Zinc and ATP Binding of the Hexameric AAA-ATPase PilF from Thermus thermophilus: ROLE IN COMPLEX STABILITY,PILIATION, ADHESION,TWITCHING MOTILITY,AND NATURAL TRANSFORMATION*

The traffic AAA-ATPase PilF is essential for pilus biogenesis and natural transformation of Thermus thermophilus HB27. Recently, we showed that PilF forms hexameric complexes containing six zinc atoms coordinated by conserved tetracysteine motifs. Here we report that zinc binding is essential for complex stability. However, zinc binding is neither required for pilus biogenesis nor natural transformation. A number of the mutants did not exhibit any pili during growth at 64 °C but still were transformable. This leads to the conclusion that type 4 pili and the DNA translocator are distinct systems. At lower growth temperatures (55 °C) the zinc-depleted multiple cysteine mutants were hyperpiliated but defective in pilus-mediated twitching motility. This provides evidence that zinc binding is essential for the role of PilF in pilus dynamics. Moreover, we found that zinc binding is essential for complex stability but dispensable for ATPase activity. In contrast to many polymerization ATPases from mesophilic bacteria, ATP binding is not required for PilF complex formation; however, it significantly increases complex stability. These data suggest that zinc and ATP binding increase complex stability that is important for functionality of PilF under extreme environmental conditions.     

Measurement of Monosaccharides and Conversion of Glucose to Acetate in Anoxic Rice Field Soil

Degradation of glucose has been implicated in acetate production in rice field soil, but the abundance of glucose, the temporal change of glucose turnover, and the relationship between glucose and acetate catabolism are not well understood. We therefore measured the pool sizes of glucose and acetate in rice field soil and investigated the turnover of [U-¹⁴C]glucose and [2-¹⁴C]acetate. Acetate accumulated up to about 2 mM during days 5 to 10 after flooding of the soil. Subsequently, methanogenesis started and the acetate concentration decreased to about 100 to 200 μM. Glucose always made up >50% of the total monosaccharides detected. Glucose concentrations decreased during the first 10 days from 90 μM initially to about 3 μM after 40 days of incubation. With the exception at day 0 when glucose consumption was slow, the glucose turnover time was in the range of minutes, while the acetate turnover time was in the range of hours. Anaerobic degradation of [U-¹⁴C]glucose released [¹⁴C]acetate and ¹⁴CO₂ as the main products, with [¹⁴C]acetate being released faster than ¹⁴CO₂. The products of [2-¹⁴C]acetate metabolism, on the other hand, were ¹⁴CO₂ during the reduction phase of soil incubation (days 0 to 15) and ¹⁴CH₄ during the methanogenic phase (after day 15). Except during the accumulation period of acetate (days 5 to 10), approximately 50 to 80% of the acetate consumed was produced from glucose catabolism. However, during the accumulation period of acetate, the rate of acetate production from glucose greatly exceeded that of acetate consumption. Under steady-state conditions, up to 67% of the CH₄ was produced from acetate, of which up to 56% was produced from glucose degradation.     

TFF3 and EGF induce different migration patterns of intestinal epithelial cells in vitro and trigger increased internalization of E-cadherin.

BACKGROUND/AIMS: TFF3, a member of the TFF (trefoil factor family) peptides, and epidermal growth factor (EGF) actively support the repair of mucosal barriers, particularly during restitution. The aim of this study was to compare the motogenic effects of TFF3 and EGF. METHODS: The influence of recombinant human TFF3 (dimeric form) and EGF on the migration of IEC-18 cells was characterized in an in vitro restitution model (scratch wound assay) with the help of time-lapse video microscopy, morphometry, and immunocytochemistry including confocal laser scanning microscopy. RESULTS: TFF3- and EGF-treated cells re-populated the wounded area via different migration patterns; TFF3 treatment resulted in the formation of continuous sheets of migrating cells with only a few gaps. In contrast, EGF-treated cells formed a network of migrating cells (often with a fibroblast-like morphology) with numerous gaps and only punctual contacts. TFF3 and EGF treatment also changed the localization of E-cadherin indicating endocytotic recycling and/or degradation of E-cadherin. CONCLUSION: TFF3, in contrast to EGF, enhanced a collective cell migration ensuring a precise coverage of the re-populated area avoiding gaps.     

