3D reconstruction and morphological analysis of electrostimulated hyperthermophile biofilms of Thermotoga neapolitana.

In this study, the morphological characteristics of the T. neapolitana biofilms on a ceramic carrier, stainless steel, graphite foil, carbon paper, carbon felt and carbon cloth using 3D reconstruction technology was investigated. This was based on the micrographs available in Squadrito et al. (Data Brief 33: 106-403, 2020). Besides the ceramic carrier, the other surfaces were conductive and slightly positively polarised (0.8 and 1.2 V). A simple drying technique was used to show the biofilm and avoid its detachment while chemical fixing with glutaraldehyde was used to better highlight the bacterial morphology within the biofilm. The latter was more suitable for investigating biofilm morphology while the former for bacteria morphology. For the ceramic carrier and stainless steel electrode surfaces, a regular undulating pattern of the biofilm was highlighted by the 3D rendering whilst the glutaraldehyde fixed sample showed a rod-like bacteria morphology. For the other surfaces, a regular undulating pattern of the biofilm and a mixture of a rod-like and a coccoid form of settled bacteria were evidenced also. Carbon cloth was the more suitable electrode for the current application due to its richer filamentous network of bacteria biofilm suggesting a better prevention of bacteria detachment from the electrode surface. Indeed, a preserved biofilm was highlighted on the surfaces of the polarised carbon cloth.

Extremophiles survival to simulated space conditions: an astrobiology model study.

In this work we investigated the ability of four extremophilic bacteria from Archaea and Bacteria domains to resist to space environment by exposing them to extreme conditions of temperature, UV radiation, desiccation coupled to low pressure generated in a Mars' conditions simulator. All the investigated extremophilic strains (namely Sulfolobus solfataricus, Haloterrigena hispanica, Thermotoga neapolitana and Geobacillus thermantarcticus) showed a good resistance to the simulation of the temperature variation in the space; on the other hand irradiation with UV at 254 nm affected only slightly the growth of H. hispanica, G. thermantarcticus and S. solfataricus; finally exposition to Mars simulated condition showed that H. hispanica and G. thermantarcticus were resistant to desiccation and low pressure.

The [4Fe-4S]-cluster coordination of [FeFe]-hydrogenase maturation protein HydF as revealed by EPR and HYSCORE spectroscopies.

[FeFe] hydrogenases are key enzymes for bio(photo)production of molecular hydrogen, and several efforts are underway to understand how their complex active site is assembled. This site contains a [4Fe-4S]-2Fe cluster and three conserved maturation proteins are required for its biosynthesis. Among them, HydF has a double task of scaffold, in which the dinuclear iron precursor is chemically modified by the two other maturases, and carrier to transfer this unit to a hydrogenase containing a preformed [4Fe-4S]-cluster. This dual role is associated with the capability of HydF to bind and dissociate an iron-sulfur center, due to the presence of the conserved FeS-cluster binding sequence CxHx(46-53)HCxxC. The recently solved three-dimensional structure of HydF from Thermotoga neapolitana described the domain containing the three cysteines which are supposed to bind the FeS cluster, and identified the position of two conserved histidines which could provide the fourth iron ligand. The functional role of two of these cysteines in the activation of [FeFe]-hydrogenases has been confirmed by site-specific mutagenesis. On the other hand, the contribution of the three cysteines to the FeS cluster coordination sphere is still to be demonstrated. Furthermore, the potential role of the two histidines in [FeFe]-hydrogenase maturation has never been addressed, and their involvement as fourth ligand for the cluster coordination is controversial. In this work we combined site-specific mutagenesis with EPR (electron paramagnetic resonance) and HYSCORE (hyperfine sublevel correlation spectroscopy) to assign a role to these conserved residues, in both cluster coordination and hydrogenase maturation/activation, in HydF proteins from different microorganisms.

Proteomic and physiological experiments to test Thermotoga neapolitana constraint-based model hypotheses of carbon source utilization.

