Prolonged stochastic single ion channel recordings in S-layer protein stabilized lipid bilayer membranes.

S-layer proteins are commonly found in bacteria and archaea as two-dimensional monomolecular crystalline arrays as the outermost cell membrane component. These proteins have the unique property that following disruption by chemical agents, monomers of the protein can re-assemble to their original lattice structure. This unique property makes S-layers interesting for utilization in bio-nanotechnological applications. Here, we show that the addition of S-layer proteins to bilayer lipid membranes increases the lifetime and the stability of the bilayer. M2delta ion channels were functionally incorporated into these S-layer stabilized membranes and we were able to record their activity for up to 20 h. Transmission electron microscopy (TEM) was used to visualize the 2D crystalline pattern of the S-layer and the M2delta ion channel characteristics in bilayer lipid membrane's were compared in the presence and absence of S-layers.

Neuro-genetic optimization of temperature control for a continuous flow polymerase chain reaction microdevice.

In this study, a hybridized neuro-genetic optimization methodology realized by embedding finite element analysis (FEA) trained artificial neural networks (ANN) into genetic algorithms (GA), is used to optimize temperature control in a ceramic based continuous flow polymerase chain reaction (CPCR) device. The CPCR device requires three thermally isolated reaction zones of 94 degrees C, 65 degrees C, and 72 degrees C for the denaturing, annealing, and extension processes, respectively, to complete a cycle of polymerase chain reaction. The most important aspect of temperature control in the CPCR is to maintain temperature distribution at each reaction zone with a precision of +/-1 degree C or better, irrespective of changing ambient conditions. Results obtained from the FEA simulation shows good comparison with published experimental work for the temperature control in each reaction zone of the microfluidic channels. The simulation data are then used to train the ANN to predict the temperature distribution of the microfluidic channel for various heater input power and fluid flow rate. Once trained, the ANN analysis is able to predict the temperature distribution in the microchannel in less than 20 min, whereas the FEA simulation takes approximately 7 h to do so. The final optimization of temperature control in the CPCR device is achieved by embedding the trained ANN results as a fitness function into GA. Finally, the GA optimized results are used to build a new FEA model for numerical simulation analysis. The simulation results for the neuro-genetic optimized CPCR model and the initial CPCR model are then compared. The neuro-genetic optimized model shows a significant improvement from the initial model, establishing the optimization method's superiority.

Electron transfer to nitrite reductase of Rhodobacter sphaeroides 2.4.3: examination of cytochromes c2 and cY.

The role of cytochrome c(2), encoded by cycA, and cytochrome c(Y), encoded by cycY, in electron transfer to the nitrite reductase of Rhodobacter sphaeroides 2.4.3 was investigated using both in vivo and in vitro approaches. Both cycA and cycY were isolated, sequenced and insertionally inactivated in strain 2.4.3. Deletion of either gene alone had no apparent effect on the ability of R. sphaeroides to reduce nitrite. In a cycA-cycY double mutant, nitrite reduction was largely inhibited. However, the expression of the nitrite reductase gene nirK from a heterologous promoter substantially restored nitrite reductase activity in the double mutant. Using purified protein, a turnover number of 5 s(-1) was observed for the oxidation of cytochrome c(2) by nitrite reductase. In contrast, oxidation of c(Y) only resulted in a turnover of approximately 0.1 s(-1). The turnover experiments indicate that c(2) is a major electron donor to nitrite reductase but c(Y) is probably not. Taken together, these results suggest that there is likely an unidentified electron donor, in addition to c(2), that transfers electrons to nitrite reductase, and that the decreased nitrite reductase activity observed in the cycA-cycY double mutant probably results from a change in nirK expression.

Site-directed mutagenesis of NnrR: a transcriptional regulator of nitrite and nitric oxide reductase in Rhodobacter sphaeroides.

NnrR, a transcriptional activator and member of the CRP/FNR family of regulators, is responsible for controlling the expression of a number of denitrification genes in Rhodobacter sphaeroides 2.4.3. The apparent effector for NnrR is nitric oxide, and in its presence NnrR activates expression of the nirK gene and the nor operon, encoding nitrite reductase and nitric oxide reductase, respectively. Whether nitric oxide directly interacts with NnrR to activate transcription is unknown. Other denitrifiers carry putative orthologs of NnrR. To gain insight into NnrR function, a number of conserved residues were mutagenized. The impact of these changes on NnrR function was assessed by monitoring expression of a nirK-lacZ fusion. In this way a region spanning from Tyr93 to Cys103 that contains residues critical for NnrR activity was identified.

Use of a green fluorescent protein-based reporter fusion for detection of nitric oxide produced by denitrifiers.

To determine if green fluorescent protein could be used as a reporter for detecting nitric oxide production, gfp was fused to nnrS from Rhodobacter sphaeroides 2.4.3. nnrS was chosen because its expression requires nitric oxide. The presence of the fusion in R. sphaeroides 2.4.3 resulted in a significant increase in fluorescent intensity of the cells, but only when nitrite reductase was active. Cells lacking nitrite reductase activity and consequently the ability to generate nitric oxide were only weakly fluorescent when grown under denitrification-inducing conditions. One of the R. sphaeroides strains unable to generate nitric oxide endogenously was used as a reporter to detect exogenously produced nitric oxide. Incubation of this strain with sodium nitroprusside, a nitric oxide generator, significantly increased its fluorescence intensity. Mixing of known denitrifiers with the reporter strain also led to significant increases in fluorescence intensity, although the level varied depending on the denitrifier used. The reporter was tested on unknown isolates capable of growing anaerobically in the presence of nitrate, and one of these was able to induce expression of the fusion. Analysis of the 16S rRNA gene sequence of this isolate placed it within the Thauera aromatica subgroup, which is known to contain denitrifiers. These experiments demonstrate that this green fluorescent protein-based assay provides a useful method for assessing the ability of bacteria to produce nitric oxide.

Involvement of the PrrB/PrrA two-component system in nitrite respiration in Rhodobacter sphaeroides 2.4.3: evidence for transcriptional regulation.

Rhodobacter sphaeroides strain 2.4.3 is capable of diverse metabolic lifestyles, including denitrification. The regulation of many Rhodobacter genes involved in redox processes is controlled, in part, by the PrrBA two-component sensor-regulator system, where PrrB serves as the sensor kinase and PrrA is the response regulator. Four strains of 2.4.3 carrying mutations within the prrB gene were isolated in a screen for mutants unable to grow anaerobically on medium containing nitrite. Studies revealed that the expression of nirK, the structural gene encoding nitrite reductase, in these strains was significantly decreased compared to its expression in 2.4.3. Disruption of prrA also eliminated the ability to grow both photosynthetically and anaerobically in the dark on nitrite-amended medium. Complementation with prrA restored the wild-type phenotype. The PrrA strain exhibited a severe decrease in both nitrite reductase activity and expression of a nirK-lacZ fusion. Nitrite reductase activity in the PrrA strain could be restored to wild-type levels by using nirK expressed from a heterologous promoter, suggesting that the loss of nitrite reductase activity in the PrrA and PrrB mutants was not due to problems with enzyme assembly or the supply of reductant. Inactivation of prrA had no effect on the expression of the gene encoding NnrR, a transcriptional activator required for the expression of nirK. Inactivation of ccoN, part of the cbb(3)-type cytochrome oxidase shown to regulate the kinase activity of PrrB, also caused a significant decrease in both nirK expression and Nir activity. This was unexpected, since PrrA-P accumulates in the ccoN strain. Together, these results demonstrate that PrrBA plays an essential role in the regulation of nirK.