Carbohydrate transporting membrane proteins of the rumen bacterium, Butyrivibrio proteoclasticus

The research was aimed at finding which membrane proteins of the rumen bacterium Butyrivibrio proteoclasticus are involved in the uptake of carbohydrates resulting from extracellular enzymatic degradation of hemicellulose and fructan. The proteomic analysis of cells grown with fructose or xylan as the sole substrate identified 13 membrane proteins predicted to function as carbohydrate transporters. One protein detected was the membrane component of a fructose-specific phosphoenolpyruvate:sugar phosphotransferase system believed to be involved in the fructose uptake following extracellular fructan breakdown. The other 12 proteins were all ABC transport system substrate-binding proteins, nine of which belong to functional category COG1653 that includes proteins predicted to transport oligosaccharides. Four of the SBPs were significantly upregulated in xylan grown cells, and three of these were found in polysaccharide utilisation loci where they are clustered with other genes involved in hemicellulose breakdown and metabolism. It is possible that the carbon source available regulates a wider network of genes. The information on the mechanisms used by rumen bacteria to take up carbohydrates from their environment may improve our understanding of the ruminant digestion and facilitate strategies for improved pasture and stored feed utilisation.     

A proteomic study of pectin‐degrading enzymes secreted by Botrytis cinerea grown in liquid culture

Botrytis cinerea is a pathogenic filamentous fungus, which infects more than 200 plant species. The enzymes secreted by B. cinerea play an important role in the successful colonization of a host plant. Some of the secreted enzymes are involved in the degradation of pectin, a major component of the plant cell wall. A total of 126 proteins secreted by B. cinerea were identified by growing the fungus on highly or partially esterified pectin, or on sucrose in liquid culture. Sixty‐seven common proteins were identified in each of the growth conditions, of which 50 proteins exhibited a SignalP motif. Thirteen B. cinerea proteins with functions related to pectin degradation were identified in both pectin growth conditions, while only four were identified in sucrose. Our results indicate it is unlikely that the activation of B. cinerea from the dormant state to active infection is solely dependent on changes in the degree of esterification of the pectin component of the plant cell wall. Further, these results suggest that future studies of the B. cinerea secretome in infections of ripe and unripe fruits will provide important information that will describe the mechanisms that the fungus employs to access nutrients and decompose tissues.     

Genetic or chemical protease inhibition causes significant changes in the Bacillus subtilis exoproteome

Bacillus subtilis is a prolific producer of enzymes and biopharmaceuticals. However, the susceptibility of heterologous proteins to degradation by (extracellular) proteases is a major limitation for use of B. subtilis as a protein cell factory. An increase in protein production levels has previously been achieved by using either protease-deficient strains or addition of protease inhibitors to B. subtilis cultures. Notably, the effects of genetic and chemical inhibition of proteases have thus far not been compared in a systematic way. In the present studies, we therefore compared the exoproteomes of cells in which extracellular proteases were genetically or chemically inactivated. The results show substantial differences in the relative abundance of various extracellular proteins. Furthermore, a comparison of the effects of genetic and/or chemical protease inhibition on the stress response triggered by (over) production of secreted proteins showed that chemical protease inhibition provoked a genuine secretion stress response. From a physiological point of view, this suggests that the deletion of protease genes is a better way to prevent product degradation than the use of protease inhibitors. Importantly however, studies with human interleukin-3 show that chemical protease inhibition can result in improved production of protease-sensitive secreted proteins even in mutant strains lacking eight extracellular proteases.     

Horizontal gene transfer of glycosyl hydrolases of the rumen fungi

By combining analyses of G + C content and patterns of codon usage and constructing phylogenetic trees, we describe the gene transfer of an endoglucanase (celA) from the rumen bacteria Fibrobacter succinogenes to the rumen fungi Orpinomyces joyonii. The strong similarity between different glycosyl hydrolases of rumen fungi and bacteria suggests that most, if not all, of the glycosyl hydrolases of rumen fungi that play an important role in the degradation of cellulose and other plant polysaccharides were acquired by horizontal gene transfer events. This acquisition allows fungi to establish a habitat within a new environmental niche: the rumen of the herbivorous mammals for which cellulose and plant hemicellulose constitute the main raw nutritive substrate.     

Acute regulation of hepatic lipase secretion by rat hepatocytes.

