The Rnf complex is a Na+ coupled respiratory enzyme in a fermenting bacterium, Thermotoga maritima.

rnf genes are widespread in bacteria and biochemical and genetic data are in line with the hypothesis that they encode a membrane-bound enzyme that oxidizes reduced ferredoxin and reduces NAD and vice versa, coupled to ion transport across the cytoplasmic membrane. The Rnf complex is of critical importance in many bacteria for energy conservation but also for reverse electron transport to drive ferredoxin reduction. However, the enzyme has never been purified and thus, ion transport could not be demonstrated yet. Here, we have purified the Rnf complex from the anaerobic, fermenting thermophilic bacterium Thermotoga maritima and show that is a primary Na+ pump. These studies provide the proof that the Rnf complex is indeed an ion (Na+) translocating, respiratory enzyme. Together with a Na+-F1FO ATP synthase it builds a simple, two-limb respiratory chain in T. maritima. The physiological role of electron transport phosphorylation in a fermenting bacterium is discussed.

Screening for Thermotoga maritima Membrane-Bound Pyrophosphatase Inhibitors.

Membrane-bound pyrophosphatases (mPPases) are dimeric enzymes that occur in bacteria, archaea, plants, and protist parasites. These proteins cleave pyrophosphate into two orthophosphate molecules, which is coupled with proton and/or sodium ion pumping across the membrane. Since no homologous proteins occur in animals and humans, mPPases are good candidates in the design of potential drug targets. Here we present a detailed protocol to screen for mPPase inhibitors utilizing the molybdenum blue reaction in a 96 well plate system. We use mPPase from the thermophilic bacterium Thermotoga maritima (TmPPase) as a model enzyme. This protocol is simple and inexpensive, producing a consistent and robust result. It takes only about one hour to complete the activity assay protocol from the start of the assay until the absorbance measurement. Since the blue color produced in this assay is stable for a long period of time, subsequent assay(s) can be performed immediately after the previous batch, and the absorbance can be measured later for all batches at once. The drawback of this protocol is that it is done manually and thus can be exhausting as well as require good skills of pipetting and time keeping. Furthermore, the arsenite-citrate solution used in this assay contains sodium arsenite, which is toxic and should be handled with necessary precautions.

Asymmetry in catalysis by Thermotoga maritima membrane-bound pyrophosphatase demonstrated by a nonphosphorus allosteric inhibitor.

Membrane-bound pyrophosphatases are homodimeric integral membrane proteins that hydrolyze pyrophosphate into orthophosphates, coupled to the active transport of protons or sodium ions across membranes. They are important in the life cycle of bacteria, archaea, plants, and parasitic protists, but no homologous proteins exist in vertebrates, making them a promising drug target. Here, we report the first nonphosphorus allosteric inhibitor of the thermophilic bacterium Thermotoga maritima membrane-bound pyrophosphatase and its bound structure together with the substrate analog imidodiphosphate. The unit cell contains two protein homodimers, each binding a single inhibitor dimer near the exit channel, creating a hydrophobic clamp that inhibits the movement of ß-strand 1-2 during pumping, and thus prevents the hydrophobic gate from opening. This asymmetry of inhibitor binding with respect to each homodimer provides the first clear structural demonstration of asymmetry in the catalytic cycle of membrane-bound pyrophosphatases.

Profiling of Indigenous Microbial Community Dynamics and Metabolic Activity During Enrichment in Molasses-Supplemented Crude Oil-Brine Mixtures for Improved Understanding of Microbial Enhanced Oil Recovery.

Anaerobic incubations using crude oil and brine from a North Sea reservoir were conducted to gain increased understanding of indigenous microbial community development, metabolite production, and the effects on the oil-brine system after addition of a complex carbon source, molasses, with or without nitrate to boost microbial growth. Growth of the indigenous microbes was stimulated by addition of molasses. Pyrosequencing showed that specifically Anaerobaculum, Petrotoga, and Methanothermococcus were enriched. Addition of nitrate favored the growth of Petrotoga over Anaerobaculum. The microbial growth caused changes in the crude oil-brine system: formation of oil emulsions, and reduction of interfacial tension (IFT). Reduction in IFT was associated with microbes being present at the oil-brine interphase. These findings suggest that stimulation of indigenous microbial growth by addition of molasses has potential as microbial enhanced oil recovery (MEOR) strategy in North Sea oil reservoirs.

