Akifumi Hosoda

Development of a PCR method for the detection and quantification of benzoyl-CoA reductase genes and its application to monitored natural attenuation.

AbstractBenzoyl coenzyme A reductase (BCR) catalyzes dearomatization of benzoyl coenzyme A (benzoyl-CoA), which is the central step in the anaerobic degradative pathways for a variety of aromatic compounds. This study developed a PCR method for the detection and quantification of BCR genes in bacterial strains and environmental samples. PCR primers were designed by aligning known BCR genes in Thauera, Azoarcus and Rhodopseudomonas species, and their utility was assessed by amplifying BCR fragments from aromatic-hydrocarbon degrading anaerobes and other bacteria. BCR fragments with the expected sizes were obtained from denitrifying and phototrophic aromatics degraders. The positive signals were also obtained from Geobacter metallireducens and xylene-degrading sulfate-reducing bacterium (strain mXyS1) but not from other aromatics-degrading sulfate-reducing bacteria and aerobic bacteria. When the PCR was used for analyzing a natural attenuation (NA) site, the positive signal was obtained only from gasoline-contaminated groundwater; sequence analysis of these amplicons revealed that most of them exhibited substantial similarities to the known BCRs. Quantitative competitive PCR analysis estimated BCR-gene copies to account for 10–40% of bacterial 16S rRNA gene copies in the contaminated groundwater, indicating that bacteria possessing BCR genes were highly enriched in the contaminated groundwater. In microcosm bioremediation tests using the contaminated groundwater, the copy number of BCR gene was approximately 10-fold increased in the course of aromatics degradation under denitrifying conditions but not under sulfidogenic conditions. These results suggest the utility of the PCR method for assessing the potential of denitrifying bacteria for aromatic-compound degradation in groundwater.

Classification of the Genus Bacillus Based on MALDI-TOF MS Analysis of Ribosomal Proteins Coded in S10 and spc Operons

A rapid bacterial identification method by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) using ribosomal proteins coded in S10 and spc operons as biomarkers, named the S10-GERMS (the S10-spc-alpha operon gene encoded ribosomal protein mass spectrum) method, was applied for the genus Bacillus a Gram-positive bacterium. The S10-GERMS method could successfully distinguish the difference between B. subtilis subsp. subtilis NBRC 13719(T) and B. subtilis subsp. spizizenii NBRC 101239(T) because of the mass difference of 2 ribosomal subunit proteins, despite the difference of only 2 bases in the 16S rRNA gene between them. The 8 selected reliable and reproducible ribosomal subunit proteins without disturbance of S/N level on MALDI-TOF MS analysis, S10, S14, S19, L18, L22, L24, L29, and L30, coded in S10 and spc operons were significantly useful biomarkers for rapid bacterial classification at species and strain levels by the S10-GERMS method of genus Bacillus strains without purification of ribosomal proteins.

Classification of genus Pseudomonas by MALDI-TOF MS based on ribosomal protein coding in S10-spc-alpha operon at strain level.

We have proposed a rapid phylogenetic classification at the strain level by MALDI-TOF MS using ribosomal protein matching profiling. In this study, the S10-spc-alpha operon, encoding half of the ribosomal subunit proteins and highly conserved in eubacterial genomes, was selected for construction of the ribosomal protein database as biomarkers for bacterial identification by MALDI-TOF MS analysis to establish a more reliable phylogenetic classification. Our method revealed that the 14 reliable and reproducible ribosomal subunit proteins with less than m/z 15,000, except for L14, coded in the S10-spc-alpha operon were significantly useful biomarkers for bacterial classification at species and strain levels by MALDI-TOF MS analysis of genus Pseudomonas strains. The obtained phylogenetic tree was consisted with that based on genetic sequence (gyrB). Since S10-spc-alpha operons of genus Pseudomonas strains were sequenced using specific primers designed based on nucleotide sequences of genome-sequenced strains, the ribosomal subunit proteins encoded in S10-spc-alpha operon were suitable biomarkers for construction and correction of the database. MALDI-TOF MS analysis using these 14 selected ribosomal proteins is a rapid, efficient, and versatile bacterial identification method with the validation procedure for the obtained results.

