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With the emergence of antibiotic-resistant bacteria, innovative approaches are needed for the treatment of urinary tract infections. Boosting antimicrobial peptide expression may provide an alternative to antibiotics. Here, we developed reporter cell lines and performed a high-throughput screen of clinically used drugs to identify compounds that boost ribonuclease 4 and 7 expression (RNase 4 and 7), peptides that have antimicrobial activity against antibiotic-resistant uropathogens. This screen identified histone deacetylase (HDAC) inhibitors as effective RNase 4 and RNase 7 inducers. Validation studies in primary human kidney and bladder cells confirmed pan-HDAC inhibitors as well as the HDAC class I inhibitor, MS-275, induce RNase 4 and RNase 7 to protect human kidney and bladder cells from uropathogenic Escherichia coli . When we administered MS-275 to mice, RNase 4 and 7 expression increased and mice were protected from acute transurethral E. coli challenge. In support of this mechanism, MS-275 treatment increased acetylated histone H3 binding to the RNASE4 and RNASE7 promoters. Overexpression and knockdown of HDAC class I proteins identified HDAC3 as a primary regulator of RNase 4 and 7. These results demonstrate the protective effects of enhancing RNase 4 and RNase 7, opening the door to repurposing medications as antibiotic conserving therapeutics for urinary tract infection.

Heterologous sensitization of adenylyl cyclase (AC) is revealed as enhanced or exaggerated AC/cAMP signaling that occurs following persistent activation of Gα(i/o)-coupled receptors. This paradoxical phenomenon was discovered more than 40 years ago and was proposed as a cellular mechanism to explain the adaptive changes that occur following chronic exposure to drugs of abuse. However, the underlying molecular mechanisms of heterologous sensitization of AC remain largely unknown. In the present study, we performed a genome-wide cell-based RNA interference screen as an unbiased approach to identify genes associated with heterologous sensitization of AC. Following a series of validation and confirmation assays, three genes that form an E3 ligase complex, cullin3 (CUL3), neural precursor-cell–expressed and developmentally downregulated 8 (NEDD8), and really interesting new gene (RING)-box protein 1 (RBX1), were identified as specific modulators of heterologous sensitization of AC. Furthermore, based on the downstream actions of these genes, we evaluated the activity of proteasome inhibitors as well as the specific NEDD8-activating enzyme inhibitor, MLN4924 (Pevonedistat), in AC sensitization. We demonstrate that MG-132 and bortezomib treatments could mimic the inhibitory effects observed with gene knockdown, and MLN4924 was potent and efficacious in blocking the development of heterologous sensitization of endogenous and recombinant AC isoforms, including AC1, AC2, AC5, and AC6. Together, by using genetic and pharmacological approaches, we identified, for the first time, cullin3-RING ligases and the protein degradation pathway as essential modulators for heterologous sensitization of AC. SIGNIFICANCE STATEMENT: Through a genome-wide cell-based RNA interference screening, we identified three genes that form an E3 ligase complex, cullin3, neural precursor-cell–expressed and developmentally downregulated 8 (NEDD8), and really interesting new gene–box protein 1, as specific modulators of heterologous sensitization of AC. The effect of cullin3, NEDD8, or really interesting new gene–box protein 1 small interfering RNAs on heterologous sensitization was recapitulated by proteasome inhibitors, MG132 and bortezomib, and the specific NEDD8-activating enzyme inhibitor, MLN4924. These results suggest a novel hypothesis in which protein degradation is involved in the sensitization of AC signaling that occurs following chronic activation of Gα(i/o)-coupled receptors.

Plant biologists are seeking new approaches for modifying lignin to improve the digestion and utilization of structural polysaccharides in crop cultivars for the production of biofuels, biochemicals, and livestock. To identify promising targets for lignin bioengineering, we artificially lignified maize (Zea mays L.) cell walls with normal monolignols plus 21 structurally diverse alternative monomers to assess their suitability for lignification and for improving fiber digestibility. Lignin formation and structure were assessed by mass balance, Klason lignin, acetyl bromide lignin, gel-state 2D-NMR and thioacidolysis procedures, and digestibility was evaluated with rumen microflora and from glucose production by fungal enzymes following mild acid or base pretreatments. Highly acidic or hydrophilic monomers proved unsuitable for lignin modification because they severely depressed cell wall lignification. By contrast, monomers designed to moderately alter hydrophobicity or introduce cleavable acetal, amide, or ester functionalities into the polymer often readily formed lignin, but most failed to improve digestibility, even after chemical pretreatment. Fortunately, several types of phenylpropanoid derivatives containing multiple ester-linked catechol or pyrogallol units were identified as desirable genetic engineering targets because they readily formed wall-bound polymers and improved digestibility, presumably by blocking cross-linking of lignin to structural polysaccharides and promoting lignin fragmentation during mild acidic and especially alkaline pretreatment.

