A Puf-strain of Rhodobacter sphaeroides (PUFB1) was constructed by deleting a portion of the proximal region of the puf operon and inserting a kanamycin resistance gene cartridge. Southern blot analysis demonstrated that in PUFB1, the defective copy of the puf operon had replaced, through homologous recombination, the normal chromosomal copy. The Puf phenotype was characterized by the inability of PUFB1 to grow photoheterotrophically (PS-), the lack of detectable puf-specific transcripts, the absence of the light-harvesting I complex and, by inference, the reaction center spectral complex, and greatly reduced levels of the light-harvesting II complex. The PS' phenotype was restored to PUFB1 when a 13-kilobase BamHI restriction endonuclease fragment containing the entire puf operon and flanking regions was cloned into the broad-host-range plasmid vector RK2 derivative pRK404 and introduced by conjugation into PUFB1. In these complemented strains, there was an increased number of copies of the puf operon (four to six copies per defective chromosomal copy) as the result of plasmid copy number. However, there was no concomitant increase in either the specific bacteriochlorophyll content or the level of puf-specific transcripts when these strains were grown photoheterotrophically.Photoheterotrophically grown Rhodobacter sphaeroides contains a specialized intracytoplasmic membrane system (ICM) (5,8,16) which houses all of the protein-pigment complexes responsible for capturing light energy and -catalyzing photosynthetic electron transport and synthesis of chemical bond energy (28,30,37).The photosynthetic unit of R. sphaeroides comprises three distinct bacteriochlorophyll (Bchl) a pigment-protein complexes: the photochemical reaction center (RC) (30) and two light-harvesting complexes (6). These two spectrally distinct light-harvesting complexes (37) contain 95% of the total Bchl in R. sphaeroides and are classified according to their absorption maxima in the near infrared (9). The B800-850 complex transfers exciton energy to the B875 complex, which in turn transfers such energy to the RC (25, 45). Both B875 and B800-850 pigment-protein complexes contain equimolar amounts of two small but unique hydrophobic polypeptides, designated a and P, which exist in the membrane as heterodimers (1,4,36).Although similarities exist between the B800-850 and B875polypeptides, the spectral differences between the two lightharvesting complexes can be attributed to differences in Bchl pigment-protein interactions within each complex. The B875 complex contains two molecules each of Bchl a and carotenoid, while three molecules of Bchl a and one carotenoid have been proposed to be associated with the B800-B850 complex (4, 17, 31). There are three molecules of Bchl a per a and ,B polypeptides in the B800-850 complex (8). Fluorescence studies suggest a topological model whereby the RC is surrounded by B875 pigment-protein complexes and the B875-RC complexes are interconnected in a lake of B800-850 spectral units (26).The stoichiometry of B...
Sympathetic hyperinnervation occurs in human ventricular tissue after myocardial infarction and may contribute to arrhythmias. Aberrant sympathetic sprouting is associated with elevated nerve growth factor (NGF) in many contexts, including ventricular hyperinnervation. However, it is unclear whether cardiomyocytes or other cell types are responsible for increased NGF synthesis. In this study, left coronary arteries were ligated and ventricular tissue examined in rats 1-28 days post-infarction. Infarct and peri-infarct tissue was essentially devoid of sensory and parasympathetic nerves at all time points. However, areas of increased sympathetic nerve density were observed in the peri-infarct zone between post-ligation days 4-14. Hyperinnervation occurred in regions containing accumulations of macrophages and myofibroblasts. To assess whether these inflammatory cells synthesize NGF, sections were processed for NGF in situ hybridization and immunohistochemistry. Both macrophage1 antigen-positive macrophages and α-smooth muscle actin immunoreactive myofibroblasts expressed NGF in areas where they were closely proximate to sympathetic nerves. To investigate whether NGF produced by peri-infarct cells induces sympathetic outgrowth, we cocultured adult sympathetic ganglia with peri-infarct explants. Neurite outgrowth from sympathetic ganglia was significantly greater at post-ligation days 7-14 as compared to control tissue. Addition of an NGF function-blocking antibody prevented the increased neurite outgrowth induced by periinfarct tissue. These findings provide evidence that inflammatory cell NGF synthesis plays a causal role in sympathetic hyperinnervation following myocardial infarction.
This manuscript calls for an international effort to generate a comprehensive catalog from genome sequences of all the archaeal and bacterial type strains.
