Microbial diversity in formation water and enrichment cultures from the Gangxi bed of the Dagang terrigenous oilfield (PRC)

Microbial diversity and biogeochemical processes of the Gangxi bed with low-mineral water and a temperature gradient from 35 to 54°C were studied. The 16S rRNA gene clone libraries (over 800 clones) were obtained from microbial DNA isolated from formation water and from the primary enrichment cultures for fermenting, sulfate-reducing, methanogenic, and aerobic organotrophic prokaryotes. While both sulfate reduction and methanogenesis were registered in formation water by radioisotope techniques, the genes of sulfate-reducing prokaryotes were not revealed in the 16S rRNA gene clone library from formation water. The 16S rRNA genes of Methanobacterium congolense and Methanococcus vannielii predominated among archaeal sequences retrieved from formation water, while the genes of Methanothermobacter thermoautotrophicus, Methanomethylovorans thermophila, and Methanoculleus sp. predominated in the combined library from enrichment cultures. In the library of Bacteria 16S rRNA genes from formation water, the genes of thermophilic fermentative bacteria of the family Thermoanaerobacteriaceae predominated; the remaining sequences belonged to mesophiles (genera Brevundimonas, Sphingomonas, Oxalicibacterium, and Stenotrophomonas), the phylum Chloroflexi, and unidentified bacteria. The combined library from enrichment cultures, contained, apart from the sequences of the family Thermoanaerobacteriaceae, the genes of fermentative bacteria (genera Anaerobaculum, Coprothermobacter, Thermanaerovibrio, Soehngenia, Bacteroides, and Aminobacterium and the order Thermotogales), of aerobic hydrocarbon-oxidizing bacteria (genera Pannonibacter and Pseudomonas), and of sulfate reducers (genera Desulfomicrobium, Thermodesulfovibrio, and Desulfotomaculum). High coverage was shown for bacterial (97.6%) and archaeal (100%) clone libraries, indicating that a significant portion of the microbial diversity in the studied communities was revealed.     

Location of the genes coding for 18S and 28S ribosomal RNA in the human genome

³H-rRNA obtained from Xenopus laevis tissue cultured cells, or a ³H-cRNA made from Xenopus ribosomal DNA, was used for heterologous in situ hybridisation with human lymphocyte metaphase chromosomes. Prior to hybridisation, chromosome spreads were stained with Quinacrine and selected cells showing good Q-banding photographed; the same cells were then rephotographed after autoradiography and pairs of photographs for each cell were used to make dual karyotypes. The chromosomes within each karyotype were divided into equal sized segments (approx. 0.7 μ), with a fixed number of segments for each chromosome type. The distribution of silver grains between segments showed that the ³H-RNAs hybridised specifically to the nucleolar organising regions of the D and G group chromosomes with no other sites of localised labelling in the complement. Control experiments showed no localisation, with insignificant labelling, when metaphase spreads were incubated in a mixture containing Xenopus ³H-rRNA and competing cold human (HeLa) rRNA. Filter hybridisation experiments on isolated human DNA showed that the Xenopus derived ³H-RNAs hybridised to a fraction of human DNA which was on the heavy side of the main DNA peak and that these RNAs were competed out in the presence of excess cold human rRNA, confirming the specificity of the heterologous hybridisation. In situ hybridisation experiments were also carried out on cells from individuals with one chromosome pair showing heteromorphism for either a very long stalk (nucleolar constriction) subtending a satellite, or a large satellite. It was shown that the chromosome with the large stalk hybridised four times as much ³H-rRNA as its homologue, whereas differences in the sizes of the subtended satellites did not materially affect hybridisation levels indicating that rDNA is located in the stalks and not the satellites. The amount of ³H-rRNA hybridised differs between chromosomes and individuals; these differences are heritable and rDNA can be detected by in situ hybridisation in all three chromosomes number 21 in cells from Down's patients and in translocated chromosomes conta.ining a nucleolar constriction. Different D and G group chromosomes which hybridised equal amounts of ³H-rRNA participated in rosette associations at metaphase in a random fashion in some individuals and in a non-random fashion in others. In all individuals studied chromosomes with large amounts of rDNA were not found to be preferentially involved in association. It was therefore concluded that the probability of a chromosome being involved in the formation of a common nucleolus is not a simple function of its rDNA content and other possible factors are considered.     

