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Tsukasa Ikeda

Publications and source records attributed to Tsukasa Ikeda.

15 recordsLinked to original sources
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 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

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

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

Identification of quorum-sensing signal molecules and the LuxRI homologs in fish pathogen Edwardsiella tarda.

Edwardsiella tarda is a gram-negative bacterium that causes septicaemia in fish and serious damage to the aquaculture industry. The virulence factors of this pathogen and control mechanisms of the expression of virulence genes have not yet been clearly elucidated. A number of gram-negative pathogenic bacteria have a quorum-sensing system. These bacteria produce N-acyl-L-homoserine lactone (AHL) that they use them as a quorum-sensing signal molecule. In this study, we found that E. tarda isolated from deceased flounder produces AHLs. Thin layer chromatography analysis indicated that the two kinds of AHL produced by E. tarda seemed to be N-hexanoyl-L-homoserine lactone (C6-HSL) and N-heptanoyl-L-homoserine lactone (C7-HSL). We have cloned and sequenced the quorum-sensing genes, luxI homolog (edwI) and luxR homolog (edwR). EdwI and EdwR showed high identity with CarI/CarR and ExpI/ExpR from Erwinia carotovora, respectively. SDS-PAGE analysis of extracellular proteins revealed that the expression of the 55-kDa protein, which was reported as a virulent-strain-specific protein, is controlled by AHLs. These results suggest that some virulence factors are regulated by the quorum-sensing system in E. tarda.

Journal Article↗
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↗
PubMed2003

Evaluation of bacterial aerotaxis for its potential use in detecting the toxicity of chemicals to microorganisms.

Bacterial aerotaxis (the movement of a cell toward oxygen) was evaluated for its potential use in detecting the toxicity of chemicals to microorganisms. The level of toxicity was determined by the concentration of test chemicals resulting in a 50% inhibition of aerotaxis of Pseudomonas aeruginosa PAO1 after 40 min of exposure. The aerotactic responses of P. aeruginosa were measured by using chemotaxis well chambers. Each clear acrylic chamber had a lower and upper well separated by a polycarbonate filter with a uniform pore size of 8.0 microm. To automatically detect bacterial cells that crossed the filter in response to a gradient of oxygen, P. aeruginosa PAO1 was marked with green fluorescent protein (GFP), and the GFP fluorescence intensity in the upper well was continuously monitored by using a fluorescence spectrometer. By using this technique, volatile chlorinated aliphatic compounds, including trichloroethylene (TCE), trichloroethane, and tetrachloroethylene, were found to be inhibitory to bacterial aerotaxis, suggesting their possible toxicity to microorganisms. We also examined more than 20 potential toxicants for their ability to inhibit the aerotaxis of P. aeruginosa. Based on these experimental results, we concluded that bacterial aerotaxis has potential for use as a fast and reliable indicator in assessing the toxicity of chemicals to microorganisms.

Biosensing Techniques↗
PubMed2003

Mutants of the nematode Caenorhabditis elegans that are defective specifically in their attraction to cycloheximide.

The nematode Caenorhabditis elegans exhibits chemotaxis toward a wide variety of chemicals including water-soluble molecules and volatile organic compounds. We have previously discovered that C. elegans wild-type strain N2 is strongly attracted by cycloheximide which has long been known as a bitter tastant for humans and other mammals. We describe here the isolation and initial characterization of the first mutants which were defective specifically in their attraction to cycloheximide. In our screenings, we selected two mutants that were defective in chemotaxis to cycloheximide but normal in their attraction to NH4Cl and histidine. These mutants also avoided quinine hydrochloride, CuSO4 and high concentrations of NaCl similar to the wild-type strain N2. Furthermore, no observable defect was detected in their attraction to volatile odorants such as isoamyl alcohol and diacetyl. Dye-filling experiments suggested that they have no morphological defect in the sensory endings of the amphid neurons.

Journal Article↗
PubMed2003

A method for screening polyphosphate-accumulating mutants which remove phosphate efficiently from synthetic wastewater.

