Polyhedral microcompartments in Clostridium phytofermentans

Clostridium phytofermentans ferments all major component of the plant cell wall to alcohols and is an emerging model organism for understanding the direct conversion of plant biomass into biofuels. Encoded in the genome of C. phytofermentans are three large genetic loci for the production of polyhedral microcompartments (PMCs) that may function as metabolic centers for the dissimilation of plant cell wall components into primary alcohols. We demonstrate that the PMC encoded by one of these loci acts in the fermentation of fucose and rhamnose to propanol and propionate. The PMC contains a propanediol dehydratase that is part of a new superfamily of enzymes that utilizes S-adenosylmethionine in place of adenosylcobalamin to catalyze radical reactions. Analysis of the C. phytofermentans genome sequence data indicated that the fucose and rhamnose pathways might converge at the point of the enzymes encoded by the PMC loci. When C. phytofermentans is grown on fucose, a 100-200 nm polyhedral body is apparent in electron micrographs, and ethanol and propanol are produced. Microarray experiments revealed that during growth on fucose, three fucose-related operons coding for (i) the PMC shell and metabolic proteins, (ii) cytoplasmic fucose dissimilatory enzymes, and (iii) an ABC transporter system become the dominant transcripts in the cell. Similarly when C. phytofermentans is grown on rhamnose, three rhamnose-related operons coding for (i) the PMC shell and metabolic proteins, (ii) cytoplasmic rhamnose dissimilatory enzymes, and (iii) an ABC transporter system become the dominant transcripts in the cell. Quite surprisingly, growth on fucose or rhamnose also led to the expression of a broader plant degradative transcriptional response. The expression of cellulose degrading enzymes on fucose and rhamnose is a fundamental difference between C. phytofermentans and its closest relatives, the human gut commensals. C. phytofermentans ISDg was cultured in anaerobic medium GS-2CB [20]. Growth on single carbon-source utilized anaerobic medium derived from GS-2CB and containing the following (g l-1): yeast extract, 6.0; urea, 2.1; KH2PO4, 4.0; Na2HPO4, 6.5; trisodium citrate dihydrate, 3.0; L-cysteine hydrochloride monohydrate, 2.0; resazurin, 1; with pH adjusted to 7.0 using KOH. This medium was supplemented with 0.3% (wt/vol) of the specific substrate (glucose, fucose and rhamnose) added as a filter-sterilized solution to the sterile medium. Broth cultures were incubated at 30˚C under anaerobic conditions (100% N2) as described in by Hungate [30]. Growth was determined spectrophotometrically by monitoring changes in optical density at 660 nm. RNA was purified from mid-exponential phase cultures at 27 hr for glucose and 62 hrs for fucose and rhamnose.