Genome Survey and Characterization of Endophytic Bacteria Exhibiting a Beneficial Effect on Growth and Development of Poplar Trees

The association of endophytic bacteria with their plant hosts has a beneficial effect for many different plant species. Our goal is to identify endophytic bacteria that improve the biomass production and the carbon sequestration potential of poplar trees (Populus spp.) when grown in marginal soil and to gain an insight in the mechanisms underlying plant growth promotion. Members of the Gammaproteobacteria dominated a collection of 78 bacterial endophytes isolated from poplar and willow trees. As representatives for the dominant genera of endophytic gammaproteobacteria, we selected Enterobacter sp. strain 638, Stenotrophomonas maltophilia R551-3, Pseudomonas putida W619, and Serratia proteamaculans 568 for genome sequencing and analysis of their plant growth-promoting effects, including root development. Derivatives of these endophytes, labeled with gfp, were also used to study the colonization of their poplar hosts. In greenhouse studies, poplar cuttings (Populus deltoides × Populus nigra DN-34) inoculated with Enterobacter sp. strain 638 repeatedly showed the highest increase in biomass production compared to cuttings of noninoculated control plants. Sequence data combined with the analysis of their metabolic properties resulted in the identification of many putative mechanisms, including carbon source utilization, that help these endophytes to thrive within a plant environment and to potentially affect the growth and development of their plant hosts. Understanding the interactions between endophytic bacteria and their host plants should ultimately result in the design of strategies for improved poplar biomass production on marginal soils as a feedstock for biofuels.Endophytic bacteria are bacteria that reside within the living tissue of their host plants without substantively harming it (19, 26). They are ubiquitous in most plant species, latently residing or actively colonizing the tissues. The diversity of cultivable bacterial endophytes is exhibited not only in the variety of plant species colonized but also in the many taxa involved, with most being members of common soil bacterial genera such as Enterobacter, Pseudomonas, Burkholderia, Bacillus, and Azospirillum (21, 23). Endophytic bacteria have several mechanisms by which they can promote plant growth and health. These mechanisms are of prime importance for the use of plants as feedstocks for biofuels and for carbon sequestration through biomass production. This is vital when considering the aim of improving biomass production of marginal soils, thus avoiding competition for agricultural resources, which is one of the critical socioeconomic issues of the increased use of biofuels.Like rhizosphere bacteria, endophytic bacteria have been shown to have plant growth-promoting activity that can be due to the production of phytohormones, enzymes involved in growth regulator metabolism, such as ethylene, 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, auxins, indole-3-acetic acid (IAA), acetoin, 2,3-butanediol, cytokinins (3, 13-15, 20, 30), or combinations thereof. They can also improve plant growth via the fixation of nitrogen (diazotrophy) (9, 38).Typical examples of marginal soils include soils that have deteriorated due to the presence of heavy metals or organic contaminants. These are often soils with a history of industrial, military, or mining activities. Endophytic bacteria can assist their host plants in overcoming phytotoxic effects caused by environmental contamination (5, 11, 12, 36), which is of direct relevance for waste management and pollution control via phytoremediation technologies. When nonsterile poplar cuttings (Populus trichocarpa × deltoides cv. Hoogvorst) were inoculated with the endophyte Burkholderia cepacia VM1468, a derivative of B. cepacia Bu72 which possesses the pTOM-Bu61 plasmid coding for a constitutively expressed toluene degradation pathway, it was observed that in addition to decreasing the phytotoxicity and releasing toluene, strain VM1468 also considerably improved the growth of poplar trees in the absence of toluene (36). This observation, which was the first of its kind for poplar trees, prompted us to further study the poplar tree-associated beneficial endophytic bacteria in order to improve the overall performance of poplar trees, as it can enhance multiple applications, including biomass production, carbon sequestration, and phytoremediation. This was done by screening endophytic bacteria for their plant growth-promoting capabilities toward poplar trees by performing colonization studies with gfp-labeled strains, by examining their metabolic properties, and by initiating the genome sequencing of several strains.     

Lipid remodeling in Rhodopseudomonas palustris TIE‐1 upon loss of hopanoids and hopanoid methylation

The sedimentary record of molecular fossils (biomarkers) can potentially provide important insights into the composition of ancient organisms; however, it only captures a small portion of their original lipid content. To interpret what remains, it is important to consider the potential for functional overlap between different lipids in living cells, and how the presence of one type might impact the abundance of another. Hopanoids are a diverse class of steroid analogs made by bacteria and found in soils, sediments, and sedimentary rocks. Here, we examine the trade‐off between hopanoid production and that of other membrane lipids. We compare lipidomes of the metabolically versatile α‐proteobacterium Rhodopseudomonas palustris TIE‐1 and two hopanoid mutants, detecting native hopanoids simultaneously with other types of polar lipids by electrospray ionization mass spectrometry. In all strains, the phospholipids contain high levels of unsaturated fatty acids (often >80 %). The degree to which unsaturated fatty acids are modified to cyclopropyl fatty acids varies by phospholipid class. Deletion of the capacity for hopanoid production is accompanied by substantive changes to the lipidome, including a several‐fold rise of cardiolipins. Deletion of the ability to make methylated hopanoids has a more subtle effect; however, under photoautotrophic growth conditions, tetrahymanols are upregulated twofold. Together, these results illustrate that the ‘lipid fingerprint’ produced by a micro‐organism can vary depending on the growth condition or loss of single genes, reminding us that the absence of a biomarker does not necessarily imply the absence of a particular source organism.     