BackgroundLignocellulosic biomass is seen as an abundant renewable source of liquid fuels and chemicals that are currently derived from petroleum. When lignocellulosic biomass is used for ethanol production, the resulting liquid residue (stillage) contains large amounts of organic material that could be further transformed into recoverable bioproducts, thus enhancing the economics of the biorefinery.ResultsHere we test the hypothesis that a bacterial community could transform the organics in stillage into valuable bioproducts. We demonstrate the ability of this microbiome to convert stillage organics into medium-chain fatty acids (MCFAs), identify the predominant community members, and perform a technoeconomic analysis of recovering MCFAs as co-products of ethanol production. Steady-state operation of a stillage-fed bioreactor showed that 18% of the organic matter in stillage was converted to MCFAs. Xylose and complex carbohydrates were the primary substrates transformed. During the MCFA production period, the five major genera represented more than 95% of the community, including Lactobacillus, Roseburia, Atopobium, Olsenella, and Pseudoramibacter. To assess the potential benefits of producing MCFAs from stillage, we modeled the economics of ethanol and MCFA co-production, at MCFA productivities observed during reactor operation.ConclusionsThe analysis predicts that production of MCFAs, ethanol, and electricity could reduce the minimum ethanol selling price from $2.15 to $1.76 gal−1 ($2.68 gal−1 gasoline equivalents) when compared to a lignocellulosic biorefinery that produces only ethanol and electricity.Electronic supplementary materialThe online version of this article (10.1186/s13068-018-1193-x) contains supplementary material, which is available to authorized users.
Glutathione-dependent formaldehyde dehydrogenases (GSH-FDH) represent a ubiquitous class of enzymes, found in both prokaryotes and eukaryotes. During the course of studying energy-generating pathways in the photosynthetic bacterium Rhodobacter sphaeroides, a gene (adhI) encoding a GSH-FDH homolog has been identified as part of an operon (adhI-cycI) that also encodes an isoform of the cytochrome c 2 family of electron transport proteins (isocytochrome c 2 ). Enzyme assays with crude Escherichia coli extracts expressing AdhI show that this protein has the characteristic substrate preference of a GSH-FDH. Ferguson plot analysis with zymograms suggests that the functional form of AdhI is a homodimer of ϳ40-kDa subunits, analogous to other GSH-FDH enzymes. These properties of AdhI were used to show that mutations which increase or decrease adhI expression change the specific activity of GSH-FDH in R. sphaeroides extracts. In addition, expression of the presumed adhI-cycI operon appears to be transcriptionally regulated, since the abundance of the major adhI-specific primer extension product is increased by the trans-acting spd-7 mutation, which increases the level of both isocytochrome c 2 and AdhI activity. While transcriptional linkage of adhI and cycI could suggest a function in a common metabolic pathway, isocytochrome c 2 (periplasm) and AdhI (cytoplasm) are localized in separate compartments of R. sphaeroides. Potential roles for AdhI in carbon and energy generation and the possible relationship of GSH-FDH activity to isocytochrome c 2 will be discussed based on the commonly accepted physiological functions of GSH-FDH enzymes in prokaryotes and eukaryotes.Most organisms have the ability, by using various metabolic pathways, to generate both carbon and energy from the oxidation of a wide spectrum of substrates. The function of these metabolic pathways often requires the coordinated expression of specific enzymes for substrate oxidation and novel electron carriers to generate cellular energy from this substrate. To understand how different metabolic pathways provide cells with both carbon and energy, we are studying the regulation and function of energy-generating systems in the purple nonsulfur photosynthetic bacterium Rhodobacter sphaeroides.While energy-generating pathways in R. sphaeroides have been extremely well analyzed from biochemical and bioenergetic standpoints (24), genetic analysis has uncovered the existence of unexpected electron transport proteins. For example, recent experiments have identified a c-type cytochrome, isocytochrome c 2 , which allows R. sphaeroides Spd mutants to grow photosynthetically in the absence of cytochrome c 2 , the normal electron donor to the light-oxidized reaction center complexes (29). While the function of isocytochrome c 2 as a photosynthetic electron carrier in Spd mutants has been confirmed both genetically (31) and biochemically (46), a physiological role for this protein in wild-type cells is not known. Because mutational and complementation analysis indicated th...
