Infection Dynamics of the Tick-Borne Pathogen “Candidatus Neoehrlichia mikurensis” and Coinfections with Borrelia afzelii in Bank Voles in Southern Sweden

The tick-borne bacterium “Candidatus Neoehrlichia mikurensis” has recently been recognized as a human pathogen. Together with Borrelia afzelii, it is one of the most common pathogens found in the tick Ixodes ricinus. Here, we compared the epidemiologies of “Ca. Neoehrlichia mikurensis” and B. afzelii by longitudinal sampling from May to September in one of their most abundant vertebrate hosts, the bank vole (Myodes glareolus), using real-time PCR for detection and quantification. The prevalences of “Ca. Neoehrlichia mikurensis” and B. afzelii were determined to be 19% (50/261) and 22% (56/261), respectively. The prevalence of “Ca. Neoehrlichia mikurensis” increased significantly during the sampling season. The clearance rate of “Ca. Neoehrlichia mikurensis” was significantly higher than that of B. afzelii. We found a high frequency of double infections; 46% of all samples infected with “Ca. Neoehrlichia mikurensis” also had a coinfection with B. afzelii. The frequency of coinfections was significantly higher than expected from the prevalence of each pathogen. The high level of coinfections can be caused by interactions between the pathogens or might reflect variation in general susceptibility among voles.     

“Candidatus Endobugula glebosa,” a Specific Bacterial Symbiont of the Marine Bryozoan Bugula simplex

The bryozoans Bugula neritina and Bugula simplex harbor bacteria in the pallial sinuses of their larvae as seen by electron microscopy. In B. neritina, the bacterial symbiont has been characterized as a gamma-proteobacterium, “Candidatus Endobugula sertula.” “Candidatus E. sertula” has been implicated as the source of the bryostatins, polyketides that provide chemical defense to the host and are also being tested for use in human cancer treatments. In this study, the bacterial symbiont in B. simplex larvae was identified by 16S rRNA-targeted PCR and sequencing as a gamma-proteobacterium closely related to and forming a monophyletic group with “Candidatus E. sertula.” In a fluorescence in situ hybridization, a 16S ribosomal DNA probe specific to the B. simplex symbiont hybridized to long rod-shaped bacteria in the pallial sinus of a B. simplex larva. The taxonomic status “Candidatus Endobugula glebosa” is proposed for the B. simplex larval symbiont. Degenerate polyketide synthase (PKS) primers amplified a gene fragment from B. simplex that closely matched a PKS gene fragment from the bryostatin PKS cluster. PCR surveys show that the symbiont and this PKS gene fragment are consistently and uniquely associated with B. simplex. Bryostatin activity assays and chemical analyses of B. simplex extracts reveal the presence of compounds similar to bryostatins. Taken together, these findings demonstrate a symbiosis in B. simplex that is similar and evolutionarily related to that in B. neritina.     

Potentiation of Ligand Binding through Cooperative Effects in Monoamine Oxidase B

Crystallographic and biochemical studies have been employed to identify the binding site and mechanism for potentiation of imidazoline binding in human monoamine oxidase B (MAO B). 2-(2-Benzofuranyl)-2-imidazoline (2-BFI) inhibits recombinant human MAO B with a K_i of 8.3 ± 0.6 μm, whereas tranylcypromine-inhibited MAO B binds 2-BFI with a K_d of 9 ± 2 nm, representing an increase in binding energy Δ(ΔG) of −3.9 kcal/mol. Crystal structures show the imidazoline ligand bound in a site that is distinct from the substrate-binding cavity. Contributions to account for the increase in binding affinity upon tranylcypromine inhibition include a conformational change in the side chain of Gln²⁰⁶ and a “closed conformation” of the side chain of Ile¹⁹⁹, forming a hydrophobic “sandwich” with the side chain of Ile³¹⁶ on each face of the benzofuran ring of 2-BFI. Data with the I199A mutant of human MAO B and failure to observe a similar binding potentiation with rat MAO B, where Ile³¹⁶ is replaced with a Val residue, support an allosteric mechanism where the increased binding affinity of 2-BFI results from a cooperative increase in H-bond strength through formation of a more hydrophobic milieu. These insights should prove valuable in the design of high affinity and specific reversible MAO B inhibitors.     

