Characterization of mutations in the two-component histidine kinase gene that confer fludioxonil resistance and osmotic sensitivity in the os-1 mutants of Neurospora crassa

Osmotic-sensitive (os-1) mutant alleles in Neurospora crassa exhibit resistance to dicarboximides, aromatic hydrocarbons and phenylpyrroles. We have previously reported that the os-1 mutants can be classified into two groups based on their resistance to fungicides and osmotic stress: type I, which are highly resistant to iprodione and fludioxonil but moderately sensitive to osmotic stress, and type II, which are highly sensitive to osmotic stress but moderately resistant to fungicides. To explain the mechanism of resistance to these fungicides, we cloned and sequenced the mutant os-1 genes that encode putative osmo-sensing histidine kinase. Within the os-1 gene product (Os1p), the type I strains, NM233t and Y256M209, carried a stop codon at amino acid position 308 and a frameshift at amino acid position 294, respectively. These mutation sites were located on the upstream of histidine kinase and the response regulator domains of Os1p, strongly suggesting that type I strains are null mutants. The null mutants, NM233t and Y256M209, were highly resistant to iprodione and fludioxonil; thus Os1p is essential for these fungicides to express their antifungal activity. The amino acid changes in Os1p, 625Pro from Leu, 578Val from Ala, and 580Arg from Gly were found in the type II strains, M16, M155-1 and P5990, respectively. Os1p is novel in having six tandem repeats of 90 amino acids in the N terminal. Each amino acid change of the type II strains was located on the fifth unit of six tandem repeats. Type II strains with single amino acid changes were more sensitive to osmotic stress than the null mutants (type I), indicating that the amino acid repeats of Os1p were responsible for an important function in osmo-regulation.     

Hyperproduction and application of alpha-agarase to enzymatic enhancement of antioxidant activity of porphyran

The nucleotide sequence of the gene for the alpha-agarase, AgaA33, from Thalassomonas sp. strain JAMB-A33 was determined. The open reading frame for AgaA33 was revealed to encode 1463 amino acid residues. We succeeded in extracellular production of recombinant -agarase (AgaA33) efficiently using Bacillus subtilis as a host. This is the first report of recombinant production of -agarase. Furthermore, we demonstrated that hydrolysis of alpha-1,3 linkages in porphyran, a sulfated polysaccharide from marine red algae, by alpha-agarase is an important step for improvement of its antioxidant activity with regard to free-radical-scavenging capacity and superoxide radical anion scavenging activity, whereas the hydrolysis of beta-1,4 linkages in porphyran by beta-agarase did not increase on the antioxidant activity markedly.     

Role of the nitric oxide-cGMP system in the regulation of ductus arteriosus patency in fetal rats

The purpose of this study was to examine the role of the nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) system in the regulation of the ductus arteriosus (DA) patency in fetal rats. Pregnant rats were administered N(G)-nitro-L-arginine methyl ester (L-NAME, 50 mg/kg, ip), an NO synthase (NOS) inhibitor; methylene blue (30, 50 and 100 mg/kg, ip), a soluble guanylate cyclase inhibitor; or indomethacin (3 mg/kg, po), a cyclooxygenase inhibitor, at various times before cesarean section. Dams were decapitated to obtain the fetuses by cesarean section, and fetuses were rapidly frozen in an acetone-dry ice mixture. Using rapid freezing and shaving methods, the calibers of the DA, pulmonary artery (PA) and descending aorta (Ao) were measured to evaluate the effects of treatment. L-NAME reduced the DA calibers to 86% of the initial values, but recovery to the control levels occurred 6 hr after the injection. Indomethacin decreased the DA calibers to 34% of the control values and sustained the DA constriction until 24 hr after the treatment. Methylene blue caused DA constriction to almost the same degree as indomethacin, but the levels normalized within 24 hr after the treatment. We conclude that L-NAME caused a slight constriction of the DA, whereas methylene blue and indomethacin caused marked constriction of the vessels, suggesting that the NO-cGMP system as well as prostaglandins contribute to the DA patency.     

The first occurrence of leaf mold of tomato caused by races 4.9 and 4.9.11 of <Emphasis Type="Italic">Passalora fulva</Emphasis> (syn. <Emphasis Type="Italic">Fulvia fulva</Emphasis>) in Japan

Tomato leaf mold caused by Passalora fulva was found on two tomato varieties carrying the Cf-9 gene in Japan, in 2007. The isolates obtained from Chiba and Fukushima were identified as race 4.9.11, and those from Gunma were races 4.9 or 4.9.11. This is the first report in Japan of tomato leaf mold caused by P. fulva strains that can overcome the Cf-9 gene.     

PCR-DGGE analysis of bacterial communities in <Emphasis Type="Italic">funazushi</Emphasis>, fermented crucian carp with rice,during fermentation

Funazushi (fermented Crucian Carp with rice) is a fermented fish product found only around Lake Biwa in Shiga Prefecture, Japan. It is characterized by a unique cheese-like flavor and characteristic sour taste. We analyzed the changes in the microbial community during funazushi fermentation by denaturing gradient gel electrophoresis of PCR-amplified 16S rDNA fragments (PCR-DGGE) and by plate counts. The plate counts showed that lactic acid bacteria reached 8.0 log₁₀ CFU/g within 7 days of fermentation initiation before decreasing slowly to 4.0 log₁₀ CFU/g during the remainder of 1-year study period. PCR-DGGE revealed that the dominant bacteria in the initial (days 14 and 30) and latter (days 90, 180, and 360) periods of fermentation were Lactobacillus plantarum and L. acetotolerans. This is the first identification of L. acetotolerans in funazushi as traditional cultivation techniques have not been sufficiently sensitive. This is the first report of PCR-DGGE being used to assess the microbial community in funazushi. This technique was also found to be effective in profiling microbial diversity.