Hepatic lipase is involved in cholesterol uptake by the liver. Although it is known that catecholamines are responsible for the daily variation of enzyme activity, the mechanisms involved are poorly understood. Rat hepatocytes incubated with adrenaline or other Ca(2+)-mobilizing hormones were used as an experimental model. Adrenaline reduced in a similar proportion the secretion of both hepatic lipase and albumin. The effect of adrenaline disappeared completely in cells exposed to cycloheximide. Adrenaline decreased incorporation of [35S]Met into cellular and secreted proteins, but it affected neither degradation of [35S]Met-prelabeled proteins nor the abundance of total and specific (albumin, hepatic lipase, beta-actin) mRNA. Other Ca(2+)-mobilizing agents had the opposite effect on hepatic lipase secretion: it was decreased by vasopressin but was increased by epidermal growth factor. Vasopressin and epidermal growth factor had the opposite effect on [35S]Met incorporation into cellular and secreted proteins, but neither affected hepatic lipase mRNA. The acute effect of adrenaline, vasopressin, and epidermal growth factor on hepatic lipase secretion is the consequence of the effect of these hormones on protein synthesis and is therefore nonspecific.     

The extracellular proteome of Bacillus licheniformis grown in different media and under different nutrient starvation conditions

The now finished genome sequence of Bacillus licheniformis DSM 13 allows the prediction of the genes involved in protein secretion into the extracellular environment as well as the prediction of the proteins which are translocated. From the sequence 296 proteins were predicted to contain an N-terminal signal peptide directing most of them to the Sec system, the main transport system in Gram-positive bacteria. Using 2-DE the extracellular proteome of B. licheniformis grown in different media was studied. From the approximately 200 spots visible on the gels, 89 were identified that either contain an N-terminal signal sequence or are known to be secreted by other mechanisms than the Sec pathway. The extracellular proteome of B. licheniformis includes proteins from different functional classes, like enzymes for the degradation of various macromolecules, proteins involved in cell wall turnover, flagellum- and phage-related proteins and some proteins of yet unknown function. Protein secretion is highest during stationary growth phase. Furthermore, cells grown in complex medium secrete considerably higher protein amounts than cells grown in minimal medium. Limitation of phosphate, carbon and nitrogen sources results in the secretion of specific proteins that may be involved in counteracting the starvation.     

Spatially resolving the secretome within the mycelium of the cell factory Aspergillus niger

Aspergillus niger is an important cell factory for the industrial production of enzymes. These enzymes are released into the culture medium, from which they can be easily isolated. Here, we determined with stable isotope dimethyl labeling the secretome of five concentric zones of 7-day-old xylose-grown colonies of A. niger that had either or not been treated with cycloheximide. As expected, cycloheximide blocked secretion of proteins at the periphery of the colony. Unexpectedly, protein release was increased by cycloheximide in the intermediate and central zones of the mycelium when compared to nontreated colonies. Electron microscopy indicated that this is due to partial degradation of the cell wall. In total, 124 proteins were identified in cycloheximide-treated colonies, of which 19 secreted proteins had not been identified before. Within the pool of 124 proteins, 53 secreted proteins were absent in nontreated colonies, and additionally, 35 proteins were released ≥4-fold in the central and subperipheral zones of cycloheximide-treated colonies when compared to nontreated colonies. The composition of the secretome in each of the five concentric zones differed. This study thus describes spatial release of proteins in A. niger, which is instrumental in understanding how fungi degrade complex substrates in nature.     

Presence of several cellulose-binding proteins in culture supernatant and cell lysate of Eubacterium cellulosolvens 5

Attempts were made to separate and characterize cellulose-binding proteins (CBPs) from both the culture supernatant and cell lysate of Eubacterium cellulosolvens 5. Once the CBPs were bound to Avicel cellulose, they were then effectively eluted with the solution containing 3.2 or 5% sodium dodecyl sulfate (SDS), but not eluted with the solution containing various kinds of carbohydrates and reagents. Namely, CBPs in both the culture supernatant and cell lysate of the bacterium bound tightly and strongly to cellulose. The SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of the eluted CBPs indicated that the CBPs contained the two major proteins having the molecular weights of approximately 160 and 84 kilodaltons (kDa) and one sub-major protein having a molecular weight of approximately 140 kDa. Zymogram analysis after the SDS-PAGE of the eluted CBPs showed that two proteins exhibited the highest levels of carboxymethyl cellulase (CMCase) activity corresponding to the molecular weights of approximately 160 and 90 kDa. A major protein having the molecular weight of approximately 160 kDa exhibited a distinct CMCase activity and was designated as CBPE1. Western immunoblot analysis indicated that the proteins prepared from 16 representative strains of rumen bacteria did not cross-react with rabbit antiserum raised against CBPE1. Thus, CBPE1 may be a unique CBP that plays an important role in the adhesion of the bacterium to cellulose.     