A Unique Carboxyl-terminal Insert Domain in the Hematopoietic-specific,GTPase-deficient Rho GTPase RhoH Regulates Post-translational Processing

RhoH is a hematopoietic-specific, GTPase-deficient member of the Rho GTPase family that was first identified as a hypermutable gene in human B lineage lymphomas. RhoH remains in a constitutively active state and thus its effects are regulated by expression levels or post-translational modifications. Similar to other small GTPases, intracellular localization of RhoH is dependent upon the conserved “CAAX” box and surrounding sequences within the carboxyl (C) terminus. However, RhoH also contains a unique C-terminal “insert” domain of yet undetermined function. RhoH serves as adaptor molecule in T cell receptor signaling and RhoH expression correlates with the unfavorable prognostic marker ZAP70 in human chronic lymphocytic leukemia. Disease progression is attenuated in a Rhoh^−/− mouse model of chronic lymphocytic leukemia and treatment of primary human chronic lymphocytic leukemia cells with Lenalidomide results in reduced RhoH protein levels. Thus, RhoH is a potential therapeutic target in B cell malignancies. In the current studies, we demonstrate that deletion of the insert domain (LFSINE) results in significant cytoplasmic protein accumulation. Using inhibitors of degradation pathways, we show that LFSINE regulates lysosomal RhoH uptake and degradation via chaperone-mediated autophagy. Whereas the C-terminal prenylation site is critical for ZAP70 interaction, subcellular localization and rescue of the Rhoh^−/− T cell defect in vivo, the insert domain appears dispensable for these functions. Taken together, our findings suggest that the insert domain regulates protein stability and activity without otherwise affecting RhoH function.     

Importance of phosphoinositide-dependent signaling pathways in the control of gene expression in resting cells and in response to phytohormones

“Phosphoinositide” refers to phosphorylated forms of phosphatidylinositol, including phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate. Both of these molecules could be in vivo substrates of plant phospholipase C. These phosphoinositides can also be biologically active “per se,” by directly binding to proteins and thus altering their location and/or activity. The use of pharmacological agents in Arabidopsis suspension cells allowed us to identify genes whose expression was positively or negatively controlled, in the basal state, by products of phosphoinositide-dependent phospholipase C. In this basal state, it seems that no genes exhibit a phosphoinositide-dependent expression “per se.” However, many genes whose expression is altered in the presence of phospholipase C inhibitors appeared to be responsive to salicylic acid. This allowed us to show that salicylic acid acts both by increasing the phosphoinositide pool and by inhibiting the phospholipase C. In response to salicylic acid it is possible to identify genes whose expression is controlled by products of PI-PLC, but also genes whose expression is controlled by phosphoinositides “per se.” Our data highlight the importance of phosphoinositide-dependent pathways in gene expression in resting cells and in response to phytohormones.     

Non-uniqueness of factors constraint on the codon usage in Bombyx mori

Background

The analysis of codon usage is a good way to understand the genetic and evolutionary characteristics of an organism. However, there are only a few reports related with the codon usage of the domesticated silkworm, Bombyx mori (B. mori). Hence, the codon usage of B. mori was analyzed here to reveal the constraint factors and it could be helpful to improve the bioreactor based on B. mori.

Results

A total of 1,097 annotated mRNA sequences from B. mori were analyzed, revealing there is only a weak codon bias. It also shows that the gene expression level is related to the GC content, and the amino acids with higher general average hydropathicity (GRAVY) and aromaticity (Aromo). And the genes on the primary axis are strongly positively correlated with the GC content, and GC3s. Meanwhile, the effective number of codons (ENc) is strongly correlated with codon adaptation index (CAI), gene length, and Aromo values. However, the ENc values are correlated with the second axis, which indicates that the codon usage in B. mori is affected by not only mutation pressure and natural selection, but also nucleotide composition and the gene expression level. It is also associated with Aromo values, and gene length. Additionally, B. mori has a greater relative discrepancy in codon preferences with Drosophila melanogaster (D. melanogaster) or Saccharomyces cerevisiae (S. cerevisiae) than with Arabidopsis thaliana (A. thaliana), Escherichia coli (E. coli), or Caenorhabditis elegans (C. elegans).

