Hisaya Kojima

Characterization of depth-related microbial community structure in lake sediment by denaturing gradient gel electrophoresis of amplified 16S rDNA and reversely transcribed 16S rRNA fragments

Vertical changes of bacterial community structure in a mesophilic lake sediment were investigated by denaturing gradient gel electrophoresis (DGGE) of amplified 16S rDNA and reversely transcribed 16S rRNA fragments. Comparison of community structure analyses based on 16S rDNA and rRNA revealed that the diversity indices from the 16S rDNA-based DGGE profiles were greater than those from the 16S rRNA-based DGGE profiles. In addition, the diversity indices based on 16S rDNA did not change drastically throughout the layers, but the diversity indices based on 16S rRNA decreased with sediment depth. This result indicates that the diversity of active bacteria decreases and inactive bacteria accumulate in association with sedimentation. Dendrograms deduced from DGGE profiles of either 16S rDNA or rRNA were different, and the rRNA-based dendrogram showed a significant difference between the upper layers (0-2, 2-5, and 5-8 cm) and lower layers (8-11, 11-14, 14-17, and 17-20 cm). The sequences of 13 DGGE bands were determined. Phylogenetic analysis of these rDNA fragments revealed that the most conspicuous band in both rDNA- and rRNA-based DGGE was closely related to the genus Nitrospira (95% sequence similarity), and the relative signal intensity was strong especially in the deep layers. Membrane hybridization with a probe targeting the genus Nitrospira also supported the observation that 16S rRNAs derived from Nitrospira-like microorganisms were abundant in this sediment (8.6-16.8% of bacterial 16S rRNA) and that the relative abundance increased with depth. Overall, our results demonstrated that parallel characterization of community structure based on both 16S rDNA and rRNA is important for assessing whole microbial populations and active microbial populations in sediments.

Sulfuritalea hydrogenivorans gen. nov., sp. nov., a facultative autotroph isolated from a freshwater lake

A novel facultatively autotrophic bacterium, designated strain sk43H(T), was isolated from water of a freshwater lake in Japan. Cells of the isolate were curved rods, motile and gram-reaction-negative. Strain sk43H(T) was facultatively anaerobic and autotrophic growth was observed only under anaerobic conditions. The isolate oxidized thiosulfate, elemental sulfur and hydrogen as sole energy sources for autotrophic growth and could utilize nitrate as an electron acceptor. Growth was observed at 8-32 °C (optimum 25 °C) and 6.4-7.6 (optimum pH 6.7-6.9). Optimum growth of the isolate occurred at NaCl concentrations of less than 50 mM. The G+C content of genomic DNA was around 67 mol%. The fatty acid profile of strain sk43H(T) when grown on acetate under aerobic conditions was characterized by the presence of C(16 : 0) and summed feature 3 (C(16 : 1)ω7c and/or iso-C(15 : 0) 2-OH) as the major components. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the strain was a member of the class Betaproteobacteria showing highest sequence similarity with Georgfuchsia toluolica G5G6(T) (94.7 %) and Denitratisoma oestradiolicum AcBE2-1(T) (94.3 %). Phylogenetic analyses were also performed using genes involved in sulfur oxidation. On the basis of its phylogenetic and phenotypic properties, strain sk43H(T) ( = DSM 22779(T)  = NBRC 105852(T)) represents a novel species of a new genus, for which the name Sulfuritalea hydrogenivorans gen. nov., sp. nov. is proposed.

Microbial community structure, pigment composition, and nitrogen source of red snow in Antarctica.

Abstract“Red snow” refers to red-colored snow, caused by bloom of cold-adapted phototrophs, so-called snow algae. The red snow found in Langhovde, Antarctica, was investigated from several viewpoints. Various sizes of rounded red cells were observed in the red snow samples under microscopy. Pigment analysis demonstrated accumulation of astaxanthin in the red snow. Community structure of microorganisms was analyzed by culture-independent methods. In the analyses of small subunit rRNA genes, several species of green algae, fungus, and various phylotypes of bacteria were detected. The detected bacteria were closely related to psychrophilic or psychrotolerant heterotrophic strains, or sequences detected from low-temperature environments. As predominant lineage of bacteria, members of the genus Hymenobacter were consistently detected from samples obtained in two different years. Nitrogen isotopic compositions analysis indicated that the red snow was significantly 15N-enriched. Based on an estimation of trophic level, it was suggested that primary nitrogen sources of the red snow were supplied from fecal pellet of seabirds including a marine top predator of Antarctica.