The biological process for phosphorus removal from wastewater is based on the use of bacteria capable of accumulating inorganic polyphosphate (polyp). We previously showed that a phoU mutation leads to polyp accumulation in Escherichia coli. The phoU mutant could be easily screened on agar plates containing 5-bromo-4-chloro-3-indolyl-phosphate (X-P(i)) after N-methyl-N'-vitro-N-nitrosoguanidine (NTG) mutagenesis. Here, we demonstrate that this method is also useful for screening polyp-accumulating mutants of bacterial strains isolated from soil and activated sludge samples.

Journal Article↗
PubMed2002

A simple method to release polyphosphate from activated sludge for phosphorus reuse and recycling.

In enhanced biological phosphorus removal processes, activated sludge microorganisms accumulate large quantities of polyphosphate (polyP). It was discovered that nearly all of the polyP could be released from activated sludge simply by heating it at 70 degrees C for about 1 h. The chain length of released polyP ranged from 100 to 200 phosphate (P(i)) residues. The addition of CaCl(2) precipitated approximately 75% of the total phosphorus without pH adjustment. The formed precipitate contained more P and less Ca than typical natural phosphorite deposits. Hence, in combination with enhanced biological phosphorus removal, the present method has potential for the development of a simple process for recovering phosphorus in a reusable form from wastewater.

Calcium Chloride↗
PubMed2002

Accumulation of inorganic polyphosphate in phoU mutants of Escherichia coli and Synechocystis sp. strain PCC6803.

The biological process for phosphate (P(i)) removal is based on the use of bacteria capable of accumulating inorganic polyphosphate (polyP). We obtained Escherichia coli mutants which accumulate a large amount of polyP. The polyP accumulation in these mutants was ascribed to a mutation of the phoU gene that encodes a negative regulator of the P(i) regulon. Insertional inactivation of the phoU gene also elevated the intracellular level of polyP in Synechocystis sp. strain PCC6803. The mutant could remove fourfold more P(i) from the medium than the wild-type strain removed.

Bacterial Proteins↗
PubMed2002

Cloning and characterization of extracellular metal protease gene of the algicidal marine bacterium Pseudoalteromonas sp. strain A28.

The gene (empI) encoding an extracellular metal protease was isolated from a Pseudoalteromonas sp. strain A28 DNA library. The recombinant EmpI protein was expressed in E. coli and purified. Paper-disk assays showed that the purified protease had potent algicidal activity. A skim milk-polyacrylamide gel electrophoresis protease assay showed that the 38-kDa band of protease activity, which co-migrated with purified EmpI and was sensitive to 1,10-phenathroline, was detected in the extracellular supernatant of A28.

Cloning, Molecular↗
PubMed2002

Isolation and characterization of cbbL and cbbS genes encoding form I ribulose-1,5-bisphosphate carboxylase/oxygenase large and small subunits in Nitrosomonas sp. strain ENI-11.

The cbbL and cbbS genes encoding form I ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) large and small subunits in the ammonia-oxidizing bacterium Nitrosomonas sp. strain ENI-11 were cloned and sequenced. The deduced gene products, CbbL and CbbS, had 93 and 87% identity with Thiobacillus intermedius CbbL and Nitrobacter winogradskyi CbbS, respectively. Expression of cbbL and cbbS in Escherichia coli led to the detection of RubisCO activity in the presence of 0.1 mM isopropyl-beta-D-thiogalactopyranoside (IPTG). To our knowledge, this is the first paper to report the genes involved in the carbon fixation reaction in chemolithotrophic ammonia-oxidizing bacteria.

Ammonia↗
PubMed2002

Behavioral responses of the ciliated protozoan Paramecium caudatum to 2,4-dichlorophenoxyacetic acid and its analogues.

We found that the ciliated protozoan Paramecium caudatum ON-1 was repelled by 2,4-dichlorophenoxyacetic acid (2,4-D). P. caudatum was also repelled by phenoxyacetic acid, while it was attracted to benzoic acid. No significant behavioral response was detected with 2,4-dichlorophenol (2,4-DP) and phenol. These results suggested that the substitution of-OH of phenol with -OCH2COOH, but not the chlorination of phenol, was important for eliciting the avoiding response to 2,4-D in P. caudatum. Electrophysiological analysis revealed that 2,4-D and phenoxyacetic acid were able to trigger the action potential in P. caudatum, whereas 2,4-DP and phenol failed to elicit electrical responses in this organism. Well test assays suggested that the avoiding reactions allowed P. caudatum to escape from the harm caused by this herbicide.

Journal Article↗