Regulation of the Cellulosomal celS (cel48A) Gene of Clostridium thermocellum Is Growth Rate Dependent

AUTOR(ES)

Dror, Tali W.

FONTE

American Society for Microbiology

RESUMO

Clostridium thermocellum produces an extracellular multienzyme complex, termed cellulosome, that allows efficient solubilization of crystalline cellulose. One of the major enzymes in this complex is the CelS (Cel48A) exoglucanase. The regulation of CelS at the protein and transcriptional levels was studied using batch and continuous cultures. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analyses indicated that the amount of CelS in the supernatant fluids of cellobiose-grown cultures is lower than that of cellulose-grown cultures. The transcriptional level of celS mRNA was determined quantitatively by RNase protection assays with batch and continuous cultures under carbon and nitrogen limitation. The amount of celS mRNA transcripts per cell was about 180 for cells grown under carbon limitation at growth rates of 0.04 to 0.21 h−1 and 80 and 30 transcripts per cell for batch cultures at growth rates of 0.23 and 0.35 h−1, respectively. Under nitrogen limitation, the corresponding levels were 110, 40, and 30 transcripts/cell for growth rates of 0.07, 0.11, and 0.14 h−1, respectively. Two major transcriptional start sites were detected at positions −140 and −145 bp, upstream of the translational start site of the celS gene. The potential promoters exhibited homology to known sigma factors (i.e., σA and σB) of Bacillus subtilis. The relative activity of the two promoters remained constant under the conditions studied and was in agreement with the results of the RNase protection assay, in which the observed transcriptional activity was inversely proportional to the growth rate.

Documentos Relacionados

• Interactions of the CelS binding ligand with various receptor domains of the Clostridium thermocellum cellulosomal scaffolding protein, CipA.
• Exoglucanase activities of the recombinant Clostridium thermocellum CelS, a major cellulosome component.
• Cloning and DNA sequence of the gene coding for Clostridium thermocellum cellulase Ss (CelS), a major cellulosome component.
• Multidomain Structure and Cellulosomal Localization of the Clostridium thermocellum Cellobiohydrolase CbhA
• Nucleotide sequence of the celG gene of Clostridium thermocellum and characterization of its product, endoglucanase CelG.