[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.

[Phylogeny and evolution of RubiCo genes in prokaryotes].

This paper reviews phylogeny and evolution of ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) protein which is the key enzyme of the autotrophic Kalvin-Benson cycle and the most abundant protein on the planet. It consists of several structural-functional forms which include fully functional forms I, II and III catalyzing carboxylation/oxygenation of ribulose-1,5-bisphosphate and "RubisCO-like" form IV without the carboxylating activity. The genome localization, the operon structure and the copy number of the RubisCO genes varies in different autotrophic organisms. The RubisCO gene phylogeny differs substantially from the phylogeny of other conservative genes including 16S rRNA gene. This is due to commonly occurred duplication/deletion and horizontal gene transfer events happened during evolution of autotrophic organisms.

[The introduction of the 9alpha-hydroxy group into androst-4-en-3,17-dione using a new actinobacterium strain].

A 9alpha-hydrolase activity of a new actinobacterium strain identified as Rhodococcus erythropolis based on the analysis of a 16S rRNA gene sequence (1417 nucleotides) towards androst-4-en-3,17-dione (AD) was studied. In the presence of glucose in the medium, this strain completely transformed AD (4-20 g/l) into 9alpha-hydroxy-AD over 20-48 h. This culture was able to grow and perform AD 9alphahydroxylation at a concentration of dimethyl formamide up to 9%. Crystalline 9alpha-hydroxy-AD was isolated with a yield of over 90%.

[AlkB homologues in thermophilic bacteria of the genus Geobacillus].

Screening of alkane hydroxylase genes (alkB) was performed in the thermophilic aerobic bacteria of the genus Geobacillus. Total DNA was extracted from the biomass of 11 strains grown on the mixture of saturated C10-C20 hydrocarbons, PCR amplification of fragments of alkB genes was performed with degenerate oligonucleotide primers, PCR products were cloned and sequenced. For the first time in the genome of thermophilic bacteria the presence of a set of alkB gene homologues was revealed. The strains each contain three to six homologues among which only two are universal for all of the strains. Comparative phylogenetic analysis of the nucleotide sequences and the inferred amino acid sequences showed close relatedness of six of the revealed variants of geobacilli sequences to the alkB4, alkB3, and alkB2 genes that had previously been revealed by other authors in Rhodococcus erythropolis strains NRRL B-16531 and Q15. The rest two variants of alkB sequences were unique. Analysis of the GC composition of all the Geobacillus alkB homologues revealed closer proximity to the rhodococcal chromosomal DNA than to the chromosomal DNA of geobacilli. This may be an indication of the introduction of the alkB genes into the Geobacillus genome by interspecies horizontal transfer; and rhodococci or other representatives of the Actinobacteria phylum were probably the donors of these genes. Analysis of the codon usage in fragments of alkB genes confirms the suggestion that the pool of these genes is common to the majority of Gram-positive and certain Gram-negative bacteria. Formation of a set of several alkB homologues in a genome of a particular microorganism may result from free gene exchange within this pool.

[The thermophilic bacteria hydrolyzing agar: characterization of thermostable agarase].

Three strains of anaerobic thermophilic bacteria capable of growing on agarose as a source of energy and carbon were isolated from hot springs near Lake Baikal (Barguzin National Park) and the caldera Uzon (Kamchatka). Cells of all the three strains were spore bacilli with peritrichous flagellation. These isolates grew at a temperature of 55-60 degrees C and pH 6.5-7.0 and fermented a wide range of organic substrates. Analysis of the 16S rRNA sequences allowed us to ascribe the strains B5 and K14 to the genus Thermoanaerobacter and the strain K67 to the genus Caldoanaerobacter. According to the results of DNA-DNA hybridization, B5 was determined as belonging to the species Thermoanaerobacter wiegelii. Agarase was isolated by preparative PAGE and subsequent gel chromatography from the culture liquid of strain B5 grown on the medium containing 0.5% agarose and 0.3% galactose. The molecular weight of this enzyme amounted to 67 kDa and pI, to 4.2. The T. wiegelii B5 agarase was active in the pH range of 3.5 to 7.0 (optimum, 5.2) and temperature range of 50 to 80 degrees C (optimum, 70 degrees C). The preincubation of this enzyme at 90 degrees C for 60 min did not reduce the agarase activity. This activity increased in the presence of metal ions; the maximal effect was observed in the presence of 5 mM Mg2+ and 25 mM Co2+.

[Raoultella planticola, a new strain degrading 2,4,5-trichlorophenoxyacetic acid].