Antibiotic resistance is a growing international threat, with 23 000 deaths, 2 million patients, and $20 billion expended in the clinical setting annually in the United States alone (1). The pharmaceutical industry's approach to this problem, combinatorial libraries, has not proven as promising as once hoped (2). Combinatorial synthesis has produced millions of compounds, but the consistent production of potent leads and eventual therapies has remained elusive (3). In an effort to identify new, promising antimicrobials, we sought to develop a high-throughput screening assay for inhibitors of prokaryotic ribosomes that we could apply to natural product extracts. Over the past decade, our laboratory has isolated phylogenetically unique pure-culture microbial strains that we have grown and extracted to create a prefractionation natural products extract library (NPEL) of more than 30 000 samples, a collection housed in the Center for Chemical Genomics (CCG) at the University of Michigan (4). The NPEL has already proven to be a potent resource in high-throughput screening, furnishing promising leads toward antivirals (4), effective inhibitors of siderophore virulence-factor biosynthesis for MRSA and Bacillus anthracis (5), and potent aggregation inhibitors for neurodegenerative disorders (6). Our intention was to develop a high-throughput screen for the identification of new antibacterial agents and apply it to the NPEL. We envisioned adapting a coupled transcription/translation assay used previously for screening industrial chemistry libraries to the NPEL (7,8). A transcription/translation screen is desirable because it enables rapid screening of libraries against the bacterial ribosome, an established target for several classes of antibiotics including macrolides, aminoglycosides, tetracyclines, and oxazolidinones (9). Because different classes of ribosome inhibitors have orthogonal molecular modes of action, resistance by a pathogen to one class does enable it to counter other classes (10,11). Hence, new chemical scaffolds uncovered by a high-throughput screen are likely to be effective against drug resistant bacterial pathogens. This study was motivated by the potential cost and throughput advantages of reducing the scale of a direct ribosome inhibitor screen using natural product extracts as the source of chemical diversity. Previous studies successfully optimized a coupled transcription/translation screen in 384-well plates at a total scale of 16 μL using a luminescence-based reporter on a small-molecule library, which resulted in the discovery of a potential lead for a new, orthogonal class of ribosome inhibitor (7,8). This type of assay is amenable for use in 1536-well plates (12), and a number of secondary and counter screens are available to enable the decoupling of transcription from translation (7,8,13), remove intercalative inhibitors (14), discriminate compounds that interfere with luminescence (7,8), and identify general (eukaryotic) ribosome inhibitors (7,8,14). Our objectives were to create an improved method by significantly increasing efficiency through a reduction in reaction scale and to apply this screening technology directly to natural product extracts.

Partial substitution of normal monolignols with phenolic precursors from other metabolic pathways may improve the susceptibility of lignified biomass to chemical pretreatment and enzymatic saccharification for biofuel production. Flavonoids and gallate esters readily undergo oxidative coupling reactions, suggesting they could serve as alternate monomers for forming lignin in plants. To test this premise, primary cell walls of Zea mays (L.) were artificially lignified with normal monolignols plus various flavan-3-ol/phenolic ester derivatives, flavonol glycoside/gallate ester derivatives, or pentagalloyl glucose added as 0 or 45 % of the precursor mixture. Most alternate monomers readily copolymerized with normal monolignols, but wall-bound lignin was most efficiently formed with epicatechin, epicatechin gallate, epigallocatechin gallate, or hyperoside. Yields of glucose from a high-throughput digestibility platform were used to examine how lignin modifications affected the susceptibility of cell walls to enzymatic hydrolysis following alkaline or acidic pretreatments of different severities. With the exception of hyperoside, incorporation of alternate monomers into lignin improved yields of enzymatically released glucose by 18–60 % after mild alkaline pretreatment and by 6–34 % after mild acid pretreatment. Responses due to lignin modification diminished as pretreatment severity increased. Overall, our results suggest that apoplastic deposition of pentagalloyl glucose or gallated flavan-3-ols such as epicatechin gallate or epigallocatechin gallate for incorporation into lignin could be promising plant genetic engineering targets for improving sugar yields from grass biomass crops that are subjected to low-temperature alkaline pretreatments.

