Overexpression, purification and immunodetection of DsrD from Desulfovibrio vulgaris Hildenborough

Dissimilatory sulfite reductase (DsrAB) of the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough is an ₂₂ tetramer of 180 kDa, encoded by the dsr operon. In addition to the dsrA and dsrB genes, this operon contains a gene (dsrD) encoding a protein of only 78 amino acids. Although, the function of DsrD is currently unknown, the presence of a dsrD gene has been demonstrated in a variety of sulfate-reducing bacteria and archaea. DsrD was expressed in Escherichia coli at a very high level and purified to homogeneity. Protein blotting experiments, using antisera raised against purified DsrD, demonstrated that it is expressed constitutively in D. vulgaris and does not copurify with DsrAB. Spectroscopic analysis of DsrD indicated that it does not bind either sulfite or sulfide, the substrate and product, respectively of the reaction catalyzed by DsrAB. Thus, although the conservation of this protein and its demonstrated presence in D. vulgaris, suggest an essential function in dissimilatory sulfite reduction, this function remains to be elucidated.     

Characterization of high density monolayers of the biofilm bacterium Caulobacter crescentus: evaluating prospects for developing immobilized cell bioreactors

Caulobacters are biofilm-forming members of the natural flora of soil and aquatic environments, which exhibit several characteristics that make them attractive for development of high surface area microbial bioreactors or biosensors. Although caulobacters are well characterized genetically, little is known about their biofilm-forming characteristics as a monoculture, or their tolerance of bioreactor-like conditions. Here we investigated the ability of caulobacters to spontaneously form high-density monolayers on artificial surfaces under a variety of environmental conditions, using phase contrast image analysis to assess biofilm density, and epifluorescence with the vital stain DiBAC to assess viability. With adequate nutrition, extremely dense monolayers formed within 24-48 h, and maintained near 100% viability in experiments ranging up to 22 days. When areas were abraded to remove cells, repopulation occurred rapidly with characteristics similar to the population of a clean surface. When established monolayers were starved for nutrients, a significant fraction of the cells detached from the surface, and cells remaining on the surface no longer tested as viable. Within 4-6 h of nutrient restoration, however, cells in the monolayer again appeared normal and tested as 100% viable. This is the first demonstration that Caulobacter crescentus is stable and amenable to high density monolayer growth and resists starvation, though some cells may express a programmed response to detach from the surface under severe nutrient limitation.     

ISD1, an Insertion Element from the Sulfate-Reducing Bacterium Desulfovibrio vulgaris Hildenborough: Structure, Transposition, and Distribution

Insertion element ISD1, discovered when its transposition caused the insertional inactivation of an introduced sacB gene, is present in two copies in the genome of Desulfovibrio vulgaris Hildenborough. Southern blot analysis indicated at least two insertion sites in the sacB gene. Cloning and sequencing of a transposed copy of ISD1 indicated a length of 1,200 bp with a pair of 44-bp imperfect inverted repeats at the ends, flanked by a direct repeat of the 4-bp target sequence. AAGG and AATT were found to function as target sequences. ISD1 encodes a transposase from two overlapping open reading frames by programmed translational frameshifting at an A₆G shifty codon motif. Sequence comparison showed that ISD1 belongs to the IS3 family. Isolation and analysis of the chromosomal copies, ISD1-A and ISD1-B, by PCR and sequencing indicated that these are not flanked by direct repeats. ISD1-A is inserted in a region of the chromosome containing the gapdh-pgk genes (encoding glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase). Active transposition to other loci in the genome was demonstrated, offering the potential of a new tool for gene cloning and mutagenesis. ISD1 is the first transposable element described for the sulfate reducers, a large and environmentally important group of bacteria. The distribution of ISD1 in genomes of sulfate-reducing bacteria is limited. A single copy is present in the genome of D. desulfuricans Norway.     

