Genome Sequence of the Cellulosome-Producing Mesophilic Organism Clostridium cellulovorans 743B

Clostridium cellulovorans 743B was isolated from a wood chip pile and is an anaerobic and mesophilic spore-forming bacterium. This organism degrades native substrates in soft biomass such as corn fiber and rice straw efficiently by producing an extracellular enzyme complex called the cellulosome. Here we report the genome sequence of C. cellulovorans 743B.The biotechnological potential of polysaccharolytic enzymes has resulted in the isolation and characterization of a large number of anaerobic, Gram-positive, spore-forming bacteria, the majority of which have been allocated to the genus Clostridium. Among clostridia, the cellulosomes produced by Clostridium species are particularly designed for efficient degradation of plant cell wall polysaccharides. The component parts of the multicomponent complex are integrated by virtue of a unique family of integrating modules, the cohesins and the dockerins, whose distribution and specificity dictate the overall cellulosome architecture (3). The cellulosome system in Clostridium cellulovorans 743B (ATCC 35296) has been studied extensively for the last 20 years and has resulted in providing basic information about mesophilic cellulosomes. This organism was isolated from a wood chip pile and is an anaerobic spore-forming bacterium whose optimal growth temperature is 37°C (9). It has the ability to utilize cellulose, xylan, pectin, cellobiose, glucose, fructose, galactose, and mannose as carbon sources for growth. Its fermentation products include H₂, CO₂, acetate, butyrate, formate, lactate, and ethanol. When it is grown in the presence of cellulose, electron micrographs have shown that large protuberances are present on its cell surface (4), while small or no protuberances are evident when cells are grown in the presence of glucose or cellobiose (5).We sequenced a total length of 101,749,598 bp and analyzed 381,514 reads by Genome Sequencer FLX 454./Roche sequencing (8) (GS-FLX version) to highly oversample the genome (20× coverage) and generated 123,892 paired-end sequence tags, to enable the assembly of all tags using the GS De Novo Assembler version 1.1.03.24 (Roche Diagnostics) and the Genome Analyzer II and sequencing kit 36-Cycle Run (Illumina). Finally, we assembled 30 scaffolds (sets of 601 ordered and oriented contigs; total length of 5,123,527 bp) to generate approximately 5.1 Mbp of nearly contiguous Clostridium botulinum E3 strain Alaska E43 (accession no. {"type":"entrez-nucleotide","attrs":{"text":"NC_010723","term_id":"188587536","term_text":"NC_010723"}}NC_010723) complete genome sequence. We analyzed a number of predicted genes included in the C. cellulovorans genome using CRITICA (version 1.05b) (2) and Glimmer 2 (version 2.10) (6) to find regions in proteins with known functions. We annotated and classified according to Gene Ontology (GO) (1). In silico Molecular Cloning Genomic Edition ver. 3.0.26 software (In Silico Biology, Co., Ltd., Japan) was used for individual genomic analysis.The C. cellulovorans 743B (ATCC 35296) genome consists of 5,123,527 bp. A total of 4,220 polypeptide-encoding open reading frames (ORFs) were identified using CRITICA, while 4,297 ORFs were identified using Glimmer 2. The number of ORFs identical between CRITICA and Glimmer 2 was 2,773. Sixty-three tRNAs and 33 anticodons were also identified using tRNAscan-SE (7). In comparison of the genome sizes among cellulosomal clostridia such as Clostridium cellulolyticum H10 (4.07 Mbp) (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"CP001348","term_id":"219997787","term_text":"CP001348"}}CP001348) and Clostridium thermocellum ATCC 27405 (3.84 Mbp) (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"CP000568","term_id":"125712750","term_text":"CP000568"}}CP000568), the C. cellulovorans genome was over 1 Mbp larger than the other genomes. Moreover, the number of predicted genes (4,220 by CRITICA) in the C. cellulovorans genome was the largest among them. On the other hand, the G+C content in C. cellulovorans was 31.1%, similar to that (30.9%) in Clostridium acetobutylicum ATCC 824 (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AE001437","term_id":"25168256","term_text":"AE001437"}}AE001437), while the G+C contents in C. cellulolyticum and C. thermocellum were 37.7% and 39.0%, respectively.A protein BLAST search against the database of clusters of orthologous groups (COGs) of proteins indicated that 4,171 genes were annotated by 4,220 predicted coding sequences using CRITICA while 4,098 genes were identified by 4,297 predicted coding sequences using Glimmer 2. On the other hand, a protein BLAST search against the NCBI nr database indicated that 4,184 genes were annotated by 4,220 predicted coding sequences using CRITICA while 4,071 genes were identified by 4,297 predicted coding sequences using Glimmer 2. Interestingly, 57 cellulosomal genes were found in the C. cellulovorans genome and coded for not only carbohydrate-active enzymes but also lipases, peptidases, and proteinase inhibitors. Moreover, two novel genes encoding a scaffolding protein were found in the genome. Thus, by examining genome sequences from multiple Clostridium species, comparative genomics offers new insight into genome evolution and the way in which natural selection molds functional DNA sequence evolution. Our analysis, coupled with the genome sequence data, will provide a road map for constructing enhanced cellulosome-producing Clostridium strains for industrial applications such as biofuel production.     

