Population Status of the Critically Endangered Preuss’s Red Colobus Monkey (Piliocolobus preussi Matschie 1900) and Recommendations for Its Conservation

International Journal of Primatology - Overhunting and habitat loss from the expansion of agriculture and extractive industries are the primary threats to primate species, 65% of which are...     

Structure,dynamics, and ionization equilibria of the tyrosine residues in <Emphasis Type="Italic">Bacillus circulans</Emphasis> xylanase

We have developed NMR spectroscopic methods to investigate the tyrosines within Bacillus circulans xylanase (BcX). Four slowly exchanging buried tyrosine hydroxyl protons with chemical shifts between 7.5 and 12.5 ppm were found using a long-range ¹³C-HSQC experiment that exploits the ³J_CH coupling between the ring ¹H^η and ¹³C^ε nuclei. The NMR signals from these protons were assigned via ¹³C-tyrosine selective labelling and a suite of scalar and ¹³C,¹⁵N-filtered/edited NOE correlation spectra. Of the fifteen tyrosines in BcX, only the buried Tyr79 and Tyr105 showed four distinct, rather than two averaged, signals from ring ¹³C–¹H pairs, indicative of slow flipping on the chemical shift timescale. Ring flipping rate constants of ~10 and ~0.2 s⁻¹ were measured for the two residues, respectively, using a ¹³C longitudinal exchange experiment. The hydrogen bonding properties of the Tyr79 and Tyr105 hydroxyls were also defined by complementary NOE and J-coupling measurements. The ¹H^η hydrogen–deuterium exchange rate constants of the buried tyrosines were determined from ¹³C/¹⁵N-filtered spectra recorded as a function of pH. These exchange rate constants correspond to estimated protection factors of ~10⁴–10⁸ relative to a random coil tyrosine. The phenolic sidechain pK _a values were also measured by monitoring their pH-dependent ¹³C^ζ chemical shifts via ¹H^ε/δ(¹³C^ε)¹³C^ζ correlation spectra. Exposed tyrosines had unperturbed pK _a values of ~10.2, whereas buried residues remained predominantly neutral at or even above pH 11. Combined with selective isotope labelling, these NMR experiments should prove useful for investigating the structural and electrostatic properties of tyrosines in many interesting proteins.     

Obituary for Professor Robert Harza Burris

Plant and Soil - Winter rapeseed (Brassica napus) is characterized by a low N recovery in seeds and requires high rates of fertilization to maintain yield. Its nutrient use efficiency could be...     

A note on a new defined medium for 'Pseudomonas tomato'

A synthetic medium based on d -galactose as a single carbon source and either l -asparagine, l -glutamine or l -threonine as a single nitrogen source has been developed for Pseudomonas tomato. The growth of Ps. tomato on this medium was equal to its growth on commonly used complex media.     

Plant Growth-Promoting Effects of Diazotrophs in the Rhizosphere

Because of their ability to transform atmospheric N₂ into ammonia that can be used by the plant, researchers were originally very optimistic about the potential of associative diazotrophic bacteria to promote the growth of many cereals and grasses. However, multiple inoculation experiments during recent decades failed to show a substantial contribution of Biological Nitrogen Fixation (BNF) to plant growth in most cases. It is now clear that associative diazotrophs exert their positive effects on plant growth directly or indirectly through (a combination of) different mechanisms. Apart from fixing N₂, diazotrophs can affect plant growth directly by the synthesis of phytohormones and vitamins, inhibition of plant ethylene synthesis, improved nutrient uptake, enhanced stress resistance, solubilization of inorganic phosphate and mineralization of organic phosphate. Indirectly, diazotrophs are able to decrease or prevent the deleterious effects of pathogenic microorganisms, mostly through the synthesis of antibiotics and/or fungicidal compounds, through competition for nutrients (for instance, by siderophore production) or by the induction of systemic resistance to pathogens. In addition, they can affect the plant indirectly by interacting with other beneficial microorganisms, for example, Azospirillum increasing nodulation of legumes by rhizobia. The further elucidation of the different mechanisms involved will help to make associative diazotrophs a valuable partner in future agriculture.     

