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Find video protocols related to scientific articles indexed in Pubmed.
Contribution of Veillonella parvula to Pseudomonas aeruginosa mediated pathogenicity in a murine tumor model system.
Infect. Immun.
PUBLISHED: 11-12-2014
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The recent finding that high numbers of strict anaerobes are present in the respiratory tract of cystic fibrosis (CF) patients has drawn attention to the pathogenic contribution of the CF microbiome to airway disease. In this study, we investigated the specific interactions of the most dominant bacterial CF-pathogen, Pseudomonas aeruginosa, and the anaerobic bacterium Veillonella parvula, that has been recovered at comparable cell numbers from the respiratory tract of CF patients. In addition to growth competition experiments, transcriptional profiling and analyses of biofilm formation by in-vitro studies, we used our recently established in-vivo murine tumor model to investigate mutual influences of the two pathogens during a biofilm-associated infection process. We found that P. aeruginosa and V. parvula colonized distinct niches within the tumor. Interestingly, from mice that were co-infected with both bacterial species significant higher cell numbers of P. aeruginosa could be recovered from the tumor tissue. Concordantly, in vivo transcriptional profiling implied that the presence of V. parvula supports P. aeruginosa growth at the infected host site, and the higher P. aeruginosa load correlated with clinical deterioration. Although many challenges must be overcome to dissect the specific interactions of co-infecting bacteria during an infection process, our findings exemplarily demonstrate that the complex interrelations between co-infecting microorganisms and the immune responses determine clinical outcome to a much greater extent than previously anticipated.
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Characterization of biofilm formation and the role of BCR1 in clinical isolates of Candida parapsilosis.
Eukaryotic Cell
PUBLISHED: 12-02-2013
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In Candida parapsilosis, biofilm formation is considered to be a major virulence factor. Previously, we determined the ability of 33 clinical isolates causing bloodstream infection to form biofilm and identified three distinct groups of biofilm forming strains (negative, low and high). Here, we establish two different biofilm structures among strains forming high amount of biofilm whereby strains with complex spider-like structure formed robust biofilms on different surface materials with increased resistance to fluconazole. Surprisingly, the transcription factor Bcr1 required for biofilm formation in Candida albicans and C. parapsilosis has an essential role only in strains with low biofilm formation, but, although leading to the formation of more and longer pseudohyphae, was not required for initial adhesion and formation of mature biofilms in strains with a high level of biofilm formation. Furthermore, an additional phenotype affected by BCR1 was the switch in colony morphology from rough to crepe, but only in strains forming high level of biofilm. All bcr1?/? mutants showed increased proteolytic activity and increased susceptibility to the antimicrobial peptides protamine and RP-1 compared to corresponding wild type and complemented strains. Taken together, our results demonstrate that biofilm formation in clinical isolates of C. parapsilosis is both dependent and independent of BCR1, but even in strains which showed a BCR1 independent biofilm phenotype BCR1 has alternative physiological functions.
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The EAL-like protein STM1697 regulates virulence phenotypes, motility and biofilm formation in Salmonella typhimurium.
Mol. Microbiol.
PUBLISHED: 10-09-2013
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The ubiquitous second messenger c-di-GMP regulates the switching of bacterial lifestyles from motility to sessility and acute to chronic virulence to adjust bacterial fitness to altered environmental conditions. Conventionally, EAL proteins being c-di-GMP phosphodiesterases promote motility and acute virulence phenotypes such as invasion into epithelial cells and inhibit biofilm formation. We report here that in contradiction, the EAL-like protein STM1697 of Salmonella typhimurium suppresses motility, invasion into HT-29 epithelial cell line and secretion of the type three secretion system 1 effector protein SipA, whereas it promotes rdar biofilm formation and CsgD expression. STM1697 can, however, functionally replace the EAL-like protein STM1344 and vice versa, whereby both proteins neither degrade nor bind c-di-GMP. Like STM1344, STM1697 suppresses the transcription of class 2 and class 3 flagella regulon genes by binding to FlhD, a component of the master regulator of the flagella regulon FlhD4 C2 and act additively under numerous conditions. Interestingly, the interaction interface of STM1697 with FlhD2 is distinct from its paralogue STM1344. We predict that the stand alone EAL domain proteins STM1697 and STM1344 belong to a subclass of EAL domain proteins in S.?typhimurium, which are all involved in motility, biofilm and virulence regulation through interaction with proteins that regulate flagella function.
