Observation of the feeding behaviour of reared Japanese eel Anguilla japonica leptocephali fed picocyanobacteria Synechococcus spp.

The authors observed the feeding behaviour of artificially reared Japanese eel Anguilla japonica leptocephali, 7.5-19 mm total length (10-61 days post-hatch), fed Synechococcus sp., which is considered a potential food source of anguilliform larvae. Three strains of Synechococcus sp. (NIES-972, 976 and 979) were tested as the food material. Larvae across the entire length range could effectively ingest a suspension of pico-sized cyanobacteria (1-3 µm in diameter). Video observations of the mid-hindgut of larvae under an epifluorescence microscope confirmed that the movement of microvilli of the intestinal epithelium allowed the cell particles to circulate in the mid-hindgut, before becoming solidified in the anal region. Significant differences in food intake were observed between larvae fed two strains of Synechococcus (NIES-972 and 976), and among different cell densities, which suggests feeding selectivity and density dependence. Comparisons of feeding behaviour under the light group (9L:15D) and the dark group (24D) showed significantly higher food intake (measured as an index of intestinal fullness) in the light group, although substantial and continuous ingestion was observed in the dark group, indicating continuous feeding by swallowing sea water. The authors hypothesise that the feeding ecology of anguilliform leptocephali is based on a survival strategy whereby the larvae do not compete with various higher-trophic-level fishes for food in an oligotrophic environment.

Tepidicaulis marinus gen. nov., sp. nov., a marine bacterium that reduces nitrate to nitrous oxide under strictly microaerobic conditions.

A moderately thermophilic, aerobic, stalked bacterium (strain MA2T) was isolated from marine sediments in Kagoshima Bay, Japan. Phylogenetic analysis of 16S rRNA gene sequences indicated that strain MA2T was most closely related to the genera Rhodobium,Parvibaculum, and Rhodoligotrophos (92-93 % similarity) within the class Alphaproteobacteria. Strain MA2T was a Gram-stain-negative and stalked dimorphic bacteria. The temperature range for growth was 16-48 °C (optimum growth at 42 °C). This strain required yeast extract and NaCl (>1 %, w/v) for growth, tolerated up to 11 % (w/v) NaCl, and was capable of utilizing various carbon sources. The major cellular fatty acid and major respiratory quinone were C18 : 1ω7c and ubiquinone-10, respectively. The DNA G+C content was 60.7 mol%. Strain MA2T performed denitrification and produced N2O from nitrate under strictly microaerobic conditions. Strain MA2T possessed periplasmic nitrate reductase (Nap) genes but not membrane-bound nitrate reductase (Nar) genes. On the basis of this morphological, physiological, biochemical and genetic information a novel genus and species, Tepidicaulis marinus gen. nov., sp. nov., are proposed, with MA2T ( = NBRC 109643T = DSM 27167T) as the type strain of the species.

Intracellular concentrations of 65 species of transcription factors with known regulatory functions in Escherichia coli.

The expression pattern of the Escherichia coli genome is controlled in part by regulating the utilization of a limited number of RNA polymerases among a total of its approximately 4,600 genes. The distribution pattern of RNA polymerase changes from modulation of two types of protein-protein interactions: the interaction of core RNA polymerase with seven species of the sigma subunit for differential promoter recognition and the interaction of RNA polymerase holoenzyme with about 300 different species of transcription factors (TFs) with regulatory functions. We have been involved in the systematic search for the target promoters recognized by each sigma factor and each TF using the newly developed Genomic SELEX system. In parallel, we developed the promoter-specific (PS)-TF screening system for identification of the whole set of TFs involved in regulation of each promoter. Understanding the regulation of genome transcription also requires knowing the intracellular concentrations of the sigma subunits and TFs under various growth conditions. This report describes the intracellular levels of 65 species of TF with known function in E. coli K-12 W3110 at various phases of cell growth and at various temperatures. The list of intracellular concentrations of the sigma factors and TFs provides a community resource for understanding the transcription regulation of E. coli under various stressful conditions in nature.

Methylocaldum marinum sp. nov., a thermotolerant, methane-oxidizing bacterium isolated from marine sediments, and emended description of the genus Methylocaldum.

