Hajime Minato

Isolation and Identification of Cellulose-Binding Proteins from Sheep Rumen Contents

ABSTRACT To extend our understanding of the mechanisms of plant cell wall degradation in the rumen, cellulose-binding proteins (CBPs) from the contents of a sheep rumen were directly isolated and identified using a metaproteomics approach. The rumen CBPs were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and some CBPs revealed endoglucanase activities toward carboxymethyl cellulose. Using mass spectrometry analyses, four CBPs were identified and annotated as known proteins from the predominant rumen cellulolytic bacterium Fibrobacter succinogenes: tetratricopeptide repeat domain protein, OmpA family protein, fibro-slime domain protein, and cellulose-binding endoglucanase F (EGF). Another CBP was identified as the cellulosomal glycosyl hydrolase family 6 exoglucanase, Cel6A, of Piromyces equi. F. succinogenes cells expressing EGF were found to be major members of the bacterial community on the surface or at the inner surface of hay stems by immunohistochemical analyses using anti-EGF antibody. The finding that four of the five CBPs isolated and identified from sheep rumen contents were from F. succinogenes indicates that F. succinogenes is significantly involved in cellulose degradation in the rumen.

Cloning, Sequencing, and Expression of a Eubacterium cellulosolvens 5 Gene Encoding an Endoglucanase (Cel5A) with Novel Carbohydrate-Binding Modules, and Properties of Cel5A

ABSTRACT A novel Eubacterium cellulosolvens 5 gene encoding an endoglucanase (Cel5A) was cloned and expressed in Escherichia coli, and its enzymatic properties were characterized. The cel5A gene consists of a 3,444-bp open reading frame and encodes a 1,148-amino-acid protein with a molecular mass of 127,047 Da. Cel5A is a modular enzyme consisting of an N-terminal signal peptide, two glycosyl hydrolase family 5 catalytic modules, two novel carbohydrate-binding modules (CBMs), two linker sequences, and a C-terminal sequence with an unknown function. The amino acid sequences of the two catalytic modules and the two CBMs are 94% and 73% identical to each other, respectively. Two regions that consisted of one CBM and one catalytic module were tandemly connected via a linker sequence. The CBMs did not exhibit significant sequence similarity with any other CBMs. Analyses of the hydrolytic activity of the recombinant Cel5A (rCel5A) comprising the CBMs and the catalytic modules showed that the enzyme is an endoglucanase with activities with carboxymethyl cellulose, lichenan, acid-swollen cellulose, and oat spelt xylan. To investigate the functions of the CBMs and the catalytic modules, truncated derivatives of rCel5A were constructed and characterized. There were no differences in the hydrolytic activities with various polysaccharides or in the hydrolytic products obtained from cellooligosaccharides between the two catalytic modules. Both CBMs had the same substrate affinity with intact rCel5A. Removal of the CBMs from rCel5A reduced the catalytic activities with various polysaccharides remarkably. These observations show that CBMs play an important role in the catalytic function of the enzyme.

Construction of a Fibrobacter succinogenes genomic map and demonstration of diversity at the genomic level.

Abstract. The genomic cleavage map of the type strain Fibrobacter succinogenes S85 was constructed. The restriction enzymes AscI, AvrII, FseI, NotI, and SfiI generated DNA fragments of suitable size distribution that could be resolved by pulsed-field gel electrophoresis (PFGE). An average genome size of 3.6 Mb was obtained by summing the total fragment sizes. The linkages between the 15 AscI fragments of the genome were determined by combining two approaches: isolation of linking clones and cross-hybridization of restriction fragments. The genome of F. succinogenes was found to be represented by the single circular DNA molecule. Southern hybridization with specific probes allowed the eight genetic markers to be located on the restriction map. The genome of this bacterium contains at least three rRNA operons. PFGE of the other three strains of F. succinogenes gave estimated genome sizes close to that of the type strain. However, RFLP patterns of these strains generated by AscI digestion are completely different. Pairwise comparison of the genomic fragment distribution between the type strain and the three isolates showed a similarity level in the region of 14.3% to 31.3%. No fragment common to all of these F. succinogenes strains could be detected by PFGE. A marked degree of genomic heterogeneity among members of this species makes genomic RFLP a highly discriminatory and useful molecular typing tool for population studies.

