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Quorum sensing provides the basis for coordinating community-wide, microbial behaviors in many mesophilic bacteria. However, little attention has been directed toward the possibility that such phenomena occur in extremely thermal microbial environments. Despite the absence of luxS in hyperthermophile genomes, autoinducer-2 (AI-2), a boronated furanone and proposed ‘universal’ interspecies mesophilic bacterial communication signal, could be formed by Thermotoga maritima and Pyrococcus furiosus through a combination of biotic and abiotic reaction steps. AI-2 did not, however, induce any detectable quorum-sensing phenotypes in these organisms, although transcriptome-based evidence of an AI-2-induced stress response was observed in T. maritima. The significance, if any, of AI-2 in hydrothermal habitats is not yet clear. Nevertheless, these results show the importance of considering environmental factors, in this case high temperatures, as abiotic causative agents of biochemical and microbiological phenomena.

Caldicellulosiruptor saccharolyticus is an extremely thermophilic, gram-positive anaerobe which ferments cellulose-, hemicellulose- and pectin-containing biomass to acetate, CO 2 , and hydrogen. Its broad substrate range, high hydrogen-producing capacity, and ability to coutilize glucose and xylose make this bacterium an attractive candidate for microbial bioenergy production. Here, the complete genome sequence of C. saccharolyticus , consisting of a 2,970,275-bp circular chromosome encoding 2,679 predicted proteins, is described. Analysis of the genome revealed that C. saccharolyticus has an extensive polysaccharide-hydrolyzing capacity for cellulose, hemicellulose, pectin, and starch, coupled to a large number of ABC transporters for monomeric and oligomeric sugar uptake. The components of the Embden-Meyerhof and nonoxidative pentose phosphate pathways are all present; however, there is no evidence that an Entner-Doudoroff pathway is present. Catabolic pathways for a range of sugars, including rhamnose, fucose, arabinose, glucuronate, fructose, and galactose, were identified. These pathways lead to the production of NADH and reduced ferredoxin. NADH and reduced ferredoxin are subsequently used by two distinct hydrogenases to generate hydrogen. Whole-genome transcriptome analysis revealed that there is significant upregulation of the glycolytic pathway and an ABC-type sugar transporter during growth on glucose and xylose, indicating that C. saccharolyticus coferments these sugars unimpeded by glucose-based catabolite repression. The capacity to simultaneously process and utilize a range of carbohydrates associated with biomass feedstocks is a highly desirable feature of this lignocellulose-utilizing, biofuel-producing bacterium.

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.

The molybdenum cofactor is ubiquitous in nature, and the pathway for Moco biosynthesis is conserved in all three domains of life. Recent work has helped to illuminate one of the most enigmatic steps in Moco biosynthesis, ligation of metal to molybdopterin (the organic component of the cofactor) to form the active cofactor. In Escherichia coli, the MoeA protein mediates ligation of Mo to molybdopterin while the MogA protein enhances this process in an ATP-dependent manner. The X-ray crystal structures for both proteins have been previously described as well as two essential MogA residues, Asp49 and Asp82. Here we describe a detailed mutational analysis of the MoeA protein. Variants of conserved residues at the putative active site of MoeA were analyzed for a loss of function in two different, previously described assays, one employing moeA{sup -} crude extracts and the other utilizing a defined system. Oddly, no correlation was observed between the activity in the two assays. In fact, our results showed a general trend toward an inverse relationship between the activity in each assay. Moco binding studies indicated a strong correlation between a variant's ability to bind Moco and its activity in the purified component assay. Crystal structures of themore » functionally characterized MoeA variants revealed no major structural changes, indicating that the functional differences observed are not due to disruption of the protein structure. On the basis of these results, two different functional areas were assigned to regions at or near the MoeA active site cleft.« less

In the genome of the hyperthermophilic bacterium Thermotoga maritima , TM0504 encodes a putative signaling peptide implicated in population density-dependent exopolysaccharide formation. Although not noted in the original genome annotation, TM0504 was found to colocate, on the opposite strand, with the gene encoding ssrA , a hybrid of tRNA and mRNA (tmRNA), which is involved in a trans- translation process related to ribosome rescue and is ubiquitous in bacteria. Specific DNA probes were designed and used in real-time PCR assays to follow the separate transcriptional responses of the colocated open reading frames (ORFs) during transition from exponential to stationary phase, chloramphenicol challenge, and syntrophic coculture with Methanococcus jannaschii . TM0504 transcription did not vary under normal growth conditions. Transcription of the tmRNA gene, however, was significantly up-regulated during chloramphenicol challenge and in T. maritima bound in exopolysaccharide aggregates during methanogenic coculture. The significance of the colocation of ORFs encoding a putative signaling peptide and tmRNA in T. maritima is intriguing, since this overlapping arrangement (tmRNA associated with putative small ORFs) was found to be conserved in at least 181 bacterial genomes sequenced to date. Whether peptides related to TM0504 in other bacteria play a role in quorum sensing is not yet known, but their ubiquitous colocalization with respect to tmRNA merits further examination.

