Genetic synergy between Acinetobacter baumannii undecaprenyl phosphate biosynthesis and the Mla system impacts cell envelope and antimicrobial resistance.

Acinetobacter baumannii is a Gram-negative bacterial pathogen that poses a major health concern due to increasing multidrug resistance. The Gram-negative cell envelope is a key barrier to antimicrobial entry and includes an inner and outer membrane. The maintenance of lipid asymmetry (Mla) system is the main homeostatic mechanism by which Gram-negative bacteria maintain outer membrane asymmetry. Loss of the Mla system in A. baumannii results in attenuated virulence and increased susceptibility to membrane stressors and some antibiotics. We recently reported two strain variants of the A. baumannii type strain ATCC 17978: 17978VU and 17978UN. Here, ∆mlaF mutants in the two ATCC 17978 strains display different phenotypes for membrane stress resistance, antibiotic resistance, and pathogenicity in a murine pneumonia model. Although allele differences in obgE were previously reported to synergize with ∆mlaF to affect growth and stringent response, obgE alleles do not affect membrane stress resistance. Instead, a single-nucleotide polymorphism (SNP) in the essential gene encoding undecaprenyl pyrophosphate (Und-PP) synthase, uppS, results in decreased enzymatic rate and decrease in total Und-P levels in 17978UN compared to 17978VU. The UppSUN variant synergizes with ∆mlaF to reduce capsule and lipooligosaccharide (LOS) levels, increase susceptibility to membrane stress and antibiotics, and reduce persistence in a mouse lung infection. Und-P is a lipid glycan carrier required for the biosynthesis of A. baumannii capsule, cell wall, and glycoproteins. These findings uncover synergy between Und-P and the Mla system in maintaining the A. baumannii cell envelope and antibiotic resistance.IMPORTANCEAcinetobacter baumannii is a critical threat to global public health due to its multidrug resistance and persistence in hospital settings. Therefore, novel therapeutic approaches are urgently needed. We report that a defective undecaprenyl pyrophosphate synthase (UppS) paired with a perturbed Mla system leads to synthetically sick cells that are more susceptible to clinically relevant antibiotics and show reduced virulence in a lung infection model. These results suggest that targeting UppS or undecaprenyl species and the Mla system may resensitize A. baumannii to antibiotics in combination therapies. This work uncovers a previously unknown synergistic relationship in cellular envelope homeostasis that could be leveraged for use in combination therapy against A. baumannii.

Genetic synergy in Acinetobacter baumannii undecaprenyl biosynthesis and maintenance of lipid asymmetry impacts outer membrane and antimicrobial resistance.

Acinetobacter baumannii is a Gram-negative healthcare-associated pathogen that poses a major health concern due to increasing multidrug resistance. The Gram-negative cell envelope is a key barrier to antimicrobial entry and includes an inner and outer membrane. The outer membrane has an asymmetric composition that is important for structural integrity and barrier to the environment. Therefore, Gram-negative bacteria have mechanisms to uphold this asymmetry such as the maintenance of lipid asymmetry system (Mla), which removes glycerophospholipids from the outer leaflet of the outer membrane and transports them to the inner membrane. Loss of this system in A. baumannii results in attenuated virulence and increased susceptibility to membrane stressors and some antibiotics. We recently reported two strain variants of the A. baumannii type strain ATCC 17978, 17978VU and 17978UN. We show here that ΔmlaF mutants in the two strains display different phenotypes for membrane stress resistance, antibiotic resistance, and pathogenicity in a murine pneumonia model. We used comparative genetics to identify interactions between ATCC 17978 strain alleles and mlaF to uncover the cause behind the phenotypic differences. Although allele differences in obgE were previously reported to synergize with ΔmlaF to affect growth and stringent response, we show that obgE alleles do not affect membrane stress resistance. Instead, a single nucleotide polymorphism (SNP) in the essential gene encoding undecaprenyl pyrophosphate (Und-PP) synthase, uppS, synergizes with ΔmlaF to increase susceptibility to membrane stress and antibiotics, and reduce persistence in a mouse lung infection. Und-P is a lipid glycan carrier known to be required for biosynthesis of A. baumannii capsule, cell wall, and glycoproteins. Our data suggest that in the absence of the Mla system, the cellular level of Und-P is critical for envelope integrity, antibiotic resistance, and lipooligosaccharide abundance. These findings uncover synergy between Und-P and the Mla system in maintaining the A. baumannii outer membrane and stress resistance.