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Highly stable monodisperse PEGylated iron oxide nanoparticle aqueous suspensions: a nontoxic tracer for homogeneous magnetic bioassays.
Nanoscale
PUBLISHED: 09-24-2013
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Uniformly sized and shaped iron oxide nanoparticles with a mean size of 25 nm were synthesized via decomposition of iron-oleate. High resolution transmission electron microscopy and Mössbauer spectroscopy investigations revealed that the particles are spheres primarily composed of Fe3O4 with a small fraction of FeO. From Mössbauer and static magnetization measurements, it was deduced that the particles are superparamagnetic at room temperature. The hydrophobic particles were successfully transferred into water via PEGylation using nitrodopamine as an anchoring group. IR spectroscopy and thermogravimetric analysis showed the success and efficiency of the phase transfer reaction. After PEGylation, the particles retained monodispersity and their magnetic core remained intact as proven by photon cross-correlation spectroscopy, ac susceptibility, and transmission electron microscopy. The particle aqueous suspensions revealed excellent water stability over a month of monitoring and also against temperature up to 40 °C. The particles exhibited a moderate cytotoxic effect on in vitro cultured bone marrow-derived macrophages and no release of inflammatory or anti-inflammatory cytokines. The PEGylated particles were functionalized with Herceptin antibodies via a conjugation chemistry, their response to a rotating magnetic field was studied using a fluxgate-based setup and was compared with the one recorded for hydrophobic and PEGylated particles. The particle phase lag rose after labeling with Herceptin, indicating the successful conjugation of Herceptin antibodies to the particles.
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Genome sequence and functional genomic analysis of the oil-degrading bacterium Oleispira antarctica.
Nat Commun
PUBLISHED: 06-18-2013
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Ubiquitous bacteria from the genus Oleispira drive oil degradation in the largest environment on Earth, the cold and deep sea. Here we report the genome sequence of Oleispira antarctica and show that compared with Alcanivorax borkumensis--the paradigm of mesophilic hydrocarbonoclastic bacteria--O. antarctica has a larger genome that has witnessed massive gene-transfer events. We identify an array of alkane monooxygenases, osmoprotectants, siderophores and micronutrient-scavenging pathways. We also show that at low temperatures, the main protein-folding machine Cpn60 functions as a single heptameric barrel that uses larger proteins as substrates compared with the classical double-barrel structure observed at higher temperatures. With 11 protein crystal structures, we further report the largest set of structures from one psychrotolerant organism. The most common structural feature is an increased content of surface-exposed negatively charged residues compared to their mesophilic counterparts. Our findings are relevant in the context of microbial cold-adaptation mechanisms and the development of strategies for oil-spill mitigation in cold environments.
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Purification of hepatitis B surface antigen virus-like particles from recombinant Pichia pastoris and in vivo analysis of their immunogenic properties.
J. Chromatogr. B Analyt. Technol. Biomed. Life Sci.
PUBLISHED: 03-15-2013
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Following earlier studies on high-level intracellular production of hepatitis B surface antigen (HBsAg) using recombinant Pichia pastoris, we present here in detail an enhanced method for the purification of recombinant HBsAg virus-like particles (VLPs). We have screened various detergents for their ability to promote the solubilization of recombinant intracellular HBsAg. In addition, we have analyzed the effect of cell disruption and extraction regarding their impact on the release of HBsAg. Our results show that introduction of the mild nonionic detergent Tween 20 in the initial process of cell lysis at ?600bars by high pressure homogenization leads to the best results. The subsequent purification steps involved polyethylene glycol precipitation of host cell contaminants, hydrophobic adsorption of HBsAg to colloidal silica followed by ion-exchange chromatography and either isopycnic density ultracentrifugation or size exclusion chromatography for the recovery of the VLPs. After final KSCN treatment and dialysis, a total yield of ?3% with a purity of >99% was reached. The pure protein was characterized by electron microscopy, showing the presence of uniform VLPs which are the pre-requisite for immunogenicity. The intramuscular co-administration of HBsAg VLPs, with either alum or a PEGylated-derivative of the toll-like receptor 2/6 agonist MALP-2, to mice resulted in the elicitation of significantly higher HBsAg-specific IgG titers as well as a stronger cellular immune response compared to mice vaccinated with a gold standard vaccine (Engerix™). These results show that P. pastoris derived HBsAg VLPs exhibit a high potential as a superior biosimilar vaccine against hepatitis B.
