Possible association of CUX1 gene polymorphisms with antidepressant response in major depressive disorder.

Association between response to antidepressant treatment and genetic polymorphisms was examined in two independent Japanese samples of patients with major depressive disorder (MDD). Genome-wide approach using the Illumina Human CNV370-quad Bead Chip was utilized in the analysis of the 92 MDD patients in the first sample. In all, 11 non-intergenic single-nucleotide polymorphisms with uncorrected allelic P-value <0.0001 were selected for the subsequent association analyses in the second sample of 136 MDD patients. Difference in allele distribution between responders and nonresponders were found in the second-stage sample for rs365836 and rs201522 of the CUX1 gene (P=0.005 and 0.004, respectively). The allelic P-values for rs365836 and rs201522 in both samples combined were 0.0000023 and 0.0000040, respectively. Our results provide the first evidence that polymorphisms of the CUX1 gene may be associated with response to antidepressant treatment in Japanese patients with MDD.

Developmental changes in the respiratory chain of Ascaris mitochondria.

The Ascaris larval respiratory chain, particularly complex II (succinate-ubiquinone oxidoreductase), was characterized in isolated mitochondria. Low-temperature difference spectra showed the presence of substrate-reducible cytochromes aa3 of complex IV, c+c1 and b of complex III (ubiquinol-cytochrome c oxidoreductase) in mitochondria from second-stage larvae (L2 mitochondria). Quinone analysis by high-performance liquid chromatography showed that, unlike adult mitochondria, which contain only rhodoquinone-9, L2 mitochondria contain ubiquinone-9 as a major component. Complex II in L2 mitochondria was kinetically different from that in adult mitochondria. The individual oxidoreductase activities comprising succinate oxidase, and fumarate reductase were determined in mitochondria from L2 larvae, from larvae cultured to later stages, and from adult nematodes. The L2 mitochondria exhibited the highest specific activity of cytochrome c oxidase, indicating that L2 larvae have the most aerobic respiratory chain among the stages studied. The Cybs subunit of complex II in L2 and cultured-larvae mitochondria exhibited different reactivities against anti-adult Cybs antibodies. Taken together, these results indicate that the complex II of larvae is different from its adult counterpart. In parallel with this change in mitochondrial biogenesis, biosynthetic conversion of quinones occurs during development in Ascaris nematodes.

Changes in quinone profiles of hot spring microbial mats with a thermal gradient

The respiratory and photosynthetic quinones of microbial mats which occurred in Japanese sulfide-containing neutral-pH hot springs at different temperatures were analyzed by spectrochromatography and mass spectrometry. All of the microbial mats that developed at high temperatures (temperatures above 68 degreesC) were so-called sulfur-turf bacterial mats and produced methionaquinones (MTKs) as the major quinones. A 78 degreesC hot spring sediment had a similar quinone profile. Chloroflexus-mixed mats occurred at temperatures of 61 to 65 degreesC and contained menaquinone 10 (MK-10) as the major component together with significant amounts of either MTKs or plastoquinone 9 (PQ-9). The sunlight-exposed biomats growing at temperatures of 45 to 56 degreesC were all cyanobacterial mats, in which the photosynthetic quinones (PQ-9 and phylloquinone) predominated and MK-10 was the next most abundant component in most cases. Ubiquinones (UQs) were not found or were detected in only small amounts in the biomats growing at temperatures of 50 degreesC and above, whereas the majority of the quinones of a purple photosynthetic mat growing at 34 degreesC were UQs. A numerical analysis of the quinone profiles was performed by using the following three parameters: dissimilarity index (D), microbial divergence index (MDq), and bioenergetic divergence index (BDq). A D matrix tree analysis showed that the hot spring mats consisting of the sulfur-turf bacteria, Chloroflexus spp., cyanobacteria, and purple phototrophic bacteria formed distinct clusters. Analyses of MDq and BDq values indicated that the microbial diversity of hot spring mats decreased as the temperature of the environment increased. The changes in quinone profiles and physiological types of microbial mats in hot springs with thermal gradients are discussed from evolutionary viewpoints.

