Application of the complex of DNA with the congo red anionic diazo dye for detection of nuclease-producing colonies of marine bacteria

This study was aimed at the development of a method for detection of colonies of nuclease-secreting marine bacteria. The BAL nuclease-producing marine bacterium Pseudoalteromonas espejiana BAL-31 was used as the test object. A new method was developed involving the congo red (CR) anionic dye. The P. espejiana culture was plated on nutrient agar with CR and denatured DNA. In such media. CR was found to form complexes with DNA. After two days of incubation at 30 degrees C, halos were found around the P. espejiana colonies. No halos appeared when DNA was not introduced, when BAL nuclease was inactivated, or when the medium was inoculated with Escherichia coli. It was concluded that the halos around the colonies indicated nuclease excretion. The halos were shown to result from the coagulation of CR released after digestion of the CR-DNA complex by the nuclease. This method for detection of nuclease-producing colonies can probably be used for all marine bacteria and possibly for halophilic bacteria as well.     

<Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> as a Probiotic Potential from Kouzeh Cheese (Traditional Iranian Cheese) and Its Antimicrobial Activity

The aim of this study is to isolate and identify Lactobacillus plantarum isolates from traditional cheese, Kouzeh, and evaluate their antimicrobial activity against some food pathogens. In total, 56 lactic acid bacteria were isolated by morphological and biochemical methods, 12 of which were identified as Lactobacillus plantarum by biochemical method and 11 were confirmed by molecular method. For analyzing the antimicrobial activity of these isolates properly, diffusion method was performed. The isolates were identified by 318 bp band dedicated for L. plantarum. The isolated L. plantarum represented an inhibitory activity against four of the pathogenic bacteria and showed different inhibition halos against each other. The larger halos were observed against Staphylococcus aureus and Staphylococcus epidermidis (15 ± 0.3 and 14.8 ± 0.7 mm, respectively). The inhibition halo of Escherichia coli was smaller than that of other pathogen and some L. plantarum did not show any inhibitory activity against E. coli, which were resistant to antimicrobial compounds produced by L. plantarum. The isolated L. plantarum isolates with the antimicrobial activity in this study had strong probiotic properties. These results indicated the nutritional value of Kouzeh cheese and usage of the isolated isolates as probiotic strains.     

Peculiarities of adhesion of epiphytic bacteria on leaves of the seagrass <Emphasis Type="Italic">Zostera marina</Emphasis> and on abiotic surfaces

A comparative study of the adhesion of epiphytic bacteria and marine free-living, saprophytic, and pathogenic bacteria on seagrass leaves and abiotic surfaces was performed to prove the occurrence of true epiphytes of Zostera marina and to elucidate the bacterium-plant symbiotrophic relationships. It was shown that in the course of adhesion to the seagrass leaves of two taxonomically different bacteria, Cytophaga sp. KMM 3552 and Pseudoalteromonas citrea KMM 461, isolated from the seagrass surface, the number of viable cells increased 3–7-fold after 60 h of incubation, reaching 1.0–2.0 × 10⁵ cells/cm²; however, in the case of adhesion of these bacteria to abiotic surfaces, such as glass or metal, virtually no viable cells were observed after 60 h of incubation. Such selectivity of cell adhesion was not observed in the case of three other bacterial species studied, viz., Vibrio alginolyticus KMM 3551, Bacillus subtilis KMM 430, and Pseudomonas aeruginosa KMM 433. The amount of viable cells of V. alginolyticus KMM 3551 absorbed on glass and metal surfaces increased twofold after 40 h of incubation. The cells of saprophytic B. subtilis KMM 430 and pathogenic P. aeruginosa KMM 433 adsorbed on three studied substrata remained viable for 36 h and died by the 60th hour of incubation.     

