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Ruminococcus flavefaciens produces a cellulosomal enzyme complex, based on the structural proteins ScaA, -B, and -C, that was recently shown to attach to the bacterial cell surface via the wall-anchored protein ScaE. ScaA, -B, -C, and -E are all cohesin-bearing proteins encoded by linked genes in the sca cluster. The product of an unknown open reading frame within the sca cluster, herein designated CttA, is similar in sequence at its C terminus to the corresponding region of ScaB, which contains an X module together with a dockerin sequence. The ScaB-XDoc dyad was shown previously to interact tenaciously with the cohesin of ScaE. Likewise, avid binding was confirmed between purified recombinant fragments of the CttA-XDoc dyad and the ScaE cohesin. In addition, the N-terminal regions of CttA were shown to bind to cellulose, thus suggesting that CttA is a cell wall-anchored, cellulose-binding protein. Proteomic analysis showed that the native CttA protein ( approximately 130 kDa) corresponds to one of the three most abundant polypeptides binding tightly to insoluble cellulose in cellulose-grown R. flavefaciens 17 cultures. Interestingly, this protein was also detected among cellulose-bound proteins in the related strain R. flavefaciens 007C but not in a mutant derivative, 007S, that was previously shown to have lost the ability to grow on dewaxed cotton fibers. In R. flavefaciens, the presence of CttA on the cell surface is likely to provide an important mechanism for substrate binding, perhaps compensating for the absence of an identified cellulose-binding module in the major cellulosomal scaffolding proteins of this species.

A 17-kb scaffoldin gene cluster in Ruminococcus flavefaciens strain FD-1 was compared with the homologous segment published for strain 17. Although the general design of the cluster is identical in the two strains, significant differences in the modular architecture of the scaffoldin proteins were discovered, implying strain-specific divergence in cellulosome organization.

"Roseburia inulinivorans" is an anaerobic polysaccharide-utilizing firmicute bacterium from the human colon that was identified as a producer of butyric acid during growth on glucose, starch, or inulin. R. inulinivorans A2-194 is also able to grow on the host-derived sugar fucose, following a lag period, producing propionate and propanol as additional fermentation products. A shotgun genomic microarray was constructed and used to investigate the switch in gene expression that is involved in changing from glucose to fucose utilization. This revealed a set of genes coding for fucose utilization, propanediol utilization, and the formation of propionate and propanol that are up-regulated during growth on fucose. These include homologues of genes that are implicated in polyhedral body formation in Salmonella enterica. Dehydration of the intermediate 1,2-propanediol involves an enzyme belonging to the new B12-independent glycerol dehydratase family, in contrast to S. enterica, which relies on a B12-dependent enzyme. A typical gram-positive agr-type quorum-sensing system was also up-regulated in R. inulinivorans during growth on fucose. Despite the lack of genome sequence information for this commensal bacterium, microarray analysis has provided a powerful tool for obtaining new information on its metabolic capabilities.

Sequence extension of the scaffoldin gene cluster from Ruminococcus flavefaciens revealed a new gene (scaE) that encodes a protein with an N-terminal cohesin domain and a C terminus with a typical gram-positive anchoring signal for sortase-mediated attachment to the bacterial cell wall. The recombinant cohesin of ScaE was recovered after expression in Escherichia coli and was shown to bind to the C-terminal domain of the cellulosomal structural protein ScaB, as well as to three unknown polypeptides derived from native cellulose-bound Ruminococcus flavefaciens protein extracts. The ScaB C terminus includes a cryptic dockerin domain that is unusual in its sequence, and considerably larger than conventional dockerins. The ScaB dockerin binds to ScaE, suggesting that this interaction occurs through a novel cohesin-dockerin pairing. The novel ScaB dockerin was expressed as a xylanase fusion protein, which was shown to bind tenaciously and selectively to a recombinant form of the ScaE cohesin. Thus, ScaE appears to play a role in anchoring the cellulosomal complex to the bacterial cell envelope via its interaction with ScaB. This sortase-mediated mechanism for covalent cell-wall anchoring of the cellulosome in R. flavefaciens differs from those reported thus far for any other cellulosome system.

The Butyrivibrio fibrisolvens tet(W) gene is located on the conjugative transposon TnB1230. TnB1230 encodes transfer proteins with 48 to 67% identity to some of those encoded by Tn1549. tet(W) is flanked by directly repeated sequences with significant homology to oxygen-insensitive nitroreductases. The 340 nucleotides upstream of tet(W) are strongly conserved and are required for tetracycline resistance.