Cytogenetics of the parthenogenetic grasshopper Warramaba virgo and its bisexual relatives

Combinations of DNA-binding fluorescent dyes and counterstains that enhance selectivity and contrast in primary stain fluorescence were used to differentiate types of C-bands in the genus Warramaba. Chromomycin A₃ (in conjunction with two A-T binding counterstains), which identifies chromosome segments enriched in G-C base pair clusters, stains only a minority of the C-bands in Warramaba species, but these include all those known to contain 18S + 26S rRNA cistrons and most of those containing 5S rRNA genes. DAPI/actinomycin D fluorescent staining is positive for a very few bands, including two (in the Standard phylad of W. virgo) that are at or adjacent to sites containing 5S rRNA cistrons. One of the latter regions is also positively stained by DAPI/distamycin A which, in addition, highlights some centromeric bands. The fluorescent staining patterns of the Standard and Boulder-Zanthus phylads of W. virgo are significantly different, confirming their independent origin by hybridization between different races of the ancestral species “P169” and “P196”.     

Size class homogeneity of repeat lengths and evolutionary divergence of ribosomal RNA genes in fishes as studied by restriction fragment length analysis

Fish ribosomal RNA genes (rDNA) have been compared by restriction endonuclease digestion followed by Southern hybridization using rRNA or cloned rRNA genes as labelled probes. In several species belonging to the orders Cypriniformes and Perciformes, the simple restriction patterns revealed a high degree of size class homogeneity among the rDNA repeats and similar restriction map within a species. Different species have different restriction patterns and fragment lengths arising mostly out of different length of the nontranscribed spacer. Polymorphic restriction sites are present in some species. The species-specific differences in fragment lengths produced in rDNA by some restriction enzymes can thus be used to study interspecific fish hybrids.     

Assessment of microbial communities associated with fermentative–methanogenic biodegradation of aromatic hydrocarbons in groundwater contaminated with a biodiesel blend (B20)

A controlled field experiment was conducted to assess the potential for fermentative–methanogenic biostimulation (by ammonium-acetate injection) to enhance biodegradation of benzene, toluene, ethylbenzene and xylenes (BTEX) as well as polycyclic aromatic hydrocarbons (PAHs) in groundwater contaminated with biodiesel B20 (20:80 v/v soybean biodiesel and diesel). Changes in microbial community structure were assessed by pyrosequencing 16S rRNA analyses. BTEX and PAH removal began 0.7 year following the release, concomitantly with the increase in the relative abundance of Desulfitobacterium and Geobacter spp. (from 5 to 52.7 % and 15.8 to 37.3 % of total Bacteria 16S rRNA, respectively), which are known to anaerobically degrade hydrocarbons. The accumulation of anaerobic metabolites acetate and hydrogen that could hinder the thermodynamic feasibility of BTEX and PAH biotransformations under fermentative/methanogenic conditions was apparently alleviated by the growing predominance of Methanosarcina. This suggests the importance of microbial population shifts that enrich microorganisms capable of interacting syntrophically to enhance the feasibility of fermentative–methanogenic bioremediation of biodiesel blend releases.     