A new strain that degrades the herbicide 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) was isolated from soil, which was exposed to factors related to the petrochemical industry. According to its physiological, biochemical, cultural, and morphological traits, together with the sequence of the 16S rRNA gene, the strain was identified as Raoultella planticola 33-4ch. The strain could consume 2,4,5-T as a sole source of carbon and energy. The amount of 2,4,5-T in the culture medium decreased by 51% after five days of incubation. Raoultella planticola 33-4ch consumes 2,4,5-T to produce 4-chlorophenoxyacetic, phenoxyacetic, and 3-methyl-2,6-dioxo-4-hexenoic acids.

[Phylogenetic position of the purple sulfur bacterium Lamprobacter modestohalophilus determined based on the data on new strains of the species].

Lamprobacter, the genus of halophilic purple sulfur bacteria (PSB) with the single species Lpb. modestohalophilus was described in 1979. Rod-shaped Lamprobacter cells contained gas vacuoles during the nonmotile growth phase; motile cells without gas vesicles were formed sometimes. Bacteria contained bacteriochlorophyll a and a carotenoid okenone. The names of this genus and species were included in the list of approved microbial names in 1988. Since the type strain Lpb. modestohalophilus ROI(T) has been lost, its 16S rRNA gene sequences have not been obtained. Based on analysis of the 16S rRNA genes, a new genus Halochromatium comprising the motile extremely halophilic Chromatium-like species was proposed in 1998. Members of this genus never contain gas vacuoles. In spite of the phenotypic differences between the genera Lamprobacter and Halochromatium, phylogenetic boundaries between these taxa remained undetermined. Description of a marine bacteria belonging to Lamprobacter according to its morphological andphysiological properties as a new Halochromatium species, Hch. roseum, resulted in additional complication of the taxonomic situation. The present work provides evidence for the preservation of two phenotypically and phylogenetically different genera, Lamprobacter and Halochromatium, Lpb. modestohalophilus is proposed, as the type species of the genus Lamprobacter. Characteristics of two Lpb. modestohalophilus strains were extensively investigated, and one of them (strain Sivash) was proposed as the neotype strain of the species. It was suggested to retain the genus Halochromatium as containing extremely halophilic species Hch. salexigens and Hch. glycolicum, while transfer of the weakly halophilic species Hch. roseum to the genus Lamprobacter is proposed, resulting in a new combination Lamprobacter roseus comb. nov.

The use of molecular hybridization to evaluate the divergence of some altered Rickettsia prowazekii strains.

Molecular DNA/DNA hybridization was used to investigate the degree of divergence of different Rickettsia prowazekii strains, namely strain Breinl, the vaccine strain E, its spontaneous erythromycin-resistant mutant, nitrosoguanidine-induced rifampicin-resistant mutant and a variant of strain E with increased virulence upon mouse lung passaging. Hybridization of highly polymerised rickettsial DNAs was carried out on nitrocellulose filters with in vitro labelled fragments of reference DNA of the Breinl strain. Nucleotide composition of the strains was also studied. The results obtained suggest the high degree of homology of nucleotide sequences in DNAs of R. prowazekii strains under study; the existing differences found are within the intraspecies range.

[Analysis of the thermostability of the hybrid DNA molecules of microorganisms as a means of increasing the resolution of the molecular hybridization technic]. / Analiz termostabil'nosti gibridnykh molekul DNK mikroorganizmov kak sredstvo povysheniia razreshaiushchei sposobnosti metoda molekuliarnoi gibridizatsii.

A comparative study of termostability of microorganisms DNA was performed in order to increase the resolution of the method of molecular hybridization. Molecular hybridization was carried out and the curve of hybrid DNA duplexes distribution, acccording to termostability of two groups of microorganisms, related to strains Echerichia coli B1 and Vibrio cholerae eltor 18647, were obtained. It was determined that the form of the curves is specie specific for the microorganisms investigated but there exists a similarity between the closely related strains which can not be distinguished by the percent of homology.

[Identification of Listeria using molecular DNA-DNA hybridization]. / Identifikatsiia listerii s pomoshch'iu metoda molekuliarnoi DNK-DNK-gibridizatsii.

The DNA-DNA hybridization method was used to study 4 species of bacteria of the genus Listeria. Concerning the DNA homology, L. monocytogenes strains may be divided into several species (in particular, the pathogenic forms may be isolated into independent taxon), in correlation with their biochemical and serological properties. Most of the studied strains of this species exhibit a high level of DNA homology--65-100%. Bacteria of the L. grayi and L. murrayi species are closely related to each other (90% of DNA homology), the reasonable suggestion being to unite them into a single species. L. denitrificans has 7% of DNA homology with the DNA of the other three species suggesting that it should be excluded from the genus Listeria.

