Kazuhiro Mori

Design of PCR primers and gene probes for the general detection of bacterial populations capable of degrading aromatic compounds via catechol cleavage pathways.

For the general detection of bacterial populations capable of degrading aromatic compounds, two PCR primer sets were designed which can, respectively, amplify specific fragments from a wide variety of catechol 1,2-dioxygenase (C12O) and catechol 2,3-dioxygenase (C23O) genes. The C12O-targeting primer set (C12O primers) was designed based on the homologous regions of 11 C12O genes listed in the GenBank, while the C23O-targeting one (C23O primers) was designed based on those of 17 known C23O genes. Oligonucleotide probes (C12Op and C23Op) were also designed from the internal homologous regions to identify the amplified fragments. The specificity of the primer sets and probes was confirmed using authentic bacterial strains known to carry the C12O and/or C23O genes used for the primer and probe design. Various authentic bacterial strains carrying neither C12O nor C23O genes were used as negative controls. PCR with the C12O primers amplified DNA fragments of the expected sizes from 5 of the 6 known C12O-carrying bacterial strains tested, and positive signals were obtained from 4 of the 5 amplified fragments on Southern hybridization with the C12Op. The C23O primers amplified DNA fragments of the expected size from all the 11 tested C23O-carrying bacterial strains used for their design, while the C23Op detected positive signals in the amplified fragments from 9 strains. On the other hand, no DNA fragments were amplified from the negative controls. To evaluate the applicability of the designed primers and probes for the general detection of aromatic compound-degrading bacteria, they were applied to wild-type phenol- and/or benzoate-degrading bacteria newly isolated from a variety of environments. The C12O and/or C23O primers amplified DNA fragments of the expected sizes from 69 of the 106 wild-type strains tested, while the C12Op and/or C23Op detected positive signals in the amplified fragments from 63 strains. These results suggest that our primer and probe systems can detect a considerable proportion of bacteria which can degrade aromatic compounds via catechol cleavage pathways.

Nutrient removal and starch production through cultivation of Wolffia arrhiza

Wolffia arrhiza, a small weed found mostly in tropical and subtropical water environments, exhibits a high growth rate and consequently absorbs large amounts of nitrogen and phosphorus. Its vegetative frond contains 40% protein on a dry weight basis and its turion, which is the dormant form, has a similar starch content. The applicability of this weed to nutrient removal from secondary-treated waste water combined with starch resource production was evaluated. The nitrogen and phosphorus removal capabilities of the vegetative frond and the optimal conditions for inducing of the formation of turions from harvested biomass of vegetative fronds for the production of starch were investigated using artificial nutrient solutions. The vegetative frond showed high contents of nitrogen (6-7% of the total dry weight) and phosphorus (1-2% of the total dry weight). The nutrient removal rates of the vegetative frond were estimated to be 126 mg-N/m(2)/d and 38 mg-P/m(2)/d under a continuous flow condition. For turion formation from the vegetative fronds, a low nutrient concentration and a high plant density were most effective. Under the optimum conditions, the starch production rate was estimated to be 6 g-starch/m(2) (nutrient removal tank)/d.

Molecular cloning and sequencing of the phenol hydroxylase gene from Pseudomonas putida BH

Abstract A genomic library of the phenol-degrading bacterium Pseudomonas putida BH was constructed in the broad host range cosmid pVK100 and introduced into Escherichia coli HB101. One of the recombinant cosmids recovered from catechol- and/or 2-hydroxymuconic semialdehyde-accumulating clones, pS10–45, had a 19.6-kb insert fragment which allowed P. putida KT2440 to grow on phenol as a sole carbon and energy source. Subcloning and expression studies indicated that the phenol hydroxylase gene cluster (pheA) is located on a 6.1-kb SacI fragment. The results of DNA sequencing of the SacI fragment revealed that the pheA gene cluster encodes a multicomponent phenol hydroxylase.