Miyuki Nishijima

Development of a Prokaryotic Universal Primer for Simultaneous Analysis of Bacteria and Archaea Using Next-Generation Sequencing

For the analysis of microbial community structure based on 16S rDNA sequence diversity, sensitive and robust PCR amplification of 16S rDNA is a critical step. To obtain accurate microbial composition data, PCR amplification must be free of bias; however, amplifying all 16S rDNA species with equal efficiency from a sample containing a large variety of microorganisms remains challenging. Here, we designed a universal primer based on the V3-V4 hypervariable region of prokaryotic 16S rDNA for the simultaneous detection of Bacteria and Archaea in fecal samples from crossbred pigs (Landrace×Large white×Duroc) using an Illumina MiSeq next-generation sequencer. In-silico analysis showed that the newly designed universal prokaryotic primers matched approximately 98.0% of Bacteria and 94.6% of Archaea rRNA gene sequences in the Ribosomal Database Project database. For each sequencing reaction performed with the prokaryotic universal primer, an average of 69,330 (±20,482) reads were obtained, of which archaeal rRNA genes comprised approximately 1.2% to 3.2% of all prokaryotic reads. In addition, the detection frequency of Bacteria belonging to the phylum Verrucomicrobia, including members of the classes Verrucomicrobiae and Opitutae, was higher in the NGS analysis using the prokaryotic universal primer than that performed with the bacterial universal primer. Importantly, this new prokaryotic universal primer set had markedly lower bias than that of most previously designed universal primers. Our findings demonstrate that the prokaryotic universal primer set designed in the present study will permit the simultaneous detection of Bacteria and Archaea, and will therefore allow for a more comprehensive understanding of microbial community structures in environmental samples.

Identification of isoprenoid quinones by frit-FAB liquid chromatography-mass spectrometry for the chemotaxonomy of microorganisms

Abstract An easy and highly sensitive analytical method for the identification of microbial isoprenoid quinones using Frit-FAB liquid chromatography–mass spectrometry was developed. The composition of ubiquinone, menaquinone, rhodoquinone and their analogs was determined directly using combined information on the high-performance liquid chromatography retention time, a UV spectrum and a mass spectrum, without any standard samples. This method can be widely applied for the chemotaxonomy of microorganisms such as bacteria, actinomycetes, fungi and yeasts.

Thermotoga petrophila sp. nov. and Thermotoga naphthophila sp. nov., two hyperthermophilic bacteria from the Kubiki oil reservoir in Niigata, Japan.

Two hyperthermophilic bacteria, strains RKU-1T and RKU-10T, which grew optimally at 80 degrees C, were isolated from the production fluid of the Kubiki oil reservoir in Niigata, Japan. They were strictly anaerobic, rod-shaped fermentative heterotrophs. Based on the presence of an outer sheath-like structure (toga) and 16S rDNA sequences, they were shown to belong to the genus Thermotoga. Cells of strain RKU-1T were 2-7 microm by 0.7-1.0 microm, with flagella. They grew at 47-88 degrees C on yeast extract, peptone, glucose, fructose, ribose, arabinose, sucrose, lactose, maltose, starch and cellulose as sole carbon sources. Cells of strain RKU-10T were 2-7 microm by 0.8-1.2 microm, with flagella. They grew at 48-86 degrees C on yeast extract, peptone, glucose, galactose, fructose, mannitol, ribose, arabinose, sucrose, lactose, maltose and starch as sole carbon sources. While strains RKU-1T and RKU-10T reduced elemental sulfur to hydrogen sulfide, their final cell yields and specific growth rates decreased in the presence of elemental sulfur. Thiosulfate also inhibited growth of strain RKU-1T but not strain RKU-10T. The G+C contents of the DNA from strains RKU-1T and RKU-10T were 46.8 and 46.1 mol%. Phenotypic characteristics and 165 rDNA sequences of the isolates were similar to those of Thermotoga maritima and Thermotoga neapolitana, both being hyperthermophilic bacteria isolated from hydrothermal fields. However, the isolates differed from these species in their minimum growth temperatures, utilization of some sugars, sensitivity to rifampicin and the effects of elemental sulfur and thiosulfate on growth. The low levels (less than 31%) of DNA reassociation between any two of these hyperthermophilic Thermotoga strains indicated that the isolates were novel species. Analysis of the gyrB gene sequences supported the view that the isolates were genotypically different from these reference species. The isolates were named Thermotoga petrophila sp. nov., with type strain RKU-1T (= DSM 13995T = JCM 10881T), and Thermotoga naphthophila sp. nov., with type strain RKU-10T (= DSM 13996T = JCM 10882T).