10-Year risk of colorectal cancer: Development and validation of a prediction model in middle-aged Japanese men

Background: To estimate an individual's probability of developing colorectal cancer (CRC) may aid health professionals and individuals in improving lifestyle behaviors or deciding the screening regimens. As fewer studies on cancer risk prediction were seen so far, we initially developed an assessment tool with synthesizing key information from a variety of CRC risk factors through a large population-based cohort study. Method: The prediction model was derived from 28,115 men in the Japan Public Health Center-based (JPHC) Prospective Study Cohort II (follow-up: 1993–2005), with risk factors selected by Cox proportion hazard regression. 18,256 men in the JPHC Study Cohort I (follow-up: 1995–2005) were used to evaluate the model's performance. Results: 543 and 398 CRCs were diagnosed during the follow-up period in Cohorts II and I, respectively. The prediction model, including age, BMI, alcohol consumption, smoking status, and the daily physical activity level, showed modest discrimination ability for CRC (C = 0.70; 95% confidential interval, 0.68–0.72) in Cohort II and well calibrated in Cohort I (Hosmer–Lemeshow χ² = 14.2, P = 0.08). Conclusion: The 10-year CRC risk prediction model may be used to estimate CRC risk in Japanese men. It may also play a role in the promotion of CRC prevention strategies.     

Periodically fluctuating protein kinases phosphorylate CLOCK, the putative target in the suprachiasmatic nucleus

We studied nuclear protein phosphorylation in the rat suprachiasmatic nucleus (SCN) and found that a nuclear fraction of the SCN contained histone H1 kinase activity that periodically fluctuated with a diurnal rhythm, reaching a maximum at the midpoint of the light phase and a minimum at the midpoint of the dark phase. A p13suc1-bound fraction from the SCN nuclear fraction also exhibited diurnally fluctuating histone H1 kinase activity. Using in situ kinase assay, three histone H1 kinases, p45PFK, p100PFK, and p200PFK (termed periodically fluctuating protein kinases, or PFKs) were found in the p13suc1-bound fractions. p45PFK exhibited the highest level of light/dark cycle phosphorylation activity fluctuation. p45PFK highly phosphorylated the Ser-Pro-rich region of CLOCK, the putative physiological target. These results suggest that PFKs, especially p45PFK, are involved in circadian clock-related signal transduction and gene expression, through the phosphorylation of target proteins such as CLOCK.     

Carboxyl-terminal fragments of presenilin-1 are closely related to cytoskeletal abnormalities in Alzheimer's brains

To clarify the role of presenilin-1 (PS-1) in the pathology of Alzheimer's disease (AD), we tested four antisera to PS-1. The specific antisera to the N-terminus (HSN-2) and C-terminus (HS-C) of PS-1 detected a 44/40kD holoprotein, a 25kD N-terminal fragment (NTF) and a 16kD C-terminal fragment (CTF) of PS-1 in COS-7 cells. The 25kD NTF and 16kD CTF were observed in human brains, and their amounts were not significantly different between the control and AD brains. The antibody HS-C labeled extensive neurofibrillary tangles, dystrophic neurites and curly fibers in the AD brains. In the paired helical filament (PHF) fraction containing A68 protein from AD brains, a smear pattern of CTFs was revealed. Antisera (HS-L292 and HS-L300) to cleavage sites of PS-1 also revealed immunoreactive neurofibrillary tangles in the AD brain sections and the smear pattern of CTFs of A68 protein fraction. The CTFs of PS-1 accumulate with PHF tau, suggesting a close relationship between PS-1 and cytoskeletal abnormalities in AD brains.     

Structural basis for the product specificity of histone lysine methyltransferases

DIM-5 is a SUV39-type histone H3 Lys9 methyltransferase that is essential for DNA methylation in N. crassa. We report the structure of a ternary complex including DIM-5, S-adenosyl-L-homocysteine, and a substrate H3 peptide. The histone tail inserts as a parallel strand between two DIM-5 strands, completing a hybrid sheet. Three post-SET cysteines coordinate a zinc atom together with Cys242 from the SET signature motif (NHXCXPN) near the active site. Consequently, a narrow channel is formed to accommodate the target Lys9 side chain. The sulfur atom of S-adenosyl-L-homocysteine, where the transferable methyl group is to be attached in S-adenosyl-L-methionine, lies at the opposite end of the channel, approximately 4 A away from the target Lys9 nitrogen. Structural comparison of the active sites of DIM-5, an H3 Lys9 trimethyltransferase, and SET7/9, an H3 Lys4 monomethyltransferase, allowed us to design substitutions in both enzymes that profoundly alter their product specificities without affecting their catalytic activities.     