Effect of flavonoids on androgen and glucocorticoid receptors based on in vitro reporter gene assay.

The effect of 32 flavonoids on androgen (AR) and glucocorticoid receptors (GR) was investigated using an MDA-kb2 human breast cancer cell line to predict potential AR and GR activities. Among them, 5-hydroxyflavone (7) had the highest AR antagonistic activity with an IC(50) value of 0.3 microM, whereas 6-methoxyflavone (11) had the highest induced luciferase activity with an EC(150) value of 0.7 microM. Genistein (2) and daizein (1) showed a sufficient increase of luciferase activities as their concentrations increased with EC(150) values of 4.4 and 10.1 microM, respectively. These findings provide evidence of a fundamental property of their structure-activity relationship with AR and/or GR.

Effect of essential oils, such as raspberry ketone and its derivatives, on antiandrogenic activity based on in vitro reporter gene assay.

The effect of essential oils, such as raspberry ketone, on androgen (AR) receptor was investigated using a MDA-kb2 human breast cancer cell line for predicting potential AR activity. Among them, eugenol had the highest AR antagonistic activity with its IC(50) value of 19 microM. Raspberry ketone, which has threefold higher anti-obese activity than that of capsaicin, also had AR antagonist activity with its IC(50) value of 252 microM. Based on these findings, a more precise CoMFA model was proposed as follows: pIC(50) [log (1/IC(50))]=3.77+[CoMFA field terms] (n=39, s=0.249, r(2)=0.834, s(cv)=0.507, q(2)=0.311 (three components).

The growth of Steroidobacter agariperforans sp. nov., a novel agar-degrading bacterium isolated from soil, is enhanced by the diffusible metabolites produced by bacteria belonging to Rhizobiales.

An agar-degrading bacterium was isolated from soil collected in a vegetable cropping field. The growth of this isolate was enhanced by supplying culture supernatants of bacteria belonging to the order Rhizobiales. Phylogenetic analysis based on 16S rRNA gene sequences indicated the novel bacterium, strain KA5–BT, belonged to the genus Steroidobacter in Gammaproteobacteria, but differed from its closest relative, Steroidobacter denitrificans FST, at the species level with 96.5% similarity. Strain KA5–BT was strictly aerobic, Gram-negative, non-motile, non-spore forming, and had a straight to slightly curved rod shape. Cytochrome oxidase and catalase activities were positive. The strain grew on media containing culture supernatants in a temperature range of 15–37°C and between pH 4.5 and 9.0, with optimal growth occurring at 30°C and pH 6.0–8.0. No growth occurred at 10 or 42°C or at NaCl concentrations more than 3% (w/v). The main cellular fatty acids were iso–C15:0, C16:1ω7c, and iso–C17:1ω9c. The main quinone was ubiquinone-8 and DNA G+C content was 62.9 mol%. In contrast, strain FST was motile, did not grow on the agar plate, and its dominant cellular fatty acids were C15:0 and C17:1ω8c. Based on its phylogenetic and phenotypic properties, strain KA5–BT (JCM 18477T = KCTC 32107T) represents a novel species in genus Steroidobacter, for which the name Steroidobacter agariperforans sp. nov. is proposed.

MALDI-TOF MS analysis of ribosomal proteins coded in S10 and spc operons rapidly classified the Sphingomonadaceae as alkylphenol polyethoxylate-degrading bacteria from the environment

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) using ribosomal subunit proteins coded in the S10-spc-alpha operon as biomarkers was applied for the classification of the Sphingomonadaceae from the environment. To construct a ribosomal protein database, S10-spc-alpha operon of type strains of the Sphingomonadaceae and their related alkylphenol polyethoxylate (APEO(n) )-degrading bacteria were sequenced using specific primers designed based on nucleotide sequences of genome-sequenced strains. The observed MALDI mass spectra of intact cells were compared with the theoretical mass of the constructed ribosomal protein database. The nine selected biomarkers coded in the S10-spc-alpha operon, L18, L22, L24, L29, L30, S08, S14, S17, and S19, could successfully distinguish the Sphingopyxis terrae NBRC 15098(T) and APEO(n) -degrading bacteria strain BSN20, despite only one base difference in the 16S rRNA gene sequence. This method, named the S10-GERMS (S10-spc-alpha operon gene-encoded ribosomal protein mass spectrum) method, is a significantly useful tool for bacterial discrimination of the Sphingomonadaceae at the strain level and can detect and monitor the main APEO(n) -degrading bacteria in the environment.

