Sequence analysis of scaffolding protein CipC and ORFXp, a new cohesin-containing protein in Clostridium cellulolyticum: Comparison of various cohesin domains and subcellular localization of ORFXp

The gene encoding the scaffolding protein of the cellulosome from Clostridi um cellulolyticum, whose partial sequence was published earlier (S. Pages, A, Belaich, C. Tardif, C. Reverbel-Leroy, C, Gaudin, and J.-P. Belaich, J, Bacteriol. 178:2279-2286, 1996; C, Reverbel-Leroy, A. Belaich, A. Bernadac, C, Gaudin, J, P, Belaich, and C, Tardif, Microbiology 142:1013-1023, 1996) , was completely sequenced. The corresponding protein, CipC, is composed of a cellulose binding domain at the N terminus followed by one hydrophilic d omain (HDL), seven highly homologous cohesin domains (cohesin domains 1 to 7), a second hydrophilic domain, and a final cohesin domain (cohesin domain 8) which is only 57 to 60% identical to the seven other cohesin domains. I n addition, a second gene located 8.89 kb downstream of cipC was found to e ncode a three-domain protein, called ORFXp, which includes a cohesin domain . By using antiserum raised against the latter, it was observed that ORFXp is associated with the membrane of C. cellulolyticum and is not detected in the cellulosome fraction. Western blot and BIAcore experiments indicate th at cohesin domains 1 and 8 from CipC recognize the same dockerins and have similar affinity for CelA (K-a = 4.8 x 10(9) M-1) whereas the cohesin from ORFXp, although it is also able to bind all cellulosome components containi ng a dockerin, has a 19-fold lower K-a for CelA (2.6 x 10(8) M-1). Taken to gether, these data suggest that ORFXp mag play a role in cellulosome assemb ly.