Search

Beta-lactamases (EC 3.5.2.6) represent a superfamily containing more than 2,000 members: it includes genetically and functionally different bacterial enzymes capable to destroy the beta-lactam antibiotics. The most common are beta-lactamases of molecular class A with serine in the active center. Among them, TEM-type beta-lactamases are of particular interest from the viewpoint of studying the mechanisms of the evolution of resistance due to their broad polymorphism. To date, more than 200 sequences of TEM-type beta-lactamases have been described and more than 60 structures of different mutant forms have been presented in Protein Data Bank. We have considered the main structural features of the enzymes of this type with particular attention to the analysis of key drug resistance and the secondary mutations, their location relative to the active center and the surface of the protein globule. We have developed the BlaSIDB database (www.blasidb.org) which is an open information resource combining available data on 3D structures, amino acid sequences and nomenclature of the corresponding forms of beta-lactamases.

The aim of research was to investigate the prevalence of complex mannan-degrading enzymes among museum and freshly isolated cultures of micromycetes, actinobacteria and bacteria. It has been shown that the producers of β-mannanases mostly extracted from sources that are rich in plant residues. We detected strains of Penicillium aculeatum, P. tardum and P. rugulosum which produce a complex of β-mannanases and α-galactosidase activity. Also, strains of thermophilic micromycetes species Corynascus sepedonium, Scytalidium thermophilum and Rhizomucor tauricus with high mannanase activity in culture liquid were detected (10 – 130 U/ml). Two strains of P. aculeatum and P. tardum showed β-mannanase, β-mannosidase and α-galactosidase activity. Actinobacteria was shown high potential, 70 % of all tested strains showed mannanase activity; their activity ranged from 2 to 55 U/ml. Among the most active bacterial cultures were representatives of soil microflora: Bacillus circulans, B. subtilis, B. mesentericus, where as among the strains isolated from sea water the active mannanases producers were not found.

Evolutionary connections were analyzed for endo-β-xylanases, which possess the GH10 family catalytic domains. A homology search yielded thrice as many proteins as are available from the Carbohydrate-Active Enzymes (CAZy) database. Lateral gene transfer was shown to play an important role in evolution of bacterial proteins of the family, especially in the phyla Acidobacteria, Cyanobacteria, Planctomycetes, Spirochaetes, and Verrucomicrobia. In the case of Verrucomicrobia, 23 lateral transfers from organisms of other phyla were detected. Evolutionary relationships were observed between the GH10 family domains and domains with the TIM-barrel tertiary structure from several other glycosidase families. The GH39 family of glycoside hydrolases showed the closest relationship. Unclassified homologs were grouped into 12 novel families of putative glycoside hydrolases (GHL51-GHL62).

Methyl-directed site-specific DNA endonucleases (MD endonucleases) form a small group of enzymes which specifically cleave only methylated DNA. There are N6-methyladenine- and 5-methylcytosine-directed enzymes in this group. In spite of limited information on the MD endonucleases they are considered to be a very interesting subject for both fundamental investigations and practical use in biotechnology and epigenomics. In this review for the first time the data on properties of MD endonucleases are summarized in particular on substate specificity of these enzymes. The role of MD endonucleases in bacterial cells and the potential of their practical use are also discussed.

The nucleotide sequence of a chromosome fragment of the thermophilic anaerobic bacterium Caldicellulosiruptor bescii (syn. Anaerocellum thermophilum) has been determined. The fragment contains four open reading frames with the second one of 749 aa encoding a multimodular endo-1,4-beta-glucanase CelD (85019 Da). N-terminal region of the protein includes the signal peptide and the catalytic module of glycoside hydrolase family 5 (GH5), followed by the substrate-binding module of family 28 (CBM28). The C-terminal region bears three SLH modules. The recombinant endoglucanase and its two separate modules, the catalytic one and CBM28, were produced in E. coli cells and purified to homogeneity. Analysis of the catalytic properties showed CelD to be endo-1,4-beta-glucanase whose maximum activity was exhibited on beta-glucan of barley at pH 6.2 and 70 degrees C. The enzyme was stable at 50 degrees C for 30 days. Upon removal of the C-terminal CBM28, the activity of GH5 decreased on cellulose substrates, and its thermostability was dropped. Binding of CBM28 to amorphous cellulose was almost irreversible as it could not be removed from this substrate in a range of pH 4-11, temperatures--of 0-75 degrees C, and NaCl concentration--of 0-5 M. Only 100% formamide or 1% SDS were able to remove the protein.

