Milk basic protein supplementation enhances fracture healing in mice.

OBJECTIVES: There is an unmet need for agents that can stimulate bone healing. The goal of this study was to evaluate the effects of basic proteins from milk whey (milk basic protein [MBP]) on fracture healing in mice. METHODS: Closed tibial transverse fractures were generated in 6-wk-old male C3 H/HeJ mice given either tap water or MBP-supplemented water for 3, 7, 14, 28, and 56 d after fracture generation. The tibial tissues were analyzed by radiography, µCT, and a three-point bending test. The expression levels of genes associated with bone metabolism were analyzed by real-time reverse transcription-polymerase chain reaction. RESULTS: Quantitative µCT analysis showed that MBP-treated fractured tibiae had a larger hard callus in the sectional area and a larger volume compared with fractured tibiae without MBP treatment. The expression levels of genes associated with chondrogenesis and osteogenesis showed greater increases in fractured tibiae with MBP treatment. Significant increases in the callus mechanical properties were found in MBP-treated tibiae. CONCLUSIONS: MBP supplementation has the potential to improve fracture healing and bone strength in mouse tibiae. MBP could be a potential safe, low-cost, and easily administered nutritional element to prevent secondary fractures in patients with bone fractures.

Archaeal diversity and distribution along thermal and geochemical gradients in hydrothermal sediments at the Yonaguni Knoll IV hydrothermal field in the Southern Okinawa trough.

A variety of archaeal lineages have been identified using culture-independent molecular phylogenetic surveys of microbial habitats occurring in deep-sea hydrothermal environments such as chimney structures, sediments, vent emissions, and chemosynthetic macrofauna. With the exception of a few taxa, most of these archaea have not yet been cultivated, and their physiological and metabolic traits remain unclear. In this study, phylogenetic diversity and distribution profiles of the archaeal genes encoding small subunit (SSU) rRNA, methyl coenzyme A (CoA) reductase subunit A, and the ammonia monooxygenase large subunit were characterized in hydrothermally influenced sediments at the Yonaguni Knoll IV hydrothermal field in the Southern Okinawa Trough. Sediment cores were collected at distances of 0.5, 2, or 5 m from a vent emission (90 degrees C). A moderate temperature gradient extends both horizontally and vertically (5 to 69 degrees C), indicating the existence of moderate mixing between the hydrothermal fluid and the ambient sediment pore water. The mixing of reductive hot hydrothermal fluid and cold ambient sediment pore water establishes a wide spectrum of physical and chemical conditions in the microbial habitats that were investigated. Under these different physico-chemical conditions, variability in archaeal phylotype composition was observed. The relationship between the physical and chemical parameters and the archaeal phylotype composition provides important insight into the ecophysiological requirements of uncultivated archaeal lineages in deep-sea hydrothermal vent environments, giving clues for approximating culture conditions to be used in future culturing efforts.

Subseafloor microbial communities associated with rapid turbidite deposition in the Gulf of Mexico continental slope (IODP Expedition 308).

The subseafloor microbial communities in the turbidite depositional basins Brazos-Trinity Basin IV (BT Basin) and the Mars-Ursa Basin (Ursa Basin) on the Gulf of Mexico continental slope (IODP holes U1319A, U1320A, U1322B and U1324B) were investigated by PCR-dependent molecular analyses targeted to the small subunit (SSU) rRNA genes, dsrA and mcrA, and hydrogenase activity measurements. Biomass at both basins was very low, with the maximum cell or the SSU rRNA gene copy number <1 x 10(7) cells mL(-1) or copies g(-1) sediments, respectively. Hydrogenase activity correlated with biomass estimated by SSU rRNA gene copy number when all data sets were combined. We detected differences in the SSU rRNA gene community structures and SSU rRNA gene copy numbers between the basin-fill and basement sediments in the BT Basin. Examination of microbial communities and hydrogenase activity in the context of geochemical and geophysical parameters and sediment depositional environments revealed that differences in microbial community composition between the basin-fill and basement sediments in the BT Basin were associated with sedimentation regimes tied to the sea-level change. This may also explain the distributions of relatively similar archaeal communities in the Ursa Basin sediments and basement sediments in the BT Basin.

