Bathy Phytochromes in Rhizobial Soil Bacteria

Phytochromes are biliprotein photoreceptors that are found in plants, bacteria, and fungi. Prototypical phytochromes have a Pr ground state that absorbs in the red spectral range and is converted by light into the Pfr form, which absorbs longer-wavelength, far-red light. Recently, some bacterial phytochromes have been described that undergo dark conversion of Pr to Pfr and thus have a Pfr ground state. We show here that such so-called bathy phytochromes are widely distributed among bacteria that belong to the order Rhizobiales. We measured in vivo spectral properties and the direction of dark conversion for species which have either one or two phytochrome genes. Agrobacterium tumefaciens C58 contains one bathy phytochrome and a second phytochrome which undergoes dark conversion of Pfr to Pr in vivo. The related species Agrobacterium vitis S4 contains also one bathy phytochrome and another phytochrome with novel spectral properties. Rhizobium leguminosarum 3841, Rhizobium etli CIAT652, and Azorhizobium caulinodans ORS571 contain a single phytochrome of the bathy type, whereas Xanthobacter autotrophicus Py2 contains a single phytochrome with dark conversion of Pfr to Pr. We propose that bathy phytochromes are adaptations to the light regime in the soil. Most bacterial phytochromes are light-regulated histidine kinases, some of which have a C-terminal response regulator subunit on the same protein. According to our phylogenetic studies, the group of phytochromes with this domain arrangement has evolved from a bathy phytochrome progenitor.Phytochromes are biological photoreceptors that were discovered in plants, where they control development throughout the life cycle in manifold ways (21, 33). Today, a large number of homologs are known also from cyanobacteria, other bacteria, and fungi, which are termed cyanobacterial phytochromes (Cphs), bacteriophytochromes (BphPs), and fungal phytochromes (Fphs), respectively (20, 24). The chromophore is autocatalytically assembled within the N-terminal part of the protein, the photosensory core module (PCM), which contains the PAS, GAF, and PHY domains (30). Typically, phytochromes are converted by light between two spectrally different forms, the red-absorbing Pr and the far-red-absorbing Pfr forms. Photoconversion is initiated by an isomerization of the covalently bound bilin chromophore (32).Plant and cyanobacterial phytochromes incorporate phytochromobilin (PΦB) and phycocyanobilin (PCB) as natural chromophores, respectively, which are covalently bound to Cys residues in the GAF domains. All characterized phytochromes that belong to these groups have a Pr ground state. Plant phytochromes can undergo dark conversion of Pfr to Pr (5), whereas the Pfr form of typical cyanobacterial phytochromes is stable in darkness (26).Bacteriophytochromes utilize biliverdin (BV) instead as a natural chromophore (1), which is covalently attached to a Cys residue in the N terminus of the PAS domain (26). Since the conjugated system of BV is longer than that of PΦB or PCB, the absorption maxima of bacteriophytochromes are found at higher wavelengths than those of cyanobacterial or plant homologs.With the discovery of a bacterial phytochrome from Bradyrhizobium sp. strain ORS278, termed BrBphP1, the first phytochrome with a Pfr ground state and dark conversion from Pr to Pfr was found (10). Thereafter, five more phytochromes with dark conversion of Pr to Pfr were described: Rhodopseudomonas palustris BphP1 (RpBphP1) from strain CEA001, RpBphP5, and RpBphP6 from strain CGA009 (11); Agrobacterium tumefaciens Agp2 (or AtBphP2) from strain C58 (18); and Pseudomonas aeruginosa BphP1 (PaBphP1) (40). These phytochromes are now termed bathy phytochromes because the absorption maxima of their ground states are bathochromically (to longer wavelengths) shifted compared to those of all other phytochromes.Moreover, some other bacterial phytochromes with unusual properties have been described. In the Ppr from Rhodospirillum centenum, a photoactive yellow protein (PYP) domain is fused to the N terminus of a phytochrome homolog. The phytochrome part of Ppr assembles with BV to form a Pr adduct. However, irradiation does not result in the formation of Pfr but in a bleaching of the Pr spectrum (23). The BV adduct of RpBphP3 from R. palustris, which has a Pr ground state, photoconverts to the so-called Pnr form with a blue-shifted absorption maximum (12). RpBphP4 from R. palustris strains Ha2 and BisB5 and Bradyrhizobium BphP3 (BrBphP3) from Bradyrhizobium BTAi1, both with a Pr ground state, photoconvert into a long-lived MetaR form (8, 42). MetaRa and MetaRc are intermediates in the photoconversion from Pr to Pfr of prototypical phytochromes (3). BphP3 from the Bradyrhizobium strain ORS 278 is an exception among bacteriophytochromes as it binds PCB as a natural chromophore. This phytochrome adopts a so-called Po (P-orange) ground state with an absorbance maximum in the orange range (11, 15). Upon irradiation, this phytochrome converts into the Pr form. RpBphP4 from R. palustris CGA009 lacks the biliverdin binding cysteine and does not bind a chromophore (42).With the rapidly growing number of bacterial genome sequences, many new bacterial phytochromes are being discovered. Thus, a large and increasing number of newly identified phytochromes remain spectroscopically uncharacterized. We established an in vivo photometry approach which allowed the rapid acquisition of spectral information about phytochromes from intact bacterial cells. In the beginning period of plant phytochrome research, in vivo photometry was extensively applied (4, 6, 29, 34). This method, in fact, allowed the identification of phytochromes for the first time in plant tissues (6), which led to the purification of phytochromes from plant extracts (37). Here, we apply in vivo photometry for the first time to organisms outside the plant kingdom. This method is especially useful for studying species with single phytochrome genes. The approach is also helpful for comparing properties of native phytochromes in vivo and of their recombinant proteins in vitro.In the present study, we concentrate on nonphotosynthetic species of the order Rhizobiales which belongs to the Alphaproteobacteria. The family Rhizobiaceae comprises plant-interacting soil bacteria. A. tumefaciens and Agrobacterium vitis can transfer genes into plants to induce plant tumors, whereas many other Rhizobiaceae can live as plant symbionts in nodules of stems or roots in which they assimilate molecular nitrogen to produce NH₄⁺, which is used by the plant for synthesis of amino acids and other nitrogen-containing molecules. A. tumefaciens C58 contains two phytochromes, termed Agp1 (or AtBphP1) and Agp2 (or AtBphP2), that have been characterized as recombinant proteins (14, 18, 26, 35) and whose spectral activities have been measured in extracts of wild-type and knockout mutants (31). A large number of phytochromes from photosynthetic Bradyrhizobium and Rhodopseudomonas species, which also belong to the order Rhizobiales, have been characterized as recombinant proteins (11), some of which have already been noted above.It turned out that most of our analyzed phytochromes undergo dark conversion of Pr to Pfr and thus belong to the group of bathy phytochromes. Such phytochromes, which absorb at around 750 nm, clearly dominate among Rhizobiales. We propose that this specific property reflects an adaptation to the light regime in the soil. Our studies also suggest that bacterial phytochromes with a C-terminal response regulator have evolved from a bathy phytochrome progenitor.     

