Uncoupling Fermentative Synthesis of Molecular Hydrogen from Biomass Formation in Thermotoga maritima.

When carbohydrates are fermented by the hyperthermophilic anaerobe Thermotoga maritima, molecular hydrogen (H2) is formed in strict proportion to substrate availability. Excretion of the organic acids acetate and lactate provide an additional sink for removal of excess reductant. However, mechanisms controlling energy management of these metabolic pathways are largely unexplored. To investigate this topic, transient gene inactivation was used to block lactate production as a strategy to produce spontaneous mutant cell lines that overproduced H2 through mutation of unpredicted genetic targets. Single-crossover homologous chromosomal recombination was used to disrupt lactate dehydrogenase (encoded by ldh) with a truncated ldh fused to a kanamycin resistance cassette expressed from a native P groESL promoter. Passage of the unstable recombinant resulted in loss of the genetic marker and recovery of evolved cell lines, including strain Tma200. Relative to the wild type, and considering the mass balance of fermentation substrate and products, Tma200 grew more slowly, produced H2 at levels above the physiologic limit, and simultaneously consumed less maltose while oxidizing it more efficiently. Whole-genome resequencing indicated that the ABC maltose transporter subunit, encoded by malK3, had undergone repeated mutation, and high-temperature anaerobic [14C]maltose transport assays demonstrated that the rate of maltose transport was reduced. Transfer of the malK3 mutation into a clean genetic background also conferred increased H2 production, confirming that the mutant allele was sufficient for increased H2 synthesis. These data indicate that a reduced rate of maltose uptake was accompanied by an increase in H2 production, changing fermentation efficiency and shifting energy management.IMPORTANCE Biorenewable energy sources are of growing interest to mitigate climate change, but like other commodities with nominal value, require innovation to maximize yields. Energetic considerations constrain production of many biofuels, such as molecular hydrogen (H2) because of the competing needs for cell mass synthesis and metabolite formation. Here we describe cell lines of the extremophile Thermotoga maritima that exceed the physiologic limits for H2 formation arising from genetic changes in fermentative metabolism. These cell lines were produced using a novel method called transient gene inactivation combined with adaptive laboratory evolution. Genome resequencing revealed unexpected changes in a maltose transport protein. Reduced rates of sugar uptake were accompanied by lower rates of growth and enhanced productivity of H2.

Identification of the ATPase Subunit of the Primary Maltose Transporter in the Hyperthermophilic Anaerobe Thermotoga maritima.

Thermotoga maritima is a hyperthermophilic anaerobic bacterium that produces molecular hydrogen (H2) by fermentation. It catabolizes a broad range of carbohydrates through the action of diverse ABC transporters. However, in T. maritima and related species, highly similar genes with ambiguous annotation obscure a precise understanding of genome function. In T. maritima, three putative malK genes, all annotated as ATPase subunits, exhibited high identity to each other. To distinguish between these genes, malK disruption mutants were constructed by gene replacement, and the resulting mutant cell lines were characterized. Only a disruption of malK3 produced a defect in maltose catabolism. To verify that the mutant phenotype arose specifically from malK3 inactivation, the malK3 mutation was repaired by recombination, and maltose catabolism was restored. This study demonstrates the importance of a maltose ABC-type transporter and its relationship to sugar metabolism in T. maritimaIMPORTANCE The application and further development of a genetic system was used here to investigate gene paralogs in the hyperthermophile Thermotoga maritima The occurrence of three ABC transporter ATPase subunits all annotated as malK was evaluated using a combination of genetic and bioinformatic approaches. The results clarify the role of only one malK gene in maltose catabolism in a nonmodel organism noted for fermentative hydrogen production.

Contribution of Pentose Catabolism to Molecular Hydrogen Formation by Targeted Disruption of Arabinose Isomerase (araA) in the Hyperthermophilic Bacterium Thermotoga maritima.

