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.

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.

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.

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.

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.

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.

Use of the MALDI BioTyper system and rapid BACpro with MALDI-TOF MS for rapid identification of microorganisms causing bacterial urinary tract infection in feline urine samples.

With the increasing number of cats kept as pets, opportunities to treat cats with lower urinary tract disease (LUTD) have recently increased in the clinical veterinary field. Urine samples collected from 50 cats with bacterial cystitis brought to Maeda Veterinary Hospital between August 10, 2015 and March 31, 2017 were used in the study. Sample preparation of the urine was performed using a MALDI Sepsityper kit and rapid BACpro. To identify the isolates, MALDI-TOF MS was performed on an AutoFlex TOF/TOF mass spectrometer. MALDI-TOF MS using rapid BACpro for pretreatment was found to be a quick and reliable method for identification of bacteria from infected urine, with a shortened analysis time enabling earlier and more accurate selection of antibiotics for treatment of feline LUTD.

Hydrocarbon Degradation and Bacterial Community Responses During Remediation of Sediment Artificially Contaminated with Heavy Oil.

　Natural biodegradation of heavy oil in the marine environment can be accelerated by the addition of nutrients or seeding of pre-selected microorganisms. In this study, a microcosm experiment was conducted to investigate the effects of inorganic nutrient supplementation (biostimulation) and bacterial consortium amendment (bioaugmentation) on the natural degradative processes of artificially contaminated sediment. Our results revealed that the addition of nutrients had greater effect on remediation than the addition of bacterial cells. Supplementation of inorganic nutrients promoted and sustained the growth of oil-degrading and heterotrophic bacteria throughout the experimental period. Highest reduction in the total petroleum hydrocarbons, and of their components, n-alkanes, polycylic aromatic hydrocarbons (PAHs) and alkyl PAHs, were obtained in the biostimulated microcosms. Changes in the bacterial community were monitored by the PCR-DGGE (polymerase chain reaction-denaturing gradient gel electrophoresis) method targeting the 16S rDNA gene. Results revealed different responses of the bacterial community to the addition of heavy oil and remediation agents. Shifts in the bacterial communities in the seawater were more dynamic than in the sediment. Results of this study showed that addition of remediation agents significantly enhanced the natural biodegradation of heavy oil in a sediment-seawater microcosm trial.

Microflora in the Soft Tissue of the Pacific Oyster Crassostrea gigas Exposed to the Harmful Microalga Heterosigma akashiwo.

　A marine raphidophyte Heterosigma akashiwo is a causative agent of harmful microalgal blooms, which often cause the massive mortality of aquacultured finfish. In the present study, the Pacific oyster Crassostrea gigas was reared with H. akashiwo, and effect of the microalga on filter-feeding behavior and microflora of the gastrointestinal tract was investigated. The intake of the raphidophyte cells inhibited the molluscan filter-feeding activities, suggesting the negative physiological effect of the microalgal cell contents. However, the bivalves ingested the H. akashiwo cells to the same extent as the diatom Chaetoceros calcitrans, a non-harmful indicator to estimate the filtration rate, showing a continuation of their non-selective ingestion of the phytoplankton. Microflora of the oyster soft tissue was dominated by bacteria affiliated with the family Rhodobacteraceae, some of which are associated with microalgae. In addition, the Bacteroidetes species, in which algicidal bacteria are included, were also found in the bivalve individuals exposed to H. akashiwo. These results suggested that the ingested phytoplankton affected the microbial flora in the gastrointestinal tracts, some constituents of which helped the mollusc assimilate the ingested red tide phytoplankton. This study will provide beneficial information to clarify mechanisms by which the oyster evades the ichthyotoxicity of harmful microalgae and the participation of the intestinal microorganisms in these processes.

Synergistic Effect of Photosynthetic Bacteria and Isolated Bacteria in Their Antifungal Activities against Root Rot Fungi.