Identification of proteolytic bacteria from the Arctic Chukchi Sea expedition cruise and characterization of cold-active proteases

Following collection of seawater samples during an Arctic Chukchi Sea expedition cruise of the Korean icebreaker Araon in 2012, a total of 15,696 bacteria were randomly isolated from Marine Broth 2216 agar plates. Of these, 2,526 (16%) showed proteolytic activity and were identified as mainly Alteromonas (31%), Staphylococcus (27%), and Pseudoalteromonas (14%). Among the proteolytic strains, seven were selected based on their significant ability to grow and produce a halo on skim milk plates at low temperatures (<5°C) owing to cold-active proteases. These strains were affiliated with the genus Pseudoalteromonas and were divided into three groups based on phylogenetic analysis of the 16S rRNA genes. Profiling cell membrane fatty acids confirmed the 16S rRNA-based differentiation and revealed the accordance between the two analyses. Seven genes for serine protease precursors were amplified from the corresponding strains, and based on sequence similarities, these genes were divided into three groups that were identical to those identified by the 16S rRNA phylogenetic analysis. Three protease genes from the representative strains of each group were composed of 2,127–2,130 bp, encoding 708–709 amino acids, and these genes yielded products with calculated molecular weights of approximately 72.3–72.8 kDa. Amino acid sequence analysis suggested that the precursors are members of the subtilase serine endo- and exo-peptidase clan and contain four domains (signal peptide, N-terminal prosequence, catalytic domain, and two pre-peptidase C-terminal domains). Upon expression in E. coli, each recombinant protease exhibited proteolytic activity on zymogram gels.     

The evolutionary pattern of aromatic amino acid biosynthesis and the emerging phylogeny of pseudomonad bacteria

Pseudomonad bacterial are a phylogenetically diverse assemblage of species named within contemporary genera that includePseudomonas, Xanthomonas andAlcaligenes. Thus far, five distinct rRNA homology groups (Groups I through V) have been established by oligonucleotide cataloging and by rRNA/DNA hybridization. A pattern of enzymic features of aromatic amino acid biosynthesis (enzymological patterning) is conserved at the level of rRNA homology, five distinct and unambiguous patterns therefore existing in correspondence with the rRNA homology groups. We sorted 87 pseudomonad strains into Groups (and Subgroups) by aromatic pathway patterning. The reliability of this methodology was tested in a blind study using coded cultures of diverse pseudomonad organisms provided by American Type Culture Collection. Fourteen of 14 correct assignments were made at the Group level (the level of rRNA homology), and 12 of 14 correct assignments were made at the finer-tuned Subgroup levels. Many strains of unknown rRNA-homology affiliation had been placed into tentative rRNA groupings based upon enzymological patterning. Positive confirmation of such strains as members of the predicted rRNA homology groups was demonstrated by DNA/rRNA hybridization in nearly every case. It seems clear that the combination of these molecular approaches will make it feasible to deduce the evolution of biochemical-pathway construction and regulation in parallel with the emerging phylogenies of microbes housing these pathways.     

Analysis of the sequences of internal transcribed spacers ITS1, ITS2 and the 5.8S ribosomal gene of species of the Amaranthus genus

Analysis of the sequence ITS1-5.8S-ITS2 in 11 samples of the amaranth species (Amaranthus caudatus, A. cruentus, A. hybridus, A. tricolor, A. paniculatus, A. hypohondriacus) was performed. It has been shown that the variability of the sequences of the intergenic spacers ITS1, ITS2 and 5.8S rRNA gene of the amaranth species analyzed is extremely low. A possible secondary structure of the 5.8S rRNA molecule was determined for the first time; three conservative motifs were identified. A single nucleotide substitution found in A. hybridus did not change the loop topology. In the sample of Celosia cristata taken as an external group, a four-nucleotide insertion in the 5′-end of the gene and a one-nucleotide deletion in the fourth hairpin not affecting the general topology of the 5.8S rRNA molecule were found.     

