A highly conserved nuclear gene for low-level phylogenetics: elongation factor-1 alpha recovers morphology-based tree for heliothine moths

Molecular systematists need increased access to nuclear genes. Highly conserved, low copy number protein-encoding nuclear genes have attractive features for phylogenetic inference but have heretofore been applied mostly to very ancient divergences. By virtue of their synonymous substitutions, such genes should contain a wealth of information about lower-level taxonomic relationships as well, with the advantage that amino acid conservatism makes both alignment and primer definition straightforward. We tested this postulate for the elongation factor-1 alpha (EF-1 alpha) gene in the noctuid moth subfamily Heliothinae, which has probably diversified since the middle Tertiary. We sequenced 1,240 bp in 18 taxa representing heliothine groupings strongly supported by previous morphological and allozyme studies. The single most parsimonious gene tree and the neighbor-joining tree for all nucleotides show almost complete concordance with the morphological tree. Homoplasy and pairwise divergence levels are low, transition/transversion ratios are high, and phylogenetic information is spread evenly across gene regions. The EF-1 alpha gene and presumably other highly conserved genes hold much promise for phylogenetics of Tertiary age eukaryote groups.      

Plant virus infection development as affected by heavy metal stress

The work was focused on the investigation of possible dependencies between the development of viral infection in plants and the presence of high heavy metal concentrations in soil. Field experiments have been conducted in order to study the development of systemic tobacco mosaic virus (TMV) infection in Lycopersicon esculentum L. cv. Miliana plants under effect of separate salts of heavy metals Cu, Zn and Pb deposited in soil. As it is shown, simultaneous effect of viral infection and heavy metals in tenfold maximum permissible concentration leads to decrease of total chlorophyll content in experiment plants mainly due to the degradation of chlorophyll a. The reduction of chlorophyll concentration under the combined influence of both stress factors was more serious comparing to the separate effect of every single factor. Plants' treatment with toxic concentrations of lead and zinc leaded to slight delay in the development of systemic TMV infection together with more than twofold increase of virus content in plants that may be an evidence of synergism between these heavy metal's and virus' effects. Contrary, copper although decreased total chlorophyll content but showed protective properties and significantly reduced amount of virus in plants.     

Microbacterium oxydans, a symbiont of Djungarian hamster, displaying probiotic properties

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.     

Phylogenetic diversity of aerobic saprotrophic bacteria isolated from the Daqing oil field

A diverse and active microbial community in the stratal waters of the Daqing oil field (China), which is exploited with the use of water-flooding, was found to contain aerobic chemoheterotrophic bacteria (including hydrocarbon-oxidizing ones) and anaerobic fermentative, sulfate-reducing, and methanogenic bacteria. The aerobic bacteria were most abundant in the near-bottom zones of injection wells. Twenty pure cultures of aerobic saprotrophic bacteria were isolated from the stratal waters. Under laboratory conditions, they grew at temperatures, pH, and salinity values typical of the stratal water from which they were isolated. These isolates were found to be able to utilize crude oil and a wide range of hydrocarbons, fatty acids, and alcohols. Phylogenetic analysis carried out with the use of complete 16S rRNA sequences showed that the isolates could be divided into three major groups: gram-positive bacteria with a high and a low G + C content of DNA and gram-negative bacteria of the gamma-subclass of the Proteobacteria. Gram-positive isolates belonged to the genera Bacillus, Brevibacillus, Rhodococcus, Dietzia, Kocuria, Gordonia, Cellulomonas, and Clavibacter. Gram-negative isolates belonged to the genera Pseudomonas and Acinetobacter. In their 16S rRNA sequences, many isolates were similar to the known microbial species and some probably represented new species.     

Phylogenetic diversity of the archaeal component in microbial mats on coral-like structures associated with methane seeps in the Black Sea

With the use of molecular ecology methods, the archaea component of microbial mats on coral-like structures associated with methane seeps occurring at a depth of about 200 m in the Black Sea was investigated without the isolation of pure cultures. Using archaea-specific 16S rRNA-targeted oligonucleotide primes, long fragments of genes were amplified, cloned, and sequenced and their phylogenetic analysis was carried out. It was shown that archaea in microbial mats on coral-like structures are represented by two dominant phylotypes that belong to the kingdoms Crenarchaeota and Euryarchaeota and are not specifically related to any described archaea species. The possible role of the revealed archaea in the process of anaerobic methane oxidation is discussed.     

Heliobacterium sulfidophilum sp. Nov. and Heliobacterium undosum sp. Nov.: sulfide-oxidizing Heliobacteria from thermal sulfidic springs