Antifungal bacteria (AB) in root rot fungus (RRF)-contaminated sweet potato farms were isolated, and seven strains were initially chosen as antagonistic candidates. An antagonistic test by using the mycelial disk placement method revealed that one AB strain by itself could inhibit the RRF growth. This AB strain was identified as Bacillus polyfermenticus based on phylogeny of 16S ribosomal RNA genes. Two AB strains (Bacillus aerophilus) displayed high levels of antifungal activity when paired with photosynthetic bacterial strain A (a purple nonsulfur photosynthetic bacterium Rhodopseudomonas faecalis). The results suggest the possible use of the isolates as agents for the biological control of the RRF infection of agricultural products in fields of cultivation.

Hypocretin-1 levels in the cerebrospinal fluid of patients with Percheron artery infarction with or without midbrain involvement: A case series.

BACKGROUND: Bilateral paramedian thalamic infarctions (BPTIs) due to artery of Percheron occlusion are known to cause hypersomnia. However, the role of hypocretin-1, a wake-promoting peptide that is located at the lateral hypothalamus, in hypersomnia in these patients remains unclear. METHODS: To clarify the role of hypocretin-1 in hypersomnia in patients with BPTIs, hypocretin-1 levels in the cerebrospinal fluid (CSF) were measured in 6 patients with BPTIs: 2 with rostral midbrain involvement (BPT+RMI) and 4 without midbrain involvement (BPT-MI). RESULTS: CSF hypocretin-1 levels were decreased in 2 patients with BPT+RMI and were within normal ranges in 4 patients with BPT-MI. Hypersomnia was noted in all the patients. In one BPT+RMI patient, hypersomnia was improved within 2 weeks and decreased CSF hypocretin-1 levels were reversed (acute phase (on day 9), 109.2 pg/mL; chronic phase (at 3 months), 323 pg/mL), whereas another BPT+RMI patient who displayed coma in the acute phase had decreased CSF orexin levels (107 pg/mL) at day 49 and exhibited severe disability. CONCLUSION: Hypocretin deficiency was not involved in hypersomnia observed in BPT-MI patients; however, CSF hypocretin-1 levels were reduced in BPT+RMI patients. Reduced CSF hypocretin-1 levels in the chronic phase may possibly predict a poor clinical outcome in patients with Percheron artery infarction.

Isolation and Characterization of a Purple Non-Sulfur Photosynthetic Bacterium Rhodopseudomonas faecalis Strain A from Swine Sewage Wastewater.

A purple non-sulfur photosynthetic bacterium (PNSB), PSB Strain A was isolated from swine sewage wastewater. Phylogenetic analysis revealed that PSB Strain A was most closely related to Rhodopseudomonas faecalis. Growth of the isolate under anaerobic-light conditions with a variety of carbon sources was investigated. Both PSB Strain A and the standard strain showed good growth with acetic acid, propionic acid, and n-butyric acid at a concentration of 20 mM. At the high concentration of 200 mM, PSB Strain A showed better growth in pyruvate, acetate, propionate, succinate and malate. By applying PSB Strain A to treat swine sewage wastewater, the concentration of VFAs, which were acetic acid and propionic acid, decreased from 158.0 mM to 120.2±2.9 mM, and 14.9 mM to 9.3±0.9 mM, respectively, after 216-h incubation. After 330-h incubation, the concentrations of TOC and ammonia nitrogen dropped from 4508.0 mg/L to 3104.0±451.5 mg/L, and 629.7 mg/L to 424.1±7.4 mg/L, respectively. The isolated PSB Strain A showed almost the same efficiency compared with the standard strain on the removal of VFAs and TOC. The results suggest the possibility of using the isolated strain to treat swine sewage wastewater.

Extrapulmonary tuberculosis presented as fever of unknown origin in two patients with endstage kidney disease not on dialysis: usefulness of 18-FDG-PET/CT in the diagnostic localization of fever of unknown origin.