Diversity of plant evolutionary lineages promotes arthropod diversity

Large‐scale habitat destruction and climate change result in the non‐random loss of evolutionary lineages, reducing the amount of evolutionary history represented in ecological communities. Yet, we have limited understanding of the consequences of evolutionary history on the structure of food webs and the services provided by biological communities. Drawing on 11 years of data from a long‐term plant diversity experiment, we show that evolutionary history of plant communities – measured as phylogenetic diversity – strongly predicts diversity and abundance of herbivorous and predatory arthropods. Effects of plant species richness on arthropods become stronger when phylogenetic diversity is high. Plant phylogenetic diversity explains predator and parasitoid richness as strongly as it does herbivore richness. Our findings indicate that accounting for evolutionary relationships is critical to understanding the severity of species loss for food webs and ecosystems, and for developing conservation and restoration policies.     

Gd-EOB-DTPA-Enhanced MR Guidance in Thermal Ablation of Liver Malignancies

Objective

To evaluate the potency of Gd-EOB-DTPA to support hepatic catheter placement in laser ablation procedures by quantifying time-dependent delineation effects for instrumentation and target tumor within liver parenchyma. Monitoring potential influence on online MR thermometry during the ablation procedure is a secondary aim.

Materials and Methods

30 cases of MR-guided laser ablation were performed after i.v. bolus injection of gadoxetic acid (0.025 mmol/Kg Gd-EOB-DTPA; Bayer Healthcare, Berlin, Germany). T₁-weighted GRE sequences were used for applicator guidance (FLASH 3D) in the catheter placement phase and for therapy monitoring (FLASH 2D) in the therapy phase. SNR and consecutive CNR values were measured for elements of interest plotted over time both for catheter placement and therapy phase and compared with a non-contrast control group of 19 earlier cases. Statistical analysis was realized using the paired Wilcoxon test.

Results

Sustainable signal elevation of liver parenchyma in the contrast-enhanced group was sufficient to silhouette both target tumor and applicator against the liver. Differences in time dependent CNR alteration were highly significant between contrast-enhanced and non-contrast interventions for parenchyma and target on the one hand (p = 0.020) and parenchyma and instrument on the other hand (p = 0.002). Effects lasted for the whole procedure (monitoring up to 60 min) and were specific for the contrast-enhanced group. Contrasting maxima were seen after median 30 (applicator) and 38 (tumor) minutes, in the potential core time of a multineedle procedure. Contrast influence on T1 thermometry for real-time monitoring of thermal impact was not significant (p = 0.068–0.715).

Conclusion

Results strongly support anticipated promotive effects of Gd-EOB-DTPA for MR-guided percutaneous liver interventions by proving and quantifying the delineating effects for therapy-relevant elements in the procedure. Time benefit, cost effectiveness and oncologic outcome of the described beneficiary effects will have to be part of further investigations.     