Application of the Duplex-Specific Nuclease Preference Method to the Analysis of Single Nucleotide Polymorphisms in Human Genes

A new modification of the single nucleotide polymorphism (SNP) analysis (DSNP, duplex-specific nuclease preference) method using the duplex-specific nuclease from the king crab was proposed. The method was used to study SNPs in the following human genes: kRAS, nRAS, hRAS, and p53, the genes of blood coagulation factor V, methyltetrahydrofolate reductase, prothrombin, and apolipoprotein E and a deletion in the BRCA1 gene. DSNP was shown to be useful for the estimation of the mutant allele content in DNA samples. A system for the simultaneous identification of several adjacent single-nucleotide polymorphisms in the kRAS gene was proposed. The approaches could be used to develop test systems for the detection of SNPs in human genes.     

Identification of the di-<Emphasis Type="Italic">n</Emphasis>-butyl phthalate-biodegrading strains and the biodegradation pathway in strain LMB-1

DNA isolated from a greenhouse soil (Nanjing, Jiangsu Province, China) was suitable for PCR amplification of gene segment coding for the 16S rRNA. Diverse PCR products were characterized by cloning and sequencing, and analysis of bacterial colonies showed the presence over 26 phyla. The most bacteria belonged to Proteobacteria, Actinobacteria, Gemmatimonadetes, Acidobacteria and Planctomycetes. Furthermore, after the enrichment procedure of DBP-degrading microorganisms, 4 strains were isolated from the soil sample with di-n-butyl phthalate (DBP) biodegradability, and they were identified to be Rhizobium sp., Streptomyces sp., Pseudomonas sp. and Acinetobacter sp. Analysis of the degradation products by LC-MS led to identification of metabolites of DBP in strain LMB-1 (identified as Rhizobium sp.) which suggests that DBP was degraded through β-oxidation, demethylation, de-esterification and cleavage of aromatic ring.     

Genetic and biochemical evidences reveal novel insights into the mechanism underlying <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> Sae2-mediated abrogation of DNA replication stress

In Saccharomyces cerevisiae, the Mre11-Rad50-Xrs2 (MRX) protein complex plays pivotal roles in double-strand break (DSB) repair, replication stress and telomere length maintenance. Another protein linked to DSB repair is Sae2, which regulates MRX persistence at DSBs. However, very little is known about its role in DNA replication stress and repair. Here, we reveal a crucial role for Sae2 in DNA replication stress. We show that different mutant alleles of SAE2 cause hypersensitivity to genotoxic agents, and when combined with Δmre11 or nuclease-defective mre11 mutant alleles, the double mutants are considerably more sensitive suggesting that the sae2 mutations synergize with mre11 mutations. Biochemical studies demonstrate that Sae2 exists as a dimer in solution, associates preferentially with single-stranded and branched DNA structures, exhibits structure-specific endonuclease activity and cleaves these substrates from the 5′ end. Furthermore, we show that the nuclease activity is indeed intrinsic to Sae2. Interestingly, sae2^G270D protein possesses DNA-binding activity, but lacks detectable nuclease activity. Altogether, our data suggest a direct role for Sae2 nuclease activity in processing of the DNA structures that arise during replication and DNA damage and provide insights into the mechanism underlying Mre11-Sae2-mediated abrogation of replication stress-related defects in S. cerevisiae.     

Photosynthetic activity and components of the electron transport chain in the aerobic bacteriochlorophyll <Emphasis Type="Italic">a</Emphasis>-containing bacterium <Emphasis Type="Italic">Roseinatronobacter thiooxidans</Emphasis>

Bioenergetics of the aerobic bacteriochlorophyll a-containing (BCl a) bacterium (ABC bacterium) Roseinatronobacter thiooxidans is a combination of photosynthesis, oxygen respiration, and oxidation of sulfur compounds under alkaliphilic conditions. The photosynthetic activity of Rna. thiooxidans cells was established by the photoinhibition of cell respiration and reversible photobleaching discoloration of the BCl a of reaction centers (RC), connected by the chain of electron transfer with cytochrome c ₅₅₁ oxidation. The species under study, like many purple bacteria and some of the known ABC bacteria, possesses a light-harvesting pigment-protein (LHI) complex with the average number of 30 molecules of antenna BCl a per one photosynthetic RC. Under microaerobic growth conditions, the cells contained bc ₁ complex and two terminal oxidases: cbb ₃-cytochrome oxidase and the alternative cytochrome oxidase of the a ₃ type. Besides, Rna. thiooxidans was shown to have several different soluble low- and high-potential cytochromes c, probably associated with the ability of utilizing sulfur compounds as additional electron donors.     