Conclusions

The codon usage bias in B. mori is relatively weak, and many influence factors are found here, such as nucleotide composition, mutation pressure, natural selection, and expression level. Additionally, it is also associated with Aromo values, and gene length. Among them, natural selection might play a major role. Moreover, the “optimal codons” of B. mori are all encoded by G and C, which provides useful information for enhancing the gene expression in B. mori through codon optimization.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1596-z) contains supplementary material, which is available to authorized users.     

Investigation of Complement Component C4 Copy Number Variation in Human Longevity

Genetic factors have been estimated to account for about 25% of the variation in an adult''s life span. The complement component C4 with the isotypes C4A and C4B is an effector protein of the immune system, and differences in the overall C4 copy number or gene size (long C4L; short C4S) may influence the strength of the immune response and disease susceptibilities. Previously, an association between C4B copy number and life span was reported for Hungarians and Icelanders, where the C4B*Q0 genotype, which is defined by C4B gene deficiency, showed a decrease in frequency with age. Additionally, one of the studies indicated that a low C4B copy number might be a genetic trait that is manifested only in the presence of the environmental risk factor “smoking”. These observations prompted us to investigate the role of the C4 alleles in our large German longevity sample (∼700 cases; 94–110 years and ∼900 younger controls). No significant differences in the number of C4A, C4B and C4S were detected. Besides, the C4B*Q0 carrier state did not decrease with age, irrespective of smoking as an interacting variable. However, for C4L*Q0 a significantly different carrier frequency was observed in the cases compared with controls (cases: 5.08%; controls: 9.12%; p = 0.003). In a replication sample of 714 German cases (91–108 years) and 890 controls this result was not replicated (p = 0.14) although a similar trend of decreased C4L*Q0 carrier frequency in cases was visible (cases: 7.84%; controls: 10.00%).     

Discrimination of Psychrotrophic and Mesophilic Strains of the Bacillus cereus Group by PCR Targeting of Major Cold Shock Protein Genes

Detection of psychrotrophic strains (those able to grow at or below 7°C) of the Bacillus cereus group (Bacillus cereus, Bacillus thuringiensis, and Bacillus mycoides) in food products is at present extremely slow with conventional microbiology. This is due to an inability to discriminate these cold-adapted strains from their mesophilic counterparts (those able to grow only above 7°C) by means other than growth at low temperature, which takes 5 to 10 days for detection. Here we report the development of a single PCR assay that, using major cold shock protein-specific primers and appropriate annealing temperatures, is capable of both rapidly identifying bacteria of the B. cereus group and discriminating between psychrotrophic and mesophilic strains. It is intended that this development help to more accurately predict the shelf life of refrigerated pasteurized food and dairy products and to reduce the incidence of food poisoning by psychrotrophic strains of the B. cereus group.     

Morals Matter in Economic Games

Contrary to predictions from Expected Utility Theory and Game Theory, when making economic decisions in interpersonal situations, people take the interest of others into account and express various forms of solidarity, even in one-shot interactions with anonymous strangers. Research in other-regarding behavior is dominated by behavioral economical and evolutionary biological approaches. Psychological theory building, which addresses mental processes underlying other-regarding behavior, is rare. Based on Relational Models Theory (RMT, [1]) and Relationship Regulation Theory (RRT, [2]) it is proposed that moral motives influence individuals’ decision behavior in interpersonal situations via conscious and unconscious (automatic) processes. To test our propositions we developed the ‘Dyadic Solidarity Game’ and its solitary equivalent, the ‘Self-Insurance Game’. Four experiments, in which the moral motives “Unity” and “Proportionality” were manipulated, support the propositions made. First, it was shown that consciously activated moral motives (via framing of the overall goal of the experiment) and unconsciously activated moral motives (via subliminal priming) influence other-regarding behavior. Second, this influence was only found in interpersonal, not in solitary situations. Third, by combining the analyses of the two experimental games the extent to which participants apply the Golden Rule (“treat others how you wish to be treated”) could be established. Individuals with a “Unity” motive treated others like themselves, whereas individuals with a “Proportionality” motive gave others less then they gave themselves. The four experiments not only support the assumption that morals matter in economic games, they also deliver new insights in how morals matter in economic decision making.     

