Hartmannella vermiformis Inhibition of Legionella pneumophila Cultivability

Hartmannella vermiformis and Acanthamoeba polyphaga are frequently isolated from drinking water and permissive to Legionella pneumophila parasitization. In this study, extracellular factor(s) produced by H. vermiformis and A. polyphaga were assessed for their effects on cultivability of L. pneumophila. Page’s amoeba saline (PAS) was used as an encystment medium for H. vermiformis and A. polyphaga monolayers, and the culture supernatants (HvS and ApS, respectively) were assessed against L. pneumophila growth. Compared to PAS and ApS, HvS significantly inhibited L. pneumophila strain Philadelphia-1 (Ph-1) cultivability by 3 log₁₀ colony forming unit (CFU) mL^?1 after 3 days of exposure compared to <0.5 log₁₀ CFU mL^?1 reduction of strain Lp02 (P?<?0.001). Flow cytometric analysis revealed changes in the percentage and cultivability of three bacterial subpopulations: intact/slightly damaged membrane (ISM), undefined membrane status (UD), and mixed type (MT). After 3 days of HvS exposure, the MT subpopulation decreased significantly (31.6 vs 67.2 %, respectively, P?<?0.001), while the ISM and UD subpopulations increased (+26.7 and +6.9 %, respectively) with the ISM subpopulation appearing as viable but nonculturable (VBNC) cells. HvS was separated into two fractions based on molecular weight, with more than 99 % of the L. pneumophila inhibition arising from the <5 kDa fraction (P?<?0.001). Liquid chromatography indicated the inhibitory molecule(s) are likely polar and elute from a Novapak C18 column between 6 and 15 min. These results demonstrate that H. vermiformis is capable of extracellular modulation of L. pneumophila cultivability and probably promote the VBNC state for this bacterium.     

Comparison of the Heterologous Expression of Trichoderma reesei Endoglucanase II and Cellobiohydrolase II in the Yeasts Pichia pastoris and Yarrowia lipolytica

The sequences encoding the genes for endoglucanase II and cellobiohydrolase II from the fungus Trichoderma reesei QM9414 were successfully cloned and expressed in Yarrowia lipolytica under the control of the POX2 or TEF promoters, and using either the native or preproLip2 secretion signals. The expression level of both recombinant enzymes was compared with that obtained using Pichia pastoris, under the control of the AOX1 promoter to evaluate the utility of Y. lipolytica as a host strain for recombinant EGII and CBHII production. Extracellular endoglucanase activity was similar between TEF-preoproLip2-eglII expressed in Y. lipolytica and P. pastoris induced by 0.5 % (v/v) methanol, but when recombinant protein expression in P. pastoris was induced with 3 % (v/v) methanol, the activity was increased by about sevenfold. In contrast, the expression level of cellobiohydrolase from the TEF-preproLip2-cbhII cassette was higher in Y. lipolytica than in P. pastoris. Transformed Y. lipolytica produced up to 15 mg/l endoglucanase and 50 mg/l cellobiohydrolase, with the specific activity of both proteins being greater than their homologs produced by P. pastoris. Partial characterization of recombinant endoglucanase II and cellobiohydrolase II expressed in both yeasts revealed their optimum pH and temperature, and their pH and temperature stabilities were identical and hyperglycosylation had little effect on their enzymatic activity and properties.     

Seed dispersal in Cakile edentula var. lacustris : decoupling the fitness effects of density and distance from the home site

A factorial design of three densities of siblings at three local distances from seed parents was employed to distinguish effects of density from effects of dispersal distance on lifespan and fruit production of Cakile edentula var. lacustris, a plant with heteromorphic seeds. The segmented fruits produce two seed types: proximal and distal, with distal seeds having greater mass and greater dispersibility. Effects of longer distances (0.5 km and 30 km) on lifespan and fruit production were investigated using plants at low density. The prediction was tested that the greater seed mass of distal seeds increases fitness when seeds are dispersed into sites of unknown quality away from the home site or when seeds are dispersed to low density. High density caused earlier mortality and lower probability of reproduction. Distance from the maternal plant did not influence lifespan or reproduction at distances of 15 m or less, but lifespan was longer 0.5 km from the home site. No interaction was detected between the effects of density and distance on either lifespan or total fitness. Environmental conditions that influence fitness did not vary as a function of dispersal distance in this system, and favorable conditions at the home site did not persist between generations. Therefore, selection on dispersion patterns in natural conditions is likely to be through effects of density rather than dispersal distance. Proximal seeds had greater reproduction than distal seeds at the home site, and distal seeds had greater reproduction at the more distant sites (but not the most distant site), as expected, but these performance differences could not be attributed to differences in mass between the two seed types. Reduced seed mass was favored at the most distant site, but larger seed mass was favored most strongly at low density. Seeds that are dispersed to low density are larger, suggesting that although kin selection may limit the effectiveness of individual selection to increase seed mass under conditions of sibling competition, density-dependent individual selection on seed mass, rather than distant-dependent selection, also contributes to the observed associations among seed type, seed mass and dispersal ability. Received: 21 October 1996 / Accepted: 4 December 1996     

