[Reactivating factor of Luteococcus japonicus subsp. casei: isolation and characterization].

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.

[Biological effect of extracellular peptide factor from Lateococcus japonicas subsp. casei on probiotic bacteria].

The biological effect of the extracellular peptide reactivating factor (RF) from Luteococcus casei on cells of probiotic cultures was studied. The RF showed the protective and reactivating effects on the Bifidobacterium bifidum cells under the action of bile salts and an acidic stress. Also, it acted as a cryoprotector during lyophilisation and long-term culture storage. The RF and the L. casei culture liquid (CL) were shown to have bifidogenic properties. The degree of protection and reactivation of lactic-acid bacteria under the action of bile salts depended on the particular strain properties. The maximum degree of protection (more than thirteen-fold) and reactivation (close to three-fold) was found in Lactobacillus casei, while the minimum values were characteristic of Lactobacillus reuterii. The resistance of lactobacilli to bile was increased in the row of L. acidophilus, L. casei, L. plantarum, L. rhamnosus, and L. reuterii correlating with the RF protection degree.

[Antistress cross-effects of extracellular metabolites of bacteria, archaea, and yeasts: a review].

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.

[Stress-protective and cross action of the extracellular reactivating factor of the microorganisms of the domains Bacteria, Archaea, and Eukaryota].

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.

[Antistress cross-protection of UV irradiated yeast cells with participation of extracellular peptide factors].

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.

[Protective and reactivating effect of the protein exometabolite on yeast cells inactivated by the ultraviolet irradiation].

It has been shown that Saccharomyces cerevisiae, Kluyveromyces lactis, and Candida utilis strains produce the protein exometabolites, which has a protective and reactivating effect on the ultraviolet irradiated yeast cells. The protective effect of the preliminary ultraviolet irradiated (activated) protein exometabolite of all strains increased 2-3 times, though its reactivating activity did not change. Yarrowia lipolytica yeast cells, isolated from the areas with the high daily irradiation, and Endomyces magnusii, the obligate fungi parasites, were characterized by the highest ultraviolet tolerance in comparison with the other strains. However, they did not produce the exometabolites with the antistress effect. Luteococcus casei reactivating factor demonstrated protective and reactivating cross-action in relation to the ultraviolet irradiated S. cerevisiae, K. lactis, and C. utilis cells and were inactive in relation to Y. lipolytica and E. magnusii. Using killer and nonkiller S. cerevisiae strain, it has been shown that the peptide exometabolite accumulation was not associated with toxin production.

[Protective action of reactivating factor of Luteococcus japonicus subsp. casei toward cells of Escherichia coli reparation mutants inactivated with UV-light].

Reactivating factor (RF) from Luteococcus japonicus subsp. casei had a protective action on UV-irradiated cells of Escherichia coli AB1157 with a native reparation system and on cells of isogenic reparation mutants of E. coli UvrA-, RecA-, and PolA-: the effect resulted in multifold increase of survivability. Defense action of L. casei exometabolite is not connected with stimulating reparation systems in E. coli, and, probably, it is mediated by involvement of the exometabolite in the mechanism of cell division. RF did not provoke the reactivation of E. coli cells inactivated by UV-light.

[The mechanism of action of reactivating factor from Luteococcus japonicus subsp. casei].

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 ofL. 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.

[Reactivating effect of Escherichia coli exometabolites on UV-irradiated cells].

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.

[Protective effect of extracellular protein metabolite of Luteococcus japonicus subsp, casei on cells subjected to heating and UV irradiation].

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.

[Cross-effects of extracellular factors of adaptation to stress in Luteococcus casei and Saccharomyces cerevisiae].

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 thereot) of the organisms studied are homologous.

[The extracellular protein of Luteococcus japonicus subsp casei reactivates cells inactivated by ultraviolet radiation or heating]. / Vnekletochnyi belok Luteococcus japonicus subsp. casei reaktiviruet kletki, inaktivirovannye ul'trafioletovym oblucheniem i nagrevaniem.

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 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.

[Extracellular protein metabolite of Luteococcus japonicus subsp. casei reactivates cells subjected to oxidative stress]. / Reaktiviruiushchee deistvie vnekletochnogo belkovogo metabolita Luteococcus japonicus subsp. casei na kletki, podvergnutye okislitel'nomu stressu.

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, the 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.

[Extracellular protein of propionic acid bacteria inhibits induced mutation in strains of Salmonella typhimurium]. / Vnekletochnyi belok propionovokislykh bakterii ingibiruet indutsiruemye mutatsii u shtammov Salmonella typhimurium.

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 based on the facts that this activity considerably decreased after the treatment of the CL with pronase, its heating at 92 degrees 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 P. shermanii cells.

[Isolation and purification of a protective protein from Propionibacterium freudenreichii subsp. Shermanii]. / Vydelenie i ochistka protektornogo belka iz kletok Propionibacterium freudenreichii substp. Shermanii.