The Role of Epibionts of Bacteria of the Genus <Emphasis Type="Italic">Pseudoalteromonas</Emphasis> and Cellular Proteasomes in the Adaptive Plasticity of Marine Cold-Water Sponges

It was found that cells of different color morphs of the cold-water marine sponges Halichondria panicea (Pallas, 1766) of the class Demospongiae differ in the content of epibionts of bacteria of the genus Pseudoalteromonas. The sponge cells with elevated levels of epibionts of bacteria of the genus Pseudoalteromonas showed an increased expression of Hsp70 proteins but had a reduced level of the proteasomal catalytic beta 5 subunit, which was accompanied by a change in their activity. Probably, epibionts of bacteria of the genus Pseudoalteromonas may affect the ubiquitin–proteasome system in the cells of cold-water marine sponges and, thereby, ensure their adaptive plasticity.     

Kinetic aspects of inhibition of the phytopathogenic fungi growth by rhizosphere bacteria

Kinetics of growth inhibition of fungi Fusarium and Bipolaris caused by bacteria Pseudomonas sp. V-6798 and Azotobacter chroococum V-2272 D on dense nutrient media, both in single-crop system and by coinoculation, was demonstrated. The speed of fungal colonies growth as a function of bacteria concentration in inoculate was shown to be in accordance with the Ierysalimskii modified equation. The degree of antagonistic activity was suggested to be assessed by the constant of inhibition (K _i ) and residual rate of fungi growth. Constant of inhibition of fungal growth by bacteria varied within 10–100 cells/ml for observed species. More effective fungistatic influence of bacterial strains in combined culture was observed. Parameters reported in the present study allow comparing the degree of bacteria antifungal activity in vitro. Suggested screening method could be used for selection of bacteria as activity biofungicide and while selecting biomedication for defined plant pathogen disruption.     

<Emphasis Type="Italic">Periconiella lecythidis</Emphasis> sp. nov., the causal agent of a zonate leaf spot disease of the Brazilian tree <Emphasis Type="Italic">Lecythis pisonis</Emphasis>

A new defoliating disease was observed on 20-year-old trees of Lecythis pisonis in Viçosa, Minas Gerais, Brazil. The disease is characterized by forming one or more large zonate epiphyllous leaf spots with a light brown to white center, followed by two to three zones gradually varying from brown to red-brown. On the corresponding abaxial leaf surface, the spots are lighter with a profuse greenish gray coloration caused by the fungal colonies. In PDA culture, the fungus is characterized by a slow growth of the colonies with brown-violet pigmentation at the reverse. Inoculation tests carried out on healthy plants of L. pisonis confirmed the pathogenicity of this fungus. Periconiella lecythidis sp. nov., the causal agent of the zonate leaf spot disease of L. pisonis is described, illustrated, discussed and compared with the other species of the genus Periconiella. The intricate generic affinity within a complex of morphologically similar hyphomycete genera consisting of Metulocladosporiella, Parapericoniella, Penidiella and Periconiella is discussed in detail.     

Intrapopulation heterogeneity of the fluorescence parameters of the marine plankton alga <Emphasis Type="Italic">Thalassiosira weissflogii</Emphasis> at various nitrogen levels

Fluorescence of the marine alga Thalassiosira weissflogii (Grunow) Fryxell et Hasle with open (F _o ) and closed (F _m ) reaction centers of photosystem 2 (PS 2) and its relative variable fluorescence (F _v/F _m ) were measured at various levels of inorganic nitrogen. A significant heterogeneity of the population in terms of these parameters was revealed. Some cells within the population were more sensitive to nitrogen deficiency, and their photosynthetic apparatus was disrupted to a greater extent. The cells within a population also differed in terms of their ability to recover after incubation at low nitrogen levels. Enhancement of nitrogen deficiency resulted in an increase in the variability of the F _o and F _v/F _m values of the cells. Fluorescence variability decreased at a less pronounced deficiency. Fluorescence variability should be taken into consideration in the studies concerning responses of algae to changes in nutrient contents.     

