Figure 1.
Structural organization of the proteins used in this study.
A. Structural organization of CipC and HycP from Clostridium cellulolyticum. Numbers above the protein correspond to the starting and final amino acids of each module in the full length molecule. The numbers in HycP indicate the order of the HYR modules starting from the N-terminus. HYR modules were identified using PFAM, SMART and by manual search (see alignments in data S1). B. Modular organization of recombinant proteins produced in the present study. The numbers of the HYR modules in recombinant proteins correspond to the same HYR modules in the wild-type protein.
Figure 2.
Amino-acid sequence alignment of CBM3a and CBM3b from various CBM3a or CBM3b containing proteins.
Alignment has been performed using ClustalW2. It is focused on residues considered to participate in planar interaction with cellulose, highlighted in grey box. Regions of secondary structure are marked with an arrow and labeled as in the structure of the CBM3a of the scaffolding protein CipA from Clostridium thermocellum (Tormo 1996). CBM amino-acid sequence aligned (accession numbers codes in parentheses) are: Cip_Ccel(YP_002505087) and HycP_Ccel (YP_002505824) from Clostridium cellulolyticum; HycP_CspBNL (YP_005147316) from Clostridium sp. BNL1100; Cip_Cpap (ZP_08194681) from Clostridium papyrosolvens; Cip_Cace (NP_347546) from Clostridium acetobutylicum; Cip_Ccvr (ZP_07630535) from Clostridium cellulovorans; Cip_Cjo (BAA32429) from Clostridium josui; Cip_Cthe (ZP_14248391) and CelI_Cthe (AAA20892) from Clostridium thermocellum; CipV_Acece (AAF06064) from Acetovibrio cellulolyticus; CelY (YP_007373484) and CelZ (CAA39010) from Clostridium stercorarium; Scaf_Bacel (AAG01230) from Bacteroides cellulosolvens.
Figure 3.
Interactions of rCBM3a and rCBM3b with straw and crystalline cellulose.
Recombinant proteins were mixed with substrates during one hour. After centrifugation the bound proteins found in the pellet (P), and the unbound proteins present in the supernatant (S), were analyzed by SDS-PAGE.
Table 1.
Dissociation constants of rCBM3a and rCBM3b to cellulosic substrates.
Figure 4.
Detection of HycP CipC and Cel48F in different Clostridium cellulolyticum strains.
Different C. cellulolyticum strains were studied: wild-type strain, MTLhycP, MTLcipC, MTLcipC(pSOS955cipC) and MTLcipC(pSOSzero-Tc) strains. Aliquot was taken from a culture of these strains at the exponential growth phase on cellobiose substrate, and centrifuged to separate the cells and the supernatant. Cell fraction and 10% TCA precipitated supernatant fraction corresponding to the same culture volume were subjected to SDS-PAGE. After transfer onto nitrocellulose membranes, membranes were probed with antibodies directed against HYR modules from HycP (Panel A), or CipC (Panel B), or Cel48F (Panel C).
Figure 5.
Cell adherence to cellulosic substrates.
Cells cultured on cellobiose were incubated one hour in anaerobic conditions with a strip of insoluble substrate. Binding percentage is calculated from the level of unbound cells measured in the supernatant by spectrophotometry (optical density at 450 nm) compared to the optical density value of an assay where no insoluble substrate was added. (A) Clostridium cellulolyticum is incubated on filter paper (FP) or on nitrocellulose (NC) strips with or without BSA saturation and compared to the binding level of Clostridium perfringens to BSA saturated filter paper. (B) Cellulose binding capacity of C. cellulolyticum wild-type strain, MTLhycP, MTLcipC, MTLcipC(pSOS955cipC), MTLcipC(pSOSzero-Tc), and cipCmut1 mutant strains. Experiments were performed in triplicates, on at least three independent experiments and two isolated clones for MTLhycP, MTLcipC, MTLcipC(pSOS955cipC) and MTLcipC(pSOSzero-Tc).
Figure 6.
Growth of Clostridium cellulolyticum wild-type and MTLhycP strains on cellulose.
Both strains were grown on rich medium containing 5 g.L−1 Sigmacell. Growth was monitored by measuring total protein content. Experiment was performed in duplicates. Growth performed in other condition as minimal medium containing Sigmacell or Avicel did not show any differences between both strains.
Figure 7.
SEM observation of Clostridium cellulolyticum and Clostridium thermocellum grown on filter paper.
Clostridium thermocellum (A) and Clostridium cellulolyticum (B) were grown on filter paper. Pictures are representative of two independent experiments. Bar represents 500 nm.
Table 2.
Summary of the compared properties of CipC and HycP.
Table 3.
Bacterial strains and vectors used in this study.