An aerobic, methane-oxidizing bacterium (strain S8(T)) was isolated from marine sediments in Kagoshima Bay, Japan. Phylogenetic analysis based on 16S rRNA gene sequences indicated that this strain is closely related to members of the genus Methylocaldum (97.6-97.9 % similarity) within the class Gammaproteobacteria. Strain S8(T) was a Gram-staining-negative, non-motile, coccoid or short rod-shaped organism. The temperature range for growth of strain S8(T) was 20-47 °C (optimum growth at 36 °C). It required NaCl (>0.5 %), tolerated up to 5 % NaCl and utilized methane and methanol. The major cellular fatty acid and major respiratory quinone were C16 : 0 and 18-methylene ubiquinone 8, respectively. The DNA G+C content was 59.7 mol%. Strain S8(T) possessed mmoX, which encodes soluble methane monooxygenase, as well as pmoA, which encodes the particulate methane monooxygenase. On the basis of this morphological, physiological, biochemical and genetic information, the first marine species in the genus Methylocaldum is proposed, with the name Methylocaldum marinum sp. nov. The type strain is S8(T) ( = NBRC 109686(T) = DSM 27392(T)). An emended description of the genus Methylocaldum is also provided.

Methyloceanibacter caenitepidi gen. nov., sp. nov., a facultatively methylotrophic bacterium isolated from marine sediments near a hydrothermal vent.

A moderately thermophilic, methanol-oxidizing bacterium (strain Gela4(T)) was isolated from methane-utilizing mixed-culture originating from marine sediment near a hydrothermal vent. Phylogenetic analysis of 16S rRNA gene sequences indicated that strain Gela4(T) was closely related to members of the genus 'Methyloligella' (94.7% similarity) within the class Alphaproteobacteria. Strain Gela4(T) was a Gram-staining-negative and aerobic organism. Cells were rod-shaped and non-motile. The temperature range for growth of strain Gela4(T) was 19-43 °C (optimal growth at 35 °C). Strain Gela4(T) tolerated up to 9% NaCl with an optimum at 1%. The organism was a facultative methylotroph that could utilize methanol, methylamine, trimethylamine and a variety of multi-carbon compounds. The major cellular fatty acid and major respiratory quinone were C18 : 1ω7c and ubiquinone-10, respectively. The predominant phospholipids were phosphatidylcholine, phosphatidylglycerol and phosphatidylethanolamine. The DNA G+C content was 63.9 mol%. On the basis of the morphological, physiological, biochemical and genetic information, a novel genus and species, Methyloceanibacter caenitepidi is proposed, with Gela4(T) ( = NBRC 109540(T) = DSM 27242(T)) as the type strain.

Development of a Transformation System for Nitratireductor sp.

Nitratireductor sp. OM-1 can accumulate butenoic acid, which is a short-chain unsaturated carboxylic acid utilized for chemical products. So far, we have predicted the thioesterase gene, te, as a candidate gene for butenoic acid biosynthesis, based on comparative transcriptome analysis. To confirm the function of te, the gene transfer system in Nitratireductor sp. OM-1 was required. Thus, in this study, we used electroporation as a transformation system and pRK415, a broad host range plasmid, and optimized the conditions. As a result, a maximum transformation efficiency of 7.9 × 104 colonies/µg DNA was obtained at 22.5 kV/cm. Moreover, an expression vector, pRK415-te, was constructed by insertion of te, which was successfully transferred into strain OM-1, using electroporation. The recombinant OM-1 strain produced butenoic acid at 26.7 mg/g of dried cell weight, which was a 254% increase compared to transformants harboring an empty vector. This is the first report of a gene transfer system for Nitratireductor sp., which showed that the te gene was responsible for butenoic acid production.

Transformation of microflora during degradation of gaseous toluene in a biofilter detected using PCR-DGGE.

