SummaryThe cydAB operon of Escherichia coli encodes the cytochrome d oxidase complex, one of two aerobic terminal oxidases that catalyses the oxidation of ubiquinol-8 and the reduction of oxygen to water. This enzyme has a higher affinity for oxygen than the cyto-

SummaryThe Escherichia coli cydAB operon encodes the highaffinity terminal oxidase of the oxygen respiratory chain, cytochrome d oxidase. The sensor±regulator pair, ArcB±ArcA, is responsible for the microaerobic activation of the cydAB operon, whereas the anaerobic regulator Fnr represses its expression in the absence of oxygen. Fnr binds in vitro at two sites within the cydAB promoter element. To discern whether these two regions have an in vivo function in the anaerobic regulation of cydAB, the Fnr-binding motifs were mutagenized individually and in combination. The effects of these mutations on in vivo gene expression were determined by lac fusion and primer extension analysis. Our results show that the Fnr-2 site is critical for Fnr-mediated anaerobic repression of the two main cydAB promoters, P1 and P2. In contrast, the Fnr-1 site has an auxiliary role in the anaerobic repression of P1, but not of P2. Transcription from P1 did not affect ArcA-mediated activation or Fnr-mediated repression of P2, indicating that oxygen regulation is exerted on both promoters in an independent fashion. In addition, three new promoters were identified in the cydAB control region, and the 5 H ends of the corresponding transcripts were mapped. Two of these promoters, designated P3 and P4, are co-ordinately regulated with P1 and P2 in response to oxygen, ArcA and Fnr. The P5 promoter is not Fnr regulated and is only weakly activated by ArcA. The contribution of these three additional promoters to the overall cydAB expression is most relevant under aerobic conditions. Our results suggest a unique repression model, in which one Fnr dimer bound to one single site (Fnr-2) is sufficient to downregulate transcription from four cydAB promoters. In conclusion, transcription of the cydAB operon is driven by a complex regulatory element containing at least five promoters that act in unison to provide adequate oxygen control of gene expression.

Force-based atomic force microscopy (AFM) was used to detect HCV (hepatitis C virus) RNA directly and to quantitatively analyse it without the need for reverse transcription or amplification. Capture and detection DNA probes were designed. The former was spotted onto a substrate with a conventional microarrayer, and the latter was immobilized on an AFM probe. To control the spacing between the immobilized DNAs on the surface, dendron self-assembly was employed. Force–distance curves showed that the mean force of the specific unbinding events was 32 ± 5 pN, and the hydrodynamic distance of the captured RNA was 30–60 nm. Adhesion force maps were generated with criteria including the mean force value, probability of obtaining the specific curves and hydrodynamic distance. The maps for the samples whose concentrations ranged from 0.76 fM to 6.0 fM showed that cluster number has a linear relationship with RNA concentration, while the difference between the observed number and the calculated one increased at low concentrations. Because the detection limit is expected to be enhanced by a factor of 10 000 when a spot of 1 micron diameter is employed, it is believed that HCV RNA of a few copy numbers can be detected by the use of AFM.

Background: Escherichia coli can respire anaerobically using dimethyl sulfoxide (DMSO) or trimethylamine-N-oxide (TMAO) as the terminal electron acceptor for anaerobic energy generation. Expression of the dmsABC genes that encode the membrane-associated DMSO/TMAO reductase is positively regulated during anaerobic conditions by the Fnr protein and negatively regulated by the NarL protein when nitrate is present.