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Biomedical subjects

Hisao Ohtake

Publications and source records attributed to Hisao Ohtake.

At least 19 recordsLinked to original sources
PubMed2006

Utilization of hydrophobic bacterium Rhodococcus opacus B-4 as whole-cell catalyst in anhydrous organic solvents.

Rhodococcus opacus strain B-4, which has recently been isolated as an organic solvent-tolerant bacterium, has a high hydrophobicity and exhibits a high affinity for hydrocarbons. This bacterium was able to survive for at least 5 days in organic solvents, including n-tetradecane, oleyl alcohol, and bis(2-ethylhexyl) phthalate (BEHP), which contained water less than 1% (w/v). The biocatalytic ability of R. opacus B-4 was demonstrated in the essentially nonaqueous BEHP using indigo production from indole as a model conversion. By the catabolism of oleic acid for NADH regeneration, indigo production increased up to 71.6 microg ml(-1) by 24 h.

Alkanes↗
PubMed2006

Effect of mineral elements on phosphorus release from heated sewage sludge.

The aim of this work was to examine the influence of mineral elements on phosphorus (P) release from heated waste sludges. Energy dispersive X-ray microanalysis suggested that P was associated with Al, Ca, and Mg on the surface of waste sludge biomass obtained from six wastewater treatment plants. The extent of P release decreased with increasing the total concentrations of Al, Mg, and Ca in waste sludges. The addition of Al2(SO4)3, Ca(OH)2, CaCl2, MgSO4, or NaAlO2 to activated sludges, which were taken from a bench-scale EBPR process, reduced the P release significantly.

Electron Probe Microanalysis↗
PubMed2006

Module-based analysis of robustness tradeoffs in the heat shock response system.

Biological systems have evolved complex regulatory mechanisms, even in situations where much simpler designs seem to be sufficient for generating nominal functionality. Using module-based analysis coupled with rigorous mathematical comparisons, we propose that in analogy to control engineering architectures, the complexity of cellular systems and the presence of hierarchical modular structures can be attributed to the necessity of achieving robustness. We employ the Escherichia coli heat shock response system, a strongly conserved cellular mechanism, as an example to explore the design principles of such modular architectures. In the heat shock response system, the sigma-factor sigma32 is a central regulator that integrates multiple feedforward and feedback modules. Each of these modules provides a different type of robustness with its inherent tradeoffs in terms of transient response and efficiency. We demonstrate how the overall architecture of the system balances such tradeoffs. An extensive mathematical exploration nevertheless points to the existence of an array of alternative strategies for the existing heat shock response that could exhibit similar behavior. We therefore deduce that the evolutionary constraints facing the system might have steered its architecture toward one of many robustly functional solutions.

Computer Simulation↗
PubMed2006

Recognition of culture state using two-dimensional gel electrophoresis with an artificial neural network.

Proteomic technologies were applied to the examination of nutrient components in culture broth. In bioprocesses, many types of media have been proposed and used on the commercial scale. Natural nutrients, the chemical components of which cannot be identified completely, are often used in fermentation processes such as in the production of baker's yeast, alcoholic beverages, amino acids, and pharmaceuticals. The catabolic activities of the microorganisms in these processes vary with the species used. We used an artificial neural network (ANN) to recognize the sufficiency of chemical elements based on the protein spots resolved in 2-DE, and we evaluated this technique using the leave-one-out method. We also attempted to reduce the number of input data for spot selection based on sensitivity analysis of the ANN, and the selected data were used to improve accuracy.

Electrophoresis, Gel, Two-Dimensional↗
PubMed2006

Identification and characterization of the chemotactic transducer in Pseudomonas aeruginosa PAO1 for positive chemotaxis to trichloroethylene.

Pseudomonas aeruginosa PAO1 is repelled by trichloroethylene (TCE), and the methyl-accepting chemotaxis proteins PctA, PctB, and PctC serve as the major chemoreceptors for negative chemotaxis to TCE. In this study, we found that the pctABC triple mutant of P. aeruginosa PAO1 was attracted by TCE. Chemotaxis assays of a set of mutants containing deletions in 26 potential mcp genes revealed that mcpA (PA0180) is the chemoreceptor for positive chemotaxis to TCE. McpA also detects tetrachloroethylene and dichloroethylene isomers as attractants.

Bacterial Proteins↗
PubMed2006

Transcriptional analysis of the multicopy hao gene coding for hydroxylamine oxidoreductase in Nitrosomonas sp. strain ENI-11.