Isolation and characterization of a new poly(3-hydroxybutyrate)-degrading, denitrifying bacterium from activated sludge.

A new denitrifying chemoorganotrophic bacterium capable of aerobic and anaerobic degradation of poly(3-hydroxybutyrate) (PHB) and poly(3-hydroxybutyrate-co-hydroxyvalerate) (PHBV) was isolated from activated sludge. A phylogenetic analysis based on 16S rDNA sequences indicated that the new isolate is a member of the beta subclass of the Proteobacteria and represents a distinct line of descent within the family Comamonadaceae. During denitrifying growth with 3-hydroxybutyrate, PHB, or PHBV as the sole carbon source, the isolate reduced nitrate to N2 without appreciable accumulation of nitrite and nitrous oxide as intermediate products. Kinetic analyses of the denitrification with different grades of PHBV indicated that approximately 0.7 g of PHBV was required to reduce 1 g of NO3-. A high denitrification rate (19 mg N-NO3- removed h(-1) x g(-1) dry wt of cells) was found with PHBV as the electron donor.

Phylogenetic position of the menaquinone-containing acidophilic chemo-organotroph Acidobacterium capsulatum.

The phylogenetic position of an acidophilic chemo-organotrophic menaquinone-containing bacterium, Acidobacterium capsulatum, was studied on the basis of 16S rRNA gene sequence information. A. capsulatum showed the highest level of sequence similarity to Heliobacterium chlorum, a member of the Gram-positive group, yet this level was only 81%. Distance matrix tree analysis suggested that A. capsulatum belongs to a unique lineage deeply branching from the Chlamydia-Planctomyces group or from the Gram-positive line.

Isoprenoid quinones as biomarkers of microbial populations in the environment.

Isoprenoid quinones are lipid molecules present in all species of respiratory and photosynthetic microorganisms and exhibit marked structural variations depending upon the microbial taxon. Taking advantage of this, quinones have been used not only as chemotaxonomic markers in microbial systematics but also as good measures of microbial populations in the environment in terms of quantity, quality, and activity. Basically, this biomarker approach, called the quinone profile method, is applicable to all environmental samples from which an absolute amount of microbial biomass > or =10(9) cells can be collected. The quinone profile method allows good measurement of both fundamental and applied aspects of ecological and environmental microbiology. In particular, numerical cluster analyses of quinone profiles are useful for monitoring microbial population shifts in an ecosystem which is not amenable to conventional culture methods and molecular techniques. The combined use of molecular techniques and the quinone profile method in this research area should provide more accurate and reliable data regarding population dynamics and community structures.

Terminal restriction pattern analysis of 16S rRNA genes for the characterization of bacterial communities of activated sludge.

A culture-independent molecular technique using terminal restriction fragment length polymorphism (T-RFLP) of 16S rRNA genes was applied to the characterization of bacterial communities of activated sludge taken from different municipal sewage treatment plants. 16S rDNA fragments from the bulk DNA of sludge were amplified by PCR with a Cy5-labeled forward primer corresponding to nucleotide positions 8 to 27 and a reverse primer complementary to positions 907 to 926 in the Escherichia coli numbering system. The 16S rDNAs thus obtained were digested with tetrameric restriction enzymes and analyzed using a Pharmacia automated DNA sequencer. A preliminary study on a model DNA mixture prepared from different bacterial species showed that the fluorescence intensity of terminal fragments (T-RFs) of 16S rDNAs amplified and detected was directly proportional to the 16S rRNA gene copy number rather than the amount of genomic DNA of each species present. 16S rDNA fragments amplified from the sludges and digested with HhaI usually generated at least 60 T-RFs, among which T-RFs of around 208 bp were the most abundant regardless of the time or area sampled. Southern blot hybridization with oligonucleotide probes specific to the 5' terminal regions of the 16S rDNA of different phylogenetic groups indicated that the T-RFs of around 208 bp were derived from members of the beta subclass of the class Proteobacteria. Hybridization with a probe specific to the class Actinobacteria failed to detect any appreciable signal. These results did not agree fully with those obtained by quinone profiling. The usefulness and limitations of the T-RFLP method for monitoring bacterial population dynamics in activated sludge were discussed.

