Abstract
The hyperthermophilic bacterium Thermotoga maritima MSB8 was grown on a variety of carbohydrates to determine the influence of carbon and energy source on differential gene expression. Despite the fact that T. maritima has been phylogenetically characterized as a primitive microorganism from an evolutionary perspective, results here suggest that it has versatile and discriminating mechanisms for regulating and effecting complex carbohydrate utilization. Growth of T. maritima on monosaccharides was found to be slower than growth on polysaccharides, although growth to cell densities of 10(8) to 10(9) cells/ml was observed on all carbohydrates tested. Differential expression of genes encoding carbohydrate-active proteins encoded in the T. maritima genome was followed using a targeted cDNA microarray in conjunction with mixed model statistical analysis. Coordinated regulation of genes responding to specific carbohydrates was noted. Although glucose generally repressed expression of all glycoside hydrolase genes, other sugars induced or repressed these genes to varying extents. Expression profiles of most endo-acting glycoside hydrolase genes correlated well with their reported biochemical properties, although exo-acting glycoside hydrolase genes displayed less specific expression patterns. Genes encoding selected putative ABC sugar transporters were found to respond to specific carbohydrates, and in some cases putative oligopeptide transporter genes were also found to respond to specific sugar substrates. Several genes encoding putative transcriptional regulators were expressed during growth on specific sugars, thus suggesting functional assignments. The transcriptional response of T. maritima to specific carbohydrate growth substrates indicated that sugar backbone- and linkage-specific regulatory networks are operational in this organism during the uptake and utilization of carbohydrate substrates. Furthermore, the wide ranging collection of such networks in T. maritima suggests that this organism is capable of adapting to a variety of growth environments containing carbohydrate growth substrates.
Highlights
The data set resulting from this study provides a rich resource for characterizing putative regulatory mechanisms in T. maritima that might have been proposed from genome sequence data [4]
The results presented here indicate the presence of carbon catabolite repression during growth on glucose
It remains to be seen whether ABC transporters identified in this study that were expressed during growth on mannose and mannan polysaccharides follow similar mechanisms
Summary
Construction of the Targeted cDNA Microarray—Open reading frames (total of 269) of known and putative genes related to sugar processing and other related metabolic functions were identified through BLAST [12] comparisons of protein sequences from the T. maritima MSB8 genome available on the World Wide Web at www.tigr.org/ tigrscripts/CMR2/GenomePage3.spl?databaseϭbtm. The selected probes were PCR-amplified in a PTC-100 Thermocycler (MJ Research, Inc., Waltham, MA) using Taq polymerase (Roche Molecular Biochemicals) and T. maritima genomic DNA, isolated as described previously [5]. Mannose, xylose, -xylan (birchwood), laminarin (Laminaria digitata), and starch (potato) were obtained from Sigma. Growth substrates were prepared as described previously [5] and included in the medium at a final concentration of 0.25% (w/v). Isolation of total RNA from T. maritima was performed on cells that were grown until early- to mid-exponential phase on the various growth substrates, as described in detail previously [5]. Labeling and Hybridization—First-strand cDNA was prepared from T. maritima total RNA using Stratascript (Stratagene, La Jolla, CA) and random hexamer primers (Invitrogen) by the incorporation of 5-[3aminoallyl]-2Ј-deoxyuridine-5Ј-triphosphate (Sigma) as described elsewhere [13]. Glucose Mannose Xylose Galactomannan Glucomannan Carboxymethyl cellulose -1,3/1,4-Glucan Laminarin Starch -Xylan a NA, not available. b Xyl, xylose. c Glr, glucuronic acid
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