Dynamics of plant and arthropod diversity during old field succession

The successional dynamics of arthropod diversity in 18 abandoned agricultural fields (age 15-54 yr) at Cedar Creek. MN. USA were determined using sweep net sampling (44833 individuals of 618 species). Total arthropod species richness and equitability (J), but not abundance, increased significantly with field successional age. Herbivore and parasite species richness, but not detritivore and predator species richness, also increased significantly with field age. All of these arthropod variables were significantly positively correlated with plant species richness in the fields. When plant species richness was included as a covariate in regressions, there were no longer any significant effects of field age. These results supported the hypothesis that increases in arthropod diversity with field age are influenced by increases in plant diversity. The additional significant positive dependence of herbivore species richness on predator species richness suggests that predator-prey interactions may also influence the successional dynamics of arthropod diversity. Nine of the ten most common arthropod species decreased in abundance with field age, two of them significantly. The abundances of these two generalist forb-feeding species, Melanoplus femurrubrum (Orthoptera: Acrididae) and Scaphytopius acutus (Homoptera: Cicadellidae). each depended significantly on amount of forbs. The average body size of arthropod species (total and herbivores) decreased significantly with field age. An efficiency vs specialization hypothesis predicts such a decrease. Because plants in later secondary succession are generally less palatable, a diversity of smaller, potentially more specialized herbivores may have an advantage over larger and more efficient herbivores in later succession.     

Tree diversity,tree height and environmental harshness in eastern and western North America

Does variation in environmental harshness explain local and regional species diversity gradients? We hypothesise that for a given life form like trees, greater harshness leads to a smaller range of traits that are viable and thereby also to lower species diversity. On the basis of a strong dependence of maximum tree height on site productivity and other measures of site quality, we propose maximum tree height as an inverse measure of environmental harshness for trees. Our results show that tree species richness is strongly positively correlated with maximum tree height across multiple spatial scales in forests of both eastern and western North America. Maximum tree height co‐varied with species richness along gradients from benign to harsh environmental conditions, which supports the hypothesis that harshness may be a general mechanism limiting local diversity and explaining diversity gradients within a biogeographic region.     

Detection in rabbit sera of blocking antibodies against staphylococcal fibronectin-binding protein by enzyme-linked immunosorbent assay

Antibody titres against fibronectin-binding protein (FnBP) of Staphylococcus aureus were determined in sera from rabbits immunized with staphylococcal whole cells or purified native fibronectin receptor. An ELISA technique for detection of antibody titres blocking the binding of soluble fibronectin to immobilized FnBP was developed. A recombinant staphylococcal FnBP fused to E. coli beta-galactosidase (gal-FnBp) was used as the immobilized antigen in this test. Serum samples from two different rabbits immunized with native fibronectin receptor gave significant blocking titres, whereas the blocking titres of antisera against staphylococcal whole cells were about 4- to 5-fold lower. Using the gal-FnBP fusion protein, the sensitivity for detection of fibronectin by ELISA was also determined. The detection limit is around 5 ng. The findings are discussed with a view to developing an anti-staphylococcal adherence vaccine and quantitating fibronectin in solution.     

Expression in Bacillus subtilis of the gene for human urogastrone using synthetic ribosome binding sites

Summary A chemically synthesised gene coding for human urogastrone which was earlier cloned in E. coli (Smith et al. 1982) has now been cloned into expression vectors for Bacillus subtilis Two types of constructs have been made, one giving production of methionylurogastrone and the other giving rise to a methionyl-urogastrone- galactosidase fusion polypeptide facilitating quantification of expression levels.The ribosome binding sites used in the expression plasmids are synthetically made oligonucleotides residing on short restriction fragments to allow easy replacement by other ribosome binding sites.Using shuttle vectors and constitutive promoters from Bacillus phages 105 and SPP1, we were able to detect levels of expression amounting to a few thousand molecules per cell during logarithmic growth in both E. coli and B. subtilis.     

Vaccination with Staphylococcus aureus fibrinogen binding proteins (FgBPs) reduces colonisation of S. aureus in a mouse mastitis model

Abstract A mouse mastitis model was used to study the effect of vaccination with fibrinogen binding proteins and collagen binding protein from Staphylococcus aureus against challenge infection with S. aureus . The mice vaccinated with fibrinogen binding proteins showed reduced rates of mastitis compared with controls. Gross examination of challenged mammary glands of mice showed that the glands of mice immunized with fibronogen binding proteins developed mild intramammary infection or had no pathological changes compared with glands from control mice. Histopathological examination of tissue sections from challenged glands showed that most glands from mice vaccinated with fibrinogen binding protein developed disseminated necrosis or had no pathological changes. A significantly reduced number of bacteria could be recovered in the glands from mice immunized with fibrinogen binding proteins as compared with controls. In a similar study, immunization of mice with collagen binding protein did not induce protection against challenge infection with S. aureus .