Direct automated sequencing of 16S rDNA amplified by polymerase chain reaction from bacterial cultures without DNA purification.

The 16S rRNA gene from various bacterial cultures was amplified by the polymerase chain reaction without DNA purification, and sequenced directly by using a laser fluorescent DNA sequencer and Tth polymerase with a cycle sequencing protocol. The described procedures provide almost complete 16S rDNA sequence data within a couple of days and facilitate systematic studies.

High-performance liquid chromatographic analysis of demethylmenaquinone and menaquinone mixtures from bacteria.

Demethylmenaquinone and menaquinone mixtures from some species of enterobacteria were analysed by reverse-phase partition high-performance liquid chromatography. This method allowed clear separation and quantitative determination of these quinone components.

Transfer of the bacteriochlorophyll b-containing phototrophic bacteria Rhodopseudomonas viridis and Rhodopseudomonas sulfoviridis to the genus Blastochloris gen. nov.

The phylogenetic positions of the bacteriochlorophyll (BChl) b-producing budding phototrophic bacteria Rhodopseudomonas viridis and Rhodopseudomonas sulfoviridis were studied on the basis of 16S rRNA gene sequence information. These bacteria formed a tight cluster with the genus Rhodoplanes as a sister group within the alpha-2 subgroup of the Proteobacteria. Genomic DNA-DNA hybridization assays showed that R. viridis and R. sulfoviridis were closely related but were different species. Creation of the genus Blastochloris gen. nov. is proposed to accommodate these BChl b-producing species of phototrophic bacteria.

Phylogenetic affiliations of Rhodoferax fermentans and related species of phototrophic bacteria as determined by automated 16S rDNA sequencing.

16S rDNA sequences of strains of Rhodoferax fermentans were analyzed and compared with those of species of the genera Rubrivivax and Rhodocyclus. Approximately 1.5-kb fragments of 16S rDNA from crude cell lysates were amplified by the polymerase chain reaction (PCR) and sequenced directly by using Tth DNA polymerase with the linear PCR sequencing protocol, followed by on-line detection with an automated laser fluorescent DNA sequencer. Pairwise sequence comparisons and distance matrix tree analysis showed that Rhodoferax fermentans, Rubrivivax gelatinosus, and Rhodocyclus species belong to three separate lineages within the beta subclass of the Proteobacteria, thereby confirming the phylogenetic validity of the genus Rhodoferax, as well as of the genera Rubrivivax and Rhodocyclus.

Isolation and characterization of Rhodovulum strictum sp. nov. and some other purple nonsulfur bacteria from colored blooms in tidal and seawater pools.

Several strains of phototrophic purple nonsulfur bacteria were isolated from colored blooms occurring in tidal and seawater pools in Japan. All of these isolates had ovoid to rod-shaped cells that were motile by means of single polar flagella and contained vesicular intracytoplasmic membranes together with bacteriochlorophyll a and carotenoids of the spheroidene series. They produced ubiquinone 10 as the major quinone and contained straight-chain fatty acids, with C18:1 predominating. They were mesophilic, halophilic, and photoheterotrophic, utilized sulfide and thiosulfate as electron donors for phototrophic growth, and photoassimilated a wide variety of organic compounds as carbon sources. Our results suggested that all of these isolates are members of the recently described genus Rhodovulum. The isolates were classified into four groups (designated groups I through IV) on the basis of phenotypic and genotypic data. The group I isolates, which were the most abundant purple nonsulfur bacteria recovered from the blooms, grew in the presence of NaCl concentrations ranging from 0.5 to 3.0% (optimum NaCl concentration, 0.8%) and at pH values ranging from 7.5 to 9.0 (optimum pH, 8.0 to 8.5). On the basis of these unique physiological traits, together with genotypic and phylogenetic data, we propose that the group I isolates should be classified as members of a new species, Rhodovulum strictum. The group II isolates were identified definitely as Rhodovulum sulfidophilum, and the group III and IV isolates were phenotypically most similar to R. sulfidophilum and Rhodovulum adriaticum, respectively, but could be differentiated from these species by DNA-DNA pairing data.