Reconstitution of the transition from a lamellipodia- to filopodia-like actin network with purified proteins.

How cells utilize complex mixtures of actin binding proteins to assemble and maintain functionally diverse actin filament networks with distinct architectures and dynamics within a common cytoplasm is a longstanding question in cell biology. A compelling example of complex and specialized actin structures in cells are filopodia which sense extracellular chemical and mechanical signals to help steer motile cells. Filopodia have distinct actin architecture, composed of long, parallel actin filaments bundled by fascin, which form finger-like membrane protrusions. Elongation of the parallel actin filaments in filopodia can be mediated by two processive actin filament elongation factors, formin and Ena/VASP, which localize to the tips of filopodia. There remains debate as to how the architecture of filopodia are generated, with one hypothesis proposing that filopodia are generated from the lamellipodia, which consists of densely packed, branched actin filaments nucleated by Arp2/3 complex and kept short by capping protein. It remains unclear if different actin filament elongation factors are necessary and sufficient to facilitate the emergence of filopodia with diverse characteristics from a highly dense network of short-branched capped filaments. To address this question, we combined bead motility and micropatterning biomimetic assays with multi-color Total Internal Reflection Fluorescence microscopy imaging, to successfully reconstitute the formation of filopodia-like networks (FLN) from densely-branched lamellipodia-like networks (LLN) with eight purified proteins (actin, profilin, Arp2/3 complex, Wasp pWA, fascin, capping protein, VASP and formin mDia2). Saturating capping protein concentrations inhibit FLN assembly, but the addition of either formin or Ena/VASP differentially rescues the formation of FLN from LLN. Specifically, we found that formin/mDia2-generated FLNs are relatively long and lack capping protein, whereas VASP-generated FLNs are comparatively short and contain capping protein, indicating that the actin elongation factor can affect the architecture and composition of FLN emerging from LLN. Our biomimetic reconstitution systems reveal that formin or VASP are necessary and sufficient to induce the transition from a LLN to a FLN, and establish robust in vitro platforms to investigate FLN assembly mechanisms.

TES-1/Tes and ZYX-1/Zyxin protect junctional actin networks under tension during epidermal morphogenesis in the C. elegans embryo.

LIM-domain-containing repeat (LCR) proteins are recruited to strained actin filaments within stress fibers in cultured cells,1,2,3 but their roles at cell-cell junctions in living organisms have not been extensively studied. Here, we show that the Caenorhabditis elegans LCR proteins TES-1/Tes and ZYX-1/Zyxin are recruited to apical junctions during embryonic elongation when junctions are under tension. In genetic backgrounds in which embryonic elongation fails, junctional recruitment is severely compromised. The two proteins display complementary patterns of expression: TES-1 is expressed in lateral (seam) epidermal cells, whereas ZYX-1 is expressed in dorsal and ventral epidermal cells. tes-1 and zyx-1 mutant embryos display junctional F-actin defects. The loss of either protein strongly enhances morphogenetic defects in hypomorphic mutant backgrounds for cadherin/catenin complex (CCC) components. The LCR regions of TES-1 and ZYX-1 are recruited to stress fiber strain sites (SFSSs) in cultured vertebrate cells. Together, these data establish TES-1 and ZYX-1 as components of a multicellular, tension-sensitive system that stabilizes the junctional actin cytoskeleton during embryonic morphogenesis.

LIM domain proteins in cell mechanobiology.