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Dengue-specific subviral nanoparticles: design, creation and characterization.
J Nanobiotechnology
PUBLISHED: 03-07-2013
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Dengue is today the most significant of arboviral diseases. Novel tools are necessary to effectively address the problem of dengue. Virus-like particles (VLP) offer a versatile nanoscale platform for developing tools with potential biomedical applications. From the perspective of a potentially useful dengue-specific tool, the dengue virus envelope protein domain III (EDIII), endowed with serotype-specificity, host receptor recognition and the capacity to elicit virus-neutralizing antibodies, is an attractive candidate.
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Genome of the R-body producing marine alphaproteobacterium Labrenzia alexandrii type strain (DFL-11(T)).
Stand Genomic Sci
PUBLISHED: 02-25-2013
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Labrenzia alexandrii Biebl et al. 2007 is a marine member of the family Rhodobacteraceae in the order Rhodobacterales, which has thus far only partially been characterized at the genome level. The bacterium is of interest because it lives in close association with the toxic dinoflagellate Alexandrium lusitanicum. Ultrastructural analysis reveals R-bodies within the bacterial cells, which are primarily known from obligate endosymbionts that trigger "killing traits" in ciliates (Paramecium spp.). Genomic traits of L. alexandrii DFL-11(T) are in accordance with these findings, as they include the reb genes putatively involved in R-body synthesis. Analysis of the two extrachromosomal elements suggests a role in heavy-metal resistance and exopolysaccharide formation, respectively. The 5,461,856 bp long genome with its 5,071 protein-coding and 73 RNA genes consists of one chromosome and two plasmids, and has been sequenced in the context of the Marine Microbial Initiative.
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Aminoalkyl functionalization of dextran for coupling of bioactive molecules and nanostructure formation.
Carbohydr Polym
PUBLISHED: 02-15-2013
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Aminopropyl dextrans and mixed aminopropyl cyanoethyl dextrans were prepared from cyanoethyl precursors by full or partial reduction with CoCl2/NaBH4. Coupling of various aldehydes to the glucan backbone by reductive amination was accomplished with 4-hydroxy-3-methoxybenzaldehyde (vanillin), 3,5-di-tert-butyl-4-hydroxy-benzaldehyde (BHT-CHO), maltose and maltotriose, and picoline borane as reducing agent. Successful coupling of these representatives for aroma compounds, antioxidants and sugar side-chains were verified by ESI-MS after hydrolysis and by 1D and 2D NMR spectroscopy. Degree of conversion (molar ratio of coupled aldehydes) was estimated from (1)H NMR spectra. Formation of secondary and tertiary amines was detectd by ESI-MS. Applying a solvent exchange process, new nanoparticles based on these modified dextrans were prepared with and without addition of iron oxide nanoparticles.
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The formation of an organic coat and the release of corrosion microparticles from metallic magnesium implants.
Acta Biomater
PUBLISHED: 02-07-2013
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Magnesium alloys have been proposed as prospective degradable implant materials. To elucidate the complex interactions between the corroding implants and the tissue, magnesium implants were analyzed in a mouse model and the response was compared to that induced by Ti and by the resorbable polymer polyglactin, respectively. One month after implantation, distinct traces of corrosion were apparent but the magnesium implants were still intact, whereas resorbable polymeric wound suture implants were already fragmented. Analysis of magnesium implants 2weeks after implantation by energy-dispersive X-ray spectroscopy indicated that magnesium, oxygen, calcium and phosphate were present at the implant surface. One month after implantation, the element composition of the outermost layer of the implant was indicative of tissue without detectable levels of magnesium, indicating a protective barrier function of this organic layer. In agreement with this notion, gene expression patterns in the surrounding tissue were highly similar for all implant materials investigated. However, high-resolution imaging using energy-filtered transmission electron microscopy revealed magnesium-containing microparticles in the tissue in the proximity of the implant. The release of such corrosion particles may contribute to the accumulation of calcium phosphate in the nearby tissue and to bone conductive activities of magnesium implants.