We have previously shown that Escherichia coli MoeA and MogA are required in vivo for the final step of molybdenum cofactor biosynthesis, the addition of the molybdenum atom to the dithiolene of molybdopterin. MoeA was also shown to facilitate the addition of molybdenum in an assay using crude extracts from E. coli moeA(-) cells. The experiments detailed in this report utilized an in vitro assay for MoeA-mediated molybdenum ligation to de novo synthesized molybdopterin using only purified components and monitoring the reconstitution of human aposulfite oxidase. In this assay, maximum activation was achieved by delaying the addition of aposulfite oxidase to allow for adequate molybdenum coordination to occur. Tungsten, which substitutes for molybdenum in hyperthermophilic organisms, could also be ligated to molybdopterin using this system, though not as efficiently as molybdenum. Addition of thiol compounds to the assay inhibited activity. Addition of MogA also inhibited the reaction. However, in the presence of ATP and magnesium, addition of MogA to the assay increased the level of aposulfite oxidase reconstitution beyond that observed with MoeA alone. This effect was not observed in the absence of MoeA. The results presented here demonstrate that MoeA is responsible for mediating molybdenum ligation to molybdopterin, whereas MogA stimulates this activity in an ATP-dependent manner.

Escherichia coli MoeA and MogA are required for molybdenum cofactor biosynthesis and are believed to function in the addition of molybdenum to the dithiolene of molybdopterin to form molybdenum cofactor. Here we show thatmoeA − and mogA − cells are able to synthesize molybdopterin, but both are deficient in molybdenum incorporation and, as a consequence, are deficient in the formation of molybdopterin-guanine dinucleotide. Human sulfite oxidase expressed in E. coli moeA − could be activated in vitro in the presence of MoeA and low concentrations of molybdate. Sulfite oxidase purified from themoeA − lysate was also activated, although to a lesser extent than observed in the presence of lysate. MogA was incapable of activating sulfite oxidase expressed in E. coli mogA −. These results demonstrate that molybdenum insertion into molybdopterin is required for molybdopterin-guanine dinucleotide formation, and that MoeA facilitates molybdenum incorporation at low levels of molybdate, but MogA has an alternative function, possibly as a carrier for molybdopterin during molybdenum incorporation.

Background: Molybdenum cofactor (Moco) biosynthesis is an evolutionarily conserved pathway present in archaea, eubacteria, and eukaryotes. In humans, genetic abnormalities in the biosynthetic pathway result in Moco deficiency, which is accompanied by severe neurological symptoms and death shortly after birth. The Escherichia coli MoeA and MogA proteins are involved in the final step of Moco biosynthesis: the incorporation of molybdenum into molybdopterin (MPT), the organic pyranopterin moiety of Moco. Results: The crystal structure of E. coli MoeA has been refined at 2 A resolution and reveals that the highly elongated MoeA monomer consists of four clearly separated domains, one of which is structurally related to MogA, indicating a divergent evolutionary relationship between both proteins. The active form of MoeA is a dimer, and a putative active site appears to be localized to a cleft formed between domain II of the first monomer and domains III and IV of the second monomer. Conclusions: In eukaryotes, MogA and MoeA are fused into a single polypeptide chain. The corresponding mammalian protein gephyrin has also been implicated in the anchoring of glycinergic receptors to the cytoskeleton at inhibitory synapses. Based on the structures of MoeA and MogA, gephyrin is surmised to be a highly organized molecule containing at least five domains. This multidomain arrangement could provide a structural basis for its functional diversity. The oligomeric states of MoeA and MogA suggest how gephyrin could assemble into a hexagonal scaffold at inhibitory synapses.

Quorum sensing provides the basis for coordinating community-wide, microbial behaviors in many mesophilic bacteria. However, little attention has been directed toward the possibility that such phenomena occur in extremely thermal microbial environments. Despite the absence of luxS in hyperthermophile genomes, autoinducer-2 (AI-2), a boronated furanone and proposed 'universal' interspecies mesophilic bacterial communication signal, could be formed by Thermotoga maritima and Pyrococcus furiosus through a combination of biotic and abiotic reaction steps. AI-2 did not, however, induce any detectable quorum-sensing phenotypes in these organisms, although transcriptome-based evidence of an AI-2-induced stress response was observed in T. maritima. The significance, if any, of AI-2 in hydrothermal habitats is not yet clear. Nevertheless, these results show the importance of considering environmental factors, in this case high temperatures, as abiotic causative agents of biochemical and microbiological phenomena.

Extremely thermophilic fermentative anaerobes (growth T(opt) > or = 70 degrees C) have the capacity to use a variety of carbohydrates as carbon and energy sources. As such, a wide variety of glycoside hydrolases and transferases have been identified in these microorganisms. The genomes of three model extreme thermophiles-an archaeon Pyrococcus furiosus (T(opt) = 98 degrees C), and two bacteria, Thermotoga maritima (T(opt) = 80 degrees C) and Caldicellulosiruptor saccharolyticus (T(opt) = 70 degrees C)-encode numerous carbohydrate-active enzymes, many of which have been characterized biochemically in their native or recombinant forms. In addition to their voracious appetite for polysaccharide degradation, polysaccharide production has also been noted for extremely thermophilic fermentative anaerobes; T. maritima generates exopolysaccharides that aid in biofilm formation, a process that appears to be driven by intraspecies and interspecies interactions.