ABSTRACT Urinary tract infections (UTIs) are a major global health problem and are caused predominantly by uropathogenic Escherichia coli (UPEC). UTIs are a leading cause of prescription antimicrobial use. Incessant increase in antimicrobial resistance in UPEC and other uropathogens poses a serious threat to the current treatment practices. Copper is an effector of nutritional immunity that impedes the growth of pathogens during infection. We hypothesized that copper would augment the toxicity of select small molecules against bacterial pathogens. We conducted a small molecule screening campaign with a library of 51,098 molecules to detect hits that inhibit a UPEC Δ tolC mutant in a copper-dependent manner. A molecule, denoted as E. coli inhibitor or ECIN, was identified as a copper-responsive inhibitor of wild-type UPEC strains. Our gene expression and metal content analysis results demonstrate that ECIN works in concert with copper to exacerbate Cu toxicity in UPEC. ECIN has a broad spectrum of activity against pathogens of medical and veterinary significance including Acinetobacter baumannii, Pseudomonas aeruginosa , and methicillin-resistant Staphylococcus aureus . Subinhibitory levels of ECIN eliminate UPEC biofilm formation. Transcriptome analysis of UPEC treated with ECIN reveals induction of multiple stress response systems. Furthermore, we demonstrate that L-cysteine rescues the growth of UPEC exposed to ECIN. In summary, we report the identification and characterization of a novel copper-responsive small molecule inhibitor of UPEC. IMPORTANCE Urinary tract infection (UTI) is a ubiquitous infectious condition affecting millions of people annually. Uropathogenic Escherichia coli (UPEC) is the predominant etiological agent of UTI. However, UTIs are becoming increasingly difficult to resolve with antimicrobials due to increased antimicrobial resistance in UPEC and other uropathogens. Here, we report the identification and characterization of a novel copper-responsive small molecule inhibitor of UPEC. In addition to E. coli , this small molecule also inhibits pathogens of medical and veterinary significance including Acinetobacter baumannii, Pseudomonas aeruginosa, and methicillin-resistant Staphylococcus aureus .

BACKGROUND: A GPCR (G protein-coupled receptor) target-based approach was applied to identify antagonists of the arthropod-specific tick kinin receptor. These small molecules were expected to reproduce the detrimental phenotypic effects that had been observed in Rhipicephalus microplus females when the kinin receptor was silenced by RNA interference. Rhipicephalus microplus, the southern cattle tick, cattle fever tick, or Asian blue tick, is the vector of pathogenic microorganisms causing the deadly bovine babesiosis and anaplasmosis. The widespread resistance to acaricides in tick populations worldwide emphasizes that exploring novel targets for effective tick control is imperative. RESULTS: Fifty-three structural analogs of previously identified tick kinin antagonists were screened in a 'dual-addition' calcium fluorescence assay using a CHO-K1 cell line expressing the tick kinin receptor. Seven molecules were validated as non-cytotoxic antagonists, four of which were partial (SACC-0428764, SACC-0428780, SACC-0428800, and SACC-0428803), and three were full antagonists (SACC-0428799, SACC-0428801, and SACC-0428815). Four of these antagonists (SACC-0428764, SACC-0428780, SACC-0428799, and SACC-0428815) also inhibited the tick midgut contractions induced by the myotropic kinin agonist analog 1728, verifying their antagonistic bioactivity. The small molecules were tested on recombinant human neurokinin (NK) receptors, the one most similar to the invertebrate kinin receptors. Most molecules were inhibitors of the NK1 receptor, except SACC-0412066, a previously identified tick kinin receptor antagonist, which inhibited the NK1 receptor only at the highest concentration tested (25 mu m). None of the molecules inhibited the NK3 human receptor. CONCLUSION: Molecules identified through this approach could be useful probes for studying the tick kinin signaling system and midgut physiology. (c) 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