Homooligomeric LonA proteases are the key components of the protein quality control system in bacteria and eukaryotes. Domain organization of the common pool of LonA proteases is determined by comparative analysis of primary and secondary structures of a number of bacterial and eukaryotic enzymes. The similarity of individual enzyme domains was estimated, domain-domain linker areas were revealed, regions that are capable to include intercalated peptide fragments were identified. LonA proteases were shown to be unique AAA+ proteins, because in addition to the classic AAA+ module they contain a part of another AAA+ module, namely the alpha-helical domain including a coiled-coil region, which is similar to the alpha-helical domain of the AAA(+)-1 module of the chaperone-disagregases ClpB/Hsp104.

F1-ATPase is a catalytic portion of the rotary molecular motor, F1Fo-ATP synthase. Cooperative ATP hydrolysis at the three catalytic sites of the F1-ATPase is connected with rotation of the central gamma-subunit inside a cylinder made of three a subunits and three beta subunits. Various experimental works have shown that the gamma-subunit rotates with irregular dwells. A simple kinetic model of this paper explains dwells during rotation as a result of the deterministic chaos. It is shown that the deterministic chaos occurs under the rate constants close to the known experimental estimations. Time duration of dwells in the model are close to those observed experimentally. Our model explains the known irregular occupancy of catalytic sites of F1-ATPase by nucleotides.

The data of local microbiologic monitoring was used to study the profiles and mechanisms of resistance to beta-lactams antibiotics of gram-negative isolates of bacteria, pathogens of hospital-acquired infections in hospital reanimation and surgical departments. The study included 210 clinical isolates of pathogens of hospital-acquired infections: Pseudomonas aeruginosa--86 (40.9%), Acinetobacter baumannii--45 (21.4%), Klebsiella pneumoniae--52 (24.8%), Escherichia coli-23 (11.0%), Enterobacter spp.--4 (1.9%). The profiles of resistance to antibiotics were analyzed using the technique of serial micro-dilutions. The detection of the most common and clinically significant gens of beta-lactamases of gram-negative bacteria was implemented using the PCR technique and sequence analysis. The most activity was detected among carbapenems and cephaperazone/sulbactam. The local characteristics of prevalence of gens coding beta-lactamases (TEM, SHV, CTX) in K. pneumoniae and E. coli were established The study detected 11 isolates of P aeruginosa resistant to carbapenems and having genetic determinants of VIM-group and coding metal-beta-lactamases. The discussed data permits to assess the indicators of resistance to beta-lactams antibiotics and basic mechanisms of resistance of causative agents of hospital-acquired infections. The studies of this kind are unique for every type of hospital-acquired infection. The results can be used in the development of the concept of etiotropic and empiric therapy.

Domains of fifteen recently found families of hypothetical glycoside hydrolases (GHL1-GHL15) have been used for iterative screening of the protein database. Evolutionary connections between representatives of these families were revealed. Also, their relationship with members of the following known families of protein domains were found: GH5, GH13, GH13_33, GH17, GH18, GH20, GH27, GH29, GH31, GH35, GH36A, GH36B, GH36C, GH36D, GH36E, GH36F, GH36G, GH36H, GH36J, GH36K, GH39, GH42, GH53, GH66, GH97, GH101, GH107, GH112, GH114, COG1082, COG1306, COG1649, COG2342, DUF3111, and PF00962. The unclassified homologues were grouped into 35 new families of hypothetical glycoside hydrolases: GHL16-GHL50. Position of GHL1-GHL15 families in the hierarchical classification of glycoside hydrolases and their homologues is discussed. Several new superfamilies of protein domains are suggested.

