Silencing and activation of ClyA cytotoxin expression in Escherichia coli.

Cytolysin A (ClyA) is a pore-forming cytotoxic protein encoded by the clyA gene of Escherichia coli K-12. Genetic analysis suggested that clyA is silenced by the nucleoid protein H-NS. Purified H-NS protein showed preferential binding to clyA sequences in the promoter region, as evidenced by DNase I footprinting and gel mobility shift assays. Transcriptional derepression and activation of a chromosomal clyA::luxAB operon fusion were seen under conditions of H-NS deficiency and SlyA overproduction, respectively. In H-NS-deficient bacteria neither the absence nor the overproduction of SlyA affected the derepressed ClyA expression any further. Therefore, we suggest that overproduction of SlyA in hns(+) E. coli derepresses clyA transcription by counteracting H-NS. The cyclic AMP receptor protein (CRP) was required for ClyA expression, and it interacted with a predicted, albeit suboptimal, CRP binding site in the clyA upstream region. Site-specific alterations of the CRP binding site to match the consensus resulted in substantially higher levels of ClyA expression, while alterations that were predicted to reduce CRP binding reduced ClyA expression. During anaerobic growth the fumarate and nitrate reduction regulator (FNR) was important for ClyA expression, and the clyA gene could be activated by overexpression of FNR. A major clyA transcript having its 5' end (+1) located 72 bp upstream of the translational start codon and 61 bp downstream of the CRP-FNR binding site was detected in the absence of H-NS. The clyA promoter was characterized as a class I promoter that could be transcriptionally activated by CRP and/or FNR. According to DNA bending analyses, the clyA promoter region has high intrinsic curvature. We suggest that it represents a regulatory region which is particularly susceptible to H-NS silencing, and its features are discussed in relation to regulation of other silenced operons.

Comparative dosimetry in narrow high-energy photon beams.

A comparison of the response of different dosimeters in narrow photon beams (phi > or = 4 mm) of 6 and 18 MV bremsstrahlung has been performed. The detectors used were a natural diamond detector, a liquid ionization chamber, a plastic scintillator and two dedicated silicon diodes. The diodes had a very small detection volume and one was a specially designed double diode using two parallel opposed active volumes with compensating interface perturbations. The characteristics of the detectors were investigated both for dose distribution measurements, such as depth-dose curves and lateral beam profiles, and for output factors. The dose rate and angular dependence of the diamond and the two diodes were also studied separately. The depth-dose distributions for small fields agree well for the diamond, the scintillator and the single diode, while the measured dose maximum for the double diode is about 1% higher and for the liquid chamber about 1% lower than the mean of the others when normalized at a depth of 10 cm. The plastic scintillator and the liquid ionization chamber detect a penumbra width that is slightly broadened due to the influence of their finite size, while the double diode may even underestimate the penumbra width due to its small size and high density. When corrected for the extension of the detector volume a good agreement with Monte Carlo calculated beam profiles was obtained for the plastic scintillator and the liquid ionization chamber. Profiles measured with the diamond show an asymmetry when positioned with the smallest dimension facing the beam, while the double diode, the scintillator and the liquid chamber measure symmetric profiles irrespective of positioning. Significant differences in the output factors were obtained with the different detectors. The natural diamond detector measures output factors close to those with an ionization chamber (less than 1% difference) for field sizes between 3 x 3 and 15 x 15 cm2, but overestimates the output factors for large fields and underestimates the output factors for the smallest field sizes. The single and double diodes overestimated the output factor for large field sizes by up to 7 and 12% respectively due to the high content of low-energy photons. The double diode, and to some extent the single diode, also showed a relative increase in response compared with the more water equivalent liquid chamber and plastic scintillator at the smallest fields where there is a lack of lateral electron equilibrium. Both the plastic scintillator and the liquid chamber also show responses that deviate from the ionization chamber for larger field sizes. The major deviations can be explained based on the characteristics of the sensitive materials and the construction of the detectors.