Yasushi Kawakami

Gene organization and low regiospecificity in aromatic-ring hydroxylation of a benzene monooxygenase of Pseudomonas aeruginosa JI104

Abstract A novel benzene monooxygenase gene cluster has been cloned from Pseudomonas aeruginosa JI104 which was isolated from soil as a benzene degrader. The nucleotide sequence of this gene cluster was found to be highly homologous to those of other toluene monooxygenase gene clusters. This multicomponent monooxygenase also has the capability to catalyze the hydroxylation of various alkylated aromatic hydrocarbons. The low regiospecific hydroxylation was observed when toluene, o-xylene, ethyl benzene and n-propyl benzene were used as substrates.

Cloning and characterization of extradiol aromatic ring-cleavage dioxygenases of Pseudomonas aeruginosa JI104

Abstract We have cloned multiple extradiol aromatic ring-cleavage dioxygenase (EDO) genes from a gene library of Pseudomonas aeruginosa JI104, which is a benzene degrader isolated from soil near a gasworks. Southern hybridization analysis revealed that P. aeruginosa JI104 possessed three homologous catechol 2,3-dioxygenase (C23O) genes. Nucleotide sequences of the cloned C23O genes, xylE JI104-1,2,3 were almost identical to that of the archetypal C23O gene ( xylE TOL ), which is carried on the TOL plasmid, pWW0. We also cloned another EDO gene, bphC JI104 , the product of which showed less activity for catechol than did XylE JI104 , but higher activity for 2,3-dihydroxy biphenyl. The nucleotide sequence of bphC JI104 was identical to that of bphC KF707 (2,3-dihydroxybiphenyl dioxygenase gene of Pseudomonas pseudoalcaligenes KF707). The substrate specificities of the four EDOs of P. aeruginosa JI104 were markedly different from each other. Although XylE JI104-1 and XylE TOL were 94% homologous, the specificities of the gene products for 4-chlorocatechol were extremely different. Results of a study of the chimeric enzymes composed of XylE JI104-1 and XylE TOL N- and C-terminal regions showed that the difference in the specificity for 4-chlorocatechol was dependent on the C-terminal amino acid sequences. All of the isofunctional homologous EDOs in P. aeruginosa JI104 seem to have been derived from a common ancestor and evolved into the present forms in which each EDO is involved in a different degradation pathway and they all coexist in one strain.

Secretion of genetically-engineered dihydrofolate reductase from Escherichia coli using an E. coli ?-hemolysin membrane translocation system

SummarySecretion of fusion proteins composed of cytoplasmic protein dihydrofolate reductase (DHFR) and the Escherichia coli α-haemolysin (HlyA) C-terminal sequence was examined through the haemolysin secretion machinery of E. coli. DHFR of various lengths was combined with the HlyA C-terminal region, and both secretion and DHFR activity of the fusions were measured. The secretion was found to be inversely correlated with the intracellular DHFR activity. Moreover, when one amino acid (Ile155) in a β-sheet of the DHFR C-terminal region was replaced with Lys, the enzymatically active DHFR fusion protein was secreted into the medium. We discuss the possibility of a relationship between folding and secretion of HlyA-fused protein in the HlyA secretion system.

cis-benzeneglycol production using a mutant Pseudomonas strain

Abstract The biotransformation of commodity aromatic chemicals into dihydroxy derivatives was studied. A strain isolated from the invironment, Pseudomonas JI104, used benzene, toluene, and other hydrocarbons as sole carbon and energy sources. We selected mutants unable to grow with benzene, and among these, screened for strains with deficient cis -benzenglycol dehydrogenase able to stably produce cis -benzeneglycol when another carbon source was co-metabolized. We exained the possibility of cis -benzeneglycol production by growing the mutant strain in the presence of benzene vapor. Ethanol was the carbon and energy source most adapted to the cis -benzeneglycol production phase, and lactate or propanol could also be used. Glucose inhibited the production of the metabolite. The growth rates were barely affected by the presence of benzene at a reduced partial pressure (less than 20% of saturation), showing that continuous culture is possible. In a batch process, 0.54g·1 −1 of a cell suspension produced 5.1 mmol·1 −1 cis -benzeneglycol in 27 h, using ethanol as the energy source.

Benzo(a)pyrene in the sediment of Osaka Bay and Keihin Canal and its estimated sources

Benzo(a)pyrene (BP) in the sediment of Osaka Bay were determined. High concentration was found at two stations near the mouth of Shin-Yodo and off Kobe respectively (0.98Μg g−1 dry mud basis, 1.1Μg g−1). The concentration decreases regularly from the mouth of the river further into the bay. The other supplemental determination was carried on the sediment and the seawater of Keihin Canal. Notable BP concentration of 89Μg g−1 was observed in the sediment beneath the effluent outlet of a gasworks. The BP in the sediment near the ironworks was also considerable, while the BP was relatively less in the sediment beneath the effluent outlet of refineries. These data suggest that coke plants working in gasworks or ironworks may be the larger sources of BP than refineries. The results of Osaka Bay coincide with this hypothesis.Cadmium, lead, copper, zinc, nickel, ignition loss and fine sand content were measured from the same sediment samples of Osaka Bay. Cd, Pb, Cu and ignition loss showed the regular distributions which resemble to BP and accordingly, good correlations with BP. No significant correlations were found between BP and Ni, Zn and fine sand content which showed no regular distributions.

Degradation of lubricating oils by marine bacteria observed by quantitative mass spectrometry

Bacterial degradation of the hydrocarbons of lubricating oils was investigated by mass spectrometric analysis which gives both total amount and the composition of hydrocarbon types of residual oil. An unused lubricating oil, which mainly consisted of hydrocarbon types with only a small percentage ofn-alkanes, was degraded by marineBacillus sp. andPseudomonas sp. with 55 % and 25 % decreases in 10 days, respectively. Susceptibility of respective hydrocarbon types to biodegradation was in the following order: alkanes > non-condensed cycloalkanes, mono-aromatics > condensed cycloalkanes. A used lubricating oil of different brand showed a larger decrease than the unused oil. Both species of bacteria degraded large portions of alkane fraction of Arabian light crude oil.

Benzene toxicity and yield improvement in a cis-benzeneglycol production processe

Abstract We carried out experiments designed to increase the production of cis -benzeneglycol from benzene by a mutant strain of Pseudomonas . Factors affecting the production were the temperature, the nitrogen source and its concentration, the concentration of iron ions, and the benzene concentration. At a benzene concentration of 33% of saturation vapor pressure, the cis -benzeneglycol production was not stable, but this benzene concentration was not toxic to the cells. In a culture under 10% of saturation vapor pressure the cells kept producing cis -benzeneglycol for over 100 h. The decrease in cis -benzeneglycol production observed production at high benzen partial pressure (33%) could not be accounted for by the combined effects of an inhibition of enzyme production and a rapid turn-over of the enzyme, but by a specific inactivation of the enzyme. Possible mechanisms of inactivation were discussed which could explain the specific inactivation of the benzene dioxygenase: generation of polyphenols or generation of oxygen radicals.