An Expression-Driven Approach to the Prediction of Carbohydrate Transport and Utilization Regulons in theHyperthermophilic BacteriumThermotoga maritima

Abstract

Comprehensive analysis of genome-wide expression patterns during growth of the hyperthermophilic bacterium Thermotoga maritima on 14 monosaccharide and polysaccharide substrates was undertaken with the goal of proposing carbohydrate specificities for transport systems and putative transcriptional regulators. Saccharide-induced regulons were predicted through the complementary use of comparative genomics, mixedmodel analysis of genome-wide microarray expression data, and examination of upstream sequence patterns. The results indicate that T. maritima relies extensively on ABC transporters for carbohydrate uptake, many of which are likely controlled by local regulators responsive to either the transport substrate or a key metabolic degradation product. Roles in uptake of specific carbohydrates were suggested for members of the expanded Opp/Dpp family of ABC transporters. In this family, phylogenetic relationships among transport systems revealed patterns of possible duplication and divergence as a strategy for the evolution of new uptake capabilities. The presence of GC-rich hairpin sequences between substrate-binding proteins and other components of Opp/Dpp family transporters offers a possible explanation for differential regulation of transporter subunit genes. Numerous improvements to T. maritima genome annotations were proposed, including the identification of ABC transport systems originally annotated as oligopeptide transporters as candidate transporters for rhamnose, xylose, ␤-xylan, and ␤-glucans and identification of genes likely to encode proteins missing from current annotations of the pentose phosphate pathway. Beyond the information obtained for T. maritima, the present study illustrates how expression-based strategies can be used for improving genome annotation in other microorganisms, especially those for which genetic systems are unavailable.Thermotoga maritima, a hyperthermophilic anaerobe with an optimal growth temperature of 80°C, has been found in diverse high-temperature locations and is capable of using a wide variety of simple and complex carbohydrate substrates for growth. The complexity of its carbohydrate utilization strategies, revealed by genome sequencing (48) and through previous work (11,12,47,51), is surprising, given the primitive features of this microorganism. Considerable genomic plasticity has been observed even within the Thermotoga genus, with respect to the gene content of carbohydrate active enzymes and transporter subunits, which may to some extent relate to lateral gene transfer events (48,49). Despite the range of sugar-active enzymes found within T. maritima MSB8 genome (Table S1 in the supplemental material) (6,9,10,23,27,34,35,37,38,39,42,45,46,54,70,71), a PTS (phosphoenolpyruvatedependent phosphotransferase system) similar to those used by other species for preferential uptake of selected sugars is apparently absent (48). No homologs of the PTS components EI and HPr (phosphocarrier proteins) and no sugar-specific EII sugar transporter subunits have been identified ...

Global analysis of carbohydrate utilization by Lactobacillus acidophilus using cDNA microarrays

“…Raw data were imported into SAS (SAS Institute), compiled, background-corrected, log 2 -transformed, and subjected to a mixed model of analysis of variance (SAS proc mixed) with two sequential linear models (44) outlined in the Supporting Materials and Methods, which is published as supporting information on the PNAS web site. ANOVA mixed models have proven successful at analyzing microarray data (41,(44)(45)(46)(47)(48)(49)(50). The array and spot effects were treated as random effects, whereas dye and treatment effect were considered fixed effects.…”

Structural Analysis of Semi-specific Oligosaccharide Recognition by a Cellulose-binding Protein of Thermotoga maritima Reveals Adaptations for Functional Diversification of the Oligopeptide Periplasmic Binding Protein Fold

“…It has been difficult to assign function reliably to the PBP components of these because of the distant sequence relationships between T. maritima and biochemically characterized model organisms. A combination of in vitro binding studies (25) and transcriptional expression profiling of cultures grown in the presence of various carbohydrate substrates (26,27) has shown that 5 of 12 PBPs that are homologous to OpBPs bind not oligopeptides but various carbohydrates. The x-ray crystal structure of one of these, the ␤-1,4-mannobiose-binding protein (Protein Data Bank code 1VR5), was recently solved in the open conformation in the absence of its cognate ligand (structure determined and deposited by the Joint Center for Structural Genomics (28) but otherwise undocumented).…”

Dynamic Metabolic Adjustments and Genome Plasticity Are Implicated in the Heat Shock Response of the Extremely Thermoacidophilic ArchaeonSulfolobus solfataricus

“…Probes were designed in OligoArray 2.0 (46), custom synthesized (Integrated DNA Technologies, Coralville, IA), and printed onto arrays following protocols previously developed for other hyperthermophiles (14,20,50); five replicates per probe were spotted on each array to fortify statistical analysis. S. solfataricus was routinely grown at 80°C and pH 4.0 on DSMZ 182 medium; cells were enumerated using epifluorescence microscopy with acridine orange stain (13).…”