Glycosylation of the phosphate binding protein, PstS, in Streptomyces coelicolor by a pathway that resembles protein O-mannosylation in eukaryotes

Previously mutations in a putative protein O -mannosyltransferase (SCO3154, Pmt) and a polyprenol phosphate mannose synthase (SCO1423, Ppm1) were found to cause resistance to phage, φC31, in the antibiotic producing bacteria Streptomyces coelicolor A3(2). It was proposed that these two enzymes were part of a protein O-glycosylation pathway that was necessary for synthesis of the phage receptor. Here we provide the evidence that Pmt and Ppm1 are indeed both required for protein O-glycosylation. The phosphate binding protein PstS was found to be glycosylated with a trihexose in the S. coelicolor parent strain, J1929, but not in the pmt ⁻ derivative, DT1025. Ppm1 was necessary for the transfer of mannose to endogenous polyprenol phosphate in membrane preparations of S. coelicolor . A mutation in ppm1 that conferred an E218V substitution in Ppm1 abolished mannose transfer and glycosylation of PstS. Mass spectrometry analysis of extracted lipids showed the presence of a glycosylated polyprenol phosphate (PP) containing nine repeated isoprenyl units (C₄₅-PP). S. coelicolor membranes were also able to catalyse the transfer of mannose to peptides derived from PstS, indicating that these could be targets for Pmt in vivo .     

The Mechanism of Supercoiled DNA Recognition by Eukaryotic Type I Topoisomerases: II. A Comparison of the Enzyme Interaction with Specific and Nonspecific Oligonucleotides

Data on the interaction of DNA type I topoisomerases from the murine and human placenta cells with specific and nonspecific oligonucleotides of various structures and lengths are summarized. The relative contributions of various contacts between the enzymes and DNA that have previously been detected by X-ray analysis to the total affinity of the topoisomerases for DNA substrates are estimated. Factors that determine the differences in the enzyme interactions with specific and nonspecific single- and double-stranded DNAs are revealed. The results of the X-ray analysis of human DNA topoisomerase I are interpreted taking into account data on the comprehensive thermodynamic and kinetic analysis of the enzyme interaction with the specific and nonspecific DNAs.     

The potential for and constraints on the evolution of compensatory ability in Asclepias syriaca

To investigate the potential for and constraints on the evolution of compensatory ability, we performed a greenhouse experiment using Asclepias syriaca in which foliar damage and soil nutrient concentration were manipulated. Under low nutrient conditions, significant genetic variation was detected for allocation patterns and for compensatory ability. Furthermore, resource allocation to storage was positively, genetically correlated both with compensatory ability and biomass when damaged, the last two being positively, genetically correlated with each other. Thus, in the low nutrient environment, compensatory ability via resource allocation to storage provided greater biomass when damaged. A negative genetic correlation between compensatory ability and plant biomass when undamaged suggests that this mechanism entailed an allocation cost, which would constrain the evolution of greater compensatory ability when nutrients are limited. Under high nutrient conditions, neither compensatory ability nor allocation patterns predicted biomass when damaged, even though genetic variation in compensatory ability existed. Instead, plant biomass when undamaged predicted biomass when damaged. The differences in outcomes between the two nutrient treatments highlight the importance of considering the possible range of environmental conditions that a genotype may experience. Furthermore, traits that conferred compensatory ability did not necessarily contribute to biomass when damaged, demonstrating that it is critical to examine both compensatory ability and biomass when damaged to determine whether selection by herbivores can favor the evolution of increased compensation. Received: 2 April 1999 / Accepted: 21 September 1999     

Essential genes for myoblast fusion in Drosophila embryogenesis.

In Drosophila, as in vertebrates, each muscle is a syncytium and arises from mesodermal cells by successive fusion. This requires cell-cell recognition, alignment, formation of prefusion complexes, followed by electron-dense plaques and membrane breakdown. Because muscle development in Drosophila is rapid and well-documented, it has been possible to identify several genes essential for fusion. Molecular analysis of two of these genes revealed the importance of cytoplasmic components. One of these, Myoblast city, is expressed in several tissues and is homologous to the mammalian protein DOCK180. Myoblast city is presumably involved in cell recognition and cell adhesion. Blown fuse, the second cytoplasmic component, is selectively expressed in the mesoderm and essential in order to proceed from the prefusion complex to electron-dense plaques at opposed membranes between adjacent myoblasts. The rolling stone gene is transiently expressed during myoblast fusion. The Rost protein is located in the membrane and thus might be a key component for cell recognition. The molecular characterization of further genes relevant for fusion such as singles bar and sticks and stones will help to elucidate the mechanism of myoblast fusion in Drosophila.     

Two-dimensional Fourier analysis of the spongy medullary keratin of structurally coloured feather barbs

We conducted two-dimensional (2D) discrete Fourier analyses of the spatial variation in refractive index of the spongy medullary keratin from four different colours of structurally coloured feather barbs from three species of bird: the rose-faced lovebird, Agapornis roseicollis (Psittacidae), the budgerigar, Melopsittacus undulatus (Psittacidae), and the Gouldian finch, Poephila guttata (Estrildidae). These results indicate that the spongy medullary keratin is a nanostructured tissue that functions as an array of coherent scatterers. The nanostructure of the medullary keratin is nearly uniform in all directions. The largest Fourier components of spatial variation in refractive index in the tissue are of the appropriate size to produce the observed colours by constructive interference alone. The peaks of the predicted reflectance spectra calculated from the 2D Fourier power spectra are congruent with the reflectance spectra measured by using microspectrophotometry. The alternative physical models for the production of these colours, the Rayleigh and Mie theories, hypothesize that medullary keratin is an incoherent array and that scattered waves are independent in phase. This assumption is falsified by the ring-like Fourier power spectra of these feathers, and the spacing of the scattering air vacuoles in the medullary keratin. Structural colours of avian feather barbs are produced by constructive interference of coherently scattered light waves from the optically heterogeneous matrix of keratin and air in the spongy medullary layer.     

Rapid determination of substrate specificity of Clostridium histolyticum beta-collagenase using an immobilized peptide library.

The molecular basis of the substrate specificity of Clostridium histolyticum beta-collagenase was investigated using a combinatorial method. An immobilized positional peptide library, which contains 24,000 sequences, was constructed with a 7-hydroxycoumarin-4-propanoyl (Cop) fluorescent group attached at the N terminus of each sequence. This immobilized peptide library was incubated with C. histolyticum beta-collagenase, releasing fluorogenic fragments in the solution phase. The relative substrate specificity (k(cat)/K(m)) for each member of the library was determined by measuring fluorescence intensity in the solution phase. Edman sequencing was used to assign structure to subsites of active substrate mixtures. Collectively, the substrate preference for subsites (P(3)-P(4)') of C. histolyticum beta-collagenase was determined. The last position on the C-terminal side in which the identity of the amino acids affects the activity of the enzyme is P(4)', and an aromatic side chain is preferred in this position. The optimal P(1)'-P(3)' extended substrate sequence is P(1)'-Gly/Ala, P(2)'-Pro/Xaa, and P(3)'-Lys/Arg/Pro/Thr/Ser. The Cop group in either the P(2) or P(3) position is required for a high substrate activity with C. histolyticum beta-collagenase. S(2) and S(3) sites of the protease play a dominant role in fixing the substrate specificity. The immobilized peptide library proved to be a powerful approach for assessing the substrate specificity of C. histolyticum beta-collagenase, so it may be applied to the study of other proteases of interest.