G protein-coupled receptors (GPCRs) represent the largest superfamily of receptors and are the targets of numerous human drugs. High-throughput screening (HTS) of random small molecule libraries against GPCRs is used by the pharmaceutical industry for target-specific drug discovery. In this study, an HTS was employed to identify novel small-molecule ligands of invertebrate-specific neuropeptide GPCRs as probes for physiological studies of vectors of deadly human and veterinary pathogens. The invertebrate-specific kinin receptor was chosen as a target because it regulates many important physiological processes in invertebrates, including diuresis, feeding, and digestion. Furthermore, the pharmacology of many invertebrate GPCRs is poorly characterized or not characterized at all; therefore, the differential pharmacology of these groups of receptors with respect to the related GPCRs in other metazoans, especially humans, adds knowledge to the structure-activity relationships of GPCRs as a superfamily. An HTS assay was developed for cells in 384-well plates for the discovery of ligands of the kinin receptor from the cattle fever tick, or southern cattle tick, Rhipicephalus microplus. The tick kinin receptor was stably expressed in CHO-K1 cells. The kinin receptor, when activated by endogenous kinin neuropeptides or other small molecule agonists, triggers Ca2+ release from calcium stores into the cytoplasm. This calcium fluorescence assay combined with a "dual-addition" approach can detect functional agonist and antagonist "hit" molecules in the same assay plate. Each assay was conducted using drug plates carrying an array of 320 random small molecules. A reliable Z' factor of 0.7 was obtained, and three agonist and two antagonist hit molecules were identified when the HTS was at a 2 µM final concentration. The calcium fluorescence assay reported here can be adapted to screen other GPCRs that activate the Ca2+ signaling cascade.

Introduction: We have discovered novel non-toxic rifamycins that are extremely potent at sensitizing many drug-resistant cancers, including DLBCL, to standard-of-care chemotherapeutics. Methods: CHOP-resistant DLBCL cell lines were derived from CHOP-sensitive cells by “on-off” cycles of CHOP treatment, analogous to clinical therapy. The CHOP-resistant cells were used in high-throughput screening of a highly diverse collection of approximately 50,000 drug-like molecules to identify small molecules that reverse CHOP-resistance. The FDA-approved drug, Rifabutin, was identified as a non-cytotoxic compound that potently reversed resistance to CHOP. Structure-Activity-Relationships (SAR) on Rifabutin were conducted, which led to the generation of a new more potent CHOP-chemosensitizing agent, designated RTI. Results: RTI was highly synergistic with variety of chemotherapeutics, including doxorubicin (DOX), epirubicin, vinblastine, etoposide in drug-resistant NHL cells. Combination therapy of DOX+RTI in mouse xenograft models of DLBCL was much more effective at repressing tumor growth than with DOX alone. RTI lowered the IC90 in a dose-dependent manner in CD20-positive B cells in bone marrow aspirates from both CHOP-naïve and CHOP-relapsed patients, and in a metastatic lymph node. RTI’s PK characteristics are similar to rifabutin, and it exhibited no overt toxicity in mice or pigs at high doses. RTI rapidly induced mitochondrial superoxide, membrane potential, and fission. The superoxide dismutase, FeTCP, antagonized RTI-induced ROS and potentiation of DOX cytotoxicity. RTI reduced the activity of the Nrf-2 antioxidant protein, upregulated proteins involved in the unfolded protein response (UPR), and induced metabolic reprogramming as indicated by Seahorse assays that showed increased glycolysis and decreased oxygen consumption from 3 to 24 hrs. Conclusions: RTI has a broad spectrum of action in both double- and triple-hit DLBCL and synergizes with many different chemotherapeutics to restore drug sensitivity. RTI-79 works by dramatically increasing intracellular superoxide through redox cycling, triggers UPR, and downregulates Nrf-2 activity. Thus, RTI-79 increases oxidative stress through the squelching of Nrf-2’s ability to respond to chemotherapeutic stress. Since the parent compound, rifabutin, binds to the aryl hydrocarbon receptor (AhR) and modulates its activity, we hypothesize that RTI mediates its unique pleiotropic chemosensitizing mechanism through targeting of AhR, and provides for a broad, safe, and novel approach to treating drug resistant cancers. Ongoing experiments are investigating the effect of RTI on the AhR-signaling pathway and the role it plays in RTI-mediated chemosensitization and potentiation of chemotherapeutics. A clinical trial of companion dogs with CHOP-relapsed DLBCL treated with combination CHOP+RTI is currently in progress. Citation Format: Steve A. Maxwell, Deeann Wallis, Nian Zhou, Dwight Baker, Seyed H. Mousavi-Fard, Kimberly Loesch, Stacy Galaviz, Liam Guthrie, Thomas Snavely, Qingan Sun, Carolina M. Rojas, David W. Threadgill, Thomas Ioerger, Wen Dong, Gwen Seemann, Theresa W. Fossum, James C. Sacchettini. Development of novel, non-toxic rifamycins that reverse drug resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 410.