Distinct patterns of marine bacterial communities in the South and North Pacific Oceans

The study of oceanic microbial communities is crucial for our understanding of the role of microbes in terms of biomass, diversity and ecosystem function. In this study, 16S rRNA gene tag pyrosequencing was used to investigate change in bacterial community structure between summer and winter water masses from Gosung Bay in the South Sea of Korea and Chuuk in Micronesia, located in the North and South Pacific Oceans, respectively. Summer and winter sampling from each water mass revealed highly diverse bacterial communities, containing ~900 Operational Taxonomic Units (OTUs). The microbial distribution and highly heterogeneous composition observed at both sampling sites were different from those of most macroorganisms. The bacterial communities in the seawater at both sites were most abundant in Proteobacteria during the summer in Gosung and in Bacterioidetes during the winter. The proportion of Cyanobacteria was higher in summer than in winter in Chuuk and similar in Gosung. Additionally, the microbial community during summer in Gosung was significantly different from other communities observed based on the unweighted UniFrac distance. These data suggest that in both oceanic areas sampled, the bacterial communities had distinct distribution patterns with spatially- and temporally-heterogeneous distributions.     

Transcription and processing of rRNA in higher plant cells (Daucus carota,Petroselinum crispum,Acer pseudoplatanus)

Actively dividing cells from parsley (Petroselinum crispum) and carrot (Daucus carota) (bothApiaceae) andAcer pseudoplatanus (Aceraceae) were used to detect the primary gene product of the rDNA in plant cells. Parsley and carrot cells were labelled with [³²P] orthophosphate. In both cases only one high molecular weight rRNA precursor was present on polyacrylamide gels under non-denaturing conditions. Its molecular weight did not exceed 2.5 × 10⁶ daltons. The component emerged from the heterogenous material after a labelling period of 5–10 min. In parsley cells 45 min after onset of incubation labelled mature rRNA (25S and 18S) arrived in the cytoplasm. InAcer pseudoplatanus (incubation period 60 min) two rapidly labelled components did emerge from polyacrylamide gels; their molecular weights were 2.3 and 3.2^? 3.4 × 10⁶ daltons. After electrophoresis under denaturing conditions the larger component was no longer present, thus indicating that it was an aggregate of different RNA molecules. The molecular weights of the rRNA precursors ofD. carota andP. crispum determined after electrophoresis in formamide gels were about 2.1 × 10⁶ daltons. From these results we have no evidence for the existence of rRNA precursors exceeding the molecular weight of 2.5 × 10⁶ daltons.     

Control of RNA level and of RNA ratios in the latex ofHevea brasiliensis Müll. Arg. effect of latex tapping and of growth regulators

The association between latex RNA and latex production was examined using MAK column chromatography techniques. In young untapped trees the introduction of tapping or the treatment of bark with growth regulators resulted in an increase of RNA level and of rRNA/tRNA ratio in the latex. In regularly tapped trees an increase in rRNA but not in tRNA was brought about by increasing the tapping frequency. Treatment with growth regulators had the same effect but essentially only through the related enhancement of latex export from latex vessels. During latex flow, the highest RNA level was registered in latex fractions originating from the most heavily drained areas of bark. Using³²P labeling, evidence was obtained that the export of latex results in an enhancement of rRNA migration into the inner latex containing space of the vessels. This is considered as the reason of the generally observed association of high RNA level and of high rRNA/tRNA ratio with high latex yield. It is proposed that in controlling the RNA level and RNA proportions in the latex an important role is played by changes in turgor pressure associated with the loss of latex which may influence the export of RNA from the nucleus through related induction of pressure disequilibrium between the nucleoplasm and the latex cytoplasm.     

Comparisons of procedures for determining ribosomal ribonucleic acid similarities

Ribosomal ribonucleic acid (rRNA) cistron similarities were determined forClostridium species with labeled preparations of 16S, 23S, or 16S plus 23S rRNA. Similarities were measured by membrane competition and by thermal stability experiments, and the results were correlated. Correlations were also made between 16S rRNA membrane competition homology values and similarities of 16S rRNA oligonucleotides. Very similar results were obtained whether 16S-or 23S-labeled rRNA was used. Clustering patterns were similar for homology values from hybridization experiments incubated at 50° or 60°C, as were the clustering patterns comparing homology values and rRNA hybrid thermal stability values. There was a linear correlation between rRNA homology values and 16S oligonucleotide S_AB values (Tanner et al. [22]) for homology values above 20%, and S_AB values above 0.45.     

