Organization of a Clostridium thermocellum gene cluster encoding the cellulosomal scaffolding protein CipA and a protein possibly involved in attachment of the cellulosome to the cell surface

Abstract

The nucleotide sequence was determined for a 9.4-kb region of Clostridium thermocellum DNA extending from the 3' end of the gene (now termed cipA), encoding the S1/SL component of the cellulosome. Three open reading frames (ORFs) belonging to two operons were detected. They encoded polypeptides of 1,664, 688, and 447 residues, termed ORF1p, ORF2p, and ORF3p, respectively. The COOH-terminal regions of the three polypeptides were highly similar and contained three reiterated segments of 60 to 70 residues each. Similar segments have been found at the NH2 terminus of the S-layer proteins of Bacillus brevis and Acetogenium kivui, suggesting that ORF1p, ORF2p, and ORF3p might also be located on the cell surface. Otherwise, the sequence of ORF1p and ORF2p gave little clue concerning their potential function. However, the NH2-terminal region of ORF3p was similar to the reiterated domains previously identified in CipA as receptors involved in binding the duplicated segment of 22 amino acids present in catalytic subunits of the cellulosome. Indeed, it was found previously that ORF3p binds 125I-labeled endoglucanase CelD containing the duplicated segment (T. Fujino, P. Béguin, and J.-P. Aubert, FEMS Microbiol. Lett. 94:165-170, 1992). These findings suggest that ORF3p might serve as an anchoring factor for the cellulosome on the cell surface by binding the duplicated segment that is present at the COOH end of CipA.

Cellulase, Clostridia, and Ethanol

“…Protein OlpA has been localized to the cell surface of C. thermocellum (286). It was hypothesized that this region may anchor the cellulosome to the cell surface (87). However, there was no binding found between the dockerin of CipA and the receptor domain of OlpA (287).…”

“…These four genes form two operons (87). All three ORFs encode C-terminal domains (three repeats of about 65 amino acid residues) responsible for the cell surface layer (S-layer) location of the encoded proteins.…”

The structure of secondary cell wall polymers: how Gram-positive bacteria stick their cell walls together

“…These studies suggest that, in general, S-layer proteins have two functional regions: a cell-wall-targeting domain, which in most of the organisms investigated thus far is located at the N-terminus, and a C-terminal self-assembly domain. The existence of a cell-wall-targeting domain in S-layer proteins was substantiated by Fujino et al (1993) and Lupas et al (1994), who identified motifs of approximately 55 amino acids, containing 10-15 conserved residues, which were designated SLH (S-layer homology) domains. SLH domains, usually composed of one to three modules, are frequently used means for targeting proteins to the cell surface.…”

Purification and characterization of endoglucanase C from Clostridium cellulolyticum

“…These particular sequences are thought to be characteristic of enzymes integrated into the cellulosome structure [4], and yet they are observed with cellulolytic systems of Clostridium thermocellum [4, 51 and Clostridium cellulovorans [6]. These organisms secrete highmolecular-mass particles named cellulosomes which are active against cellulose, and they possess an assembly factor, CbpA in C. cellulovorans [7] and CipA in C. thermocellum [8, 91. Four genes, celCCA, celCCC, coding for enzymes of the cellulolytic system of C. cellulolyticum have been completely sequenced and the four corresponding proteins are members of three different fami-…”