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Transcription of Clostridium cellulovorans cellulosomal cellulase and hemicellulase genes.

ABSTRACT: Transcription of the cellulosomal cellulase/hemicellulase genes of Clostridium cellulovorans has been investigated by Northern blot, reverse transcriptase PCR (RT-PCR), primer extension, and S1 nuclease analysis. Northern hybridizations revealed that the cellulosomal cbpA gene cluster is transcribed as polycistronic mRNAs of 8 and 12 kb. The 8-kb mRNA coded for cbpA and exgS, and the 12-kb mRNA coded for cbpA, exgS, engH, and engK. The sizes of the mRNAs were about 3 kb for engE, 1.8 kb for manA, 2.7 kb for xynA, and 4 kb for pelA, indicating monocistronic transcription of these genes. Primer extension and S1 nuclease analysis of C. cellulovorans RNA showed that the transcriptional start sites of cbpA, engE, manA, and hbpA were located 233, 97, 64, and 61 bp upstream from the first nucleotide of each of the respective translation initiation codons. Alignment of the cbpA, engE, manA, and hbpA promoter regions provided evidence for highly conserved sequences that exhibited strong similarity to the sigma(A) consensus promoter sequences of gram-positive bacteria.

SUBMITTER: Han SO

PROVIDER: S-EPMC152600 | biostudies-literature | 2003 Apr

REPOSITORIES: biostudies-literature

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Transcription of Clostridium cellulovorans cellulosomal cellulase and hemicellulase genes.

Han Sung Ok SO Yukawa Hideaki H Inui Masayuki M Doi Roy H RH

Journal of bacteriology 20030401 8

Transcription of the cellulosomal cellulase/hemicellulase genes of Clostridium cellulovorans has been investigated by Northern blot, reverse transcriptase PCR (RT-PCR), primer extension, and S1 nuclease analysis. Northern hybridizations revealed that the cellulosomal cbpA gene cluster is transcribed as polycistronic mRNAs of 8 and 12 kb. The 8-kb mRNA coded for cbpA and exgS, and the 12-kb mRNA coded for cbpA, exgS, engH, and engK. The sizes of the mRNAs were about 3 kb for engE, 1.8 kb for manA ...[more]

PMID: 12670976

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Project description:BackgroundGoat rumen microbial communities are perceived as one of the most potential biochemical reservoirs of multi-functional enzymes, which are applicable to enhance wide array of bioprocesses such as the hydrolysis of cellulose and hemi-cellulose into fermentable sugar for biofuel and other value-added biochemical production. Even though, the limited understanding of rumen microbial genetic diversity and the absence of effective screening culture methods have impeded the full utilization of these potential enzymes. In this study, we applied culture independent metagenomics sequencing approach to isolate, and identify microbial communities in goat rumen, meanwhile, clone and functionally characterize novel cellulase and xylanase genes in goat rumen bacterial communities.ResultsBacterial DNA samples were extracted from goat rumen fluid. Three genomic libraries were sequenced using Illumina HiSeq 2000 for paired-end 100-bp (PE100) and Illumina HiSeq 2500 for paired-end 125-bp (PE125). A total of 435gb raw reads were generated. Taxonomic analysis using Graphlan revealed that Fibrobacter, Prevotella, and Ruminococcus are the most abundant genera of bacteria in goat rumen. SPAdes assembly and prodigal annotation were performed. The contigs were also annotated using the DOE-JGI pipeline. In total, 117,502 CAZymes, comprising endoglucanases, exoglucanases, beta-glucosidases, xylosidases, and xylanases, were detected in all three samples. Two genes with predicted cellulolytic/xylanolytic activities were cloned and expressed in E. coli BL21(DE3). The endoglucanases and xylanase enzymatic activities of the recombinant proteins were confirmed using substrate plate assay and dinitrosalicylic acid (DNS) analysis. The 3D structures of endoglucanase A and endo-1,4-beta xylanase was predicted using the Swiss Model. Based on the 3D structure analysis, the two enzymes isolated from goat's rumen metagenome are unique with only 56-59% similarities to those homologous proteins in protein data bank (PDB) meanwhile, the structures of the enzymes also displayed greater stability, and higher catalytic activity.ConclusionsIn summary, this study provided the database resources of bacterial metagenomes from goat's rumen fluid, including gene sequences with annotated functions and methods for gene isolation and over-expression of cellulolytic enzymes; and a wealth of genes in the metabolic pathways affecting food and nutrition of ruminant animals.

Dataset Information

Transcription of Clostridium cellulovorans cellulosomal cellulase and hemicellulase genes.

Publications

Transcription of Clostridium cellulovorans cellulosomal cellulase and hemicellulase genes.

Similar Datasets

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