Molecular dynamics simulations of Trp side-chain conformational flexibility in the gramicidin A channel

Gramicidin A (gA) is prototypical peptide antibiotic and a model ion channel former. Configured in the solid-state NMR beta(6.5)-helix channel conformation, gA was subjected to 1-ns molecular dynamics (MD) gas phase simulations using the all-atom charmm22 force field to ascertain the conformational stability of the Trp side chains as governed by backbone and neighboring side-chain contacts. Three microcanonical trajectories were computed using different initial atomic velocities for each of twenty different initial structures. For each set, one of the four Trp side chains in each monomer was initially positioned in one of the five non-native conformations (A. E. Dorigo et al., Biophysical Journal, 1999, Vol. 76, 1897-1908), the other Trps being positioned in the native state, o1. In three additional control simulations, all Trps were initiated in the native conformation. After equilibration, constraints were removed and subsequent conformational changes of the initially constrained Trp were measured. The chi(1) was more flexible than chi(2.1). The energetically optimal orientation, o1 (Dorigo et al., 1999), was the most stable in all four Trp positions (9, 11, 13, 15) and remained unchanged for the entire 1 ns simulation in 19 of 24 trials. Changes in chi(1) from each of the 5 suboptimal states occur readily. Two of the non-native conformations reverted readily to o1, whereas the other three converted to an intermediate state, i2. There were frequent interconversions between i2 and o1. We speculate that experimentally observed Trp stability is caused by interactions with the lipid-water interface, and that stabilization of one of the suboptimal conformations in gA, such as i2, by lipid headgroups could produce a secondary, metastable conformational state. This could explain recent experimental studies of differences in the channel conductance dispersity between gA and a Trp-to-Phe gA analog, gramicidin M (gM, J. C. Markham et al., Biochimica et Biophysica Acta, 2001, Vol. 1513, 185-192).     

Novel 3-O-carbamoyl erythromycin A derivatives (carbamolides) with activity against resistant staphylococcal and streptococcal isolates

A novel series of 3-O-carbamoyl erythromycin A derived analogs, labeled carbamolides, with activity versus resistant bacterial isolates of staphylococci (including macrolide and oxazolidinone resistant strains) and streptococci are reported. An (R)-2-aryl substituent on a pyrrolidine carbamate appeared to be critical for achieving potency against resistant strains. Crystal structures showed a distinct aromatic interaction between the (R)-2-aryl (3-pyridyl for 4d) substituent on the pyrrolidine and G2484 (G2505, Escherichia coli) of the Deinococcus radiodurans 50S ribosome (3.2 Å resolution).     

Seasonal variation in nutrient limitation of microbial biofilms colonizing organic and inorganic substrata in streams

Humans have increased the availability of nutrients including nitrogen and phosphorus worldwide; therefore, understanding how microbes process nutrients is critical for environmental conservation. We examined nutrient limitation of biofilms colonizing inorganic (fritted glass) and organic (cellulose sponge) substrata in spring, summer, and autumn in three streams in Michigan, USA. Biofilms were enriched with nitrate (NO₃ ⁻), phosphate (PO₄ ³⁻), ammonium (NH₄ ⁺), NO₃ ⁻ + PO₄ ³⁻, NH₄ ⁺ + PO₄ ³⁻, or none (control). We quantified biofilm structure and function as chlorophyll a (i.e., primary producer biomass) and community respiration on all substrata. In one stream, we characterized bacterial and fungal communities on cellulose in autumn using clone library sequencing and denaturing gradient gel electrophoresis to determine if community structure was linked to nutrient limitation status. Despite oligotrophic conditions, primary producer biomass was infrequently nutrient limited. In contrast, respiration on organic substrata was frequently limited by N + P combinations. We found no difference between biofilm response to NH₄ ⁺ versus NO₃ ⁻ enrichment, although the response to both N-species was positively related to water column PO₄ ³⁻ concentrations and temperature. Molecular analysis for fungal community composition suggested no relationship to nutrient limitation, but the dominant members of the bacterial community on cellulose were different on NO₃ ⁻, PO₄³, and NO₃ ⁻ + PO₄ ³⁻ treatments relative to control, NH₄ ⁺, and NH₄ ⁺ + PO₄ ³⁻ treatments, which matched patterns for biofilm respiration rates from each treatment. Our results show discrete patterns of nutrient limitation dependent upon substratum type and season, and imply changes in bacterial community structure and function may be linked following nutrient enrichment in streams.     

