerevisiae RF1 into individual components by means of semi-preparative RP-HPLC was used for structural characterization of the active component of the RF1 fraction. It was found to contain two pentapeptides (

Protective effect of the extracellular peptide fraction (reactivating factors, RF) produced by yeasts of various taxonomic groups (Saccharomyces cerevisiae, Kluyveromyces lactis, Candida utilis, and Yarrowia li- polytica) on probiotic lactic acid bacteria (LAB) Lactobacillus casei, L. acidophilus,'and L. reuteri under bile salt (BS)-induced stress was shown. RF of all yeasts were shown to be of peptide nature; the active component of the S. cerevisiae RF was identified as a combination of low-molecular polypeptides with molecular masses of 0.6 to 1.5 kDa. The protective and reactivating effects of the yeast factors were not species-specific and were similar to those of the Luteococcusjaponicus subsp. casei R. In BS-treated cells of the tester bacteria, a pro- tective effect was observed after 10-min preincubation of the LAB cell suspension with yeast RE: the number of surviving cells (CFU) was 2 to 4.5 times higher than in the control. The reactivating effect was observed when RF was added to LAB cell suspensions not later than 15 min after stress treatment. It was less pro- nounced than the protector effect, with the CFU number I to 3 times that of the control. Both the protector and the reactivating effects were most pronounced in the S. cerevisiae and decreased in the row: C. utilis > K. lactis > Y lipolytica. The efficiency of protective action of yeast RF was found to depend on the properties of recepient LAB cells, with the L. casei strain being most sensitive to BS treatment. In both variants, the highest protective effect of RF (increase in the CFU number) was observed for L. acidophilus, while the least pronounced one, for L. casei. The reasons for application of the LAB strains combining high stress resistance and high response to stress-protecting metabolites, including RF factors, as probiotics, is discussed.

Protective effect of Luteococcus japonicus subsp. casei exometabolites on the cells of transient probiotic Propionibacterium strains, Enterococcus faecium, and the yeasts Saccharomyces cerevisiae Boulardii under exposure to bile salts (BS) and acid stress was studied. The extracellular peptide reactivating factor (RF) and the peptide component of the culture liquid (CL) after RF removal possessed a protective effect. Protective (preventive) and reactivating (after stress impact) application of RF resulted in 1.5- to 2.0-fold increased survival of the human transient probiotics P. freudenreichii, P. acidipropionici and E. faecium subjected to BS treatment of acid stress. The CL peptide fraction had a stronger protective effect. Its application for preincubation of P. acidipropionici cells resulted in 14-fold (BS treatment) and 8-fold (acid stress) increased survival compared to the control. Yeasts exhibited very high resistance to the stress factors used. Two active glycopeptide fractions with molecular masses of 1.8 and 2.4 kDa were found in RF. Analysis of their amino acid composition revealed the residues of glycine, leucine, proline, arginine, and aspartate/asparagine, while no residues of aromatic and sulfur-containing amino acids, or the disulfide bridge-type posttranslational modifications, were found. The possible mechanisms of reactivating and the protective effect of RF are discussed.

It has been shown that a producer strain of reactivating factor (RF) is identical to a typical strain of Luteococcus japonicus DSM 10546 from the Propionibacteriaceae family according to the physiological and biochemical properties and the sequencing of 16S rRNA fragments. A number of phenotypical differences from the model strain allowed the producer strain to be considered a subspecies of Luteococcus japonicus, and it was named Luteococcus japonicus subsp. casei. At cultivation of the producer, RF is secreted into the medium and plays the role of a signaling molecule. RF antioxidant activities towards various organic radicals may be a possible mechanism of its protective and reactivating effects. Metabolites secreted by the L. casei producer strain into the culture medium were separated by a combination of liquid chromatographies. Four components possessing biological activities were found. The most active one was studied by MALDI-TOF mass spectrometry, which revealed that it is a polypeptide. Primary identification of some amino acid residues was performed. Sugar residues were found in the structure.

