Strategies to enhance stress tolerance in lactic acid bacteria across diverse stress conditions.

Lactic acid bacteria (LAB) hold significant importance in diverse fields, including food technology, industrial biotechnology, and medicine. As basic components of starter cultures, probiotics, immunomodulators, and live vaccines, LAB cells resist a variety of stressors, including temperature fluctuations, osmotic and pH shocks, exposure to oxidants and ultraviolet radiation, substrate deprivation, mechanical damage, and more. To stay alive in these adversities, LAB employ a wide range of stress response strategies supported by various mechanisms, for example rearrangement of metabolism, expression of specialized biomolecules (e.g., chaperones and antioxidants), exopolysaccharide synthesis, and complex repair and regulatory systems. LAB can coordinate responses to various stressors using global regulators. In this review, we summarize current knowledge about stress response strategies used by LAB and consider mechanisms of response to specific stressful factors, supported by illustrative examples. In addition, we discuss technical approaches to increase the stress resistance of LAB, including pre-adaptation, genetic modification of strains, and adjustment of cultivation conditions. A critical analysis of the recent findings in this field augments comprehension of stress tolerance mechanisms in LAB, paving the way for prospective research directions with implications in fundamental and practical areas.

[CHARACTERISTICS OF FUNGAL STRAINS PRODUCING THERMOSTABLE XYLOGLUCANASES FROM THE RUSSIAN NATIONAL COLLECTION OF INDUSTRIAL MICROORGANISMS.]

Fungal strains degrading plant biomass available from the Russian National Collection of Industrial Microorganisms (VKPM) have been screened for the xyloglucanase activity. Under conditions of submerged cultivation, the thermophilic strains Sporotrichum thermophile VKPM F-972, Myceliophthora thermophila VKPM F-244, and Sporotrichum pruinosum VKPM F-235 produced extracellular xyloglucanases with optimal activity at 60°C, pH 5.0. 88-100% of the initial enzyme activity was retained after l-h incubation at 50°C; 79-84% of the activity was retained after l-h incubation at 60°C. S. thermophile VKPM F-972, M. thermophila VKPM F-244, and S. pruinosum VKPM F-235 strains may be used as the gene sources for construction of highly active producers of the thermostable xyloglucanases.

Analysis of the association of polymorphic loci rs917997 in IL18RAP gene and rs187238 in IL18 gene with the risk for non-Hodgkin's malignant lymphomas in Novosibirsk population.

We analyzed the association of polymorphic variants of rs917997 (G/A) locus in IL18RAP gene and rs187238 (G/C) locus in IL18 gene with the risk of malignant non-Hodgkin's lymphomas in Novosibirsk population. Allele and genotype frequencies of the above loci were determined in patients (243 persons) and control group (371 persons) and compared using χ(2) test. None of the analyzed loci showed statistically significant association with the risk of malignant non-Hodgkin's lymphomas.

Role of polymorphic loci in HLA-region rs2647012 and rs805288 in the development of non-Hodgkin's malignant lymphomas in Western Siberia.

The data of genome-wide association analysis suggest that human 6p21.3 chromosomal region (localization of HLA genes) contains polymorphic loci influencing the risk of developing non-Hodgkin's lymphomas. We analyzed association of rs2647012 and rs805288 loci with the risk for non-Hodgkin's malignant lymphomas in the population of Western Siberia. Allele and genotype frequencies were determined in the group of 298 patients and in the control group including 551 individuals. Subgroups of diffuse large B-cell lymphoma (86 patients) and follicular lymphoma (25 patients) were analyzed separately. An association of rs2647012 А/А genotype with increased risk of the disease (OR = 2.78, p = 0.002) was detected in the subgroup of diffuse large B-cell lymphoma.

[Expression of the genes CelA and XylA isolated from a fragment of metagenomic DNA in Escherichia coli].