A laboratory-scale biofiltration system, the rotatory-switching biofilter (RSB), was operated for 199 days using toluene as a model pollutant. The target gaseous pollutant for the biofiltration experiment was approximately 300 ppmv of toluene. Toluene removal efficiency (RE, %) was initially approximately 20% with a 247-ppmv concentration (0.9 g m(-3)) of toluene during the first 10 days. Although the RE decreased several times whenever nitrogen was consumed, it again reached almost 100% when the nitrogen source was in sufficient supply. Denaturing gradient gel electrophoresis (DGGE) analysis was employed to assess the transformation ofmicroflora during operation of the biofilter The results based on a 16S rRNA gene profile showed that the microbial community structure changed with operation time. Although the microflora changed during the initial period (before day 40), transformation of the bacterial component was hardly observed after day 51. Statistical analyses of the DGGE profiles indicated that the bacterial community was almost unaffected by the environmental factors, such as adding ozone, high-level nitrogen supply, increase of loading toluene, and the shutdown of the RSB. The DGGE profile using tmoA-like genes, which encode proteins belonging to the hydroxylase component mono-oxygenases involved in the initial attack of aerobic benzene, toluene, ethylbenzene, and xylene degradation, confirmed the existence of toluene-degrading bacteria. There were at least four kinds of toluene-degradable bacteria having tmoA-like genes up to day 36, which decreased to two species after day 40. Sequence analysis after DGGE profiling revealed that Burkholderia cepacia, Sphingobacterium multivorum, and Pseudomonas putida were present in the biofilter. Only Alicycliphilus denitrificans was present throughout the whole operation period. In the initial stage of operating the RSB, many types of bacteria may have tried to adapt to the conditions, and subsequently, only selected bacteria were able to grow and to degrade toluene.

Extracellular neutral lipids produced by the marine bacteria Marinobacter sp.

We analyzed the production of neutral lipids by the marine hydrocarbonoclastic bacteria Marinobacter sp. strain PAD-2 using hexadecane or succinate as the sole carbon source. Results showed that strain PAD-2 was able to grow and reduce the surface tension to 33±1.5 mN m(-1) and 58±1.5 mN m(-1) when n-hexadecane or succinate was used as the sole carbon source, respectively. The lipophilic compounds produced by Marinobacter sp. strain PAD-2 were extracted, and then crude lipophilic compounds, expected to be wax ester-like lipids, were analyzed by thin layer chromatography (TLC) . Furthermore, the lipophilic compound demonstrating surface activity was purified and subjected to gas chromatography/mass spectrometry (GC/MS) analysis. Although these did not give definite structural information due to the weak molecular ion peak (M(+)) , one component Ma-1 showed almost the same mass spectrum as that of component Fa-2, which represented a biosurfactant derived from Dietzia maris reported previously. Cell hydrophobicity was measured by a test of bacterial adhesion to hydrocarbons. A higher hydrophobic cell surface was observed in strain PAD-2. Extracellular wax ester-like compounds seem to be one type of the surface active compounds when bacteria grow on hexadecane or succinate as the sole carbon source.

Bacterial diversity associated with the rotifer Brachionus plicatilis sp. complex determined by culture-dependent and -independent methods.

The bacterial communities associated with rotifers (Brachionus plicatilis sp. complex) and their culture water were determined using culture-dependent and -independent methods (16S rRNA gene clone library). The bacterial communities determined by the culture-independent method were more diverse than those determined by the culture-dependent method. Although the culture-dependent method indicated the bacterial community of rotifers was relatively similar to that of the culture water, 16S rRNA gene clone library analyses revealed a great difference between the two microbiotas. Our results suggest that most bacteria associated with rotifers are not easily cultured using conventional methods, and that the microbiota of rotifers do not correspond with that of the culture water completely.

Wax ester-like compounds as biosurfactants produced by Dietzia maris from n-alkane as a sole carbon source.