Larvae from deep-sea methane seeps disperse in surface waters

Many species endemic to deep-sea methane seeps have broad geographical distributions, suggesting that they produce larvae with at least episodic long-distance dispersal. Cold-seep communities on both sides of the Atlantic share species or species complexes, yet larval dispersal across the Atlantic is expected to take prohibitively long at adult depths. Here, we provide direct evidence that the long-lived larvae of two cold-seep molluscs migrate hundreds of metres above the ocean floor, allowing them to take advantage of faster surface currents that may facilitate long-distance dispersal. We collected larvae of the ubiquitous seep mussel “Bathymodiolus” childressi and an associated gastropod, Bathynerita naticoidea, using remote-control plankton nets towed in the euphotic zone of the Gulf of Mexico. The timing of collections suggested that the larvae might disperse in the water column for more than a year, where they feed and grow to more than triple their original sizes. Ontogenetic vertical migration during a long larval life suggests teleplanic dispersal, a plausible explanation for the amphi-Atlantic distribution of “B.” mauritanicus and the broad western Atlantic distribution of B. naticoidea. These are the first empirical data to demonstrate a biological mechanism that might explain the genetic similarities between eastern and western Atlantic seep fauna.     

Human 2-Oxoglutarate Dehydrogenase Complex E1 Component Forms a Thiamin-derived Radical by Aerobic Oxidation of the Enamine Intermediate

Herein are reported unique properties of the human 2-oxoglutarate dehydrogenase multienzyme complex (OGDHc), a rate-limiting enzyme in the Krebs (citric acid) cycle. (a) Functionally competent 2-oxoglutarate dehydrogenase (E1o-h) and dihydrolipoyl succinyltransferase components have been expressed according to kinetic and spectroscopic evidence. (b) A stable free radical, consistent with the C2-(C2α-hydroxy)-γ-carboxypropylidene thiamin diphosphate (ThDP) cation radical was detected by electron spin resonance upon reaction of the E1o-h with 2-oxoglutarate (OG) by itself or when assembled from individual components into OGDHc. (c) An unusual stability of the E1o-h-bound C2-(2α-hydroxy)-γ-carboxypropylidene thiamin diphosphate (the “ThDP-enamine”/C2α-carbanion, the first postdecarboxylation intermediate) was observed, probably stabilized by the 5-carboxyl group of OG, not reported before. (d) The reaction of OG with the E1o-h gave rise to superoxide anion and hydrogen peroxide (reactive oxygen species (ROS)). (e) The relatively stable enzyme-bound enamine is the likely substrate for oxidation by O₂, leading to the superoxide anion radical (in d) and the radical (in b). (f) The specific activity assessed for ROS formation compared with the NADH (overall complex) activity, as well as the fraction of radical intermediate occupying active centers of E1o-h are consistent with each other and indicate that radical/ROS formation is an “off-pathway” side reaction comprising less than 1% of the “on-pathway” reactivity. However, the nearly ubiquitous presence of OGDHc in human tissues, including the brain, makes these findings of considerable importance in human metabolism and perhaps disease.     

Role of Carbon Dioxide in Catabolism of Propane by “Nocardia paraffinicum” (Rhodococcus rhodochrous)

The catabolism of propane by “Nocardia paraffinicum” (Rhodococcus rhodochrous) has been shown to involve CO₂ fixation after its oxidation to propionic acid. “N. paraffinicum” failed to grow on either propane or 1-propanol in the absence of CO₂. The rate of propane utilization was directly related to the initial CO₂ concentration, and Warburg respirometry suggested that CO₂ was required for the catabolism of 1-propanol, propionaldehyde, and propionate but not for 2-propanol. These data also suggested that the predominant pathway for the utilization of propane by “N. paraffinicum” was through 1-propanol. The use of [2-¹⁴C]propane and ¹⁴CO₂ confirmed the catabolism of propane and the fixation of CO₂. Through the use of these isotopes and the pyruvate carboxylase inhibitor sodium arsenite, the labeled 2,4-dinitrophenylhydrazine derivative of pyruvate was trapped and isolated via thin-layer chromatography. The trapping of [¹⁴C]pyruvate in this manner was considered to be indicative of the presence of the methylmalonyl coenzyme A pathway for CO₂ fixation.     

