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Nanotechnology should produce numerous new materials in the coming years. Because of the novel design of nanomaterials with new physicochemical characteristics, their potential adverse impact on the environment and human health must be addressed. In the present study, agglomerates of pristine Csub 60 fullerenes (50 nm to km-size) were applied to soil at 0, 5, 25, and 50 mg/kg dry soil to assess their effect on the soil microbiota by measuring total respiration; biomass, number, and diversity of bacteria; and total number and diversity of protozoans during 14 d. Respiration and microbial biomass were unaffected by the fullerenes at any time, whereas the number of fast-growing bacteria was decreased by three- to fourfold just after incorporation of the nanomaterial. Protozoans seemed not to be very sensitive to Csub 60, because their number decreased only slightly in the beginning of the experiment. With polymerase chain reaction and denaturing gradient gel electrophoresis analysis of eubacteria and kinetoplastids from the soil, however, a difference between the fullerene treatments and nonamended controls was demonstrated. The fullerenes did not induce more than 20 to 30% of relative dissimilarity (with both bacteria and protozoans) between treatments, but this effect was persistent throughout the experiment. It therefore is recommended that fullerene nanomaterial not be spread deliberately in the environment and that their ecotoxicology be further clarified. [PUBLICATION ABSTRACT]

Effects of C sub(60) fullerene nanoparticles on soil bacteria and protozoans, are investigated. The techniques used allow production of nanometer- to micrometer-sized C sub(60) agglomerates. The environmental soil test only permits monitoring part of the bacterial and protozoan community. The results indicate that some members of these taxonomic groups are affected by the agglomerates of the supplied, pristine C sub(60) fullerenes. The soil environment is very complex and it is not possible to determine whether the organisms are sensitive to the fullerenes and affected directly or indirectly. The perturbations are minor, but they seem to persist during the two-week experimental period, as shown by the PCR-DGGE profiles in particular.

A previous bioinformatics-based search for riboswitches yielded several candidate motifs in eubacteria. One of these motifs commonly resides in the 5' untranslated regions of genes involved in the biosynthesis of queuosine (Q), a hypermodified nucleoside occupying the anticodon wobble position of certain transfer RNAs. Here we show that this structured RNA is part of a riboswitch selective for 7-aminomethyl-7-deazaguanine (preQ sub(1)), an intermediate in queuosine biosynthesis. Compared with other natural metabolite-binding RNAs, the preQ sub(1) aptamer appears to have a simple structure, consisting of a single stem-loop and a short tail sequence that together are formed from as few as 34 nucleotides. Despite its small size, this aptamer is highly selective for its cognate ligand in vitro and has an affinity for preQ sub(1) in the low nanomolar range. Relatively compact RNA structures can therefore serve effectively as metabolite receptors to regulate gene expression.

Mucus production is initiated before birth and provides mucin glycans to the infant gut microbiota. Bifidobacteria are the major bacterial group in the feces of vaginally delivered and breast milk-fed infants. Among the bifidobacteria, only Bifidobacterium bifidum is able to degrade mucin and to release monosaccharides which can be used by other gut microbes colonizing the infant gut. Eubacterium hallii is an early occurring commensal that produces butyrate and propionate from fermentation metabolites but that cannot degrade complex oligo-and polysaccharides. We aimed to demonstrate that mucin crossfeeding initiated by B. bifidum enables growth and metabolite formation of E. hallii leading to short-chain fatty acid (SCFA) formation. Growth and metabolite formation of co-cultures of B. bifidum, of Bifidobacterium breve or Bifidobacterium infantis, which use mucin-derived hexoses and fucose, and of E. hallii were determined. Growth of E. hallii in the presence of lactose and mucin monosaccharides was tested. In co-culture fermentations, the presence of B. bifidum enabled growth of the other strains. B. bifidum/B. infantis co-cultures yielded acetate, formate, and lactate while co-cultures of B. bifidum and E. hallii formed acetate, formate, and butyrate. In three-strain co-cultures, B. bifidum, E. hallii, and B. breve or B. infantis produced up to 16 mM acetate, 5 mM formate, and 4 mM butyrate. The formation of propionate (approximately 1 mM) indicated cross-feeding on fucose. Lactose, galactose, and GlcNAc were identified as substrates of E. hallii. This study shows that trophic interactions of bifidobacteria and E. hallii lead to the formation of acetate, butyrate, propionate, and formate, potentially contributing to intestinal SCFA formation with potential benefits for the host and for microbial colonization of the infant gut. The ratios of SCFA formed differed depending on the microbial species involved in mucin cross-feeding.

