Role of novel polysaccharide layers in assembly of the exosporium, the outermost protein layer of the Bacillus anthracis spore.

A fundamental question in cell biology is how cells assemble their outer layers. The bacterial endospore is a well-established model for cell layer assembly. However, the assembly of the exosporium, a complex protein shell comprising the outermost layer in the pathogen Bacillus anthracis, remains poorly understood. Exosporium assembly begins with the deposition of proteins at one side of the spore surface, followed by the progressive encirclement of the spore. We seek to resolve a major open question: the mechanism directing exosporium assembly to the spore, and then into a closed shell. We hypothesized that material directly underneath the exosporium (the interspace) directs exosporium assembly to the spore and drives encirclement. In support of this, we show that the interspace possesses at least two distinct layers of polysaccharide. Secondly, we show that putative polysaccharide biosynthetic genes are required for exosporium encirclement, suggesting a direct role for the interspace. These results not only significantly clarify the mechanism of assembly of the exosporium, an especially widespread bacterial outer layer, but also suggest a novel mechanism in which polysaccharide layers drive the assembly of a protein shell.

Chemostat cultivation and transcriptional analyses of Clostridium acetobutylicum mutants with defects in the acid and acetone biosynthetic pathways.

Clostridium acetobutylicum is a model organism for the biotechnologically important acetone-butanol-ethanol (ABE) fermentation. With the objective to rationally develop strains with improved butanol production, detailed insights into the physiological and genetic mechanisms of solvent production are required. Therefore, pH-controlled phosphate-limited chemostat cultivation and DNA microarray technology were employed for an in-depth analysis of knockout mutants with defects in the central fermentative metabolism. The set of studied mutants included strains with inactivated phosphotransacetylase (pta), phosphotransbutyrylase (ptb), and acetoacetate decarboxylase (adc) encoding genes, as well as an adc/pta double knockout mutant. A comprehensive physiological characterization of the mutants was performed by continuous cultivation, allowing for a well-defined separation of acidogenic and solventogenic growth, combined with the advantage of the high reproducibility of steady-state conditions. The ptb-negative strain C. acetobutylicum ptb::int(87) exhibited the most striking metabolite profile: Sizable amounts of butanol (29 ± 1.3 mM) were already produced during acidogenic growth. The product patterns of the mutants as well as accompanying transcriptomic data are presented and discussed.

New insights into the butyric acid metabolism of Clostridium acetobutylicum.

Biosynthesis of acetone and n-butanol is naturally restricted to the group of solventogenic clostridia with Clostridium acetobutylicum being the model organism for acetone-butanol-ethanol (ABE) fermentation. According to limited genetic tools, only a few rational metabolic engineering approaches were conducted in the past to improve the production of butanol, an advanced biofuel. In this study, a phosphotransbutyrylase-(Ptb) negative mutant, C. acetobutylicum ptb::int(87), was generated using the ClosTron methodology for targeted gene knock-out and resulted in a distinct butyrate-negative phenotype. The major end products of fermentation experiments without pH control were acetate (3.2 g/l), lactate (4.0 g/l), and butanol (3.4 g/l). The product pattern of the ptb mutant was altered to high ethanol (12.1 g/l) and butanol (8.0 g/l) titers in pH ≥ 5.0-regulated fermentations. Glucose fed-batch cultivation elevated the ethanol concentration to 32.4 g/l, yielding a more than fourfold increased alcohol to acetone ratio as compared to the wildtype. Although butyrate was never detected in cultures of C. acetobutylicum ptb::int(87), the mutant was still capable to take up butyrate when externally added during the late exponential growth phase. These findings suggest that alternative pathways of butyrate re-assimilation exist in C. acetobutylicum, supposably mediated by acetoacetyl-CoA:acyl-CoA transferase and acetoacetate decarboxylase, as well as reverse reactions of butyrate kinase and Ptb with respect to previous studies.

Modifying the product pattern of Clostridium acetobutylicum: physiological effects of disrupting the acetate and acetone formation pathways.