Quantitative in-depth analysis of the dynamic secretome of activated Jurkat T-cells

Proteins secreted by cells are of the highest biomedical relevance since they play a significant role in the progression of numerous diseases. However, characterization of the proteins specifically secreted in response to precise stimuli is challenging, since these proteins are contaminated by cellular byproducts. Here we present a method to characterize a dynamic secretome and demonstrate its utility by performing the deepest quantitative analysis to date of proteins secreted by lymphoid Jurkat T-cells upon activation. Cell-free supernatant proteins were analyzed by using an optimized protocol for differential (18)O/(16)O-labeling and LC-MS/MS, followed by statistical analysis using a random-effects model. More than 4000 unique peptides belonging to 1288 proteins were identified and a large proportion could be quantified. To determine the proteins whose secretion was up-regulated upon T-cell activation, protein variance of the null hypothesis was estimated after protein classification in terms of secretion and ontology using bioinformatic tools. 62 proteins showed a statistically significant change in abundance upon cell activation and most of them (49 proteins) were up-regulated. These proteins were functionally involved mainly in inflammatory response, signal transduction, cell growth and differentiation and cell redox homeostasis. Our approach provides a promising technology for the high-throughput quantitative study of dynamic secretomes.     

A proteomic analysis of the Pichia pastoris secretome in methanol-induced cultures

The secreted proteome of Pichia pastoris X-33 was investigated in methanol-induced cultures with a goal to enhance the secretion and purification of recombinant proteins. In a fed-batch fermentation at 30 °C, more host proteins were found in greater concentrations compared to cultures grown at 25 °C. Protein samples collected directly from the culture media at 25 °C, as well as separated by two-dimensional (2D) gel, were subjected to ESI-MS/MS analysis. A total of 75 proteins were identified in the media from different conditions including pre- and post-methanol induction and in a strain overexpressing a recombinant schistosomiasis vaccine, Sm14-C62V. The identified proteins include native secreted proteins and some intracellular proteins, most of which have low isoelectric points (pI < 6). 2D gel analyses further revealed important characteristics, such as abundance, degradation, and glycosylation of these identified proteins in this proteome. Cell wall-associated proteins involved in cell wall biogenesis, structure, and modification comprised the majority of the secreted proteins which have been identified. Intracellular proteins such as alcohol oxidase and superoxide dismutase were also found in the proteome, suggesting some degree of cell lysis. However, both protocols show that their concentrations are significantly lower than the native secreted proteins. This study identifies proteins secreted or released into the culture media in the methanol-induced fermentation cultures of P. pastoris X-33 and suggests potential biotechnology applications based on the discovery of this proteome.     

The Glycobiome of the Rumen Bacterium Butyrivibrio proteoclasticus B316T Highlights Adaptation to a Polysaccharide-Rich Environment

Determining the role of rumen microbes and their enzymes in plant polysaccharide breakdown is fundamental to understanding digestion and maximising productivity in ruminant animals. Butyrivibrio proteoclasticus B316^T is a Gram-positive, butyrate-forming rumen bacterium with a key role in plant polysaccharide degradation. The 4.4Mb genome consists of 4 replicons; a chromosome, a chromid and two megaplasmids. The chromid is the smallest reported for all bacteria, and the first identified from the phylum Firmicutes. B316 devotes a large proportion of its genome to the breakdown and reassembly of complex polysaccharides and has a highly developed glycobiome when compared to other sequenced bacteria. The secretion of a range of polysaccharide-degrading enzymes which initiate the breakdown of pectin, starch and xylan, a subtilisin family protease active against plant proteins, and diverse intracellular enzymes to break down oligosaccharides constitute the degradative capability of this organism. A prominent feature of the genome is the presence of multiple gene clusters predicted to be involved in polysaccharide biosynthesis. Metabolic reconstruction reveals the absence of an identifiable gene for enolase, a conserved enzyme of the glycolytic pathway. To our knowledge this is the first report of an organism lacking an enolase. Our analysis of the B316 genome shows how one organism can contribute to the multi-organism complex that rapidly breaks down plant material in the rumen. It can be concluded that B316, and similar organisms with broad polysaccharide-degrading capability, are well suited to being early colonizers and degraders of plant polysaccharides in the rumen environment.     