Dominant Microbial Composition and Its Vertical Distribution in Saline Meromictic Lake Kaiike (Japan) as Revealed by Quantitative Oligonucleotide Probe Membrane Hybridization

ABSTRACT Vertical distributions of dominant bacterial populations in saline meromictic Lake Kaiike were investigated throughout the water column and sediment by quantitative oligonucleotide probe membrane hybridization. Three oligonucleotide probes specific for the small-subunit (SSU) rRNA of three groups of Chlorobiaceae were newly designed. In addition, three general domain (Bacteria, Archaea, and Eukarya)-specific probes, two δ-Proteobacteria-specific probes, a Chlorobiaceae-specific probe, and a Chloroflexi-specific probe were used after optimization of their washing conditions. The abundance of the sum of SSU rRNAs hybridizing with probes specific for three groups of Chlorobiaceae relative to total SSU rRNA peaked in the chemocline, accounting for up to 68%. The abundance of the δ-proteobacterial SSU rRNA relative to total SSU rRNA rapidly increased just below the chemocline up to 29% in anoxic water and peaked at the 2- to 3-cm sediment depth at ca. 34%. The abundance of SSU rRNAs hybridizing with the probe specific for the phylum Chloroflexi relative to total SSU rRNA was highest (31 to 54%) in the top of the sediment but then steeply declined with depth and became stable at 11 to 19%, indicating the robust coexistence of sulfate-reducing bacteria and Chloroflexi in the top of the sediment. Any SSU rRNA of Chloroflexi in the water column was under the detection limit. The summation of the signals of group-specific probes used in this study accounted for up to 89% of total SSU rRNA, suggesting that the DGGE-oligonucleotide probe hybridization approach, in contrast to conventional culture-dependent approaches, was very effective in covering dominant populations.

Complete genomes of freshwater sulfur oxidizers Sulfuricella denitrificans skB26 and Sulfuritalea hydrogenivorans sk43H: genetic insights into the sulfur oxidation pathway of betaproteobacteria.

Despite detailed studies of marine sulfur-oxidizing bacteria, our knowledge concerning their counterparts in freshwater lake ecosystems is limited. Genome sequencing of the freshwater sulfur-oxidizing betaproteobacteria Sulfuricella denitrificans skB26 and Sulfuritalea hydrogenivorans sk43H have been completed. Strain skB26 possessed a circular plasmid of 86.6-kbp in addition to its chromosome, and an approximate 18-kbp region of the plasmid was occupied by an arxA-like operon, encoding a new clade of anaerobic arsenite oxidase. Multilocus sequence analysis showed that strain skB26 could not be assigned to any existing order; thus a novel order, Sulfuricellales, is proposed. The genomes of strains skB26 and sk43H were examined, focusing on the composition and the phylogeny of genes involved in the oxidation of inorganic sulfur compounds. Strains skB26 and sk43H shared a common pathway, which consisted of Sqr, SoxEF, SoxXYZAB, Dsr proteins, AprBA, Sat, and SoeABC. Comparative genomics of betaproteobacterial sulfur oxidizers showed that this pathway was also shared by the freshwater sulfur oxidizers Thiobacillus denitrificans and Sideroxydans lithotrophicus. It also revealed the presence of a conserved gene cluster, which was located immediately upstream of the betaproteobacterial dsr operon.

Morphological and Phylogenetic Characterizations of Freshwater Thioploca Species from Lake Biwa, Japan, and Lake Constance, Germany

ABSTRACT Filamentous, gliding, sulfide-oxidizing bacteria of the genus Thioploca were found on sediments in profundal areas of Lake Biwa, a Japanese freshwater mesotrophic lake, and were characterized morphologically and phylogenetically. The Lake Biwa Thioploca resembled morphologically Thioploca ingrica, a brackish water species from a Danish fjord. The diameters of individual trichomes were 3 to 5.6 μm; the diameters of complete Thioploca filaments ranged from 18 to 75 μm. The cell lengths ranged from 1.2 to 3.8 μm. In transmission electron microscope specimens stained with uranyl acetate, dense intracellular particles were found, which did not show any positive signals for phosphorus and sulfur in an X-ray analysis. The 16S rRNA gene of the Thioploca from Lake Biwa was amplified by using newly designed Thioploca-specific primers (706-Thioploca, Biwa160F, and Biwa829R) in combination with general bacterial primers in order to avoid nonspecific amplification of contaminating bacterial DNA. Denaturing gradient gel electrophoresis (DGGE) analysis of the three overlapping PCR products resulted in single DGGE bands, indicating that a single 16S rRNA gene had been amplified. With the same method, the Thioploca from Lake Constance was examined. The 16S rRNA sequence was verified by performing fluorescence in situ hybridization targeted at specific motifs of the Lake Biwa Thioploca. Positive signals were obtained with the bacterial probe EUB-338, the γ-proteobacterial probe GAM42a, and probe Biwa829 targeting the Lake Biwa Thioploca. Based on the nearly complete 16S rRNA sequence and on morphological similarities, the Thioploca from Lake Biwa and the Thioploca from Lake Constance are closely related to T. ingrica and to each other.