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Schwertmannite formation adjacent to bacterial cells in a mine water treatment plant and in pure cultures of Ferrovum myxofaciens.
Environ. Sci. Technol.
PUBLISHED: 08-29-2011
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Schwertmannite has previously been found in iron- and sulfate-rich mine waters at pH 2.8-4.5. In the present study, schwertmannite (Fe(8)O(8)(OH)(6)SO(4)) was shown to be the major mineral in a mine water treatment plant at pH 3, in which ferrous iron is mainly oxidized by bacteria belonging to the species Ferrovum myxofaciens. Strain EHS6, which is closely related to the type strain of Fv. myxofaciens, was isolated from the pilot plant and characterized as an acidophilic, iron-oxidizing bacterium. In contrast to the pilot plant, the mineral phase formed by a pure culture of Fv. myxofaciens EHS6 was a mixture of schwertmannite and jarosite (KFe(3)(SO(4))(2)(OH)(6)). In contrast to other reports of neutrophilic, iron-oxidizing bacteria, acidophilic microorganisms in the pilot plant and cultures of strain EHS6 did not show encrustation of the cell surface or deposition of minerals inside the cell, though a few cells appeared to be in contact with jarosite crystals. It was concluded that no direct biomineralization occurred in the pilot plant or in laboratory cultures. The lack of encrustation of bacterial cells in the pilot plant is considered advantageous since the cells are still able to get in contact with ferrous iron and the iron oxidation process in the mine water treatment plant can proceed.
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Gulosibacter molinativorax ON4T molinate hydrolase, a novel cobalt-dependent amidohydrolase.
J. Bacteriol.
PUBLISHED: 08-12-2011
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A new pathway of molinate mineralization has recently been described. Among the five members of the mixed culture able to promote such a process, Gulosibacter molinativorax ON4(T) has been observed to promote the initial breakdown of the herbicide into ethanethiol and azepane-1-carboxylate. In the current study, the gene encoding the enzyme responsible for molinate hydrolysis was identified and heterologously expressed, and the resultant active protein was purified and characterized. Nucleotide sequence analysis revealed that the gene encodes a 465-amino-acid protein of the metal-dependent hydrolase A subfamily of the amidohydrolase superfamily with a predicted molecular mass of 50.9 kDa. Molinate hydrolase shares the highest amino acid sequence identity (48 to 50%) with phenylurea hydrolases of Arthrobacter globiformis and Mycobacterium brisbanense. However, in contrast to previously described members of the metal-dependent hydrolase A subfamily, molinate hydrolase contains cobalt as the only active-site metal.
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An angiotensin I-converting enzyme mutation (Y465D) causes a dramatic increase in blood ACE via accelerated ACE shedding.
PLoS ONE
PUBLISHED: 05-12-2011
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Angiotensin I-converting enzyme (ACE) metabolizes a range of peptidic substrates and plays a key role in blood pressure regulation and vascular remodeling. Thus, elevated ACE levels may be associated with an increased risk for different cardiovascular or respiratory diseases. Previously, a striking familial elevation in blood ACE was explained by mutations in the ACE juxtamembrane region that enhanced the cleavage-secretion process. Recently, we found a family whose affected members had a 6-fold increase in blood ACE and a Tyr465Asp (Y465D) substitution, distal to the stalk region, in the N domain of ACE.
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Nanoscale copper sulfide hollow spheres with phase-engineered composition: covellite (CuS), digenite (Cu1.8S), chalcocite (Cu2S).
Nanoscale
PUBLISHED: 05-09-2011
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Covellite (CuS), digenite (Cu(1.8)S) and chalcocite (Cu(2)S) are prepared as nanoscaled hollow spheres by reaction at the liquid-to-liquid phase boundary of a w/o-microemulsion. According to electron microscopy (SEM, STEM, TEM, HRTEM) the hollow spheres exhibit an outer diameter of 32-36 nm, a wall thickness of 8-12 nm and an inner cavity of 8-16 nm in diameter. The phase composition is determined based on HRTEM, electron-energy loss spectroscopy, X-ray powder diffraction and thermal analysis. In face of the advanced morphology of the hollow spheres, precise control of its phase composition is nevertheless possible by adjusting the experimental conditions (i.e. type and concentration of the copper precursor, concentration of ammonia inside of the micelle). Such phase-engineering of nanoscale hollow spheres is firstly observed and might allow adjusting even further compositions/structures as well as tailoring of phase-specific properties in the future.