Thermotoga maritima ferments a broad range of sugars to form acetate, carbon dioxide, traces of lactate, and near theoretic yields of molecular hydrogen (H2). In this organism, the catabolism of pentose sugars such as arabinose depends on the interaction of the pentose phosphate pathway with the Embden-Myerhoff and Entner-Doudoroff pathways. Although the values for H2 yield have been determined using pentose-supplemented complex medium and predicted by metabolic pathway reconstruction, the actual effect of pathway elimination on hydrogen production has not been reported due to the lack of a genetic method for the creation of targeted mutations. Here, a spontaneous and genetically stable pyrE deletion mutant was isolated and used as a recipient to refine transformation methods for its repair by homologous recombination. To verify the occurrence of recombination and to assess the frequency of crossover events flanking the deleted region, a synthetic pyrE allele, encoding synonymous nucleotide substitutions, was used. Targeted inactivation of araA (encoding arabinose isomerase) in the pyrE mutant was accomplished using a divergent, codon-optimized Thermosipho africanus pyrE allele fused to the T. maritima groES promoter as a genetic marker. Mutants lacking araA were unable to catabolize arabinose in a defined medium. The araA mutation was then repaired using targeted recombination. Levels of synthesis of H2 using arabinose-supplemented complex medium by wild-type and araA mutant cell lines were compared. The difference between strains provided a direct measurement of H2 production that was dependent on arabinose consumption. Development of a targeted recombination system for genetic manipulation of T. maritima provides a new strategy to explore H2 formation and life at an extremely high temperature in the bacterial domain. IMPORTANCE: We describe here the development of a genetic system for manipulation of Thermotoga maritima T. maritima is a hyperthermophilic anaerobic bacterium that is well known for its efficient synthesis of molecular hydrogen (H2) from the fermentation of sugars. Despite considerable efforts to advance compatible genetic methods, chromosome manipulation has remained elusive and hindered use of T. maritima or its close relatives as model hyperthermophiles. Lack of a genetic method also prevented efforts to manipulate specific metabolic pathways to measure their contributions to H2 yield. To overcome this barrier, a homologous chromosomal recombination method was developed and used to characterize the contribution of arabinose catabolism to H2 formation. We report here a stable genetic method for a hyperthermophilic bacterium that will advance studies on the basic and synthetic biology of Thermotogales.

Identification of an archaeal mercury regulon by chromatin immunoprecipitation.

Mercury is a heavy metal and toxic to all forms of life. Metal exposure can invoke a response to improve survival. In archaea, several components of a mercury response system have been identified, but it is not known whether metal transport is a member of this system. To identify such missing components, a peptide-tagged MerR transcription factor was used to localize enriched chromosome regions by chromosome immunoprecipitation combined with DNA sequence analysis. Such regions could serve as secondary regulatory binding sites to control the expression of additional genes associated with mercury detoxification. Among the 31 highly enriched loci, a subset of five was pursued as potential candidates based on their current annotations. Quantitative reverse transcription-PCR analysis of these regions with and without mercury treatment in WT and mutant strains lacking merR indicated significant regulatory responses under these conditions. Of these, a Family 5 extracellular solute-binding protein and the MarR transcription factor shown previously to control responses to oxidation were most strongly affected. Inactivation of the solute-binding protein by gene disruption increased the resistance of mutant cells to mercury challenge. Inductively coupled plasma-MS analysis of the mutant cell line following metal challenge indicated there was less intracellular mercury compared with the isogenic WT strain. Together, these regulated genes comprise new members of the archaeal MerR regulon and reveal a cascade of transcriptional control not previously demonstrated in this model organism.

Effects of walking with a shopping trolley on spinal posture and loading in subjects with neurogenic claudication.

OBJECTIVES: To explore possible mechanisms underpinning symptom relief and improved walking tolerance in patients with neurogenic claudication (NC) when pushing a shopping trolley by evaluating the effects of a shopping trolley on spinal posture and loading patterns. DESIGN: An exploratory study of kinematic and kinetic changes in walking with and without pushing a shopping trolley in persons with NC symptoms and a comparison with asymptomatic control subjects. SETTING: A primary care-based musculoskeletal service. PARTICIPANTS: Participants (n=8) with NC symptoms who have anecdotally reported symptomatic improvement when walking with a shopping trolley and a control group of asymptomatic persons (n=8). INTERVENTIONS: Shopping trolley. MAIN OUTCOME MEASURES: Changes in lumbar spinal sagittal posture and ground reaction force. RESULTS: Subjects with NC and asymptomatic controls walked with significantly more flexed spinal posture (increase in flexion, 3.40°; z=3.516; P<.001) and reduced mean ground reaction forces (-6.9% of body weight; z=-3.46; P=.001) when walking with a shopping trolley. However, at the midstance point of the gait cycle, controls showed minimal reliance on the trolley, whereas, people with NC showed continued offloading. CONCLUSIONS: Both posture and loading are affected by pushing a shopping trolley; however, patients with NC were found to offload the spine throughout the stance phase of gait, whereas asymptomatic controls did not.

Validation of Physical Performance Tests in Individuals with Advanced Knee Osteoarthritis.