Engineered platform for bioethylene production by a cyanobacterium expressing a chimeric complex of plant enzymes.

Ethylene is an industrially important compound, but more sustainable production methods are desirable. Since cellulosomes increase the ability of cellulolytic enzymes by physically linking the relevant enzymes via dockerin-cohesin interactions, in this study, we genetically engineered a chimeric cellulosome-like complex of two ethylene-generating enzymes from tomato using cohesin-dockerins from the bacteria Clostridium thermocellum and Acetivibrio cellulolyticus. This complex was transformed into Escherichia coli to analyze kinetic parameters and enzyme complex formation and into the cyanobacterium Synechococcus elongatus PCC 7942, which was then grown with and without 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) induction. Only at minimal protein expression levels (without IPTG), the chimeric complex produced 3.7 times more ethylene in vivo than did uncomplexed enzymes. Thus, cyanobacteria can be used to sustainably generate ethylene, and the synthetic enzyme complex greatly enhanced production efficiency. Artificial synthetic enzyme complexes hold great promise for improving the production efficiency of other industrial compounds.

Ecotoxicity by the Biodegradation of Alkylphenol Polyethoxylates Depends on the Effect of Trace Elements

The bacteria Sphingomonas sp. strain BSN22, isolated from bean fields, degraded octylphenol polyethoxylates (OPEO(n)) to octylphenol (OP) under aerobic conditions. This biodegradation mechanism proceeded by the following two-step degradation process: (1) degradation of OPEO(n) to octylphenol triethoxylate (OPEO(3)), (2) degradation from OPEO(3) to OP via octylphenoxy acetic acid (OPEC(1)). The chemical structure of OPEC(1) was confirmed by analysis using (18)O-labeled water. Quantitative studies revealed that magnesium (Mg(2+)) and calcium (Ca(2+)) ions were essential for the biodegradation of OPEO(n). Furthermore, the rate of biodegradation was especially accelerated by ferric ions (Fe(3+)), and the accumulated amounts of endocrine active chemicals, such as OP, OPEO(1), and OPEC(1), significantly increased to the concentration of 22.8, 221.7, and 961.1 microM in the presence of 37.0 microM Fe(3+), respectively. This suggests that environmental elements significantly influence the resultant ecotoxicity as well as the rate of their biodegradation in the environment. This study on the mechanism of OPEO(n) biodegradation may play an important role in understanding and managing environmental safety, including drinking water safety.

Identification of the Electron Transfer Flavoprotein as an Upregulated Enzyme in the Benzoate Utilization of Desulfotignum balticum

Desulfotignum balticum utilizes benzoate coupled to sulfate reduction. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) analysis was conducted to detect proteins that increased more after growth on benzoate than on butyrate. A comparison of proteins on 2D gels showed that at least six proteins were expressed. The N-terminal sequences of three proteins exhibited significant identities with the α and β subunits of electron transfer flavoprotein (ETF) from anaerobic aromatic-degraders. By sequence analysis of the fosmid clone insert (37,590 bp) containing the genes encoding the ETF subunits, we identified three genes, whose deduced amino acid sequences showed 58%, 74%, and 62% identity with those of Gmet_2267 (Fe-S oxidoreductase), Gmet_2266 (ETF β subunit), and Gmet_2265 (ETF α subunit) respectively, which exist within the 300-kb genomic island of aromatic-degradation genes from Geobacter metallireducens GS-15. The genes encoding ETF subunits found in this study were upregulated in benzoate utilization.