Structure-based design and screening of inhibitors for an essential bacterial GTPase, Der.

Der is an essential and widely conserved GTPase that assists assembly of a large ribosomal subunit in bacteria. Der associates specifically with the 50S subunit in a GTP-dependent manner and the cells depleted of Der accumulate the structurally unstable 50S subunit, which dissociates into an aberrant subunit at a lower Mg(2+) concentration. As Der is an essential and ubiquitous protein in bacteria, it may prove to be an ideal cellular target against which new antibiotics can be developed. In the present study, we describe our attempts to identify novel antibiotics specifically targeting Der GTPase. We performed the structure-based design of Der inhibitors using the X-ray crystal structure of Thermotoga maritima Der (TmDer). Virtual screening of commercially available chemical library retrieved 257 small molecules that potentially inhibit Der GTPase activity. These 257 chemicals were tested for their in vitro effects on TmDer GTPase and in vivo antibacterial activities. We identified three structurally diverse compounds, SBI-34462, -34566 and -34612, that are both biologically active against bacterial cells and putative enzymatic inhibitors of Der GTPase homologs. We also presented the possible interactions of each compound with the Der GTP-binding site to understand the mechanism of inhibition. Therefore, our lead compounds inhibiting Der GTPase provide scaffolds for the development of novel antibiotics against antibiotic-resistant pathogenic bacteria.

Construction and transformation of a Thermotoga-E. coli shuttle vector.

BACKGROUND: Thermotoga spp. are attractive candidates for producing biohydrogen, green chemicals, and thermostable enzymes. They may also serve as model systems for understanding life sustainability under hyperthermophilic conditions. A lack of genetic tools has hampered the investigation and application of these organisms. This study aims to develop a genetic transfer system for Thermotoga spp. RESULTS: Methods for preparing and handling Thermotoga solid cultures under aerobic conditions were optimized. A plating efficiency of ~50% was achieved when the bacterial cells were embedded in 0.3% Gelrite. A Thermotoga-E. coli shuttle vector pDH10 was constructed using pRQ7, a cryptic mini-plasmid found in T. sp. RQ7. Plasmid pDH10 was introduced to T. maritima and T. sp. RQ7 by electroporation and liposome-mediated transformation. Transformants were isolated, and the transformed kanamycin resistance gene (kan) was detected from the plasmid DNA extracts of the recombinant strains by PCR and was confirmed by restriction digestions. The transformed DNA was stably maintained in both Thermotoga and E. coli even without the selective pressure. CONCLUSIONS: Thermotoga are transformable by multiple means. Recombinant Thermotoga strains have been isolated for the first time. A heterologous kan gene is functionally expressed and stably maintained in Thermotoga.

A22 disrupts the bacterial actin cytoskeleton by directly binding and inducing a low-affinity state in MreB.

S-(3,4-Dichlorobenzyl)isothiourea (A22) disrupts the actin cytoskeleton of bacteria, causing defects of morphology and chromosome segregation. Previous studies have suggested that the actin homologue MreB itself is the target of A22, but there has been no direct observation of A22 binding to MreB and no mechanistic explanation of its mode of action. We show that A22 binds MreB with at least micromolar affinity in its nucleotide-binding pocket in a manner that is sterically incompatible with simultaneous ATP binding. A22 negatively affects both the time course and extent of MreB polymerization in vitro in the presence of ATP. A22 prevents assembly of MreB into long, rigid polymers, as determined by both fluorescence microscopy and sedimentation assays. A22 increases the critical concentration of ATP-bound MreB assembly from 500 nM to approximately 2000 nM. We therefore conclude that A22 is a competitive inhibitor of ATP binding to MreB. A22-bound MreB is capable of polymerization, but with assembly properties that more closely resemble those of the ADP-bound state. Because the cellular concentration of MreB is in the low micromolar range, this mechanism explains the ability of A22 to largely disassemble the actin cytoskeleton in bacterial cells. It also represents a novel mode of action for a cytoskeletal drug and the first biochemical characterization of the interaction between a small molecule inhibitor of the bacterial cytoskeleton and its target.

Functional properties of the thermostable mutL from Thermotoga maritima.

