Ubiquitous late competence genes in Bacillus species indicate the presence of functional DNA uptake machineries

Natural competence for genetic transformation, i.e. the ability to take up DNA and stably integrate it in the genome, has so far only been observed in the bacterial kingdom (both in Gram-negative and Gram-positive species) and may contribute to survival under adverse growth conditions. Bacillus subtilis , the model organism for the Bacillus genus, possesses a well-characterized competence machinery. Phylogenetic analysis of several genome sequences of different Bacillus species reveals the presence of many, but not all genes potentially involved in competence and its regulation. The recent demonstration of functional DNA uptake by B. cereus supports the significance of our genome analyses and shows that the ability for functional DNA uptake might be widespread among Bacilli .     

Generation of a tightly regulated doxycycline-inducible model for studying mouse intestinal biology

To develop a sensitive and inducible system to study intestinal biology, we generated a transgenic mouse model expressing the reverse tetracycline transactivator rtTA2-M2 under control of the 12.4 kb murine Villin promoter. The newly generated Villin-rtTA2-M2 mice were then bred with the previously developed tetO-HIST1H2BJ/GFP model to assess inducibility and tissue-specificity. Expression of the histone H2B-GFP fusion protein was observed exclusively upon doxycycline induction and was uniformly distributed throughout the intestinal epithelium. The Villin-rtTA2-M2 was also found to drive transgene expression in the developing mouse intestine. Furthermore, we could detect transgene expression in the proximal tubules of the kidney and in a population of alleged gastric progenitor cells. By administering different concentrations of doxycycline, we show that the Villin-rtTA2-M2 system drives transgene expression in a dosage-dependent fashion. Thus, we have generated a novel doxycycline-inducible mouse model, providing a valuable tool to study the effect of different gene dosages on intestinal physiology and pathology.     

The role of fungi in biogenic weathering in boreal forest soils

In this article we discuss the possible significance of biological processes, and of fungi in particular, in weathering of minerals. We consider biological activity to be a significant driver of mineral weathering in forest ecosystems. In these environments fungi play key roles in organic matter decomposition, uptake, transfer and cycling of organic and inorganic nutrients, biogenic mineral formation, as well as transformation and accumulation of metals. The ability of lichens, mutualistic symbioses between fungi and photobionts such as algae or cyanobacteria, to weather minerals is well documented. The role of mycorrhizal fungi forming symbioses with forest trees is less well understood, but the mineral horizons of boreal forests are intensively colonised by mycorrhizal mycelia which transfer protons and organic metabolites derived from plant photosynthates to mineral surfaces, resulting in mineral dissolution and mobilisation and redistribution of anionic nutrients and metal cations. The mycorrhizal mycelia, in turn provide efficient systems for the uptake and direct transport of mobilised essential nutrients to their host plants which are large sinks. Since almost all (99.99 %) non-suberised lateral plant roots involved in nutrient uptake are covered by ectomycorrhizal fungi, most of this exchange of metabolites must take place through the plant–fungus interface. This idea is still consistent with a linear relationship between soil mineral surface area and weathering rate since the mycelia that emanate from the tree roots will have a larger area of contact with minerals if the mineral surface area is higher. Although empirical models based on bulk soil solution chemistry may fit field data, we argue that biological processes make an important contribution to mineral weathering and that a more detailed mechanistic understanding of these must be developed in order to predict responses to environmental changes and anthropogenic impact.     

Role of CLASP2 in microtubule stabilization and the regulation of persistent motility

In motile fibroblasts, stable microtubules (MTs) are oriented toward the leading edge of cells. How these polarized MT arrays are established and maintained, and the cellular processes they control, have been the subject of many investigations. Several MT "plus-end-tracking proteins," or +TIPs, have been proposed to regulate selective MT stabilization, including the CLASPs, a complex of CLIP-170, IQGAP1, activated Cdc42 or Rac1, a complex of APC, EB1, and mDia1, and the actin-MT crosslinking factor ACF7. By using mouse embryonic fibroblasts (MEFs) in a wound-healing assay, we show here that CLASP2 is required for the formation of a stable, polarized MT array but that CLIP-170 and an APC-EB1 interaction are not essential. Persistent motility is also hampered in CLASP2-deficient MEFs. We find that ACF7 regulates cortical CLASP localization in HeLa cells, indicating it acts upstream of CLASP2. Fluorescence-based approaches show that GFP-CLASP2 is immobilized in a bimodal manner in regions near cell edges. Our results suggest that the regional immobilization of CLASP2 allows MT stabilization and promotes directionally persistent motility in fibroblasts.     

Inoculation of selenium hyperaccumulator Stanleya pinnata and related non‐accumulator Stanleya elata with hyperaccumulator rhizosphere fungi—investigation of effects on Se accumulation and speciation

Little is known about how fungi affect elemental accumulation in hyperaccumulators (HAs). Here, two rhizosphere fungi from selenium (Se) HA Stanleya pinnata, Alternaria seleniiphila (A1) and Aspergillus leporis (AS117), were used to inoculate S. pinnata and related non‐HA Stanleya elata. Growth and Se and sulfur (S) accumulation were analyzed. Furthermore, X‐ray microprobe analysis was used to investigate elemental distribution and speciation. Growth of S. pinnata was not affected by inoculation or by Se. Stanleya elata growth was negatively affected by AS117 and by Se, but combination of both did not reduce growth. Selenium translocation was reduced in inoculated S. pinnata, and inoculation reduced S translocation in both species. Root Se distribution and speciation were not affected by inoculation in either species; both species accumulated mainly (90%) organic Se. Sulfur, in contrast, was present equally in organic and inorganic forms in S. pinnata roots. Thus, these rhizosphere fungi can affect growth and Se and/or S accumulation, depending on host species. They generally enhanced root accumulation and reduced translocation. These effects cannot be attributed to altered plant Se speciation but may involve altered rhizosphere speciation, as these fungi are known to produce elemental Se. Reduced Se translocation may be useful in applications where toxicity to herbivores and movement of Se into the food chain is a concern. The finding that fungal inoculation can enhance root Se accumulation may be useful in Se biofortification or phytoremediation using root crop species.     

