The NreABC system of Staphylococcus carnosus combines nitrate and oxygen sensing by an NreA,NreB sensor complex

Staphylococcus carnosus is a facultative anaerobic bacterium which features the cytoplasmic NreABC system. It is necessary for regulation of nitrate respiration and the nitrate reductase gene narG in response to oxygen and nitrate availability. NreB is a sensor kinase of a two-component system and represents the oxygen sensor of the system. It binds an oxygen labile [4Fe-4S]2+ cluster under anaerobic conditions. NreB autophosphorylates and phosphoryl transfer activates the response regulator NreC which induces narG expression. The third component of the Nre system is the nitrate receptor NreA. In this study the role of the nitrate receptor protein NreA in nitrate regulation and its functional and physiological effect on oxygen regulation and interaction with the NreBC two-component system were detected. In vivo, a reporter gene assay for measuring expression of the NreABC regulated nitrate reductase gene narG was used for quantitative evaluation of NreA function. Maximal narG expression in wild type S. carnosus required anaerobic conditions and the presence of nitrate. Deletion of nreA allowed expression of narG under aerobic conditions, and under anaerobic conditions nitrate was no longer required for maximal induction. This indicates that NreA is a nitrate regulated inhibitor of narG expression. Purified NreA and variant NreA(Y95A) inhibited the autophosphorylation of anaerobic NreB in part and completely, respectively. Neither NreA nor NreA(Y95A) stimulated dephosphorylation of NreB-phosphate, however. Inhibition of phosphorylation was relieved completely when NreA with bound nitrate (NreA•[NO3-]) was used. The same effects of NreA were monitored with aerobically isolated Fe-S-less NreB, which indicates that NreA does not have an influence on the iron-sulfur cluster of NreB. In summary, the data of this study show that NreA interacts with the oxygen sensor NreB and controls its phosphorylation level in a nitrate dependent manner. This modulation of NreB-function by NreA and nitrate results in nitrate/oxygen co-sensing by an NreA/NreB sensory unit. It transmits the regulatory signal from oxygen and nitrate in a joint signal to target promoters. Therefore, nitrate and oxygen regulation of nitrate dissimilation follows a new mode of regulation not present in other facultative anaerobic bacteria.

Signalerkennung und Signaltransduktion im DctA,DcuS-Sensor-Regulatorkomplex von Escherichia coli.