BACKGROUND: The southern cattle tick, Rhipicephalus microplus, is a primary vector of the deadly bovine disease babesiosis. Worldwide populations of ticks have developed resistance to acaricides, underscoring the need for novel target discovery for tick control. The arthropod-specific R. microplus kinin receptor is such a target, previously validated by silencing, which resulted in female reproductive fitness costs, including a reduced percentage of eggs hatching. RESULTS: In order to identify potent small molecules that bind and activate or inhibit the kinin receptor, a high-throughput screening (HTS) assay was developed using a CHO-K1 cell line expressing the recombinant tick kinin receptor (BMLK3). A total of similar to 20 000 molecules from a random in-house small molecule library were screened in a 'dual-addition' calcium fluorescence assay. This was followed by dose-response validation of the hit molecules identified both from HTS and an in silico screen of similar to 390 000 molecules. We validated 29 antagonists, 11 of them were full antagonists with IC50 values between 0.67 and 8 mu mol L-1. To explore the structure-activity relationships (SAR) of the small molecules, we tested the activities of seven analogs of the most potent identified antagonist, additionally discovering three full antagonists and four partial antagonists. These three potent antagonists (IC50 < 3.2 mu mol L-1) were validated in vitro using the recombinant mosquito kinin receptor and showed similar antagonistic activities. In vivo, these three compounds also inhibited the mosquito hindgut contraction rate induced by a myotropic kinin agonist analog 1728. CONCLUSION: Antagonists identified in this study could become pesticide leads and are reagents for probing the kinin signaling system. (c) 2021 Society of Chemical Industry

The success of the acaricide amitraz, a ligand of the tick tyramine/octopamine receptor (a G protein-coupled receptor; GPCR), stimulated interest on arthropod-specific GPCRs as targets to control tick populations. This search advances tick physiology because little is known about the pharmacology of tick GPCRs, their endogenous ligands or their physiological functions. Here we explored the tick kinin receptor, a neuropeptide GPCR, and its ligands. Kinins are pleiotropic insect neuropeptides but their function in ticks is unknown. The endogenous tick kinins are unknown and their cDNAs have not been cloned in any species. In contrast, more than 271 insect kinin sequences are available in the DINeR database. To fill this gap, we cloned the kinin cDNA from the cattle fever tick, Rhipicephalus microplus, which encodes 17 predicted kinins, and verified the kinin gene structure. We predicted the kinin precursor sequences from additional seven tick species, including Ixodes scapularis. All species showed an expansion of kinin paracopies. The "kinin core" (minimal active sequence) of tick kinins FX1X2WGamide is similar to those in insects. Pro was predominant at the X2 position in tick kinins. Toward accelerating the discovery of kinin function in ticks we searched for novel synthetic receptor ligands. We developed a dual-addition assay for functional screens of small molecules and/or peptidomimetics that uses a fluorescent calcium reporter. A commercial library of fourteen small molecules antagonists of mammalian neurokinin (NK) receptors was screened using this endpoint assay. One acted as full antagonist (TKSM02) with inhibitory concentration fifty (IC50) of ∼45 μM, and three were partial antagonists. A subsequent calcium bioluminescence assay tested these four antagonists through kinetic curves and confirmed TKSM02 as full antagonist and one as partial antagonist (TKSM14). Antagonists of NK receptors displayed selectivity (>10,000-fold) on the tick kinin receptor. Three peptidomimetic ligands of the mammalian NK receptors (hemokinin 1, antagonist G, and spantide I) were tested in the bioluminescence assay but none were active. Forward approaches may accelerate discovery of kinin ligands, either as reagents for tick physiological research or as lead molecules for acaricide development, and they demonstrate that selectivity is achievable between mammalian and tick neuropeptide systems.

