Molecular analysis of a novel family of complex glycoinositolphosphoryl ceramides from Cryptococcus neoformans: structural differences between encapsulated and acapsular yeast forms

Complex glycoinositolphosphoryl ceramides (GIPCs) have been purified from a pathogenic encapsulated wild-type (WT) strain of Cryptococcus neoformans var. neoformans and from an acapsular mutant (Cap67). The structures of the GIPCs were determined by a combination of tandem mass spectrometry, nuclear magnetic resonance spectroscopy, methylation analysis, gas chromatography-mass spectrometry, and chemical degradation. The main GIPC from the WT strain had the structure Manp(alpha1-3)[Xylp(beta1-2)] Manp(alpha1-4)Galp(beta1-6)Manp(alpha1-2)Ins-1-phosphoryl ceramide (GIPC A), whereas the compounds from the acapsular mutant were more heterogeneous in their glycan chains, and variants with Manp(alpha1-6) (GIPC B), Manp(alpha1-6) Manp(alpha1-6) (GIPC C), and Manp(alpha1-2)Manp(alpha1-6)Manp(alpha1-6) (GIPC D) substituents linked to the nonreducing terminal mannose residue found in the WT GIPC A were abundant. The ceramide moieties of C. neoformans GIPCs were composed of a C(18) phytosphingosine long-chain base mainly N-acylated with 2-hydroxy-tetracosanoic acid in the WT GIPC while in the acapsular Cap67 mutant GIPCs, as well as 2-hydroxy-tetracosanoic acid, the unusual 2,3-dihydroxy-tetracosanoic acid was characterized. In addition, structural analysis revealed that the amount of GIPC in the WT cells was fourfold less of that in the acapsular mutant.     

Proteome turnover in the green alga Ostreococcus tauri by time course 15N metabolic labeling mass spectrometry

Protein synthesis and degradation determine the cellular levels of proteins, and their control hence enables organisms to respond to environmental change. Experimentally, these are little known proteome parameters; however, recently, SILAC-based mass spectrometry studies have begun to quantify turnover in the proteomes of cell lines, yeast, and animals. Here, we present a proteome-scale method to quantify turnover and calculate synthesis and degradation rate constants of individual proteins in autotrophic organisms such as algae and plants. The workflow is based on the automated analysis of partial stable isotope incorporation with (15)N. We applied it in a study of the unicellular pico-alga Ostreococcus tauri and observed high relative turnover in chloroplast-encoded ATPases (0.42-0.58% h(-1)), core photosystem II proteins (0.34-0.51% h(-1)), and RbcL (0.47% h(-1)), while nuclear-encoded RbcS2 is more stable (0.23% h(-1)). Mitochondrial targeted ATPases (0.14-0.16% h(-1)), photosystem antennae (0.09-0.14% h(-1)), and histones (0.07-0.1% h(-1)) were comparatively stable. The calculation of degradation and synthesis rate constants k(deg) and k(syn) confirms RbcL as the bulk contributor to overall protein turnover. This study performed over 144 h of incorporation reveals dynamics of protein complex subunits as well as isoforms targeted to different organelles.     

Identification of a novel protonation pattern for carboxylic acids upon Q(B) photoreduction in Rhodobacter sphaeroides reaction center mutants at Asp-L213 and Glu-L212 sites

In the reaction center from the photosynthetic purple bacterium Rhodobacter sphaeroides, light energy is rapidly converted to chemical energy through coupled electron-proton transfer to a buried quinone molecule Q(B). Involved in the proton uptake steps are carboxylic acids, which have characteristic infrared vibrations that are observable using light-induced Fourier transform infrared (FTIR) difference spectroscopy. Upon formation, Q(B)(-) induces protonation of Glu-L212, located within 5 A of Q(B), resulting in a IR signal at 1728 cm(-1). However, no other IR signal is observed within the classic absorption range of protonated carboxylic acids (1770-1700 cm(-1)). In particular, no signal for Asp-L213 is found despite its juxtaposition to Q(B) and importance for proton uptake on the second electron-transfer step. In an attempt to uncover the reason behind this lack of signal, the microscopic electrostatic environment in the vicinity of Q(B) was modified by interchanging Asp and Glu at the L213 and L212 positions. The Q(B)(-)/Q(B) FTIR spectrum of the Asp-L212/Glu-L213 swap mutant in the 1770-1700 cm(-1) range shows several distinct new signals, which are sensitive to (1)H/(2)H isotopic exchange, indicating that the reduction of Q(B) results in the change of the protonation state of several carboxylic acids. The new bands at 1752 and 1747 cm(-1) were assigned to an increase of protonation in response to Q(B) reduction of Glu-L213 and Asp-L212, respectively, based on the effect of replacing them with their amine analogues. Since other carboxylic acid signals were observed, it is concluded that the swap mutations at L212 and L213 affect a cluster of carboxylic acids larger than the L212/L213 acid pair. Implications for the native reaction center are discussed.     