Analysis of Cell Division and Elongation Underlying the Developmental Acceleration of Root Growth in Arabidopsis thaliana

To investigate the relation between cell division and expansion in the regulation of organ growth rate, we used Arabidopsis thaliana primary roots grown vertically at 20°C with an elongation rate that increased steadily during the first 14 d after germination. We measured spatial profiles of longitudinal velocity and cell length and calculated parameters of cell expansion and division, including rates of local cell production (cells mm⁻¹ h⁻¹) and cell division (cells cell⁻¹ h⁻¹). Data were obtained for the root cortex and also for the two types of epidermal cell, trichoblasts and atrichoblasts. Accelerating root elongation was caused by an increasingly longer growth zone, while maximal strain rates remained unchanged. The enlargement of the growth zone and, hence, the accelerating root elongation rate, were accompanied by a nearly proportionally increased cell production. This increased production was caused by increasingly numerous dividing cells, whereas their rates of division remained approximately constant. Additionally, the spatial profile of cell division rate was essentially constant. The meristem was longer than generally assumed, extending well into the region where cells elongated rapidly. In the two epidermal cell types, meristem length and cell division rate were both very similar to that of cortical cells, and differences in cell length between the two epidermal cell types originated at the apex of the meristem. These results highlight the importance of controlling the number of dividing cells, both to generate tissues with different cell lengths and to regulate the rate of organ enlargement.     

Self-Transmissible Mercury Resistance Plasmids with Gene-Mobilizing Capacity in Soil Bacterial Populations: Influence of Wheat Roots and Mercury Addition

A set of mercury resistance plasmids was obtained from wheat rhizosphere soil amended or not amended with mercuric chloride via exogenous plasmid isolation by using Pseudomonas fluorescens R2f, Pseudomonas putida UWC1, and Enterobacter cloacae BE1 as recipient strains. The isolation frequencies were highest from soil amended with high levels of mercury, and the isolation frequencies from unamended soil were low. With P. putida UWC1 as the recipient, the isolation frequency was significantly enhanced in wheat rhizosphere compared to bulk soil. Twenty transconjugants were analyzed per recipient strain. All of the transconjugants contained plasmids which were between 40 and 50 kb long. Eight selected plasmids were distributed among five groups, as shown by restriction digestion coupled with a similarity matrix analysis. However, all of the plasmids formed a tight group, as judged by hybridization with two whole-plasmid probes and comparisons with other plasmids in dot blot hybridization analyses. The results of replicon typing and broad-host-range incompatibility (Inc) group-specific PCR suggested that the plasmid isolates were not related to any previously described Inc group. Although resistance to copper, resistance to streptomycin, and/or resistance to chloramphenicol was found in several plasmids, catabolic sequences were generally not identified. One plasmid, pEC10, transferred into a variety of bacteria belonging to the β and γ subdivisions of the class Proteobacteria and mobilized as well as retromobilized the IncQ plasmid pSUP104. A PCR method for detection of pEC10-like replicons was used, in conjunction with other methods, to monitor pEC10-homologous sequences in mercury-polluted and unpolluted soils. The presence of mercury enhanced the prevalence of pEC10-like replicons in soil and rhizosphere bacterial populations.The potential use of genetically modified bacteria in agriculture has raised questions pertaining to the spread of introduced recombinant DNA through soil bacterial communities. Gene transfer in soil via conjugation has received much attention, and the focus of most studies has been the transfer and fate of introduced plasmids (6, 22, 27–29, 39). Under favorable conditions, in specific soil microhabitats, or under selection conditions, both self-transmissible and mobilizable plasmids present in introduced hosts can be transferred to introduced recipients, as well as to a variety of indigenous bacteria (15, 20, 27, 28, 33). In particular, rhizospheres of crop plants, such as wheat and sugar beet, provide conditions conducive to conjugal plasmid transfer between bacterial inhabitants (15, 36). When genetically modified bacteria are developed as inoculants for the rhizosphere, insertion of heterologous DNA into non-self-transmissible plasmids or the chromosome might restrict conjugal transfer of this DNA to members of the indigenous bacterial community. However, mobilizing or retromobilizing (33) plasmids present in indigenous soil bacteria could potentially still effect the transfer of the less mobile heterologous DNA via chromosome or plasmid mobilization, which may involve cointegration (9, 19, 31). Such plasmids might thus be responsible for the escape of heterologous DNA from genetically modified bacteria introduced into soil.There is a paucity of knowledge concerning the incidence of plasmids with mobilizing capacity in soils and rhizospheres, as well as concerning the effects of soil factors, such as stresses resulting from pollution or from natural causes (e.g., rhizosphere acidity), on plasmid prevalence and transfer (e.g., reference 38). Whereas it has been suggested that chemical stress often does not enhance plasmid incidence in selected soil bacterial populations (40), pollution in river water or mines (in particular mercury pollution) has been found to exert a selective (enhancing) effect (4, 13).Plasmids of environmental bacteria have classically been obtained by endogenous isolation procedures (20). Endogenous isolation implies that putative plasmid hosts with the phenotype of interest are isolated from soil, after which plasmids are extracted from pure cultures of these strains. On the other hand, pioneering studies performed with river stone epilithon (9) and later extended to soil and sediment (32) have shown that plasmids can be obtained directly from indigenous bacterial communities in new hosts by exogenous isolation. In this approach, plasmids are captured in selectable recipient strains by using mating between these strains and the total bacterial community obtained from an environmental sample. Following incubation, the mating mixture is plated with selection for the recipient and an additional marker gene presumedly located on a plasmid present in the indigenous bacteria (6). The advantage of the exogenous isolation procedure is that no culturing step is required in the mating, which thus allows isolation of plasmids from nonculturable hosts. Furthermore, plasmids are directly selected for their transfer capacity, in addition to the presence of a specific selectable marker.In this study, exogenous plasmid isolation was employed to obtain transferable plasmids from soil bacteria by using mercury resistance as the selectable marker. The objective of this work was to gain insight into the potential present in soil bacterial populations to (retro)mobilize genes out of introduced bacteria into members of the soil bacterial community. Since the incidence of plasmids in soil bacteria is likely influenced by soil ecological factors and selection pressure, the presence of wheat roots and selection by mercury (25) were studied as experimental variables.     