Investigation of 15 different glycoside activities of 64 strains isolated from water and invertebra of the Black Sea has shown that 64% of the studied strains displayed the capacity to synthesize enzymes with alpha-L-ramnosidase activity which varied from 0.01 to 0.20 un/ml depending on the strain. The greatest number of the enzyme producers was found in representatives of Alteromonas macleodii. Other investigated glycosidase activities: alpha-amylase, beta-N-acetyl-D-glucosaminidase, beta-D-glucuronide, alpha-N-acetyl-D-galactosaminidase, beta-N-acetyl-D-galactosaminidase, beta-D-galactosidase, alpha-D-galactosidase, beta-D-glucosidase, KM-cellulase activities though have been found, but mainly with inconsiderable indices, alpha-D-glucosidase, alpha-D-mannosidase, alpha-L-fucosidase, beta-D-xylosidase and alpha-D-xylosidase activities were found in neither of the studied strains. Strains with rather high proteolytic activity were found among marine species of bacteria. It has been established that 18 strains (28%) of 64 marine isolates were characterized by rather high level of total proteolytic activity (from 0.1 to 05 un/ml), 43.75% of them displayed inconsiderable (up to 0.1 un/ml) or only trace (up to 0.01 un/ml), 18.75% did not display any hydrolytic activity in respect of casein. Investigation of substrate specificity to a number of fibrillar and globular proteins of 9 studied strains, which displayed considerable general (caseinolytic) activity has shown that 8 of them displayed fibrinolytic activity from 0.15 to 2.175 un/ml. All 9 strains were characterized by gelatin activity. Collagenase and keratinase activity was also revealed. Neither of 9 studied strains displayed elastase activity.

Endo-alpha-1,4-polygalactosaminidase is a rare enzyme. Its catalytic domain belongs to the GH114 family of glycoside hydrolases. Phylogenetic analysis of the family proteins allowed us to show an important role of duplications, eliminations, and horizontal transfer in the evolution of their genes. Domain structure, the secondary structure, and proposed structure of the active center of the endo-alpha-1,4-polygalactosaminidases are discussed. Evolutionary connections of the GH114 family with GH13, GH18, GH20, GH27, GH29, GH31, GH35, GH36, and GH66 families of glycoside hydrolases, as well as, with COG1306, COG1649, COG2342, GHL3, and GHL4 families of enzymatically uncharacterized proteins have been revealed by iterative screening of the protein database. The unclassified homologues have been grouped into 13 new families of hypothetical glycoside hydrolases: GHL5 - GHL15, GH36J, and GH36K.

The influence of a number of coordinative compounds of zinc with N-substituted thiocarbamoil-N'-pentamethylensulfenamides on activity of elastase, alpha-L-rhamnosidase and alpha-galactosidases evidence for a possibility of their usage as stimulators or inhibitors of enzymes tested have been studied. It was shown that all the compounds in concentration of 0.1 and 0.01% inhibited by 90-100% Bacillus thuringiensis 27-88Els+ elastase activity. [Zn(L2)Br2], [Zn(L1)(NCS)2] and [Zn(L3)(NCS)2] at 20 h exposition activated Cryptococcus albidus 1001 alpha-L-rhamnosidase activity. The rest of compounds influenced it on the control level or inhibited it by 7-23%. The obtained results testify that essential role is not played by separate fragments (L-ligand and anions), but by molecules of zinc complexes as a whole. All the studied complexes, exept for [Zn(L3)(NCS)2], induced alpha-L-rhamnosidase activity of Eupenicillium erubescens 248 (7 to 60%). All zinc compounds (concentration 0.01%, exposition time - 60 min) influenced at the control level Aspergillus niger and Cladosporium cladosporioides alpha-galactosidases activity, however inhibited (up to 20%) activity of Penicillium canescens alpha-galactosidase. The increasing of exposition time of the compounds tested with enzymes up to 20 h testify to selective action of separate compounds on enzymes tested. The data obtained prove, that the character of interaction of zinc complexes is changed depending on the enzyme tested and its strain-producer.