BACKGROUND: Individuals with advanced osteoarthritis (OA) of the knee experience significant impairments in balance and in essential physical functions such as walking and rising from a chair. There is limited evidence on valid outcome measures to capture these impairments. QUESTIONS/PURPOSES: We sought to examine the construct validity of three physical performance measures in patients with advanced knee OA: a gait speed (GS) test, the Short Physical Performance Battery (SPPB), and the Timed Up and Go (TUG) test. METHODS: We designed a cross-sectional clinical measurement study in which patients with advanced knee OA completed two self-reported measures: the Knee Injury and Osteoarthritis Outcome Score-Physical Function Shortform (KOOS-PS) and a four-part numeric pain rating scale (Q-NPRS). They were also administered the GS test, TUG test, and SPPB. Convergent and divergent construct validity were assessed by examining relationships between the GS test, the SPPB, the TUG test, the KOOS-PS, and the Q-NPRS and calculating Pearson correlation coefficients (r). The scores for the GS, TUG test, and SPPB were compared with established normative values for age-matched healthy controls. RESULTS: Forty-four subjects (mean age, 66.9 ± 8.1 years) participated in the study. The GS test showed low concordance with the SPPB component tests and the TUG test. The relationships between the physical performance measures and the self-reported measures were low. The scores for the GS test, TUG test, and SPPB in our sample were significantly worse when compared with age-matched normative values, indicating impairments in physical performance. CONCLUSION: These results advance the understanding of the validity of the GS test, TUG test, and SPPB in demonstrating the impairments in physical performance that patients with advanced knee OA experience in walking, balancing, and rising from a chair. Future research should examine the reproducibility and responsiveness of the GS test, TUG test, and SPPB in patients with advanced knee OA, in order to facilitate the integration of these measures into clinical practice.

Complete Genome Sequence of an Evolved Thermotoga maritima Isolate.

Thermotoga maritima is a hyperthermophilic bacterium with a small genome (1.86 Mbp). Genome resequencing of Tma200, a derivative produced by experimental microbial evolution, revealed the occurrence of deletions and substitution mutations. Their identification contributes to a better understanding of genome instability in this organism.

Concurrent validation of activity monitors in patients with rheumatoid arthritis.

BACKGROUND: Physical activity is frequently reported in rheumatology but it is difficult to measure objectively outside the gait laboratory. A new generation of activity monitors offers this potential but it has not yet been evaluated in patients with rheumatoid arthritis. This study aimed to evaluate three types of activity monitors in patients with rheumatoid arthritis. METHODS: The Step-N-Tune, Activ4Life Pro V3.8, and the Intelligent Device for Energy Expenditure and Activity activity monitors were tested concurrently in 12 patients with rheumatoid arthritis as well as in a healthy control group of 12 volunteers. Participants walked at a self selected speed for two minutes and were filmed for later review. Temporal and spatial gait parameters were also validated against the GAITRite walkway and the total number of steps recorded by each activity monitor was compared to a gold standard derived from half speed video replays. FINDINGS: Activity monitor performance varied between devices but all showed poorer performance when used in the group with rheumatoid arthritis. Bland-Altman plots demonstrated wider 95% limits of agreement in the group with rheumatoid arthritis and a systematic decrease in agreement between activity monitors and the gold standard with decreasing functional ability. INTERPRETATION: Despite some variation between devices, all the activity monitors tested performed reasonably well in healthy young volunteers. All except the Activ4Life showed a marked decrease in performance in patients with rheumatoid arthritis, suggesting Activ4Life could be the most suitable for use in this patient group. The marked between group difference in functional ability, and systematic decrease in device performance with deteriorating gait, indicate that activity monitors require specific validation in target clinical populations.

Modelling the propagation of ultrasound in the joint space of a human knee.

There is strong evidence to support the clinical use of low-intensity pulsed ultrasound (LIPUS) to augment fracture healing. A previous experimental study showed that ultrasound can propagate in the joint space of a single human cadaveric knee. A full experimental investigation of this propagation is not possible due to poor reproducibility, the scarcity of human cadaveric tissues and the practical difficulties in making ultrasound measurements in the knee. The aim of the present work is to investigate whether a computer simulation (Wave2000 Pro®; Cyberlogic Inc., New York, NY, USA) can give a good representation of the experimental model. The simulations provided a good agreement with the experimental data, giving some confidence in the application of this computer simulation method as a means of determining whether ultrasound can propagate through different anatomical regions where bone is present.