The nitrifying bacterium Nitrosomonas sp. strain ENI-11 has three copies of the gene encoding hydroxylamine oxidoreductase (hao(1), hao(2), and hao(3)) on its genome. Broad-host-range reporter plasmids containing transcriptional fusion genes between hao copies and lacZ were constructed to analyze the expression of each hydroxylamine oxidoreductase gene (hao) copy individually and quantitatively. beta-Galactosidase assays of ENI-11 harboring reporter plasmids revealed that all hao copies were transcribed in the wild-type strain. Promoter analysis of hao copies revealed that transcription of hao(3) was highest among the hao copies. Expression levels of hao(1) and hao(2) were 40% and 62% of that of hao(3) respectively. Transcription of hao(1) was negatively regulated, whereas a portion of hao(3) transcription was read through transcription from the rpsT promoter. When energy-depleted cells were incubated in the growth medium, only hao(3) expression increased. This result suggests that it is hao(3) that is responsible for recovery from energy-depleted conditions in Nitrosomonas sp. strain ENI-11.

DNA, Bacterial↗
PubMed2005

Isolation and characterization of solvent-tolerant Pseudomonas putida strain T-57, and its application to biotransformation of toluene to cresol in a two-phase (organic-aqueous) system.

Pseudomonas putida T-57 was isolated from an activated sludge sample after enrichment on mineral salts basal medium with toluene as a sole source of carbon. P. putida T-57 utilizes n-butanol, toluene, styrene, m-xylene, ethylbenzene, n-hexane, and propylbenzene as growth substrates. The strain was able to grow on toluene when liquid toluene was added to mineral salts basal medium at 10-90% (v/v), and was tolerant to organic solvents whose log P(ow) (1-octanol/water partition coefficient) was higher than 2.5. Enzymatic and genetic analysis revealed that P. putida T-57 used the toluene dioxygenase pathway to catabolize toluene. A cis-toluene dihydrodiol dehydrogenase gene (todD) mutant of T-57 was constructed using a gene replacement technique. The todD mutant accumulated o-cresol (maximum 1.7 g/L in the aqueous phase) when cultivated in minimal salts basal medium supplemented with 3% (v/v) toluene and 7% (v/v) 1-octanol. Thus, T-57 is thought to be a good candidate host strain for bioconversion of hydrophobic substrates in two-phase (organic-aqueous) systems.

Biotechnology↗
PubMed2005

Effect of sulfur sources on specific desulfurization activity of Rhodococcus erythropolis KA2-5-1 in exponential fed-batch culture.

The effects of sulfur sources on the desulfurization activity of Rhodococcus erythropolis KA2-5-1 were investigated by using an exponential fed-batch culture technique. The feed rate of a sulfur source was controlled independently of the feed rate of ethanol, which was used as a carbon and energy source. Among the sulfur sources examined were dibenzothiophene (DBT), ammonium sulfate, L-cysteine, L-methionine, and 2-amino-ethanesulfonic acid. When the fed-medium contained DBT as the sole sulfur source, KA2-5-1 cells showed a maximum desulfurization activity of approximately 130 mmol 2-HBP kg-cell(-1) h(-1). Similar levels of enzyme activity were also achieved with inexpensive ammonium sulfate by using the exponential fed-batch culture technique. In addition, higher levels of desulfurization activity were achieved by increasing the dosage of the DBT desulfurization (dsz) operon and dszD gene in R. erythropolis KA2-5-1. The recombinant strain showed a maximum desulfurization activity of approximately 250 mmol 2-HBP kg-cell(-1) h(-1) in the exponential fed-batch cultures.

Biodegradation, Environmental↗
PubMed2005

Isolation and characterization of benzene-tolerant Rhodococcus opacus strains.

Twenty-two benzene-utilizing bacteria were isolated from soil samples. Among them, three isolates were highly tolerant to benzene. They grew on benzene when liquid benzene was added to the basal salt medium at 10--90% (v/v). Taxonomical analysis identified the benzene-tolerant isolates as Rhodococcus opacus. One of the benzene-tolerant isolates, designated B-4, could utilize many aromatic and aliphatic hydrocarbons including benzene, toluene, styrene, xylene, ethylbenzene, propylbenzene, n-octane and n-decane as sole sources of carbon and energy. Strain B-4 grew well in the presence of 10% (v/v) organic solvents that it was capable of using as growth substrates. Genetic analysis revealed the benzene dioxygenase pathway is involved in benzene catabolism in strain B-4. A deletion-insertion mutant defective in the benzene dioxygenase large and small subunits genes (bnz A 1 and bnz A 2) was as tolerant to organic solvents as the wild-type strain B-4, suggesting that utilization or degradation of organic solvents is not essential for the organic solvent tolerance of R. opacus B-4.