The methyl-directed mismatch repair (MMR) mechanism has been extensively studied in vitro and in vivo, but one of the difficulties in determining the biological relationships between the MMR-related proteins is the tendency of MutL to self-aggregate. The properties of a stable MutL homologue were investigated using a thermostable MutL (TmL) from Thermotoga maritima MSB8 and whose size exclusion chromatographic and crosslinking analyses were compatible with a dimeric form of TmL. TmL underwent conformational changes in the presence of nucleotides and single-stranded DNA (ssDNA) with ATP binding not requiring ssDNA binding activity of TmL, while ADPnP-stimulated TmL showed a high ssDNA binding affinity. Finally, TmL interacted with the T. maritima MutS (TmS), increasing the affinity of TmS to mismatched DNA base pairs and suggesting that the role of TmL in the formation of a mismatched DNA-TmS complex may be a pivotal observation for the study of the initial MMR system. [BMB reports 2009; 42(1): 53-58].

Polymerization properties of the Thermotoga maritima actin MreB: roles of temperature, nucleotides, and ions.

MreB is a bacterial orthologue of actin that affects cell shape, polarity, and chromosome segregation. Although a significant body of work has explored its cellular functions, we know very little about the biochemical behavior of MreB. We have cloned, overexpressed in Escherichia coli, and purified untagged MreB1 from Thermotoga maritima. We have characterized the conditions that regulate its monomer-to-polymer assembly reaction, the critical concentrations of that reaction, the manner in which MreB uses nucleotides, its stability, and the structure of the assembled polymer. MreB requires a bound purine nucleotide for polymerization and rapidly hydrolyzes it following assembly. MreB assembly contains two distinct components, one that does not require divalent cations and one that does, which may comprise the nucleation and elongation phases of assembly, respectively. MreB assembly is strongly favored by increasing temperature or protein concentration but inhibited differentially by high concentrations of monovalent salts. The polymerization rate increases and the bulk critical concentration decreases with increasing temperature, but in contrast to previous reports, MreB is capable of polymerizing across a broad range of temperatures. MreB polymers are shorter and stiffer and scatter more light than eukaryotic actin filaments. Due to rapid ATP hydrolysis and phosphate release, we suggest that most assembled MreB in cells is in the ADP-bound state. Because of only moderate differences between the ATP and ADP critical concentrations, treadmilling may occur, but we do not predict dynamic instability in cells. Because of the relatively low cellular concentration of MreB and the observed structural properties of the polymer, a single MreB assembly may exist in cells.

Responses of wild-type and resistant strains of the hyperthermophilic bacterium Thermotoga maritima to chloramphenicol challenge.

Transcriptomes and growth physiologies of the hyperthermophile Thermotoga maritima and an antibiotic-resistant spontaneous mutant were compared prior to and following exposure to chloramphenicol. While the wild-type response was similar to that of mesophilic bacteria, reduced susceptibility of the mutant was attributed to five mutations in 23S rRNA and phenotypic preconditioning to chloramphenicol.

A highly efficient method for liquid and solid cultivation of the anaerobic hyperthermophilic eubacterium Thermotoga maritima.

An efficient and economical medium--Thermotoga maritima basal medium (TMB)--was designed for the cultivation of T. maritima under either liquid or solid conditions. When the broth was flushed with N2 or CO2 throughout cell growth in a 10-L fermentor (pH controlled to 6.5), the maximum cell density (OD600) on TMB containing 1% glucose rose to 2.0 or higher (1.63 x 10(9) cells mL(-1)). Sheath-less cells observed by electron microscopy were captured during growth in the fermentor. Using a two-layer plating method, isolated single-well colonies were consistently obtained within 24 h on the TMB in modified tissue culture flasks. The minimal inhibitory chloramphenicol concentrations for T. maritima on TMB agar were 5 microg mL(-1) after 24 h and 48 h, and 25 microg mL(-1) at 72 h.

Hyperthermophilic alpha-L: -arabinofuranosidase from Thermotoga maritima MSB8: molecular cloning, gene expression, and characterization of the recombinant protein.