Thermosulfidibacter takaii gen. nov., sp. nov., a thermophilic, hydrogen-oxidizing, sulfur-reducing chemolithoautotroph isolated from a deep-sea hydrothermal field in the Southern Okinawa Trough.

A novel thermophilic, sulfur-reducing chemolithoautotroph, strain ABI70S6(T), was isolated from a deep-sea hydrothermal field at the Yonaguni Knoll IV, Southern Okinawa Trough. Cells of strain ABI70S6(T) were motile rods, 0.9-2.0 microm in length and 0.4-0.8 microm in width. Strain ABI70S6(T) was an obligately anaerobic chemolithotroph, exhibiting hydrogen oxidation coupled with sulfur reduction. Growth was observed at 55-78 degrees C (optimum, 70 degrees C), pH 5.0-7.5 (optimum, pH 5.5-6.0) and 0.5-4.5 % NaCl (optimum, 3.0 % NaCl). H(2) and elemental sulfur were utilized as electron donor and acceptor, respectively. The major fatty acids were C(16 : 0) (40.0 %) and C(20 : 1) (60.0 %). The G+C content of genomic DNA was 44.2 mol%. The physiological attributes of strain ABI70S6(T) are similar to those of species of genera within the family Desulfurobacteriaceae, most of which are thermophilic and chemolithoautotrophic sulfur reducers. However, 16S rRNA gene sequence similarities between the novel isolate and type strains of all species within the family Desulfurobacteriaceae were <87 %, which is close to the similarities found between the novel isolate and members of the family Thermodesulfobacteriaceae (<85 %). Based on physiological and phylogenetic features of the novel isolate, it is proposed that it represents a novel species in a novel genus, Thermosulfidibacter takaii gen. nov., sp. nov., within the phylum Aquificae. The type strain of T. takaii is ABI70S6(T) (=JCM 13301(T)=DSM 17441(T)).

Quantification of mcrA by fluorescent PCR in methanogenic and methanotrophic microbial communities.

A quantitative fluorogenic PCR method for detecting methanogenic and methanotrophic orders was established using a refined primer set for the methyl coenzyme M reductase subunit A gene (mcrA). The method developed was applied to several microbial communities in which diversity and abundance of methanogens or anaerobic methanotrophs (ANMEs) was identified by 16S rRNA gene clone analysis, and strong correlations between the copy numbers of mcrA with those of archaeal 16S rRNA genes in the communities were observed. The assay can be applied to detecting and assessing the abundance of methanogens and/or ANMEs in anoxic environments that could not be detected by 16S rRNA gene sequence analyses.

Culture-dependent and -independent characterization of microbial communities associated with a shallow submarine hydrothermal system occurring within a coral reef off Taketomi Island, Japan.

Microbial communities in a shallow submarine hydrothermal system near Taketomi Island, Japan, were investigated using cultivation-based and molecular techniques. The main hydrothermal activity occurred in a craterlike basin (depth, approximately 23 m) on the coral reef seafloor. The vent fluid (maximum temperature, >52 degrees C) contained 175 microM H2S and gas bubbles mainly composed of CH4 (69%) and N2 (29%). A liquid serial dilution cultivation technique targeting a variety of metabolism types quantified each population in the vent fluid and in a white microbial mat located near the vent. The most abundant microorganisms cultivated from both the fluid and the mat were autotrophic sulfur oxidizers, including mesophilic Thiomicrospira spp. and thermophilic Sulfurivirga caldicuralii. Methane oxidizers were the second most abundant organisms in the fluid; one novel type I methanotroph exhibited optimum growth at 37 degrees C, and another novel type I methanotroph exhibited optimum growth at 45 degrees C. The number of hydrogen oxidizers cultivated only from the mat was less than the number of sulfur and methane oxidizers, although a novel mesophilic hydrogen-oxidizing member of the Epsilonproteobacteria was isolated. Various mesophilic to hyperthermophilic heterotrophs, including sulfate-reducing Desulfovibrio spp., iron-reducing Deferribacter sp., and sulfur-reducing Thermococcus spp., were also cultivated. Culture-independent 16S rRNA gene clone analysis of the vent fluid and mat revealed highly diverse archaeal communities. In the bacterial community, S. caldicuralii was identified as the predominant phylotype in the fluid (clonal frequency, 25%). Both bacterial clone libraries indicated that there were bacterial communities involved in sulfur, hydrogen, and methane oxidation and sulfate reduction. Our results indicate that there are unique microbial communities that are sustained by active chemosynthetic primary production rather than by photosynthetic production in a shallow hydrothermal system where sunlight is abundant.