The glycosyl hydrolase genes cel5A and xyl3A previously isolated by ourselves within a fragment of DNA from the methagenomic library of cow rumen microflora DNA were sub-cloned and expressed in E. coli. The recombinant proteins Cel5A and Xyl3A were purified and characterized. Cellulase Cel5A belongs to the Family 5 glycosyl hydrolases and is a one-module 38.2 kDa enzyme that hydrolyses the 1,4-glycoside bonds of soluble cellulose substrates and amorphous cellulose, showing its maximal activity (31200 u/mg) on lichenan, a soluble substrate with mixed (beta-1,3-1,4) bonds. The end product of the amorphous cellulose hydrolysis is cellobiose. Cel5A is inactive toward the crystal forms of cellulose. Cel5A is an endoglucanase capable of exohydrolysis. The molecular mass of beta-xylosidase Xyl3A belonging to the Family 3 glycosyl hydrolases is 83.7 kDa. The enzyme is active only on xylooligosaccharides, with the maximal activity shown on xylobiose, the end product of the reaction being xylose. No activity on xylane was hitherto observed. Recombinant Cel5A and Xyl3A are stable over a wide range of pH and temperatures, their maximal activity being observed at pH 6.5 and at 55 degrees C.

[Autoimmune reactions in diseases of the digestive system organs].

AIM: To examine content, diagnostic and prognostic role of autologous antibodies in gastrointestinal diseases (GID). MATERIAL AND METHODS: Enzyme immunoassay was used to measure content of autologous antibodies to N+/K+ ATPase (Aab) of gastric parietal cells, mitochondria, microsomes, tissue transglutaminase in blood serum of 196 patients with gastric, gallbladder, small and large intestinal diseases. Aab relations with heterologous antibodies were studied with kits provided by Bektor-Best (Novosibirsk), DRG-Diagnostics, Orgentec (Germany) and others. RESULTS: In GID high circulation of Aab to parietal cells (Ab-PC) was detected in 42% cases, mean content being 217 +/- 32.4 U/ml, 10 U/ml in the control. Maximal concentration (180 = 340 U/ml) occurred in hepatic cirrhosis, celiac disease, atrophic gastritis. In exacerbations of pancreatitis, colelithiasis and duodenal ulcer ab-PC concentration was 190-210 U/ml, in remission--6-12 U/ml. Minimal concentration (8-38 U/ml) was seen in polyps, gastric cancer, nonspecific ulcerative colitis. For primary biliary cirrhosis more typical was high A/ab concentration to mitochondria (in 83%; 200 U.ml), for autoimmune hepatitis--Aab to microsomes (in 81%; 170 U/ml), in celial disease--Aab to tissue transglutaminase (93%, 75 U/ml). High autoantibodies concentration in GID is accompanied with overcirculation of heteroantibodies to infectious-toxic agents confirming their role in development of autoimmune processes. CONCLUSION: GID are associated with high circulation of autologous antibodies--markers of systemic humoral autoimmune reactions differing in duration, severity, site of lesion, form, stage, disease duration. Maximal detection rate and concentration of serum autologous antibodies were observed in hepatic cirrhosis, active hepatitis, celiac disease, atrophic gastritis, exacerbations of cholelithiasis, ulcer, pancreatitis. Estimation of Aab concentration is essential for diagnosis, prognosis of autoimmune diseases, it reflects intensity and duration of autoimmune reactions in GID.

[Cloning and characterization of a large metagenomic DNA fragment containing glycosyl-hydrolase genes].

The problem of search for and characterization of enzymes synthesized by non-cultivated microorganisms is presently being settled by creating metagenomic libraries. A 6000-clone library with the average size of its inserts amounting to 15 bp has been constructed on the basis of total DNA isolated from cow rumen microorganisms. As the result of the screening of the library on plates with different substrates, a clone was selected that efficiently hydrolyzed lichenan and carboxymethylcellulose. The clone contained the recombinant plasmid pBlue-13 bearing a 12071 bp.-long metagenomic fragment carrying ten open reading frames, two of them being identified as glycosyl hydrolase genes. No homology of the metagenomic DNA with any known microorganism genomes was revealed. The amino acid sequence, deduced on the basis of frame 4 and denoted by Xyl3A, bears resemblance with beta-xylosidases of glycosyl hydrolase Family 3. Frame 6 encodes polypeptide Cel5A homologous to cellulases of glycosyl hydrolase Family 5. The amino acid sequences deduced on the basis of seven out of ten open reading frames were homologous to proteins of microorganisms belonging to the Bacteroides sp. family, the bacteria inhabiting mammalian intestines.

[Extracellular glycosyl hydrolase activity of the clostridia producing acetone, butanol, and ethanol].