Transglutaminasc (protein-glutamine gamma-glutamyltransferase, EC 2.3.2.13, TG) catalyzes reactions of the acyl transfer, which introduce the epsilon-(gamma-glutamyl)lysine bonds between proteins to create polymers of high mol. mass. Properties of the TG enzyme are described. Its structure is considered: there are characterized items of the TG life cycle and stability, its biological (physiological) role, and significance in pathology and medicine as well as obtaining of the purified enzyme preparations and their use. There are compared TG from different sources: of animal and microbial origin. Mechanism of catalysis of microbial TG is discussed. There are presented characteristics of isoenzymes from different biological sources.

Influence of nitrogen source on proteinases synthesis in aerobic alkalotolerant and halotolerant bacteria from soda-salt lakes of Transbaikalia was studied. Maximal accumulation of proteinases was revealed on medium with peptones. Introduction of various sources of nitrogen in the medium did not result in increase of enzyme activity in cultural liquid. It was indicated that secreting proteinases of the studied bacteria strains possess narrow substrate specificity, hydrolyze proteins and n-nitroanilide substrates have maximal activity during GlpAALpNA hydrolysis. Data of inhibitory analysis and substrate specificity of studied extracellular enzymes indicate that they belong to a class of serine proteinases of subtilisin-like type.

Experimental rationale is provided for the parameters of a rapid (1-2-min) test to concurrently determine the oxidase activity of all bacteria grown on the membrane filter after water filtration. Oxidase reagents that are the aqueous solutions of tetramethyl-p-phenylenediamine dihydrochloride and demethyl-p-phenylenediamine dihydrochloride have been first ascertained to exert no effect on the viability and enzymatic activity of bacteria after one-hour contact. An algorithm has been improved for the rapid oxidase activity test: the allowable time for bacteria to contact oxidase reagents and procedures for minimizing the effect on bacterial biochemical activity following the contact. An accelerated method based on lactose medium with tergitol 7 and Endo agar has been devised to determine coliform bacteria, by applying the rapid oxidase test: the time of a final response is 18-24 hours. The method has been included into GOST 52426-2005.

The paper gives the results of studying the enzymatic activity of bacteria in the microbial community of the Lena River in the central part of Yakutia (the town of Yakutsk and its adjoining areas). The study properties were found in 31.8% of the bacteria. Aeromonas had the highest enzymatic activity. High (22.0%) DNAase production rates were noted in Enterobactericeae.

The analysis of modern data on biochemical adaptation of microorganisms for living in extreme conditions is presented in this review. Special attention has been paid to the analysis of adaptive responses of microorganisms to the conditions of increased radiation at the molecular and cellular levels. The data on the practical application of extremophils and extremoenzymes, synthesized by them, biologically active substances, biopolymers and so on, have been systematized.

The bacteria capable of producing the enzymes hydrolyzing carbohydrates of various degrees of complexity (from starch to sucrose) were found to be associated with the intestinal mucosa of fishes and tegument of cestodes. Presence of the bacteria displaying the sucrase activity is especially important for macroorganisms, as bacteriogenous glucose can be used by all members of the arising community. The greatest contribution to the hydrolysis of carbohydrates (both in host and parasite) is obviously made by those microorganisms which are more closely connected with the digestive-transport surfaces and are hardly removable from the intestines by peristalsis. The levels of total amylolytic activity of bacteriogenous enzymes and activity of their a-amylase under the experimental conditions are comparable to those of the enzymes involved in membrane digestion of the host and parasite, which can be evidence of the significant contribution of enzymes produced by symbiotic microflora to the digestive processes in macroorganisms.