Transformation efficiency of RasQ61 mutants linked to structural features of the switch regions in the presence of Raf

Transformation efficiencies of Ras mutants at residue 61 range over three orders of magnitude, but the in vitro GTPase activity decreases 10-fold for all mutants. We show that Raf impairs the GTPase activity of RasQ61L, suggesting that the Ras/Raf complex differentially modulates transformation. Our crystal structures show that, in transforming mutants, switch II takes part in a network of hydrophobic interactions burying the nucleotide and precatalytic water molecule. Our results suggest that Y32 and a water molecule bridging it to the gamma-phosphate in the wild-type structure play a role in GTP hydrolysis in lieu of the Arg finger in the absence of GAP. The bridging water molecule is absent in the transforming mutants, contributing to the burying of the nucleotide. We propose a mechanism for intrinsic hydrolysis in Raf-bound Ras and elucidate structural features in the Q61 mutants that correlate with their potency to transform cells.     

Development,characterization, validation,and mapping of SSRs derived from <Emphasis Type="Italic">Theobroma cacao</Emphasis> L.<Emphasis Type="Italic">–Moniliophthora perniciosa</Emphasis> interaction ESTs

In this study, we report results of the detection and analysis of SSR markers derived of cacao–Moniliophthora perniciosa expressed sequence tags (ESTs) in relation to cacao resistance to witches’ broom disease (WBD), and we compare the polymorphism of those ESTs (EST-simple sequence repeat (SSR)) with classical neutral SSR markers. A total of 3,487 ESTs was used in this investigation. SSRs were identified in 430 sequences: 277 from the resistant genotype TSH 1188 and 153 from the susceptible one Catongo, totalizing 505 EST-SSRs with three types of motifs: dinucleotides (72.1%), trinucleotides (27.3%), and tetranucleotides (0.6%). EST-SSRs were classified into 16 main categories; most of the EST-SSRs belonged to “Unknown function” and “No homology” categories (45.82%). A high frequency of SSRs was found in the 5’UTR and in the ORF (about 27%) and a low frequency was observed in the 3’UTR (about 8%). Forty-nine EST-SSR primers were designed and evaluated in 21 cacao accessions, 12 revealed polymorphism, having 47 alleles in total, with an average of 3.92 alleles per locus. On the other hand, the 11 genomic SSR markers revealed a total of 47 alleles, with an average of 5.22 alleles per locus. The association of EST-SSR with the genomic SSR enhanced the analysis of genetic distance among the genotypes. Among the 12 polymorphic EST-SSR markers, two were mapped on the F₂ Sca 6 × ICS 1 population reference for WBD resistance.     

Isoflavonoid exudation from white lupin roots is influenced by phosphate supply, root type and cluster-root stage

The internal concentration of isoflavonoids in white lupin (Lupinus albus) cluster roots and the exudation of isoflavonoids by these roots were investigated with respect to the effects of phosphorus (P) supply, root type and cluster-root developmental stage.To identify and quantify the major isoflavonoids exuded by white lupin roots, we used high-pressure liquid chromatography (HPLC) coupled to electrospray ionization (ESI) in mass spectrometry (MS).The major exuded isoflavonoids were identified as genistein and hydroxygenistein and their corresponding mono- and diglucoside conjugates. Exudation of isoflavonoids during the incubation period used was higher in P-deficient than in P-sufficient plants and higher in cluster roots than in noncluster roots. The peak of exudation occurred in juvenile and immature cluster roots, while exudation decreased in mature cluster roots.Cluster-root exudation activity was characterized by a burst of isoflavonoids at the stage preceding the peak of organic acid exudation. The potential involvement of ATP-citrate lyase in controlling citrate and isoflavonoid exudation is discussed, as well as the possible impact of phenolics in repelling rhizosphere microbial citrate consumers.     

Replacement of potassium ions by ammonium ions in different micro-organisms grown in potassium-limited chemostat culture

The biomass concentration extant in potassiumlimited cultures of either Klebsiella pneumoniae or Bacillus stearothermophilus (when growing at a fixed temperature and dilution rate in a glucose/ammonium salts medium) increased progressively as the medium pH value was raised step-wise from 7.0 to 8.5. Because the macromolecular composition of the organisms did not vary significantly, this increase in biomass could not be attributed to an accumulation of storage-type polymers but appeared to reflect a pH-dependent decrease in the cells' minimum K⁺ requirement. Significantly, this effect of pH was not eviden with cultures in which no ammonium salts were present and in which either glutamate or nitrate was added as the sole nitrogen source; however, it was again manifest when various concentrations of NH₄Cl were added to the glutamate-containing medium. This suggested a functional replacement of K⁺ by NH ₄ ⁺ , a proposition consistent with the close similarity of the ionic radii of the potassium ion (1.33 Å) and the ammonium ion (1.43 Å). At pH 8.0, and with a medium containing both glutamate (30 mM) and NH₄Cl (100 mM), cultures of B. stearothermophilus would grow without added potassium at a maximum rate of 0.7 h^-1. Under these conditions the cells contained maximally 0.1% (w/w) potassium (derived from contaminating amounts of this element in the medium constituents), a value which should be compared with one of 1.4% (w/w) for cells growing in a potassiumlimited medium containing initially 0.5 mM K⁺. Qualitatively similar findings were made with cultures of K. pneumoniae; and whereas one may not conclude that NH ₄ ⁺ can totally replace K⁺ in the growth of these bacteria, it can clearly do so very extensively.