Characterization of Recombinant Rhamnogalacturonan α-l-Rhamnopyranosyl-(1,4)-α-d-Galactopyranosyluronide Lyase from Aspergillus aculeatus : An Enzyme That Fragments Rhamnogalacturonan I Regions of Pectin

The four major oligomeric reaction products from saponified modified hairy regions (MHR-S) from apple, produced by recombinant rhamnogalacturonan (RG) α-l-rhamnopyranosyl-(1,4)-α-d-galactopyranosyluronide lyase (rRG-lyase) from Aspergillus aculeatus, were isolated and characterized by ¹H-nuclear magnetic resonance spectroscopy. They contain an alternating RG backbone with a degree of polymerization of 4, 6, 8, and 10 and with an α-Δ-(4,5)-unsaturated d-galactopyranosyluronic acid at the nonreducing end and an l-rhamnopyranose at the reducing end. l-Rhamnopyranose units are substituted at C-4 with β-galactose. The maximum reaction rate of rRG-lyase toward MHR-S at pH 6.0 and 31°C was 28 units mg⁻¹. rRG-lyase and RG-hydrolase cleave the same alternating RG I subunit in MHR. Both of these enzymes fragment MHR by a multiple attack mechanism. The catalytic efficiency of rRG-lyase for MHR increases with decreasing degree of acetylation. Removal of arabinose side chains improves the action of rRG-lyase toward MHR-S. In contrast, removal of galactose side chains decreased the catalytic efficiency of rRG-lyase. Native RG-lyase was purified from A. aculeatus, characterized, and found to be similar to the rRG-lyase expressed in Aspergillus oryzae.     