Cross protection of members of the domains Bacteria, Archaea, and lower Eukaryota from stress factors due to the action of extracellular low-molecular metabolites with adaptogenic functions was shown. The adaptogen produced by Luteococcus japonicus subsp. casei and described previously as a reactivating factor (RF) was shown to protect the yeasts Saccharomyces cerevisiae, archaea Haloarcula marismorti, and the cells of higher eukaryotes (HeLa) against weak stressor impacts. Production of an archaeal extracellular metabolite with a weak adaptogenic effect of the producer cells and capable of a threefold increase in survival of heat-inactivated yeast cells was discovered. Our results confirm the similarity of the compensatory adaptive reactions in prokaryotes (bacteria and archaea) and eukaryotes.

This paper reviews examples of specific and global responses of microorganisms and the characteristics of stress responses involving extracellular signaling metabolites. Information regarding the protective and reactivating effects produced by active exometabolites of representatives of domains of bacteria, archaea, and eukaryotes is summarized, and interdomain cross-responses to stressors are demonstrated.

The effect of the extracellular peptide reactivating factor (RF) synthesized by Luteococcus casei on stress response of Escherichia coli cells subjected to UV irradiation was studied. For these studies, we constructed a test strain carrying the umuD-lacZ operon. The expression rate of this operon reflects the rate of SOS response. Protective effect of RF, defined as the number of cells retaining the colony-forming activity (CFU) after UV irradiation (49–1166 J/m2), was dose-dependent, species-nonspecific, and increasing with increase of the stress load. RF was demonstrated to possess the properties of a direct adaptogen: 15 min of preincubation with RF caused a 1.5–6-fold decrease in expression of the umuD SOS response gene in UV-treated cells, concurrently with a 1.2–7.5 times increase in the number of viable cells (those having retained their colony-forming activity). The probable mechanisms of the protective effect of RF are being discussed.

It was shown that matrix-assisted laser desorption/ionization (MALDI) mass spectrometry could be used for the diagnostic characterization of propionic acid bacteria (PABs). The spectra of proteins (whole PAB cells) with a molecular mass of 3000 to 11 000 were obtained and analyzed using three matrices: sinapinic (SA), 2,5-dihydroxibenzoic (DHB), and α-cyano-4-hydroxycinnamic acid (HCCA). The MALDI spectra of PAB revealed the protein peaks characteristic of (1) the genus Propionibacterium (3496, 5386, 5605, 10 470), (2) the groups of species sharing the common composition of their cell walls and fatty acids, and (3) a species (four species were investigated). Exemplified by the P. shermanii strains (the collection and mutant ones) producing and not producing vitamin B12, the possibility of using MALDI profiles for strain differentiation was confirmed. The MALDI profiles of the propionic acid cocci of the genus Luteococcus differ substantially from the profiles of PAB strains of the genus Propionibacterium, which is an additional proof of the validity of whole-cell MALDI spectra for generic differentiation of bacteria. Our investigation shows that the bacterial groups determined using the MALDI profiles correlate with the phylogenetic 16S rRNA gene groups, thus demonstrating the high resolution of this method for the differentiation of intraspecific differences (subspecies, strains).

Antistress effect of extracellular peptides on UV-irradiated yeast of different phylogenetic groups was studied. Yeast from different ecotopes and taxonomic groups exposed to UV radiation of a lethal intensity showed a protective effect and reactivating effect with participation of extracellular peptides. The highest protective activity was found in peptide reactivation factors (RFs) of bakery yeast—Saccharomyces cerevisiae, Kluyveromyces fragilis, and Candida utilis; the highest reactivating activity was exhibited by factors of the above-mentioned cultures and Debariomyces hansenii. Cross-protective and reactivating effects of RFs of yeast belonging to different taxonomic groups were demonstrated. Cross-protection increased two to three times after preexposure of reactivation factors to UV light (activation) in contrast to their reactivating effect.

