Yeast communities associated with stingless bees

The yeast communities associated with the stingless bees Tetragonisca angustula, Melipona quadrifasciata and Frieseomelitta varia were studied. The bees T. angustula and F. varia showed a strong association with the yeast Starmerella meliponinorum. M. quadrifasciata more frequently carried a species related to Candida apicola, but also vectored low numbers of S. meliponinorum. Some of the yeasts isolated from adult bees were typical of species known to occur in flowers. Other yeast species found in adult bees were more typical of those found in the phylloplane. S. meliponinorum and the species in the C. apicola complex, also part of the Starmerella clade, may have a mutualistic relationship with the bees studied. Many yeasts in that group are often found in bees or substrates visited by bees, suggesting that a mutually beneficial interaction exists between them.     

Carbohydrate uptake in Advenella mimigardefordensis strain DPN7T is mediated by periplasmic sugar oxidation and a TRAP‐transport system

In this study, we investigated an SBP (DctP_Am) of a tripartite ATP‐independent periplasmic transport system (TRAP) in Advenella mimigardefordensis strain DPN7^T. Deletion of dctP_Am as well as of the two transmembrane compounds of the tripartite transporter, dctQ and dctM, impaired growth of A. mimigardefordensis strain DPN7^T, if cultivated on mineral salt medium supplemented with d ‐glucose, d ‐galactose, l ‐arabinose, d ‐fucose, d ‐xylose or d ‐gluconic acid, respectively. The wild type phenotype was restored during complementation studies of A. mimigardefordensis ΔdctP_Am using the broad host vector pBBR1MCS‐5::dctP_Am. Furthermore, an uptake assay with radiolabeled [¹⁴C(U)]‐d ‐glucose clearly showed that the deletion of dctP_Am, dctQ and dctM, respectively, disabled the uptake of this aldoses in cells of either mutant strain. Determination of K_D performing thermal shift assays showed a shift in the melting temperature of DctP_Am in the presence of d ‐gluconic acid (K_D 11.76 ± 1.3 µM) and the corresponding aldonic acids to the above‐mentioned carbohydrates d ‐galactonate (K_D 10.72 ± 1.4 µM), d ‐fuconic acid (K_D 13.50 ± 1.6 µM) and d ‐xylonic acid (K_D 8.44 ± 1.0 µM). The sugar (glucose) dehydrogenase activity (E.C.1.1.5.2) in the membrane fraction was shown for all relevant sugars, proving oxidation of the molecules in the periplasm, prior to transport.     

Estimation of the quantity of bacteria encapsulated in Lentikats Biocatalyst via phospholipid fatty acids content: a preliminary study

The content of phospholipid fatty acids (PLFA) was determined in samples of polyvinyl alcohol lenses (Lentikats Biocatalyst, LB) with encapsulated Paracoccus denitrificans withdrawn during long-term denitrification experiments. The total PLFA content correlated highly with specific denitrification activities of LB as well as biomass estimation based on image analyses of microscopic photos. The results confirmed the applicability of PLFA determination for estimation of the amount of living encapsulated microbial biomass during biotechnological applications.     

Enantioselective synthesis of amines via reductive amination with a dehydrogenase mutant from Exigobacterium sibiricum: Substrate scope,co-solvent tolerance and biocatalyst immobilization

In recent years, the reductive amination of ketones in the presence of amine dehydrogenases emerged as an attractive synthetic strategy for the enantioselective preparation of amines starting from ketones, an ammonia source, a reducing reagent and a cofactor, which is recycled in situ by means of a second enzyme. Current challenges in this field consists of providing a broad synthetic platform as well as process development including enzyme immobilization. In this contribution these issues are addressed. Utilizing the amine dehydrogenase EsLeuDH-DM as a mutant of the leucine dehydrogenase from Exigobacterium sibiricum, a range of aryl-substituted ketones were tested as substrates revealing a broad substrate tolerance. Kinetics as well as inhibition effects were also studied and the suitability of this method for synthetic purpose was demonstrated with acetophenone as a model substrate. Even at an elevated substrate concentration of 50?mM, excellent conversion was achieved. In addition, the impact of water-miscible co-solvents was examined, and good activities were found when using DMSO of up to 30% (v/v). Furthermore, a successful immobilization of the EsLeuDH-DM was demonstrated utilizing a hydrophobic support and a support for covalent binding, respectively, as a carrier.     

