Kohzo Kanda

Single amino acid mutation in an ATP-binding cassette transporter gene causes resistance to Bt toxin Cry1Ab in the silkworm, Bombyx mori

Bt toxins derived from the arthropod bacterial pathogen Bacillus thuringiensis are widely used for insect control as insecticides or in transgenic crops. Bt resistance has been found in field populations of several lepidopteran pests and in laboratory strains selected with Bt toxin. Widespread planting of crops expressing Bt toxins has raised concerns about the potential increase of resistance mutations in targeted insects. By using Bombyx mori as a model, we identified a candidate gene for a recessive form of resistance to Cry1Ab toxin on chromosome 15 by positional cloning. BGIBMGA007792-93, which encodes an ATP-binding cassette transporter similar to human multidrug resistance protein 4 and orthologous to genes associated with recessive resistance to Cry1Ac in Heliothis virescens and two other lepidopteran species, was expressed in the midgut. Sequences of 10 susceptible and seven resistant silkworm strains revealed a common tyrosine insertion in an outer loop of the predicted transmembrane structure of resistant alleles. We confirmed the role of this ATP-binding cassette transporter gene in Bt resistance by converting a resistant silkworm strain into a susceptible one by using germline transformation. This study represents a direct demonstration of Bt resistance gene function in insects with the use of transgenesis.

Wastewater Treatment with Bacteria Immobilized onto a Ceramic Carrier in an Aerated System.

Biological treatment of the wastewater discharged from a food processing factory was continuously carried out in a packed bed bioreactor under aerobic conditions. The bacterium isolated from the wastewater was immobilized onto a new type of ceramic carrier by a vacuum method and high numbers of bacteria were colonized onto the carrier (2.9 x 10(9) cfu/g of dry ceramic carrier). The effect of the hydraulic retention time (HRT) and aeration rate on the removal of the chemical oxygen demand (COD) was investigated. The system was able on average to remove more than 82% of the influent COD during 160 d of operation and more than 87% of the influent COD on average was removed when the HRT was 30.17 h and the aeration rate was 2.0 vvm. Aeration rates in the range of 0.4 to 2.0 vvm do not affect the COD removal efficiency.

Denitrification activity of the bacterium Pseudomonas sp. ASM-2-3 isolated from the Ariake Sea tideland.

A new denitrifying bacterium strain ASM-2-3 was isolated from the Ariake Sea tideland, Japan. The isolate had the capability to fully remove as high as 225.8 mg nitrate-nitrogen.l(-1) under stationary culture conditions without accumulation of nitrite as an intermediate. From biochemical tests and 16S rDNA sequencing analysis, the genus of the bacterium was identified as Pseudomonas and close to stutzeri species. The nitrate removal efficiency of the isolate was faster than that of the control strain Pseudomonas stutzeri NBRC 14165, using succinate as the sole carbon source. The isolate could grow in up to 10% (w/v) of NaCl containing medium. The enzymatic tests showed that the activity of enzymes responsible for the reduction of nitrate and nitrite in strain ASM-2-3 was 1.4 and 2.3 times higher than that of the control strain. The feasibility of application of the isolate strain ASM-2-3 in a packed bed bioreactor was investigated for 40 d.

Distribution of virulence markers among Vibrio vulnificus isolates of clinical and environmental origin and regional characteristics in Japan.

Background Vibrio vulnificus is an opportunistic human pathogen that is widely distributed in estuarine environments and is capable of causing necrotizing fasciitis and sepsis. In Japan, based on epidemiological research, the incidences of V. vulnificus were concentrated in Kyusyu, mainly in coastal areas of the Ariake Sea. To examine the virulence potential, various genotyping methods have recently been developed. This study aimed to investigate the distribution of virulence markers among V. vulnificus isolates of clinical and environmental origin in three coastal areas with different infection incidences and to determine whether these isolates have the siderophore encoding gene viuB. Methodology/Principal Findings We examined the distribution of genotypes of the 16S ribosomal ribonucleic acid (rRNA) gene, vvhA, vcg, and capsular polysaccharide (CPS), and the presence of viuB in 156 isolates collected from patients and environmental samples in Japan. The environmental samples were collected from three coastal areas: the Ariake Sea, Ise & Mikawa Bay, and Karatsu Bay. The results showed disparity in the ratios of genotypes depending on the sample origins. V. vulnificus isolates obtained from patients were classified into the clinical type for all genotypes. In the environmental isolates, the ratios of the clinical type for genotypes of the 16S rRNA gene, vvhA, and vcg were in the order of the Ariake Sea>Ise & Mikawa Bay>Karatsu Bay. Meanwhile, CPS analysis showed no significant difference. Most isolates possessed viuB. Conclusions Many V. vulnificus belonging to the clinical type existed in the Ariake Sea. Three coastal areas with different infection incidences showed distinct ratios of genotypes. This may indicate that the distribution of clinical isolates correlates with the incidence of V. vulnificus infection.

An extrachromosomal prophage naturally associated with Bacillus thuringiensis serovar israelensis

Bacillus thuringiensis serovar israelensis, an entomopathogen for mosquito larvae, was demonstrated to be lysogenized by temperate phage SU‐11 whose genome was located extrachromosomally in the cell. The prophage SU‐11 was cured at high frequency from the parental strain by continuous sub‐culture at high temperature, but the ability to produce δ‐endotoxin remained in the prophage cured strain. Moreover, phage induction was found to occur after mating of serovar israelensis with its prophage cured strain, as well as with B. thuringiensis serovar thuringiensis, B. cereus and B. subtilis.

Isolation and characteristics of new phages from a serine-producing Escherichia coli K-12

Abstract Two lytic phages, designated S1 and S2, were isolated from culture lysates of a genetically modified serine-producing Escherichia coli K-12. Both phages were highly species-specific for E. coli . S1 was specific for strains of K-12, while S2 attacked strains B and C in addition to K-12 strains. S1 had an icosahedral head 75 nm in diameter and a contractile tail 150 nm long. S2 had an icosahedral head 60 nm in diameter and a noncontractile tail 160 nm long. They were serologically unrelated. Their serotypes were different from those of the other E. coli phages. The latent period and burst size were 28 min and 450, respectively, for S1, and 15 min and 100, respectively, for S2. The phages contained double-stranded DNA with four normal bases. The G+C contents were about 31% for S1 DNA and about 37% for S2 DNA. Restriction patterns of their DNAs were different from each other. The genome sizes were 52 kbp for S1 and 49 kbp for S2. No homology was observed between their genomes. Furthermore, the structural proteins of the two phages also differend. W conclude that phages S1 and S2, differing from each other, could be new phages for E. coli .

Phage-inactivating Effect of Iron(II) : Ascorbate Complex

The effect of iron(II)-ascorbate complex on various phages was investigated. At 10- (6) M, the complex inactivated all nine phages examined. The mechanism of the inactivation was studied with phage J1, the most sensitive to the complex. The addition of H2O2 or Cu(2+) to the reaction mixture increased the inactivation. Bubbling of nitrogen through the reaction mixture and the addition of Fe(3+), a reducing agent, a chelating agent, or a radical scavenger prevented inactivation. These findings suggest the involvement of oxygen radicals in the inactivation. The complex had no effects on the SDS-PAGE pattern or amino acid composition of bovine serum albumin, or the structural protein of phage J1. The complex nicked the supercoiled form of pUC18 DNA, giving first single-stranded breaks (the open circular form) and then double-stranded breaks (the linear form). Strands of M13mp8 DNA, λDNA, and J1 DNA were also broken. The breaks could account for the inactivation.