Breeding has transformed wild plant species into modern crops, increasing the allocation of their photosynthetic assimilate into grain, fiber, and other products for human use. Despite progress in increasing the harvest index, much of the biomass of crop plants is not utilized. Potential uses for the large amounts of agricultural residues that accumulate are animal fodder or bioenergy, though these may not be economically viable without additional efforts such as targeted breeding or improved processing. We characterized leaf and stem tissue from a diverse set of rice genotypes (varieties) grown in two environments (greenhouse and field) and report bioenergy-related traits across these variables. Among the 16 traits measured, cellulose, hemicelluloses, lignin, ash, total glucose, and glucose yield changed across environments, irrespective of the genotypes. Stem and leaf tissue composition differed for most traits, consistent with their unique functional contributions and suggesting that they are under separate genetic control. Plant variety had the least influence on the measured traits. High glucose yield was associated with high total glucose and hemicelluloses, but low lignin and ash content. Bioenergy yield of greenhouse-grown biomass was higher than field-grown biomass, suggesting that greenhouse studies overestimate bioenergy potential. Nevertheless, glucose yield in the greenhouse predicts glucose yield in the field (ρ = 0.85, p < 0.01) and could be used to optimize greenhouse (GH) and field breeding trials. Overall, efforts to improve cell wall composition for bioenergy require consideration of production environment, tissue type, and variety.

article en open access; International audience; In the search for renewable energy sources, genetic engineering is a promising strategy to improve plant cell wall composition for biofuel and bioproducts generation. Lignin is a major factor determining saccharification efficiency and, therefore, is a prime target to engineer. Here, lignin content and composition were modified in poplar (Populus tremula 3 Populus alba) by specifically down-regulating CINNAMYL ALCOHOL DEHYDROGENASE1 (CAD1) by a hairpin-RNA-mediated silencing approach, which resulted in only 5% residual CAD1 transcript abundance. These transgenic lines showed no biomass penalty despite a 10% reduction in Klason lignin content and severe shifts in lignin composition. Nuclear magnetic resonance spectroscopy and thioacidolysis revealed a strong increase (up to 20-fold) in sinapaldehyde incorporation into lignin, whereas coniferaldehyde was not increased markedly. Accordingly, ultra-high-performance liquid chromatography-mass spectrometry-based phenolic profiling revealed a more than 24,000-fold accumulation of a newly identified compound made from 8-8 coupling of two sinapaldehyde radicals. However, no additional cinnamaldehyde coupling products could be detected in the CAD1-deficient poplars. Instead, the transgenic lines accumulated a range of hydroxycinnamate-derived metabolites, of which the most prominent accumulation (over 8,500-fold) was observed for a compound that was identified by purification and nuclear magnetic resonance as syringyl lactic acid hexoside. Our data suggest that, upon down-regulation of CAD1, coniferaldehyde is converted into ferulic acid and derivatives, whereas sinapaldehyde is either oxidatively coupled into S'(8-8) S' and lignin or converted to sinapic acid and derivatives. The most prominent sink of the increased flux to hydroxycinnamates is syringyl lactic acid hexoside. Furthermore, low-extent saccharification assays, under different pretreatment conditions, showed strongly increased glucose (up to +81%) and xylose (up to +153%) release, suggesting that down-regulating CAD1 is a promising strategy for improving lignocellulosic biomass for the sugar platform industry.

Grass lignins contain substantial amounts of p-coumarate (pCA) that acylate the side-chains of the phenylpropanoid polymer backbone. An acyltransferase, named p-coumaroyl-CoA:monolignol transferase (OsPMT), that could acylate monolignols with pCA in vitro was recently identified from rice. In planta, such monolignol-pCA conjugates become incorporated into lignin via oxidative radical coupling, thereby generating the observed pCA appendages; however p-coumarates also acylate arabinoxylans in grasses. To test the authenticity of PMT as a lignin biosynthetic pathway enzyme, we examined Brachypodium distachyon plants with altered BdPMT gene function. Using newly developed cell wall analytical methods, we determined that the transferase was involved specifically in monolignol acylation. A sodium azide-generated Bdpmt-1 missense mutant had no (

