Yoshifumi Itoh

The CbrA–CbrB two‐component regulatory system controls the utilization of multiple carbon and nitrogen sources in Pseudomonas aeruginosa

A novel two‐component system, CbrA–CbrB, was discovered in Pseudomonas aeruginosa;cbrA and cbrB mutants of strain PAO were found to be unable to use several amino acids (such as arginine, histidine and proline), polyamines and agmatine as sole carbon and nitrogen sources. These mutants were also unable to use, or used poorly, many other carbon sources, including mannitol, glucose, pyruvate and citrate. A 7u2003kb EcoRI fragment carrying the cbrA and cbrB genes was cloned and sequenced. The cbrA and cbrB genes encode a sensor/histidine kinase (Mr 108u2003379, 983 residues) and a cognate response regulator (Mr 52u2003254, 478 residues) respectively. The amino‐terminal half (490 residues) of CbrA appears to be a sensor membrane domain, as predicted by 12 possible transmembrane helices, whereas the carboxy‐terminal part shares homology with the histidine kinases of the NtrB family. The CbrB response regulator shows similarity to the NtrC family members. Complementation and primer extension experiments indicated that cbrA and cbrB are transcribed from separate promoters. In cbrA or cbrB mutants, as well as in the allelic argR9901 and argR9902 mutants, the aot–argR operon was not induced by arginine, indicating an essential role for this two‐component system in the expression of the ArgR‐dependent catabolic pathways, including the aruCFGDB operon specifying the major aerobic arginine catabolic pathway. The histidine catabolic enzyme histidase was not expressed in cbrAB mutants, even in the presence of histidine. In contrast, proline dehydrogenase, responsible for proline utilization (Pru), was expressed in a cbrB mutant at a level comparable with that of the wild‐type strain. When succinate or other C4‐dicarboxylates were added to proline medium at 1u2003mM, the cbrB mutant was restored to a Pru+ phenotype. Such a succinate‐dependent Pru+ property was almost abolished by 20u2003mM ammonia. In conclusion, the CbrA–CbrB system controls the expression of several catabolic pathways and, perhaps together with the NtrB–NtrC system, appears to ensure the intracellular carbon: nitrogen balance in P. aeruginosa.

Family 19 chitinase from rice (Oryza sativa L.) : substrate-binding subsites demonstrated by kinetic and molecular modeling studies

A family 19 chitinase (OsChia1c, class I) from rice, Oryza sativa L., and its chitin-binding domain-truncated mutant (OsChia1cΔCBD, class II) were produced by the Pichia expression system, and the hydrolytic mechanism toward N-acetylglucosamine hexasaccharide [(GlcNAc)6] was investigated by HPLC analysis of the reaction products. The profile of the time-course of (GlcNAc)6 degradation obtained by OsChia1c was identical to that obtained by OsChia1cΔCBD, indicating that the chitin-binding domain does not significantly participate in oligosaccharide hydrolysis. From the theoretical analysis of the reaction time-course of OsChia1cΔCBD, the free energy changes of sugar residue binding were estimated to be −0.4, −4.7, +3.4, −0.5, −2.3, and −1.0 kcal/mol for the individual subsites of (−3), (−2), (−1), (+1), (+2), and (+3), respectively. The hexasaccharide substrate appears to bind to the enzyme through interactions at the high-affinity sites, (−2) and (+2), and the sugar residues at both ends more loosely bind to the corresponding subsites, (−3) and (+3). The docking study of (GlcNAc)6 with the modeled structure of OsChia1cΔCBD supported the subsite structure estimated from the experimental time-course of hexasaccharide degradation. Since the class II chitinase from barley seeds was reported to possess a similar subsite structure from (−3) to (+3) and a similar free energy distribution, substrate-binding mode of plant chitinases of this class would be similar to each other.

Heterologous expression and characterization of class III chitinases from rice (Oryza sativa L.)

Abstract Acidic (OsChib1a) and basic (OsChib1b) class III chitinases from rice ( Oryza sativa L.), sharing 70% of the identical amino acid residues each other, were expressed from the corresponding cDNAs in Pichia pastoris and purified homogenously. Both OsChib1a and OsChib1b degraded actively glycol chitin over colloidal chitin at the optimum pH of 4.3 and 8.3, respectively. OsChib1b had lower specific chitinase activity than OsChib1a, but it showed a strong lytic activity and significant antifungal activity: no lytic and antifungal activity was observed for OsChib1a. Experiments with N -acetyl chitooligosaccharides showed that OsChib1a hydrolyzed (GlcNAc) 6 efficiently to yield (GlcNAc) 2 as the major product, while OsChib1b hydrolyzed (GlcNAc) 6 to yield both (GlcNAc) 2 and (GlcNAc) 4 . Possible structures responsible for their distinct catalytic properties are discussed.