Effects of age and altrenogest treatment on conceptus development and secretion of LH, progesterone and eCG in early-pregnant mares

The treatment of early pregnant mares with a history of repeated early embryonic loss with the progestin altrenogest has become routine; however no controlled studies on the efficiency of altrenogest to prevent embryonic losses are available so far. In the present study, we have investigated effects of altrenogest treatment in mares on conceptus development and the secretion of LH, progesterone, and eCG until day 100 of pregnancy. In addition, differences related to age of mares were assessed. Mares were treated with altrenogest (0.044 mg/kg per os once daily) or sunflower oil (10 ml per os once daily) from day 6 to day 100 after ovulation. Blood samples for analysis of LH, progesterone, and eCG were collected. The size of the embryonic vesicle and embryo/fetus was determined by ultrasound. No difference in the per cycle pregnancy rate between altrenogest-treated (75%) and sunflower oil-treated mares (74%) was detected (n.s.). A significant effect of age but not of altrenogest treatment on mean diameter of the embryonic vesicle was found between days 12 and 22 of pregnancy (e.g. day 15: control, 4-8 years: 22.9 ± 1.0 mm, >8 years: 22.0 ± 1.7 mm, altrenogest, 4-8 years: 26.1 ± 2.0 mm, >8 years: 20.4 ± 1.0 mm, P < 0.05). A significant effect of age and treatment on size of the embryo proper between days 30 and 45 was detected (P < 0.05). In the control group but not in the altrenogest group, size of the embryo proper respective fetus was negatively correlated with age of the mares (day 30: r = −0.834, P < 0.05; day 35: r = −0.506, P < 0.05). Plasma concentrations of LH and progesterone were neither effected by age nor by treatment of mares, but significant effects of age and altrenogest treatment on eCG concentrations between days 40 and 130 were detected (P < 0.05). The present study demonstrates for the first time a positive influence of altrenogest-treatment on a retarded development of the embryo respective fetus around the beginning of placentation in mares older than 8 years.     

nAChR‐induced octopamine release mediates the effect of nicotine on a startle response in Drosophila melanogaster

Biogenic amines (BAs) play a central role in the generation of complex behaviors in vertebrates and invertebrates, including the fly Drosophila melanogaster. The comparative advantages of Drosophila as a genetic model to study the contribution of BAs to behaviors stumble upon the difficulty to access the fly brain to ask relevant physiological questions. For instance, it is not known whether the activation of nicotinic acetylcholine receptors (nAChRs) induces the release of BAs in fly brain, a phenomenon associated to several behaviors in vertebrates. Here, we describe a new preparation to study the efflux of BAs in the adult fly brain by in vitro chronoamperometry. Using this preparation we show that nAChR agonists including nicotine induce a fast, transient, dose‐dependent efflux of endogenous BAs, an effect mediated by α‐bungarotoxin‐sensitive nAChRs. By using different genetic tools we demonstrate that the BA whose efflux is induced by nAChR activation is octopamine (Oct). Furthermore, we show that the impairment of a mechanically induced startle response after nicotine exposure is not observed in flies deficient in Oct transmission. Thus, our data show that the efflux of BAs in Drosophila brain is increased by nAChR activation as in vertebrates, and that then AChR‐induced Oct release could have implications in a nicotine‐induced behavioral response.     

Platform Engineering of Corynebacterium glutamicum with Reduced Pyruvate Dehydrogenase Complex Activity for Improved Production of l-Lysine,l-Valine,and 2-Ketoisovalerate