Carbohydrates stored in vegetative organs, particularly stems, of grasses are a very important source of energy. We examined carbohydrate accumulation in adult sorghum and maize hybrids with distinct phenology and different end uses (grain, silage, sucrose or sweetness in stalk juice, and biomass). Remarkable variation was observed for non-structural carbohydrates and structural polysaccharides during three key developmental stages both between and within hybrids developed for distinct end use in both species. At the onset of the reproductive phase (average 65 days after planting, DAP), a wide range for accumulation of non-structural carbohydrates (free glucose and sucrose combined), was observed in internodes of maize (11–24%) and sorghum (7–36%) indicating substantial variation for transient storage of excess photosynthate during periods of low grain or vegetative sink strength. Remobilization of these reserves for supporting grain fill or vegetative growth was evident from lower amounts in maize (8–19%) and sorghum (9–27%) near the end of the reproductive period (average 95 DAP). At physiological maturity of grain hybrids (average 120 DAP), amounts of these carbohydrates were generally unchanged in maize (9–21%) and sorghum (16–27%) suggesting a loss of photosynthetic assimilation due to weakening sink demand. Nonetheless, high amounts of non-structural carbohydrates at maturity even in grain maize and sorghum (15–18%) highlight the potential for developing dual-purpose (grain/stover) crops. For both species, the amounts of structural polysaccharides in the cell wall, measured as monomeric components (glucose and pentose), decreased during grain fill but remained unchanged thereafter with maize biomass possessing slightly higher amounts than sorghum. Availability of carbohydrates in maize and sorghum highlights the potential for developing energy-rich dedicated biofuel or dual-purpose (grain/stover) crops.

Brachypodium distachyon (Brachypodium) has emerged as a useful model system for studying traits unique to graminaceous species including bioenergy crop grasses owing to its amenability to laboratory experimentation and the availability of extensive genetic and germplasm resources. Considerable natural variation has been uncovered for a variety of traits including flowering time, vernalization responsiveness, and above-ground growth characteristics. However, cell wall composition differences remain underexplored. Therefore, we assessed cell wall-related traits relevant to biomass conversion to biofuels in seven Brachypodium inbred lines that were chosen based on their high level of genotypic diversity as well as available genome sequences and recombinant inbred line (RIL) populations. Senesced stems plus leaf sheaths from these lines exhibited significant differences in acetyl bromide soluble lignin (ABSL), cell wall polysaccharide-derived sugars, hydroxycinnamates content, and syringyl:guaiacyl:p-hydroxyphenyl (S:G:H) lignin ratios. Free glucose, sucrose, and starch content also differed significantly in senesced stems, as did the amounts of sugars released from cell wall polysaccharides (digestibility) upon exposure to a panel of thermochemical pretreatments followed by hydrolytic enzymatic digestion. Correlations were identified between inbred line lignin compositions and plant growth characteristics such as biomass accumulation and heading date (HD), and between amounts of cell wall polysaccharides and biomass digestibility. Finally, stem cell wall p-coumarate and ferulate contents and free-sugars content changed significantly with increased duration of vernalization for some inbred lines. Taken together, these results show that Brachypodium displays substantial phenotypic variation with respect to cell wall composition and biomass digestibility, with some compositional differences correlating with growth characteristics. Moreover, besides influencing HD and biomass accumulation, vernalization was found to affect cell wall composition and free sugars accumulation in some Brachypodium inbred lines, suggesting genetic differences in how vernalization affects carbon flux to polysaccharides. The availability of related RIL populations will allow for the genetic and molecular dissection of this natural variation, the knowledge of which may inform ways to genetically improve bioenergy crop grasses.

The development of a viable lignocellulosic ethanol industry requires multiple improvements in the process of converting biomass to ethanol. A key step is the improvement of the plants that are to be used as biomass feedstocks. To facilitate the identification and evaluation of feedstock plants, it would be useful to have a method to screen large numbers of individual plants for enhanced digestibility in response to combinations of specific pretreatments and enzymes. This paper describes a high-throughput digestibility platform (HTDP) for screening collections of germplasm for improved digestibility, which was developed under the auspices of the Department of Energy-Great Lakes Bioenergy Research Center (DOE-GLBRC). A key component of this platform is a custom-designed workstation that can grind and dispense 1–5 mg quantities of more than 250 different plant tissue samples in 16 h. The other steps in the processing (pretreatment, enzyme digestion, and sugar analysis) have also been largely automated and require 36 h. The process is adaptable to diverse acidic and basic, low-temperature pretreatments. Total throughput of the HTDP is 972 independent biomass samples per week. Validation of the platform was performed on brown midrib mutants of maize, which are known to have enhanced digestibility. Additional validation was performed by screening approximately 1,200 Arabidopsis mutant lines with T-DNA insertions in genes known or suspected to be involved in cell wall biosynthesis. Several lines showed highly significant (p