Clostridial acetone-butanol-ethanol (ABE) fermentation is a natural source for microbial n-butanol production and regained much interest in academia and industry in the past years. Due to the difficult genetic accessibility of Clostridium acetobutylicum and other solventogenic clostridia, successful metabolic engineering approaches are still rare. In this study, a set of five knock-out mutants with defects in the central fermentative metabolism were generated using the ClosTron technology, including the construction of targeted double knock-out mutants of C. acetobtuylicum ATCC 824. While disruption of the acetate biosynthetic pathway had no significant impact on the metabolite distribution, mutants with defects in the acetone pathway, including both acetoacetate decarboxylase (Adc)-negative and acetoacetyl-CoA:acyl-CoA transferase (CtfAB)-negative mutants, exhibited high amounts of acetate in the fermentation broth. Distinct butyrate increase and decrease patterns during the course of fermentations provided experimental evidence that butyrate, but not acetate, is re-assimilated via an Adc/CtfAB-independent pathway in C. acetobutylicum. Interestingly, combining the adc and ctfA mutations with a knock-out of the phosphotransacetylase (Pta)-encoding gene, acetate production was drastically reduced, resulting in an increased flux towards butyrate. Except for the Pta-negative single mutant, all mutants exhibited a significantly reduced solvent production.

Switching Clostridium acetobutylicum to an ethanol producer by disruption of the butyrate/butanol fermentative pathway.

Solventogenic clostridia are well-known since almost a century due to their unique capability to biosynthesize the solvents acetone and butanol. Based on recently developed genetic engineering tools, a targeted 3-hydroxybutyryl-CoA dehydrogenase (Hbd)-negative mutant of Clostridium acetobutylicum was generated. Interestingly, the entire butyrate/butanol (C(4)) metabolic pathway of C. acetobutylicum could be inactivated without a severe growth limitation and indicated the general feasibility to manipulate the central fermentative metabolism for product pattern alteration. Cell extracts of the mutant C. acetobutylicum hbd::int(69) revealed clearly reduced thiolase, Hbd and crotonase but increased NADH-dependent alcohol dehydrogenase enzyme activities as compared to the wildtype strain. Neither butyrate nor butanol were detected in cultures of C. acetobutylicum hbd::int(69), and the formation of molecular hydrogen was significantly reduced. Instead up to 16 and 20g/l ethanol were produced in glucose and xylose batch cultures, respectively. Further sugar addition in glucose fed-batch fermentations increased the ethanol production to a final titer of 33g/l, resulting in an ethanol to glucose yield of 0.38g/g.

Human coronavirus NL63 open reading frame 3 encodes a virion-incorporated N-glycosylated membrane protein.

BACKGROUND: Human pathogenic coronavirus NL63 (hCoV-NL63) is a group 1 (alpha) coronavirus commonly associated with respiratory tract infections. In addition to known non-structural and structural proteins all coronaviruses have one or more accessory proteins whose functions are mostly unknown. Our study focuses on hCoV-NL63 open reading frame 3 (ORF 3) which is a highly conserved accessory protein among coronaviruses. RESULTS: In-silico analysis of the 225 amino acid sequence of hCoV-NL63 ORF 3 predicted a triple membrane-spanning protein. Expression in infected CaCo-2 and LLC-MK2 cells was confirmed by immunofluorescence and Western blot analysis. The protein was detected within the endoplasmatic reticulum/Golgi intermediate compartment (ERGIC) where coronavirus assembly and budding takes place. Subcellular localization studies using recombinant ORF 3 protein transfected in Huh-7 cells revealed occurrence in ERGIC, Golgi- and lysosomal compartments. By fluorescence microscopy of differently tagged envelope (E), membrane (M) and nucleocapsid (N) proteins it was shown that ORF 3 protein colocalizes extensively with E and M within the ERGIC. Using N-terminally FLAG-tagged ORF 3 protein and an antiserum specific to the C-terminus we verified the proposed topology of an extracellular N-terminus and a cytosolic C-terminus. By in-vitro translation analysis and subsequent endoglycosidase H digestion we showed that ORF 3 protein is N-glycosylated at the N-terminus. Analysis of purified viral particles revealed that ORF 3 protein is incorporated into virions and is therefore an additional structural protein. CONCLUSIONS: This study is the first extensive expression analysis of a group 1 hCoV-ORF 3 protein. We give evidence that ORF 3 protein is a structural N-glycosylated and virion-incorporated protein.

