Homolog of FlhDC, a master regulator for flagellum synthesis: required for pathogenicity in Erwinia carotovora subsp. carotovora

 Erwinia carotovora subsp. carotovora (Ecc) is a causal agent of soft-rot diseases in a wide variety of plants. Here, we have isolated nonmotile mutants in Ecc by in vivo insertional mutagenesis using a transposon Tn5. The sequence disrupted by the Tn5 insertion in YMU1 and YMU5 mutants was highly homologous to that of flhC and flhD genes, respectively. They are involved in the initiation of the expression of flagellum-related genes in many gram-negative bacteria such as Escherichia coli and Salmonella. With electron microscopy, the flhC and the flhD homolog mutants were shown to be aflagellate. Furthermore, the virulence of these mutants was greatly reduced in Chinese cabbage and potato compared to that of the parental strain. These results suggest that flagellar formation is required for the pathogenicity of Ecc. Received: November 5, 2002 / Accepted: December 2, 2002 Acknowledgments This research was supported in part by Grant-in-Aid (12052210) and by a grant from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (13073).     

Identification of the core rumen bacterial taxa and their population dynamics during the fattening period in Japanese Black cattle

The rumen microbiota comprises a vast range of bacterial taxa, which may affect the production of high-quality meat in Japanese Black cattle. The aim of this study was to identify core rumen microbiota in rumen fluid samples collected from 74 Japanese Black cattle raised under different dietary conditions using 16S rRNA gene amplicon sequencing. In the rumen of fattening Japanese Black cattle, 10 bacterial taxa, showing >1% average relative abundance and >95% prevalence, irrespective of the dietary conditions and the fattening periods, were identified as the core rumen bacterial taxa, which accounted for approximately 80% of the rumen microbiota in Japanese Black cattle. Additionally, population dynamics of the core rumen bacterial taxa revealed two distinct patterns: Prevotella spp. and unclassified Bacteroidales decreased in the mid-fattening period, whereas unclassified Clostridiales, unclassified Ruminococcaceae, Ruminococcus spp., and unclassified Christensenellaceae increased during the same period. Therefore, the present study reports the wide distribution of the core rumen bacterial taxa in Japanese Black cattle, and the complementary nature of the population dynamics of these core taxa, which may ensure stable rumen fermentation during the fattening period.     

Seasonal changes in semen characteristics, composition of seminal plasma and frequency of acrosome reaction induced by calcium and calcium ionophore A23187 in Large White boars

This study attempted to explain the mechanisms regulating boar fertility by examining seasonal changes in semen characteristics, the composition of seminal plasma and responsiveness of sperm acrosomes to Ca(2+) and the Ca(2+) ionophore A23187 (Ca(2+)/A23187). Sperm-rich and sperm-poor fractions were separately collected from 3 mature fertile Large White boars once a month over a one-year period. During the period of study, ambient temperature and relative humidity were recorded for within the stall in which the boars were kept and the semen characteristics, composition of the seminal plasma of sperm-rich fractions, and occurrence of the acrosome reaction in response to Ca(2+) (3 mM)/A23187 (0.3 microM) were examined. The highest mean maximum and minimum ambient temperatures were recorded in August-September, whereas the lowest mean maximum and minimum ambient temperatures were recorded in December and January, respectively. There was a moderate peak in relative humidity from July to October. The lowest percentages of motile spermatozoa and of spermatozoa with intact acrosomes and highest percentage of spermatozoa with abnormal morphology and strongest agglutination were seen in August-September. The total protein and albumin concentrations were lowest in August-September. Testosterone levels increased gradually as day length decreased after the summer solstice (June) and peaked in October-November. The percentage of acrosome reactions in response to Ca(2+)/A23187 was highest with the quickest response in August-September, as shown by the shortest time required for 50% of relative acrosome reactions. The farrowing rates were lowest in these same 2 months. These results suggest that seasonal infertility in Large White boars may be due, at least in part, to a combination of low motility, abnormal morphology including acrosomal abnormality, and early occurrence of the acrosome reaction in response to stimulus, possibly resulting from a decrease in acrosomal stabilizing proteins in the seminal plasma during summer. These changes may be modulated by heat/humidity stress and/or photoperiod-regulated testosterone.     