The F sub(1)F sub(0)-adenosine triphosphate (ATP) synthase rotational motor synthesizes most of the ATP required for living from adenosine diphosphate, Pi, and a proton electrochemical gradient across energy-transducing membranes of bacteria, chloroplasts, and mitochondria. However, as a reversible nanomotor, it also hydrolyzes ATP during de-energized conditions in all energy-transducing systems. Thus, different subunits and mechanisms have emerged in nature to control the intrinsic rotation of the enzyme to favor the ATP synthase activity over its opposite and commonly wasteful ATPase turnover. Recent advances in the structural analysis of the bacterial and mitochondrial ATP synthases are summarized to review the distribution and mechanism of the subunits that are part of the central rotor and regulate its gyration. In eubacteria, the e subunit works as a ratchet to favor the rotation of the central stalk in the ATP synthase direction by extending and contracting two a-helixes of its C-terminal side and also by binding ATP with low affinity in thermophilic bacteria. On the other hand, in bovine heart mitochondria, the so-called inhibitor protein (IF sub(1)) interferes with the intrinsic rotational mechanism of the central g subunit and with the opening and closing of the catalytic b-subunits to inhibit its ATPase activity. Besides its inhibitory role, the IF sub(1) protein also promotes the dimerization of the bovine and rat mitochondrial enzymes, albeit it is not essential for dimerization of the yeast F sub(1)F sub(0) mitochondrial complex. High-resolution electron microscopy of the dimeric enzyme in its bovine and yeast forms shows a conical shape that is compatible with the role of the ATP synthase dimer in the formation of tubular the cristae membrane of mitochondria after further oligomerization. Dimerization of the mitochondrial ATP synthase diminishes the rotational drag of the central rotor that would decrease the coupling efficiency between rotation of the central stalk and ATP synthesis taking place at the F sub(1) portion. In addition, F sub(1)F sub(0) dimerization and its further oligomerization also increase the stability of the enzyme to natural or experimentally induced destabilizing conditions.

The involvement of the gut microbiota in metabolic disorders, and the ability of whole grains to affect both host metabolism and gut microbial ecology, suggest that some benefits of whole grains are mediated through their effects on the gut microbiome. Nutritional studies that assess the effect of whole grains on both the gut microbiome and human physiology are needed. We conducted a randomized cross-over trial with four-week treatments in which 28 healthy humans consumed a daily dose of 60 g of whole-grain barley (WGB), brown rice (BR), or an equal mixture of the two (BR+WGB), and characterized their impact on fecal microbial ecology and blood markers of inflammation, glucose and lipid metabolism. All treatments increased microbial diversity, the Firmicutes/Bacteroidetes ratio, and the abundance of the genus Blautia in fecal samples. The inclusion of WGB enriched the genera Roseburia , Bifidobacterium and Dialister , and the species Eubacterium rectale , Roseburia faecis and Roseburia intestinalis . Whole grains, and especially the BR+WGB treatment, reduced plasma interleukin-6 (IL-6) and peak postprandial glucose. Shifts in the abundance of Eubacterium rectale were associated with changes in the glucose and insulin postprandial response. Interestingly, subjects with greater improvements in IL-6 levels harbored significantly higher proportions of Dialister and lower abundance of Coriobacteriaceae. In conclusion, this study revealed that a short-term intake of whole grains induced compositional alterations of the gut microbiota that coincided with improvements in host physiological measures related to metabolic dysfunctions in humans.