Exchange of the native Corynebacterium glutamicum promoter of the aceE gene, encoding the E1p subunit of the pyruvate dehydrogenase complex (PDHC), with mutated dapA promoter variants led to a series of C. glutamicum strains with gradually reduced growth rates and PDHC activities. Upon overexpression of the l-valine biosynthetic genes ilvBNCE, all strains produced l-valine. Among these strains, C. glutamicum aceE A16 (pJC4 ilvBNCE) showed the highest biomass and product yields, and thus it was further improved by additional deletion of the pqo and ppc genes, encoding pyruvate:quinone oxidoreductase and phosphoenolpyruvate carboxylase, respectively. In fed-batch fermentations at high cell densities, C. glutamicum aceE A16 Δpqo Δppc (pJC4 ilvBNCE) produced up to 738 mM (i.e., 86.5 g/liter) l-valine with an overall yield (Y_P/S) of 0.36 mol per mol of glucose and a volumetric productivity (Q_P) of 13.6 mM per h [1.6 g/(liter × h)]. Additional inactivation of the transaminase B gene (ilvE) and overexpression of ilvBNCD instead of ilvBNCE transformed the l-valine-producing strain into a 2-ketoisovalerate producer, excreting up to 303 mM (35 g/liter) 2-ketoisovalerate with a Y_P/S of 0.24 mol per mol of glucose and a Q_P of 6.9 mM per h [0.8 g/(liter × h)]. The replacement of the aceE promoter by the dapA-A16 promoter in the two C. glutamicum l-lysine producers DM1800 and DM1933 improved the production by 100% and 44%, respectively. These results demonstrate that C. glutamicum strains with reduced PDHC activity are an excellent platform for the production of pyruvate-derived products.     

Degradation of the fluoroquinolone enrofloxacin by wood-rotting fungi.

The veterinary fluoroquinolone enrofloxacin was degraded in vitro by four species of wood-rotting fungi growing on wetted wheat straw containing carbonyl-14C-labeled drug. A maximum 14CO2 production of 17% per week was observed with the brown rot fungus Gloeophyllum striatum, resulting in up to 53% after 8 weeks. However, rates reached at most 0.2 and 0.9% per week, if enrofloxacin was preadsorbed to native or gamma ray-sterilized soil, respectively.     

Diversity of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Large-Subunit Genes from Groundwater and Aquifer Microorganisms

To test our hypothesis that microbial autotrophic CO2 fixation plays an important role in subsurface systems of two large groundwater remediation projects, several anaerobic/microaerobic aquifer and groundwater samples were taken and used to investigate the distribution and phylogenetic diversity of ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) large-subunit genes. Two primer sets were designed for amplifying partial-subunit genes of RubisCO forms I and II from the DNA, directly extracted from the samples. PCR products were used to construct five clone libraries with putative RubisCO form I sequences, and two libraries of DNA amplified by form II primers. Selected clones were screened for variation by restriction fragment length polymorphism analysis, and a total of 28 clone inserts were sequenced and further analyzed. The phylogenies constructed from amino acid sequences derived from the partial RubisCO large-subunit sequences showed a distinct pattern. Diverse sequences affiliated to the cluster of green-like type IA RubisCO sequences were found, representing various obligate and facultative chemolithoautotrophic Proteobacteria, whereas type II RubisCO sequences detected were most closely related to those of thiobacilli species. An isolate obtained from aquifer enrichment culture, which has been provisionally named Halothiobacillus sp. RA13 on the basis of its 16S rDNA sequence, was found to contain both types of RubisCO genes, i.e., forms I and II. Physiological and ecological considerations are discussed in the context of additional microbial data and physicochemical properties.     

Kinetic screening of antibody-Im7 conjugates by capture on a colicin E7 DNase domain using optical biosensors

Antibody generation by phage display and related in vitro display technologies routinely yields large panels of clones detected in primary end-point screenings such as enzyme-linked immunosorbent assay (ELISA). However, for the development of clinical lead candidates, rapid determination of secondary characteristics such as kinetics and thermodynamics is of nearly equal importance. Surface plasmon resonance-based biosensors are ideal tools for carrying out such high-throughput secondary screenings, allowing preliminary but confident ranking and identification of lead clones. A key feature of these assays is the stable and reversible capture of antibody fragments from crude samples leading to high-resolution kinetic analysis of library outputs. Here we exploit the high-affinity interaction between the naturally occurring nuclease domain of E. coli colicin E7 (DNaseE7) and its cognate partner, the immunity protein 7 (Im7), to develop a ligand capture system suitable for accurate kinetic ranking of library clones. We demonstrate generic applicability for a range of antibody formats: scFv antibodies, diabodies, antigen binding fragments (Fabs), and shark V_NAR single domain antibodies. The system is adaptable and reproducible, with comparable results achieved for both the Biacore T100 and ProteOn XPR36 array biosensors.