Lignin-degrading activity of edible mushroom <Emphasis Type="Italic">Strobilurus ohshimae</Emphasis> that forms fruiting bodies on buried sugi (<Emphasis Type="Italic">Cryptomeria japonica</Emphasis>) twigs

Strobilurus ohshimae is an edible mushroom, and it specifically forms its fruiting bodies on buried sugi (Cryptomeria japonica) twigs. In this research, we studied lignindegrading activity of S. ohshimae. We isolated 18 strains of S. ohshimae from various regions of Japan, and determined their lignin degradation rates on sugi wood meal medium. All the strains of S. ohshimae degraded approximately 6%–12% of sugi lignin in 30 days, and these lignin degradation rates were 1.5–3 times higher than those of Trametes versicolor, which is a typical lignin-degrading fungus. Among the three main lignin-degrading enzymes, activity of lignin peroxidase and manganese peroxidase was not observed, while 4340U/g of laccase was produced in 30 days. To investigate the effect of wood species on lignin degradation by S. ohshimae, the lignin degradation rate and laccase productivity on sugi wood meal medium were compared with those on beech (Fagus crenata). In T. versicolor, both lignin degradation rate and laccase productivity were higher on beech than on sugi. Conversely, in S. ohshimae, lignin degradation rate and laccase productivity were higher on sugi than on beech. Therefore, it was suggested that coniferous lignin is not always difficult to degrade for the fungi that inhabit softwood. Part of this article presented at the 54th Annual Meeting of the Japan Wood Research Society, Sapporo, August 2004     

Comparative Evaluation on Nitrogen Saturation of Forest Catchments in Japan and Northeastern United States

To analyze the differences in the status and processes of nitrogen saturation in Japan and northeastern United States, we examined the hydrobiogeochemistry of nitrogen of forested watersheds in these regions. Two distinct differences were found between watersheds in Japan compared with those in US. 1) In Japanese watersheds, marked decreases of NO₃ ^? concentration in surface waters during the summer growing season were not found and NO₃ ^? concentrations sometimes increased especially in the summer at nitrogen saturated sites. This contrast with watersheds in US where decreases in NO₃ ^? concentration during the summer are commonly observed except those watersheds in advanced stages of nitrogen saturation. These differences in NO₃ ^? concentration relationships can be attributed to climatic differences, with Japan having high precipitation and high discharge during the summer, while in many regions of North America lowest discharges are found in the summer. The climatic regime in Japan leads to high rates of mineralization and the rapid transport of NO₃ ^? to streams in summer. 2) Japanese watersheds, even those with high NO₃ ^? concentrations in surface waters, show little evidence of acidification. This is in contrast to sites in US where increased NO₃ ^? concentrations, especially during episodic events, result in surface water acidification.     

Isolation and characterization of aminopeptidase (Jc-peptidase) from Japanese cedar pollen (Cryptomeria japonica)

An aminopeptidase, Jc-peptidase, was purified from Japanese cedar pollen by seven steps, including precipitation with ammonium sulfate, ion-exchange chromatography, gel filtration, hydrophobic interaction chromatography on phenyl-agarose, and high-performance liquid chromatography. Purified Jc-peptidease has a molecular weight of 42 kDa and hydrolyzes the synthetic substrates of L-phenylalanyl-4-methylcoumaryl-7-amide (Phe-MCA) with Km = 5 x 10(-5) M, Tyr-MCA with Km = 7 x 10(-4) M, Leu-MCA with Km = 1 x 10(-3) M, and Met-MCA with Km = 1 x 10(-3) M. Other MCA analogues such as Arg-MCA or Glu-MCA failed to serve as its substrates. The activity was inhibited in the presence of phebestin, [(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl-L-valyl]-L-phenylalanine, with Ki = 4.7 x 10(-5) M, or bestatin, [(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl]-L-leucine, with Ki = 1.1 x 10(-4) M. According to amino acid sequence analysis, the N-terminal amino group seems to be blocked. The physiological function of the aminopeptidase (Jc-peptidase) has not been clarified in vivo.     

Comparative efficacy of two single-dose bacterins in the control of Mycoplasma hyopneumoniae in swine raised under commercial conditions in Brazil

A field study was conducted in Brazil to evaluate the efficacy of single vaccination of pigs with two bacterins to prevent Mycoplasma hyopneumoniae lung lesions. The first (T1) treatment group (174 pigs) was injected with 2 mL of saline solution; group T2 (177 pigs) with 2 mL of bacterin A, and group T3 (174 pigs) with 2 mL of bacterin B. On days-on-test (DOT) 0, 35, 66, 97 and 125, blood samples and tonsil swabs were collected from selected pigs for antibody determination (indirect ELISA) and PCR assay for the presence of M. hyopneumoniae. Pigs were slaughtered on DOT 126-129 and lung lesions were scored blindly. Bacterin A vaccinated pigs had significantly (P < or = 0.05) lower lung lesion scores (0.2%) than bacterin B (0.4%) or saline-treated pigs (1.2%); there was also a significantly lower (P < or = 0.05) number of pigs with lung lesions (27.1%), than bacterin B (38.2%) or saline-treated (55.4%) pigs. The two vaccines had similar (P>0.05) results in terms of mean weight gain, average daily weight gain, feed efficiency, frequency of PCR positives, and there was similar antibody conversion (ELISA). It was concluded that although the productivity parameters and antibody conversions were similar, bacterin A was more effective in preventing and reducing the severity of lung lesions than bacterin B.