Establishment of introduced antagonistic bacteria in the rhizosphere of transgenic potatoes and their effect on the bacterial community

In a field release experiment, rifampicin resistant mutants of two antagonistic plant-associated bacteria were used for seed tuber inoculation of transgenic T4 lysozyme expressing potatoes, transgenic control potatoes and non-transgenic parental potatoes. The T4 lysozyme tolerant Pseudomonas putida QC14-3-8 was originally isolated from the tuber surface (geocaulosphere) of T4 lysozyme producing plants and showed in vitro antibacterial activity to the bacterial pathogen Erwinia carotovora ssp. atroseptica. The T4 lysozyme sensitive Serratia grimesii L16-3-3 was originally isolated from the rhizosphere of parental potatoes and showed in vitro antagonism toward the plant pathogenic fungus Verticillium dahliae. The establishment of the inoculated bacteria in the rhizosphere and geocaulosphere of the different plant lines was monitored over one growing season to assess the effect of T4 lysozyme produced by transgenic potato plants on the survival of both inoculants. Both introduced isolates were able to colonize the rhizo- and geocaulosphere of transgenic plants and non-transgenic parental plants, and established in the rhizosphere at levels of ca. log(10) 5 colony forming units g(-1) fresh weight of root. During flowering of plants, significantly more colony counts of the T4 lysozyme tolerant P. putida were recovered from transgenic T4 lysozyme plants than from the transgenic control and the parental line. At this time, the highest level of T4 lysozyme (% of total soluble protein) was detected. Effects of the inoculants on the indigenous microbial community were monitored by analysis of PCR-amplified fragments of the 16S rRNA genes of the whole bacterial community after separation by denaturing gradient gel electrophoresis (DGGE). At any sampling time, the DGGE pattern of rhizosphere and geocaulosphere communities did not show differences between the inoculated and non-inoculated potatoes. Neither of the introduced strains became a dominant member of the bacterial community. This work was the first approach to assess the establishment of plant growth promoting rhizobacteria and potential biocontrol agents on transgenic plants.     

Cations and hydration in catalytic RNA: molecular dynamics of the hepatitis delta virus ribozyme

The hepatitis delta virus (HDV) ribozyme is an RNA enzyme from the human pathogenic HDV. Cations play a crucial role in self-cleavage of the HDV ribozyme, by promoting both folding and chemistry. Experimental studies have revealed limited but intriguing details on the location and structural and catalytic functions of metal ions. Here, we analyze a total of approximately 200 ns of explicit-solvent molecular dynamics simulations to provide a complementary atomistic view of the binding of monovalent and divalent cations as well as water molecules to reaction precursor and product forms of the HDV ribozyme. Our simulations find that an Mg2+ cation binds stably, by both inner- and outer-sphere contacts, to the electronegative catalytic pocket of the reaction precursor, in a position to potentially support chemistry. In contrast, protonation of the catalytically involved C75 in the precursor or artificial placement of this Mg2+ into the product structure result in its swift expulsion from the active site. These findings are consistent with a concerted reaction mechanism in which C75 and hydrated Mg2+ act as general base and acid, respectively. Monovalent cations bind to the active site and elsewhere assisted by structurally bridging long-residency water molecules, but are generally delocalized.     

Contribution of leaves of different ages to plant carbon balance as affected by potassium supply and water stress

The carbon balances of whole, 21-d old French bean plants (Phaseolus vulgaris L.) grown in standard nutrient solution (1K) and its modifications without (OK) or surplus (2K) potassium were calculated from the daily photosynthetic carbon inputs of individual leaves, and the daily respiratory carbon losses by individual leaves, stalks and petioles, and roots. Under the three K concentrations, maximum net photosynthetic rates (P_n) were found in the 2nd or in the 3rd trifoliate leaves, maximum respiratory rates (R_d) in the youngest, 4th trifoliate leaves; the P_n/R_d ratio decreased with leaf age. In all leaves of 2K plants, leaf dry masses and thicknesses, P_n, P_n/P_d ratios, and stomatal and intracellular conductances were lower than in OK and IK plants. Daily whole-plant net carbon gain was highest in IK plants, whereas in OK and 2K plants it was 98.0 and 81.3 % of IK, respectively. Similar values were found in the parameters of growth analysis, namely in net assimilation rates and relative growth rates. No differences were found in water potential (Ψ _w ) or water saturation deficit (W_sat) in the OK, 1K and 2K plants sufficiently supplied with water or during wilting and resaturation. The decrease in Ψ_w to −0.97 MPa was associated with a 19.9 %, 31.4 % and 23.4 % decrease in P_n of OK, 1K and 2K plants, respectively, but no effect on R_d was found. In the three variants, the short-time effect of mild water stress was fully reversible.     