Rare hybridization and introgression in smooth and palmate newts (Salamandridae: Triturus vulgaris and T. helveticus)

This paper describes the occurrence of hybridization and introgression in two species of amphibians (the newts Triturus helveticus and Triturus vulgaris ) in mid-Wales, northern France and western France. A single aberrant adult male with intermediate phenotype was found. The multivariate analysis of 14 morphometric and two meristic characters supported its hybrid status. Electrophoretic analysis of 42 protein loci showed a genetic distance of 0.57 ± 0.14 Nei units between the species and revealed 15 diagnostic loci. The aberrant specimen was heterozygous at 11 of those and most likely to be a F₁ hybrid. Four marker loci appeared homozygous, suggesting the presence of enzymatically non-active ('null') alleles. The analysis of (maternally inherited) mitochondrial DNA showed the hybrid to be the offspring of a T. helveticus mother (and a T. vulgaris father). This observation does not conform to expectations based on the species composition in the pond from which the hybrid was collected. No F₁ hybrids were observed in a large sample (n > 5000) of larvae, recently metamorphosed newts and adults using two diagnostic protein loci. Occasionally alleles characteristic for one species were observed in the gene pool of the other species, suggesting the presence of bidirectional introgression. However, the frequency of alien genes was low (maximally 0.07%) which renders it difficult to rule out alternative explanations conclusively. The increase in total genetic variation in T. helveticus and T. vulgaris due to gene flow between them is negligible.     

A constitutive model for the time-dependent,nonlinear stress response of fibrin networks

Blood clot formation is important to prevent blood loss in case of a vascular injury but disastrous when it occludes the vessel. As the mechanical properties of the clot are reported to be related to many diseases, it is important to have a good understanding of their characteristics. In this study, a constitutive model is presented that describes the nonlinear viscoelastic properties of the fibrin network, the main structural component of blood clots. The model is developed using results of experiments in which the fibrin network is subjected to a large amplitude oscillatory shear (LAOS) deformation. The results show three dominating nonlinear features: softening over multiple deformation cycles, strain stiffening and increasing viscous dissipation during a deformation cycle. These features are incorporated in a constitutive model based on the Kelvin–Voigt model. A network state parameter is introduced that takes into account the influence of the deformation history of the network. Furthermore, in the period following the LAOS deformation, the stiffness of the networks increases which is also incorporated in the model. The influence of cross-links created by factor XIII is investigated by comparing fibrin networks that have polymerized for 1 and 2 h. A sensitivity analysis provides insights into the influence of the eight fit parameters. The model developed is able to describe the rich, time-dependent, nonlinear behavior of the fibrin network. The model is relatively simple which makes it suitable for computational simulations of blood clot formation and is general enough to be used for other materials showing similar behavior.     

Infection with Soil-Transmitted Helminths Is Associated with Increased Insulin Sensitivity

Objective

Given that helminth infections have been shown to improve insulin sensitivity in animal studies, which may be explained by beneficial effects on energy balance or by a shift in the immune system to an anti-inflammatory profile, we investigated whether soil-transmitted helminth (STH)-infected subjects are more insulin sensitive than STH-uninfected subjects.

Design

We performed a cross-sectional study on Flores island, Indonesia, an area with high prevalence of STH infections.

Methods

From 646 adults, stool samples were screened for Trichuris trichiura by microscopy and for Ascaris lumbricoides, Necator americanus, Ancylostoma duodenale, and Strongyloides stercoralis by qPCR. No other helminth was found. We collected data on body mass index (BMI, kg/m²), waist-to-hip ratio (WHR), fasting blood glucose (FBG, mmol/L), insulin (pmol/L), high sensitive C-reactive protein (ng/ml) and Immunoglobulin E (IU/ml). The homeostatic model assessment for insulin resistance (HOMAIR) was calculated and regression models were used to assess the association between STH infection status and insulin resistance.

Results

424 (66%) participants had at least one STH infection. STH infected participants had lower BMI (23.2 vs 22.5 kg/m², p value = 0.03) and lower HOMAIR (0.97 vs 0.81, p value = 0.05). In an age-, sex- and BMI-adjusted model a significant association was seen between the number of infections and HOMAIR: for every additional infection with STH species, the HOMAIR decreased by 0.10 (p for linear trend 0.01). This effect was mainly accounted for by a decrease in insulin of 4.9 pmol/L for every infection (p for trend = 0.07).

Conclusion

STH infections are associated with a modest improvement of insulin sensitivity, which is not accounted for by STH effects on BMI alone.