Introduction: Refractory/relapsed disease remains a primary cause of morbidity and mortality in DLBCL patients. We have discovered novel non-toxic rifamycins that potently reverse drug-resistance in DLBCL cells. Methods: CHOP-resistant DLBCL cells were generated from the CRL2631 cell line by “on-off” cycles of CHOP treatment, analogous to clinical therapy. The CHOP-resistant cells were used in high-throughput screening of a collection of 50,000 drug-like molecules to identify compounds that reverse CHOP-resistance. The FDA-approved drug, rifabutin, was identified as a non-cytotoxic compound that potently reversed resistance to CHOP. Structure-Activity-Relationship (SAR) studies on rifabutin were conducted, which led to the generation of a new more potent CHOP-chemosensitizing agent, designated RTI-79. Results: RTI-79 was highly synergistic with CHOP in a variety of DLBCL cells. Combination therapy of doxorubicin + RTI-79 in mouse xenograft models of DLBCL was more effective at repressing tumor growth than doxorubicin alone. Two companion dogs afflicted with CHOP-relapsed DLBCL treated with combination CHOP + rifabutin showed regression of DLBCL. RTI-79 lowered the doxorubicin IC90 and IC99 in a dose-dependent manner in CD20-positive B cells in bone marrow aspirates from both CHOP-naïve and CHOP-relapsed human patients, and in a metastatic DLBCL-diseased lymph node. RTI-79 potentiated the activity of many chemotherapeutics, including daunorubicin, epirubicin, vinblastine, etoposide, paclitaxel, and topotecan. RTI-79's PK characteristics are similar to rifabutin and exhibited no overt toxicity in mice at high doses. RTI-79 did not exacerbate the known cardiotoxicity associated with doxorubicin as evaluated by body weight, CBCs, and heart function via echocardiograms. RTI-79 rapidly induced intracellular ROS and increased both mitochondrial membrane potential and fission. The anti-oxidant quercetin antagonized both RTI-79-induced ROS and potentiation of doxorubicin cytotoxicity. RTI-79 reduced expression of the anti-oxidant regulator protein, Nrf-2, potentially through upregulation of SYVN1, an E3 ubiquitin ligase that interacts directly with Nrf-2. Moreover, RTI-79 caused upregulation of proteins involved in the unfolded protein response (UPR). Conclusions: RTI-79 has a broad spectrum of action in both double- and triple-hit DLBCL and synergizes with many different chemotherapeutics to restore drug sensitivity. RTI-79 works by increasing intracellular ROS, primarily superoxide, through redox cycling. RTI-79 triggers the UPR that results in increased ubiquitination and loss of Nrf-2. Thus, RTI-79 induces oxidative stress by increasing ROS and reducing Nrf-2's ability to respond to ROS. This unique pleiotropic chemosensitizing mechanism provides a novel approach for treating drug resistant cancers. Keywords: diffuse large B-cell lymphoma (DLBCL); doxorubicin; non-Hodgkin lymphoma (NHL).

New and improved drugs against tuberculosis are urgently needed as multi-drug-resistant forms of the disease become more prevalent. Mycobacterium tuberculosis cytidylate kinase is an attractive target for screening due to its essentiality and different substrate specificity to the human orthologue. However, we selected the Mycobacterium smegmatis cytidylate kinase for screening because of the availability of high-resolution X-ray crystallographic data defining its structure and the high likelihood of active site structural similarity to the M. tuberculosis orthologue. We report the development and implementation of a high-throughput luciferase-based activity assay and screening of 19,920 compounds derived from small-molecule libraries and an in silico screen predicting likely inhibitors of the cytidylate kinase enzyme. Hit validation included a counterscreen for luciferase inhibitors that would result in false positives in the initial screen. Results of this counterscreen ruled out all of the putative cytidylate kinase inhibitors identified in the initial screening, leaving no compounds as candidates for drug development. Although a negative result, this study indicates that this important drug target may in fact be undruggable and serve as a warning for future investigations.

The twin arginine translocation (Tat) pathway transports fully-folded and assembled proteins in bacteria, archaea and plant thylakoids. The Tat pathway contributes to the virulence of numerous bacterial pathogens that cause disease in humans, cattle and poultry. Thus, the Tat pathway has the potential to be a novel therapeutic target. Deciphering the Tat protein transport mechanism has been challenging since the active translocon only assembles transiently in the presence of substrate and a proton motive force. To identify inhibitors of Tat transport that could be used as biochemical tools and possibly as drug development leads, we developed a high throughput screen (HTS) to assay the effects of compounds in chemical libraries against protein export by the Escherichia coli Tat pathway. The primary screen is a live cell assay based on a fluorescent Tat substrate that becomes degraded in the cytoplasm when Tat transport is inhibited. Consequently, low fluorescence in the presence of a putative Tat inhibitor was scored as a hit. Two diverse chemical libraries were screened, yielding average Z'-factors of 0.74 and 0.44, and hit rates of ~0.5% and 0.04%, respectively. Hits were evaluated by a series of secondary screens. Electric field gradient (Δψ) measurements were particularly important since the bacterial Tat transport requires a Δψ. Seven low IC50 hits were eliminated by Δψ assays, suggesting ionophore activity. As Δψ collapse is generally toxic to animal cells and efficient membrane permeability is generally favored during the selection of library compounds, these results suggest that secondary screening of hits against electrochemical effects should be done early during hit validation. Though none of the short-listed compounds inhibited Tat transport directly, the screening and follow-up assays developed provide a roadmap to pursue Tat transport inhibitors.