BACTERIA THAT INDUCE MORPHOGENESIS IN ULVA PERTUSA (CHLOROPHYTA) GROWN UNDER AXENIC CONDITIONS1

Marine foliaceous green macroalgae such as Ulva lose their typical morphology when cultured aseptically in defined synthetic media. However, after reinfection by certain marine bacteria (isolated from unialgal cultures of Ulva pertusa Kjellman), the organisms regain their typical foliaceous or tubular morphology. To investigate the morphogenesis (MG) induced in U. pertusa by bacteria, we isolated and identified bacteria with MG activity on U. pertusa and studied the distribution of such bacteria in seawater and on various marine macroalgae. We isolated 1555 bacterial strains from 18 species of marine macroalgae (six Chlorophyta, five Phaeophyta, and seven Rhodophyta), from seawater and from sediment collected at the beach at Omaezaki, Shizuoka Prefecture; Japan. Of these, 676 bacterial strains (43.5%) showed MG activity. They were classified into six bacterial groups, Flavobacterium, Vibrio, Pseudomonas, Deleya, Escherichia, and gram‐positive cocci. These bacteria were ubiquitous among the samples and were not specific to U. pertusa. Several plant growth regulators had no MG activity. Filter‐sterilized supernatants of culture media of MG‐active bacteria strains did not induce MG. Cocultivation of Ulva with active bacterial strains is so far the only way to induce the MG effect, which suggests that for MG direct contact between Ulva and the bacterial strain is necessary.

Distribution and physiological characteristics of hyperthermophiles in the Kubiki oil reservoir in Niigata, Japan

ABSTRACT The distribution of culturable hyperthermophiles was studied in relation to environmental conditions in the Kubiki oil reservoir in Japan, where the temperature was between 50 and 58°C. Dominant hyperthermophilic cocci and rods were isolated and shown to belong to the genera Thermococcus and Thermotoga, respectively, by 16S rDNA analyses. Using the most-probable-number method, we found that hyperthermophilic cocci were widely distributed in several unconnected fault blocks in the Kubiki oil reservoir. In 1996 to 1997, their populations in the production waters from oil wells were 9.2 × 103 to 4.6 × 104cells/ml, or 10 to 42% of total cocci. On the other hand, hyperthermophilic rods were found in only one fault block of the reservoir with populations less than 10 cells/ml. DominantThermococcus and Thermotoga spp. grew at reservoir temperatures and utilized amino acids and sugars, respectively, as sole carbon sources. While organic carbon was plentiful in the environment, these hyperthermophiles were unable to grow in the formation water due to lack of essential nutrients. Concentrations of some organic and inorganic substances differed among fault blocks, indicating that the movement of formation water between fault blocks was restricted. This finding suggests that the supply of nutrients via fluid current is limited in this subterranean environment and that the organisms are starved in the oil reservoir. Under starved conditions at 50°C, culturable cells of Thermococcus sp. remained around the initial cell density for about 200 days, while those of Thermotoga sp. decreased exponentially to 0.01% of the initial cell density after incubation for the same period. The difference in survivability between these two hyperthermophiles seems to reflect their populations in the fault blocks. These results indicate that hyperthermophilic cocci and rods adapt to the subterranean environment of the Kubiki oil reservoir by developing an ability to survive under starved conditions.

