Electrochemical Antigenic Sensor for the Diagnosis of Chronic Q Fever

In this work, we report the development of an impedimetric biosensor for the direct, quick, and easy diagnosis of chronic Q fever. The biosensor is based on highly sensitive antigens that can selectively recognize antibodies against Coxiella burnetii. The biosensor is based on the immobilization of antigens onto a gold electrode using the EDC/NHS immobilization methodology. The detection is performed by impedance spectroscopy that monitors specific frequencies which provide the maximum sensitivity for the biosensor. Q fever antibodies that are present in the sera of patients interact selectively with the biosensor antigens, thereby altering the impedance of the biosensor surfaceand generating a large impedance change within a few seconds. The biosensor allows for the specific serological detection of chronic Q fever, while the developed system can also be modified for the detection of other biomarkers, such as the ones against acute Q fever

Quantitative Proteome Profiling of C. burnetii under Tetracycline Stress Conditions

The recommended antibiotic regimen against Coxiella burnetii, the etiological agent of Q fever, is based on a semi-synthetic, second-generation tetracycline, doxycycline. Here, we report on the comparison of the proteomes of a C. burnetii reference strain either cultured under control conditions or under tetracycline stress conditions. Using the MS-driven combined fractional diagonal chromatography proteomics technique, out of the 531 proteins identified, 5 and 19 proteins were found significantly up- and down-regulated respectively, under tetracycline stress. Although the predicted cellular functions of these regulated proteins did not point to known tetracycline resistance mechanisms, our data clearly reveal the plasticity of the proteome of C. burnetii to battle tetracycline stress. Finally, we raise several plausible hypotheses that could further lead to more focused experiments on studying tetracycline resistance in C. burnetii and thus reduced treatment failures of Q fever

Complete Genome Sequence of Pseudomonas sp. Strain phDV1, an Isolate Capable of Efficient Degradation of Aromatic Hydrocarbons

Pseudomonas sp. strain phDV1 is a Gram-negative bacterium capable of degrading aromatic hydrocarbons. Here, we present the complete genome sequence of this strain, which consists of 4,727,682 bp, with a 62.3% G+C content and 4,574 genes. Multiple genes responsible for the degradation of aromatics are present in this strai

Isolation and structural characterization of trimeric cyanobacterial photosystem I complex with the help of recombinant antibody fragments

A monoclonal antibody was derived from mice immunized with the native trimeric, photosystem I (PSI) complex from the cyanobacterium Synechococcus PCC 7002 which reacts with a conformational epitope of the PSI complex. As seen by immunoelectron microscopy, the mAb bound to the stromal side of the thylakoid membranes. The DNA sequence encoding variable regions of the mAb was cloned into recombinant plasmids, sequenced and expressed in Escherichia coli. ELISA, Western blots and immunoelectron microscopy provided evidence that the expressed paired variable domain (Fv) fragments bind to the antigen in the same way as the parent mAb. A one-step purification was applied to purify the trimeric PSI complex using an affinity tag attached to the Fv fragment. Analysis by gel electrophoresis and N-terminal sequencing revealed the presence of the psaA, psaB, psaC, psaD, psaE, psaF and psaL gene products. The antenna size of the isolated PSI/Fv was 139 +/- 9 chlorophyll a/primary electron donor. Flash-induced absorption-change measurements showed that the complex exhibited electron transfer from the primary electron donor, P700, to the Fe-S center, FA/FB. The position of the bound Fv fragment on the trimeric PSI surface was determined by high-resolution electron microscopy and digital image processing

Purification and crystallization of Photosystem I complex from a phycobilisome-less mutant of the cyanobacterium Synechococcus PCC 7002

An active photosystem (PSI) complex was isolated from a phycobilisome-less mutant of the mesophilic cyanobacterium Synechococcus PCC 7002 by a mild procedure. Purification of PS I was achieved using a sucrose density gradient and an isoelectric focussing subsequent to the extraction of PSI from thylakoids with dodecyl-β-maltoside. Electron microscopy and gel filtration HPLC suggested that the isolated complex represents a trimeric form of PSI. The trimeric form was resistant to pH or detergent exchange. A ‘molecular weight’ of 690 kDa to 760 kDa has been determined for the complex by gel filtration HPLC in several detergents or mixtures of detergents. The PSI complex contains the polypeptides of the psaA, psaB, psaC, psaD, psaE, psaL gene products and two small polypeptides as determined by SDS-PAGE and N-terminal sequencing; its antenna size is 77±2 Chl a/P700. The full set of Fe-S clusters (FA, FB and FX) was observed by EPR-spectroscopy. A preliminary characterization of crystals obtained from this preparation was carried out using SDS-PAGE, optical and EPR spectroscopy

Proteomic analysis of chlorosome-depleted membranes of the green sulfur bacterium Chlorobium tepidum

Green sulfur bacteria are obligate anaerobic phototrophs, which in addition to outer and plasma membranes contain chlorosomes. The analysis of the membrane proteome of Chlorobium tepidum from chlorosome-depleted membranes is described in this study. The membranes were purified by sucrose density centrifugation and characterized by 1-DE and 2-DE coupled with MS, absorption spectroscopy, and electron microscopy. 1-DE and 2-DE were employed to analyze the membrane proteins and to characterize the capabilities of the methods. Solubilization of the membrane proteins prior to 2-DE was improved by using a series of zwitterionic detergents. Based on the resolved spots after 2-DE, the combination of amidosulfobetaine 14 with Triton X-100 is more efficient than the combination of CHAPS, N-decyl-N,N-dimethyl-3-ammonio-1-propane sulfonate, and Triton X-100. From the application of 1-DE and 2-DE, 167 and 202 unique proteins were identified, respectively, using PMF by MALDI-TOF MS. Both methods resulted in the detection of 291 different proteins of which only 88 were predicted membrane proteins, indicating the limitation of membrane protein detection after separation with electrophoresis methods. In addition, 53 of these proteins were identified as outer membrane protein

Highly ordered two-dimensional crystals of photosystem i reaction center from synechococcus sp - functional and structural analyses

The photosystem I reaction center complex from the thermophilic cyanobacterium Synechococcus sp. was isolated by Triton X-100 solubilization and fractional precipitation with polyethylene glycol. As shown by gel electrophoresis, the isolated complex was composed of the 83 kDa subunits A and B, and at least six other subunits with molecular mass below 20 kDa. Electron transfer from the primary electron donor P700 to the F-A/F-B centers was demonstrated by flash-induced absorption change of the isolated complex, while electron paramagnetic resonance (EPR) spectroscopy showed that the complex contained a full set of Fe-S clusters. Isolated complexes were reconstituted into two-dimensional crystals in the presence of phospholipids and different cations. The crystals were found to be active by flash-induced separation and EPR spectroscopy. Electron microscopy and digital image processing of negatively stained and frozen-hydrated specimens revealed orthorhombic crystals with unit cell dimensions a = 138 Angstrom, b = 145 Angstrom and p12(1) symmetry. A three-dimensional map was calculated for negatively stained crystals to 19 Angstrom resolution, whereas the projection map of frozen-hydrated crystals exhibited 8 Angstrom resolution. (C) 1996 Academic Press Limited [References: 41