Identification of a microspordium isolated from Megacopta cribraria (Hemiptera: Plataspidae) and characterization of its pathogenicity in silkworms

A new microsporidium isolated from Megacopta cribraria was characterized by both biological characteristics and phylogenetic analysis. Moreover, its pathogenicity to silkworms was also studied. The spores are oval in shape and measured 3.64 ± 0.2 × 2.20 ± 0.2 μm in size. Its ultrastructure is characteristic of the genus Nosema: a diplokaryon, 13–14 polar filament coils and posterior vacuole. Its life cycle includes meronts, sporonts, sporoblasts and mature spores, with a typical diplokaryon in each stage and propagation in a binary fission. A phylogenetic tree based on SSU rRNA and rRNA ITS gene sequence analysis further indicated that the parasite is closely related to Nosema bombycis and should be placed in the genus Nosema and sub-group ‘true’ Nosema. Furthermore, the microsporidium heavily infects lepidopteran silkworm insect and can be transmitted per os (horizontally) and transovarially (vertically). Our findings showed that the microsporidium belongs to the ‘true’ Nosema group within the genus Nosema and heavily infects silkworms. Based on the information obtained during this study, we named this new microsporidium isolated from M. cribraria as Nosema sp. MC.     

Streptosporangium subfuscum sp. nov., isolated from the rhizosphere of marigold (Tagetes erecta L.)

A Gram-stain positive, filamentous bacterial strain, designated strain NEAU-TWSJ13^T, was isolated from the rhizosphere of a marigold (Tagetes erecta L.) plant collected in Heilongjiang Province, northeast China, and characterized using a polyphasic approach. The strain was observed to form abundant aerial hyphae differentiated into spherical sporangia. 16S rRNA gene sequence similarity studies showed that strain NEAU-TWSJ13^T belongs to the genus Streptosporangium, being most closely related to Streptosporangium fragile DSM 43847^T (98.6 %). Phylogenetic analysis of the 16S rRNA gene sequence indicated that it formed a phyletic line with S. fragile DSM 43847^T, Streptosporangium jomthongense NBRC 110047^T (98.4 % 16S rRNA gene similarity) and Streptosporangium violaceochromogenes DSM 43849^T (97.6 % 16S rRNA gene similarity). A combination of DNA–DNA hybridization results and some phenotypic characteristics indicated that strain NEAU-TWSJ13^T can be distinguished from S. fragile DSM 43847^T and S. jomthongense NBRC 110047^T. Moreover, strain NEAU-TWSJ13^T can also be differentiated from S. violaceochromogenes DSM 43849^T and other Streptosporangium species showing high 16S rRNA gene sequence similarity (>98.0 %) by morphological and physiological characteristics. Therefore, it is proposed that strain NEAU-TWSJ13^T represents a novel species of the genus Streptosporangium, for which the name Streptosporangium subfuscum sp. nov. is proposed. The type strain is NEAU-TWSJ13^T ( = CGMCC 4.7146^T = DSM = 46724^T).     

Changes in Sediment Bacterial Community in Response to Long-Term Nutrient Enrichment in a Subtropical Seagrass-Dominated Estuary

Florida Bay exhibits a natural gradient of strong P limitation in the east which shifts to weak P or even N limitation at the western boundary. This nutrient gradient greatly affects seagrass abundance and productivity across the bay. We assessed the effects of N and P additions on sediment bacterial community structure in relation to the existing nutrient gradient in Florida Bay. Sediment samples from 24 permanent 0.25 m² plots in each of six sites across Florida Bay were fertilized with granular N and P in a factorial design for 26 months. Sediment bacterial community structure was analyzed using PCR-denaturing gradient gel electrophoresis (DGGE) analysis of 16S ribosomal RNA (rRNA) genes and a cloning strategy from DGGE bands. The phylogenetic positions of 16S rRNA sequences mostly fell into common members found in marine sediments such as sulfate-reducing Deltaproteobacteria, Gammaproteobacteria, Spirochaetes, and Bacteriodetes. Twenty-eight common DGGE bands were found in all sediment samples; however, some DGGE bands were only found or were better represented in eastern sites. Bacterial community diversity (Shannon-Weiner index) showed similar values throughout all sediment samples. The N treatment had no effect on the bacterial community structures across the bay. Conversely, the addition of P significantly influenced the bacterial community structure at all but the most western site, where P is least limiting due to inputs from the Gulf of Mexico. P additions enhanced DGGE band sequences related to Cytophagales, Ectothiorhodospiraceae, and Desulfobulbaceae, suggesting a shift toward bacterial communities with increased capability to degrade polymeric organic matter. In addition, a band related to Deferribacteres was enhanced in eastern sites. Thus, indigenous environmental conditions were the primary determining factors controlling the bacterial communities, while the addition of P was a secondary determining factor. This P-induced change in community composition tended to be proportional to the amount of P limitation obviated by the nutrient additions.     