Reactivating factor (RF) from Luteococcus japonicus subsp. casei was shown to be constitutively synthesized and to act a by one-step mechanism, being activated independently from stress. Cell reactivation (reversion of a cell’s ability to form macrocolonies) might be ensured by the membrane mechanism of RF action, which is proved with the dependence of antistress activity from the condition of the cytoplasmic membrane and with the form of concentration dependence. The incubation of UV-treated L. casei suspension with RF increased the number of cells with intact barrier membrane (1.6–1.8-fold increase compared to RF-untreated cells) and the number of colony-forming cells. Cross defensive and reactivating RF effects on both L. casei and yeast Saccharomyces cerevisiae cells were described. Bacterial and yeast’s RF compete for membrane receptors. Matrix Assisted Laser Desorption/Ionization time-of-flight (MALDI-TOF) spectrometry revealed that RF of L. casei contained two major peptides of 5.8 and 7.6 kDa, while RF of S. cerevisiae was represented by a single peptide of 5.8 kDa. The presence of 5.8 kDa peptide in RF from bacteria and yeasts might ensure cross responses in these organisms.

The colony-forming ability of long (3–9 months) incubated cystlike resting cells (CRC) of the nonspore-forming gram-positive bacteria Micrococcus luteus and Arthrobacter globiformis was studied in this work. The preservation of the CRC proliferative potential as assayed by plating on standard LB agar was shown to depend on the conditions of the formation of the dormant cells. In aged post-stationary cultures of micrococci and arthrobacters grown under carbon and phosphorus limitation the number of colony-forming units (CFU/ml) of CRC decreased in the course of 3–9 month incubation to the level of 106–107 CFU/ml. However, M. luteus CRC obtained under carbon and nitrogen limitation and A. globiformis CRC obtained under nitrogen limitation and starvation completely lost their ability to form colonies on standard solid medium after 4–6 months of incubation and turned into a ‘non-culturable’ (non-platable) state. In this case, the ratio of live cells in the population of M. luteus and A. globiformis ‘non-culturable’ CRCs (determined by the Live/Dead staining test) was 10–44% of the total cell number. To study the possible preservation of proliferative potential in non-platable CRCs, various methods of their reactivation were applied. Although preincubation of CRC suspensions in a buffer solution of 0.1 M K2HPO4 (pH 7.4) or in the presence of lysozyme (1 or 10 μg/ml) resulted in increased numbers of live cells (determined by the Live/Dead test) or in disruption of the cell conglomerates, it did not increase considerably the CFU titer on LB medium. Variations in the medium composition, such as addition of sodium pyruvate as an antioxidant or dilution of the medium, promoted the formation of macrocolonies by a small portion of nonplateable CRC of M. luteus (50−80 CFU/ml), whereas the number of the cells capable of microcolony formation (mCFU) was 1.8–6.8 × 105 mCFU/ml, exceeding the CFU titers by four orders of magnitude. The application of semisolid agar and the most probable number (MPN) method was the most efficient for determination of the mCFU titer, and an almost complete reversion of ‘non-culturable’ micrococcal CRCs to microcolony formation was observed (up to 2.3 × 107 mCFU/ml). The usefulness of diluted complete media for the restoration of the colony-forming ability of the dormant forms was confirmed in experiments with ‘nonculturable’ CRCs of A. globiformis. The development of special procedures and methods for determining actively proliferating cells not detected by ordinary methods is of great importance for advanced monitoring studies.

Wild-type and mutant (AB 1157 and K-12) strains of Escherichia coli were shown to synthesize the logarithmic growth phase, exometabolites reactivating UV-irradiated cells of producer strains. The exometabolites of the strain K-12 were of protein nature and had a molecular weight of no more than 10 kDa. The reactivating activity of these exometabolites was inversely related to bacterial survival and slightly increased under the influence of stress factors. The reactivating factor of Luteococcus casei had a cross-reactivating and protective effect on UV-irradiated cells of E. coli strain K-12. Due to activation of the reactivating factor after UV irradiation and heating, the cross-protective effect increased more than threefold. The reactivating effect remained unchanged under these conditions. The protein exometabolites of E. coli did not induce cross-stress response in L. casei.

Extracellular protein metabolite isolated from the culture liquid of Luteococcus japonicus subsp. casei had reactivating and protective effects on UV-irradiated and heated cells. The extracellular metabolite, produced by cells in the logarithmic growth phase, was present in culture liquid in minuscule amounts. Mass spectral analysis showed that, along with the major component with a molecular weight of 7.6 kDa, the preparation contained low quantities of three minor proteins. Apparently, the biological activity of the exometabolite is determined by the major polypeptide component.