β-cyanoalanine production by marine bacteria on cyanide-free medium and its specific inhibitory activity toward cyanobacteria

ABSTRACT In screening the culture broth of marine bacteria collected at Yap (Micronesia), Palau (Belau), and Okinawa (the southwest islands of Japan) for antimicroalgal activity, 37 out of 2,594 bacterial isolates tested were found to produce anticyanobacterial substances against Oscillatoria amphibia NIES-361. One strain, C-979, identified as a Vibrio sp., was selected and cultured in 2.4 liters of marine broth 2216 to identify the bioactive compound produced by the strain. The purified very hydrophilic compound (16.4 mg) was determined to be β-cyano-l-alanine (l-CNAla) by instrumental analyses and the application of the advanced Marfey method. l-CNAla did not inhibit the growth of bacteria, yeast, or eukaryotic microalgae, but some cyanobacteria were found to be sensitive to l-CNAla at a concentration of 0.4 to 25 μg/ml. The effect of l-CNAla on some other environmental organisms, including invertebrates and a macroalgae, is discussed. CNAla production in marine broth was examined by thin-layer chromatography for the 37 bacterial isolates which produced an anticyanobacterial substance. The broth of 36 of these strains contained CNAla, suggesting the wide distribution of CNAla production by marine bacteria. This is the first report on bacteria that produce CNAla without a supply of the cyanide ion in the medium.

Endozoicomonas numazuensis sp. nov., a gammaproteobacterium isolated from marine sponges, and emended description of the genus Endozoicomonas Kurahashi and Yokota 2007.

Two non-motile, rod-shaped gammaproteobacteria were isolated from marine sponges collected from the coast of Japan at Numazu. The isolates were oxidase- and catalase-positive facultative anaerobes that fermented carbohydrates. They required sodium ions for growth and were slightly halophilic, growing in the presence of 1.0-5.0 % (w/v) NaCl (optimum of 2.0 % NaCl). Under aerobic conditions, the major isoprenoid quinones were ubiquinone-9 and menaquinone-9 and the minor quinones were ubiquinone-8 and menaquinone-8. The major cellular fatty acids were C(18 : 1)ω7c, C(16 : 1)ω7c and C(16 : 0) and the hydroxy acids were C(10 : 0) 3-OH and C(12 : 0) 3-OH. The DNA G+C content was 48.3-48.7 mol%. Phylogenetic analysis of 16S rRNA gene sequences placed the isolates within the radiation of the genus Endozoicomonas in a broad clade of uncultured clones recovered from various marine invertebrates. The isolates exhibited 96.5-96.9 % 16S rRNA gene sequence similarity with Endozoicomonas elysicola MKT110(T) and Endozoicomonas montiporae CL-33(T), with which the isolates formed a monophyletic cluster with 100 % bootstrap support. The phenotypic features (carbohydrate fermentation, quinone system and some major cellular fatty acids) differed from those of members of the genus Endozoicomonas, which are aerobic, produce little or no menaquinone under aerobic conditions and possess different amounts of C(14 : 0) and C(18 : 1)ω7c. Although some phenotypic differences were identified, the isolates should be assigned to the genus Endozoicomonas on the basis of congruity of phylogeny and should be classified as representatives of a novel species, for which the name Endozoicomonas numazuensis sp. nov. is proposed. The type strain is HC50(T) ( = NBRC 108893(T)  = DSM 25634(T)). An emended description of the genus Endozoicomonas is presented.