Constraint-based models of biochemical reaction networks require experimental validation to test model-derived hypotheses and iteratively improve the model. Physiological and proteomic analysis of Thermotoga neapolitana growth on cellotetraose was conducted to identify gene products related to growth on cellotetraose to improve a constraint-based model of T. neapolitana central carbon metabolism with incomplete cellotetraose pathways. In physiological experiments comparing cellotetraose to cellobiose and glucose as growth substrates, product formation yields on cellotetraose, cellobiose, and glucose were similar; however cell yields per mol carbon consumed were higher on cellotetraose than on cellobiose or glucose. Proteomic analysis showed increased expression of several proteins from cells grown on cellotetraose compared with glucose cell cultures, including cellobiose phosphorylase (CTN_0783), endo-1,4-ß-glucosidase (CTN_1106), and an ATP-binding protein (CTN_1296). The CTN_1296 gene product should be evaluated further for participation in cellotetraose metabolism and is included as one of two hypothetical gene-protein-reaction associations in the T. neapolitana constraint-based model to reinstate cellotetraose metabolism in model simulations.

Structural and functional analyses of beta-glucosidase 3B from Thermotoga neapolitana: a thermostable three-domain representative of glycoside hydrolase 3.

Based on sequence and phylogenetic analyses, glycoside hydrolase (GH) family 3 can be divided into several clusters that differ in the length of their primary sequences. However, structural data on representatives of GH3 are still scarce, since only three of their structures are known and only one of them has been thoroughly characterized-that of an exohydrolase from barley. To allow a deeper structural understanding of the GH3 family, we have determined the crystal structure of the thermostable beta-glucosidase from Thermotoga neapolitana, which has potentially important applications in environmentally friendly industrial biosynthesis at a resolution of 2.05 A. Selected active-site mutants have been characterized kinetically, and the structure of the mutant D242A is presented at 2.1 A resolution. Bgl3B from Th. neapolitana is the first example of a GH3 glucosidase with a three-domain structure. It is composed of an (alpha/beta)(8) domain similar to a triose phosphate isomerase barrel, a five-stranded alpha/beta sandwich domain (both of which are important for active-site organization), and a C-terminal fibronectin type III domain of unknown function. Remarkably, the direction of the second beta-strand of the triose phosphate isomerase barrel domain is reversed, which has implications for the active-site shape. The active site, at the interface of domains 1 and 2, is much more open to solvent than the corresponding site in the structurally homologous enzyme from barley, and only the -1 site is well defined. The structures, in combination with kinetic studies of active-site variants, allow the identification of essential catalytic residues (the nucleophile D242 and the acid/base E458), as well as other residues at the -1 subsite, including D58 and W243, which, by mutagenesis, are shown to be important for substrate accommodation/interaction. The position of the fibronectin type III domain excludes a direct participation of this domain in the recognition of small substrates, although it may be involved in the anchoring of the enzyme on large polymeric substrates and in thermostability.

Hydrogen production of the hyperthermophilic eubacterium, Thermotoga neapolitana under N2 sparging condition.

Gas sparging was found to be a useful technique to reduce hydrogen partial pressure in the liquid phase to enhance the hydrogen yields of strictly anaerobically fermentative bacteria. The effect of nitrogen (N(2)) sparging on hydrogen yield was investigated in sterile and non-sterile conditions using a pure strain of the hyperthermophilic eubacteria, Thermotoga neapolitana with glucose or xylose as a carbon source. The maximum hydrogen accumulations reached 41% of the gaseous mixtures after 30-40 h. Two applications of N(2) sparging after the H(2) content in the headspace reached the maximum levels gave an increase of H(2) production by 78% from 1.82 to 3.24 mol H(2)/mol glucose and by 56% from 1.41 to 2.20 mol H(2)/mol xylose. This result suggested that the removal of the produced H(2) from the gas headspace of the limited-volume, closed culture vial when it achieves the maximum level of H(2) tolerance of the bacterium is a necessary technique to improve its H(2) yield.

