Significant natural product biosynthetic potential of actinorhizal symbionts of the genus frankia, as revealed by comparative genomic and proteomic analyses

Bacteria of the genus Frankia are mycelium-forming actinomycetes that are found as nitrogen-fixing facultative symbionts of actinorhizal plants. Although soil-dwelling actinomycetes are well-known producers of bioactive compounds, the genus Frankia has largely gone uninvestigated for this potential. Bioinformatic analysis of the genome sequences of Frankia strains ACN14a, CcI3, and EAN1pec revealed an unexpected number of secondary metabolic biosynthesis gene clusters. Our analysis led to the identification of at least 65 biosynthetic gene clusters, the vast majority of which appear to be unique and for which products have not been observed or characterized. More than 25 secondary metabolite structures or structure fragments were predicted, and these are expected to include cyclic peptides, siderophores, pigments, signaling molecules, and specialized lipids. Outside the hopanoid gene locus, no cluster could be convincingly demonstrated to be responsible for the few secondary metabolites previously isolated from other Frankia strains. Few clusters were shared among the three species, demonstrating species-specific biosynthetic diversity. Proteomic analysis of Frankia sp. strains CcI3 and EAN1pec showed that significant and diverse secondary metabolic activity was expressed in laboratory cultures. In addition, several prominent signals in the mass range of peptide natural products were observed in Frankia sp. CcI3 by intact-cell matrix-assisted laser desorption-ionization mass spectrometry (MALDI-MS). This work supports the value of bioinformatic investigation in natural products biosynthesis using genomic information and presents a clear roadmap for natural products discovery in the Frankia genus.     

Effect of flywheel-based resistance exercise on processes contributing to muscle atrophy during unloading in adult rats.

Flywheel-based resistance exercise (RE) attenuates muscle atrophy during hindlimb suspension. We have previously shown that protein synthesis is elevated in response to RE, but the effect on protein degradation, cell proliferation, or apoptosis was not investigated. We hypothesized that, in addition to affecting protein synthesis, RE inhibits processes that actively contribute to muscle atrophy during hindlimb suspension. Male rats were housed in regular cages (control), tail suspended for 2 wk (HS), or HS with RE every other day for 2 wk (HSRE). Although RE attenuated soleus muscle atrophy during HS, the observed fivefold elevation in apoptosis and the 53% decrease in cell proliferation observed with HS were unaffected by RE. Expression of genes encoding components of the ubiquitin-proteasome pathway of protein degradation were elevated with HS, including ubiquitin, MAFbx, Murf-1, Nedd4, and XIAP, and proteasome subunits C2 and C9. Total ubiquitinated protein was increased with HS, but proteasome activity was not different from control. RE selectively altered the expression of different components of this pathway: MAFbx, Murf-1, and ubiquitin mRNA abundance were downregulated, whereas C2 and C9 subunits remained elevated. Similarly, Nedd4 and XIAP continued to be upregulated, potentially accounting for the observed augmentation in total ubiquitinated protein with RE. Thus a different constellation of proteins is likely ubiquitinated with RE due to altered ubiquitin ligase composition. In summary, the flywheel-based resistance exercise paradigm used in this study is associated with the inhibition of some mechanisms associated with muscle atrophy, such as the increase in MAFbx and Murf-1, but not with others, such as proteasome subunit remodeling, apoptosis, and decreased proliferation, potentially accounting for the inability to completely restore muscle mass. Identifying specific exercise parameters that affect these latter processes may be useful in designing effective exercise strategies in the elderly or during spaceflight.     

Hydrodynamic Radii of Intrinsically Disordered Proteins Determined from Experimental Polyproline II Propensities

The properties of disordered proteins are thought to depend on intrinsic conformational propensities for polyproline II (PP _II) structure. While intrinsic PP _II propensities have been measured for the common biological amino acids in short peptides, the ability of these experimentally determined propensities to quantitatively reproduce structural behavior in intrinsically disordered proteins (IDPs) has not been established. Presented here are results from molecular simulations of disordered proteins showing that the hydrodynamic radius (R _h) can be predicted from experimental PP _II propensities with good agreement, even when charge-based considerations are omitted. The simulations demonstrate that R _h and chain propensity for PP _II structure are linked via a simple power-law scaling relationship, which was tested using the experimental R _h of 22 IDPs covering a wide range of peptide lengths, net charge, and sequence composition. Charge effects on R _h were found to be generally weak when compared to PP _II effects on R _h. Results from this study indicate that the hydrodynamic dimensions of IDPs are evidence of considerable sequence-dependent backbone propensities for PP _II structure that qualitatively, if not quantitatively, match conformational propensities measured in peptides.     

Host plant selection of Chrysolina clathrata (Coleoptera: Chrysomelidae) from Mpumalanga, South Africa

Hyperaccumulated elements such as Ni may defend plants against some natural enemies whereas other enemies may circumvent this defense. The Ni hyperaccumulator Berkheya coddii Roessler (Asteraceae) is a host plant species for Chrysolina clathrata (Clark), which suffers no apparent harm by consuming its leaf tissue. Beetle specimens collected from B. coddii had a whole body Ni concentration of 260 μg/g dry weight, despite consuming leaf material containing 15 100 μg Ni/g. Two experiments were conducted with adults of this beetle species: a no-choice experiment and a choice experiment. In the no-choice experiment we offered beetles foliage of one of four species of Berkheya: B. coddii, B. rehmannii Thell. var. rogersiana Thell., B. echinacea (Harv.) O. Hoffm. ex Burtt Davey, and B. insignis (Harv.) Thell. The two former species are Ni hyperaccumulators (defined as having leaf Ni concentration > 1 000 μg/g) whereas the latter have low Ni levels (< 200 μg/g) in their leaves. Masses of beetles were monitored for 6 days. Choice experiments used growing stem tips from the same Berkheya species, placed into Petri dishes with five Chrysolina beetles in each, and the amount of feeding damage caused on each of the four species was recorded. Beetles in the no-choice experiment gained mass when offered B. coddii , maintained mass on leaves of the other Ni hyperaccumulator ( B. rehmannii var. rogersiana ), and lost mass when offered non-hyperaccumulator leaves. In the choice test, beetles strongly preferred B. coddii to other Berkheya species. We conclude that C. clathrata may be host-specific on B. coddii.     

A new polymorphic marker (D10S97) tightly linked to the multiple endocrine neoplasia type 2A (MEN2A) locus

Familial multiple endocrine neoplasia type 2A (MEN 2A) is a cancer syndrome that is inherited as an autosomal dominant with high penetrance. Its clinical features are medullary carcinoma of the thyroid, pheochromocytomas, and hyperparathyroidism. A new polymorphic locus D10S97 (probe: KW6SacI) detects a codominant EcoRI polymorphism that is tightly linked to the MEN2A locus. The peak lod score for linkage between D10S97 with MEN2A is 13.03 at =0.00. The polymorphic locus D10S97 maps, by linkage analysis, into the previously defined interval between FNRB and RBP3 to which MEN2A has been assigned. We present physical mapping data showing that the probe pKW6 originates from 10p13 and that the polymorphic locus D10S97 in 10q11.2 is detected by cross-hybridization.