A new gene, designated scaC and encoding a protein carrying a single cohesin, was identified in the cellulolytic rumen anaerobe Ruminococcus flavefaciens 17 as part of a gene cluster that also codes for the cellulosome structural components ScaA and ScaB. Phylogenetic analysis showed that the sequence of the ScaC cohesin is distinct from the sequences of other cohesins, including the sequences of R. flavefaciens ScaA and ScaB. The scaC gene product also includes at its C terminus a dockerin module that closely resembles those found in R. flavefaciens enzymes that bind to the cohesins of the primary ScaA scaffoldin. The putative cohesin domain and the C-terminal dockerin module were cloned and overexpressed in Escherichia coli as His(6)-tagged products (ScaC-Coh and ScaC-Doc, respectively). Affinity probing of protein extracts of R. flavefaciens 17 separated in one-dimensional and two-dimensional gels with recombinant cohesins from ScaC and ScaA revealed that two distinct subsets of native proteins interact with ScaC-Coh and ScaA-Coh. Furthermore, ScaC-Coh failed to interact with the recombinant dockerin module from the enzyme EndB that is recognized by ScaA cohesins. On the other hand, ScaC-Doc was shown to interact specifically with the recombinant cohesin domain from ScaA, and the ScaA-Coh probe was shown to interact with a native 29-kDa protein spot identified as ScaC by matrix-assisted laser desorption ionization-time of flight mass spectrometry. These results suggest that ScaC plays the role of an adaptor scaffoldin that is bound to ScaA via the ScaC dockerin module, which, via the distinctive ScaC cohesin, expands the range of proteins that can bind to the ScaA-based enzyme complex.

The final steps in butyrate synthesis by anaerobic bacteria can occur via butyrate kinase and phosphotransbutyrylase or via butyryl-coenzyme A (CoA):acetate CoA-transferase. Degenerate PCR and enzymatic assays were used to assess the presence of butyrate kinase among 38 anaerobic butyrate-producing bacterial isolates from human feces that represent three different clostridial clusters (IV, XIVa, and XVI). Only four strains were found to possess detectable butyrate kinase activity. These were also the only strains to give PCR products (verifiable by sequencing) with degenerate primer pairs designed within the butyrate kinase gene or between the linked butyrate kinase/phosphotransbutyrylase genes. Further analysis of the butyrate kinase/phosphotransbutyrylase genes of one isolate, L2-50, revealed similar organization to that described previously from different groups of clostridia, along with differences in flanking sequences and phylogenetic relationships. Butyryl-CoA:acetate CoA-transferase activity was detected in all 38 strains examined, suggesting that it, rather than butyrate kinase, provides the dominant route for butyrate formation in the human colonic ecosystem that contains a constantly high concentration of acetate.

The xylanase gene cluster from the rumen anaerobe Prevotella bryantii B(1)4 was found to include a gene (xynR) that encodes a multidomain regulatory protein and is downstream from the xylanase and beta-xylosidase genes xynA and xynB. Additional genes identified upstream of xynA and xynB include xynD, which encodes an integral membrane protein that has homology with Na:solute symporters; xynE, which is related to the genes encoding acylhydrolases and arylesterases; and xynF, which has homology with the genes encoding alpha-glucuronidases. XynR includes, in a single 833-amino-acid polypeptide, a putative input domain unrelated to other database sequences, a likely transmembrane domain, histidine kinase motifs, response regulator sequences, and a C-terminal AraC-type helix-turn-helix DNA binding domain. Two transcripts (3.7 and 5.8 kb) were detected with a xynA probe, and the start site of the 3.7-kb transcript encoding xynABD was mapped to a position upstream of xynD. The DNA binding domain of XynR was purified after amplification and overexpression in Escherichia coli and was found to bind to a 141-bp DNA fragment from the region immediately upstream of xynD. In vitro transcription assays demonstrated that XynR stimulates transcription of the 3.7-kb transcript. We concluded that XynR acts as a positive regulator that activates expression of xynABD in P. bryantii B(1)4. This is the first regulatory protein that demonstrates significant homology with the two-component regulatory protein superfamily and has been shown to be involved in the regulation of polysaccharidase gene expression.