The article describes a story of the discovery and properties of bacterial metalloproteinase ECP32 that cleaves actin at the only site between Gly42 and Val43. This site is intensively involved in the monomer-monomer contacts within actin filament. Cleavage with ECP32 results in a reversible loss of actin polymerizability. Therefore ECP cleaved actin has been a unique model to study mechanisms of actin polymerization and the filament dynamics. and the effects of actin-binding proteins on these processes, as well as to reveal allosteric effects in actin molecule and to determine three-dimensional actin structure that was previously determined only for the actin-ligand complexes. Furthermore, the non-pathogenic bacteria synthesizing ECP32 were shown to penetrate in eukaryotic cells rearranging their cytoskeleton. Biochemical analysis using the Vitek-2 system and sequencing of the 16S rRNA gene reidentified the ECP32-producing strain, previously identified as E. coli, as Serratia grimesii. Bacteria of a reference strain S. grimesii were found to express the gene of metalloprotease that cleaves actin similarly to ECP32. The gene was cloned, sequenced and expressed in E. coli. The protein encoded by this gene was named grimelysin. Grimelysin shared essential characteristics of ECP32: molecular weight, limited actin proteolysis, inhibition by chelating agents, cleavage site, and the N-terminal amino acids of the active enzyme published for ECP32. These data show that grimelysin and ECP32 seem to be the same protein. As it was demonstrated by confocal microscopy the bacteria capable of synthesizing natural or recombinant grimelysin acquire invasive phenotype. The data described here allow us to suggest that actin may be a target protein which proteolysis promotes bacterial invasion.

Type II restriction-modification systems are comprised of a restriction endonuclease and methyltransferase. The enzymes are coded by individual genes and recognize the same DNA sequence. Endonuclease makes a double-stranded break in the recognition site, and methyltransferase covalently modifies the DNA bases within the recognition site, thereby down-regulating endonuclease activity. Coordinated action of these enzymes plays a role of primitive immune system and protects bacterial host cell from the invasion of foreign (for example, viral) DNA. However, uncontrolled expression of the restriction-modification system genes can result in the death of bacterial host cell because of the endonuclease cleavage of host DNA. In the present review, the data on the expression regulation of the type II restriction-modification enzymes are discussed.

Microbiological and biotechnological characteristics of intensification of aerobic processing of organic waste have been reviewed, with a view for revealing two types of correlations: (1) between the quality of the composts obtained and the microorganisms involved in composting and (2) between physicochemical parameters and consumer properties of the composts.

The ability of bacterial DNA methyltransferases Alu I, Cfr I, Cfr 6, Cfr 10, Eco RI, Eco RII, Msp I, Mva I, Pvu I, Pvu II, and Sau 3A to use methyl-cobalamine and methyl-methionine as cofactors of DNA methylation in vitro. These bacterial DNA methyl transferase used methyl-cobalamine, but not methylmethionine for DNA methylation.

The characteristics of xylose isomerase biosynthesis in the bacteria Arthrobacter nicotianae BIM B-5, Erwinia carotovota subsp atroseptica jn42xylA, and Escherichia coli HB101xylA have been studied. The bacteria formed the enzyme constitutively. Out of the carbon sources studied, D-glucose and D-xylose were most favorable for the biosynthesis of xylose isomerase in E. carotovota subsp atroseptica, but the least appropriate in terms of the enzyme production efficiency in E. coli. Minimum and maximum levels of xylose isomerase formation in A. nicotianae were noted, respectively, during D-xylose and sucrose utilization. An addition to the nutrient medium of 0.1-1.5% D-glucose (together with D-xylose) did not affect the enzyme synthesis in A. nicotianae, but suppressed it in Erwinia carotovota subsp atroseptica (by 7% at the highest concentration) and Escherichia coli (by 63 and 75% at concentrations of 0.1 and 1.0%, respectively). The enzyme proteins produced by the bacteria exhibited the same substrate specificity and electrophoretic mobility (PAGE) as xylose isomerase A. nicotianae, although insignificant differences in the major physicochemical properties were noted.