The actin cytoskeleton is important for maintaining mechanical homeostasis in adherent cells, largely through its regulation of adhesion and cortical tension. The LIM (Lin-11, Isl1, MEC-3) domain-containing proteins are involved in a myriad of cellular mechanosensitive pathways. Recent work has discovered that LIM domains bind to mechanically stressed actin filaments, suggesting a novel and widely conserved mechanism of mechanosensing. This review summarizes the current state of knowledge of LIM protein mechanosensitivity.

Evolutionarily diverse LIM domain-containing proteins bind stressed actin filaments through a conserved mechanism.

The actin cytoskeleton assembles into diverse load-bearing networks, including stress fibers (SFs), muscle sarcomeres, and the cytokinetic ring to both generate and sense mechanical forces. The LIM (Lin11, Isl- 1, and Mec-3) domain family is functionally diverse, but most members can associate with the actin cytoskeleton with apparent force sensitivity. Zyxin rapidly localizes via its LIM domains to failing SFs in cells, known as strain sites, to initiate SF repair and maintain mechanical homeostasis. The mechanism by which these LIM domains associate with stress fiber strain sites (SFSS) is not known. Additionally, it is unknown how widespread strain sensing is within the LIM protein family. We identify that the LIM domain-containing region of 18 proteins from the Zyxin, Paxillin, Tes, and Enigma proteins accumulate to SFSS. Moreover, the LIM domain region from the fission yeast protein paxillin like 1 (Pxl1) also localizes to SFSS in mammalian cells, suggesting that the strain sensing mechanism is ancient and highly conserved. We then used sequence and domain analysis to demonstrate that tandem LIM domains contribute additively, for SFSS localization. Employing in vitro reconstitution, we show that the LIM domain-containing region from mammalian zyxin and fission yeast Pxl1 binds to mechanically stressed F-actin networks but does not associate with relaxed actin filaments. We propose that tandem LIM domains recognize an F-actin conformation that is rare in the relaxed state but is enriched in the presence of mechanical stress.

Dynamin regulates the dynamics and mechanical strength of the actin cytoskeleton as a multifilament actin-bundling protein.

The dynamin GTPase is known to bundle actin filaments, but the underlying molecular mechanism and physiological relevance remain unclear. Our genetic analyses revealed a function of dynamin in propelling invasive membrane protrusions during myoblast fusion in vivo. Using biochemistry, total internal reflection fluorescence microscopy, electron microscopy and cryo-electron tomography, we show that dynamin bundles actin while forming a helical structure. At its full capacity, each dynamin helix captures 12-16 actin filaments on the outer rim of the helix. GTP hydrolysis by dynamin triggers disassembly of fully assembled dynamin helices, releasing free dynamin dimers/tetramers and facilitating Arp2/3-mediated branched actin polymerization. The assembly/disassembly cycles of dynamin promote continuous actin bundling to generate mechanically stiff actin super-bundles. Super-resolution and immunogold platinum replica electron microscopy revealed dynamin along actin bundles at the fusogenic synapse. These findings implicate dynamin as a unique multifilament actin-bundling protein that regulates the dynamics and mechanical strength of the actin cytoskeletal network.

Mechanical and kinetic factors drive sorting of F-actin cross-linkers on bundles.

In cells, actin-binding proteins (ABPs) sort to different regions to establish F-actin networks with diverse functions, including filopodia used for cell migration and contractile rings required for cell division. Recent experimental work uncovered a competition-based mechanism that may facilitate spatial localization of ABPs: binding of a short cross-linker protein to 2 actin filaments promotes the binding of other short cross-linkers and inhibits the binding of longer cross-linkers (and vice versa). We hypothesize this sorting arises because F-actin is semiflexible and cannot bend over short distances. We develop a mathematical theory and lattice models encompassing the most important physical parameters for this process and use coarse-grained simulations with explicit cross-linkers to characterize and test our predictions. Our theory and data predict an explicit dependence of cross-linker separation on bundle polymerization rate. We perform experiments that confirm this dependence, but with an unexpected cross-over in dominance of one cross-linker at high growth rates to the other at slow growth rates, and we investigate the origin of this cross-over with further simulations. The nonequilibrium mechanism that we describe can allow cells to organize molecular material to drive biological processes, and our results can guide the choice and design of cross-linkers for engineered protein-based materials.