Mechanotransduction by Membrane Proteins: Worms find PEZO-1’s function easy to swallow

JGP study finds that the C. elegans orthologue of the PIEZO family is a mechanosensitive ion channel that regulates pharyngeal pumping and food sensation.

The PIEZO family of mechanosensitive cation channels has been implicated in a wide variety of physiological processes in mammals and is also associated with human disease. Mammalian genomes encode two family members, known as Piezo1 and Piezo2, but invertebrates such as the nematode Caenorhabditis elegans only possess a single Piezo-related gene (1). The function of the C. elegans orthologue, known as pezo-1, has largely remained obscure, but, in this issue of JGP, Millet et al. reveal that it encodes a bona fide mechanosensitive ion channel that regulates pharyngeal activity (2).Jonathan Millet (left), Valeria Vásquez (center), and colleagues reveal that pezo-1, the sole PIEZO family member in C. elegans, is a mechanosensitive ion channel that regulates pharyngeal pumping and food sensation, particularly when worms are fed with large and stiff bacterial filaments that are difficult to swallow (graphic created with BioRender.com).In 2020, an elegant study demonstrated that pezo-1 controls C. elegans ovulation and fertilization (3). However, explains Valeria Vásquez from the University of Tennessee Health Science Center, whether pezo-1 encodes for a mechanosensitive ion channel was unknown. “PEZO-1 is expressed in many tissues, including the pharynx, which is the organ we decided to concentrate on in our study,” Vásquez says.Muscle cells in the C. elegans pharynx rhythmically contract and relax to pump food into the worm’s intestine. Vásquez and colleagues, including first author Jonathan Millet, found that PEZO-1 is expressed in several different pharyngeal cell types (2), including the gland cells whose secretions lubricate the pharynx, and the proprioceptive NSM neurons that are thought to sense the presence of food within the pharynx lumen and release serotonin to increase the rate of pharyngeal pumping.Millet et al. analyzed pharyngeal pumping in worms lacking pezo-1, as well as in animals expressing a pezo-1 point mutant that, in human Piezo1, increases channel function by slowing channel deactivation and inactivation. Loss or gain of pezo-1 function had surprisingly little effect on pharyngeal activity, causing only mild alterations in the duration and frequency of pumping induced by serotonin, and more obvious effects when challenged with high osmolarity solutions.Worms cultured in the laboratory are usually fed a diet of small, easily ingested Escherichia coli cells and, both loss and gain of pezo-1 function increased the pharynx’s response to this type of food. In their natural habitat, however, C. elegans encounter bacteria of various shapes and sizes, some of which might be harder to swallow. “It occurred to me that it might make a difference if we fed the worms with bacteria that were stiffer and longer,” Vásquez says.The researchers therefore provided their pezo-1 mutants with E. coli treated with cephalexin, an antibiotic that inhibits cell separation and causes the bacteria to form long, spaghetti-like filaments. Compared with wild-type worms fed with this diet, pharyngeal activity was markedly enhanced by the gain-of-function pezo-1 mutant, but substantially reduced in the absence of pezo-1, almost as if the worms were “choking” on the bacterial filaments.Crucially, by performing patch-clamp experiments on both cultured C. elegans cells and insect cells expressing recombinant pezo-1, Millet et al. confirmed that PEZO-1 is, indeed, a mechanosensitive ion channel. However, it remains to be seen exactly how PEZO-1 helps the pharynx sense the physical parameters of food and adjust its pumping activity accordingly. One possibility is that the channel acts within the proprioceptive neurons to regulate the release of serotonin.Intriguingly, the Drosophila PIEZO orthologue controls feeding behavior in flies (4). “However, it’s not known which mechanosensitive channels are important in the pharyngeal system of mammals,” Vásquez says. “Our studies in C. elegans could therefore open an opportunity to understand food sensation in humans.”     