A putative alpha-L: -arabinofuranosidase (AFase) gene belonging to family 51 of glycosyl hydrolases of a hyperthermophilic bacterium Thermotoga maritima MSB8 was cloned, sequenced, and overexpressed in Escherichia coli. The recombinant protein (Tm-AFase) was purified to apparent homogeneity by heat treatment (80 degrees C, 30 min), followed by hydrophobic interaction, anion-exchange, and gel permeation column chromatography. Tm-AFase had a molecular mass of 55,284 Da on matrix assisted laser desorption ionization time-of-flight mass spectrometry and approximately 332 kDa on gel permeation column chromatography. Therefore, Tm-AFase comprised six identical subunits as in the case of homologous AFase from Geobacillus stearothermophilus. Regarding substrate specificity, Tm-AFase was active with p-nitrophenyl alpha-L: -arabinofuranoside but not with p-nitrophenyl alpha-L: -arabinopyranoside. Regarding polysaccharides, Tm-AFase hydrolyzed arabinan and debranched arabinan but not arabinoxylan, arabinogalactan, and carboxymethyl cellulose. Tm-AFase was extremely thermophilic, displaying an optimal reaction temperature of 90 degrees C in a 10 min assay. When Tm-AFase was heated at 90 degrees C, no loss of activity was observed for at least 24 h. At 100 degrees C, the activity dropped to approximately 50% in 20 min; thereafter, inactivation occurred very slowly exhibiting a half-life of approximately 2.7 h, characterizing the enzyme to be the most thermophilic AFase reported thus far.

Kinetic and mechanistic analyses of new classes of inhibitors of two-component signal transduction systems using a coupled assay containing HpkA-DrrA from Thermotoga maritima.

Two-component signal transduction systems (TCSs) play fundamental roles in bacterial survival and pathogenesis and have been proposed as targets for the development of novel classes of antibiotics. A new coupled assay was developed and applied to analyse the kinetic mechanisms of three new kinds of inhibitors of TCS function. The assay exploits the biochemical properties of the cognate HpkA-DrrA histidine kinase-response regulator pair from Thermotoga maritima and allows multiple turnovers of HpkA, linear formation of phosphorylated DrrA, and Michaelis-Menten analysis of inhibitors. The assay was validated in several ways, including confirmation of competitive inhibition by adenosine 5'-beta,gamma-imidotriphosphate (AMP-PNP). The coupled assay, autophosphorylation and chemical cross-linking were used to determine the mechanisms by which several compounds inhibit TCS function. A cyanoacetoacetamide showed non-competitive inhibition with respect to ATP concentration in the coupled assay. The cyanoacetoacetamide also inhibited autophosphorylation of histidine kinases from other bacteria, indicating that the coupled assay could detect general inhibitors of histidine kinase function. Inhibition of HpkA autophosphorylation by this compound was probably caused by aggregation of HpkA, consistent with a previous model for other hydrophobic compounds. In contrast, ethodin was a potent inhibitor of the combined assay, did not inhibit HpkA autophosphorylation, but still led to aggregation of HpkA. These data suggest that ethodin bound to the HpkA kinase and inhibited transfer of the phosphoryl group to DrrA. A peptide corresponding to the phosphorylation site of DrrA appeared to inhibit TCS function by a mechanism similar to that of ethodin, except that autophosphorylation was inhibited at high peptide concentrations. The latter mechanism of inhibition of TCS function is unusual and its analysis demonstrates the utility of these approaches to the kinetic analyses of additional new classes of inhibitors of TCS function.

Liposome-mediated DNA uptake and transient expression in Thermotoga.

We report here the successful application of a PCR-based method to detect genetic transformation of Thermotoga neapolitana and Thermotoga maritima. Plasmid vectors were constructed using pRQ7, an 846-bp plasmid found in Thermotoga species strain RQ7, which replicates by a rolling circle mechanism. The vector pJY1 was constructed by placing a gene encoding a thermostable chloramphenicol acetyltransferase from Stacphylococcus aureus under the control of the tac promoter and joining this with pRQ7 in a pBluescript vector. A second vector, pJY2, was similarly constructed using a gene encoding a kanamycin nucleotidyltransferase previously engineered for thermostability. Genetic transformation of T. neapolitana and T. maritima spheroplasts was achieved using cationic liposomes. The transforming DNA was detected in cells grown in liquid cultures using polymerase chain reaction amplification of the cat or kan genes. T. neapolitana could maintain pJY1 for at least 25 generations in liquid medium containing chloramphenicol. The pJY2 vector conferred kanamycin resistance to T. maritima cells grown in liquid culture. Isolation of stable transformants on solid media after 2-3 days of incubation at 77 degrees C was not possible with either vector, probably because of the instability of both vectors and antibiotics under these conditions. However, this transformation procedure provides, for the first time, a method to introduce DNA into this hyperthermophilic bacterium for potential applications such as targeted gene disruption analyses.