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Transcriptional profiling of the marine oil-degrading bacterium Alcanivorax borkumensis during growth on n-alkanes.
FEMS Microbiol. Lett.
PUBLISHED: 04-20-2011
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The marine oil-degrading bacterium Alcanivorax borkumensis SK2 has attracted significant interest due to its hydrocarbonoclastic lifestyle, its alkane-centered metabolism, and for playing an important ecological role in cleaning up marine oil spills. In this study, we used microarray technology to characterize the transcriptional responses of A. borkumensis to n-hexadecane exposure as opposed to pyruvate, which led to the identification of a total of 220 differentially expressed genes, with 109 genes being upregulated and 111 genes being downregulated. Among the genes upregulated on alkanes are systems predicted to be involved in the terminal oxidation of alkanes, biofilm formation, signal transduction, and regulation.
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Virus-like particle production with yeast: ultrastructural and immunocytochemical insights into Pichia pastoris producing high levels of the hepatitis B surface antigen.
Microb. Cell Fact.
PUBLISHED: 03-18-2011
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A protective immune response against Hepatitis B infection can be obtained through the administration of a single viral polypeptide, the Hepatitis B surface antigen (HBsAg). Thus, the Hepatitis B vaccine is generated through the utilization of recombinant DNA technology, preferentially by using yeast-based expression systems. However, the polypeptide needs to assemble into spherical particles, so-called virus-like particles (VLPs), to elicit the required protective immune response. So far, no clear evidence has been presented showing whether HBsAg assembles in vivo inside the yeast cell into VLPs or later in vitro during down-stream processing and purification.
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Porcine pulmonary angiotensin I-converting enzyme--biochemical characterization and spatial arrangement of the N- and C-domains by three-dimensional electron microscopic reconstruction.
Micron
PUBLISHED: 03-17-2010
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The somatic angiotensin I-converting enzyme (sACE; peptidyl-dipeptidase A; EC 3.4.15.1) was isolated from pig lung and purified to homogeneity. The purified enzyme has a molecular mass of about 180 kDa. Upon proteolytic cleavage, two approximately 90 kDa fragments were obtained and identified by amino-terminal sequence analysis as the N- and C-domains of sACE. Both purified domains were shown to be catalytically active. A 2.3 nm resolution model of sACE was obtained by three-dimensional electron microscopic reconstruction of negatively stained sACE particles, based on atomic X-ray data fitting. Our model shows for the first time the relative orientation of the sACE catalytically active domains and their spatial distance.
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Characteristics of biofilms from urinary tract catheters and presence of biofilm-related components in Escherichia coli.
Curr. Microbiol.
PUBLISHED: 09-21-2009
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Long term catheterization of the urinary tract leads to bacterial colonization of the urine, whereby adherence to the catheter surface is a major determinative factor for colonization. Collection of bacterial isolates from urine and urinary catheters of 45 patients showed multi-species catheter-colonization, while Escherichia coli isolates were frequently found in the urine in high numbers. Biofilm formation of catheter and urine-derived E. coli isolates was associated with the presence of the fluA gene, loss of O-antigen, and expression of type 1 fimbriae. The second messenger cyclic di-GMP (cdiGMP), a major regulator of biofilm formation, regulated adherence to the catheter surface in a selected clinical isolate suggesting that the cdiGMP second messenger pathway may be a target for anti-biofilm therapeutic approaches.
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Polynucleobacter cosmopolitanus sp. nov., free-living planktonic bacteria inhabiting freshwater lakes and rivers.
Int. J. Syst. Evol. Microbiol.
