Filamentous bacteriophages of vibrios are integrated into the dif-like site of the host chromosome

The dif site is located in the replication terminus region of bacterial chromosomes, having a function of resolving dimeric chromosomes formed during replication. We demonstrate that filamentous bacteriophages of vibrios, such as f237 (Vibrio parahaemolyticus) and CTXphi (V. cholerae), are integrated into the dif-like site of host chromosome.     

Genetic diversity and origin of Potamogeton anguillanus (Potamogetonaceae) in Lake Biwa,Japan

We analyzed the genetic variation in Potamogeton anguillanus Koidz. and its putative parents, P. malaianus Miq. and P. perfoliatus L., at five allozyme loci of four enzymes to test the hypothesis of a hybrid origin for P. anguillanus, collected in Lake Biwa, Japan. Alleles diagnostic for either P. malaianus or P. perfoliatus were present at four loci. Of 13 single locus phenotypes (SLPs) of P. anguillanus, eight were phenotypes that were expected in F(1) hybrids between P. malaianus and P. perfoliatus. Two SLPs were different from those expected in F(1) hybrids but could have resulted from segregation of parental alleles in later generation hybrids. Each of the remaining three SLPs possessed one allele unique to P. anguillanus. Allozyme analyses thus supported the view that P. anguillanus was derived from hybridization between P. malaianus and P. perfoliatus. It seems likely that the genetic diversity of P. anguillanus found previously originated through multiple hybridizations and sexual processes in P. anguillanus. Other processes such as intragenic recombination, mutation, or hybridization with another lineage are also discussed with reference to the origin of unique alleles.     

Preparation of chiral 4-benzyloxymethyldihydrofuran-2-one using lipase-catalyzed kinetic resolution: synthesis of (-)-virginiae butanolide C (VB C)

Lipase-catalyzed kinetic resolution of the N,N-dialkyl-3-benzyloxymethyl-4-hydroxybutanamide 10a,b afforded the acetate 11a,b with (R) configuration, whereas the N-monoalkyl-3-benzyloxymethyl-4-hydroxybutanamide 10c-e gave the acetate 11c-e with (S) configuration. The butanamide 10 smoothly cyclized to give chiral 4-benzyloxymethyldihydrofuran-2-one 9 without racemization, which was effectively transformed into highly stereocontrolled virginiae butanolide C (VB C).     

Endogenous alpha-ketol linolenic acid levels in short day-induced cotyledons are closely related to flower induction in Pharbitis nil

Alpha-ketol linolenic acid [KODA, 9,10-ketol-octadecadienoic acid, that is 9-hydroxy-10-oxo-12(Z),15(Z)-octadecadienoic acid] is a signal compound found in Lemna paucicostata after exposure to stress, such as drought, heat or osmotic stress. KODA reacts with catecholamines to generate products that strongly induce flowering, although KODA itself is inactive [Yokoyama et al. (2000) Plant Cell Physiol. 41: 110; Yamaguchi et al. (2001) Plant Cell Physiol. 42: 1201]. We examined the role of KODA in the flower-induction process of Pharbitis nil (violet). KODA was identified for the first time in seedlings of P. nil grown under a flower-inductive condition (16-h dark exposure), by means of LC-SIM and LC-MS/MS. In addition, the changes in endogenous KODA levels (evaluated after esterification of KODA with 9-anthryldiazomethane) during the flower-inductive phase in short day-induced cotyledons were closely related to flower induction. The KODA concentration sharply increased in seedlings during the last 2 h of a 16-h dark period, while the KODA level showed no significant elevation under continuous light. The increase of KODA level occurred in cotyledonal blades, but not in other parts (petiole, hypocotyls and shoot tip). When the 16-h dark period was interrupted with a 10-min light exposure at the 8th h, flower induction was blocked and KODA level also failed to increase. The degree of elevation of KODA concentration in response to 16-h dark exposure was the highest when the cotyledons had just unfolded, and gradually decreased in seedlings grown under continuous light for longer periods, reaching the basal level at the 3rd day after unfolding. Flower-inducing ability also decreased in a similar manner. These results suggest that KODA may be involved in flower induction in P. nil.     

Efficient release of overproduced gene products from Escherichia coli BL21(DE3) by lytic infection with newly isolated bacteriophages

Overproduced proteins from Escherichia coli BL21(DE3) were efficiently released with virulent bacteriophages. Leviviridae-like bacteriophages were isolated from soil and used to lyse BL21(DE3) cells transformed with beta-glucosidase, chitinase, or chitosanase genes. This method caused lysis of bacterial cells similar to that by conventional sonication and enabled us to effectively recover and purify the enzymes.     

Profile of a nonylphenol-degrading microflora and its potential for bioremedial applications

Nonylphenol (NP) is an important intermediate in the production of various commercial and industrial materials, but is also known as a ubiquitous pollutant in urban aquatic environments. We recently studied the NP-degrading activities of microflora in several aquatic environments, and found a notable degrading activity for wastewater from a sewage treatment plant in Tokyo. This result led us to isolate NP-degrading microbes and identify biodegradation products. Using conventional plate culture techniques and molecular biological methods, Pseudomonas and Sphingomonas species, which are known for their degradation activities of many aromatic compounds, have been isolated. But it has also been found that Sphingomonas sp. (S-strain) is necessary and sufficient for the degradation of NP. Although the role of Pseudomonas sp. (P-strain) remains unclear, P-strain seems to provide some co-nutrients for the growth of S-strain. The degradation products were analyzed by GC/MS and NMR. More than 95% of NP was degraded within 10 days and aromatic compounds other than NP were not found, suggesting that the phenolic part of NP was completely degraded. We also examined the potential of S-strain for bioremedial applications. S-strain cells immobilized on chitosan or alginate beads retain their NP-degrading activity in flask-scale experiments. Furthermore, the chitosan-bound cells in a lab-scale bioreactor have been found to be persistent for repeated use, suggesting that S-strain is applicable to the treatment of NP-contaminated wastewater.     

Mechanism of superoxide anion generation in the toxic red tide phytoplankton Chattonella marina: possible involvement of NAD(P)H oxidase

Red tide phytoplankton Chattonella marina is known to produce reactive oxygen species (ROS), such as superoxide anion (O(2)(-)), hydrogen peroxide (H(2)O(2)) and hydroxyl radical (&z.rad;OH), under normal physiological conditions. Although several lines of evidence suggest that ROS are involved in the mortality of fish exposed to C. marina, the mechanism of ROS generation in C. marina remains to be clarified. In this study, we found that the cell-free supernatant prepared from C. marina cells showed NAD(P)H-dependent O(2)(-) generation, and this response was inhibited by diphenyleneiodonium, an inhibitor of mammalian NADPH oxidase. When the cell-free supernatant of C. marina was analyzed by immunoblotting using antibody raised against the human neutrophil cytochrome b558 large subunit (gp91phox), a main band of approximately 110 kDa was detected. The cell surface localization of the epitope recognized with this antibody was also demonstrated in C. marina by indirect immunofluorescence. Furthermore, Southern blot analysis performed on genomic DNA of C. marina with a probe covering the C-terminal region of gp91phox suggested the presence of a single-copy gene coding for gp91phox homologous protein in C. marina. These results provide evidence for the involvement of an enzymatic system analogous to the neutrophil NADPH oxidase as a source of O(2)(-) production in C. marina.