Benzene↗
PubMed2005

Identification of chemosensory proteins for trichloroethylene in Pseudomonas aeruginosa.

The involvement of the chemotaxis gene cluster 1 (cheYZABW) and cheR in repellent responses of Pseudomonas aeruginosa to trichloroethylene (TCE) is described and three methyl-accepting chemotaxis proteins (MCPs) for TCE are identified. TCE chemotaxis assays of a number of deletion-insertion mutants of P. aeruginosa PAO1 revealed that the chemotaxis gene cluster 1 and cheR are required for negative chemotaxis to TCE. Mutant strains which contained deletions in pctA, pctB and pctC showed decreased responses to TCE. The pctA, pctB and pctC genes have been reported to encode MCPs for amino acids [K. Taguchi et al., Microbiology, 143, 3223--3229 (2000)]. The pctA mutation more severely impaired chemotactic responses to TCE than did those of pctB and pctC, suggesting that PctA is the major MCP for TCE among the three MCPs. The pctA, pctB and pctC mutant strains showed decreased responses to chloroform and methylthiocyanate. This result demonstrates that PctA, PctB and PctC are also involved in repellent responses to chloroform and methylthiocyanate.

Bacterial Proteins↗
PubMed2005

Development of a genetic transformation system for benzene-tolerant Rhodococcus opacus strains.

Rhodococcus opacus B-4 and B-9 are tolerant to various organic solvents including benzene, toluene, ethylbenzene, xylenes and styrene, and are suitable bacterial hosts for the production of chemical products from hydrophobic substrates. A 4.4-kb endogenous plasmid (pKNR 01) was isolated from R. opacus B-4 and sequenced completely. Plasmid pKNR 01 encodes proteins that share similarity to replication proteins from the enteric bacterial and actinomycete theta-replication plasmids. A 7.4-kb chimeric plasmid, designated pKNR 01.1, was constructed by fusing XhoI-digested pKNR 01 and Escherichia coli vector pSTV 28. Plasmid pKNR 01.1 had the ability to replicate in B-4 and B-9. A protocol for transformation of B-9 by electroporation was optimized employing pKNR 01.1. Frequencies of 4.1 x 10(5) transformants per mug of plasmid DNA were obtained for B-9 cells, whereas B-4 harboring naturally occurring pKNR 01 was transformed at lower frequencies (approximately 1 x 10(4) transformants per mug of plasmid DNA). Deletion analysis of pKNR 01.1 showed that the 1.9-kb SphI-XhoI region containing the repA and rep B genes and the 0.6-kb region upstream of repA was essential for plasmid maintenance in R. opacus strains.

Benzene↗
PubMed2004

Effects of inorganic polyphosphate on the proteolytic and DNA-binding activities of Lon in Escherichia coli.

Lon belongs to a unique group of proteases that bind to DNA and is involved in the regulation of several important cellular functions, including adaptation to nutritional downshift. Previously, we revealed that inorganic polyphosphate (polyP) increases in Escherichia coli in response to amino acid starvation and that it stimulates the degradation of free ribosomal proteins by Lon. In this work, we examined the effects of polyP on the proteolytic and DNA-binding activities of Lon. An order-of-addition experiment suggested that polyP first binds to Lon, which stimulates Lon-mediated degradation of ribosomal proteins. A polyP-binding assay using Lon deletion mutants showed that the polyP-binding site of Lon is localized in the ATPase domain. Because the same ATPase domain also contains the DNA-binding site, polyP can compete with DNA for binding to Lon. In fact, an equimolar amount of polyP almost completely inhibited DNA-Lon complex formation, suggesting that Lon binds to polyP with a higher affinity than it binds to DNA. Collectively, our results showed that polyP may control the cellular activity of Lon not only as a protease but also as a DNA-binding protein.

ATP-Dependent Proteases↗
PubMed2004

Chemotaxis proteins and transducers for aerotaxis in Pseudomonas aeruginosa.