The fitness benefits of germinating later than neighbors

Premise of the Study

Phenology, the seasonal timing of development, can alter biotic interactions. Emergence from dormant or quiescent stages often occurs earlier when neighbors are present, which may reduce the neighbors' competitive effects. Delayed emergence in response to neighbors also has been observed, but the potential benefits of such delays are unclear. Further, emergence time may respond to neighbors experienced by parents, which may predict future competition in offspring.

Methods

In the annual plant Arabidopsis thaliana (Brassicaceae), we quantified seed germination responses to neighbors in parental and offspring (seed) environments. To examine how observed changes in germination affect interactions with neighbors, we performed an outdoor experiment using neighbors of different sizes to represent different germination times.

Key Results

Seeds were more likely to germinate if their parent had neighbors, but they were less likely to germinate if they themselves experienced a neighbor cue (canopy). As seeds lost dormancy over time, they gained the ability to germinate under a canopy, which suggests that they germinate later in the presence of neighbors. Neighbors of both sizes reduced growth, survival to reproduction, fecundity, and total fitness, but large neighbors increased seedling survival. Smaller neighbors provided no such benefit and had stronger negative effects.

Conclusions

Delayed germination in response to neighbors can reduce negative interactions and promote positive ones if it occurs late enough to expose seedlings to larger neighbors. By altering relative phenologies and, in turn, the outcomes of biotic interactions, phenological responses to environmental change may influence species interactions and community dynamics.     

Environmental effects on pollen-pistil compatibility between Phlox cuspidata and P. drummondii (Polemoniaceae): implications for hybridization dynamics

Postpollination mechanisms of reproductive isolation can critically influence the amount of gene flow between hybridizing species. While much evidence exists for genetically based pollen-pistil incompatibility, we show that environmental variation also influences the postpollination performance of heterospecific pollen in the annual Phlox hybrid system. Thus, the environmental segregation of species can influence hybridization dynamics. We found that P. cuspidata was restricted to soils of low Ca concentrations in the field and performed better under experimentally low Ca; P. drummondii was able to inhabit high-Ca soils and sometimes performed better in this environment. To determine whether soil Ca influenced pollen-pistil compatibility in a manner that alters pollen siring success, single-donor pollinations were performed in a completely factorial crossing design between species, maternal Ca environments, and paternal Ca environments. Maternal and paternal environments interacted in their effects on pollen-pistil compatibility for both inter- and intraspecific crosses, such that pollen performance was highest when mothers and fathers were grown in different soil Ca environments. These results suggest that when Phlox species predictably inhabit different environments, environmental heterogeneity can impede the processes of speciation and local adaptation by enhancing the performance of pollen dispersed across species and environments.     

Proteomic characterization of the Rhodobacter sphaeroides 2.4.1 photosynthetic membrane: identification of new proteins

The Rhodobacter sphaeroides intracytoplasmic membrane (ICM) is an inducible membrane that is dedicated to the major events of bacterial photosynthesis, including harvesting light energy, separating primary charges, and transporting electrons. In this study, multichromatographic methods coupled with Fourier transform ion cyclotron resonance mass spectrometry, combined with subcellular fractionation, was used to test the hypothesis that the photosynthetic membrane of R. sphaeroides 2.4.1 contains a significant number of heretofore unidentified proteins in addition to the integral membrane pigment-protein complexes, including light-harvesting complexes 1 and 2, the photochemical reaction center, and the cytochrome bc(1) complex described previously. Purified ICM vesicles are shown to be enriched in several abundant, newly identified membrane proteins, including a protein of unknown function (AffyChip designation RSP1760) and a possible alkane hydroxylase (RSP1467). When the genes encoding these proteins are mutated, specific photosynthetic phenotypes are noted, illustrating the potential new insights into solar energy utilization to be gained by this proteomic blueprint of the ICM. In addition, proteins necessary for other cellular functions, such as ATP synthesis, respiration, solute transport, protein translocation, and other physiological processes, were also identified to be in association with the ICM. This study is the first to provide a more global view of the protein composition of a photosynthetic membrane from any source. This protein blueprint also provides insights into potential mechanisms for the assembly of the pigment-protein complexes of the photosynthetic apparatus, the formation of the lipid bilayer that houses these integral membrane proteins, and the possible functional interactions of ICM proteins with activities that reside in domains outside this specialized bioenergetic membrane.