Culturable symbionts associated with the reproductive and digestive tissues of the Neotropical brown stinkbug <Emphasis Type="Italic">Euschistus heros</Emphasis>

Symbionts are widely distributed in eukaryotes, and potentially affect the physiology, ecology and evolution of their host. Most insects harbour free-living bacteria in their haemocoel and gut lumen, intracellular-living bacteria in a range of tissues or bacteria in host-derived specialized cells. Stinkbugs, as do many arthropods, harbour extracellular bacteria in the gut that may affect the fitness of their host. This study identified the culturable symbionts associated with the ovaries, spermatheca, seminal vesicle and posterior midgut region (V4) of males and females of Euschistus heros (F.) (Hemiptera: Pentatomidae). Several culture media were used to isolate the bacteria associated with these structures. The selected colonies (morphotypes) were cultured in liquid medium, subjected to genomic DNA extraction, 16S rRNA gene amplification, and restriction fragment length polymorphism (RFLP) analyses. Morphotypes with distinct RFLP patterns were purified and sequenced, and the sequences obtained were used for putative identification and phylogenetic analysis. Comparison of the sequences with those available in the EzTaxon-e database and the use of a matrix of paired distances grouped the isolates in phylotypes belonging to the Phylum Proteobacteria. Proteobacteria was represented by γ-Proteobacteria phylotypes belonging to Enterobacteriaceae, while Firmicutes had Bacilli phylotypes distributed in Enterococcaceae and Staphylococcaceae. Some of the phylotypes identified were associated exclusively with single structures, such as ovaries, spermatheca and the V4 midgut region of males and females. All culturable bacteria associated with the seminal vesicle were also associated with other tissues.     

Phylogenetic in situ/ex situ analysis of a sulfur mat microbial community from a thermal sulfide spring in the north Caucasus

A phylogenetic in situ/ex situ analysis of a sulfur mat formed by colorless filamentous sulfur bacteria in a thermal sulfide spring (northern spur of the main Caucasian ridge) was carried out. Nine phylotypes were revealed in the mat. Thiothrix sp. and Sphaerotilus sp. were the dominant phylotypes (66.3% and 26.3%, respectively). The 16S rRNA gene nucleotide sequence of Sphaerotilus sp. phylotype from the clone library was identical to the sequences of the seven Sphaerotilus strains isolated from the same source. A very high degree of similarity of Sphaerotilus strains revealed by ERIC-PCR fingerprints indicated little or no population diversity of this species in the mat. Thiothrix phylotype from the clone library and two Thiothrix strains isolated from the same mat sample differed in one to three nucleotides of 16S rRNA genes; this is an indication of this organism’s population variability in the mat. 16S rRNA genes of the strains and clones of Thiothrix sp. exhibited the highest similarity (ca. 99%) with Thiothrix unzii; the strains and clones of Sphaerotilus had 99% similarity with the type species Sphaerotilus natans (the only species of this genus) and therefore can be assigned to this species. The minor seven components belong to the phylotypes from the Proteobacteria (3%), as well as the Chlorobia, Cyanobacteria, Clostridia, and Bacteroidetes phylogenetic groups, each of them constituting not more than 1%. Intracellular accumulation of elemental sulfur by Sphaerotilus similar to other filamentous sulfur bacteria was demonstrated for the first time (both in the population of the sulfur spring and in cultures with sulfide). Although mass growth of Sphaerotilus and Thiothrix is typical of bacterial populations of anthropogenic ecosystems (the activated sludge of treatment facilities), stable communities of these bacteria have not been previously found in the sulfur mats or “threads” of natural sulfide springs.     