Interactive effects of seed availability,water depth,and phosphorus enrichment on cattail colonization in an Everglades wetland

The relative importance of seed availability, waterdepth, and soil phosphorus (P) concentrations oncattail (Typha domingensis pers.) earlyestablishment in an Everglades wetland area wasexamined using seed bank analysis and controlledexperiments. The experiments measured seed germinationand seedling growth in tanks with cattail seedaddition subjected to two P concentrations(un-enriched vs. enriched) and water depth (saturatedvs. flooded soils). A limited seed bank (223 ± 69m²) of cattail was found in the surface soil ofthe area studied. The germination of added seeds wasinhibited under flooded conditions, and only 0.6% ofthe germination was found. In contrast,under-saturated soil conditions, a maximum of 6% and15% germination was observed in P-un-enriched andP-enriched treatments, respectively. High mortality ofseedlings occurred regardless of P treatments followinga cold spell. However, P enrichment resulted inincreased seedling growth and asexual propagation.These results suggested the importance of theconcurrence of appropriate hydrologic regimes, Penrichment, and air temperature on the recruitment ofplant species.     

Effect of Omega-3 Fatty Acid Ethyl Esters on the Oxylipin Composition of Lipoproteins in Hypertriglyceridemic,Statin-Treated Subjects

Background

Oxylipins mediate inflammation, vascular tension, and more. Their presence in lipoproteins could explain why lipoproteins mediate nearly identical activities.

Methods

To determine how oxylipins are distributed in the lipoproteins of hypertriglyceridemic subjects, and whether omega-3 fatty acids alter them in a manner consistent with improved cardiovascular health, we recruited 15 dyslipidemic subjects whose levels of low density lipoprotein cholesterol (LDL-C) were at goal but who remained hypertriglyceridemic (200–499 mg/dL). They were treated them with the indicated dose of 4 g/d omega-3 acid ethyl esters (P-OM3) for 8 weeks. Measured oxylipins included mid-chain alcohols (HETEs, HEPEs and HDoHEs), ketones (KETEs), epoxides (as EpETrEs, EpETEs, and EpDPEs).

Results

At baseline, arachidonate-oxylipins (HETEs, KETEs, and EpETrEs) were most abundant in plasma with the greatest fraction of total abundance (mean |95% CI|) being carried in high density lipoproteins (HDL); 42% |31, 57| followed by very low density lipoproteins (VLDL); 27% |20, 36|; and LDL 21% |16, 28|. EPA- and DHA-derived oxylipins constituted less than 11% of total. HDL carried alcohols and epoxides but VLDL was also rich in ketones. Treatment decreased AA-derived oxylipins across lipoprotein classes (−23% |−33, −12|, p = 0.0003), and expanded EPA−(322% |241, 422|, p<0.0001) and DHA-derived oxylipins (123% |80, 176|, p<0.0001).

Conclusions

Each lipoprotein class carries a unique oxylipin complement. P-OM3 treatment alters the oxylipin content of all classes, reducing pro-inflammatory and increasing anti-inflammatory species, consistent with the improved inflammatory and vascular status associated with the treatment.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00959842","term_id":"NCT00959842"}}NCT00959842     

Structural and compositional changes associated with chlorine inactivation of polioviruses.

Chlorine inactivation of polioviruses resulted in the loss of viral ribonucleic acid, converting the viruses from 156S particles to 80S particles. However, it was found that virus inactivation occurred before the ribonucleic acid was released from the virions. Extraction of ribonucleic acid from partially inactivated virus suspensions indicated that chlorine inactivation was due to degradation of the ribonucleic acid before release and that ribonucleic acid loss was a secondary event. The empty 80S capsids had the same isoelectric point and ability to attach to host cells as infective virions. Thus, no major capsid conformational changes occurred during chlorine inactivation.     

Metabolomics analysis of the Lolium perenne–Neotyphodium lolii symbiosis: more than just alkaloids?