The Visible Integral-Field Replicable Unit Spectrograph (VIRUS) instrument will be installed at the Hobby-Eberly Telescope† in the near future. The instrument will be housed in two enclosures that are mounted adjacent to the telescope, via the VIRUS Support Structure (VSS). We have designed the enclosures to support and protect the instrument, to enable servicing of the instrument, and to cool the instrument appropriately while not adversely affecting the dome environment. The system uses simple HVAC air handling techniques in conjunction with thermoelectric and standard glycol heat exchangers to provide efficient heat removal. The enclosures also provide power and data transfer to and from each VIRUS unit, liquid nitrogen cooling to the detectors, and environmental monitoring of the instrument and dome environments. In this paper, we describe the design and fabrication of the VIRUS enclosures and their subsystems.

Natural products have provided considerable value to the pharmaceutical industry over the past half century. In particular, the therapeutic areas of infectious diseases and oncology have benefited from numerous drug classes derived from natural product sources. Unfortunately, pharmaceutical companies have significantly decreased activities in natural product discovery during the past several years. Biotechnology companies working in the fields of combinatorial biosynthesis, genetic engineering and metagenomic approaches to identify novel natural product lead molecules have had limited success. Despite what appears to be a slow death of natural product discovery research, many new and interesting molecules with biological activity have been published in the past few years. If natural product materials continue to be tested for desirable therapeutic activities, we believe that significant progress in identifying new antibiotics, oncology therapeutics and other useful medicines will be made.

Natural product compounds are the source of numerous therapeutic agents. Recent progress to discover drugs from natural product sources has resulted in compounds that are being developed to treat cancer, resistant bacteria and viruses and immunosuppressive disorders. Many of these compounds were discovered by applying recent advances in understanding the genetics of secondary metabolism in actinomycetes, exploring the marine environment and applying new screening technologies. In many instances, the discovery of a novel natural product serves as a tool to better understand targets and pathways in the disease process. This review describes recent progress in drug discovery from natural sources including several examples of compounds that inhibit novel drug targets.

A wide variety of novel small-molecule natural products has recently been reported. These compounds were isolated from marine and terrestrial sources, and from a variety of animals, plants and microorganisms. With the breadth of diversity represented in these bioactive small molecules, the future of natural product drug discovery looks bright.

The extent of transfer of fixed N between N2-fixing and non-N2-fixing plant species is largely unknown in successional studies. In order to redress this deficiency at a locale intensively studied ecologically, leaf tissue samples were collected from actinorhizal N2-fixing (Alnus, Shepherdia, and Dryas) and two non-N2-fixing (Salix) woody species within research plots located along a chronosequence of deglaciated fjord in Glacier Bay National Park, Alaska. The tissue samples were analyzed for δ15N content, and the resulting data analyzed for trends in plant tissue N. Among the non-N2-fixing Salix species, δ15N values increased from the most recently deglaciated sites to converge with the temporally more-stable values for the symbiotic N2-fixing species on sites at about 40 years after deglaciation. The lower δ15N values of sequestered N in plant tissues suggested that N derived from N2-fixing plants accounts for the major portion of N in associated plants up to 40 years after deglaciation. The 15N isotopic data also suggested that Shepherdia canadensis depends least on soil N, D. drummondii the most, and A. viridis ssp. sinuata somewhere between those two species. The presence of a sere dominated by dense thickets of A. viridis ssp. sinuata at the convergence of δ15N values for the N2-fixing and non-N2-fixing species indicated that this species is most responsible for accumulation of fixed N in soil at Glacier Bay. This paper is dedicated to the memory of Steven J. Kohls who died prior to publication of this research.