Psychosocial and Demographic Characteristics of Children and Adolescents With Headache Presenting for Treatment in a Headache Infusion Center.

BACKGROUND: Approximately 10% of pediatric patients have recurrent headaches, with migraine being the most common headache type. If untreated, migraine may progress to status migrainosus, a debilitating condition of prolonged duration, high pain severity, and significant disability. There is high variability in the treatment of status migrainosus including medications used and treatment setting, which may occur in the emergency room, as an inpatient admission, or, less often, in an outpatient infusion center. The paucity of research on the treatment of status migrainosus is a limitation to treatment effectiveness. OBJECTIVE: The objective of the study was twofold. First, we sought to examine the demographic characteristics of children and adolescents accessing our outpatient infusion center for prolonged headache. Second, we sought to determine whether any demographic or psychosocial differences exist between patients who access infusion therapy compared to patients who do not access infusion therapy for their headaches. METHODS: We conducted a retrospective chart review of all patients between the ages of 6 and 19 years who were treated in our outpatient headache infusion center. A subset of these patients completed a behavioral health evaluation (treatment group) and they were compared to a control group of similar age (birthdate within 6 months) and gender to patients not seeking infusion treatment. Variables of interest included patient demographics, headache type and characteristics, and scores on the Pediatric Quality of Life Inventory (PedsQL), Functional Disability Inventory (FDI), Pediatric Pain Coping Inventory (PPCI), and the Behavior Assessment System for Children - Second Edition (BASC-2). RESULTS: A total of 284 patients were included in the study (n = 227 receiving infusion treatment and n = 57 controls). Patients were primarily female (224/286; 78.9%), Caucasian (254/286; 90.1%), and had a mean age of 15 years. Findings suggest a promising difference in the PPCI Distraction subscale, χ2 (1) = 3.7, P = .054, with a mean rank score of 61.90 for the treatment group and 50.21 for the control group. Additionally, a statistically significant difference was noted on the Social Support subscale, χ2 (1) = 10.6, P = .001, with a mean rank score of 65.92 for the treatment group and 46.26 for the control group. Results also indicated a statistically significant difference in disability scores, χ2 (1) = 10.0, P = .002, with a mean rank FDI score of 66.83 for the treatment group and 47.34 for the control group. Patients in the infusion group also reported lower quality of life on the PedsQL Total score (F[1, 109] = 5.0, P = .028; partial η2  = 0.044), and on the Physical (F[1, 109] = 7.9, P = .006; partial η2  = 0.069) and School (F[1, 109] = 4.6, P = .035; partial η2  = 0.041) subscales. No significant differences were found on the BASC-2. Parent reported data also revealed a significantly higher level of disability among patients seeking infusion treatment compared to the non-infusion group χ2 (1) = 11.7, P = .001. However, there were no significant differences on the PedsQL, PPCI, or BASC-2. CONCLUSIONS: Our findings support the disabling nature of migraine among children and adolescents, with higher levels of disability and lower quality of life reported in the group of patients utilizing infusion treatment. Developing concrete treatment plans and goals combined with bio-behavioral therapy are necessary to reduce functional disability and increase quality of life among these patients. Awareness of this patient group's pain-related coping strategies may help health care providers tailor treatment recommendations and develop or refine cognitive-behavioral headache treatment techniques.

Fascin- and α-Actinin-Bundled Networks Contain Intrinsic Structural Features that Drive Protein Sorting.