The hydrocarbon-degrading bacterium Dietzia maris WR-3 was isolated from a consortium comprising ammonia-oxidizing and denitrifying bacteria derived from marine sediments. Here, we examined biosurfactant production by strain WR-3 when cultured using several different carbon (D-glucose, n -decane, n -hexadecane, motor oil, olive oil, and rapeseed oil) and nitrogen (NH(4) )(2) SO(4) , NaNO(3) , yeast extract, and polypeptone) sources as growth substrates. Strain WR-3 was able to grow and reduce the surface tension of culture broth to 31±1.0 mN m(-1) when cultured using n -hexadecane and nitrate ions. The surface-active compounds produced by strain WR-3 were extracted and analyzed by thin layer chromatography. Moreover, the main components in the extract were further purified and subjected to gas chromatography/mass spectrometry (GC/MS). From the analysis, the surface-active compounds were tentatively identified as wax ester-like compounds, which were synthesized from the degradation process of n -alkane. The production of surface-active compounds by strain WR-3 promoted attachment of cells to hydrocarbon droplets via increased cell hydrophobicity, thus allowing enhanced degradation of water immiscible substrates. As Dietzia spp. can grow and produce wax esters from the degradation process of hydrocarbons, these marine bacteria are potentially useful for the bioremediation of hydrocarbon-contaminated environments.

Cloning and analysis of the ggpS gene from Cyanobacteria Arthrospira spp. involved in the synthesis of an osmolyte glucosylglycerol.

The genus Arthrospira is a nonheterocystous filamentous cyanobacterium inhabiting diverse environments including those of high salinity. In the present study, Arthrospira strains were isolated from freshwater and brackish lakes in Myanmar, and their osmoprotective adaptation was investigated as it was for the genus Synechococcus strains. The Arthrospira strains showed satisfactory growth up to 1.0 M sodium chloride, suggesting their acclimation to high salinity stress. Cloning and phylogenetic analysis of ggpS, which encodes an osmolyte glucosylglycerol-phosphate synthase (GgpS), showed that the cyanobacterial strains possess a GgpS-based osmoprotective mechanism, except for Synechococcus strains of freshwater origin. The Arthrospira spp. strains fell into the same cluster in the GgpS phylogeny, suggesting their close taxonomic relationship. One exception was Arthrospira sp. TT-1 (II); the ggpS (II), possibly a paralogue of the ggpS (I), branched out from the cyanobacterial cluster. These findings suggest the wide distribution of the genus Arthrospira in freshwater to brackish environments is ascribed to its glucosylglycerol production as an osmoprotectant and resulting in salt acclimation.

Effect of salinity on denitrification under limited single carbon source by Marinobacter sp. isolated from marine sediment.

Marinobacter comprises Gram-negative, aerobic, motile, and rod-shaped bacteria within the gamma-subclass of the Proteobacteria and is known to be halophilic or halotolerant, heterotrophic neutrophile. Two strains classified as belonging to Marinobacter, named PAD-2 and SeT-1, were isolated from marine sediment. The most closely related species of PAD-2 and SeT-1 are M. alkaliphilus and M. guinea, respectively. The strain PAD-2 exhibited remarkably higher denitrification at concentrations of 0.5 to 1 M NaCl (3-6% w/w) than at other salinities (2 and 3 M NaCl, 12-18% w/w), and optimal denitrification was observed in media with 0.5 M NaCl. The effect of pH on denitrification by strain PAD-2 was also examined, and the optimum denitrification occurred at neutral pH rather than under alkaline conditions. Overall, strain PAD-2 appears to be a novel halotolerant species belonging to the genus Marinobacter that shares many characteristics, such as substrate utilization profile and optimum NaCl concentration for growth with M. alkaliphilus.

Draft Genome Sequence of Thermanaeromonas sp. Strain C210, Isolated in the Presence of Carbon Monoxide.

The genus Thermanaeromonas comprises two species of thermophilic, strictly anaerobic, spore-forming bacteria. Here, we report the draft genome sequence of Thermanaeromonas sp. strain C210, which was first isolated in the presence of carbon monoxide. The genome sequence provides insight into carbon monoxide-dependent metabolism for members of the genus Thermanaeromonas.

Diversity of the Photosynthetic Bacterial Communities in Highly Eutrophicated Yamagawa Bay Sediments.