Cis-acting regulatory regions of the soybean seed storage 11S globulin gene and their interactions with seed embryo factors

A 2.2 kb fragment containing the 5′-flanking region of the soybean glycinin A2B1a gene and its successive deletions with a shorter 5′-flanking sequence were fused, in frame, to the β-glucuronidase (GUS) reporter gene. The resultant fusions were introduced into tobacco plants via Agrobacterium tumefaciens. Assays of the GUS activity in seeds of transgenic tobacco showed that the upstream region, −657 to −327 (relative to the transcription initiation site [+1]), of the glycinin gene is required for optimal expression of the transformed gene. Interactions between embryo nuclear factors and DNA fragments covering the downstream region of −326, in which are included the TATA box and legumin boxes, were not apparent. The embryo factors capable of binding specifically to three subregions, −653 to −527, −526 to −422, and −427 to −321, of the upstream regulatory region were detected. Such factors appeared to be organ-specific and could be found solely in developing seeds at the early middle stage of embryogenesis (around 24 days after flowering). Evidence obtained by characterizing the nature of the binding proteins and by gel mobility shift assays established that the same factor does interact with a consensus motif 5′-ATA/TATTTCN-/CTA-3′ which occurs four times in the cis-acting regulatory region between −657 and −327. Moreover, this conserved motif could also be found in the 5′ regulatory region of another glycinin A1aB1b gene. Thus it is likely that the observed interaction between the nuclear factor and the conserved motifs would lead to activation of transcription from the glycinin genes in maturing soybean seeds.

Mutations affecting regulation of the anabolic argF and the catabolic aru genes in Pseudomonas aeruginosa PAO

SummaryThe nucleotide sequence required for a fully functional promoter and operator of the Pseudomonas aeruginosa argF gene (argFpo), the arginine-repressible gene for anabolic ornithine carbamoyltransferase, was defined within a 160 by region. The streptomycin (Sm) resistance genes strAB of plasmid RSF1010 were fused to argFpo. This construct in P. aeruginosa strain PAO conferred resistance to Sm. Mutants of strain PAO were selected which were resistant to Sm in the presence of arginine due to constitutive expression of argFpo—strAB. These mutants were designated argR. They were unable to grow or grew poorly on arginine or ornithine as the sole carbon and nitrogen source. This growth defect (Aru−/Oru− phenotype) was correlated with a reduced level of N-succinylornithine aminotransferase, an enzyme participating in the major aerobic pathway for arginine and ornithine catabolism in this organism. The argR mutants were classified into four groups by transduction analysis and three argR mutations were mapped on the PAO chromosome. argR9901 and argR9902 were co-transducible with car-9 (at 1 min) and thus close to the oru-310 locus; argR9906 was localized in the oruI (=aru) gene cluster (67 min). Some aru mutants, which have been isolated previously and which produce very low amounts of all enzymes in the arginine succinyltransferase pathway, were unable to repress the argF gene in an arginine medium. Thus, P. aeruginosa PAO appears to have multiple genes that are involved in the regulation of both the anabolic argF and the catabolic aru genes.

The glycinin box: a soybean embryo factor binding motif within the quantitative regulatory region of the 11S seed storage globulin promoter

The soybean embryo factor binding sequence in the glycinin A2B1a gene promoter was delimited to an A/T-rich 9 bp sequence, 5′-TAATAATTT-3′, designated as the glycinin box, by DNA footprinting and gel mobility shift assay using synthetic oligonucleotides. It was shown that the interaction with the factor takes place at a defined DNA sequence rather than at random A/T-rich sequence blocks in the glycinin 5′ flanking region. There are four glycinin boxes in the quantitative regulatory region between positions − 545 and − 378 of the glycinin A2B1a promoter. Multiple nonamer motifs similar to the glycinin box were also found in the equivalent regions of other glycinin and legumin promoters, suggesting that they must be conserved as a binding site for the embryo factor that activates the differential and stage-specific expression of seed 11S globulin genes in leguminous plants.