Cells assemble and maintain functionally distinct actin cytoskeleton networks with various actin filament organizations and dynamics through the coordinated action of different sets of actin-binding proteins. The biochemical and functional properties of diverse actin-binding proteins, both alone and in combination, have been increasingly well studied. Conversely, how different sets of actin-binding proteins properly sort to distinct actin filament networks in the first place is not nearly as well understood. Actin-binding protein sorting is critical for the self-organization of diverse dynamic actin cytoskeleton networks within a common cytoplasm. Using in vitro reconstitution techniques including biomimetic assays and single-molecule multi-color total internal reflection fluorescence microscopy, we discovered that sorting of the prominent actin-bundling proteins fascin and α-actinin to distinct networks is an intrinsic behavior, free of complicated cellular signaling cascades. When mixed, fascin and α-actinin mutually exclude each other by promoting their own recruitment and inhibiting recruitment of the other, resulting in the formation of distinct fascin- or α-actinin-bundled domains. Subdiffraction-resolution light microscopy and negative-staining electron microscopy revealed that fascin domains are densely packed, whereas α-actinin domains consist of widely spaced parallel actin filaments. Importantly, other actin-binding proteins such as fimbrin and espin show high specificity between these two bundle types within the same reaction. Here we directly observe that fascin and α-actinin intrinsically segregate to discrete bundled domains that are specifically recognized by other actin-binding proteins.

ACTIN-DIRECTED TOXIN. ACD toxin-produced actin oligomers poison formin-controlled actin polymerization.

The actin cross-linking domain (ACD) is an actin-specific toxin produced by several pathogens, including life-threatening spp. of Vibrio cholerae, Vibrio vulnificus, and Aeromonas hydrophila. Actin cross-linking by ACD is thought to lead to slow cytoskeleton failure owing to a gradual sequestration of actin in the form of nonfunctional oligomers. Here, we found that ACD converted cytoplasmic actin into highly toxic oligomers that potently "poisoned" the ability of major actin assembly proteins, formins, to sustain actin polymerization. Thus, ACD can target the most abundant cellular protein by using actin oligomers as secondary toxins to efficiently subvert cellular functions of actin while functioning at very low doses.

Profilin-1 serves as a gatekeeper for actin assembly by Arp2/3-dependent and -independent pathways.

Cells contain multiple F-actin assembly pathways, including the Arp2/3 complex, formins, and Ena/VASP, which have largely been analyzed separately. They collectively generate the bulk of F-actin from a common pool of G-actin; however, the interplay and/or competition between these pathways remains poorly understood. Using fibroblast lines derived from an Arpc2 conditional knockout mouse, we established matched-pair cells with and without the Arp2/3 complex. Arpc2(-/-) cells lack lamellipodia and migrate more slowly than WT cells but have F-actin levels indistinguishable from controls. Actin assembly in Arpc2(-/-) cells was resistant to cytochalasin-D and was highly dependent on profilin-1 and Ena/VASP but not formins. Profilin-1 depletion in WT cells increased F-actin and Arp2/3 complex in lamellipodia. Conversely, addition of exogenous profilin-1 inhibited Arp2/3 complex actin nucleation in vitro and in vivo. Antagonism of the Arp2/3 complex by profilin-1 in cells appears to maintain actin homeostasis by balancing Arp2/3 complex-dependent and -independent actin assembly pathways.

Ena/VASP Enabled is a highly processive actin polymerase tailored to self-assemble parallel-bundled F-actin networks with Fascin.

