Shannon B. Conners

Carbohydrate-induced Differential Gene Expression Patterns in the Hyperthermophilic Bacterium Thermotoga maritima ,

The hyperthermophilic bacteriumThermotoga 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 ofT. maritima on monosaccharides was found to be slower than growth on polysaccharides, although growth to cell densities of 108 to 109 cells/ml was observed on all carbohydrates tested. Differential expression of genes encoding carbohydrate-active proteins encoded in the T. maritimagenome 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 ofT. 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.

Heat Shock Response by the Hyperthermophilic Archaeon Pyrococcus furiosus

ABSTRACT Collective transcriptional analysis of heat shock response in the hyperthermophilic archaeon Pyrococcus furiosus was examined by using a targeted cDNA microarray in conjunction with Northern analyses. Differential gene expression suggests that P. furiosus relies on a cooperative strategy of rescue (thermosome [Hsp60], small heat shock protein [Hsp20], and two VAT-related chaperones), proteolysis (proteasome), and stabilization (compatible solute formation) to cope with polypeptide processing during thermal stress.

Population density-dependent regulation of exopolysaccharide formation in the hyperthermophilic bacterium Thermotoga maritima

Co‐cultivation of the hyperthermophiles Thermotoga maritima and Methanococcus jannaschii resulted in fivefold higher T. maritima cell densities when compared with monoculture as well as concomitant formation of exopolysaccharide and flocculation of heterotroph‐methanogen cellular aggregates. Transcriptional analysis of T. maritima cells from these aggregates using a whole genome cDNA microarray revealed the induction of a putative exopolysaccharide synthesis pathway, regulated by intracellular levels of cyclic diguanosine 3′,5′‐(cyclic)phosphate (cyclic di‐GMP) and mediated by the action of several GGDEF proteins, including a putative diguanylate cyclase (TM1163) and a putative phosphodiesterase (TM1184). Transcriptional analysis also showed that TM0504, which encodes a polypeptide containing a motif common to known peptide‐signalling molecules in mesophilic bacteria, was strongly upregulated in the co‐culture. Indeed, when a synthetically produced peptide based on TM0504 was dosed into the culture at ecologically relevant levels, the production of exopolysaccharide was induced at significantly lower cell densities than was observed in cultures lacking added peptide. In addition to identifying a pathway for polysaccharide formation in T. maritima, these results point to the existence of peptide‐based quorum sensing in hyperthermophilic bacteria and indicate that cellular communication should be considered as a component of the microbial ecology within hydrothermal habitats.

An Expression-Driven Approach to the Prediction of Carbohydrate Transport and Utilization Regulons in the Hyperthermophilic Bacterium Thermotoga maritima†

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, mixed-model 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, beta-xylan, and beta-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.

Transcriptional Analysis of Biofilm Formation Processes in the Anaerobic, Hyperthermophilic Bacterium Thermotoga maritima

ABSTRACT Thermotoga maritima, a fermentative, anaerobic, hyperthermophilic bacterium, was found to attach to bioreactor glass walls, nylon mesh, and polycarbonate filters during chemostat cultivation on maltose-based media at 80°C. A whole-genome cDNA microarray was used to examine differential expression patterns between biofilm and planktonic populations. Mixed-model statistical analysis revealed differential expression (twofold or more) of 114 open reading frames in sessile cells (6% of the genome), over a third of which were initially annotated as hypothetical proteins in the T. maritima genome. Among the previously annotated genes in the T. maritima genome, which showed expression changes during biofilm growth, were several that corresponded to biofilm formation genes identified in mesophilic bacteria (i.e., Pseudomonas species, Escherichia coli, and Staphylococcus epidermidis). Most notably, T. maritima biofilm-bound cells exhibited increased transcription of genes involved in iron and sulfur transport, as well as in biosynthesis of cysteine, thiamine, NAD, and isoprenoid side chains of quinones. These findings were all consistent with the up-regulation of iron-sulfur cluster assembly and repair functions in biofilm cells. Significant up-regulation of several β-specific glycosidases was also noted in biofilm cells, despite the fact that maltose was the primary carbon source fed to the chemostat. The reasons for increased β-glycosidase levels are unclear but are likely related to the processing of biofilm-based polysaccharides. In addition to revealing insights into the phenotype of sessile T. maritima communities, the methodology developed here can be extended to study other anaerobic biofilm formation processes as well as to examine aspects of microbial ecology in hydrothermal environments.

The Thermotoga maritima phenotype is impacted by syntrophic interaction with Methanococcus jannaschii in hyperthermophilic coculture

ABSTRACT Significant growth phase-dependent differences were noted in the transcriptome of the hyperthermophilic bacterium Thermotoga maritima when it was cocultured with the hyperthermophilic archaeon Methanococcus jannaschii. For the mid-log-to-early-stationary-phase transition of a T. maritima monoculture, 24 genes (1.3% of the genome) were differentially expressed twofold or more. In contrast, methanogenic coculture gave rise to 292 genes differentially expressed in T. maritima at this level (15.5% of the genome) for the same growth phase transition. Interspecies H2 transfer resulted in three- to fivefold-higher T. maritima cell densities than in the monoculture, with concomitant formation of exopolysaccharide (EPS)-based cell aggregates. Differential expression of specific sigma factors and genes related to the ppGpp-dependent stringent response suggests involvement in the transition into stationary phase and aggregate formation. Cell aggregation was growth phase dependent, such that it was most prominent during mid-log phase and decayed as cells entered stationary phase. The reduction in cell aggregation was coincidental with down-regulation of genes encoding EPS-forming glycosyltranferases and up-regulation of genes encoding β-specific glycosyl hydrolases; the latter were presumably involved in hydrolysis of β-linked EPS to release cells from aggregates. Detachment of aggregates may facilitate colonization of new locations in natural environments where T. maritima coexists with other organisms. Taken together, these results demonstrate that syntrophic interactions can impact the transcriptome of heterotrophs in methanogenic coculture, and this factor should be considered in examining the microbial ecology in anaerobic environments.

