Synthesis of Clostridium cellulovorans minicellulosomes by intercellular complementation

Abstract

The ability of two strains of bacteria to cooperate in the synthesis of an enzyme complex (a minicellulosome) was examined. Three strains of Bacillus subtilis were constructed to express Clostridium cellulovorans genes engB, xynB, and minicbpA. MiniCbpA, EngB, and XynB were synthesized and secreted into the medium by B. subtilis. When the strains with the minicbpA and engB genes or with xynB were cocultured, minicellulosomes were synthesized, consisting in one case of miniCbpA and EngB and in the second case of miniCbpA and XynB. Both minicellulosomes showed their respective enzymatic activities. We call this phenomenon "intercellular complementation." Interesting implications concerning bacterial cooperation are suggested from these results.

Fig. 1.

Molecular architecture of EngB, XynB, miniCbpA, and their derivatives. CBM, carbohydrate-binding module; Cat., catalytic domain.

Fig. 2.

Production of EngB, Xyn B, miniCbpA, and their derivatives by B. subtilis. (A) Purified proteins analyzed by SDS/PAGE and stained with Coomassie blue. Each lane contained 10 μg of protein. The numbers on the left are in kilodaltons. Lane 1, molecular weight markers; lane 2, EngB-His; lane 3, XynB-His; lane 4, ST-miniCbpA-His. (B) Secreted proteins in growth medium of B. subtilis analyzed by Western blots. Lane 1, EngB; lane 2, EngBΔdoc; lane 3, ST-XynB; lane 4, ST- XynBΔdoc; and lane 5, ST-miniCbpA-His.

Fig. 3.

Growth curves and Western immunoblots of culture supernatants of B. subtilis grown at 30°C. Growth curves of cultures expressing EngB-His (A), XynB-His (B), and ST-miniCbpA-His (C). Western blot analyses of proteins from culture supernatants containing EngB-His with anti-EngB (D), XynB-His with anti-XynB (E), and ST-miniCbpA-His with anti-CbpA (E) at various times during growth.

Fig. 4.

Analysis of minicellulosomes from cocultures of B. subtilis expressing ST-mini-CbpA and EngB. Lane M, molecular mass marker. Lane 1, SDS/PAGE of Ni-NTA-bound minicellulosomes containing EngB and ST-miniCbpA-His from a coculture of B. subtilis strains containing pWB980-ST-minicbpA-his and pWB980-engB. Lane 2, Western blot analysis of minicellulosome with anti-CbpA; the strains are described in the legend of lane 1. Lane 3, Western blot analysis of minicellulosome with anti-EngB; the strains are described in the legend of lane 1. Lane 4, Zymogram analysis for endoglucanase activity of lane 3; the strains are described in the legend of lane 1.

Fig. 5.

Analysis of cocultures ST-miniCbpA-His and ST-XynB and of ST-miniCbpA-His and ST-XynBΔDoc. (A) Western blot of minicellulosomes probed with antibodies raised against CbpA (Upper) and ST-XynB (Lower), respectively. Lane 1, supernatant of ST-XynB and ST-miniCbpA-His cultures. Lane 2, purified minicellulosomes with ST-XynB and ST-miniCbpA-His. Lane 3, supernatant of ST-XynBΔDoc and ST-miniCbpA-His cultures. Lane 4, ST-miniCbpA-His bound to Ni-NTA column; note no ST-XynBΔDoc was bound to ST-miniCbpA-His in lower frame. (B) (Upper) M, molecular mass marker. Lane 1 (Upper), SDS/PAGE of purified minicellulosome from cocultures containing ST-miniCbpA-His and ST-XynB (both of these proteins have the same molecular weight, so there is one band); zymogram (Lower) shows the presence of xylanase activity in this band. Lane 2, SDS/PAGE of ST-miniCbpA-His bound to Ni-NTA column (Upper). No ST-XynBΔDoc was bound to ST-miniCbpA-His, and therefore no xylanase zymogram was obtained (Lower).