[Similarity of the DNA mucleotide sequences of vibrios]. / Skhodstvo nukleotidnykh posledovatel'nostei DNK nekotorykh vibrionov.

The systematic position of some Vibrio species was ascertained by the method of molecular DNA -- DNA hybridization. The DNA of the brine vibrios V. costicola and V. fischeri were shown to have about 10% of sequences homologous with DNA of a typical cholera vibrio (V. cholerae eltor No. 334). Similarity between the genomes of other representatives of the Vibrionaceae family, as well as in DNA hybridization of V. costicola and V. fischeri, was found to be approximately on the same level. All species included into the genus, Vibrio on account of their phenotypic characteristics may be considered to have essential differences in the structures of their genomes.

[Genome characteristics of a new group of microorganisms in the Vibrionaceae family]. / Genomnaia kharakteristika novoi gruppy mikroorganizmov semeistva Vibrionaceae.

A new group of microorganisms isolated from river and sewage water was studied by the method of molecular DNA-DNA hybridization and included into the family Vibrionaceae on account of their phenotypical properties. The genomes of the microorganisms belonging to this group proved to be highly similar (41-92% of homologies), which suggested that they were related on the genus level. The degree of similarity between the nucleotide sequences in the DNA of the bacteria under study and the DNA of other representatives of the family Vibrionaceae (the genera Vibrio, Beneckea, Aeromonas, Plesiomonas) amounting to 10-20% of homology indicates that the group of microorganisms under study forms a separate genus within the family Vibrionaceae.

[Allomonads--a new group of microorganisms of the family Vibrionaceae. V. Taxonomic position of the allomonads based on a study of their DNA]. / Allomonady--novaia gruppa mikroorganizmov semeistva Vibrionaceae. Soobshchenie V. Taksonomicheskoe polozhenie allomonad na osnove izucheniia ikh DNK.

The DNA of Allomonas, a new group of microorganisms, has been studied to determine the taxonomic status of this group. In the nucleotide composition of the DNA of a typical representative of this group 57.4 mol % G + C have been revealed, which permits to sharply differentiate Allomonas from vibrios and Plesiomonas. A relatively low level of similarity between the DNA of Allomonas and that of other representatives of the family Vibrionaceae has been determined by the method of molecular DNA hybridization (6-18% of homologies), this level being approximately the same in vibrios, Plesiomonas and Aeromonas. These data indicate the necessity of regarding Allomonas as a separate genus of the family Vibrionaceae.

[Microbacterium oxydans, a symbiont of Djungarian hamster, displaying probiotic properties]. / Microbacterium oxydans--simbiont khomiachka Kémpbella, obladaiushchii probioticheskimi svoistvami.

A resident microorganism (strain Kho-17) was isolated from secretions of the specific glandular structures at the angles of mouth of Djungarian hamster (Phodopus campbelli). According to cultural, morphological, and physiological properties as well as to the phylogenetic analysis basing on the sequences of 16S rRNA gene and analysis of the cell wall the strain was assigned to the species Microbacterium oxydans. The bacterium isolated displayed probiotic properties when administered orally as a suspension of live cells for 20 days to Syrian hamster (Mesocricetus auratus), which manifested themselves in increased body weight and weights of several organs and stimulation of both cell-mediated and humoral immunities.

[More precise identification of the systematic position of marine bacteria by the method of DNA-DNA hybridization]. / Ob utochenii sistematicheskogo polozheniia nekotorykh morskikh bakterii metodom gibridizatsii DNK-DNK

In the present work by the method of molecular DNA hybridization there was shown a low degree of affinity of the standard museum strains of cholera vibrios to the respresentatives of the sea species V. parahaemolyticus and V. alginolyticus, and also halophilic vibrios identified earlier on the basis of phenotypical characteristics of the nucleotide DNA composition as Marinovibrio. The presence of only 20--30% of homology in the DNA successiveness in cholera vibrios and the mentioned sea bacteria pointed to the necessity of exclusion of the latter from the Vibrio genus.

[Identification of a new steroid transforming strain of mycobacteria as Mycobacterium neoaurum]. / Identifikatsiia novogo shtamma transformiruiushchikh steroidy mikobakterii kak Mycobacterium neoaurum.

The ability to utilize sterols as a sole source of carbon was studied in 80 strains and consortia of hydrocarbon-oxidizing bacteria. One of the strains, which efficiently transformed both individual sterols and their mixtures, was identified as Mycobacterium neoaurum, based on the analysis of the sequence of the 16S rRNA gene.