Coordinated Assembly of the Bacillus anthracis Coat and Exosporium during Bacterial Spore Outer Layer Formation.

Bacterial spores produced by the Bacillales are composed of concentric shells, each of which contributes to spore function. Spores from all species possess a cortex and coat, but spores from many species possess additional outer layers. The outermost layer of Bacillus anthracis spores, the exosporium, is separated from the coat by a gap known as the interspace. Exosporium and interspace assembly remains largely mysterious. As a result, we have a poor understanding of the overarching mechanisms driving the assembly of one of the most ubiquitous cell types in nature. To elucidate the mechanisms directing exosporium assembly, we generated strains bearing mutations in candidate exosporium-controlling genes and analyzed the effect on exosporium formation. Biochemical and cell biological analyses argue that CotE directs the assembly of CotO into the spore and that CotO might be located at or close to the interior side of the cap. Taken together with data showing that CotE and CotO interact directly in vitro, we propose a model in which CotE and CotO are important components of a protein interaction network that connects the exosporium to the forespore during cap formation and exosporium elongation. Our data also suggest that the cap interferes with coat assembly at one pole of the spore, altering the pattern of coat deposition compared to the model organism Bacillus subtilis We propose that the difference in coat assembly patterns between these two species is due to an inherent flexibility in coat assembly, which may facilitate the evolution of spore outer layer complexity.IMPORTANCE This work dramatically improves our understanding of the assembly of the outermost layer of the B. anthracis spore, the exosporium, a layer that encases spores from many bacterial species and likely plays important roles in the spore's interactions with the environment, including host tissues. Nonetheless, the mechanisms directing exosporium assembly into a shell surrounding the spore are still very poorly understood. In this study, we clarify these mechanisms by the identification of a novel protein interaction network that directs assembly to initiate at a specific subcellular location in the developing cell. Our results further suggest that the presence or absence of an exosporium has a major impact on the assembly of other more interior spore layers, thereby potentially explaining long-noted differences in spore assembly between B. anthracis and the model organism B. subtilis.

Effects of different perfusates on functional parameters of isolated perfused dog kidneys.

BACKGROUND: The isolated perfused canine kidney has been established as a valid model for conducting both renal physiology and transplantation research. This model is of particular importance for developing new strategies to improve graft function after renal transplantation. In the present study, a newly developed method using isolated haemoperfused porcine kidneys was adapted for use in canine kidneys. In contrast to haemoperfusion, synthetic perfusion media can be standardized and can prevent the initiation of blood-mediated reperfusion reactions. Thus, an additional aim was to determine whether blood could be replaced by synthetic cell-free perfusion solutions. METHODS: Canine kidneys (n = 30) were harvested from donors euthanized in veterinary practices for causes unrelated to the present study. The kidneys were isolated and perfused with autologous blood or cell-free synthetic electrolyte buffer (Tyrode solution). During perfusion, we monitored renal perfusate flow (RPF), glomerular filtration rate (GFR), electrolyte and glucose reabsorption, oxygen consumption and urine concentration. RESULTS: Changes in perfusion medium did not affect the RPF. In contrast, GFR, urine concentration and oxygen consumption were significantly higher, whereas fractional excretion of sodium and glucose were significantly lower in blood- than in Tyrode-perfused kidneys. CONCLUSIONS: This system offers a simple model for studying whole-organ functional alterations after acute renal ischaemia. Renal function indicators were below values reported during in vivo physiological conditions. These functions were better conserved when kidneys were perfused with autologous blood than with Tyrode.