A protein responsible for the protective and reactivating activities of two active fractions (AF1 and AF2) of the cells of Propionibacterium freudenreichii subsp. shermanii was isolated. The active fraction AF1 was obtained by fractional precipitation of the cell-free extract of propionic acid bacteria between 20 and 40% ammonium sulfate saturation, whereas fraction AF2 was precipitated between 60 and 80% saturation. Further fractionation of AF1 and AF2 by gel filtration on Sephacryl S-200 and by ion-exchange chromatography on DEAE-Sepharose yielded seven active subfractions, as revealed by testing for their protective activity on UV-inactivated cells of Escherichia coli. Analysis of subfraction AF2-2.5 by SDS-electrophoresis and HPLC showed that it contained an apparently homogeneous protein with a molecular mass of 44 +/- 2 kDa. The concentrational dependence of the protective activity of this protein was derived. Peptides of subfractions AF2-2.1 and AF2-2.2 with molecular masses lower than 15 kDa also exhibited protective activity.

[Protective and reactive effect of a cellular extract from propionic acid bacteria on Candida guilliermondii and Escherichia coli, inactivated by ultraviolet radiation]. / Protektornoe i reaktiviruiushchee deistvie kletochnogo ékstrakta propionovo-kislykh bakterii na Candida guilliermondii i Escherichia coli, inaktivirovannye ul'trafioletovym izlucheniem.

Dialyzed soluble proteins of Propionibacterium freudenreichii subsp. shermanii cell extract (dialyzate) were found to exert a protective and reactivating effect on Candida guilliermondii and Escherichia coli cells inactivated by UV light of C and B ranges. Reactivation occurred when dialyzate was added to irradiated bacterial suspensions immediately after irradiation or 15 min afterwards. Some common features of dialyzate effect on irradiated prokaryotic and eukaryotic cells were established. No reactivation was observed when the cells were irradiated with the light of visible (400-600 nm) or entire optical (> 290 nm) ranges. The possible mechanisms of a reactivating effect of dialyzate are discussed.

[Mechanism of reactivating Escherichia coli inactivated by ultraviolet light with cell extracts of propionic acid bacteria]. / Mekhanizm reaktivatsii inaktivirovannykh ul'trafioletovym svetom Escherichia coli kletochnymi ékstraktami propionovo-kislykh bakterii.

Two mechanisms of reactivation of UV-inactivated Escherichia coli cells--photoreactivation (PhR) and reactivation by the dialysates of propionic acid bacteria--are shown to be different but not completely additive. PhR displays an insignificant negative effect on the reactivation by active substances (peptides) of the dialysate, whereas reactivation by dialysate inhibits PhR. The maximal reactivation can be attained under complete PhR followed by the protective action of dialysate. The dialysate protects UV-irradiated E. coli cells with PolA, UvrA, and RecA mutations and Salmonella typhimurium TA 100 (UvrB) cells and also exerts an antimutagenic effect on S. typhimurium TA 100. Protection by dialysate is suggested to be due to restoration of the cell division mechanism damaged by UV irradiation.

[Reactivation of UV inactivated Escherichia coli cell extracts of propionic acid bacteria: fractionation of the extract]. / Reaktivatsiia inaktivirovannykh ul'trafioletovym svetom Escherichia coli kletochnym ékstraktami propionovo-kislykh bakterii fraktsionirovenie ékstrakta.

Cell free extract from Propionibacterium shermanii VKM-101 partially reactivates Escherichia coli AB 1157 irradiated by UV light. Fractionation of extract followed by the estimation of protective effect of fractions showed that this effect is linked to two fractions of soluble proteins. The fraction of cell walls, ribosomes and nucleic acids were poorly effective. Two active protein fractions (I-20-40% (NH4)SO4 and II-60-80% (NH4)2SO4) were separated by HPLC chromatography into 7 and 8 subfractions respectively. The activity was localised in subfraction N4 (fraction I) and N5.6 (fraction II).

[Reactivation of Escherichia coli inactivated by ultraviolet light by cell extracts of propionic acid bacteria]. / Reaktivatsiia inaktivirovannykh ul'travioletovym svetom Escherichia coli kletochnymi ékstraktami propionovo-kislykh bakterii.

For the first time reactivation of cell extract of three strains of Propionibacterium shermanii in UV inactivated not filament-forming strain Escherichia colli AB 1157 is shown. Reactivation was demonstrated in preincubated and postincubated test-culture and increased as survival of E. coli decreased in a range 1.8-0.006%. The factor (factors) of defense is dialysable, thermolabile and is present as in a fraction of nucleoproteins and nucleic acids so in a fraction of soluble proteins. The extracts were inactivated by incubation with proteinase K and trypsin, partly decreased activity by incubation with alpha-amylase and selected nuclease but not with lipase. Polypeptide nature of reactivating factor is supposed.