Phylogenetic Diversity of the Sulfur Cycle Bacteria in the Bottom Sediments of the Chersonesus Bay

The Black Sea is the largest meromictic basin, in the bottom sediments of which a powerful biogenic process of sulfide production occurs. The goal of the present work was to obtain data on phylogenetic diversity of the sulfur cycle microorganisms (sulfate-reducing and sulfur-oxidizing bacteria) in the Black Sea coastal gas-saturated bottom sediments. The samples were collected in the Chersonesus (Blue) Bay near Sevastopol from whitish bacterial mats of sulfurettes, and from the upper layer of the nearby seabed. Using DNA isolated from the native samples and obtained enrichment cultures, PCR analysis was performed with oligonucleotide primers specific to the fragments of the 16S rRNA genes of the main subgroups of sulfatereducing bacteria (SRB) and to the fragments of the dsrB gene (both reductive and oxidative types), encoding the β-subunit of dissimilatory (bi)sulfite reductase, the key enzyme in the sulfur cycle, inherent in both sulfate- reducing and sulfur-oxidizing microorganisms. The presence of 16S rRNA gene fragments specific to the genera Desulfobacterium, Desulfobacter, Desulfococcus–Desulfonema–Desulfosarcina, and Desulfovibrio–Desulfomicrobium was detected in the DNA samples isolated from coastal bottom bacterial mats. Usage of denaturing gradient gel electrophoresis (DGGE) with subsequent sequencing of reamplified dsrB gene fragments revealed that according to deduced amino acid sequences encoded by the dsrB gene (reductive type), SRB from the coastal gas-saturated bottom sediments of the Black Sea had the highest homology (92?99%) with the dsrB gene of cultured SRB belonging to the genera Desulfovibrio, Desulfatitalea, Desulfobacter, and Desulfobacterium, as well as with uncultured SRB strains from various marine habitats, such as bottom sediments of the Northern and Japanese seas. Deduced amino acid sequences encoded by the oxidative dsrB gene had the highest homology (90?99%) with the relevant sequences of the genera Thiocapsa, Thiobaca, Thioflavicoccus, and Thiorhodococcus.     

Analysis of the diversity of diazotrophic bacteria in peat soil by cloning of the <Emphasis Type="Italic">nifH</Emphasis> gene

The diversity of nitrogen-fixing microorganisms in the soil of an oligotrophic Sphagnum peat bog was studied by molecular cloning of fragments of the nifH gene encoding one of the main components of the nitrogenase complex. The fragments were amplified from the DNA isolated from the peat samples collected at the same site in January (library I) and November (library II), 2005. Analysis of the nifH sequence libraries revealed high diversity of diazotrophic bacteria in peat soil: the first library consisted of 237 clones and 55 unique sequence types, the second one included 171 clones and 52 sequence types. Comparison of the two clone libraries showed that the composition and population structure of the nitrogen-fixing community depended greatly on the sampling time; they shared only 11 phylotypes. The sequences of representatives of the class Alphaproteobacteria prevailed in both libraries (27% and 57% of clones in libraries I and II, respectively). Representatives of the classes Deltaproteobacteria and Chlorobea were minor components of library I (6% and 7% of clones, respectively), whereas they prevailed in library II (18% and 24% of clones, respectively). Members of the class Chloroflexi were present only in library I, while members of the classes Bacilli, Clostridia, and Methanomicrobia were present only in library II. Our studies demonstrated that, for complete evaluation of the diversity of natural nitrogen-fixing communities, nifH libraries should consist of at least 200–300 clones.     