The DNA sequence coding for putative cellulosomal scaffolding protein ScaA from the rumen cellulolytic anaerobe Ruminococcus flavefaciens 17 was completed. The mature protein exhibits a calculated molecular mass of 90,198 Da and comprises three cohesin domains, a C-terminal dockerin, and a unique N-terminal X domain of unknown function. A novel feature of ScaA is the absence of an identifiable cellulose-binding module. Nevertheless, native ScaA was detected among proteins that attach to cellulose and appeared as a glycosylated band migrating at around 130 kDa. The ScaA dockerin was previously shown to interact with the cohesin-containing putative surface-anchoring protein ScaB. Here, six of the seven cohesins from ScaB were overexpressed as histidine-tagged products in E. coli; despite their considerable sequence differences, each ScaB cohesin specifically recognized the native 130-kDa ScaA protein. The binding specificities of dockerins found in R. flavefaciens plant cell wall-degrading enzymes were examined next. The dockerin sequences of the enzymes EndA, EndB, XynB, and XynD are all closely related but differ from those of XynE and CesA. A recombinant ScaA cohesin bound selectively to dockerin-containing fragments of EndB, but not to those of XynE or CesA. Furthermore, dockerin-containing EndB and XynB, but not XynE or CesA, constructs bound specifically to native ScaA. XynE- and CesA-derived probes did however bind a number of alternative R. flavefaciens bands, including an approximately 110-kDa supernatant protein expressed selectively in cultures grown on xylan. Our findings indicate that in addition to the ScaA dockerin-ScaB cohesin interaction, at least two distinct dockerin-binding specificities are involved in the novel organization of plant cell wall-degrading enzymes in this species and suggest that different scaffoldins and perhaps multiple enzyme complexes may exist in R. flavefaciens.

Two tandem cellulosome-associated genes were identified in the cellulolytic rumen bacterium, Ruminococcus flavefaciens. The deduced gene products represent multimodular scaffoldin-related proteins (termed ScaA and ScaB), both of which include several copies of explicit cellulosome signature sequences. The scaB gene was completely sequenced, and its upstream neighbor scaA was partially sequenced. The sequenced portion of scaA contains repeating cohesin modules and a C-terminal dockerin domain. ScaB contains seven relatively divergent cohesin modules, two extremely long T-rich linkers, and a C-terminal domain of unknown function. Collectively, the cohesins of ScaA and ScaB are phylogenetically distinct from the previously described type I and type II cohesins, and we propose that they define a new group, which we designated here type III cohesins. Selected modules from both genes were overexpressed in Escherichia coli, and the recombinant proteins were used as probes in affinity-blotting experiments. The results strongly indicate that ScaA serves as a cellulosomal scaffoldin-like protein for several R. flavefaciens enzymes. The data are supported by the direct interaction of a recombinant ScaA cohesin with an expressed dockerin-containing enzyme construct from the same bacterium. The evidence also demonstrates that the ScaA dockerin binds to a specialized cohesin(s) on ScaB, suggesting that ScaB may act as an anchoring protein, linked either directly or indirectly to the bacterial cell surface. This study is the first direct demonstration in a cellulolytic rumen bacterium of a cellulosome system, mediated by distinctive cohesin-dockerin interactions.

Adjacent regions of a Ruminococcus flavefaciens 17 DNA fragment were found to encode xylanase and beta(1,3-1,4)-glucanase activities. Sequencing of this fragment showed that both activities are encoded by a single 2,406-bp open reading frame corresponding to the xynD gene. The predicted product has a characteristic signal sequence that is followed by an amino-terminal domain related to family G xylanases, while the carboxyterminal domain is related to beta(1,3-1,4)-glucanases from several other bacterial species. These two domains are connected by a region of unknown function that consists of 309 amino acids and includes a 30-amino-acid threonine-rich sequence. A polypeptide having a molecular weight of approximately 90,000 and exhibiting xylanase and beta(1,3-1,4)-glucanase activities was detected in Escherichia coli cells carrying the cloned xynD gene. This is one of the first cases in which a microbial polysaccharidase has been shown to carry separate catalytic domains active against different plant cell wall polysaccharides within the same polypeptide. xynD is one of a family of related genes in R. flavefaciens that encode enzymes having multiple catalytic domains, and the amino terminus of XYLD exhibits a high degree of similarity with the corresponding regions of another xylanase, XYLA, which carries two different xylanase catalytic domains.