Characterization of the bacterial population structure in an anaerobic-aerobic activated sludge system on the basis of respiratory quinone profiles.

Bacterial respiratory quinones were used as biomarkers for studying the bacterial population structure, especially the content of Acinetobacter species, in a laboratory-scale anaerobic-aerobic activated sludge system and in the standard aerobic system. All tested sludges contained both ubiquinone and menaquinone, with a molar ratio of about 1:0.5. High-performance liquid chromatography showed that ubiquinone with eight isoprene units (Q-8) was present as the predominant ubiquinone, Q-10 was the second most common type, and Q-9 and other homologs were minor components in the anaerobic-aerobic sludge and the standard aerobic sludge. Bacteriological examination indicated that, in both sludge systems, Q-8-containing bacteria constituted a large proportion of the aerobic heterotrophic bacterial flora, but only a few strains with Q-9 were found. These findings demonstrate that the population of Acinetobacter species, which contain Q-9 as the major quinone, is negligible in those environments. The present results suggest that the introduction of anaerobic conditions into the aerobic batch process has little influence on the bacterial community structure.

Proposal of the genus Sphingomonas sensu stricto and three new genera, Sphingobium, Novosphingobium and Sphingopyxis, on the basis of phylogenetic and chemotaxonomic analyses.

Phylogenetic analyses of 16S rRNA gene sequences by distance matrix and parsimony methods indicated that the currently known species of the genus Sphingomonas can be divided into four clusters. Some chemotaxonomic and phenotypic differences were noted among these clusters. Three new genera, Sphingobium, Novosphingobium and Sphingopyxis, are proposed in addition to the genus Sphingomonas sensu stricto. The genus Sphingobium is proposed to accommodate Sphingomonas chlorophenolica, Sphingomonas herbicidovorans and Sphingomonas yanoikuyae. The genus Novosphingobium is proposed for Sphingomonas aromaticivorans, Sphingomonas capsulata, Sphingomonas rosa, Sphingomonas stygia, Sphingomonas subarctica and Sphingomonas subterranea. Sphingomonas macrogoltabidus and Sphingomonas terrae are reclassified in the genus Sphingopyxis. The type species of Sphingobium, Novosphingobium and Sphingopyxis are Sphingobium yanoikuyae, Novosphingobium capsulatum and Sphingopyxis macrogoltabida, respectively.

Emendation of the description of Blastomonas natatoria (Sly 1985) Sly and Cahill 1997 as an aerobic photosynthetic bacterium and reclassification of Erythromonas ursincola Yurkov et al. 1997 as Blastomonas ursincola comb. nov.

Photosynthetic properties of Blastomonas natatoria (Sly 1985) Sly and Cahill 1997, which had been recognized as being non-photosynthetic, were examined and compared with those of its close relative, the aerobic photosynthetic bacterium, Erythromonas ursincola Yurkov et al. 1997. HPLC experiments demonstrated that bacteriochlorophyll a was present in a detectable amount in the lipid extract from B. natatoria DSM 3183T as well as that from E. ursincola DSM 9006T. The puf genes, encoding the proteins of the photosynthetic reaction centre and core light-harvesting complexes, were detected by PCR from both the organisms. 16S rDNA sequence comparisons and DNA-DNA hybridization studies confirmed that B. natatoria and E. ursincola were closely related genetically in a single genus. On the basis of phenotypic, chemotaxonomic and phylogenetic data, it is proposed that the description of B. natatoria is emended as a species of aerobic photosynthetic bacteria and that E. ursincola is reclassified as Blastomonas ursincola comb. nov.

A new genus of marine budding phototrophic bacteria, Rhodobium gen. nov., which includes Rhodobium orientis sp. nov. and Rhodobium marinum comb. nov.