Yamagawa Bay, located in Ibusuki, Kagoshima Prefecture, Japan, is a geographically enclosed coastal marine inlet, and its deteriorating seabed sediments are under an anoxic, reductive, sulfide-rich condition. In order to gain insight into diversity of anoxygenic photosynthetic bacteria (AnPBs) and their ecophysiological roles in the sediments, three approaches were adopted: isolation of AnPBs, PCR-DGGE of 16S rDNA, and PCR-DGGE of pufM. Among the bacterial isolates, relatives of Rhodobacter sphaeroides were most dominant, possibly contributing to transforming organic pollutants in the sediments. Abundance of Chlorobium phaeobacteroides BS1 was suggested by 16S rDNA PCR-DGGE. It could reflect intensive stratification and resultant formation of the anoxic, sulfide-rich layer in addition to extreme low-light adaptation of this strain. Diverse purple non-sulfur or sulfur bacteria as well as aerobic anoxygenic photoheterotrophs were also detected by pufM PCR-DGGE, which could be associated with organic or inorganic sulfur cycling. The outcome of the present study highlights ecophysiologically important roles of AnPBs in the organically polluted marine sediments.

Use of Purple Non-Sulfur Photosynthetic Bacteria (Rhodobacter sphaeroides) in Promoting Ciliated Protozoa Growth.

Photosynthetic bacterium (PSB) was isolated from sediment samples of Yamagawa Bay, Kagoshima, Japan. Phylogenetic analysis results of PSB isolate were closely related to Rhodobacter sphaeroides, purple non-sulfur photosynthetic bacteria (PNSB). Pink-colored smooth edges of single bacterial colonies were observed after 3-5 days of incubation period on Basic I medium agar plates. Rhodobacter sphaeroides microscopic examination showed a short rod cell (1-2 µm length) with round ends. Sediment and water samples used for ciliates cultivation were collected from Kuwano-ura Bay, Koshiki Island, Japan. Ciliates were cultivated using fish meal with radish leaves medium (MI), with sediment into MI (MII) and algae media (MIII). The use of the algae media (MIII) in cultivation mixture produced the highest total number of ciliates. Big size ciliates were identified as Euplotes minuta and Cyclidium varibonneti, while small size was identified as Micrometopion nutans, based on PCR-DGGE. When ciliates were cultured with the PSB isolate, Rhodobacter sphaeroides as a feed, ciliates grow to 2,081 individual ml-1 72 hrs later. These findings indicate that PNSB can be used to promote ciliates growth.

Severe anasarca due to beriberi heart disease and diabetic nephropathy.

A 40-year-old man was transferred to our hospital because of severe anasarca. He was a heavy drinker for more than 20 years, and diagnosed with diabetes mellitus 8 years earlier and treated with retinal photocoagulation 8 months earlier. He reported loss of appetite after divorce 10 months prior to admission. On admission, he presented with systemic edema and dyspnea. Chest radiography showed massive pleural effusion and cardiomegaly. Serum total protein was 5.6 g/dl, albumin 2.6 g/dl, and urinary protein excretion was 5.3 g/day. Glucose tolerance test showed normal pattern. Ultrafiltration and continuous hemofiltration resulted in loss of 40 kg body weight in 5 days. Echocardiography revealed high-output heart failure and blood tests showed low serum thiamine level of 12 ng/ml (normal, >28 ng/ml). Accordingly, the diagnosis was established as beriberi heart disease complicated with nephrotic syndrome. Treatment with 50 mg/day thiamine intravenously and 80 mg/day furosemide resulted in increase in urine output, decrease in cardiac output, resolution of pulmonary effusion, and about 70 kg body weight loss. Percutaneous renal biopsy showed nodular glomerulosclerosis, mesangial matrix expansion, and thickening of glomerular basement membrane (GBM). Immunofluorescence study showed no glomerular deposition of immunoglobulin or complement. Electron microscopy showed GBM thickening and mesangial matrix deposition without electron-dense deposits or fibrils. These findings were compatible with diabetic glomerulosclerosis. In this patient, extreme malnutrition altered glucose tolerance but, on the other hand, nephrotic syndrome associated with diabetic nephropathy made the diagnosis of beriberi heart disease difficult.

Denitrifying activity and homologous enzyme analysis of Alcanivorax dieselolei strain N1203.