Tungsten metabolism was found to be prevalent in the human gut microbiome, which is involved in the detoxification of food and antimicrobial aldehydes, as well as in the production of beneficial SCFAs. In this study, we characterized a protein in the human gut microbe, Eubacterium limosum , that stores tungstate in preparation for its use in enzymes involved in SCFA generation. This revealed several families of tungstate binding proteins that are also involved in tungstate transport and tungstate-dependent regulation and are widely distributed in the human gut microbiome. Elucidating how tungsten is stored and transported in the human gut microbes contributes to our understanding of the human gut microbiome and its impact on human health.

Abstract Eubacterium limosum is an acetogenic bacterium of potential industrial relevance for its ability to efficiently metabolize a range of single carbon compounds. However, extracellular polymeric substance (EPS) produced by the type strain ATCC 8486 is a serious impediment to bioprocessing and genetic engineering. To remove these barriers, here we bioinformatically identified genes involved in EPS biosynthesis, and targeted several of the most promising candidates for inactivation, using a homologous recombination-based approach. Deletion of a single genomic region encoding homologues for epsABC, ptkA, and tmkA resulted in a strain incapable of producing EPS. This strain is significantly easier to handle by pipetting and centrifugation, and retains important wild-type phenotypes including the ability to grow on methanol and carbon dioxide and limited oxygen tolerance. Additionally, this strain is also more genetically tractable with a 2-fold increase in transformation efficiency compared to the highest previous reports. This work advances a simple, rapid protocol for gene knockouts in E. limosum using only the native homologous recombination machinery. These results will hasten the development of this organism as a workhorse for valorization of single carbon substrates, as well as facilitate exploration of its role in the human gut microbiota. We developed a rapid, simple protocol for gene deletion in the gas-fermenting microbe Eubacterium limosum, and used this to abolish biofilm formation to improve handling and genetic engineering.

Background: Sigma54, or RpoN, is an alternative [sigma] factor found widely in eubacteria. A significant complication in analysis of the global [sigma] super(54) regulon in a bacterium is that the [sigma] super(54) RNA polymerase holoenzyme requires interaction with an active bacterial enhancer-binding protein (bEBP) to initiate transcription at a [sigma] super(54)-dependent promoter. Many bacteria possess multiple bEBPs, which are activated by diverse environmental stimuli. In this work, we assess the ability of a promiscuous, constitutively-active bEBP-the AAA+ ATPase domain of DctD from Sinorhizobium meliloti-to activate transcription from all [sigma] super(54)-dependent promoters for the characterization of the [sigma] super(54) regulon of Salmonella Typhimurium LT2. Results: The AAA+ ATPase domain of DctD was able to drive transcription from nearly all previously characterized or predicted [sigma] super(54)-dependent promoters in Salmonella under a single condition. These promoters are controlled by a variety of native activators and, under the condition tested, are not transcribed in the absence of the DctD AAA+ ATPase domain. We also identified a novel [sigma] super(54)-dependent promoter upstream of STM2939, a homolog of the cas1 component of a CRISPR system. ChIP-chip analysis revealed at least 70 [sigma] super(54) binding sites in the chromosome, of which 58% are located within coding sequences. Promoter-lacZ fusions with selected intragenic [sigma] super(54) binding sites suggest that many of these sites are capable of functioning as [sigma] super(54)-dependent promoters. Conclusion: Since the DctD AAA+ ATPase domain proved effective in activating transcription from the diverse [sigma] super(54)-dependent promoters of the S. Typhimurium LT2 [sigma] super(54) regulon under a single growth condition, this approach is likely to be valuable for examining [sigma] super(54) regulons in other bacterial species. The S. Typhimurium [sigma] super(54) regulon included a high number of intragenic [sigma] super(54) binding sites/promoters, suggesting that [sigma] super(54) may have multiple regulatory roles beyond the initiation of transcription at the start of an operon.