A Rhodobacter splaeroides mutant (CYCAl) lacking cytochrome c2 (cyt c2) was previouly comtructed (T. J. Donohue, A. G. McEwan, S. Van Doren, A. R. Crofts, and S. Kaplan, Biochemistry, 27: 1918-1924) by a combination of in vivo and in vitro moleular genetic techniques. CYCAl was inc b of photosynthetic growth (PS-); in this presentation, we show that chemoheterotrophically grown CYCAl contained si ant quantities of a high potential soluble c-type cytochrome(s) with an alpha band of -554nm which had previously gone undetected under these physiologkal conditions in wild-type cells. In addition, the PS phenotype of CYCAl can be complemented in trans with stable low-copy-number (-5 to 9 per R. spkaeroides genome) broad-host-range plamis containing the wid-type cyt c2 structural gene (cycA) and upstrem regulatory sequences. cyt c2 and cycA-specific mRNA levels were elevated in both the wfld type and CYCAl derivatives harboring intact cycA genes in rans, presumably as a result of Increased gene dosage. Although photosyntheticafly grown wid-type cells contained approximately twofold more cycA-specific tr s t chemoheterotrophicaly grown cells, there was an approximately four-to sevenfold increase in cyt c2 leels under photosynthetic conditions. Simiarly, complemented CYCAl strains contained between 1.3-and 2.3-fold more cycA mRNA under photosynthetic conditions than under chemoheterotrophic conditions and had 6-to 12-fold higher steady-state levels of cyt c2 under the same physiological conditions. These data are dicu in tenus of possible posttranscriptional control over cyt c2.Rhodobacter sphaeroides is a purple nonsulfur photosynthetic bacterium possessing the capacity to grow aerobically, anaerobically in the light (34), or anaerobically in the dark in the presence of external electron acceptors such as dimethyl sulfoxide. During aerobic growth, the R. sphaeroides cell envelope resembles that of other gram-negative bacteria (13, 31), and energy is generated by a branched aerobic respiratory chain whose components are structurally and functionally similar to those of mitochondria (1,37,38). Anaerobiosis induces differentiation of the cell membrane through a process of invagination, resulting in synthesis of the intracytoplasmic membrane (20). The intracytoplasmic membrane, which is structurally continuous with but functionally distinct from the cell membrane, contains the pigment-protein complexes and redox components necessary for the capture of light energy and its conversion to cellular energy (10,20).Cytochrome c2 (cyt c2) is a soluble electron carrier located in the periplasm of R. sphaeroides (29) which is common to both the respiratory and the cyclic photosynthetic redox chains. In aerobically grown cells, cyt c2 transfers electrons from the membrane-bound ubiquinol-cyt c2 oxidoreductase (cyt b-cl) complex (15) to a terminal cyt a-a3 oxidase similar to that of mitochondria (16). Under photoheterotrophic conditions, cyt c2 functions to complete the cyclic photosynthetic redox chain (4, 28, 30) by transferring an ...
The genes for the Rhodobacter sphaeroi4es light-harvesting B875-I1, and B875-a polypeptides (puJB and pufA) are closely linlked to the genes for the reaction center L and reaction center M polypeptides (puJL and pufiM) on what has been termed the puf operon (gene order, pufB, A, L, M). The DNA sequence of the pufB and pufA structural genes from wild-type R. sphaeroides 2.4.1 was determined and aligned with the available amino acid sequence of the wild-type B875-0 and B875-a polypeptides. The relative levels of the B875-0 and B875-a and the reaction center L and reaction center M polypeptides synthesized in a homologous cell-free transcription-translation system were compared with those found in vivo. Analysis of the gene products produced in vitro with plasmids containing deletions upstream of the pufB structural gene identified a region of DNA required for expression of the B875-0 and B875-a polypeptides. These results support the hypothesis that the mapped 5' termini of the large and small pufoperon transcripts represent transcription initiation sites.In the purple nonsulfur photosynthetic bacteria, bacteriochlorophyll a molecules complexed with specific integral membrane proteins are responsible for the absorption and entrapment of light energy. These reactions represent the initial steps in the conversion of light energy to chemical energy after charge separation (7,12). In Rhodobacter sphaeroides, there are three distinct pigment-protein complexes, and these are uniquely associated with the photosynthetic intracytoplasmic membrane (ICM) system (16). The study of these pigment-protein complexes has been instrumental in understanding the reactions of primary photochemistry, as well as elucidating the mechanisms of the synthesis and assembly of integral membrane protein complexes (15).The most abundant of these pigment-protein complexes in R. sphaeroides are the light-harvesting complexes that have been designated B875 and B800-850, based on their nearinfrared absorption maxima (7). Analysis of the organization of the light-harvesting pigment-protein complexes within the ICM of both wild-type and mutant strains suggests that the B875 complex accepts light energy from a pool of peripherally arranged B800-850 complexes and may be obligatory in energy transfer to a centrally located reaction center (RC) complex (23,25). The amount of the B800-850 complex relative to the RC complex in the ICM varies inversely with incident light intensity, whereas the ratio of B875 to RC complexes is independent of light intensity and is approximately 10 to 15:1 (1 5,441 and 6,809, respectively (33, 34). However, neither B875-P nor B875-a migrate to their true molecular weights during sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The RC complex contains three polypeptides, RC-H, RC-M, and RC-L (based on their relative mobilities from SDS-PAGE of 28,000, 24,000, and 21,000 daltons) in a 1:1:1 stoichiometric ratio, four molecules of bacteriochlorophyll, two molecules of bacteriopheophytin, two molecules of...
Identification of unknown compounds is of critical importance in GC/MS applications (metabolomics, environmental toxin identification, sports doping, petroleomics, and biofuel analysis, among many others) and remains a technological challenge. Derivation of elemental composition is the first step to determining the identity of an unknown compound by MS, for which high accuracy mass and isotopomer distribution measurements are critical. Here, we report on the development of a dedicated, applications-grade GC/MS employing an Orbitrap mass analyzer, the GC/Quadrupole-Orbitrap. Built from the basis of the benchtop Orbitrap LC/MS, the GC/Quadrupole-Orbitrap maintains the performance characteristics of the Orbitrap, enables quadrupole-based isolation for sensitive analyte detection, and includes numerous analysis modalities to facilitate structural elucidation. We detail the design and construction of the instrument, discuss its key figures-of-merit, and demonstrate its performance for the characterization of unknown compounds and environmental toxins.
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