Quaternary structure of the hydroxylamine oxidoreductase from Nitrosomonas europaea

The hydroxylamine oxidoreductase from Nitrosomonas europaea was prepared to apparent electrophoretic homogeneity. Electron microscopy of negatively stained preparations of the sample revealed an overall diameter of about 8.8 nm of the enzyme particle. The native structure was determined as a tetrahedron-like assembly of identical subunits exhibiting four protein masses.Abbreviations ESI Electron spectroscopic imaging - HAO Hydroxylamine oxidoreductase     

Oviposition and feeding preferences of a flower-feeding weevil,Coelocephalapion aculeatum, in relation to conspecific damage to its host-plant

The oviposition and feeding preferences ofCoelocephalapion aculeatum Fall (Coleoptera: Apionidae), a host specific florivore ofMimosa pigra L. (Mimosaceae), were studied in relation to conspecific damage to its hostplant. Adults ofC. aculeatum cease ovipositing in inflorescences when the egg load reaches a number consistent with the larval carrying capacity of the inflorescence. The basis for this oviposition deterrence was examined by offering inflorescences damaged by adult feeding alone, larval feeding alone and a combination of adult feeding and oviposition. Adults preferred to oviposit on inflorescences which are not damaged by either adult feeding, larval feeding, or oviposition. No evidence for the existence of an oviposition deterring pheromone (ODP) was found. I suggest that the ability of a single host inflorescence to support the development of many larvae causes selection for the use of these oviposition deterring cues which can convey more quantitative information about the level of previous infestation than can ODPs. Adults fed a similar amount on damaged compared to undamaged inflorescences. These results assisted in the design of host range testing trials and allows predictions to be made about the effectiveness of this insect as a biological control agent.     

Latitudinal patterns of range size and species richness of New World woody plants

Aim  Relationships between range size and species richness are contentious, yet they are key to testing the various hypotheses that attempt to explain latitudinal diversity gradients. Our goal is to utilize the largest data set yet compiled for New World woody plant biogeography to describe and assess these relationships between species richness and range size.
Location  North and South America.
Methods  We estimated the latitudinal extent of 12,980 species of woody plants (trees, shrubs, lianas). From these estimates we quantified latitudinal patterns of species richness and range size. We compared our observations with expectations derived from two null models.
Results   Peak richness and the smallest- and largest-ranged species are generally found close to the equator. In contrast to prominent diversity hypotheses: (1) mean latitudinal extent of tropical species is greater than expected; (2) latitudinal extent appears to be decoupled from species richness across New World latitudes, with abrupt transitions across subtropical latitudes; and (3) mean latitudinal extents show equatorial and north temperate peaks and subtropical minima. Our results suggest that patterns of range size and richness appear to be influenced by three broadly overlapping biotic domains (biotic provinces) for New World woody plants.
Main conclusions  Hypotheses that assume a direct relationship between range size and species richness may explain richness patterns within these domains, but cannot explain gradients in richness across the New World.     

Targeting the role of a key conserved motif for substrate selection and catalysis by 3-deoxy-D-manno-octulosonate 8-phosphate synthase

3-Deoxy-D-manno-octulosonate 8-phosphate synthase (KDO8PS) catalyzes the reaction between three-carbon phosphoenolpyruvate (PEP) and five-carbon d-arabinose 5-phosphate (A5P), generating KDO8P, a key intermediate in the biosynthetic pathway to 3-deoxy-D-manno-octulosonate, a component of the lipopolysaccharide of the Gram-negative bacterial cell wall. Both metal-dependent and metal-independent forms of KDO8PS have been characterized. KDO8PS is evolutionarily and mechanistically related to the first enzyme of the shikimate pathway, the obligately divalent metal ion-dependent 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS) that couples PEP and four-carbon D-erythrose 4-phosphate (E4P) to give DAH7P. In KDO8PS, an absolutely conserved KANRS motif forms part of the A5P binding site, whereas in DAH7PS, an absolutely conserved KPR(S/T) motif accommodates E4P. Here, we have characterized four mutants of this motif (AANRS, KAARS, KARS, and KPRS) in metal-dependent KDO8PS from Acidithiobacillus ferrooxidans and metal-independent KDO8PS from Neisseria meningitidis to test the roles of the universal Lys and the Ala-Asn portion of the KANRS motif. The X-ray structures, determined for the N. meningitidis KDO8PS mutants, indicated no gross structural penalty resulting from mutation, but the subtle changes observed in the active sites of these mutant proteins correlated with their altered catalytic function. (1) The AANRS mutations destroyed catalytic activity. (2) The KAARS mutations lowered substrate selectivity, as well as activity. (3) Replacing KANRS with KARS or KPRS destroyed KDO8PS activity but did not produce a functional DAH7PS. Thus, Lys is critical to catalysis, and other changes are necessary to switch substrate specificity for both the metal-independent and metal-dependent forms of these enzymes.