Highlight Reducing the function of PAL, the first enzyme in the phenylpropanoid pathway, in Brachypodium distachyon alters cell wall composition, increases fungal susceptibility, but minimally affects caterpillar herbivory and abiotic stress tolerance., The phenylpropanoid pathway in plants synthesizes a variety of structural and defence compounds, and is an important target in efforts to reduce cell wall lignin for improved biomass conversion to biofuels. Little is known concerning the trade-offs in grasses when perturbing the function of the first gene family in the pathway, PHENYLALANINE AMMONIA LYASE (PAL). Therefore, PAL isoforms in the model grass Brachypodium distachyon were targeted, by RNA interference (RNAi), and large reductions (up to 85%) in stem tissue transcript abundance for two of the eight putative BdPAL genes were identified. The cell walls of stems of BdPAL-knockdown plants had reductions of 43% in lignin and 57% in cell wall-bound ferulate, and a nearly 2-fold increase in the amounts of polysaccharide-derived carbohydrates released by thermochemical and hydrolytic enzymic partial digestion. PAL-knockdown plants exhibited delayed development and reduced root growth, along with increased susceptibilities to the fungal pathogens Fusarium culmorum and Magnaporthe oryzae. Surprisingly, these plants generally had wild-type (WT) resistances to caterpillar herbivory, drought, and ultraviolet light. RNA sequencing analyses revealed that the expression of genes associated with stress responses including ethylene biosynthesis and signalling were significantly altered in PAL knocked-down plants under non-challenging conditions. These data reveal that, although an attenuation of the phenylpropanoid pathway increases carbohydrate availability for biofuel, it can adversely affect plant growth and disease resistance to fungal pathogens. The data identify notable differences between the stress responses of these monocot pal mutants versus Arabidopsis (a dicot) pal mutants and provide insights into the challenges that may arise when deploying phenylpropanoid pathway-altered bioenergy crops.

We thank the technical staff for managing and sampling both field trials, Ingeborg Stals and Dieter Depuydt for help in SSF analyses, and René Custers for requesting regulatory permission for the Belgian field trial.; Lignin is one of the main factors determining recalcitrance to enzymatic processing of lignocellulosic biomass. Poplars (Populus tremula x Populus alba) down-regulated for cinnamoyl-CoA reductase (CCR), the enzyme catalyzing the first step in the monolignol-specific branch of the lignin biosynthetic pathway, were grown in field trials in Belgium and France under short-rotation coppice culture. Wood samples were classified according to the intensity of the red xylem coloration typically associated with CCR down-regulation. Saccharification assays under different pretreatment conditions (none, two alkaline, and one acid pretreatment) and simultaneous saccharification and fermentation assays showed that wood from the most affected transgenic trees had up to 161% increased ethanol yield. Fermentations of combined material from the complete set of 20-mo-old CCR-down-regulated trees, including bark and less efficiently down-regulated trees, still yielded ∼ 20% more ethanol on a weight basis. However, strong down-regulation of CCR also affected biomass yield. We conclude that CCR down-regulation may become a successful strategy to improve biomass processing if the variability in down-regulation and the yield penalty can be overcome.

The yeast Candida glabrata rapidly autoactivates transcription of the AMT1 gene in response to potentially toxic copper levels through the copper-inducible binding of the Amt1 transcription factor to a metal response element (MRE) within a positioned nucleosome. Our previous studies have characterized the role of a 16 bp homopolymeric dA:dT DNA structural element in facilitating rapid Amt1 access to the AMT1 promoter nucleosomal MRE. In this study, we have used the genetically more facile yeast Saccharomyces cerevisiae to identify additional cellular factors that are important for promoting rapid autoactivation of the AMT1 gene in response to toxic copper levels. We demonstrate that the Swi/Snf nucleosome remodelling complex and the histone acetyltransferase Gcn5 are both essential for AMT1 gene autoregulation, and that the requirement for these chromatin remodelling factors is target gene specific. Chromatin accessibility measurements performed in vitro and in vivo indicate that part of the absolute requirement for these factors is derived from their involvement in facilitating nucleosomal access to the AMT1 promoter MRE. Additionally, these data implicate the involvement of Swi/Snf and Gcn5 at multiple levels of AMT1 gene autoregulation.

Transcriptional and metabolic changes were evaluated during senescence induced by preventing pollination in the B73 genotype of maize (Zea mays). Accumulation of free glucose and starch and loss of chlorophyll in leaf was manifested early at 12 d after anthesis (DAA), while global transcriptional and phenotypic changes were evident only at 24 DAA. Internodes exhibited major transcriptomic changes only at 30 DAA. Overlaying expression data onto metabolic pathways revealed involvement of many novel pathways, including those involved in cell wall biosynthesis. To investigate the overlap between induced and natural senescence, transcriptional data from induced senescence in maize was compared with that reported for Arabidopsis (Arabidopsis thaliana) undergoing natural and sugar-induced senescence. Notable similarities with natural senescence in Arabidopsis included up-regulation of senescence-associated genes (SAGs), ethylene and jasmonic acid biosynthetic genes, APETALA2, ethylene-responsive element binding protein, and no apical meristem transcription factors. However, differences from natural senescence were highlighted by unaltered expression of a subset of the SAGs, and cytokinin, abscisic acid, and salicylic acid biosynthesis genes. Key genes up-regulated during sugar-induced senescence in Arabidopsis, including a cysteine protease (SAG12) and three flavonoid biosynthesis genes (PRODUCTION OF ANTHOCYANIN PIGMENT1 (PAP1), PAP2, and LEUCOANTHOCYANIDIN DIOXYGENASE), were also induced, suggesting similarities in senescence induced by pollination prevention and sugar application. Coexpression analysis revealed networks involving known senescence-related genes and novel candidates; 82 of these were shared between leaf and internode networks, highlighting similarities in induced senescence in these tissues. Insights from this study will be valuable in systems biology of senescence in maize and other grasses.