Latitudinal Variation of a Defensive Symbiosis in the Bugula neritina (Bryozoa) Sibling Species Complex

Mutualistic relationships are beneficial for both partners and are often studied within a single environment. However, when the range of the partners is large, geographical differences in selective pressure may shift the relationship outcome from positive to negative. The marine bryozoan Bugula neritina is a colonial invertebrate common in temperate waters worldwide. It is the source of bioactive polyketide metabolites, the bryostatins. Evidence suggests that an uncultured vertically transmitted symbiont, “Candidatus Endobugula sertula”, hosted by B. neritina produces the bryostatins, which protect the vulnerable larvae from predation. Studies of B. neritina along the North American Atlantic coast revealed a complex of two morphologically similar sibling species separated by an apparent biogeographic barrier: the Type S sibling species was found below Cape Hatteras, North Carolina, while Type N was found above. Interestingly, the Type N colonies lack “Ca. Endobugula sertula” and, subsequently, defensive bryostatins; their documented northern distribution was consistent with traditional biogeographical paradigms of latitudinal variation in predation pressure. Upon further sampling of B. neritina populations, we found that both host types occur in wider distribution, with Type N colonies living south of Cape Hatteras, and Type S to the north. Distribution of the symbiont, however, was not restricted to Type S hosts. Genetic and microscopic evidence demonstrates the presence of the symbiont in some Type N colonies, and larvae from these colonies are endowed with defensive bryostatins and contain “Ca. Endobugula sertula”. Molecular analysis of the symbiont from Type N colonies suggests an evolutionarily recent acquisition, which is remarkable for a symbiont thought to be transmitted only vertically. Furthermore, most Type S colonies found at higher latitudes lack the symbiont, suggesting that this host-symbiont relationship is more flexible than previously thought. Our data suggest that the symbiont, but not the host, is restricted by biogeographical boundaries.     

Life in hot carbon monoxide: the complete genome sequence of Carboxydothermus hydrogenoformans Z-2901

We report here the sequencing and analysis of the genome of the thermophilic bacterium Carboxydothermus hydrogenoformans Z-2901. This species is a model for studies of hydrogenogens, which are diverse bacteria and archaea that grow anaerobically utilizing carbon monoxide (CO) as their sole carbon source and water as an electron acceptor, producing carbon dioxide and hydrogen as waste products. Organisms that make use of CO do so through carbon monoxide dehydrogenase complexes. Remarkably, analysis of the genome of C. hydrogenoformans reveals the presence of at least five highly differentiated anaerobic carbon monoxide dehydrogenase complexes, which may in part explain how this species is able to grow so much more rapidly on CO than many other species. Analysis of the genome also has provided many general insights into the metabolism of this organism which should make it easier to use it as a source of biologically produced hydrogen gas. One surprising finding is the presence of many genes previously found only in sporulating species in the Firmicutes Phylum. Although this species is also a Firmicutes, it was not known to sporulate previously. Here we show that it does sporulate and because it is missing many of the genes involved in sporulation in other species, this organism may serve as a “minimal” model for sporulation studies. In addition, using phylogenetic profile analysis, we have identified many uncharacterized gene families found in all known sporulating Firmicutes, but not in any non-sporulating bacteria, including a sigma factor not known to be involved in sporulation previously.     

Blind Flight? A New Troglobiotic Orthoclad (Diptera,Chironomidae) from the Lukina Jama – Trojama Cave in Croatia

The genus Troglocladius Andersen, Baranov et Hagenlund, gen. n. is erected based on T. hajdi Andersen, Baranov et Hagenlund, sp. n. collected at 980 m depth in the Lukina jama—Trojama cave system in Croatia. Morphological features such as pale color, strongly reduced eyes and very long legs make it a typical cave animal. Surprisingly, it has also retained large wings and appears to be capable of flight which would make T. hajdi the first flying troglobiont worldwide, disproving previous beliefs that bats are the only animals capable of flying in complete darkness. Morphologically the new species does not readily fit within any described genus, but shares characteristics with genera both in the tribes “Metriocnemini” and “Orthocladiini”. Bayesian molecular phylogenetic analysis using the markers COI, 18S rDNAs, 28S rDNA, CADI, and CADIV groups it with the genera Tvetenia, Cardiocladius and Eukiefferiella in the tribe “Metriocnemini”. Troglocladius hajdi may be parthenogenetic, as only females were collected. The discovery confirms the position of the Dinaric arch as a highly important hotspot of subterranean biodiversity.     