Dimethylglycine Provides Salt and Temperature Stress Protection to Bacillus subtilis

Glycine betaine is a potent osmotic and thermal stress protectant of many microorganisms. Its synthesis from glycine results in the formation of the intermediates monomethylglycine (sarcosine) and dimethylglycine (DMG), and these compounds are also produced when it is catabolized. Bacillus subtilis does not produce sarcosine or DMG, and it cannot metabolize these compounds. Here we have studied the potential of sarcosine and DMG to protect B. subtilis against osmotic, heat, and cold stress. Sarcosine, a compatible solute that possesses considerable protein-stabilizing properties, did not serve as a stress protectant of B. subtilis. DMG, on the other hand, proved to be only moderately effective as an osmotic stress protectant, but it exhibited good heat stress-relieving and excellent cold stress-relieving properties. DMG is imported into B. subtilis cells primarily under osmotic and temperature stress conditions via OpuA, a member of the ABC family of transporters. Ligand-binding studies with the extracellular solute receptor (OpuAC) of the OpuA system showed that OpuAC possesses a moderate affinity for DMG, with a K_d value of approximate 172 μM; its K_d for glycine betaine is about 26 μM. Docking studies using the crystal structures of the OpuAC protein with the sulfur analog of DMG, dimethylsulfonioacetate, as a template suggest a model of how the DMG molecule can be stably accommodated within the aromatic cage of the OpuAC ligand-binding pocket. Collectively, our data show that the ability to acquire DMG from exogenous sources under stressful environmental conditions helps the B. subtilis cell to cope with growth-restricting osmotic and temperature challenges.     

Colorectal cancers choosing sides

In contrast to the majority of sporadic colorectal cancer which predominantly occur in the distal colon, most mismatch repair deficient tumours arise at the proximal side. At present, these regional preferences have not been explained properly. Recently, we have screened colorectal tumours for mutations in Wnt-related genes focusing specifically on colorectal location. Combining this analysis with published data, we propose a mechanism underlying the side-related preferences of colorectal cancers, based on the specific acquired genetic defects in β-catenin signalling.     

Correlations between molecular and operational parameters in continuous lab-scale anaerobic reactors

In this study, the microbial community characteristics in continuous lab-scale anaerobic reactors were correlated to reactor functionality using the microbial resource management (MRM) approach. Two molecular techniques, denaturing gradient gel electrophoresis (DGGE) and terminal-restriction fragment length polymorphism (T-RFLP), were applied to analyze the bacterial and archaeal communities, and the results obtained have been compared. Clustering analyses showed a similar discrimination of samples with DGGE and T-RFLP data, with a clear separation between the meso- and thermophilic communities. Both techniques indicate that bacterial and mesophilic communities were richer and more even than archaeal and thermophilic communities, respectively. Remarkably, the community composition was highly dynamic for both Bacteria and Archaea, with a rate of change between 30% and 75% per 18 days, also in stable performing periods. A hypothesis to explain the latter in the context of the converging metabolism in anaerobic processes is proposed. Finally, a more even and diverse bacterial community was found to be statistically representative for a well-functioning reactor as evidenced by a low Ripley index and high biogas production.     

A novel bisphosphonate inhibitor of squalene synthase combined with a statin or a nitrogenous bisphosphonate in vitro

Statins and nitrogenous bisphosphonates (NBP) inhibit 3-hydroxy-3-methylglutaryl-coenzyme-A reductase (HMGCR) and farnesyl diphosphate synthase (FDPS), respectively, leading to depletion of farnesyl diphosphate (FPP) and disruption of protein prenylation. Squalene synthase (SQS) utilizes FPP in the first committed step from the mevalonate pathway toward cholesterol biosynthesis. Herein, we have identified novel bisphosphonates as potent and specific inhibitors of SQS, including the tetrasodium salt of 9-biphenyl-4,8-dimethyl-nona-3,7-dienyl-1,1-bisphosphonic acid (compound 5). Compound 5 reduced cholesterol biosynthesis and lead to a substantial intracellular accumulation of FPP without reducing cell viability in HepG2 cells. At high concentrations, lovastatin and zoledronate impaired protein prenylation and decreased cell viability, which limits their potential use for cholesterol depletion. When combined with lovastatin, compound 5 prevented lovastatin-induced FPP depletion and impairment of protein farnesylation. Compound 5 in combination with the NBP zoledronate completely prevented zoledronate-induced impairment of both protein farnesylation and geranylgeranylation. Cotreatment of cells with compound 5 and either lovastatin or zoledronate was able to significantly prevent the reduction of cell viability caused by lovastatin or zoledronate alone. The combination of an SQS inhibitor with an HMGCR or FDPS inhibitor provides a rational approach for reducing cholesterol synthesis while preventing nonsterol isoprenoid depletion.