5.8S rDNA variability in Allium species belonging to the third evolutionary group

Sequence analysis of 5.8S rDNA in 67 accessions of the subgenus Allium and six other subgenera belonging to the third evolutionary group of Allium genus (Friesen et al., 2006) was performed. Nucleotide substitutions in 5.8S rDNA sequences of Allium accessions were identified and studied for the first time. The probable secondary structure of 5.8S rRNA was constructed. It was shown that mutations in 5.8S rDNA do not involve conserved motifs, and they did not significantly affect the secondary structure of the RNA molecule in Allium accessions.     

Litoribrevibacter albus gen. nov. sp. nov., isolated from coastal seawater,Fujian province,China

A Gram-stain negative, short rod-shaped aerobic bacterium with flagella, designated strain Y32^T, was isolated from coastal seawater in Xiamen, Fujian Province of China. 16S rRNA gene sequence comparisons showed that strain Y32^T is a member of the family Oceanospirillaceae, forming a distinct lineage with species of the genus Litoribacillus. The 16S rRNA gene sequence similarities between strain Y32^T and other strains were all less than 94.0 %. Strain Y32^T was found to grow optimally at 28 °C, at pH 7.0–8.0 and in the presence of 4–5 % (w/v) NaCl. The major fatty acids were identified as Summed Feature 3 (comprising C_16:1 ω7c and/or C_16:1 ω6c, 49.4 %), C_16:0 (17.7 %), C_14:0 (6.9 %) and C_18:1 ω9c (5.4 %). The major respiratory quinone was identified as ubiquinone-8 (Q-8). The major polar lipids were identified as diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. The DNA G+C content of strain Y32^T was determined to be 55.6 mol%. According to its morphology, physiology, fatty acid composition, polar lipids composition and 16S rRNA gene sequence data, strain Y32^T represents a novel species of a new genus in the family Oceanospirillaceae, for which the name Litoribrevibacter albus gen. nov. sp. nov. is proposed. The type strain of Litoribrevibacter albus is Y32^T (=MCCC 1F01211^T=NBRC 110071^T).     

Identification of nucleolus-localized PTEN and its function in regulating ribosome biogenesis

The tumor suppressor PTEN is a lipid phosphatase that is found mutated in different types of human cancers. PTEN suppresses cell proliferation by inhibiting the PI3K-Akt signaling pathway at the cell membrane. However, PTEN is also demonstrated to localize in the cell nucleus where it exhibits tumor suppressive activity via a different, unknown mechanism. In this study we report that PTEN also localizes to the nucleolus and that nucleolar PTEN plays an important role in regulating nucleolar homeostasis and maintaining nucleolar morphology. Overexpression of nuclear PTEN in PTEN null cells inhibits Akt phosphorylation and reduces cell size. Knockdown of PTEN in PTEN positive cells leads to nucleolar morphologic changes and an increase in the proportion of cells with a greater number of nucleoli. In addition, knockdown of PTEN in PTEN positive cells increased ribosome biogenesis. These findings expand current understanding of function and relevance of nuclear localized PTEN and provide a foundation for the development of novel therapies targeting PTEN.