PUBLISHED: 07-31-2009
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Five heterotrophic, aerobic, catalase- and oxidase-positive, non-motile strains were characterized from freshwater habitats located in Austria, France, Uganda, P. R. China and New Zealand. The strains shared 16S rRNA gene similarities of >/=99.3 %. The novel strains grew on NSY medium over a temperature range of 10-35 degrees C (two strains also grew at 5 degrees C and one strain grew at 38 degrees C) and a NaCl tolerance range of 0.0-0.3 % (four strains grew up to 0.5 % NaCl). The predominant fatty acids were C(16 : 0), C(18 : 1)omega7c, C(12 : 0) 3-OH, and summed feature 3 (including C(16 : 1)omega7c). The DNA G+C content of strain MWH-MoIso2(T) was 44.9 mol%. Phylogenetic analysis of 16S rRNA gene sequences demonstrated that the five new strains formed a monophyletic cluster closely related to Polynucleobacter necessarius (96-97 % sequence similarity). This cluster also harboured other isolates as well as environmental sequences which have been obtained from several habitats. Investigations with taxon-specific FISH probes demonstrated that the novel bacteria dwell as free-living, planktonic cells in freshwater systems. Based on the revealed phylogeny and pronounced chemotaxonomic differences to P. necessarius (presence of >7 % C(12 : 0) 3-OH and absence of C(12 : 0) and C(12 : 0) 2-OH), the new strains are suggested to represent a novel species, for which the name Polynucleobacter cosmopolitanus sp. nov. is proposed. The type strain is MWH-MoIso2(T) (=DSM 21490(T)=CIP 109840(T)=LMG 25212(T)). The novel species belongs to the minority of described species of free-living bacteria for which both in situ data from their natural environments and culture-based knowledge are available.
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Acidiplasma aeolicum gen. nov., sp. nov., a euryarchaeon of the family Ferroplasmaceae isolated from a hydrothermal pool, and transfer of Ferroplasma cupricumulans to Acidiplasma cupricumulans comb. nov.
Int. J. Syst. Evol. Microbiol.
PUBLISHED: 07-23-2009
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A novel acidophilic, cell-wall-less archaeon, strain V(T), was isolated from a hydrothermal pool on Vulcano Island, Italy. The morphology of cells was observed to vary from pleomorphic to coccoid. The temperature range for growth of strain V(T) was 15-65 degrees C with an optimum at 45 degrees C. The pH for growth ranged from pH 0 to 4 with an optimal at pH 1.4-1.6. Strain V(T) was able to grow aerobically and anaerobically, oxidizing ferrous iron and reducing ferric iron, respectively. The isolate grew chemo-organotrophically with yeast extract and yeast extract with glucose as the sources of energy and carbon. The molar G+C content in the DNA was 36 mol%. 16S rRNA gene sequence analysis demonstrated that strain V(T) was a member of the family Ferroplasmaceae, order Thermoplasmatales, phylum Euryarchaeota, showing sequence identities of 100 % with Ferroplasma cupricumulans BH2(T), 95.4 % with Ferroplasma acidiphilum Y(T), 94 % with Picrophilus torridus DSM 9790(T) and 92 % with Picrophilus oshimae DSM 9789(T). 16S rRNA gene sequence-based phylogenetic analysis showed that strain V(T) formed a monophyletic cluster together with F. cupricumulans BH2(T) and all other thermophilic isolates with available 16S rRNA gene sequences, whereas F. acidiphilum Y(T) formed another cluster with mesophilic isolates within the family Ferroplasmaceae. DNA-DNA hybridization values between strain V(T) and F. cupricumulans BH2(T) were well below 70 %, indicating that the two strains belong to separate species. Principal membrane lipids of strain V(T) were dibiphytanyl-based tetraether lipids containing pentacyclic rings. The polar lipids were dominated by a single phosphoglycolipid derivative based on a galactosyl dibiphytanyl phosphoglycerol tetraether, together with smaller amounts of monoglycosyl and diglycosyl dibiphytanyl ether lipids and the corresponding phosphoglycerol derivatives. The major respiratory quinones present were naphthoquinone derivatives. Given the notable physiological and chemical differences as well as the distinct phylogenetic placement of the new isolate relative to the type species of the genus Ferroplasma, we propose strain V(T) as a member of a new genus and species, Acidiplasma aeolicum gen. nov., sp. nov. The type strain of Acidiplasma aeolicum is strain V(T) (=DSM 18409(T) =JCM 14615(T)). In addition, we propose to transfer Ferroplasma cupricumulans Hawkes et al. 2008 to the genus Acidiplasma as Acidiplasma cupricumulans comb. nov. (type strain BH2(T) =DSM 16551(T) =JCM 13668(T)).