Biodiversity of aerobic methylobacteria associated with the phyllosphere of the southern Moscow region

During the summer period (15–25°C), 34 strains of methylotrophic bacteria associated with different species of herbs, shrub, and trees in Pushchino (Moscow oblast, Russia) were isolated on the medium with methanol. Predominance of pink-colored Methylobacterium strains in the phyllosphere of many plants was confirmed by microscopy, enumeration of the colonies from grass leaves, and sequencing of the 16S rRNA genes. Colorless and yellow-pigmented methylotrophs belonged to the genera Methylophilus, Methylobacillus, Hansschlegelia, Methylopila, Xanthobacter, and Paracoccus. All isolates were able to synthesize plant hormones auxins from L-tryptophan (5?50 μg/mL) and are probably plant symbionts.     

A cellulose-decomposing bacterial association

The structure of a cellulose-decomposing bacterial association was described using phenotypic and phylogenetic characteristics. Based on their morphological, physiological, and biochemical characteristics, the bacteria isolated from the association were identified as Sporocytophaga sp., Xanthomonas sp, and Pseudomonas sp. The phylogenetic analysis based on comparison of 16S rRNA gene fragments obtained from the association revealed six bacterial species belonging to the clusters of Alcaligenes sp., Ochrobactrum sp., Sphingomonas sp., Achromobacter sp., Pseudomonas sp., and Flexibacteriaceae (Sporocytophaga).     

Bacteria of the sulfur cycle in the sediments of gold mine tailings,Kuznetsk Basin,Russia

The number and diversity of culturable microorganisms involved in sulfur oxidation and sulfate reduction were investigated in the oxidized sediments of gold mine tailings, Kuznetsk Basin, Russia. The sediments had a low pH (2.4–2.8), high SO ₄ ^2? content (up to 22 g/l), and high concentrations of dissolved metals. The arsenic content was as high as 1.9 g/l. Bacterial phylogeny in microcosms was investigated by amplification of 16S rRNA gene fragments with subsequent denaturing gradient gel electrophoresis (DGGE). Spore-forming bacteria Desulfosporosinus were the only bacteria revealed for which the capacity for dissimilatory sulfate reduction is known. Strain Desulfosporosinus sp. DB was obtained in pure culture, and it was phylogenetically remote from other cultured and uncultured members of the genus. No sulfate-reducing members of the Deltaproteobacteria were detected. The Firmicutes members were the most numerous phylotypes in the microcosms, including a separate cluster with the similarity to Pelotomaculum not exceeding 94%. Acidithiobacillus ferrooxidans and A. caldus were found in anaerobic and microaerophilic microcosms. The number of sulfate reducers did not exceed 9.5 × 10² cells/ml.     

Characterization of biofilm-forming marine bacteria and their effect on attachment and germination of algal spores

In this work, 37 bacterial strains isolated from biofouling of marine organisms and from the Museum of Heterotrophic Bacteria of the National Scientific Center of Marine Biology were studied. The strains were identified based on their phenotypic characteristics and on the fatty acid composition of their cell wall lipids. Members of the genus Pseudoalteromonas prevailed both in associated microflora of two dinoflagellate clones and in the biofilms from marine hydrobionts. Associated microflora included also members of the CFB cluster, Bacillus, Sulfitobacter, Acinetobacter, Shewanella, and Psychrobacter. A considerable portion of strains (48.6%) exhibited antimicrobial activity. Antifouling activity against algal spores was studied using single-species bacterial biofilms and the spores of Ulva lactuca и Undaria pinnatifida, the algae most common in the Sea of Japan. Strong inhibitory effect on attachment of Ulva and Undaria spores was observed for 75 and 51% of the strains, respectively. Attached spores were, however, less sensitive to the inhibitory action of biofilms. Species specificity of algal response to bacteria was shown, with a strain having different effect on the spores of different algal species. Biotechnologically promising strains were determined, which exhibited high activity against the spores of macroalgae and could probably be used as producers of antifouling substances and as components of antifouling coatings. No relation was found between antifouling activity of bacteria and the source of their isolation. Our results indicate wide occurrence of bacteria with antifouling activity among associated microflora of marine hydrobionts and demonstrate the extent of complexity and diversity of relations between bacterial biofilms and algal spores.