Transcriptional and Biochemical Analysis of Starch Metabolism in the Hyperthermophilic Archaeon Pyrococcus furiosus

Pyrococcus furiosus utilizes starch and its degradation products, such as maltose, as primary carbon sources, but the pathways by which these alpha-glucans are processed have yet to be defined. For example, its genome contains genes proposed to encode five amylolytic enzymes (including a cyclodextrin glucanotransferase [CGTase] and amylopullulanase), as well as two transporters for maltose and maltodextrins (Mal-I and Mal-II), and a range of intracellular enzymes have been purified that reportedly metabolize maltodextrins and maltose. However, precisely which of these enzymes are involved in starch processing is not clear. In this study, starch metabolism in P. furiosus was examined by biochemical analyses in conjunction with global transcriptional response data for cells grown on a variety of glucans. In addition, DNA sequencing led to the correction of two key errors in the genome sequence, and these change the predicted properties of amylopullulanase (now designated PF1935*) and CGTase (PF0478*). Based on all of these data, a pathway is proposed that is specific for starch utilization that involves one transporter (Mal-II [PF1933 to PF1939]) and only three enzymes, amylopullulanase (PF1935*), 4-alpha-glucanotransferase (PF0272), and maltodextrin phosphorylase (PF1535). Their expression is upregulated on starch, and together they generate glucose and glucose-1-phosphate, which then feed into the novel glycolytic pathway of this organism. In addition, the results indicate that several hypothetical proteins encoded by three gene clusters are also involved in the transport and processing of alpha-glucan substrates by P. furiosus.

Proteolysis in hyperthermophilic microorganisms

Proteases are found in every cell, where they recognize and break down unneeded or abnormal polypeptides or peptide-based nutrients within or outside the cell. Genome sequence data can be used to compare proteolytic enzyme inventories of different organisms as they relate to physiological needs for protein modification and hydrolysis. In this review, we exploit genome sequence data to compare hyperthermophilic microorganisms from the euryarchaeotal genus Pyrococcus, the crenarchaeote Sulfolobus solfataricus, and the bacterium Thermotoga maritima. An overview of the proteases in these organisms is given based on those proteases that have been characterized and on putative proteases that have been identified from genomic sequences, but have yet to be characterized. The analysis revealed both similarities and differences in the mechanisms utilized for proteolysis by each of these hyperthermophiles and indicated how these mechanisms relate to proteolysis in less thermophilic cells and organisms.

Impact of Substrate Glycoside Linkage and Elemental Sulfur on Bioenergetics of and Hydrogen Production by the Hyperthermophilic Archaeon Pyrococcus furiosus

ABSTRACT Glycoside linkage (cellobiose versus maltose) dramatically influenced bioenergetics to different extents and by different mechanisms in the hyperthermophilic archaeon Pyrococcus furiosus when it was grown in continuous culture at a dilution rate of 0.45 h−1 at 90°C. In the absence of S0, cellobiose-grown cells generated twice as much protein and had 50%-higher specific H2 generation rates than maltose-grown cultures. Addition of S0 to maltose-grown cultures boosted cell protein production fourfold and shifted gas production completely from H2 to H2S. In contrast, the presence of S0 in cellobiose-grown cells caused only a 1.3-fold increase in protein production and an incomplete shift from H2 to H2S production, with 2.5 times more H2 than H2S formed. Transcriptional response analysis revealed that many genes and operons known to be involved in α- or β-glucan uptake and processing were up-regulated in an S0-independent manner. Most differentially transcribed open reading frames (ORFs) responding to S0 in cellobiose-grown cells also responded to S0 in maltose-grown cells; these ORFs included ORFs encoding a membrane-bound oxidoreductase complex (MBX) and two hypothetical proteins (PF2025 and PF2026). However, additional genes (242 genes; 108 genes were up-regulated and 134 genes were down-regulated) were differentially transcribed when S0 was present in the medium of maltose-grown cells, indicating that there were different cellular responses to the two sugars. These results indicate that carbohydrate characteristics (e.g., glycoside linkage) have a major impact on S0 metabolism and hydrogen production in P. furiosus. Furthermore, such issues need to be considered in designing and implementing metabolic strategies for production of biofuel by fermentative anaerobes.

Strategic biocatalysis with hyperthermophilic enzymes

With the advent of genome sequence information, in addition to capabilities for cloning and expressing genes of interest in foreign hosts, a wide range of hyperthermophilic enzymes have become accessible for potential applications for biocatalytic processes. Not only can these enzymes be useful for strategic opportunities at high temperatures, but there may also be advantages that derive from their relatively low activity at suboptimal temperatures. Examples of several possible ways in which hyperthermophilic enzymes could be used are presented, including cases where they could serve as environmentally benign alternatives in existing industrial processes.