Effect of divalent cations on the structure of the antibiotic daptomycin

Daptomycin, a cyclic anionic lipopeptide antibiotic, whose three-dimensional structure was recently solved using solution state NMR (Ball et al. 2004; Jung et al. 2004; Rotondi and Gierasch 2005), requires calcium for function. To date, the exact nature of the interaction between divalent cations, such as Ca²⁺ or Mg²⁺, has not been fully characterized. It has, however, been suggested that addition of Ca²⁺ to daptomycin in a 1:1 molar ratio induces aggregation. Moreover, it has been suggested that certain residues, e.g. Asp3 and Asp7, which are essential for activity (Grunewald et al. 2004; Kopp et al. 2006), may also be important for Ca²⁺ binding (Jung et al. 2004). In this work, we have tried: (1) to further pinpoint how Ca²⁺ affects daptomycin structure/oligomerization using analytical ultracentrifugation; and (2) to determine whether a specific calcium binding site exists, based on one-dimensional ¹³C NMR spectra and molecular dynamics (MD) simulations. The centrifugation results indicated that daptomycin formed micelles of between 14 and 16 monomers in the presence of a 1:1 molar ratio of Ca²⁺ and daptomycin. The ¹³C NMR data indicated that addition of calcium had a significant effect on the Trp1 and Kyn13 residues, indicating that either calcium binds in this region or that these residues may be important for oligomerization. Finally, the molecular dynamics simulation results indicated that the conformational change of daptomycin upon calcium binding might not be as significant as originally proposed. Similar studies on the divalent cation Mg²⁺ are also presented. The implication of these results for the biological function of daptomycin is discussed. Electronic supplementary material The online version of this article (doi:) contains Supplemental Material, which is available to authorized users. Steven W. Ho and David Jung have contributed equally to this work.     

Structural characterization of the type-III pilot-secretin complex from Shigella flexneri

Assembly of the type-III secretion apparatus, which translocates proteins through both membranes of Gram-negative bacterial pathogens into host cells, requires the formation of an integral outer-membrane secretin ring. Typically, a small lipidated pilot protein is necessary for the stabilization and localization of this ring. Using NMR spectroscopy, we demonstrate that the C-terminal residues 553-570 of the Shigella flexneri secretin MxiD encompass the minimal binding domain for its cognate pilot MxiM. Although unstructured in isolation, upon complex formation with MxiM, these residues fold into an amphipathic turn-helix motif that caps the elongated hydrophobic cavity of the cracked beta-barrel pilot. Along with a rearrangement of core aromatic residues, this prevents the binding of lipids within the cavity. The mutually exclusive association of lipids and MxiD with MxiM establishes a framework for understanding the role of a pilot in the outer-membrane insertion and multimerization of the secretin ring.     

Identification and characterization of a periplasmic nitrate reductase in Azospirillum brasilense Sp245

The Azospirillum brasilense Sp245 napABC genes, encoding nitrate reductase activity, were isolated and sequenced. The derived protein sequences are very similar throughout the whole Nap segment to the NapABC protein sequences of Escherichia coli, Pseudomonas sp. G-179, Ralstonia eutropha, Rhodobacter sphaeroides, and Paracoccus denitrificans. Based on whole-cell nitrate reductase assays with the artificial electron donors benzyl viologen and methyl viologen, and assays with periplasmic cell-free extracts, it was concluded that the napABC-encoded enzyme activity in Azospirillum brasilense Sp245 corresponds to a periplasmic dissimilatory nitrate reductase, which was expressed under anoxic conditions and oxic conditions. A kanamycin-resistant Azospirillum brasilense Sp245 napA insertion mutant was constructed. The mutant still expressed assimilatory nitrate reductase activity, but was devoid of its periplasmic dissimilatory nitrate reductase activity.