[Diversity of diazotrophs in the sediments of hypersaline salt and soda lakes analyzed with the use of the nifH gene as a molecular marker].

Phylogenetic analysis of the nifH genes, encoding the Fe protein of the nitrogenas enzymatic complex, was carried out for pure cultures of anoxygenic phototrophic bacteria of diverse origin, as well as for heterotrophic alkaliphilic sulfate reducers isolated from saline and soda lakes. Topology of the nitrogenase tree correlated with that of the 16S rRNAgene tree to a considerable degree; which niade it possible to use the nifH gene as a molecular marker for investigation of diazotrophic bacterialcommunities in silty sediments of saline and sodalakes. Although diazotrophs were revealed in all environmentalsamples, their phylogenetic diversity was relatively low. Sulfate-reducing deltaproteobacteria and photo- and chemotrophicgammaproteobacteria were predominant in samples integrated over sediment thickness. Analysis of samples fromthe upper sediment layers revealed predominance of phototrophic diazotrophs of various phyla, including purple sulfur and nonsulfur proteobacteria, green nonsulfur bacteria, heliobacteria; and cyanobacteria. Some phylotypes could not be identified, probably indicating the presence of bacterial groups which have not yet been studied by conventional microbiological techniques.

[Use of genes of carbon metabolism enzymes as molecular markers of Chlorobi Phylum representatives].

This work examined the feasibility of using certain genes of carbon metabolism enzymes as molecular markers adequate for studying phylogeny and ecology of green sulfur bacteria (GSB) of the Chlorobi phylum. Primers designed to amplify the genes of ATP citrate lyase (aclB) and citrate synthase (gltA) revealed the respective genes in the genomes of all of the newly studied GSB strains. The phylogenetic trees constructed based on nucleotide sequences of these genes and amino acid sequences of the conceptually translated proteins were on the whole congruent with the 16S rRNA gene tree, with the single exception of GltA of Chloroherpeton thalassium, which formed a separate branch beyond the cluster comprised by other representatives of the Chlorobi phylum. Thus, the aclB genes but not gltA genes proved to be suitable for the design of primers specific to all Chlorobi representatives. Therefore, it was the aclB gene that was further used asa molecular marker to detect GSB in enrichment cultures and environmental samples. AclB phylotypes of GSB were revealed in all of the samples studied, with the exception of environmental samples from soda lakes. The identification of the revealed phylotypes was in agreement with the identification based on the FMO protein gene (fmo), is a well-known Chlorobi-specific molecular marker.

[Activity and structure of the sulfate-reducing bacterial community in the sediments of the southern part of Lake Baikal].

The rates of sulfate reduction (SR) and the diversity of sulfate-reducing bacteria (SRB) were studied in the sediments of the Posol'skaya banka elevation in the southern part of Lake Baikal. SR rates varied from 1.2 to 1641 nmol/(dm3 day), with high rates (> 600 nmol/(dm3 day)) observed at both deep-water stations and in subsurface silts. Integral SR rates calculated for the uppermost 50 cm of the sediments were higher for gas-saturated and gas hydrate-bearing sediments than in those with low methane content. Enrichment SRB cultures were obtained in Widdel medium for freshwater SRB. Analysis of the 16S rRNA gene fragments from clone libraries obtained from the enrichments revealed the presence of SRB belonged to Desulfosporosinus genus, with D. lacus as the most closely related member (capable of sulfate, sulfite, and thiosulfate reduction), as well as members of the order Clostridiales.

[Detection and analysis of sulfur metabolism genes in Sphaerotilus natans subsp. sulfidivorans representatives].

The lithotrophic capacity of the betaproteobacteria Sphaerotilus natans subsp. sulfidivorans was confirmed at genetic level: functional genes of sulfur metabolism were detected (aprBA, soxB, and sqr, coding for adenylyl phosphosulfate reductase, thiosulfate-cleaving enzyme, and sulfide:quinone oxidoreductase, respectively), and the expression of aprA and soxB genes was demonstrated. An evolutionary scenario for soxB genes in Sphaerotilus representatives is suggested based on comparative analysis of codon occurrence frequency, DNA base composition (G + C content), and topology of phylogenetic trees. The ancestor bacterium of the Sphaerotilus-Leptothrix group was capable of lithotrophic growth in the presence of reduced sulfur compounds. However, in the course of further evolution, the sulfur metabolism genes, including the soxB gene, were lost by some Sphaerotilus strains. As a result, the lithotrophic Sphaerotilus-Leptothrix group split into two phylogenetic lineages, lithotrophic and organotrophic ones.