Background Global warming, environmental disasters, and increasing oil prices have catalyzed a worldwide trend to use plant biomass as a renewable source for liquid biofuels and bio-based materials. Plant biomass can be processed into fermentable sugars by enzymatic depolymerization of the cell wall polysaccharides, followed by fermentation. However, the presence of lignin in the cell wall constitutes a major recalcitrance factor because it limits the accessibility of polysaccharidases to the cellulose microfibrils. To overcome this hurdle, plant biomass is pretreated in a costly and energyrequiring process. One approach to overcome the recalcitrance problem is to engineer lignin amount or alter its composition to make lignin more susceptible to chemical degradation [1]. Cinnamoyl-CoA reductase (CCR), the enzyme that converts feruloyl-CoA into coniferaldehyde, is considered the first enzyme in the monolignol-specific branch of the phenylpropanoid pathway. Poplar trees down-regulated in CCR have been produced in the early nineties and planted in a field trial in France to produce sufficient wood for small scale chemical pulping tests [2]. These trees had 20% lower lignin levels and relatively more cellulose per gram of wood [2]. Given that lignin is one of the main limiting factors limiting the conversion of plant biomass into fermentable sugars, and that poplar is considered as a promising second generation biofuel crop, we have re-grown these trees in the greenhouse and in the field, and evaluated wood produced from these trees by saccharification experiments. Methods Cinnamoyl-CoA reductase (CCR) expression was downregulated in poplar by sense and antisense strategies [2]. Transgenic trees were evaluated for lignin amount and composition [2] and for sugar release by saccharification assays [3]. After obtaining permission from the regulatory authorities, two transgenic lines were planted in a field trial in Belgium and two in a field trial in France, both under short rotation coppice culture to maximize biomass production. Wood was saccharified with and without acid pretreatment.

One consequence of the dramatic rise of antibiotic-resistant pathogenic bacteria is the need for new targets for antibiotics. Because membrane lipid biogenesis is essential for bacterial growth, enzymes of the fatty acid biosynthetic pathway offer attractive possibilities for the development of new antibiotics. Acetyl-coenzyme A carboxylase (ACC) catalyzes the first committed and regulated step in fatty acid biosynthesis in bacteria and thus is a prime target for development of antibiotics. ACC is a multifunctional enzyme composed of three separate proteins. The biotin carboxylase component catalyzes the ATP-dependent carboxylation of biotin. The biotin carboxyl carrier protein features a biotin molecule covalently attached at Lys122 of the Escherichia coli enzyme. The carboxyltransferase subunit catalyzes the transfer of a carboxyl group from biotin to acetyl-coenzyme A (acetyl-CoA) to form malonyl-CoA. The objective of this study was to develop an assay for high-throughput screening for inhibitors of the carboxyltransferase subunit. The carboxyltransferase reaction was assayed in the reverse direction in which malonyl-CoA reacts with biocytin (an analog of the biotin carboxyl carrier protein) to form acetyl-CoA and carboxybiotin. The production of acetyl-CoA was coupled to citrate synthase, which produced citrate and coenzyme A. The amount of coenzyme A formed was detected using 5,5′-dithiobis(2-nitrobenzoic acid) (Ellman’s reagent). The assay has been developed for use in both 96- and 384-well microplate formats and was validated using a known bisubstrate analog inhibitor of carboxyltransferase. The spectrophotometric readout in the visible absorbance range used in this assay does not generate the number of false negatives associated with frequently used NAD/NADH assay systems that rely on detection of NADH using UV absorbance.