Anaplastic large cell lymphoma of the mediastinum diagnosed by transbronchial scratch cytology. A case report

BACKGROUND: Anaplastic large cell lymphoma (ALCL) is a subtype of non-Hodgkin's lymphoma characterized by CD30 antigen-positive, large neoplastic cells. We describe a case of ALCL suggested by cytologic examination of the tumor cells obtained from bronchial scratch preparations. CASE: A 26-year-old woman had had a dry cough since November 1996. Chest radiography in May 1997 revealed an abnormal shadow in the mediastinum extending to the pulmonary hilar region. The patient was hospitalized in June 1997. Computed tomography revealed a neoplastic lesion in the anterior mediastinum invading the right lung. Transbronchial scratch cytology revealed large, atypical lymphoid cells expressing CD30 and CD3 on immunocytochemical examination. A transcutaneous mediastinal biopsy was performed and a diagnosis of ALCL made. CONCLUSION: Differentiation from Hodgkin's disease was the most difficult point in this case. Detailed cytologic observation and CD3-positive immunocytology led to the correct diagnosis. The cell transfer technique of Sherman et al was very useful for immunocytologic staining. Thus, transbronchial scratch cytology was an especially valuable and effective procedure in this case.     

A cysteine protease inhibitor prevents suspension-induced declines in bone weight and strength in rats

In this study, we examined the effects of a potent cysteine protease inhibitor, N-(L-3-trans-carboxyoxirane-2-cabonyl)-L-leucine-4-aminobutylamide (E-64a), on bone weight and strength in tail-suspended rats. We first administered a vehicle or 4 or 8 mg/rat of E-64a to rats fed with a low calcium diet for 7 wks to determine effective doses of E-64a on bone resorption in vivo. Femoral cathepsin K-like activity and serum hydroxyproline level in rats fed with a low calcium diet were significantly higher than those in rats fed with a standard diet. The intraperitoneal injection of 8 mg/rat of E-64a to rats decreased their serum calcium and hydroxyproline concentrations after 3 to 6 hrs in parallel with changes in femoral cathepsin K-like activity, while 4 mg/rat of E-64a had weaker effects on these parameters. Based on these results, we injected 8 mg/rat of E-64a to tail-suspended rats twice a day for 2 wks and compared the results with those of treatment with 1 mg/rat of etidronate, a bisphosphonate, twice a week. In tail-suspended rats, femoral weight and strength, assessed by three-point bending test, significantly decreased from Day 5 to 21, while femoral cathepsin K-like activity and serum calcium and hydroxyproline concentrations did not change. E-64a inhibited femoral cathepsin K-like activity in tail-suspended rats, but etidronate did not. E-64a as well as etidronate significantly prevented the suspension-induced declines in bone weight and strength. However, more frequent injection and higher doses were required for E-64a to exhibit significant efficacy of antiresorption, compared with those of etidronate. Our results suggest that a cysteine protease inhibitor could improve suspension-induced osteopenia by inhibiting cathepsin K-like activity in bone; however, it needs several improvements in the effect as a clinical drug.     

Formation of Protoplasts from Cultured Tobacco Cells and Arabidopsis thaliana by the Action of Cellulosomes and Pectate Lyase from Clostridium cellulovorans

The crude culture supernatants from Clostridium cellulovorans were tested for their ability to convert plant cells to protoplasts. The supernatants readily released protoplasts from cultured tobacco cells and Arabidopsis thaliana. The crude culture supernatant from pectin-grown cells was more active than supernatants from glucose-, cellobiose-, xylan-, and locust bean gum-grown cells. After removal of cellulosomes, the crude culture supernatant lost its protoplast formation activity. The protoplast formation activity of the crude culture supernatant from C. cellulovorans was more effective than those of commercial enzymes based on protein content.     

Analysis of shoot-forming capacity in vitro in two lines of tomato (Lycopersicon esculentum Mill.) and their hybrids

Shoot differentiation in vitro of tomato plants and transmissionof this capacity into hybrids was studied. Most of the resultswere obtained with hypocotyls. The highest level of shoot formationoccurred when IAA and IPA were given, but the optimum concentrationof these growth regulators varied depending on lines and hybrids,on organs treated (hypocotyl and leaf), and on the positionof explants in these organs. Histological study of hypocotyltissues indicated that shoots were differentiated from the callusoriginating from superficial tissues. The organogenetic capacityof hypocotyls (in terms of the number of shoots per explant)of two lines and their reciprocal F₁ hybrids was modified byexogenous growth regulators. However, one parental line consistentlyshowed a higher organogenetic capacity than the other. Withregards to shoot-forming capacity, the reciprocal hybrids showed:

1. maternal effect and
2. heterosis

¹ This work was done in Laboratoire de Physiologie Pluricellulaire,C.N.R.S., Gif-sur-Yvette, France, with courtesy of Dr. H. Haradaand Dr. J. P. Pernes. (Received July 25, 1977; )