Identification and molecular cloning of a heparosan synthase from Pasteurella multocida type D.

Pasteurella multocida Type D, a causative agent of atrophic rhinitis in swine and pasteurellosis in other domestic animals, produces an extracellular polysaccharide capsule that is a putative virulence factor. It was reported previously that the capsule was removed by treating microbes with heparin lyase III. We molecularly cloned a 617-residue enzyme, pmHS, which is a heparosan (nonsulfated, unepimerized heparin) synthase. Recombinant Escherichia coli-derived pmHS catalyzes the polymerization of the monosaccharides from UDP-GlcNAc and UDP-GlcUA. Other structurally related sugar nucleotides did not substitute. Synthase activity was stimulated about 7-25-fold by the addition of an exogenous polymer acceptor. Molecules composed of approximately 500-3,000 sugar residues were produced in vitro. The polysaccharide was sensitive to the action of heparin lyase III but resistant to hyaluronan lyase. The sequence of the pmHS enzyme is not very similar to the vertebrate heparin/heparan sulfate glycosyltransferases, EXT1 and 2, or to other Pasteurella glycosaminoglycan synthases that produce hyaluronan or chondroitin. The pmHS enzyme is the first microbial dual-action glycosyltransferase to be described that forms a polysaccharide composed of beta4GlcUA-alpha4GlcNAc disaccharide repeats. In contrast, heparosan biosynthesis in E. coli K5 requires at least two separate polypeptides, KfiA and KfiC, to catalyze the same polymerization reaction.     

Analysis of the membrane-interacting domain of the sea urchin sperm adhesive protein bindin

We have investigated the domain of the bindin polypeptide that selectively associates with gel-phase phospholipid vesicles. We found that small trypsin fragments of bindin retain the ability to selectively associate with gel-phase vesicles. The primary amino acid sequence of bindin suggests that these peptides are derived from the central portion of the polypeptide between residues 77 and 126, which is the most hydrophobic region of bindin. We have also employed 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine (TID) and novel, radioiodinated, photoactivatable derivatives of the polar head group of phosphatidylethanolamine (ASD-PE and ASA-PE) to identify membrane-associated polypeptide segments after the transfer of radiolabel from the probe to the bindin polypeptide. After photolysis, bindin was selectively labeled only from probes incorporated in gel-phase vesicles. The labeling of bindin was much more efficient from the head group probes ASA-PE and ASD-PE (8 and 2% of the total label, respectively) in comparison to the hydrophobic probe TID (less than 0.02% of the total label), suggesting that bindin is localized within the polar part of the bilayer. Protease mapping experiments with V8 protease, trypsin, and endoprotease Lys-C suggest that some of the probe label is distributed along the amino-terminal portion of bindin between residues 1 and 76 and the rest of the label is restricted to the segments between residues 77 and 126 which also selectively bind to gel-phase vesicles. The carboxyl-terminal portion of bindin between residues 127 and 236 is not labeled.     

Resource allocation in offspring provisioning: an evaluation of the conditions favoring the evolution of matrotrophy

We used analytic and simulation models to determine the ecological conditions favoring evolution of a matrotrophic fish from a lecithotrophic ancestor given a complex set of trade-offs. Matrotrophy is the nourishment of viviparous embryos by resources provided between fertilization and parturition, while lecithotrophy describes embryo nourishment provided before fertilization. In fishes and reptiles, embryo nourishment encompasses a continuum from solely lecithotrophic to primarily matrotrophic. Matrotrophy has evolved independently from lecithotrophic ancestors many times in many groups. We assumed matrotrophy increased the number of offspring a viviparous female could gestate and evaluated conditions of food availability favoring lecithotrophy or matrotrophy. The matrotrophic strategy was superior when food resources exceeded demand during gestation but at a risk of overproduction and reproductive failure if food intake was limited. Matrotrophic females were leaner during gestation than lecithotrophic females, yielding shorter life spans. Our models suggest that matrotrophic embryo nourishment evolved in environments with high food availability, consistently exceeding energy requirements for maintaining relatively large broods. Embryo abortion with some resorption of invested energy is a necessary preadaptation to the evolution of matrotrophy. Future work should explore trade-offs of age-specific mortality and reproductive output for females maintaining different levels of fat storage during gestation.     

Using data from an encounter sampler to model fish dispersal

A method to estimate speed of free-ranging fishes using a passive sampling device is described and illustrated with data from the Everglades, U.S.A. Catch per unit effort (CPUE) from minnow traps embedded in drift fences was treated as an encounter rate and used to estimate speed, when combined with an independent estimate of density obtained by use of throw traps that enclose 1 m(2) of marsh habitat. Underwater video was used to evaluate capture efficiency and species-specific bias of minnow traps and two sampling studies were used to estimate trap saturation and diel-movement patterns; these results were used to optimize sampling and derive correction factors to adjust species-specific encounter rates for bias and capture efficiency. Sailfin mollies Poecilia latipinna displayed a high frequency of escape from traps, whereas eastern mosquitofish Gambusia holbrooki were most likely to avoid a trap once they encountered it; dollar sunfish Lepomis marginatus were least likely to avoid the trap once they encountered it or to escape once they were captured. Length of sampling and time of day affected CPUE; fishes generally had a very low retention rate over a 24 h sample time and only the Everglades pygmy sunfish Elassoma evergladei were commonly captured at night. Dispersal speed of fishes in the Florida Everglades, U.S.A., was shown to vary seasonally and among species, ranging from 0· 05 to 0· 15 m s(-1) for small poeciliids and fundulids to 0· 1 to 1· 8 m s(-1) for L. marginatus. Speed was generally highest late in the wet season and lowest in the dry season, possibly tied to dispersal behaviours linked to finding and remaining in dry-season refuges. These speed estimates can be used to estimate the diffusive movement rate, which is commonly employed in spatial ecological models.     