E. coli ist in der Lage unter aeroben sowie anaeroben Bedingungen C4-Dicarbonsäuren zur Energiekonservierung zu nutzen. Das DcuS/DcuR-Zweikomponentensystem detektiert diese und reguliert die Gene für den C4-Dicarboxylat-Transport und Metabolismus. Dabei hängt die Sensitivität der Sensorkinase DcuS für C4-Dicarbonsäuren von der Anwesenheit des aeroben Symporters DctA oder des anaeroben Antiporters DcuB ab. Diese bifunktionalen Transporter bilden mit DcuS über direkte Protein-Protein-Wechselwirkungen Sensoreinheiten. In dieser Arbeit wurden die Funktionen von DctA und DcuS im DctA/DcuS-Sensorkomplex analysiert. Mit DctA(S380D) wurde eine Variante des Transporters identifiziert, in der die regulatorische Eigenschaft von der katalytischen Funktion entkoppelt ist. Stämme von E. coli, die den DctA(S380D)/DcuS-Sensorkomplex enthielten, waren in der Lage C4-Dicarbonsäuren wahrzunehmen, obwohl die Transportfunktion von DctA inaktiviert war. Zudem wurden Unterschiede in den Substratspektren von DctA und DcuS festgestellt. Citrat, ein guter Effektor des DctA/DcuS-Sensorkomplexes, wurde durch DctA nicht gebunden oder transportiert. Anhand von Titrationsexperimenten mit variierenden DctA-Mengen wurde außerdem nachgewiesen, dass die Sensitivität von DcuS für seine Effektoren von der DctA-Konzentration abhängig ist. Es konnte gezeigt werden, dass DctA im DctA/DcuS-Sensorkomplex nicht an der Erkennung von C4-Dicarbonsäuren beteiligt ist. DcuS stellt die Signaleingangsstelle des Komplexes dar, während DctA durch seine Anwesenheit die Sensorkinase in eine funktionsbereite oder sensitive Form überführt, die auf Effektoren reagieren kann. Darüber hinaus wurde die Rolle der Transmembranhelices TM1 und TM2 von DcuS für die Funktion und Dimerisierung der Sensorkinase untersucht. Durch Sequenzanalysen wurden „SmallxxxSmall“-Motive, deren Relevanz als Dimerisierungsschnittstellen bereits in Transmembranhelices anderer Proteine nachgewiesen wurde, in TM1 sowie TM2 identifiziert. Die Homodimerisierung beider Transmembrandomänen wurde im GALLEX Two-Hybrid System nachgewiesen, wobei die TM2-TM2-Interaktion stärker war. Die Substitution G190A/G194A im SxxxGxxxG-Tandemmotiv von TM2 rief zudem einen deutlichen Funktionsverlust der Sensorkinase hervor. Dieser Aktivitätsverlust korrelierte mit Störungen der Homodimerisierung von TM2(G190A/G194A) sowie DcuS(G190A/G194A) bei bakteriellen Two-Hybrid Messungen im GALLEX- bzw. BACTH-System. Demzufolge agiert Transmembranhelix 2 mit seinem SxxxGxxxG-Sequenzmotiv als wesentliche Homodimerisierungsstelle in DcuS. Die Dimerisierung von DcuS ist essentiell für die Funktion der Histidinkinase. Zusätzlich wurde bei fluoreszenzmikroskopischen Studien durch Koexpression von DcuS bzw. DctA die zelluläre Kolokalisierung von DctA und DcuR mit DcuS sowie DauA mit DctA nachgewiesen. Die DctA/DcuS-Sensoreinheit kann demnach zum DauA/DctA/DcuS/DcuR-Komplex erweitert werden.

Signaltransduktion in der membranständigen Sensor-Histidinkinase DcuS von Escherichia coli

Escherichia coli kann unter aeroben und anaeroben Bedingungen mit C4-Dicarboxylaten wachsen, die Regulation des Stoffwechsels erfolgt durch das Zwei-Komponenten-System DcuSR. Die C4-Dicarboxylattransporter DctA (aerob) bzw. DcuB (anaerob) agieren als Co-Regulatoren und bilden gemeinsam mit der Sensor-Histidinkinase DcuS einen Sensorkomplex, in dem DcuS den Sensor darstellt und DctA bzw. DcuB diesen in seine rezeptive Form überführen. DcuS ist membranständig und verknüpft die Bindung von C4-Dicarboxylaten im Periplasma mit der Autophosphorylierung seiner Kinasedomäne im Cytoplasma. Dies stellt den Beginn einer Signalkaskade vom extrazellulären Reiz zum cytoplasmatischen Responseregulator DcuR dar.rnIn dieser Arbeit wurde die intramolekulare Signaltransduktion in DcuS und über die Membran untersucht. Der Fokus lag auf der Funktion der beiden Transmembranhelices TM1 und TM2 und der cytoplasmatischen PAS-Domäne, die die sensorische PASp- mit der effektorischen Kinasedomäne verbinden. Konformationsänderungen dieser Signalweiterleitung wurden durch Cysteinzugänglichkeitsstudien, oxidatives Cystein-Crosslinking und Mutageneseexperimente analysiert. rnTM2 wurde als der Überträger eines transmembranen Signals identifiziert, während TM1 als Membrananker fungiert. Der aktive Signalzustand von TM2 wird unabhängig von der Art der DcuS-Aktivierung (Effektorbindung, Deletion des Co-Regulators DctA oder PASc-ON-Mutationen) eingenommen. Der Signaltransduktion liegt eine Verschiebung von TM2 entlang ihrer Längsachse (Kolbenhub) in Richtung Periplasma zu Grunde. Cystein-Crosslinking offenbarte eine durchgehende Helix aus PASp-α6 und TM2, die im Dimer parallel mit ihrem Pendant verschoben wird. Die Amplitude des Kolbenhubs wurde anhand von Zugänglichkeitsveränderungen, der Lage verankernder Tryptophanreste, Strukturvergleichen und energetischen Berechnungen auf max. 4 - 6 Å festgelegt. Sie ist von der Effektorstärke abhängig und koppelt so die metabolische Bevorzugung einzelner Substrate an das Ausmaß des Kolbenhubs und der Genexpression. Für die cytoplasmatische PAS-Domäne wurde ein Zusammenhang zwischen lokaler Dimerisierung und Kontrolle der Sensorfunktion nachgewiesen. Schwächung der Dimerisierung führt zu einer Aktivierung der Sensorkinase. Es wurde eine hydrophobe Region identifiziert, deren strukturelle Integrität für diese Dimerisierung essentiell ist. Mit N248 wurde ein funktionell bedeutender Rest beschrieben, der auf Grund seiner Lage und seiner Eigenschaft mehrere Sekundärstrukturelemente zu verknüpfen, als Scharnier innerhalb der Domäne an der Umsetzung des Kolbenhubs in eine veränderte Quartärstruktur von PASc beteiligt sein könnte.