Gene expression in response to heat shock is mediated by the heat shock transcription factor (HSF), which in yeast harbors both amino- and carboxyl-terminal transcriptional activation domains. Yeast cells bearing a truncated form of HSF in which the carboxyl-terminal transcriptional activation domain has been deleted [HSF(1-583)] are temperature sensitive for growth at 37 degreesC, demonstrating a requirement for this domain for sustained viability during thermal stress. Here we demonstrate that HSF(1-583) cells undergo reversible cell cycle arrest at 37 degreesC in the G2/M phase of the cell cycle and exhibit marked reduction in levels of the molecular chaperone Hsp90. As in higher eukaryotes, yeast possesses two nearly identical isoforms of Hsp90: one constitutively expressed and one highly heat inducible. When expressed at physiological levels in HSF(1-583) cells, the inducible Hsp90 isoform encoded by HSP82 more efficiently suppressed the temperature sensitivity of this strain than the constitutively expressed gene HSC82, suggesting that different functional roles may exist for these chaperones. Consistent with a defect in Hsp90 production, HSF(1-583) cells also exhibited hypersensitivity to the Hsp90-binding ansamycin antibiotic geldanamycin. Depletion of Hsp90 from yeast cells wild type for HSF results in cell cycle arrest in both G1/S and G2/M phases, suggesting a complex requirement for chaperone function in mitotic division during stress.

The baker’s yeast Saccharomyces cerevisiae possesses a single gene encoding heat shock transcription factor (HSF), which is required for the activation of genes that participate in stress protection as well as normal growth and viability. Yeast HSF (yHSF) contains two distinct transcriptional activation regions located at the amino and carboxyl termini. Activation of the yeast metallothionein gene, CUP1, depends on a nonconsensus heat shock element (HSE), occurs at higher temperatures than other heat shock-responsive genes, and is highly dependent on the carboxyl-terminal transactivation domain (CTA) of yHSF. The results described here show that the noncanonical (or gapped) spacing of GAA units in the CUP1 HSE (HSE1) functions to limit the magnitude of CUP1 transcriptional activation in response to heat and oxidative stress. The spacing in HSE1 modulates the dependence for transcriptional activation by both stresses on the yHSF CTA. Furthermore, a previously uncharacterized HSE in the CUP1 promoter, HSE2, modulates the magnitude of the transcriptional activation of CUP1, via HSE1, in response to stress. In vitro DNase I footprinting experiments suggest that the occupation of HSE2 by yHSF strongly influences the manner in which yHSF occupies HSE1. Limited proteolysis assays show that HSF adopts a distinct protease-sensitive conformation when bound to the CUP1 HSE1, providing evidence that the HSE influences DNA-bound HSF conformation. Together, these results suggest that CUP1 regulation is distinct from that of other classic heat shock genes through the interaction of yHSF with two nonconsensus HSEs. Consistent with this view, we have identified other gene targets of yHSF containing HSEs with sequence and spacing features similar to those of CUP1 HSE1 and show a correlation between the spacing of the GAA units and the relative dependence on the yHSF CTA.

Heat shock factors (HSF) are important eukaryotic stress responsive transcription factors which are highly structurally conserved from yeast to mammals. HSFs bind as homotrimers to conserved promoter DNA recognition sites called HSEs. The baker's yeast Saccharomyces cerevisiae possesses a single essential HSF gene, while distinct HSF isoforms have been identified in humans. To ascertain the degree of functional similarity between the yeast and human HSF proteins, human HSF1 and HSF2 were expressed in yeast cells lacking the endogenous HSF gene. We demonstrate that human HSF2, but not HSF1, homotrimerizes and functionally complements the viability defect associated with a deletion of the yeast HSF gene. However, derivatives of hHSF1 that give rise to a trimerized protein, through disruption of a carboxyl- or aminoterminal coiled-coil domain thought to engage in intramolecular interactions that maintain the protein in a monomeric state, functionally substitute for yeast HSF. Surprisingly, hHSF2 expressed in yeast activates target gene transcription in response to thermal stress. Moreover, hHSF1 and hHSF2 exhibit selectivity for transcriptional activation of two distinct yeast heat shock responsive genes, which correlate with previously established mammalian HSF DNA binding preferences in vitro. These results provide new insight into the function of human HSF isoforms, and demonstrate the remarkable functional conservation between yeast and human HSFs, critical transcription factors required for responses to physiological, pharmacological and environmental stresses.