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Gordonibacter pamelaeae gen. nov., sp. nov., a new member of the Coriobacteriaceae isolated from a patient with Crohns disease, and reclassification of Eggerthella hongkongensis Lau et al. 2006 as Paraeggerthella hongkongensis gen. nov., comb. nov.
Int. J. Syst. Evol. Microbiol.
PUBLISHED: 06-09-2009
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A strictly anaerobic, Gram-positive, short-rod/coccobacillus-shaped bacterial strain, designated 7-10-1-b(T), was isolated from the colon of a patient suffering from acute Crohns disease. The isolate formed small, pale-white, semi-translucent colonies on solid cultivation media. The strain was catalase-positive and metabolized only a small number of carbon sources. Whole-cell fatty acids consisted predominantly of saturated fatty acids (89 %), of which 15 : 0 anteiso was the major component. The polar lipids phosphatidylglycerol and diphosphatidylglycerol as well as four glycolipids were identified. 16S rRNA gene sequence analysis revealed that the isolate represents a distinct lineage within the family Coriobacteriaceae and has 94.6 % identity to the type strain of [Eggerthella] hongkongensis, the phylogenetically closest bacterial species. On the basis of the analyses performed, the new genus and species Gordonibacter pamelaeae gen. nov., sp. nov. is described, with strain 7-10-1-b(T) (=DSM 19378(T) =CCUG 55131(T)) as the type and only strain of Gordonibacter pamelaeae. Also, based on the chemotaxonomic data obtained for all type strains of the neighbouring genus Eggerthella, we propose that Eggerthella hongkongensis Lau et al. 2006 be transferred to a new genus as Paraeggerthella hongkongensis gen. nov., comb. nov.; the type strain of Paraeggerthella hongkongensis is HKU10(T) (=DSM 16106(T) =CCUG 49250(T)).
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The photosynthetic apparatus and its regulation in the aerobic gammaproteobacterium Congregibacter litoralis gen. nov., sp. nov.
PLoS ONE
PUBLISHED: 02-16-2009
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There is accumulating evidence that in some marine environments aerobic bacteriochlorophyll a-producing bacteria represent a significant part of the microbial population. The interaction of photosynthesis and carbon metabolism in these interesting bacteria is still largely unknown and requires further investigation in order to estimate their contribution to the marine carbon cycle.
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Simple high-cell density fed-batch technique for high-level recombinant protein production with Pichia pastoris: Application to intracellular production of Hepatitis B surface antigen.
Microb. Cell Fact.
PUBLISHED: 02-10-2009
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Hepatitis B is a serious global public health concern. Though a safe and efficacious recombinant vaccine is available, its use in several resource-poor countries is limited by cost. We have investigated the production of Hepatitis B virus surface antigen (HBsAg) using the yeast Pichia pastoris GS115 by inserting the HBsAg gene into the alcohol oxidase 1 locus.
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Cauliform bacteria lacking phospholipids from an abyssal hydrothermal vent: proposal of Glycocaulis abyssi gen. nov., sp. nov., belonging to the family Hyphomonadaceae.
Int. J. Syst. Evol. Microbiol.