Production of acetone, butanol, ethanol, acetic acid, and butyric acid by three strains of anaerobic bacteria, which we identified as Clostridium acetobutylicum, was studied. The yield of acetone and alcohols in 6% flour medium amounted to 12.7-15 g/l with butanol constituting 51.0-55.6%. Activities of these strains towards xylan, beta-glucan, carboxymethylcellulose, and crystalline and amorphous celluloses were studied. C. acertobutylicum 6, C. acetoburylicum 7, and C. acertobutylicum VKPM B-4786 produced larger amounts of acetone and alcohols and displayed higher cellulase and hemicellulase activities than the type strain C. acetobutylicum ATCC 824. It was demonstrated that starch in the medium could be partially substituted with plant biomass.

Glucose-1-phosphatase (AgpE) from Enterobacter cloacae displays enhanced phytase activity.

Using a screening procedure developed for detection of phytate hydrolysing enzymes, the gene agpE encoding glucose-1-phosphatase was cloned from an Enterobacter cloacae VKPM B2254 plasmid library. Sequence analysis revealed 78% identity on nucleotide and 79% identity on peptide level to Escherichia coli glucose-1-phosphatase characterising the respective gene product as a representative of acid histidine phosphatases harbouring the RH(G/N)RXRP motif. The purified recombinant protein displayed maximum specific activity of 196 U mg(-1) protein against glucose-1-phosphate but was also active against other sugar phosphates and p-nitrophenyl phosphate. High-performance ion chromatography of hydrolysis products revealed that AgpE can act as a 3-phytase but is only able to cleave off the third phosphate group from the myo-inositol sugar ring. Based on sequence comparison and catalytic behaviour against phytate, we propose to classify bacterial acid histidine phosphatases/phytases in the three following subclasses: (1) AppA-related phytases, (2) PhyK-related phytases and (3) Agp-related phytases. A distinguished activity of 32 U mg(-1) of protein towards myo-inositol-hexa-phosphate, which is two times higher than that of E. coli Agp, suggests that possibly functional differences in terms of phytase activity between Agp- and AppA-like acid histidine phosphatases are fluent.

[Thermoanaerobacter ethanolicus gene cluster containing the alpha- and beta-galactosidases genes melA and lacA, and properties of recombinant lacA].

The nucleotide sequence of a 4936 bp Thermoanaerobacter ethanolicus genomic DNA fragment containing the thermostable beta-galactosidase gene lacA and two incomplete open reading frames has been determined. The product of the first frame is highly homologous to alpha-galactosidases (melibiases), the product of the third frame is homologous to the alpha-D-mannosidases. The terminal area of the lacA, immediately following the stop-codon, harbors presumably a transcription termination site. Based on the location of the putative alpha-galactosidase gene melA and of the beta-galactosidase gene lacA on the T. ethanolicus chromosome, their combined transcription could be presumed. The calculated molecular mass of LacA is 86 kDa. LacA belongs to GH family 2 (GH2). Maximal activity of the purified recombinant enzyme was observed between pH values of 5.7 and 6.0 and temperatures of 75-80 degrees C. The highest activity, 480 units mg(-1), was found on lactose (Km 30 mM), the activities on pNPhGal and oNPhGal amounting to 330 and 420 units mg(-1), respectively. Immobilization on aldehyde silochrome increases the thermostability of the enzyme and keeps its high activity.

[Phytase activity in some groups of bacteria. Search for and cloning of genes for bacterial phytases]. / Fitaznaia aktivnost' u nekotorykh grupp bakterii. Poisk i klonirovanie genov bakterial'nykh fitaz.