To get better appreciation of the margins of phenotypic adaptation and genotypic changes in bacteria-fungi associations within the typical microbiota residing on structural materials of space-flown equipment, developed were a program and hardware for a series of experiments under the general name BIORISK. Protocol of each experimental cycle is based on the well-proven method of exposure of "passive" samples of materials (Biorisk-KM), microorganisms-materials systems inside the ISS service module (Biorisk-MSV), and microorganisms-materials systems on the outside of the ISS SM (Biorisk-MSN). Each six months the samples are returned to the laboratory in conjunction with crew rotation. Already the first in-hand data from the experiment point to the dramatic effect of space flight on growth, reproduction, and biological properties of test microbes and fungi. Thus, the activity of enzymes that characterize the pathogenic potential (RNA-ase and DNA-ase), and resistance of microorganisms to aseptic agents were found increased.

Bacteria capable to secrete enzymes hydrolyzing proteins and carbohydrates proved to be associated with the digestive-transport surfaces of the bream intestine and tegument of cestode Caryophyllaeus laticeps. Apparently, the contribution of symbiotic digestion to the hydrolysis of polymers increases during active feeding of fishes, while at low feeding rate, bacteria can compete with the host and its parasite for the available monomers. Secretion of bacterial enzymes hydrolyzing both complex carbohydrates and disaccharides reduces the metabolic expenditures of the macroorganisms for the synthesis of their own hydrolases. Glucose resulting from hydrolytic activity of bacterial enzymes can be used by all members of the established community. While bacteria colonizing the digestive-transport surfaces of the host and parasite were generally similar, some specific features were also observed probably due to the structural peculiarities of the intestinal mucosa and cestode tegument.

Results of the research performed at the Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, on designing immunobiosensors for detection of toxic compounds and microbial cells enzyme-based biosensors for detection of hydrocarbons and alcohols, and microbial biosensors for aromatic compounds, surfactants, and biological oxygen consumption are briefed. Parameters of the mediator electrodes involving microbial cells and data on the properties of microbial biofuel cells--devices based on biosensor principle and representing alternative sources of electric energy--are given.

Genes for catechol 1,2- and 2,3-dioxygenases were cloned. These enzymes hold important positions in the ortho and meta pathways of the metabolism of aromatic carbons by microbial associations that consume the following volatile organic compounds in pilot minireactors: toluene, styrene, ethyl benzene, o-xylene, m-xylene, and naphthalene. Genes of both pathways were found in an association consuming m-xylene; only genes of the ortho pathway were found in associations consuming o-xylene, styrene, and ethyl benzene, and only genes of the meta pathway were found in associations consuming naphthalene and toluene. Genes of the ortho pathway (C120) cloned from associations consuming o-xylene and ethyl benzene were similar to corresponding genes located on the pND6 plasmid of Pseudomonas putida. Genes of the ortho pathway from associations consuming o-xylene and m-xylene were similar to chromosomal genes of P. putida. Genes of the meta pathway (C230) from associations consuming toluene and naphthalene were similar to corresponding genes formerly found in plasmids pWWO and pTOL.

The symbiotic microflora associated with the digestive-transport surfaces of the pike intestine and the parasitic cestodes Triaenophorus nodulosus proved capable of the initial stages of carbohydrate hydrolysis mediated by alpha-amylase. The products of hydrolysis by alpha-amylase can be used by both the host and the parasite, which decreases energy expenditures of the macroorganisms. The levels of the bacterial alpha-amylase activity are comparable to those of the analogous enzyme absorbed on the mucosa of the intestine and on the cestode tegument, which indicates a considerable contribution of enzymes of the symbiotic microflora to digestion by the host and the parasite. Apparently, this contribution depends on the fish diet.