Transitions of interaction outcomes in a uni-directional consumer-resource system

A uni-directional consumer-resource system of two species is analyzed. Our aim is to understand the mechanisms that determine how the interaction outcomes depend on the context of the interaction; that is, on the model parameters. The dynamic behavior of the model is described and, in particular, it is demonstrated that no periodic orbits exist. Then the parameter (factor) space is shown to be divided into four regions, which correspond to the four forms of interaction outcomes; i.e. mutualism, commensalism, parasitism and amensalism. It is shown that the interaction outcomes of the system transition smoothly among these four forms when the parameters of the system are varied continuously. Varying each parameter individually or varying pairs of parameters can also lead to smooth transitions between the interaction outcomes. The analysis leads to both conditions for which each species achieves its maximal density, and situations in which periodic oscillations of the interaction outcomes emerge.     

Application of phenotypic microarrays to environmental microbiology

Environmental organisms are extremely diverse and only a small fraction has been successfully cultured in the laboratory. Culture in micro wells provides a method for rapid screening of a wide variety of growth conditions and commercially available plates contain a large number of substrates, nutrient sources, and inhibitors, which can provide an assessment of the phenotype of an organism. This review describes applications of phenotype arrays to anaerobic and thermophilic microorganisms, use of the plates in stress response studies, in development of culture media for newly discovered strains, and for assessment of phenotype of environmental communities. Also discussed are considerations and challenges in data interpretation and visualization, including data normalization, statistics, and curve fitting.     

Preparation of soybean seed samples for FT-IR microspectroscopy

Typical preparation of seed samples for infrared (IR) microspectroscopy involves imbibition of the seed for varying time periods followed by cryosectioning. Imbibition, however, may initiate germination even at 4° C with associated changes in the chemistry of the sample. We have found that it is possible to section seeds that are sufficiently hard, such as soybeans, on a standard laboratory microtome without imbibition. The use of dry sectioning of unimbibed seeds is reported here, as well as a comparison of different mounting media and modes of analysis. Glycerol, Tissue-Tek, and ethanol were used as mounting media, and the quality of the resulting spectra was assessed. Ethanol was the preferred mountant, because it dried quickly with no residue and thus did not interfere with the spectrum of interest. Analysis in transmission mode using barium fluoride windows to hold the samples was compared with transmission-reflection analysis with sections mounted on special infrared-reflecting slides. The two modes of analysis performed well in different regions of the spectrum. The mode of analysis (transmission vs. transmission-reflection) should be based on the components of greatest interest in the sample.     

Soil carbon cycling proxies: Understanding their critical role in predicting climate change feedbacks

The complexity of processes and interactions that drive soil C dynamics necessitate the use of proxy variables to represent soil characteristics that cannot be directly measured (correlative proxies), or that aggregate information about multiple soil characteristics into one variable (integrative proxies). These proxies have proven useful for understanding the soil C cycle, which is highly variable in both space and time, and are now being used to make predictions of the fate and persistence of C under future climate scenarios. However, the C pools and processes that proxies represent must be thoughtfully considered in order to minimize uncertainties in empirical understanding. This is necessary to capture the full value of a proxy in model parameters and in model outcomes. Here, we provide specific examples of proxy variables that could improve decision‐making, and modeling skill, while also encouraging continued work on their mechanistic underpinnings. We explore the use of three common soil proxies used to study soil C cycling: metabolic quotient, clay content, and physical fractionation. We also consider how emerging data types, such as genome‐sequence data, can serve as proxies for microbial community activities. By examining some broad assumptions in soil C cycling with the proxies already in use, we can develop new hypotheses and specify criteria for new and needed proxies.     

Failure of the bovine papillomavirus to transform mouse embryo fibroblasts with a targeted disruption of the insulin-like growth factor I receptor genes.

Mouse embryo cells with a targeted disruption of the insulin-like growth factor I receptor (IGF-IR) genes (R- cells) are refractory to transformation by the simian virus 40 large T antigen and/or an activated and overexpressed Ras, both of which readily transform cells from wild-type littermate embryos and other 3T3-like cells. R- cells are also refractory to transformation induced by overexpressed epidermal growth factor receptor and platelet-derived growth factor receptor beta. Since the platelet-derived growth factor receptor beta is required for transformation by bovine papillomavirus, we inquired whether the IGF-IR was also required for transformation by bovine papillomavirus E5 oncoprotein. We show here that R- cells are refractory to transformation by E5; reintroduction into R- cells of a human IGF-IR restores the susceptibility to transformation.