Seasonal Appearance of Prochlorococcus in Suruga Bay, Japan in 1992-1993

Seasonal appearance ofProchlorococcus was studied by flow cytometry in Suruga Bay, Japan in 1992–1993.Prochlorococcus cells were in high concentrations (>1×104 cells ml−1) from July to October 1992 and September 1993, when the water temperature was over 20°C. The 16S rRNA of the isolated cells showed 98.5% sequence homology with that ofP. marinus (Sargasso strain), indicating that they are the same species. The former has a high divinyl-chlorophyll (DV-Chl.)a/b ratio similar to the Mediterranean strain and different from the Sargasso strain. Maximum concentration ofProchlorococcus at the surface water was 2.5×104 cells ml−1 in August 1992 and their DV-Chl.a accounted for 4.0% of the total chlorophylla. A decrease in cell density to less than 5×103 cells ml−1 was observed from December to May with an exceptional rise in January 1993. WhileProchlorococcus showed a maximum concentration of 3.6×104 cells ml−1 at 10 m depth in September 1992, phycoerythrin (PE)-richSynechococcus spp. were dominant with their maximum concentration of 2.2×105 cells ml−1 in the same water body. On the other hand, phycocyanin (PC)-richSynechococcus spp. and the larger phytoplankters showed maximum concentrations in the surface waters in May and June. BothProchlorococcus and PE-richSynechococcus showed their lowest concentrations in April. A significant positive correlation was obtained between cell concentrations of the PE-richSynechococcus andProchlorococcus.

Purification and properties of two chitinases from Vibrio alginolyticus H-8☆

Abstract Chitinases C1 and C3 from Vibrio alginolyticus H-8 were purified by column chromatography on DEAE-Toyopearl 650M and Superdex 200HR. The molecular weights were estimated by SDS-gel electrophoresis (SDS-PAGE) to be 81,000 Da and 68,000 Da for C1 and C3, respectively. The pIs for C1 and C3 were 3.9 and 3.6, respectively. The activities of both enzymes were inhibited by Ag + and Hg 2+ .

Microbulbifer variabilis sp. nov. and Microbulbifer epialgicus sp. nov., isolated from Pacific marine algae, possess a rod―coccus cell cycle in association with the growth phase

Phylogenetic and taxonomic characterization was performed for 14 strains of bacteria that produce anticancer antibiotics (pelagiomicins) (represented by strain Ni-2088(T)) and one strain that produces UV-absorbing substances (strain F-104(T)), isolated from marine algae and seagrass collected from coastal areas of tropical Pacific islands and a subtropical island of Japan. All 15 isolates were Gram-negative, strictly aerobic, non-motile and non-spore-forming. Sequence analysis of the 16S rRNA gene showed that the isolates occupied positions in the phylogenetic radiation of the genus Microbulbifer, with similarities of 93.6-97.6 %. The cells possessed a clearly discernible rod-coccus cell cycle in association with the growth phase; cells were rods during the growth phase and all converted to coccoid-ovoid cells when proliferation ceased. The coccoid-ovoid cells were optically denser than the rod cells and were viable for extended periods. They were considered to constitute a resting form. The type strains of described species of Microbulbifer were also found to possess identical rod-coccus cell cycles. The G+C content of the DNA was 48.1-49.7 mol%. The major respiratory quinone system was ubiquinone-8. The major fatty acids were C(18 : 1)omega7c and C(16 : 0), and the hydroxy acids comprised C(10 : 0) 3-OH, C(12 : 0) 3-OH and iso-C(11 : 0) 3-OH. The polar lipids comprised phosphatidylethanolamine, phosphatidylglycerol and phosphatidylserine. The group of 14 pelagiomicin-producing strains and strain F-104(T) each constituted a single genomic species. Based on phylogenetic affiliation, phenotypic characteristics and genomic distinctness, the isolates represent two novel species in the genus Microbulbifer, for which the names Microbulbifer variabilis sp. nov. (type strain Ni-2088(T) =MBIC01082(T) =ATCC 700307(T)) and Microbulbifer epialgicus sp. nov. (type strain F-104(T) =MBIC03330(T) =DSM 18651(T)) are proposed.