An Alcanivorax dieselolei strain, termed strain N1203, was isolated from the consortia of ammonia-oxidizing bacteria (AOB) combined with denitrifying bacteria from our previous study and was shown to have ability to reduce nitrate to nitrite to either nitrous oxide or molecular nitrogen. Analysis of 16S rRNA gene sequences established strain N1203 as a member of the species Alcanivorax dieselolei. In addition, the ability of strain N1203 to utilize various organic substrates as the sole carbon source, supplemented with carbohydrates, amino acids, and n-alkane compounds, was investigated, and this strain was found to have a narrow substrate range of growth such as grycerol, succinate, ethanol and n-alkane hydrocarbon. Furthermore, N1203's stepwise denitrifying activity, utilizing succinate and hexadecane as sole carbon sources, was measured. Gene fragments of nirK and qnorB genes, which are involved in denitrifying activities, were obtained, cloned and sequenced. Phylogenetic analysis for these two genes showed that both the nirK and qnorB sequences, although found in separate branches within clusters, formed subclusters branching from uncultured environmental clones. This demonstrated the typical uniqueness of these genes from any cultivated denitrifiers. Thus, strain N1203 is novel type of denitrifying bacteria that demonstrated denitrifying activities when cultivated using succinate as the sole carbon source.

Draft Genome Sequence of Parageobacillus thermoglucosidasius Strain TG4, a Hydrogenogenic Carboxydotrophic Bacterium Isolated from a Marine Sediment.

Parageobacillus thermoglucosidasius possesses biotechnological potential for fuel generation. Here, we report the draft genome sequence of P. thermoglucosidasius strain TG4, which was first isolated from a marine sediment. The genome sequence provides insight into the plasmid diversity and carbon monoxide-dependent hydrogen production capacity of P. thermoglucosidasius.

Assessment of microbial adherent carriers for possible application in nitrogen removal: a fundamental study.

Microbes have been used for cleaning polluted environments and degrading unwanted compounds. This study assessed the feasibility of using three microbial adherent carriers, Isolite, activated carbon fiber (ACF), and a marine sediment solidified carrier, MSSC, which was newly developed from dredged waste marine sediment, to remove nitrogen in sediment. The properties of three carriers were investigated, and the extent of microbe adhesion to the carriers was observed by using scanning electron microscopy. The amount of nitrogen removed by MSSC was higher than that removed by Isolite and ACF. We concluded that MSSC may be useful as a microbial adherent carrier.

Construction of a consortium comprising ammonia-oxidizing bacteria and denitrifying bacteria isolated from marine sediment.

A unique consortium of ammonia-oxidizing bacteria (AOB) and denitrifying bacteria was obtained via a long-term, 3-step cultivation of isolates from organically-enriched marine sediment. We developed this microbial consortium for possible applications in the remediation of degraded habitats in closed aquaculture or other aquatic environments via microbial degradation. Analysis of media components found definitive evidence of nitrogen removal via the coupling of ammonia-oxidation and denitrification. The phylogenetic diversity of the consortium was investigated by performing polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) that targeted the 16S rRNA gene, and the functional genes involved in ammonia-oxidation (amoA) and denitrification (nirS, nirK, norB and nosZ). Consequently, no significant divergence was observed, and thus it is suggested microbial populations were selected via a long-term, 3-step incubation process. All of the 16S rRNA clones fell into three phylogenetic groups, namely, gamma-proteobacteria, Actinobacteria and Flavobacteria. For almost half of the clones, the closest relatives in the database were identified as Alcanivorax spp. and these clones were present at all cultivation stages. The presence of these species as the dominant clones is significant since these bacterial species are known to reduce nitrate to nitrite. Accordingly, their abundance in our microbial consortium may have been responsible for the observed stepwise denitrification. All sequences of the amoA gene were identified to be Nitrosomonas lineage. Half of the nirS clones were identified to be from one major group of well-known denitrifying bacteria, Pseudomonas sp. Furthermore, 70% of the nirK clones were closely related to the nirK sequences of uncultured bacterial clones isolated from arable soil. The qnorB clones consisted of clusters exclusively, and formed a distinct cluster from the novel sequences of cultivated species. The nosZ clones also were not found in any of the closest relatives in the database including the uncultured bacterium from marine sediment. The unique clones obtained from the functional genes were related to each denitrification step.