Filopodia are exploratory finger-like projections composed of multiple long, straight, parallel-bundled actin filaments that protrude from the leading edge of migrating cells. Drosophila melanogaster Enabled (Ena) is a member of the Ena/vasodilator-stimulated phosphoprotein protein family, which facilitates the assembly of filopodial actin filaments that are bundled by Fascin. However, the mechanism by which Ena and Fascin promote the assembly of uniformly thick F-actin bundles that are capable of producing coordinated protrusive forces without buckling is not well understood. We used multicolor evanescent wave fluorescence microscopy imaging to follow individual Ena molecules on both single and Fascin-bundled F-actin in vitro. Individual Ena tetramers increase the elongation rate approximately two- to threefold and inhibit capping protein by remaining processively associated with the barbed end for an average of ∼10 s in solution, for ∼60 s when immobilized on a surface, and for ∼110 s when multiple Ena tetramers are clustered on a surface. Ena also can gather and simultaneously elongate multiple barbed ends. Collectively, these properties could facilitate the recruitment of Fascin and initiate filopodia formation. Remarkably, we found that Ena's actin-assembly properties are tunable on Fascin-bundled filaments, facilitating the formation of filopodia-like F-actin networks without tapered barbed ends. Ena-associated trailing barbed ends in Fascin-bundled actin filaments have approximately twofold more frequent and approximately fivefold longer processive runs, allowing them to catch up with leading barbed ends efficiently. Therefore, Fascin and Ena cooperate to extend and maintain robust filopodia of uniform thickness with aligned barbed ends by a unique mechanistic cycle.

Enabled negatively regulates diaphanous-driven actin dynamics in vitro and in vivo.

Actin regulators facilitate cell migration by controlling cell protrusion architecture and dynamics. As the behavior of individual actin regulators becomes clear, we must address why cells require multiple regulators with similar functions and how they cooperate to create diverse protrusions. We characterized Diaphanous (Dia) and Enabled (Ena) as a model, using complementary approaches: cell culture, biophysical analysis, and Drosophila morphogenesis. We found that Dia and Ena have distinct biochemical properties that contribute to the different protrusion morphologies each induces. Dia is a more processive, faster elongator, paralleling the long, stable filopodia it induces in vivo, while Ena promotes filopodia with more dynamic changes in number, length, and lifetime. Acting together, Ena and Dia induce protrusions distinct from those induced by either alone, with Ena reducing Dia-driven protrusion length and number. Consistent with this, EnaEVH1 binds Dia directly and inhibits DiaFH1FH2-mediated nucleation in vitro. Finally, Ena rescues hemocyte migration defects caused by activated Dia.

Elevated atmospheric CO2 impacts abundance and diversity of nitrogen cycling functional genes in soil.

The concentration of CO(2) in the Earth's atmosphere has increased over the last century. Although this increase is unlikely to have direct effects on soil microbial communities, increased atmospheric CO(2) may impact soil ecosystems indirectly through plant responses. This study tested the hypothesis that exposure of plants to elevated CO(2) would impact soil microorganisms responsible for key nitrogen cycling processes, specifically denitrification and nitrification. We grew trembling aspen (Populus tremuloides) trees in outdoor chambers under ambient (360 ppm) or elevated (720 ppm) levels of CO(2) for 5 years and analyzed the microbial communities in the soils below the trees using quantitative polymerase chain reaction and clone library sequencing targeting the nitrite reductase (nirK) and ammonia monooxygenase (amoA) genes. We observed a more than twofold increase in copy numbers of nirK and a decrease in nirK diversity with CO(2) enrichment, with an increased predominance of Bradyrhizobia-like nirK sequences. We suggest that this dramatic increase in nirK-containing bacteria may have contributed to the significant loss of soil N in the CO(2)-treated chambers. Elevated CO(2) also resulted in a significant decrease in copy numbers of bacterial amoA, but no change in archaeal amoA copy numbers. The decrease in abundance of bacterial amoA was likely a result of the loss of soil N in the CO(2)-treated chambers, while the lack of response for archaeal amoA supports the hypothesis that physiological differences in these two groups of ammonia oxidizers may enable them to occupy distinct ecological niches and respond differently to environmental change.

Mechanism of actin filament nucleation by Vibrio VopL and implications for tandem W domain nucleation.