Application of active heat pulse method with fiber optic temperature sensing for estimation of wetting bulbs and water distribution in drip emitters.

Through the use of the Distributed Fiber Optic Temperature Measurement (DFOT) method, it is possible to measure the temperature in small intervals (on the order of centimeters) for long distances (on the order of kilometers) with a high temporal frequency and great accuracy. The heat pulse method consists of applying a known amount of heat to the soil and monitoring the temperature evolution, which is primarily dependent on the soil moisture content. The use of both methods, which is called the active heat pulse method with fiber optic temperature sensing (AHFO), allows accurate soil moisture content measurements. In order to experimentally study the wetting patterns, i.e. shape, size, and the water distribution, from a drip irrigation emitter, a soil column of 0.5 m of diameter and 0.6 m high was built. Inside the column, a fiber optic cable with a stainless steel sheath was placed forming three concentric helixes of diameters 0.2 m, 0.4 m and 0.6 m, leading to a 148 measurement point network. Before, during, and after the irrigation event, heat pulses were performed supplying electrical power of 20 W/m to the steel. The soil moisture content was measured with a capacitive sensor in one location at depths of 0.1 m, 0.2 m, 0.3 m and 0.4 m during the irrigation. It was also determined by the gravimetric method in several locations and depths before and right after the irrigation. The emitter bulb dimensions and shape evolution was satisfactorily measured during infiltration. Furthermore, some bulb's characteristics difficult to predict (e.g. preferential flow) were detected. The results point out that the AHFO is a useful tool to estimate the wetting pattern of drip irrigation emitters in soil columns and show a high potential for its use in the field.

Sensor magneto-óptico de corriente con supresión de birrefringencia lineal mediante espejo de Faraday

Fiber optic sensors have some advantages in subjects related with electrical current and magnetic field measurement. In spite of the optical fiber utilization advantages we have to take into account undesirable effects, which are present in real non-ideal optical fibers. In telecommunication and sensor application fields the presence of inherent and induced birefringence is crucial. The presence of birefringence may cause an undesirable change in the polarization state. In order to compensate the linear birefringence a promising method has been chosen. This method employs orthogonal polarization conjugation in the back propagation direction of the light wave in the fiber. A study and a simulation of an experimental setup are realized with the advantage of a significant sensitivity improvement.

Adaptive pulse shaping through BER Feedback

We propose a simplified approach to optical signal pre-distortion based on adaptive pulse shaping through unconventional use of a MZ modulator. The scheme allows natural tailoring of transmitted pulses by optimising the received pulse.