The CssRS two-component regulatory system controls a general secretion stress response in Bacillus subtilis

Bacillus species are valuable producers of industrial enzymes and biopharmaceuticals, because they can secrete large quantities of high-quality proteins directly into the growth medium. This requires the concerted action of quality control factors, such as folding catalysts and 'cleaning proteases'. The expression of two important cleaning proteases, HtrA and HtrB, of Bacillus subtilis is controlled by the CssRS two-component regulatory system. The induced CssRS-dependent expression of htrA and htrB has been defined as a protein secretion stress response, because it can be triggered by high-level production of secreted alpha-amylases. It was not known whether translocation of these alpha-amylases across the membrane is required to trigger a secretion stress response or whether other secretory proteins can also activate this response. These studies show for the first time that the CssRS-dependent response is a general secretion stress response which can be triggered by both homologous and heterologous secretory proteins. As demonstrated by high-level production of a nontranslocated variant of the alpha-amylase, AmyQ, membrane translocation of secretory proteins is required to elicit this general protein secretion stress response. Studies with two other secretory reporter proteins, lipase A of B. subtilis and human interleukin-3, show that the intensity of the protein secretion stress response only partly reflects the production levels of the respective proteins. Importantly, degradation of human interleukin-3 by extracellular proteases has a major impact on the production level, but only a minor effect on the intensity of the secretion stress response.     

Use of label-free quantitative proteomics to distinguish the secreted cellulolytic systems of Caldicellulosiruptor bescii and Caldicellulosiruptor obsidiansis

The extremely thermophilic, Gram-positive bacteria Caldicellulosiruptor bescii and Caldicellulosiruptor obsidiansis efficiently degrade both cellulose and hemicellulose, which makes them relevant models for lignocellulosic biomass deconstruction to produce sustainable biofuels. To identify the shared and unique features of secreted cellulolytic apparatuses from C. bescii and C. obsidiansis, label-free quantitative proteomics was used to analyze protein abundance over the course of fermentative growth on crystalline cellulose. Both organisms' secretomes consisted of more than 400 proteins, of which the most abundant were multidomain glycosidases, extracellular solute-binding proteins, flagellin, putative pectate lyases, and uncharacterized proteins with predicted secretion signals. Among the identified proteins, 53 to 57 significantly changed in abundance during cellulose fermentation in favor of glycosidases and extracellular binding proteins. Mass spectrometric characterizations, together with cellulase activity measurements, revealed a substantial abundance increase of a few bifunctional multidomain glycosidases composed of glycosidase (GH) domain family 5, 9, 10, 44, or 48 and family 3 carbohydrate binding (CBM3) modules. In addition to their orthologous cellulases, the organisms expressed unique glycosidases with different domain organizations: C. obsidiansis expressed the COB47_1671 protein with GH10/5 domains, while C. bescii expressed the Athe_1857 (GH10/48) and Athe_1859 (GH5/44) proteins. Glycosidases containing CBM3 domains were selectively enriched via binding to amorphous cellulose. Preparations from both bacteria contained highly thermostable enzymes with optimal cellulase activities at 85°C and pH 5. The C. obsidiansis preparation, however, had higher cellulase specific activity and greater thermostability. The C. bescii culture produced more extracellular protein and additional SDS-PAGE bands that demonstrated glycosidase activity.     

A time course analysis of the extracellular proteome of Aspergillus nidulans growing on sorghum stover

Background

Fungi are important players in the turnover of plant biomass because they produce a broad range of degradative enzymes. Aspergillus nidulans, a well-studied saprophyte and close homologue to industrially important species such as A. niger and A. oryzae, was selected for this study.

Results

A. nidulans was grown on sorghum stover under solid-state culture conditions for 1, 2, 3, 5, 7 and 14?days. Based on analysis of chitin content, A. nidulans grew to be 4-5% of the total biomass in the culture after 2?days and then maintained a steady state of 4% of the total biomass for the next 12?days. A hyphal mat developed on the surface of the sorghum by day one and as seen by scanning electron microscopy the hyphae enmeshed the sorghum particles by day 5. After 14?days hyphae had penetrated the entire sorghum slurry. Analysis (1-D PAGE LC-MS/MS) of the secretome of A. nidulans, and analysis of the breakdown products from the sorghum stover showed a wide range of enzymes secreted. A total of 294 extracellular proteins were identified with hemicellulases, cellulases, polygalacturonases, chitinases, esterases and lipases predominating the secretome. Time course analysis revealed a total of 196, 166, 172 and 182 proteins on day 1, 3, 7 and 14 respectively. The fungus used 20% of the xylan and cellulose by day 7 and 30% by day 14. Cellobiose dehydrogenase, feruloyl esterases, and CAZy family 61 endoglucanases, all of which are thought to reduce the recalcitrance of biomass to hydrolysis, were found in high abundance.