Saccharomyces cerevisiae yeasts (lower eukaryotes) were shown to produce a protein exometabolite with reactivation activity. We demonstrated cross-effects of extracellular protein factors of adaptation to stress (heat and UV irradiation) in yeasts and Luteococcus casei bacteria. The possibility for isolation and partial purification of protein exometabolites from the culture liquid of yeasts and bacteria by similar methods, as well as the similarity of elution profiles for the active proteins in high-performance liquid chromatography, suggests that the proteins (or fragments thereof) of the organisms studied are homologous.

Recent data on the molecular mechanisms of the stress responses of bacteria are reviewed, with emphasis on their reactions to a variety of stressors (heat, oxidation, cold, osmotic shock, etc.). The mechanisms underlying the phenomenon of sensoring are discussed. It is shown that cross-resistance to stressors and cell-to-cell communication, mediated by chemical metabolites, affect bacterial survival in food products. The stress-antagonizing activity of bacteria is discussed in relation to food product biotechnology.

The culture liquid of Luteococcus japonicus subsp. casei was found to be able to reactivate cells of this bacterium inactivated by UV irradiation or heat shock. The antistress activity of the culture liquid was due to the presence of an extracellular exometabolite of a protein nature with a molecular mass of more than 10 kDa. When the bacterium was grown in a nutrient broth or glucose-containing mineral medium, the antistress protein was secreted by cells in the logarithmic growth phase. The reactivating effect of the antistress protein was inversely proportional to the survival rate of stressed cells.

A protein exometabolite isolated from the culture liquid of Luteococcus japonicus subsp. casei reactivates the cells of this microorganism following H2O2- or paraquat-induced oxidative stress. The resistance of L. casei cells to these oxidizers is accounted for by the high activity of superoxide dismutase and catalase. The effect of the protein exometabolite is universal, in that it reactivates the cells after UV irradiation, heating, or oxidative stress. However, cells subjected to oxidative stress are significantly less susceptible to the reactivating effect as compared to their UV-irradiated or heated counterparts. Possible causes of these differences are discussed.

Lactic acid and propionic acid bacteria, bifidobacteria, and fecal enterococci associated with the activity of humans and animals caused antimutagenic effects (AME) on many test systems designed for detecting point mutations and chromosomal aberrations. Bacterial cells and some of their metabolites attenuate the mutagenic action of several genotoxic agents, and this effect is mediated by the mechanism of dysmutagenesis and/or bioantimutagenesis. Possible mechanisms of various AMEs and possible practical applications of antimutagenic properties of bacteria are discussed.

A culture of propionic acid bacteria grown in a glucose-containing minimal medium, as well as the culture liquid and logarithmic-phase cells obtained from this culture, were found to inhibit the base pair substitution mutations induced by 4-nitroquinoline N-oxide, N-methyl-N"-nitro-N-nitrosoguanidine, and sodium azide and the frameshift mutations induced by 9-aminoacridine. The antimutagenic activity of the culture liquid (CL) was presumably due to the presence of an extracellular thermolabile protein with a molecular mass of no more than 12 kDa, as evidenced by the facts that this activity considerably decreased after the treatment of the CL with pronase, its heating at 92°C, and its dialysis in a cellulose sack, which retains substances with molecular masses greater than 12 kDa. The residual antimutagenic activity of the dialyzed culture liquid was probably related to the interaction of the mutagen with thiols, rather than to the presence of organic acids (acetic or propionic). Thiols may also contribute to the antimutagenic activity of the Propionibacterium shermanii cells.

The microbiological transformation of 1-benzoylpiperidine and 1-benzoylpyrrolidine by cultures of the fungi Aspergillus niger VKM F-1119 and Cunninghamella verticillala VKPM F-430 proceeds regio- and stereospecificall and leads respectively to the 4- and 3-hydroxy derivatives in 40–80% yield. The spectrometric properties of these compounds were studied.