Airborne myxomycete spores: detection using molecular techniques

Myxomycetes are organisms characterized by a life cycle that includes a fruiting body stage. Myxomycete fruiting bodies contain spores, and wind dispersal of the spores is considered important for this organism to colonize new areas. In this study, the presence of airborne myxomycetes and the temporal changes in the myxomycete composition of atmospheric particles (aerosols) were investigated with a polymerase chain reaction (PCR)-based method for Didymiaceae and Physaraceae. Twenty-one aerosol samples were collected on the roof of a three-story building located in Sapporo, Hokkaido Island, northern Japan. PCR analysis of DNA extracts from the aerosol samples indicated the presence of airborne myxomycetes in all the samples, except for the one collected during the snowfall season. Denaturing gradient gel electrophoresis (DGGE) analysis of the PCR products showed seasonally varying banding patterns. The detected DGGE bands were subjected to sequence analyses, and four out of nine obtained sequences were identical to those of fruiting body samples collected in Hokkaido Island. It appears that the difference in the fruiting period of each species was correlated with the seasonal changes in the myxomycete composition of the aerosols. Molecular evidence shows that newly formed spores are released and dispersed in the air, suggesting that wind-driven dispersal of spores is an important process in the life history of myxomycetes. This study is the first to detect airborne myxomycetes with the use of molecular ecological analyses and to characterize their seasonal distribution.

Draft Genome Sequence of a Psychrotolerant Sulfur-Oxidizing Bacterium, Sulfuricella denitrificans skB26, and Proteomic Insights into Cold Adaptation

ABSTRACT Except for several conspicuous cases, very little is known about sulfur oxidizers living in natural freshwater environments. Sulfuricella denitrificans skB26 is a psychrotolerant sulfur oxidizer recently isolated from a freshwater lake as a representative of a new genus in the class Betaproteobacteria. In this study, an approximately 3.2-Mb draft genome sequence of strain skB26 was obtained. In the draft genome, consisting of 23 contigs, a single rRNA operon, 43 tRNA genes, and 3,133 coding sequences were identified. The identified genes include those required for sulfur oxidation, denitrification, and carbon fixation. Comparative proteomic analysis was conducted to assess cold adaptation mechanisms of this organism. From cells grown at 22°C and 5°C, proteins were extracted for analysis by nano-liquid chromatography–electrospray ionization–tandem mass spectrometry. In the cells cultured at 5°C, relative abundances of ribosomal proteins, cold shock proteins, and DEAD/DEAH box RNA helicases were increased in comparison to those at 22°C. These results suggest that maintenance of proper translation is critical for growth under low-temperature conditions, similar to the case for other cold-adapted prokaryotes.

Phylogenetic analysis of Beggiatoa spp. from organic rich sediment of Tokyo Bay, Japan

Nitrate-accumulating filamentous bacteria from organic rich sediment of Tokyo Bay, morphologically similar to Beggiatoa species, were phylogenetically analyzed. From several sites in Tokyo Bay, Beggiatoa-like bacteria were collected. Each sample contained narrower or wider type (10 and 30 microns, respectively) of trichomes. With the nested PCR using specific primers for Beggiatoa, fragments of 16S rRNA gene were amplified and then subjected to denaturing gradient gel electrophoresis (DGGE) analysis. Sequencing and the following phylogenetic analysis indicated that they are related to large Beggiatoa species. The wider type was related to uncultured Beggiatoa clones of other geographical localities and distinct from the narrower type in Tokyo Bay. Among the narrower types, a sample from a tidal flat was genetically distinct from the sample from sites of 10 and 20 m water depth. These narrower types form a new branch in Beggiatoa/Thioploca cluster. The result of phylogenetic analysis was in accordance with the previous studies that indicate possession of nitrate-accumulation capability is congruent with phylogeny based on 16S rRNA sequences.

Temperature-dependent differences in community structure of bacteria involved in degradation of petroleum hydrocarbons under sulfate-reducing conditions

Aim:  The aim of this study was to characterize the microbial community involved in anaerobic degradation of petroleum hydrocarbon under low‐ and moderate‐temperature conditions.