The fermentation stoichiometry of Thermotoga neapolitana and influence of temperature, oxygen, and pH on hydrogen production.

The hyperthermophilic bacterium, Thermotoga neapolitana, has potential for use in biological hydrogen (H(2)) production. The objectives of this study were to (1) determine the fermentation stoichiometry of Thermotoga neapolitana and examine H(2) production at various growth temperatures, (2) investigate the effect of oxygen (O(2)) on H(2) production, and (3) determine the cause of glucose consumption inhibition. Batch fermentation experiments were conducted at temperatures of 60, 65, 70, 77, and 85 degrees C to determine product yield coefficients and volumetric productivity rates. Yield coefficients did not show significant changes with respect to growth temperature and the rate of H(2) production reached maximum levels in both the 77 degrees C and 85 degrees C experiments. The fermentation stoichiometry for T. neapolitana at 85 degrees C was 3.8 mol H(2), 2 mol CO(2), 1.8 mol acetate, and 0.1 mol lactate produced per mol of glucose consumed. Under microaerobic conditions H(2) production did not increase when compared to anaerobic conditions, which supports other evidence in the literature that T. neapolitana does not produce H(2) through microaerobic metabolism. Glucose consumption was inhibited by a decrease in pH. When pH was adjusted with buffer addition cultures completely consumed available glucose.

Screening of thermophilic anaerobic bacteria for solid substrate cultivation on lignocellulosic substrates.

Interest in solid substrate cultivation (SSC) techniques is gaining for biochemical production from renewable resources; however, heat and mass transfer problems may limit application of this technique. The use of anaerobic thermophiles in SSC offers a unique solution to overcoming these challenges. The production potential of nine thermophilic anaerobic bacteria was examined on corn stover, sugar cane bagasse, paper pulp sludge, and wheat bran in submerged liquid cultivation (SmC) and SSC. Production of acetate, ethanol, and lactate was measured over a 10 day period, and total product concentrations were used to compare the performance of different organism-substrate combinations using the two cultivation methods. Overall microbial activity in SmC and SSC was dependent on the organism and growth substrate. Clostridium thermocellum strains JW20, LQRI, and 27405 performed significantly better in SSC when grown on sugar cane bagasse and paper pulp sludge, producing at least 70 and 170 mM of total products, respectively. Growth of C. thermocellum strains in SSC on paper pulp sludge proved to be most favorable, generating at least twice the concentration of total products produced in SmC (p-value < 0.05). Clostridium thermolacticum TC21 demonstrated growth on all substrates producing 30-80 and 60-116 mM of total product in SmC and SSC, respectively. Bacterial species with optimal growth temperatures of 70 degrees C grew best on wheat bran in SmC, producing total product concentrations of 45-75 mM. For some of the organism-substrate combinations total end product concentrations in SSC exceeded those in SmC, indicating that SSC may be a promising alternative for microbial activity and value-added biochemical production.

H(2) production and carbon utilization by Thermotoga neapolitana under anaerobic and microaerobic growth conditions.

H(2) production by Petrotoga miotherma, Thermosipho africanus, Thermotoga elfii, Fervidobacterium pennavorans, and Thermotoga neapolitana was compared under microaerobic conditions. Contrary to these previously reported strains being strict anaerobes, all tested strains grew and produced H(2) in the presence of micromolar levels of O(2). T. neapolitana showed the highest H(2) production under these conditions. Microscopic counting techniques were used to determine growth curves and doubling times, which were subsequently correlated with optical density measurements. The Biolog anaerobic microtiter plate system was used to analyze the carbon source utilization spectrum of T. neapolitana and to select non-metabolized or poorly metabolized carbohydrates as physiological buffers. Itaconic acid was successfully used as a buffer to overcome pH-induced limitations of cell growth and to facilitate enhanced production of CO-free H(2).