Endolysin of bacteriophage BFK20: evidence of a catalytic and a cell wall binding domain

A gene product of ORF24' was identified on the genome of corynephage BFK20 as a putative phage endolysin. The protein of endolysin BFK20 (gp24') has a modular structure consisting of an N-terminal amidase_2 domain (gp24CD) and a C-terminal cell wall binding domain (gp24BD). The C-terminal domain is unrelated to any of the known cell wall binding domains of phage endolysins. The whole endolysin gene and the sequences of its N-terminal and C-terminal domains were cloned; proteins were expressed in Escherichia coli and purified to homogeneity. The lytic activities of endolysin and its catalytic domain were demonstrated on corynebacteria and bacillus substrates. The binding activity of cell wall binding domain alone and in fusion with green fluorescent protein (gp24BD-GFP) were shown by specific binding assays to the cell surface of BFK20 host Brevibacterium flavum CCM 251 as well as those of other corynebacteria.     

Imbalanced circulating matrix metalloproteinases in polycystic ovary syndrome

Altered levels of matrix metalloproteinases (MMPs) may reflect relevant pathogenetic mechanisms of disease conditions. The objective of this study was to compare the plasma levels of MMPs and tissue inhibitors of MMPs (TIMPs) in polycystic ovary syndrome (PCOS) patients with those found in healthy ovulatory controls and to examine whether the levels of these biomarkers are associated with clinical and biochemical features of this syndrome. Sixty-five healthy ovulatory subjects (controls) and 80 patients with PCOS were include in this study. MMP-2, MMP-8, MMP-9, TIMP-1, TIMP-2 concentrations were measured in plasma samples by gelatin zymography or enzyme-linked immunoassays. MMP-2, MMP-8, MMP-9, and TIMP-1 levels were similar in PCOS patients and in healthy controls (P > 0.05). PCOS patients had lower plasma TIMP-2 levels than healthy controls (P < 0.05). We found higher MMP-2/TIMP-2 and MMP-9/TIMP-1 ratios in PCOS patients than in healthy controls (all P < 0.05). Testosterone levels correlated positively with the MMP-9/TIMP-1 ratio and negatively with TIMP-2 levels (r = 0.26, P < 0.01 and r = -0.21, P = 0.02, respectively). In addition, only testosterone was an independent predictor of TIMP-2 levels (estimate = -0.35, P = 0.04) and the MMP-9/TIMP-1 ratio (estimate = 0.01, P = 0.04). We found evidence indicating that the balance between MMPs and TIMPs in women with PCOS is altered, probably due to androgen excess found in these women.     

Extinction Risk Escalates in the Tropics

The latitudinal biodiversity gradient remains one of the most widely recognized yet puzzling patterns in nature [1]. Presently, the high level of extinction of tropical species, referred to as the “tropical biodiversity crisis”, has the potential to erode this pattern. While the connection between species richness, extinction, and speciation has long intrigued biologists [2], [3], these interactions have experienced increased poignancy due to their relevancy to where we should concentrate our conservation efforts. Natural extinction is a phenomenon thought to have its own latitudinal gradient, with lower extinction rates in the tropics being reported in beetles, birds, mammals, and bivalves [4]–[7]. Processes that have buffered ecosystems from high extinction rates in the past may also buffer ecosystems against disturbance of anthropogenic origin. While potential parallels between historical and present-day extinction patterns have been acknowledged, they remain only superficially explored and plant extinction patterns have been particularly neglected. Studies on the disappearances of animal species have reached conflicting conclusions, with the rate of extinction appearing either higher [8] or lower [9] in species richness hotspots. Our global study of extinction risk in vascular plants finds disproportionately higher extinction risk in tropical countries, even when indicators of human pressure (GDP, population density, forest cover change) are taken into account. Our results are at odds with the notion that the tropics represent a museum of plant biodiversity (places of historically lowered extinction) and we discuss mechanisms that may reconcile this apparent contradiction.