Pathogen proteins targeting the actin cytoskeleton often serve as model systems to understand their more complex eukaryotic analogs. We show that the strong actin filament nucleation activity of Vibrio parahaemolyticus VopL depends on its three W domains and on its dimerization through a unique VopL C-terminal domain (VCD). The VCD shows a previously unknown all-helical fold and interacts with the pointed end of the actin nucleus, contributing to the nucleation activity directly and through duplication of the W domain repeat. VopL promotes rapid cycles of filament nucleation and detachment but generally has no effect on elongation. Profilin inhibits VopL-induced nucleation by competing for actin binding to the W domains. Combined, the results suggest that VopL stabilizes a hexameric double-stranded pointed end nucleus. Analysis of hybrid constructs of VopL and the eukaryotic nucleator Spire suggest that Spire may also function as a dimer in cells.

Actin filament bundling by fimbrin is important for endocytosis, cytokinesis, and polarization in fission yeast.

Through the coordinated action of diverse actin-binding proteins, cells simultaneously assemble actin filaments with distinct architectures and dynamics to drive different processes. Actin filament cross-linking proteins organize filaments into higher order networks, although the requirement of cross-linking activity in cells has largely been assumed rather than directly tested. Fission yeast Schizosaccharomyces pombe assembles actin into three discrete structures: endocytic actin patches, polarizing actin cables, and the cytokinetic contractile ring. The fission yeast filament cross-linker fimbrin Fim1 primarily localizes to Arp2/3 complex-nucleated branched filaments of the actin patch and by a lesser amount to bundles of linear antiparallel filaments in the contractile ring. It is unclear whether Fim1 associates with bundles of parallel filaments in actin cables. We previously discovered that a principal role of Fim1 is to control localization of tropomyosin Cdc8, thereby facilitating cofilin-mediated filament turnover. Therefore, we hypothesized that the bundling ability of Fim1 is dispensable for actin patches but is important for the contractile ring and possibly actin cables. By directly visualizing actin filament assembly using total internal reflection fluorescence microscopy, we determined that Fim1 bundles filaments in both parallel and antiparallel orientations and efficiently bundles Arp2/3 complex-branched filaments in the absence but not the presence of actin capping protein. Examination of cells exclusively expressing a truncated version of Fim1 that can bind but not bundle actin filaments revealed that bundling activity of Fim1 is in fact important for all three actin structures. Therefore, fimbrin Fim1 has diverse roles as both a filament "gatekeeper" and as a filament cross-linker.

Alteration of microbial communities colonizing leaf litter in a temperate woodland stream by growth of trees under conditions of elevated atmospheric CO2.

Elevated atmospheric CO(2) can cause increased carbon fixation and altered foliar chemical composition in a variety of plants, which has the potential to impact forested headwater streams because they are detritus-based ecosystems that rely on leaf litter as their primary source of organic carbon. Fungi and bacteria play key roles in the entry of terrestrial carbon into aquatic food webs, as they decompose leaf litter and serve as a source of nutrition for invertebrate consumers. This study tested the hypothesis that changes in leaf chemistry caused by elevated atmospheric CO(2) would result in changes in the size and composition of microbial communities colonizing leaves in a woodland stream. Three tree species, Populus tremuloides, Salix alba, and Acer saccharum, were grown under ambient (360 ppm) or elevated (720 ppm) CO(2), and their leaves were incubated in a woodland stream. Elevated-CO(2) treatment resulted in significant increases in the phenolic and tannin contents and C/N ratios of leaves. Microbial effects, which occurred only for P. tremuloides leaves, included decreased fungal biomass and decreased bacterial counts. Analysis of fungal and bacterial communities on P. tremuloides leaves via terminal restriction fragment length polymorphism (T-RFLP) and clone library sequencing revealed that fungal community composition was mostly unchanged by the elevated-CO(2) treatment, whereas bacterial communities showed a significant shift in composition and a significant increase in diversity. Specific changes in bacterial communities included increased numbers of alphaproteobacterial and cytophaga-flavobacter-bacteroides (CFB) group sequences and decreased numbers of betaproteobacterial and firmicutes sequences, as well as a pronounced decrease in overall gram-positive bacterial sequences.