Modified hydrogel matrices in fibre optic sensors

Common problems encountered in clinical sensing are those of non-biocompatibility, and slow response time of the device. The latter, also applying to chemical sensors, is possibly due to a lack of understanding of polymer support or membrane properties and hence failure to optimise membranes chosen for specific sensor applications. Hydrogels can be described as polymers which swell in water. In addition to this, the presence of water in the polymer matrix offers some control of biocompatibility. They thus provide a medium through which rapid transport of a sensed species to an incorporated reagent could occur. This work considers the feasibility of such a system, leading to the design and construction of an optical sensor test bed. The development of suitable membrane systems and of suitable coating techniques in order to apply them to the fibre optics is described. Initial results obtained from hydrogel coatings implied that the refractive index change in the polymer matrix, due to a change in water content with pH is the major factor contributing to the sensor response. However the presence of the colourimetric reagent was also altering the output signal obtained. An analysis of factors contributing to the overall response, such as colour change and membrane composition were made on both the test bed, via optical response, and on whole membranes via measurement of water content change. The investigation of coatings with low equilibrium water contents, of less than 10% was carried out and in fact a clearer signal response from the test bed was noted. Again these membranes were suprisingly responding via refractive index change, with the reagent playing a primary role in obtaining a sensible or non-random response, although not in a colourimetric fashion. A photographic study of these coatings revealed some clues as to the physical nature of these coatings and hence partially explained this phenomenon. A study of the transport properties of the most successful membrane, on a coated wire electrode and also on the fibre optic test bed, in a series of test environments, indicated that the reagent was possibly acting as an ion exchanger and hence having a major influence on transport and therefore sensor characteristics.

Optic sensors of high refractive-index responsivity and low thermal cross sensitivity that use fiber Bragg gratings of >80° tilted structures

For the first time to the authors' knowledge, fiber Bragg gratings (FBGs) with >80° tilted structures have been fabricated and characterized. Their performance in sensing temperature, strain, and the surrounding medium's refractive index was investigated. In comparison with normal FBGs and long-period gratings (LPGs), >80° tilted FBGs exhibit significantly higher refractive-index responsivity and lower thermal cross sensitivity. When the grating sensor was used to detect changes in refractive index, a responsivity as high as 340nm/refractive-index unit near an index of 1.33 was demonstrated, which is three times higher than that of conventional LPGs.

Fibre optic sensors for high speed hypervelocity impact studies and low velocity drop tests

The initial aim of this project was to develop a non-contact fibre optic based displacement sensor to operate in the harsh environment of a 'Light Gas Gun' (LGG), which can 'fire' small particles at velocities ranging from 1-8.4 km/s. The LGG is used extensively for research in aerospace to analyze the effects of high speed impacts on materials. Ideally the measurement should be made close to the centre of the impact to minimise corruption of the data from edge effects and survive the impact. A further requirement is that it should operate at a stand-off distance of ~ 8cm. For these reasons we chose to develop a pseudo con-focal intensity sensor, which demonstrated resolution comparable with conventional PVDF sensors combined with high survivability and low cost. A second sensor was developed based on 'Fibre Bragg Gratings' (FBG) which although requiring contact with the target the low weight and very small contact area had minimal effect on the dynamics of the target. The FBG was mounted either on the surface of the target or tangentially between a fixed location. The output signals from the FBG were interrogated in time by a new method. Measurements were made on composite and aluminium plates in the LGG and on low speed drop tests. The particle momentum for the drop tests was chosen to be similar to that of the particles used in the LGG.