Above ground plant parts of Lolium perenne often harbour endophytic Neotyphodium lolii fungi. These occur both naturally and commercially, as variant strains are introduced to modify the grass metabolic profile. They reside in the apoplastic spaces and rarely cause visible symptoms of infection. The vast majority of literature has focussed on the biosynthesis, accumulation, and ecological relevance of a limited number of alkaloids produced by N. lolii which have been shown to negatively affect insect pests and vertebrate herbivores. Much less is known about the effects of other metabolites in these interactions or the role of resource supply on metabolic profiles, nor critically on the metabolic consequences of differences in the amount (concentration) of endophyte present. Here, we provide a synthesis of some of our recently published studies on effects of resource supply (nitrogen, carbohydrates) on concentrations of endophytes and endophyte specific metabolites in the L. perenne–N. lolii association. We present results of both quantitative PCR and targeted metabolomics studies, using contrasting endophyte strains in two perennial ryegrass cultivars. We also present and discuss a hypothetical schematic representation of possible links between plant and fungal metabolic networks. A multiple regression analysis of numerical insect responses and metabolic profiles indicates that effects of endophyte infection on insect population sizes could be predicted by concentrations of a range of metabolites other than alkaloids and depended on insect species, fungal strain, and nitrogen supply.     

Effect of minocycline on subgingival plaque bacteria

The effects of minocycline on subgingival plaque samples from patients with chronic periodontitis were investigated in vitro. Minocycline concentrations as low as 1.0 μg/ml inhibited 95.7% of the cultivable bacteria in the samples but 256 μg/ml was necessary to inhibit all of the cultivable bacteria in the samples. Although up to 99.9% of bacteria in the plaque samples were killed by a 6 h exposure to 8.0 μg/ml of minocycline, large numbers of viable bacteria remained. These results imply that adequate reductions in the numbers of viable subgingival plaque bacteria are unlikely to occur after exposure to minocycline at concentrations attainable in gin-gival crevicular fluid after systemic administration.     

A new role for platelet-endothelial cell adhesion molecule-1 (CD31): inhibition of TCR-mediated signal transduction.

Platelet-endothelial cell adhesion molecule-1 (PECAM-1) is a 130-kDa transmembrane glycoprotein expressed by endothelial cells, platelets, monocytes, neutrophils, and certain T cell subsets. The PECAM-1 extracellular domain has six Ig-homology domains that share sequence similarity with cellular adhesion molecules. The PECAM-1 cytoplasmic domain contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) that, when appropriately engaged, becomes phosphorylated on tyrosine residues, creating docking sites for nontransmembrane, Src homology 2 domain-bearing protein tyrosine phosphatase (SHP)-1 and SHP-2. The purpose of the present study was to determine whether PECAM-1 inhibits protein tyrosine kinase (PTK)-dependent signal transduction mediated by the immunoreceptor tyrosine-based activation motif-containing TCR. Jurkat cells, which coexpress PECAM-1 and the TCR/CD3 complex, were INDO-1AM-labeled and then incubated with anti-CD3epsilon mAbs, anti-PECAM-1 mAbs, or both, and goat anti-mouse IgG was used to cross-link surface-bound mAbs. Calcium mobilization induced by CD3 cross-linking was found to be attenuated by coligation of PECAM-1 in a dose-dependent manner. PECAM-1-mediated inhibition of TCR signaling was attributable, at least in part, to inhibition of release of calcium from intracellular stores. These data provide evidence that PECAM-1 can dampen signals transduced by ITAM-containing receptors and support inclusion of PECAM-1 within the family of ITIM-containing inhibitors of PTK-dependent signal transduction.     

Fluxes of h and k in corn roots : characterization and stoichiometries using ion-selective microelectrodes

We report here on an experimental system that utilizes ion-selective microelectrodes to measure the electrochemical potential gradients for H⁺ and K⁺ ions within the unstirred layer near the root surface of both intact 4-day-old corn seedlings and corn root segments. Analysis of the steady state H⁺ and K⁺ electrochemical potential gradients provided a simultaneous measure of the fluxes crossing a localized region of the root surface. Net K⁺ influx values obtained by this method were compared with unidirectional K⁺ (⁸⁶Rb⁺) influx kinetic data; at any particular K⁺ concentration, similar values were obtained by either technique. The ionspecific microelectrode system was then used to investigate the association between net H⁺ efflux and net K⁺ influx. Although the computed H⁺:K⁺ stoichiometry is dependent upon the choice of diffusion coefficients, the values obtained were extremely variable, and net K⁺ influx rarely appeared to be charge-balanced by H⁺ efflux. In contrast to earlier studies, we found the cortical membrane potential to be highly K⁺ sensitive within the micromolar K⁺ concentration range. Simultaneous measurements of membrane potential and K⁺ influx, as a function of K⁺ concentration, revealed similar K_m values for the depolarization of the potential (K_m 6-9 micromolar K⁺) and net K⁺ influx (K_m 4-7 micromolar K⁺). These data suggest that K⁺ may enter corn roots via a K⁺-H⁺ cotransport system rather than a K⁺/H⁺ antiporter.