Conclusions

Our results show that A. nidulans secretes a wide array of enzymes to degrade the major polysaccharides and lipids (but probably not lignin) by 1?day of growth on sorghum. The data suggests simultaneous breakdown of hemicellulose, cellulose and pectin. Despite secretion of most of the enzymes on day 1, changes in the relative abundances of enzymes over the time course indicates that the set of enzymes secreted is tailored to the specific substrates available. Our findings reveal that A. nidulans is capable of degrading the major polysaccharides in sorghum without any chemical pre-treatment.     

Exosome-associated tau is secreted in tauopathy models and is selectively phosphorylated in cerebrospinal fluid in early Alzheimer disease

Recent demonstrations that the secretion, uptake, and interneuronal transfer of tau can be modulated by disease-associated tau modifications suggest that secretion may be an important element in tau-induced neurodegeneration. Here, we show that much of the tau secreted by M1C cells occurs via exosomal release, a widely characterized mechanism that mediates unconventional secretion of other aggregation-prone proteins (α-synuclein, prion protein, and β-amyloid) in neurodegenerative disease. Exosome-associated tau is also present in human CSF samples and is phosphorylated at Thr-181 (AT270), an established phosphotau biomarker for Alzheimer disease (AD), in both M1C cells and in CSF samples from patients with mild (Braak stage 3) AD. A preliminary analysis of proteins co-purified with tau in secreted exosomes identified several that are known to be involved in disease-associated tau misprocessing. Our results suggest that exosome-mediated secretion of phosphorylated tau may play a significant role in the abnormal processing of tau and in the genesis of elevated CSF tau in early AD.     

The rumen anaerobic fungi

The anaerobic fungi represent a new group of organisms inhabiting the rumen ecosystem and possess a life cycle alternating between a motile flagellated form (zoospore) and a non-motile vegetative reproductive form (thallus). In vivo studies show extensive colonization of plant material suspended in the rumen indicating the fungi have a role in fiber digestion. Pure cultures of anaerobic fungi ferment cellulose to give lactate, acetate, CO₂ and H₂ as the major products. Ethanol and formate may also be produced. Fermentation of cellulose by the fungi in coculture with H₂-utilizing methanogens results in a shift in the fermentation pattern favouring the production of H₂ (utilized in the formation of CH₄) and acetate at the expense of the electron-sink products, lactate and ethanol. It is postulated that the methanogens in reducing the partial pressure of H₂, facilitate an increased passage of reducing equivalents towards the production of H₂ via a pyridine-nucleotide (PN)-linked hydrogenase reaction. H₂ is believed to be produced in microbodies of the fungi called hydrogenosomes which possess all of the enzymes necessary for this function including PN-linked hydrogenase. Absence of mitochondria and key electron transport components in these organisms indicate a dependence wholly on fermentative processes for growth. Anaerobic fungi also participate in hemicellulose and starch degredation but it is not yet clear whether they have a role in the degradation of lignin. Simple sugars (mono- and disaccharides) are readily utilized and their uptake is subject to similar regulatory constraints such as is found with other micro-organisms.Enzymological studies have revealed that anaerobic fungi release substantial amounts of endo-acting cellulase and protease, possibly giving them a competitive advantage over rumen bacteria in the degradation of plant structural material.     

Bacillus spp. in the rumen ecosystem. Hemicellulose depolymerases and glycoside hydrolases of Bacillus spp. and rumen isolates grown under anaerobic conditions

Bacillus spp. were isolated from the rumen contents of animals fed on hay. The bacilli were present in the vegetative form in the microbial populations of the liquid phase and in those associated with the particulate material in the digesta. Thirty-six isolates were identified as strains of B. licheniformis (14), B. circulans (14), B. coagulans (2), B. laterosporus (3) and three isolates were facultative anaerobes that resembled B. pumilus. All were able to utilize isolated Lolium perenne hemicellulose B as a carbon source when grown aerobically or anaerobically at 39°C. A survey of 17 Bacillus species (24 strains including 5 rumen isolates) confirmed that enzymes capable of releasing reducing sugars from hemicelullosic polysaccharides were present in many anaerobically grown bacilli; in addition, the range and specific activities of 20 glycoside hydrolases were also monitored in these crude enzyme preparations. The presence and possible role of Bacillus spp. in the rumen digesta particle-associated microbial population is discussed.