Strains of a previously undescribed species of purple nonsulfur phototrophic bacteria were isolated from coastal seawater in Japan. These new isolates were gram-negative, motile, budding rods that contained lamellar intracytoplasmic membranes and produced pink to red cultures. Cell extracts of photosynthetic cultures exhibited absorption maxima at 377, 468, 500, 530, 591, 802, and 870 nm, indicating that bacterio-chlorophyll a and carotenoids of the spirilloxanthin series were present. The new isolates were halophilic, facultatively aerobic photoheterotrophs that grew anaerobically in the light or aerobically in the dark. Maximum growth occurred in the presence of 4 to 5% NaCl. Anaerobic growth in the dark with nitrate as a terminal electron acceptor also occurred. Various organic compounds were used as photosynthetic electron donors and carbon sources. Sulfate was used as a sulfur source. Both menaquinone 10 and ubiquinone 10 were produced; these quinones were the major quinones. A phylogenetic analysis based on 16S rRNA gene sequences revealed that strain MB312T (T = type strain), a representative of the new phototrophs, was a member of a lineage that was distinct from members of the genus Rhodopseudomonas; Rhodopseudomonas marina was the closest relative. On the basis of the data described above, we propose the name Rhodobium orientis gen. nov., sp. nov. for the new isolates. We also propose that Rhodopseudomonas marina Imhoff 1983 should be transferred to the genus Rhodobium as Rhodobium marinum comb. nov.

Application of polyhydroxyalkanoates for denitrification in water and wastewater treatment.

Application of polyhydroxyalkanoates (PHAs) and related biodegradable polymers has gained momentum in various areas of biotechnology. A promising application that started appearing in the past decade is the use of PHAs as the solid substrate for denitrification of water and wastewater. This type of denitrification, termed here "solid-phase denitrification", has several advantages over the conventional system supplemented with liquid organic substrate. PHAs serve not only as constant sources of reducing power for denitrification but also as solid matrices favorable for development of microbial films. In addition, in contrast to conventional processes, the use of PHAs has no potential risk of release of dissolved organic carbon with the resultant deterioration of effluent water quality. If the production cost of PHAs can be brought down, its application to the denitrification process will become economically more promising. A number of PHA-degrading denitrifying bacteria have been isolated and characterized from activated sludge and continuous flow-bed reactors for denitrification with PHAs. Most of these isolates have been assigned phylogenetically to members of beta-Proteobacteria, especially those of the family Comamonadaceae. The metabolic and regulatory relationships between PHA degradation and denitrification, and the interactive relationship between PHA-degrading cells and the solid surface structure are important subjects awaiting future studies, which would provide a new insight into our comprehensive understanding of the solid-phase denitrification process.

Quinone profiling of bacterial communities in natural and synthetic sewage activated sludge for enhanced phosphate removal.

Respiratory quinones were used as biomarkers to study bacterial community structures in activated sludge reactors used for enhanced biological phosphate removal (EBPR). We compared the quinone profiles of EBPR sludges and standard sludges, of natural sewage and synthetic sewage, and of plant scale and laboratory scale systems. Ubiquinone (Q) and menaquinone (MK) components were detected in all sludges tested at molar MK/Q ratios of 0.455 to 0.981. The differences in MK/Q ratios were much larger when we compared different wastewater sludges (i.e., raw sewage and synthetic sewage) than when we compared sludges from the EBPR and standard processes or plant scale and laboratory scale systems. In all sludges tested a Q with eight isoprene units (Q-8) was the most abundant quinone. In the MK fraction, either tetrahydrogenated MK-8 or MK-7 was the predominant type, and there was also a significant proportion of MK-6 to MK-8 in most cases. A numerical cluster analysis of the profiles showed that the sludges tested fell into two major clusters; one included all raw sewage sludges, and the other consisted of all synthetic sewage sludges, independent of the operational mode and scale of the reactors and the phosphate accumulation. These data suggested that Q-8-containing species belonging to the class Proteobacteria (i.e., species belonging to the beta subclass) were the major constituents of the bacterial populations in the EBPR sludge, as well as in standard activated sludge. Members of the class Actinobacteria (gram-positive bacteria with high DNA G+C contents) were the second most abundant group in both types of sludge. The bacterial community structures in activated sludge processes may be affected more by the nature of the influent wastewater than by the introduction of an anaerobic stage into the process or by the scale of the reactors.