Acetate Oxidation Coupled to Fe(III) Reduction in Hyperthermophilic Microorganisms

No hyperthermophilic microorganisms have previously been shown to anaerobically oxidize acetate, the key extracellular intermediate in the anaerobic oxidation of organic matter. Here we report that two hyperthermophiles, Ferroglobus placidus and “Geoglobus ahangari,” grow at 85°C by oxidizing acetate to carbon dioxide, with Fe(III) serving as the electron acceptor. These results demonstrate that acetate could potentially be metabolized within the hot microbial ecosystems in which hyperthermophiles predominate, rather than diffusing to cooler environments prior to degradation as has been previously proposed.     

Microbial Conversion of Glycerol to 1,3-Propanediol: Physiological Comparison of a Natural Producer, Clostridium butyricum VPI 3266, and an Engineered Strain, Clostridium acetobutylicum DG1(pSPD5)

Clostridium acetobutylicum is not able to grow on glycerol as the sole carbon source since it cannot reoxidize the excess of NADH generated by glycerol catabolism. Nevertheless, when the pSPD5 plasmid, carrying the NADH-consuming 1,3-propanediol pathway from C. butyricum VPI 3266, was introduced into C. acetobutylicum DG1, growth on glycerol was achieved, and 1,3-propanediol was produced. In order to compare the physiological behavior of the recombinant C. acetobutylicum DG1(pSPD5) strain with that of the natural 1,3-propanediol producer C. butyricum VPI 3266, both strains were grown in chemostat cultures with glycerol as the sole carbon source. The same “global behavior” was observed for both strains: 1,3-propanediol was the main fermentation product, and the qH₂ flux was very low. However, when looking at key intracellular enzyme levels, significant differences were observed. Firstly, the pathway for glycerol oxidation was different: C. butyricum uses a glycerol dehydrogenase and a dihydroxyacetone kinase, while C. acetobutylicum uses a glycerol kinase and a glycerol-3-phosphate dehydrogenase. Secondly, the electron flow is differentially regulated: (i) in C. butyricum VPI 3266, the in vitro hydrogenase activity is 10-fold lower than that in C. acetobutylicum DG1(pSPD5), and (ii) while the ferredoxin-NAD⁺ reductase activity is high and the NADH-ferredoxin reductase activity is low in C. acetobutylicum DG1(pSPD5), the reverse is observed for C. butyricum VPI 3266. Thirdly, lactate dehydrogenase activity is only detected in the C. acetobutylicum DG1(pSPD5) culture, explaining why this microorganism produces lactate.     

Bisecting Galactose as a Feature of N-Glycans of Wild-type and Mutant Caenorhabditis elegans