It was previously shown that the chemotaxis gene cluster 1 (cheYZABW) was required for chemotaxis. In this study, the involvement of the same cluster in aerotaxis is described and two transducer genes for aerotaxis are identified. Aerotaxis assays of a number of deletion-insertion mutants of Pseudomonas aeruginosa PAO1 revealed that the chemotaxis gene cluster 1 and cheR are required for aerotaxis. Mutant strains which contained deletions in the methyl-accepting chemotaxis protein-like genes tlpC and tlpG showed decreased aerotaxis. A double mutant deficient in tlpC and tlpG was negative for aerotaxis. TlpC has 45% amino acid identity with the Escherichia coli aerotactic transducer Aer. The TlpG protein has a predicted C-terminal segment with 89% identity to the highly conserved domain of the E. coli serine chemoreceptor Tsr. A hydropathy plot of TlpG indicated that hydrophobic membrane-spanning regions are missing in TlpG. A PAS motif was found in the N-terminal domains of TlpC and TlpG. On this basis, the tlpC and tlpG genes were renamed aer and aer-2, respectively. No significant homology other than the PAS motif was detected in the N-terminal domains between Aer and Aer-2.

Bacterial Proteins↗
PubMed2004

The aerotaxis transducer gene aer, but not aer-2, is transcriptionally regulated by the anaerobic regulator ANR in Pseudomonas aeruginosa.

The regulation of aerotaxis in Pseudomonas aeruginosa is reported. P. aeruginosa possesses two aerotaxis transducers, Aer and Aer-2. The aerotactic responses of P. aeruginosa cells were induced during the transition from exponential to stationary growth phase. A deletion mutant for the anaerobic transcriptional regulator ANR showed decreased aerotaxis. The anr mutation eliminated Aer-mediated aerotaxis, but not Aer-2-mediated aerotaxis. Expression of an aer-lacZ transcriptional fusion was also induced during the transition from exponential to stationary growth phase. The anr mutant showed only background levels of aer-lacZ expression. Rapid amplification of cDNA ends (RACE) and DNA sequencing revealed that the 5' end of the mRNA was located at an A nucleotide -67 nt upstream of aer. The aer promoter contained two putative FNR/ANR boxes at -42.5 and -93.5 bp upstream of the transcriptional start site of aer. Mutational analysis of the aer promoter region revealed that both FNR/ANR boxes were essential for the expression of the aer gene. These results indicate that ANR is required for the activation of aer expression but it is not essential for Aer-2-mediated aerotaxis in P. aeruginosa.

Journal Article↗
PubMed2004

A laboratory-scale test of anaerobic digestion and methane production after phosphorus recovery from waste activated sludge.

In enhanced biological phosphorus removal (EBPR) processes, activated sludge microorganisms accumulate large quantities of polyphosphate (polyP) intracellularly. We previously discovered that nearly all of polyP could be released from waste activated sludge simply by heating it at 70 degrees C for about 1 h. We also demonstrated that this simple method was applicable to phosphorus (P) recovery from waste activated sludge in a pilot plant-scale EBPR process. In the present study, we evaluated the effect of this sludge processing (heat treatment followed by calcium phosphate precipitation) on anaerobic digestion in laboratory-scale experiments. The results suggested that the sludge processing for P recovery could improve digestive efficiency and methane productivity at both mesophilic (37 degrees C) and thermophilic (53 degrees C) temperatures. In addition, heat-treated waste sludge released far less P into the digested sludge liquor than did untreated waste sludge. It is likely that the P recovery step prior to anaerobic digestion has a potential advantage for controlling struvite (magnesium ammonium phosphate) deposit problems in sludge handling processes.

Journal Article↗
PubMed2004

ATP amplification for ultrasensitive bioluminescence assay: detection of a single bacterial cell.

We developed an ultrasensitive bioluminescence assay of ATP by employing (i) adenylate kinase (ADK) for converting AMP + ATP to two molecules of ADP, (ii) polyphosphate (polyP) kinase (PPK) for converting ADP back to ATP (ATP amplification), and (iii) a commercially available firefly luciferase. A highly purified PPK-ADK fusion protein efficiently amplified ATP, resulting in high levels of bioluminescence in the firefly luciferase reaction. The present method, which was approximately 10,000-fold more sensitive to ATP than the conventional bioluminescence assay, allowed us to detect bacterial contamination as low as one colony-forming unit (CFU) of Escherichia coli per assay.

Adenosine Triphosphate↗