Desulfothermus okinawensis sp. nov., a thermophilic and heterotrophic sulfate-reducing bacterium isolated from a deep-sea hydrothermal field.

A novel thermophilic and heterotrophic sulfate-reducing bacterium, strain TFISO9(T), was isolated from a deep-sea hydrothermal field at the Yonaguni Knoll IV in the Southern Okinawa Trough. The cells were motile rods 2.5-5.0 microm in length and 0.6-0.9 microm in width. Strain TFISO9(T) was an obligate heterotroph and reduced sulfate. It grew between 35 and 60 degrees C (optimum 50 degrees C), at pH 5.4-7.9 (optimum pH 5.9-6.4) and with 1.5-4.5 % NaCl (optimum 2.5 %). The fatty acid composition was C(16 : 0) (61.5 %) and 12Me(16 : 0) (38.5 %). The DNA G+C content was 34.9 mol%. The 16S rRNA gene sequence analysis indicated that strain TFISO9(T) belonged to the genus Desulfothermus. Based on physiological and phylogenetic characteristics, strain TFISO9(T) represents a novel species for which the name Desulfothermus okinawensis sp. nov. is proposed. The type strain is TFISO9(T) (=JCM 13304(T)=DSM 17375(T)).

Marinitoga okinawensis sp. nov., a novel thermophilic and anaerobic heterotroph isolated from a deep-sea hydrothermal field, Southern Okinawa Trough.

A novel thermophilic and sulfur-reducing heterotrophic bacterium, strain TFS10-5(T), was isolated from a deep-sea hydrothermal field in Yonaguni Knoll IV, Southern Okinawa Trough. Cells of strain TFS10-5(T) were motile rods, 1.5-5 microm in length and 0.5-0.8 microm in width. Strain TFS10-5(T) was an obligately anaerobic heterotroph and sulfur-reduction stimulated growth. Growth was observed between 30 and 70 degrees C (optimum at 55-60 degrees C), pH 5.0-7.4 (optimum at pH 5.5-5.8), 1.0-5.5 NaCl % (optimum at 3.0-3.5 %). The fatty acid content was C(16 : 0) (71.0 %), C(16 : 1) (6.0 %), C(18 : 0) (21.4 %) and C(18 : 1) (1.6 %). The G+C content of the genomic DNA was 28 mol%. 16S rRNA gene sequence analysis indicated that strain TFS10-5(T) belongs to the genus Marinitoga. Based on the physiological and phylogenetic features of the new isolate, strain TFS10-5(T) represents a novel species in the genus Marinitoga for which the name Marinitoga okinawensis sp. nov. is proposed. The type strain is TFS10-5(T) (=JCM 13303(T)=DSM 17373(T)).

Sulfurivirga caldicuralii gen. nov., sp. nov., a novel microaerobic, thermophilic, thiosulfate-oxidizing chemolithoautotroph, isolated from a shallow marine hydrothermal system occurring in a coral reef, Japan.

Novel thermophilic bacteria, designated strains VW1 and MM1(T), were isolated from hydrothermal fluid and microbial mat samples, respectively, collected from a shallow marine hydrothermal system (water depth 22 m) occurring in coral reefs off Taketomi Island, Okinawa, Japan. Cells of the two novel strains were motile rods with a single polar flagellum in the exponential growth phase. In a medium that included elemental sulfur, cells of the two strains became non-motile with oval to spherical cell shapes. For both strains, growth occurred at between 30 and 60 degrees C (optimum temperature of 50-55 degrees C; 60-80 min doubling time) and between pH 5.5 and 7.1 (optimum pH 6.0). The isolates were microaerobic chemolithoautotrophs capable of using thiosulfate or tetrathionate as the sole energy source, O(2) as the sole electron acceptor and CO(2) as the sole carbon source. Organic substrates, such as yeast extract and tryptone, inhibited growth of both strains. The G+C contents of genomic DNA were 51.3 and 49.5 mol% for strains VW1 and MM1(T), respectively. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the two strains were closely related to each other (99.9 % sequence similarity) and were distantly related to other previously described genera within the Gammaproteobacteria. The novel isolates could also be differentiated from other gammaproteobacterial genera on the basis of their physiological properties. It is suggested that the novel isolates represent the type species of a new genus, for which the name Sulfurivirga caldicuralii gen. nov., sp. nov. (type strain MM1(T)=JCM 13439(T)=DSM 17737(T)) is proposed.

