Mutations in the Scaffoldin Gene, cipA , of Clostridium thermocellum with Impaired Cellulosome Formation and Cellulose Hydrolysis: Insertions of a New Transposable Element, IS 1447 , and Implications for Cellulase Synergism on Crystalline Cellulose

Abstract

Mutants of Clostridium thermocellum that had lost the ability to adhere to microcrystalline cellulose were isolated. Six of them that showed diminished ability to depolymerize crystalline cellulose were selected. Size exclusion chromatography of the proteins from the culture supernatant revealed the loss of the supramolecular enzyme complex, the cellulosome. However, denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis resulted in extracellular protein patterns comparable to those of isolated cellulosomes, except for a missing CipA band. Sequencing of the six mutant cipA genes revealed a new insertion (IS) element, IS1447, belonging to the IS3 family. It was inserted into the cipA reading frame in four different locations: cohesin module 1, two different positions in the carbohydrate binding module, and cohesin module 3. The IS sequences were identical and consisted of a transposase gene and the inverted repeats IRR and IRS. The insertion resulted in an obviously nonspecific duplication of 3 base pairs within the target sequence. This lack of specificity allows transposition without the need of a defined target DNA sequence. Eighteen copies of IS1447 were identified in the genomic sequence of C. thermocellum ATCC 27405. At least one of them can be activated for transposition. Compared to the wild type, the mutant culture supernatant, with a completely defective CipA protein, showed equal specific hydrolytic activity against soluble ␤-glucan but a 15-fold reduction in specific activity with crystalline cellulose. These results identify a genetic basis for the synergistic effect of complex formation on crystalline-cellulose degradation.

Role of the CipA Scaffoldin Protein in Cellulose Solubilization, as Determined by Targeted Gene Deletion and Complementation in Clostridium thermocellum

“…In agreement with Zverlov et al, we have shown that cipA is essential for rapid solubilization of crystalline cellulose (13). However, contrary to what was reported previously, we observed that cipA deletion strains are able to solubilize Avicel microcrystalline cellulose (which is similar to the MN300 microcrystalline cellulose used by Zverlov et al).…”

“…Previous work has shown that the abilities to bind and to solubilize cellulose are linked (11,13). Thus, mutants deficient in cellulose binding are also deficient in cellulose solubilization.…”

“…Strain SM1 had an insertion in the first type I cohesin and appeared to be completely lacking functional type I cohesins. This strain was unable to grow on MN300 cellulose and exhibited a 15-fold reduction in enzymatic activity compared to the wild-type (WT) strain (13).…”

“…Strains SM1, SM4, SM5, and SM6, which also are deficient in cellulase activity, were isolated using a procedure similar to that used for strain AD2, though augmented by an initial chemical mutagenesis step followed by a screen on cellobiose plates with an Avicel overlay (13). This final screen was designed to identify cells that were deficient in cellulose solubilization.…”

Recombinant Cellulase and Cellulosome Systems

“…In natural and recombinant systems, these synergistic effects are further augmented by an extra level of synergy resulting from the cellulosome's association with the surface of cells, yielding cellulose-enzyme-microbe (CEM) ternary complexes [89,[112][113][114][115][116][117][118]. CEM ternary complexes benefit from the effects of microbe-enzyme synergy, ultimately limiting the escape of hydrolysis products and enzymes, increasing access to substrate hydrolysis products, minimizing the distance products must diffuse before cellular uptake occurs, concentrating enzymes at the substrate surface, protecting hydrolytic enzymes from proteases and thermal degradation, as well as optimizing the chemical environment at the substrate-microbe interface [89,[112][113][114][115][116]. In several cellulosome-producing bacteria, including C. thermocellum, the cellulosome is anchored to the surface of cells, resulting in one of the most efficient systems for bacterial cellulose hydrolysis [4,116].…”