Two new species of heliobacteria isolated from cyanobacterial mats of two alkaline sulfidic hot springs are formally described. Strains BR4 and BG29 are assigned to anoxygenic phototrophic bacteria of the family Heliobacteriaceae, since they possess the unique properties of this taxon: strict anaerobiosis, formation of bacteriochlorophyll g, the lack of extensive intracytoplasmic membranes and chlorosomes, an unusual cell wall structure, and phylogenetic relatedness to the low G + C gram-positive eubacteria. Based on the 16S rDNA sequence similarity, strains BR4 and BG29 are assigned to the genus Heliobacterium and described as two new species of this genus: Heliobacterium sulfidophilum sp. nov. and Heliobacterium undosum sp. nov. The G + C content of the DNA is 51.3 mol % in Hbt. sulfidophilum and 57.2-57.7 mol % in Hbt. undosum. The cells of Hbt. sulfidophilum are rods, and the cells of Hbt. undosum are slightly twisted spirilla or short rods. Both new bacteria are motile by peritrichous flagella. Hbt. sulfidophilum produces endospores. The new bacteria are strict anaerobes growing photoheterotrophically on a limited range of organic compounds. In the dark, they can switch from photosynthesis to the slow fermentation of pyruvate. Biotin is required as a growth factor. Both species are highly tolerant to sulfide (up to 2 mM at pH 7.5) and oxidize it photoheterotrophically to elemental sulfur; photoautotrophic growth was not observed. The temperature optimal for growth of Hbt. sulfidophilum and Hbt. undosum is 30-35 degrees C, and the optimal pH is 7-8.     

Clusters of nonsolvent water in partially destroyed Saccharomyces cerevisiae yeast cells

The state of water in partially destroyed dry yeast cells has been studied using low-temperature ¹H NMR spectroscopy. It has been shown that the residual water is in the form of clusters of strongly and weakly associated water (SAW and WAW, respectively). Three or more types of SAW different in the chemical shift values have been found. It has been established that the interfacial water poorly dissolves hydrochloric and trifluoroacetic acids as well as DMSO and CD₃CN. Hydrochloric acid on a surface of biomaterials can be separated into HCl and water. This process is stabilized by polar co-solvents (DMSO and CD₃CN) added to the CDCl₃ dispersion medium.     

Description of Desulfotomaculum nigrificans subsp. salinus as a new species, Desulfotomaculum salinum sp. nov

This study focused on the physiological, chemotaxonomic, and genotypic characteristics of two thermophilic spore-forming sulfate-reducing bacterial strains, 435T and 781, of which the former has previously been assigned to the subspecies Desulfotomaculum nigrificans subsp. salinus. Both strains reduced sulfate with the resulting production of H2S on media supplemented with H2 + CO2, formate, lactate, pyruvate, malate, fumarate, succinate, methanol, ethanol, propanol, butanol, butyrate, valerate, or palmitate. Lactate oxidation resulted in acetate accumulation; butyrate was oxidized completely, with acetate as an intermediate product. Growth on acetate was slow and weak. Sulfate, sulfite, thiosulfate, and elemental sulfur, but not nitrate, served as electron acceptors for growth with lactate. The bacteria performed dismutation of thiosulfate to sulfate and hydrogen sulfide. In the absence of sulfate, pyruvate but not lactate was fermented. Cytochromes of b and c types were present. The temperature and pH optima for both strains were 60-65 degrees C and pH 7.0. Bacteria grew at 0 to 4.5-6.0% NaCl in the medium, with the optimum being at 0.5-1.0%. Phylogenetic analysis based on a comparison of incomplete 16S rRNA sequences revealed that both strains belonged to the C cluster of the genus Desulfotomaculum, exhibiting 95.5-98.3% homology with the previously described species. The level of DNA-DNA hybridization of strains 435T and 781 with each other was 97%, while that with closely related species D. kuznetsovii 17T was 51-52%. Based on the phenotypic and genotypic properties of strains 435T and 781, it is suggested that they be assigned to a new species: Desulfotomaculum salinum sp. nov., comb. nov. (type strain 435T = VKM B 1492T).     

An oligonucleotide primer system for amplification of the ribulose-1,5-bisphosphate carboxylase/oxygenase genes of bacteria of various taxonomic groups

Based on the analysis of GenBank nucleotide sequences of the cbbL and cbbM genes, coding for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPC), the key enzyme of the Calvin cycle, a primer system was designed that allows about 800-bp-long fragments of these genes to be PCR-ampliflied in various photo- and chemotrophic bacteria. The efficiency of the designed primer system in detection of RuBPC genes was demonstrated in PCR with DNA of taxonomically diverse bacteria possessing RuBPC genes with a known primary structure. Nucleotide sequences of RuBPC gene fragments of bacteria belonging to the genera Acidithiobacillus. Ectothiorhodospira, Magnetospirillum, Methylocapsa, Thioalkalispira, Rhodobacter, and Rhodospirillum were determined to be deposited with GenBank and to be translated into amino acid sequences and subjected to phylogenetic analysis.     

Detection of a cultivated hyperthermophilic archaeon of the genus Sulfophobococcus in a methane tank operated in a thermophilic regime

A stable association of hyperthermophilic microorganisms (82 degrees C), which contained mostly cocci and a minor amount of non-spore-forming rods, was obtained from the fermented sludge of a methane tank used to process municipal wastewaters under thermophilic conditions (50 degrees C). PCR amplification of 16S rRNA genes using total DNA isolated from this association and archaea-specific primers, followed by sequencing of the product obtained, showed that the archaeal component was represented by a single nucleotide sequence, which was 99.9% homologous to 16S rRNA gene of Sulfophobococcus zilligii. Thus, a hyperthermophilic archaeon was for the first time detected in a system of anaerobic biological treatment of wastewater. In addition, this is the first report on the detection of a cultivated member of Crenarchaeota in anthropogenic habitats with neutral pH.