Tuberculosis is one of the common causes of fever of unknown origin in patients with chronic kidney disease (CKD). Extrapulmonary tuberculosis is more common in CKD patients, and is, unfortunately, often underdiagnosed despite extensive assessments. Recently, fluorine-18-deoxyglucose-positron emission tomography/computed tomography (FDG-PET/CT) has been available in the diagnosis of malignancy, inflammatory and infectious diseases, and has become a useful diagnostic tool. Here, we present two cases of endstage kidney disease who presented with fever of unknown origin at the time of dialysis initiation. In both cases, although interferon-gamma-releasing assay was positive, combined conventional diagnostic modalities such as computed tomography and gallium-citrate scintigraphy failed to detect the sites infected with tuberculosis. By contrast, extrapulmonary lesions were detected by FDG-PET/CT and successfully treated with combined anti-tuberculous drugs. Diagnosis of extrapulmonary tuberculosis was confirmed by biopsy of the affected lymph node and lumbar spine, followed by PCR of the biopsied specimen. These cases highlight the importance of considering tuberculosis as one of the differential diagnoses in pre-dialysis CKD patients with persistent fever, and the usefulness of FDG-PET/CT in the detection of infectious sites of extrapulmonary tuberculosis.

Urinary albumin and transferrin as early diagnostic markers of chronic kidney disease.

Feline renal diseases are increasingly noted in veterinary practice. It is important to diagnose and identify the pathological basis of renal dysfunction accurately at an early stage, but there are only a few reports on this area in clinical veterinary medicine. We investigated the efficacy of measurement of urinary albumin (u-Alb) and urinary transferrin (u-Tf) for early diagnosis using 5-µl urine samples collected noninvasively by catheterization from normal (IRIS stage I) cats and cats with stage I chronic kidney disease (CKD). The u-Alb levels in normal and stage I CKD cats were 6.0 ± 4.5 and 11.2 ± 8.4 mg/dl, respectively, and the u-Tf levels were 0.09 ± 0.42 and 0.52 ± 0.79 mg/dl, respectively. Based on ROC curve analysis, the sensitivity and specificity of u-Alb and u-Tf were higher than those of the currently used biomarker, the plasma creatinine level. The sensitivity of u-Alb was higher than that of u-Tf, whereas the specificity of u-Tf was higher than that of u-Alb. The validity of the threshold albumin level (20 mg/dl) was confirmed by measurements using SDS-PAGE. Since leakage of u-Tf in urine precedes leakage of u-Alb, inclusion of u-Tf in biochemistry tests may be appropriate for IRIS staging as a diagnostic marker of early diagnosis of renal disorder in cats.

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.

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.

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.

Bioremediation of heavily oil-polluted seawater by a bacterial consortium immobilized in cocopeat and rice hull powder.

We examined the effectiveness of cocopeat and rice hull powder obtained from agricultural wastes as biocarriers for an oil-degrading bacterial consortium. Scanning electron microscopy revealed colonization and strong attachment of bacterial cells on the surface of both carriers. Results of a 60-day in vitro seawater bioremediation trial showed significant oil reduction and high cultivable bacterial counts in treatments augmented with the carrier-attached bacterial consortia compared to treatments supplemented with the same consortium in free living and encapsulated forms. Significant degradations in both aliphatic and aromatic fractions were obtained in treatments augmented with carrier-immobilized consortia. The developed immobilized cells showed sustained activities and viabilities during storage for six months. Results of this study demonstrated that inexpensive waste materials can be utilized as biocarriers of an oil-degrading consortium and that immobilization on biocarriers can enhance the bioremediation of oil-contaminated seawater.

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.

Characterization of the bacterial community in the sediment of a brackish lake with oyster aquaculture.

The physicochemical properties and bacterial community in sediments of Lake Shiraishi, a lake with brackish water, were characterized to elucidate the influence of oyster farming and seawater and freshwater inflow. Physicochemical analyses suggested the marine origin of the sediment at the mouth of the lake, while higher organic matter load and the resultant anaerobic, reductive condition of the sediments of the inner part were observed. The bacterial community in the sediments reflects these sediment environments: the bacterial community in the vicinities of oyster farms included sulfate-reducing bacteria (SRB) , although sulfur-oxidizing bacteria (SOB) were found at all the sampling sites. In addition, similarity of the band profiles obtained with 16S ribosomal RNA gene (16S rDNA) -denaturing gradient gel electrophoresis (DGGE) decreased in proportion to the distance from the mouth of the lake to the oyster farms in the inner part. This study was able to characterize the microbial community shift in brackish lake sediments with an oyster aquaculture system through the molecular fingerprinting technique, DGGE, in relation to their physicochemical characteristics.