Protein tyrosine phosphatases (PTPases) share a number of conserved amino acid residues, including the active site sequence HCXXGXXRS(T), which are strongly implicated in catalysis. The roles of two conserved active site residues, Asp-181 and Ser-222, were investigated using a combination of site-directed mutagenesis and kinetic analysis in the mammalian PTPase PTP1. The pH profiles for k(cat)/K(m) and k(cat) of the wild-type enzyme indicate that two ionizable groups, of pK(a) values 5.1 and 5.44, must be deprotonated and one group with a pK(a) value of 4.93 must be protonated for maximal activity. The group of pK(a) value 5.1 is the second ionization of the substrate phosphate moiety. Selective thiolate anion inactivation indicates the residue with pK(a) value of 5.44 is C215. The pH-dependent profiles of the D181N mutant during establish the residue with pK(a) value of 4.93 to be Asp-181 and suggest that it functions as a general acid phosphoryl transfer to the enzyme. Rapid reaction kinetics of wild-type and D181N mutant enzymes indicate that the formation of the phospho-enzyme intermediate is rate-limiting at pH 7.0 and 30 degrees C. Enzymes containing the S222A mutation exhibited rapid reaction burst kinetics, strongly suggesting that phospho-enzyme intermediate hydrolysis is fully rate-limiting. The role of the active site S222 is to accelerate the rate of phospho-enzyme intermediate hydrolysis. The kinetic analysis of a third mutant, containing both the D181N and S222A mutations, suggests that D181 also serves as a general base in the breakdown of the phospho-enzyme intermediate.

Sediments from FeSO4 · 7H2O solutions oxidized by resting cell suspensions of Thiobacillus ferrooxidans in the absence of additional monovalent cations consisted of amorphous hydrated Fe(III) sulfate possessing reticular or filamentous microstructure. In the presence of additional sulfate supplied as Li2SO4, MgSO4, (CH3NH3)2SO4, or (CH3NH3)HSO4 at pH 2.4, hydronium jarosite crystallized in the form of pseudocubic rhombohedra. Other cations produced ammonio‐ and natrojarosites as pyramidal and bipyramidal rhombohedra or potassium and rubidium jarosites in rounded and botryoidal forms. Addition of Na2SeO4 yielded the selenium analog of amorphous hydrated Fe(III) sulfate, but in the presence of NH4 + or Rb+, selenojarosites formed instead. Addition of organic ligands, together with (NH4)2SO4, produced ammoniojarosite in the form of irregular granules and rounded and spheroidal crystals. Despite delayed precipitation, the infrared spectra of sediments were usually the same in the presence or absence ...

Gas-liquid chromatography (GLC) and high-performance liquid chromatography (HPLC) were used to separate organic acids in spent culture media. Organic acid components were identified on the basis of their retention times and confirmed by gas chromatography/mass spectrometry (GLC/MS). The sensitivity of the analytical procedures varied considerably with each organic acid. GLC analysis of spent culture media esterified by the H2SO,4/methanol procedure failed to detect fumarate, malate and lactate. The BF3/methanol derivatization was particularly sensitive to pyruvate and lactate. GLC/MS, using the BF3/methanol technique, was insensitive to acetate. Cation-exchange HPLC, using a Bio-Rad acid analysis column, did not require prior derivatization of the sample, and proved to be a rapid and non-destructive assay technique for organic acids. This procedure was used to analyse the spent culture media of aerobically grown Azospirillum brasilense and those of Desulfovibrio desulfuricans grown on pyruvate fermentatively or with sulphate as the electron acceptor. Organic acid profiles of the spent culture media obtained by cation-exchange HPLC analysis were compared with those obtained by GLC analysis using both the H2SO4/methanol and the BF3/methanol esterification procedures.

Background A major challenge in the identification and development of superior feedstocks for the production of second generation biofuels is the rapid assessment of biomass composition in a large number of samples. Currently, highly accurate and precise robotic analysis systems are available for the evaluation of biomass composition, on a large number of samples, with a variety of pretreatments. However, the lack of an inexpensive and high-throughput process for large scale sampling of biomass resources is still an important limiting factor. Our goal was to develop a simple mechanical maize stalk core sampling device that can be utilized to collect uniform samples of a dimension compatible with robotic processing and analysis, while allowing the collection of hundreds to thousands of samples per day. Results We have developed a core sampling device (CSD) to collect maize stalk samples compatible with robotic processing and analysis. The CSD facilitates the collection of thousands of uniform tissue cores consistent with high-throughput analysis required for breeding, genetics, and production studies. With a single CSD operated by one person with minimal training, more than 1,000 biomass samples were obtained in an eight-hour period. One of the main advantages of using cores is the high level of homogeneity of the samples obtained and the minimal opportunity for sample contamination. In addition, the samples obtained with the CSD can be placed directly into a bath of ice, dry ice, or liquid nitrogen maintaining the composition of the biomass sample for relatively long periods of time. Conclusions The CSD has been demonstrated to successfully produce homogeneous stalk core samples in a repeatable manner with a throughput substantially superior to the currently available sampling methods. Given the variety of maize developmental stages and the diversity of stalk diameter evaluated, it is expected that the CSD will have utility for other bioenergy crops as well.