The N-glycosylation of the model nematode Caenorhabditis elegans has proven to be highly variable and rather complex; it is an example to contradict the existing impression that “simple” organisms possess also a rather simple glycomic capacity. In previous studies in a number of laboratories, N-glycans with up to four fucose residues have been detected. However, although the linkage of three fucose residues to the N,N′-diacetylchitobiosyl core has been proven by structural and enzymatic analyses, the nature of the fourth fucose has remained uncertain. By constructing a triple mutant with deletions in the three genes responsible for core fucosylation (fut-1, fut-6 and fut-8), we have produced a nematode strain lacking products of these enzymes, but still retaining maximally one fucose residue on its N-glycans. Using mass spectrometry and HPLC in conjunction with chemical and enzymatic treatments as well as NMR, we examined a set of α-mannosidase-resistant N-glycans. Within this glycomic subpool, we can reveal that the core β-mannose can be trisubstituted and so carries not only the ubiquitous α1,3- and α1,6-mannose residues, but also a “bisecting” β-galactose, which is substoichiometrically modified with fucose or methylfucose. In addition, the α1,3-mannose can also be α-galactosylated. Our data, showing the presence of novel N-glycan modifications, will enable more targeted studies to understand the biological functions and interactions of nematode glycans.Nematodes represent, along with arthropods, one of the largest groups of animals to exist on the planet; 25.000 species are described, but the existence of up to one million has been estimated (1, 2). They have various ecological niches and include free-living “worms” in the soil, fungivorous, entomopathogenic, and necromenic species as well as parasites of plants and mammals, which share the basic conserved body plan (more-or-less a digestive tube surrounded with muscle, whether larger or smaller). There are five major clades (Rhabditina, Enoplia, Spirurina, Tylenchina, and Dorylaimia) (2), yet the glycosylation of only a few nematode species has been studied with an inevitable focus on the model nematode Caenorhabditis elegans and parasitic species (3). Thereby, the use of C. elegans mutants has been highly valuable in dissecting aspects of nematode N-glycan biosynthesis and revealing the in vivo substrates for certain glycosyltransferases (4).As many nematodes are parasites, their interactions with the immune systems of their hosts have attracted attention; particularly, there are relationships between autoimmunity, allergy, vaccination, and helminth infections. The “old friends” hypothesis seeks to understand the evolutionary factors that have shaped the immune system and to explain correlations between lifestyles in the developed world and modern “epidemics,” which are due to immunological misbalance (5–7). Promising data have suggested that “worm therapy” may bring advantages to some patients with Crohn''s disease or allergies (8, 9); however, such approaches are controversial. Nevertheless, crude extracts even of Caenorhabditis elegans were shown to induce a glycan-dependent Th2 response (10), whereas the excretory-secretory products of some nematodes also have immunomodulatory activity (11). Furthermore, the native glycoproteins of some nematodes have proven effective in vaccination trials, whereas recombinant forms are not, which is suggestive that post-translational modifications may have a role in an efficacious immune response (12).As at least some of the molecules relevant to nematode immunomodulation or vaccination are glycoproteins, a proper understanding of nematode glycosylation is of biomedical and veterinary relevance. Over the years, it has become apparent that the core chitobiosyl region of nematode N-glycans is subject to a range of modifications, with up to three core fucose residues being present (α1,3- and α1,6-linked on the reducing-terminal “proximal” GlcNAc and α1,3-linked on the second “distal” GlcNAc). However, up to four fucose residues have been detected on C. elegans N-glycans and the exact nature of the linkage of the fourth fucose has remained obscure despite work in our own and other laboratories (3, 13–15). Combined with the latest knowledge regarding the specificity of C. elegans core fucosyltransferases (13, 16, 17) as well as our recent data regarding the exact structures of N-glycans from the C. elegans double hexosaminidase mutant and other nematodes (18–20), we concluded that some models for the tri- and tetrafucosylated N-glycans were incorrect. By preparing a triple mutant unable to core fucosylate its N-glycans, we generated a C. elegans strain containing maximally one fucose residue on the N-linked oligosaccharides. Thereby a pool of unusual mannosidase-resistant N-glycans was identified and, using mass spectrometry (MS) and NMR, we reveal their modification with bisecting galactose frequently capped with fucose or methylfucose.     

Isolation of a Bacterial Strain Able To Degrade Branched Nonylphenol

Conventional enrichment of microorganisms on branched nonylphenol (NP) as only carbon and energy source yielded mixed cultures able to grow on the organic compound. However, plating yielded no single colonies capable, alone or in combination with other isolates, of degrading the NP in liquid culture. Therefore, a special approach was used, referred to as “serial dilution-plate resuspension,” to reduce culture complexity. In this way, one isolate, TTNP3, tentatively identified as a Sphingomonas sp., was found to be able to grow on NP in liquid culture. Remarkably, this isolate was able to be filtered through a 0.45-μm-pore-diameter filter. Moreover, isolate TTNP3 did not form visible colonies on mineral medium with NP, and it formed visible colonies on R₂A agar only after a prolonged incubation of 1 week. High-performance liquid chromatography and gas chromatography-mass spectroscopy analysis of the culture media indicated that the strain starts the degradation of NP with a fission of the phenol ring and preferably uses the para isomer of NP and not the ortho isomer. No distinct accumulation of an intermediary product could be observed.