Restriction endonucleases (RENs) were detected in 650 microbial strains isolated from water columns and bottom sediments of deep rift lakes, Baikal (Russia) and Nyasa (Southeastern Africa). They enzymes included unique (Fan I, Aca I, and Sse 91) and very rare (Bsi I, and Cci N I) species not typical of aquatic ecosystems. Water columns, deep cores, and bottom sediments of pure areas of the lakes contained no microorganisms with new RENs. Thus, inshore areas of Lake Baikal exposed to anthropogenic influence may contain mutant bacterial strains expressing RENs that have not been described previously.

More than 600 bacterial strains isolated from different horizons of steppe biogeocenoses and zoogenous loci (diplopod intestines and feces) were tested for the ability to lyse yeast cell walls. About half of the strains that were isolated from biotopes with active degradation of plant debris (steppe litters and diplopod intestines and feces) were found to possess yeast-lytic activity. Most of the yeast-lytic strains belonged to the genera Streptomyces, Promicromonospora, Oerskovia, and Agromyces. The yeast-lytic activity of actinobacteria from the genera Agromyces, Mycobacterium, and Micrococcus has not previously been reported.

A study was made of the refolding of bacterial luciferases of Vibrio fischeri, V. harveyi, Photobacterium phosphoreum, and Photorhabdus luminescens. By reaction rate, luciferases were divided into two groups. The reaction rate constants of fast luciferases of V. fischeri and Ph. phosphoreum were about tenfold higher than those of slow luciferases of Ph. luminescens and V. harveyi. The order of increasing luciferase thermostability was Ph. phosphoreum, V. fischeri, V. harveyi, and Ph. luminescens. The refolding of thermoinactivated luciferases completely depended on the active DnaK-DnaJ-GrpE chaperone system. Thermolabile fast luciferases of V. fischeri and Ph. phosphoreum showed highly efficient rapid refolding. Slower and less efficient refolding was characteristic of thermostable slow luciferases of V. harveyi and Ph. luminescens. Chaperones of the Clp family were tested for effect on the efficiency of DnaK-dependent refolding of bacterial luciferases in Escherichia coli cells. The rate and extent of refolding were considerably lower in the clpB mutant than in wild-type cells. In E. coli cells with mutant clpA, clpP, of clpX showed a substantially lower luciferase refolding after heat shock.

The results of the qualitative and quantitative analysis of the microflora of the large intestine in 45 healthy children aged 6, 8, 10 months and 1 year, living in Moscow, are presented. During the first year of life high concentrations and detection rate of not only bifidobacteria, enterococci, nonpathogenic Escherichia, but also bacteria, commonly regarded as opportunistic were typical. The latter include lecithinase positive Clostridium, citrate assimilating enterobacteria, as well as Escherichia with low biochemical activity and capacity for hemolysin production. In addition, this group also includes coagulase-positive staphylococci. Later on, as these children become older, opportunistic bacteria are partially or completely eliminated under normal conditions. Suggestion is made that the composition of intestinal microflora depends on protective and other systems of the host at different periods of life, as well as on the character of nutrition and the microbial contamination of the environment.

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.

Sulfur oxygenase, sulfite oxidase, adenylyl sulfate reductase, rhodanase, sulfur:Fe(III) oxidoreductase, and sulfite:Fe(III) oxidoreductase were found in cells of aerobic thermoacidophilic bacteria Sulfobacillus sibiricus strains N1 and SSO. Enzyme activity was revealed in cells grown on the medium with elemental sulfur or in the presence of various sulfide elements and concentrates of sulfide ores. The activity of sulfur-metabolizing enzymes depended little on the degree of aeration during bacterial growth.

The review summarizes characteristics of pathogenic microbial collagenolytic enzymes. Th collagenases usually represent the main factors of virulence; they provide microbe penetration int host tissues and supply microorganisms with nutritients. The study of these enzymes is of great valu for scientific, medical and biochemical purposes.

