The genus Thermotoga comprises extremely thermophilic (Topt ≥ 70°C) and hyperthermophilic (Topt ≥ 80°C) bacteria that have been extensively studied for insights into the basis for life at elevated temperatures and for biotechnological opportunities (e.g., biohydrogen production, biocatalysis). Over the past decade, genome sequences have become available for a number of Thermotoga species, leading to functional genomics efforts to understand growth physiology as well as genomics-based identification and characterization of novel high temperature biocatalysts. Discussed here are recent developments along these lines for this novel group of microorganisms.

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The proteome of extremely thermophilic microorganisms affords a glimpse into the dynamics of microbial ecology of high temperature environments. The secretome, or extracellular proteome of these microorganisms no doubt harbors technologically important enzymes and other thermostable biomolecules that to date have been characterized only to a limited extent. In the first of a two part study on selected thermophiles, defining the secretome requires a sample preparation method that has no negative impact on all downstream experiments. Following efficient secretome purification, GeLC-MS2 analysis and prediction servers suggest probable protein secretion to complement experimental data. In an effort to define the extracellular proteome of the extreme thermophilic bacterium Caldicellulosiruptor saccharolyticus, several techniques were considered regarding sample processing to achieve the most in-depth analysis of secreted proteins. Order of operation experiments all including the C18 bead technique demonstrated that two levels of sample purification were necessary to effectively de-salt the sample and provide sufficient protein identifications. Five sample preparation combinations yielded 71 proteins and the majority described as enzymatic and putative uncharacterized proteins anticipating consolidated bioprocessing applications. Nineteen proteins were predicted by Phobius, SignalP, SecretomeP, or TatP for extracellular secretion and 11 contain transmembrane domain stretches suggested by Phobius and TMHMM. The sample preparation technique demonstrating the most effective outcome for C. saccharolyticus secreted proteins in this study involves acetone precipitation followed by the C18 bead method in which 2.4% (63 proteins) of the predicted proteome was indentified including proteins suggested to have secretion and transmembrane moieties.

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A mutant(‘lab strain’) of the hyperthermophilicarchaeonPyrococcusfuriosusDSM3638 exhibited an extended exponential phase andatypical cell aggregation behavior. Genomic DNA from the mutant culturewas sequenced and compared to wild-type (WT) DSM3638, revealing145genes with one or more insertions, deletions, or substitutions (12 silent, 33amino acid substitutions, and 100 frame-shifts). Approximately half of the mutated genes weretransposases or hypothetical proteins.The WT transcriptomerevealednumerous changes in amino acid and pyrimidine biosynthesis pathways coincidental with growth phase transitions, unlike the mutant whose transcriptome reflected the observedprolonged exponential phase. Targeted gene deletions,based on frame-shifted ORFs in the mutant genome, in a genetically tractable strain of P. furiosus (COM1) could not generate the extended exponential phase behavior observed for the mutant. For example, a putative radical SAM family protein (PF2064) was the most highly up-regulated ORF (>25-fold) in the WT between exponential and stationary phase, although this ORF was unresponsive in the mutant; deletion of this gene in P. furiosus COM1 resulted in no apparent phenotype. On the other hand, frame-shifting mutations in the mutantgenome negatively impacted transcription ofa flagellar biosynthesis operon (PF0329-PF0338).Consequently, cells in the mutant culture lacked flagella and, unlike the WT, showed minimal evidence of exopolysaccharide-based cell aggregation in post-exponential phase. Electron microscopyofPF0331-PF0337 deletionsin P. furiosus COM1 showed that absence of flagella impacted normal cell aggregation behavior and, furthermore,indicatedthat flagella play a key role, beyond motility, in thegrowthphysiology ofP. furiosus.