Cel5I, a SLH-Containing Glycoside Hydrolase: Characterization and Investigation on Its Role in Ruminiclostridium cellulolyticum.

Ruminiclostridium cellulolyticum (Clostridium cellulolyticum) is a mesophilic cellulolytic anaerobic bacterium that produces a multi-enzymatic system composed of cellulosomes and non-cellulosomal enzymes to degrade plant cell wall polysaccharides. We characterized one of the non-cellulosomal enzymes, Cel5I, composed of a Family-5 Glycoside Hydrolase catalytic module (GH5), a tandem of Family-17 and -28 Carbohydrate Binding Modules (CBM), and three S-layer homologous (SLH) modules, where the latter are expected to anchor the protein on the cell surface. Cel5I is the only putative endoglucanase targeting the cell surface as well as the only putative protein in R. cellulolyticum containing CBM17 and/or CBM28 modules. We characterized different recombinant structural variants from Cel5I. We showed that Cel5I has an affinity for insoluble cellulosic substrates through its CBMs, that it is the most active endoglucanase on crystalline cellulose of R. cellulolyticum characterized to date and mostly localized in the cell envelope of R. cellulolyticum. Its role in vivo was analyzed using a R. cellulolyticum cel5I mutant strain. Absence of Cel5I in the cell envelope did not lead to a significant variation of the phenotype compared to the wild type strain. Neither in terms of cell binding to cellulose, nor for its growth on crystalline cellulose, thus indicating that the protein has a rather subtle role in tested conditions. Cel5I might be more important in a natural environment, at low concentration of degradable glucose polymers, where its role might be to generate higher concentration of short cellodextrins close to the cell surface, facilitating their uptake or for signalization purpose.

Are cellulosome scaffolding protein CipC and CBM3-containing protein HycP, involved in adherence of Clostridium cellulolyticum to cellulose?

Clostridium cellulolyticum, a mesophilic anaerobic bacterium, produces highly active enzymatic complexes called cellulosomes. This strain was already shown to bind to cellulose, however the molecular mechanism(s) involved is not known. In this context we focused on the gene named hycP, encoding a 250-kDa protein of unknown function, containing a Family-3 Carbohydrate Binding Module (CBM3) along with 23 hyaline repeat modules (HYR modules). In the microbial kingdom the gene hycP is only found in C. cellulolyticum and the very close strain recently sequenced Clostridium sp BNL1100. Its presence in C. cellulolyticum guided us to analyze its function and its putative role in adhesion of the cells to cellulose. The CBM3 of HycP was shown to bind to crystalline cellulose and was assigned to the CBM3b subfamily. No hydrolytic activity on cellulose was found with a mini-protein displaying representative domains of HycP. A C. cellulolyticum inactivated hycP mutant strain was constructed, and we found that HycP is neither involved in binding of the cells to cellulose nor that the protein has an obvious role in cell growth on cellulose. We also characterized the role of the cellulosome scaffolding protein CipC in adhesion of C. cellulolyticum to cellulose, since cellulosome scaffolding protein has been proposed to mediate binding of other cellulolytic bacteria to cellulose. A second mutant was constructed, where cipC was inactivated. We unexpectedly found that CipC is only partly involved in binding of C. cellulolyticum to cellulose. Other mechanisms for cellulose adhesion may therefore exist in C. cellulolyticum. In addition, no cellulosomal protuberances were observed at the cellular surface of C. cellulolyticum, what is in contrast to reports from several other cellulosomes producing strains. These findings may suggest that C. cellulolyticum has no dedicated molecular mechanism to aggregate the cellulosomes at the cellular surface.

Thermal wavelength stabilization of Bragg gratings photowritten in hole-filled microstructured optical fibers.

We demonstrate that the resonance wavelength of fiber Bragg gratings photowritten in the core of microstructured optical fibers can be efficiently stabilized versus temperature by inserting suitable refractive index materials with a negative thermal sensitivity into the holes. By these means, the effective index of the guided mode undergoes thermal variations which counterbalance the effect of the grating period thermal drift. The residual excursion of the resonance wavelength can be limited to less than +/- 10 pm over a 70 degrees C range of temperature into Microstructured Optical Fibers (MOFs) having realistic geometrical parameters, and using existing refractive index materials. Low cost passively stabilized reflectors with insertion loss lower than 0.3 dB can be realized by splicing single mode fibers at both ends of a short length of a filled MOF including the fiber Bragg grating.

Biofunctionalized tilted Fiber Bragg Gratings for label-free immunosensing.

We present a study aimed at developing a label-free optical fiber biosensor for detection and quantification of biomolecules in real-time. The biosensor based on a Tilted Fiber Bragg Grating (TFBG) transduces a binding event between the probe and target molecules into a change in the refractive index of the medium surrounding the fiber. This work describes the experimental results obtained with three methods for immobilizing biomolecular probes on a TFBG silica cladding surface. Bovine serum albumin (BSA) and anti-BSA are used to assess the performances of the TFBG based biosensor in each configuration.

Three-hole microstructured optical fiber for efficient fiber Bragg grating refractometer.

We present a photosensitive three-hole microstructured optical fiber specifically designed to improve the refractive index sensitivity of a standard fiber Bragg grating (FBG) sensor photowritten in the suspended Ge-doped silica core. We describe the specific photowriting procedure used to realize gratings in such a fiber. We then determine their spectral sensitivity to the refractive index changes of material filling the holes surrounding the core. The sensitivity is compared with that of standard FBGs photowritten in a six-hole fiber with a larger core diameter. We demonstrate an improvement in the sensitivity by two orders of magnitude and reach a resolution of 3 x 10(-5) and 6 x 10(-6) around mean refractive index values of 1.33 and 1.40, respectively.

Tilted Fiber Bragg Grating photowritten in microstructured optical fiber for improved refractive index measurement.

We report what we believe to be the first Tilted short-period Fiber Bragg Grating photowritten in a microstructured optical fiber for refractive index measurement. We investigate the spectral sensitivity of Tilted Fiber Bragg Grating to refractive index liquid inserted into the holes of a multimode microstructured fiber. We measure the wavelength shift of the first four modes experimentally observed when calibrated oils are inserted into the fiber holes, and thus we determine the refractive index resolution for each of these modes. Moreover, a cross comparison between experimental and simulation results of a modal analysis is performed. Two simulation tools are used, respectively based on the localized functions method and on a finite element method. All results are in very good agreement.

Apodized fiber Bragg gratings manufactured with the phase plate process.

We present a manufacturing method based on the dynamic use of phase plates to photowrite Bragg gratings. This process allows for control of the local value of the index modulation envelope in the grating. The application to apodized fiber Bragg gratings is discussed.