Quantification of mcrA by quantitative fluorescent PCR in sediments from methane seep of the Nankai Trough.

A quantitative fluorogenic PCR method for group-specific methyl coenzyme M reductase subunit A genes (mcrA) from methanotrophic archaea was established and applied to the characterization of microbial communities in anoxic methane seep sediments at the accretionary prism of the Nankai Trough. All of the previously identified subgroups of anaerobic methanotroph (ANME) mcrA genes were detected in the cores up to 25 cm below the seafloor, but distributional patterns of mcrA genes were found to differ according to depth. These findings suggest a distinct distribution of phylogenetically and physiologically diverse methanotrophic archaea that mediate methane oxidation in the anoxic sediments. This quantification method will contribute to future investigations of methanotrophic microbial ecosystems in anoxic marine sediments.

Genetic and functional properties of uncultivated thermophilic crenarchaeotes from a subsurface gold mine as revealed by analysis of genome fragments.

Within a phylum Crenarchaeota, only some members of the hyperthermophilic class Thermoprotei, have been cultivated and characterized. In this study, we have constructed a metagenomic library from a microbial mat formation in a subsurface hot water stream of the Hishikari gold mine, Japan, and sequenced genome fragments of two different phylogroups of uncultivated thermophilic Crenarchaeota: (i) hot water crenarchaeotic group (HWCG) I (41.2 kb), and (ii) HWCG III (49.3 kb). The genome fragment of HWCG I contained a 16S rRNA gene, two tRNA genes and 35 genes encoding proteins but no 23S rRNA gene. Among the genes encoding proteins, several genes for putative aerobic-type carbon monoxide dehydrogenase represented a potential clue with regard to the yet unknown metabolism of HWCG I Archaea. The genome fragment of HWCG III contained a 16S/23S rRNA operon and 44 genes encoding proteins. In the 23S rRNA gene, we detected a homing-endonuclease encoding a group I intron similar to those detected in hyperthermophilic Crenarchaeota and Bacteria, as well as eukaryotic organelles. The reconstructed phylogenetic tree based on the 23S rRNA gene sequence reinforced the intermediate phylogenetic affiliation of HWCG III bridging the hyperthermophilic and non-thermophilic uncultivated Crenarchaeota.

Enzymatic and genetic characterization of carbon and energy metabolisms by deep-sea hydrothermal chemolithoautotrophic isolates of Epsilonproteobacteria.

The carbon and energy metabolisms of a variety of cultured chemolithoautotrophic Epsilonproteobacteria from deep-sea hydrothermal environments were characterized by both enzymatic and genetic analyses. All the Epsilonproteobacteria tested had all three key reductive tricarboxylic acid (rTCA) cycle enzymatic activities--ATP-dependent citrate lyase, pyruvate:ferredoxin oxidoreductase, and 2-oxoglutarate:ferredoxin oxidoreductase--while they had no ribulose 1,5-bisphosphate carboxylase (RubisCO) activity, the key enzyme in the Calvin-Benson cycle. These results paralleled the successful amplification of the key rTCA cycle genes aclB, porAB, and oorAB and the lack of success at amplifying the form I and II RubisCO genes, cbbL and cbbM. The combination of enzymatic and genetic analyses demonstrates that the Epsilonproteobacteria tested use the rTCA cycle for carbon assimilation. The energy metabolisms of deep-sea Epsilonproteobacteria were also well specified by the enzymatic and genetic characterization: hydrogen-oxidizing strains had evident soluble acceptor:methyl viologen hydrogenase activity and hydrogen uptake hydrogenase genes (hyn operon), while sulfur-oxidizing strains lacked both the enzyme activity and the genes. Although the energy metabolism of reduced sulfur compounds was not genetically analyzed and was not fully clarified, sulfur-oxidizing Epsilonproteobacteria showed enzyme activity of a potential sulfite:acceptor oxidoreductase for a direct oxidation pathway to sulfate but no activity of AMP-dependent adenosine 5'-phosphate sulfate reductase for a indirect oxidation pathway. No activity of thiosulfate-oxidizing enzymes was detected. The enzymatic and genetic characteristics described here were consistent with cellular carbon and energy metabolisms and suggest that molecular tools may have great potential for in situ elucidation of the ecophysiological roles of deep-sea Epsilonproteobacteria.