The role of enzyme--substrate interactions for the survival of bacteria in natural biocenoses has been analyzed with the systems "lysozyme-antilysozyme", "histon-antihiston" used as models. The conception of a possible universal mechanism for supporting the circulation of pathogenic and opportunistic microorganisms among eukaryotes, irrespective of their evolutionary status and environment, has been formulated. This mechanism is ensured by the natural resistance of the eukaryotic cells and by persistence factors on the part of the pathogen; as a result, the dynamic system of interactions is formed which facilitates the survival of microorganisms due to their persistence potential. New knowledge thus obtained opens prospects in the study of sanitary and ecological aspects of water biocenoses.

Modern data (collected mainly in the 1998-2001 studies) about the transport of carbohydrates in bacteria, about the regulation of utilization of sugars via the glycolytic pathway as well as about the regulation of transformation of pyruvat into the products of secondary metabolism and of tricarboxylic acid cycle are presented in the survey. Issues, related with the regulation of synthesis of enzymes involved in the last mentioned process, are discussed in detail. Besides, the key pathways pertaining to the regulation of synthesis and activity of adenylate cyclase; elimination of the inductor in the gram-negative bacteria and entry of phage lambda DNA into E. coli are described. As for the gram-positive bacteria, properties of their main components (involved in catabolic repression), i.e. HPr, K/P, CcpA, CCpB and CcpC, cre, are presented. The mechanisms of catabolic repressions and of catabolic activation in bacteria are in the focus of attention. Finally, issues related with the structural organization of PTS as well as molecular-and-biological aspects of the interaction of proteins of the mentioned system are considered in the survey.

Modern published data on the folding of microbial proteolytic enzymes mediated through their pro-parts, or on a so-called pro-dependent folding, are surveyed. Various aspects related with the functioning of pro-sequences are under discussion. The possibility to build up, on the basis of one and the same amino-acid sequence, the special protein conformers involving different pro-peptides is in the focus of attention.

Prolyl endopeptidases, or post-proline-cleaving enzymes, are the specific endopeptidases that hydrolyze peptide substrates at the carbonyl of the internal Pro residue. All the currently known prolyl endopeptidases from animals, microorganisms, fungi, and plants as well as the post-proline-cleaving enzymes that do not exhibit the strict specificity to Pro are reviewed. The data on their physicochemical and catalytic properties, substrate specificity, inhibitors, sequences, and three-dimensional structures are discussed.

The composition of the fecal microflora in somatic patients and patients with enteric infections under the conditions of surpluscolonization by yeast-like fungi of the genus Candida was analyzed. The study revealed that the high level of fungal contamination was linked with decreased colonization resistance of the intestine (deficiency in bifidoflora) and with the presence of opportunistic microorganisms: Staphylococcus aureus, hemolytic and lactose-negative Escherichia coli, as well as nonfermenting Gram negative bacteria. The antilysozyme activity of enterobacteria was found to increase in the course of their joint cultivation with fungi of the genus Candida, that may be regarded as one of the mechanisms of the formation and maintenance of pathobiocenosis.

Major pathways of carbon and sulfur metabolisms were studied in representatives of two clusters of bacteria: Leucothrix thiophila (cluster I, strains 2WS, 4WS, and 6WS) and Leucothrix sp. (cluster II, strains 1WS, 3WS, and 5WS). All strains were capable of chemoorganoheterotrophic growth, as well as of chemolithoheterotrophic growth in the presence of reduced sulfur compounds. The bacteria were found to possess a complete set of the enzymes of the tricarboxylic acid cycle and glyoxylate cycle. The hydrogenase activity in cells of cluster I strains was an order of magnitude lower than in cluster II strains and in other known heterotrophic bacteria. Cells of bacteria of both clusters exhibited high activity levels of enzymes involved in the energy metabolism of sulfur. The oxidation of sulfur compounds and the operation of the electron-transport chain were shown to be related. Cluster II bacteria more efficiently use organic compounds in their energy metabolism, whereas cluster I bacteria are characterized by more efficient utilization of reduced sulfur compounds. During sulfite oxidation, cluster I bacteria can synthesize ATP both via substrate-level phosphorylation and oxidative phosphorylation, whereas cluster II bacteria synthesize ATP only via the latter process.