Fibre optics sensors in tear electrolyte analysis:towards a novel point of care potassium sensor

The diagnosis of ocular disease is increasingly important in optometric practice and there is a need for cost effective point of care assays to assist in that. Although tears are a potentially valuable source of diagnostic information difficulties associated with sample collection and limited sample size together with sample storage and transport have proved major limitations. Progressive developments in electronics and fibre optics together with innovation in sensing technology mean that the construction of inexpensive point of care fibre optic sensing devices is now possible. Tear electrolytes are an obvious family of target analytes, not least to complement the availability of devices that make the routine measurement of tear osmolarity possible in the clinic. In this paper we describe the design, fabrication and calibration of a fibre-optic based electrolyte sensor for the quantification of potassium in tears using the ex vivo contact lens as the sample source. The technology is generic and the same principles can be used in the development of calcium and magnesium sensors. An important objective of this sensor technology development is to provide information at the point of routine optometric examination, which would provide supportive evidence of tear abnormality.

An intrinsic biochemical concentration sensor using a polymer optical fibre Bragg grating

A new type of fibre-optic biochemical concentration sensor based on a polymer optical fibre Bragg grating (POFBG) is proposed. The wavelength of the POFBG varies as a function of analyte concentration. The feasibility of this sensing concept is demonstrated by a saline concentration sensor. When polymer fibre is placed in a water based solution the process of osmosis takes place in this water-fibre system. An osmotic pressure which is proportional to the solution concentration, will apply to the fibre in addition to the hydraulic pressure. It tends to drive the water content out of the fibre and into the surrounding solution. When the surrounding solution concentration increases the osmotic pressure increases to drive the water content out of the fibre, consequently increasing the differential hydraulic pressure and reducing the POFBG wavelength. This process will stop once there is a balance between the osmotic pressure and the differential hydraulic pressure. Similarly when the solution concentration decreases the osmotic pressure decreases, leading to a dominant differential hydraulic pressure which drives the water into the fibre till a new pressure balance is established. Therefore the water content in the polymer fibre - and consequently the POFBG wavelength - depends directly on the solution concentration. A POFBG wavelength change of 0.9 nm was measured for saline concentration varying from 0 to 22%. For a wavelength interrogation system with a resolution of 1 pm, a measurement of solution concentration of 0.03% can be expected.

Respiratory function monitoring using a real-time three-dimensional fiber-optic shaping sensing scheme based upon fiber Bragg gratings

An array of in-line curvature sensors on a garment is used to monitor the thoracic and abdominal movements of a human during respiration. The results are used to obtain volumetric changes of the human torso in agreement with a spirometer used simultaneously at the mouth. The array of 40 in-line fiber Bragg gratings is used to produce 20 curvature sensors at different locations, each sensor consisting of two fiber Bragg gratings. The 20 curvature sensors and adjoining fiber are encapsulated into a low-temperature-cured synthetic silicone. The sensors are wavelength interrogated by a commercially available system from Moog Insensys, and the wavelength changes are calibrated to recover curvature. A three-dimensional algorithm is used to generate shape changes during respiration that allow the measurement of absolute volume changes at various sections of the torso. It is shown that the sensing scheme yields a volumetric error of 6%. Comparing the volume data obtained from the spirometer with the volume estimated with the synchronous data from the shape-sensing array yielded a correlation value 0.86 with a Pearson's correlation coefficient p <0.01.

Free water in fuel sensor using fiber long period grating

A fiber optic free water in fuel (WIF) sensor is proposed by utilizing a long period fiber grating (LPFG). The existence of free water in fuel is indicated by the appearance of a characteristic loss band. The free water level in fuel can be determined by measuring the transmissions of two characteristic loss bands.

Acousto-optic modulation in a microstructured plastic optical fibre Bragg grating

The present work addresses the control of the mPOF Bragg grating spectrum properties through acousto-optic modulation. For the first time, the interaction of a flexural acoustic wave, generated by longitudinal excitation of different frequencies, with the Bragg grating will be presented. Also it will be demonstrated the quasi linear relationship between PZT load and maximum reflected power/ 3dB bandwidth of the reflected spectrum.

Adaptive pulse shaping through BER Feedback

We propose a simplified approach to optical signal pre-distortion based on adaptive pulse shaping through unconventional use of a MZ modulator. The scheme allows natural tailoring of transmitted pulses by optimising the received pulse.