Spatial distribution of marine crenarchaeota group I in the vicinity of deep-sea hydrothermal systems.

Distribution profiles of marine crenarchaeota group I in the vicinity of deep-sea hydrothermal systems were mapped with culture-independent molecular techniques. Planktonic samples were obtained from the waters surrounding two geographically and geologically distinct hydrothermal systems, and the abundance of marine crenarchaeota group I was examined by 16S ribosomal DNA clone analysis, quantitative PCR, and whole-cell fluorescence in situ hybridization. A much higher proportion of marine crenarchaeota group I within the microbial community was detected in deep-sea hydrothermal environments than in normal deep and surface seawaters. The highest proportion was always obtained from the ambient seawater adjacent to hydrothermal emissions and chimneys but not from the hydrothermal plumes. These profiles were markedly different from the profiles of epsilon-Proteobacteria, which are abundant in the low temperatures of deep-sea hydrothermal environments.

Microbial communities associated with geological horizons in coastal subseafloor sediments from the sea of okhotsk.

Microbial communities from a subseafloor sediment core from the southwestern Sea of Okhotsk were evaluated by performing both cultivation-dependent and cultivation-independent (molecular) analyses. The core, which extended 58.1 m below the seafloor, was composed of pelagic clays with several volcanic ash layers containing fine pumice grains. Direct cell counting and quantitative PCR analysis of archaeal and bacterial 16S rRNA gene fragments indicated that the bacterial populations in the ash layers were approximately 2 to 10 times larger than those in the clays. Partial sequences of 1,210 rRNA gene clones revealed that there were qualitative differences in the microbial communities from the two different types of layers. Two phylogenetically distinct archaeal assemblages in the Crenarchaeota, the miscellaneous crenarchaeotic group and the deep-sea archaeal group, were the most predominant archaeal 16S rRNA gene components in the ash layers and the pelagic clays, respectively. Clones of 16S rRNA gene sequences from members of the gamma subclass of the class Proteobacteria dominated the ash layers, whereas sequences from members of the candidate division OP9 and the green nonsulfur bacteria dominated the pelagic clay environments. Molecular (16S rRNA gene sequence) analysis of 181 isolated colonies revealed that there was regional proliferation of viable heterotrophic mesophiles in the volcanic ash layers, along with some gram-positive bacteria and actinobacteria. The porous ash layers, which ranged in age from tens of thousands of years to hundreds of thousands of years, thus appear to be discrete microbial habitats within the coastal subseafloor clay sediment, which are capable of harboring microbial communities that are very distinct from the communities in the more abundant pelagic clays.

Expression cloning and characterization of a novel gene that encodes the RNA-binding protein FAU-1 from Pyrococcus furiosus.

We systematically screened a genomic DNA library to identify proteins of the hyperthermophilic archaeon Pyrococcus furiosus using an expression cloning method. One gene product, which we named FAU-1 (P. furiosus AU-binding), demonstrated the strongest binding activity of all the genomic library-derived proteins tested against an AU-rich RNA sequence. The protein was purified to near homogeneity as a 54 kDa single polypeptide, and the gene locus corresponding to this FAU-1 activity was also sequenced. The FAU-1 gene encoded a 472-amino-acid protein that was characterized by highly charged domains consisting of both acidic and basic amino acids. The N-terminal half of the gene had a degree of similarity (25%) with RNase E from Escherichia coli. Five rounds of RNA-binding-site selection and footprinting analysis showed that the FAU-1 protein binds specifically to the AU-rich sequence in a loop region of a possible RNA ligand. Moreover, we demonstrated that the FAU-1 protein acts as an oligomer, and mainly as a trimer. These results showed that the FAU-1 protein is a novel heat-stable protein with an RNA loop-binding characteristic.