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Cauliform bacteria are prosthecate bacteria often specialized for oligotrophic environments. A polyphasic approach, comprising 16S rRNA gene sequencing, lipid analysis and salt tolerance characterizations, was used to clarify the taxonomy of one isolate, strain MCS 33(T), obtained from above the hot water plume of a deep-sea hydrothermal vent near Vancouver island, Canada. Cells contained no detectable phospholipids or sulpholipids, but did contain 1,2-di-O-acyl-3-O-?-D-glucopyranosylglycerol, 1,2-di-O-acyl-3-O-?-D-glucopyranuronosylglycerol and the novel lipid 1,2-di-O-acyl-3-[O-?-D-glucopyranuronosyl]glycerol-6-N-glycine. It is assumed that the various glucoronosyl lipids are replacing, at least partially, the phospholipids in their various tasks in the cell cycle. The G+C content of the genomic DNA of strain MCS 33(T) was 62.8 mol%, and Q10 was the predominant respiratory ubiquinone. The 16S rRNA gene sequence of this chemoheterotrophic, aerobic, moderately halophilic strain showed only a low similarity of 94.4% to that of Oceanicaulis alexandrii C116-18(T), and both strains also differed based on their lipids. Although the novel strain was isolated from seawater sampled near a hydrothermal vent, its optimum temperature for growth was 30 °C. The main cellular fatty acids were C18:1?7c, C18:0 and the unknown fatty acid ECL 11.798, and the main hydroxy fatty acid was C12:0 3-OH. The strain is proposed to represent a novel species of a new genus, Glycocaulis abyssi gen. nov., sp. nov. The type strain of the type species is MCS 33(T) (=LMG 27140(T)=CCUG 62981(T)).
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Physiological response of Pichia pastoris GS115 to methanol-induced high level production of the Hepatitis B surface antigen: catabolic adaptation, stress responses, and autophagic processes.
Microb. Cell Fact.
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Pichia pastoris is an established eukaryotic host for the production of recombinant proteins. Most often, protein production is under the control of the strong methanol-inducible aox1 promoter. However, detailed information about the physiological alterations in P. pastoris accompanying the shift from growth on glycerol to methanol-induced protein production under industrial relevant conditions is missing. Here, we provide an analysis of the physiological response of P. pastoris GS115 to methanol-induced high-level production of the Hepatitis B virus surface antigen (HBsAg). High product titers and the retention of the protein in the endoplasmic reticulum (ER) are supposedly of major impact on the host physiology. For a more detailed understanding of the cellular response to methanol-induced HBsAg production, the time-dependent changes in the yeast proteome and ultrastructural cell morphology were analyzed during the production process.
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The Tat system for membrane translocation of folded proteins recruits the membrane-stabilizing Psp machinery in Escherichia coli.
J. Biol. Chem.
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Tat systems transport folded proteins across energized membranes of bacteria, archaea, and plant plastids. In Escherichia coli, TatBC complexes recognize the transported proteins, and TatA complexes are recruited to facilitate transport. We achieved an abstraction of TatA from membranes without use of detergents and observed a co-purification of PspA, a membrane-stress response protein. The N-terminal transmembrane domain of TatA was required for the interaction. Electron microscopy displayed TatA complexes in direct contact with PspA. PspB and PspC were important for the TatA-PspA contact. The activator protein PspF was not involved in the PspA-TatA interaction, demonstrating that basal levels of PspA already interact with TatA. Elevated TatA levels caused membrane stress that induced a strictly PspBC- and PspF-dependent up-regulation of PspA. TatA complexes were found to destabilize membranes under these conditions. At native TatA levels, PspA deficiency clearly affected anaerobic TMAO respiratory growth, suggesting that energetic costs for transport of large Tat substrates such as TMAO reductase can become growth limiting in the absence of PspA. The physiological role of PspA recruitment to TatA may therefore be the control of membrane stress at active translocons.
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Cyanoethylation of the glucans dextran and pullulan: Substitution pattern and formation of nanostructures and entrapment of magnetic nanoparticles.
Beilstein J Org Chem
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Cyanoethylglucans with a degree of substitution in the range of 0.74 to 2.40 for dextran and 0.84 to 2.42 for pullulan were obtained by Michael addition of acrylonitrile to the glucans under various conditions. Products were thoroughly characterized, comprising elementary analysis, NMR and ATR-IR spectroscopy, and analysis of the substituent distribution in the glucosyl units by GC-FID and GC-MS of the constituting monosaccharide derivatives. Nanostructuring of the highly substituted cyanoethylpolysaccharides was performed by dialysis against a non-solvent. In the presence of ferromagnetic iron-oxide nanoparticles, multicore cyanoethylglucan-coated ferromagnetic nanoparticles were formed by selective entrapment. The specific interaction between cyano groups and iron could be proven. The size distribution and morphology of the nanoparticles were analyzed by dynamic light scattering (DLS), scanning electron microscopy (SEM) and energy-filtered transmission electron microscopy (EF-TEM) with parallel electron energy loss spectroscopy (PEELS).
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