Investigations into the location of Treponema hyodysenteriae in the cecum of experimentally infected young broiler chicks by light- and electronmicroscopy

Numerous Treponema hyodysenteriae were present both on the mucosal surface and in the deep crypts of the cecum of young broiler chicks 7 and 14 days after inoculation with the treponemes. The treponemes in the ceca of chicks inoculated with 10(8) cells were observed more frequently than those of chicks inoculated with 10(7) cells. The treponemes in the ceca were observed by light microscopy, scanning electron microscopy and transmission electron microscopy. The lesions were primarily confined to the cecum. Desquamation of epithelial cells, edema, leukocytic infiltration and hemorrhage were observed in the mucosae.

Influence of repeated ochratoxin A ingestion on milk production and its carry-over into the milk, blood and tissues of lactating cows

An experiment was conducted to investigate the influence of repeated ingestion of ochratoxin A (OTA) on milk production of lactating Holstein cows over 28 days, and the carry-over of OTA from the diets into the milk and tissues of the cows. Nine cows were divided into three groups, labeled OTA5, OTA50 and OTA100, and fed a diet containing 5, 50 and 100 µg OTA/kg of dry matter, respectively. Body weight, feed intake and daily milk yield in cows were not different among the three groups during the OTA-intake period. OTA residues were neither detected in the tissues, such as liver, kidney, muscles, fat and jejunoileum, nor in the milk of any cows in the OTA intake groups. In contrast, a small amount of OTA (0.1 µg/kg) was detected in the blood plasma of one sample in the OTA50 group and multiple samples in the OTA100 group. The results of this study show that the ingestion of diets containing up to 100 µg/kg of OTA over 28 days does not affect feed intake or milk production of cows, and the dietary OTA is not carried over into milk and edible tissues such as the liver, muscles and fat.

Biochemical Characterization of Cellulose-Binding Proteins (CBPA and CBPB) from the Rumen Cellulolytic Bacterium Eubacterium cellulosolvens 5

The cellulose-binding proteins, CBPA and CBPB, of rumen cellulolytic bacterium Eubacterium cellulosolvens 5 were biochemically characterized, and their properties were compared. Recombinant CBPA and CBPB were a typical 1,4-β-endoglucanase. Both proteins bound to insoluble polysaccharides such as Avicel cellulose, acid swollen cellulose, lichenan, chitin, and oat spelt xylan. On the other hand, only recombinant CBPB bound to agarose and starch.

Effect of the Administration of Dietary Microbes to Pigs on Intestinal Microbial Population

微生物資材(ラクトヒロックス)を添加した飼料および無添加飼料を給与した子豚間での糞便中の発酵産物および微生物構成を比較した.60日齢時では,微生物資材投与区の豚糞便中総VFA濃度は対照区のブタに比べて有意に高く,VFA組成比でも両区のブタ間で有意な差が認められた.しかし,90日齢時には,総VFA濃度とVFA組成比は両区のブタ間で有意な差は認められなかった.腸内微生物構成については,60日齢時では微生物資材投与区のブタでveillonellaeの菌密度が対照区のブタに比べて有意に低かった.90日齢時には微生物資材投与区のブタでstreptococciの菌密度が対照区のブタに比べて有意に高かった.また,90日齢時における微生物資材投与区のブタでの最優勢菌群はBacteroides属菌とLactobacillus属菌であったが,対照区のブタでの最優勢菌群はBacteroides属菌とFusobacterium属菌であった.最優勢菌群として分離されたLactobacillus属菌の菌種構成では,微生物資材投与区のブタではL. gasseriが多数を占め,対照区のブタではL. acidophillusが多数を占めていた.