A search for phytase genes in 9 Bacillus strains from the collection of IMGAN was implemented. The growth optimum of strains IX-22, IX-12B, K17-2, K18, IMG I, IMG II, M4 and M8 was 50-60 degrees C; the optimal growth temperature for Bacillus sp. 790 was 45-47 degrees C. According to the sequence data of 16S RNA genes, Bacillus sp. 790 belongs to the B. subtilis/amyloliquefaciens group. The other 8 strains were identified as B. licheniformis. Selection of Bacillus strains, potentially containing the phytase genes, was performed via PCR with primers designed on the basis of the conserved sequence regions of the phyA gene from B. amyloliquefaciens FZB45 with chromosomal DNA being used as the template. The nucleotide sequences of all PCR fragments showed a high level of homology to the known Bacillus phytase genes. The gene libraries of B. licheniformis M8 and B. amyloliquefaciens 790 in E. coli were constructed and phytase-containing clones were selected from them. Twenty-four Pseudomonas strains of different species, 5 Xanthomonas maltophilia strains and 1 Xanthomonas malvacearum (all from the mentioned collection) were tested for phytase activity. Such activity was found in 13 Pseudomonas strains and in 6 Xanthomonas strains. The accumulation of phytase in Pseudomonas was shown to take place at later (over 2 days') growth stages. The optimum pH for phytase from 3 Pseudomonas strains were established. The enzymes were found to be most active at pH 5.5.

[Thermotoga neopolitina gene cluster, participating in degradation of starch and maltodextrins: expression of aglB and aglA gene in Escherichia coli, properties of recombinant enzymes]. / Klaster genov Thermotoga neapolitana, uchastvuiushchikh v degradatsii krakhmala i mal'todekstrinov: éksptressiia genov aglB i aglA v Escherichia coli, svoistva rekombinantykh fermentov.

The aglB and aglA genes from the starch/maltodextrin utilization gene cluster of Thermotoga neapolitana were subcloned into pQE vectors for expression in Escherichia coli. The recombinant proteins AglB and AglA were purified to homogeneity and characterized. Both enzymes are hyperthermostable, the highest activity was observed at 85 degrees C. AglB is an oligomer of identical 55-kDa subunits capable of aggregation. This protein hydrolyses cyclodextrins and linear maltodextrins to glucose and maltose by liberating glucose from the reducing end of the molecules, and it is a cyclodextrinase with alpha-glucosidase activity. The pseudo-tetrasaccharide acarbose, a potent alpha-amylase and alpha-glucosidase inhibitor, does not inhibit AglB but, on the contrary, acarbose is degraded quantitatively by AglB. Recombinant AglB is activated in the presence of CaCl2, KCl, and EDTA, as well as after heating of the enzyme. AglA is a dimer of two identical 54-kDa subunits, and it hydrolyses the alpha-glycoside bonds of disaccharides and short maltooligosaccharides, acting on the substrate from the non-reducing end of the chain. It is a cofactor-dependent alpha-glucosidase with a wide action range, hydrolysing both oligoglucosides and galactosides with alpha-link. Thereby, the enzyme is not specific with respect to the configuration at the C4 position of its substrate. For the enzyme to be active, the presence of NAD+, DTT, and Mn2+ is required. Enzymes AglB and AglA supplement one another in substrate specificity and ensure complete hydrolysis to glucose for the intermediate products of starch degradation.

[Thermotoga neapolitana gene clusters participating in degradation of starch and maltodextins: molecular structure of the locus]. / Klaster genov Thermotoga neapolitana, uchastvuiushchikh v degradatsii krakhmala i mal'todekstrinov: molekuliarnaia struktura lokusa.

A 5451-bp genome fragment of the hyperthermophilic anaerobic eubacterium Thermotoga neapolitana has been cloned and sequenced. The fragment contains one truncated and three complete open reading frames highly homologous to the starch/maltodextrin utilization gene cluster from Thermotoga maritima whose genome sequence is known. The incomplete product of the first frame is highly homologous to MalG, the E. coli protein of starch and maltodextrin transport. The product of the second frame, AglB, is highly homologous to cyclomaltodextrinase with the alpha-glucosidase activity TMG belonging to family 13 of glycosyl hydrolases (GH13). The product of the third frame, AglA, is homologous to the Thermotoga maritima cofactor-dependent alpha-glucosidase from the GH4 family. The two enzymes form a separate branch on the phylogenetic tree of the family. The AglA and AglB proteins supplement each other in substrate specificity and can ensure complete hydrolysis to glucose of cyclic and linear maltodextrins, the intermediate products of starch degradation. The product of the fourth reading frame has sequence similarity with the riboflavin-specific deaminase RibD from T. maritima. The homologous locus of this bacterium, between the aglA and ribD genes, has five open reading frames missing in T. neapolitana. The nucleotide sequences of two frames are homologous to transposase genes. The deletion size is 2.9 kb.