Two new trichothecenes, 14'-hydroxymytoxin B (1) and 16-hydroxyroridin E (3), were isolated from a fermentation extract of Myrothecium roridum. The structures of 1 and 3 were determined by spectral data interpretation. Both compounds showed potent cytotoxic activity against primary soft-tissue sarcoma cells.

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Actinorhizal plants form a nodular, nitrogen-fixing root symbiosis with the actinomycete Frankia and are economically and ecologically important due to their ability to improve the nitrogen fertility of disturbed and infertile substrates. In this study, water-retentive polymer inoculum carriers were applied as a root dip. This treatment significantly increased nodulation and in some cases early growth of Alnus glutinosa (L.) Gaertn. and Casuarina equisetifolia var. equisetifolia Forst. & Forst. in a controlled environment and also of A. glutinosa under field conditions. Nodule number and nodule dry weight per plant were at least two to three times greater after 56 to 140 days for plants inoculated with Frankia carried in a water-retentive polymer base compared with plants inoculated with Frankia in water. Nodules on the roots of the plants that were inoculated with Frankia in a polymer slurry were distributed throughout the entire root system, rather than concentrated near the root collar. When amended with water-retentive polymers, actinorhizal plants inoculated with 5- to 10-fold lower titers of Frankia exhibited early growth and nodule numbers equal to or greater than those plants inoculated with standard titers without polymers. The water-retentive, superabsorbent polymers clearly increased the nodulation of two actinorhizal plant species.

Root nodules of the actinorhizal shrub Ceanothus were collected from seven sites from its native range. DNA was extracted from individual nodule lobes using a cetyl trimethyl ammonium bromide (CTAB) extraction procedure. Three DNA probes were used in combination with two restriction endonucleases to evaluate the extent of restriction fragment length polymorphism (RFLP) diversity within and among populations of Ceanothus endophytes. We observed some diversity using a nif DH gene probe; however there was no correlation of RFLP pattern and geographic site. Using two random Frankia probes, we observed more diversity among the Ceanothus endophytes than with the nif probe. Differences in RFLP patterns were observed among plants at a single geographic site and between geographical sites. The results demonstrated that considerable diversity exists among Frankia strains symbiotic with Ceanothus, as has been shown for pure-cultured Frankia strains isolated from other actinorhizal genera. We have also demonstrated the usefulness of this method for the study of Frankia ecology in planta.

The role of both actinorhizal and leguminous N2-flxing plants during primary succession within glacial forelands has received much attention, but there are few estimates of the contribution of fixed N to neo-glacial substrates. The main objectives of our work were (i) to assess the N2-fixing ability of three Dryas taxa across a chronosequence of ca 135 yr of post neo-glacial recession, and (ii) to establish whether Dryas serves as a source of N for non-N2-fixing species. The mineral N pool was highly limited across the sere and increased from 0.3 to 1.3 μg g−1 of soil over the chronosequence. The vascular non-N2-fixing species had negative mean δ15N values over the sere, ranging from −6.4 ± 0.4 to −3.3 ± 0.4. Dryas drummondii, present over the entire chronosequence, showed δ15N values ranging from −6.0 ± 0.5 to 0.32 ± 0.4, and appeared not to fix N2 until the mid to late stages of the sere. Mean estimated values of percent N derived from the atmosphere for D. drummondii ranged from 81 to 89%. The observed δ15N mean values for D. octopetala and D. integrifolia were −3.5 ± 0.5 and −4.9 ± 0.3, respectively. Those values were close to the δ 15N values of the non-N2-fixing species, therefore, at this site, these species did not appear to fix N2. Four non-N2-fixing woody vascular taxa that grew adjacent to N2-fixing plants had significantly greater foliar N contents and higher (less negative) δ15N values than non-N2-fixing woody vascular taxa grown at a distance from the N2-fixing Dryas. The δ15N values from non-N2-fixing plants that grew adjacent to N2-fixing D. drummondii appear to contradict the widely held hypothesis that pioneer N2-fixing species facilitate the establishment of later successional species during primary succession. The absence of N2-fixing activity by D. drummondii during the early stages of the sere, the higher concentration of N, and the less negative δ15N values for foliar N content in plants adjacent to this taxon, suggest an alternative mechanism for the spatial and temporal vegetation change during primary succession. These observations confirm and refine a hypothesis suggested earlier that N2-fixing plants stimulate, through the process of N transfer, adjacent non-N2-fixing plants that are already established.