Diagnostics and characterisation of preocclusive stenoses and occlusions of the internal carotid artery with B-flow.

The purpose was to evaluate whether B-flow can improve the ultrasonographic diagnosis of preocclusive stenosis and occlusion of the internal carotid artery (ICA) compared with colour-coded Doppler and power Doppler. Ninety patients with occlusions or preocclusive stenoses of the ICA suspected by Doppler sonography were examined with B-flow in comparison with colour-coded Doppler sonography (CCDS), power Doppler (PD) and intra-arterial digital subtraction angiography (DSA). Intrastenotic flow detection and lengths of stenoses were the main criteria. Ulcerated plaques found by surgery in 42/90 patients were compared by ultrasonography (US). Diagnosis of ICA occlusion with CCDS, PD and B-flow was correct in all 42 cases. A preocclusive ICA stenosis in DSA was detected correctly in all 48/48 cases (100%) for B-flow, in 44/48 (92%) for PD and in 39/48 (81%) for CCDS. Surgical findings showed in 17/42 cases ulcerated plaques; 15/17 (89%) of these cases were detected with B-flow, 12/17 (71%) with PD, 10/17 (59%) with CCDS, and 8/17 (47%) with DSA. With B-flow the extent of stenosis was appraised more precisely than with PD and CCDS (P<0.0001). In conclusion, B-flow is a reliable method for preocclusive stenosis of the ICA with less intrastenotic flow artefacts. B-flow facilitates the characterization of plaque morphologies.

Color Doppler, power Doppler and B-flow ultrasound in the assessment of ICA stenosis: Comparison with 64-MD-CT angiography.

The purpose of this study is to investigate the diagnostic potential of color-coded Doppler sonography (CCDS), power-Doppler (PD) and B-flow ultrasound in assessing the degree of extracranial internal carotid artery (ICA) stenosis in comparison to CT-angiography (MD-CTA). Thirty-two consecutive patients referred for CTA with 41 ICA-stenoses were included in this prospective study. MD-CTA was performed using a 64 row scanner with a CTDIvol of 13.1 mGy/cm. In CTA, CCDS, PD and B-flow, the degree of stenosis was evaluated by the minimal intrastenotic diameter in comparison to the poststenotic diameter. Two radiologists performed a quantitative evaluation of the stenoses in consensus blinded to the results of ultrasound. These were correlated to CTA, CCDS, PD and B-flow, intraoperative findings and clinical follow-up. Grading of the stenoses in B-flow ultrasound outperformed the other techniques in terms of accuracy with a correlation coefficient to CTA of 0.88, while PD and CCDS measurements yield coefficients of 0.74 and 0.70. Bland-Altman analysis additionally shows a very little bias of the three US methods between 0.5 and 3.2 %. There is excellent correlation (coefficient 0.88, CI 0.77-0.93) with 64-MD-CTA and B-flow ultrasound in terms of accuracy for intrastenotic and poststenotic diameter. Duplex sonography is useful for screening purposes.

Binding of transcriptional activators to sigma 54 in the presence of the transition state analog ADP-aluminum fluoride: insights into activator mechanochemical action.

Conformational changes in sigma 54 (sigma(54)) and sigma(54)-holoenzyme depend on nucleotide hydrolysis by an activator. We now show that sigma(54) and its holoenzyme bind to the central ATP-hydrolyzing domains of the transcriptional activators PspF and NifA in the presence of ADP-aluminum fluoride, an analog of ATP in the transition state for hydrolysis. Direct binding of sigma(54) Region I to activator in the presence of ADP-aluminum fluoride was shown and inferred from in vivo suppression genetics. Energy transduction appears to occur through activator contacts to sigma(54) Region I. ADP-aluminum fluoride-dependent interactions and consideration of other AAA+ proteins provide insight into activator mechanochemical action.

Responses of Gram-negative bacteria to certain environmental stressors.

Bacteria in nature are exposed to variations in temperature, and are affected by the availability of nutrients and water and the presence of toxic molecules. Their reactions to these changes require a series of rapid adaptive responses. Although transcriptional regulation is of primary importance in these responses, translational regulation and even activation of 'silenced' enzymes are critical for survival in changing environments. Bacteria have developed a series of mechanisms at the membrane structure level to cope with high concentrations of solvents. In addition, solvent-tolerant strains express highly effective efflux pumps to remove solvents from the cytoplasm. Desiccation tolerance is based on the synthesis and accumulation of osmoprotectants together with changes in fatty acid composition to preserve membrane structure. Both cold shock and heat shock responses are mainly regulated at a post-transcriptional level, translation efficiency in the case of cold shock and mRNA half-life and sigma32 stability in the case of heat shock.

DNA melting within a binary sigma(54)-promoter DNA complex.

The final sigma(54) subunit of the bacterial RNA polymerase requires the action of specialized enhancer-binding activators to initiate transcription. Here we show that final sigma(54) is able to melt promoter DNA when it is bound to a DNA structure representing the initial nucleation of DNA opening found in closed complexes. Melting occurs in response to activator in a nucleotide-hydrolyzing reaction and appears to spread downstream from the nucleation point toward the transcription start site. We show that final sigma(54) contains some weak determinants for DNA melting that are masked by the Region I sequences and some strong ones that require Region I. It seems that final sigma(54) binds to DNA in a self-inhibited state, and one function of the activator is therefore to promote a conformational change in final sigma(54) to reveal its DNA-melting activity. Results with the holoenzyme bound to early melted DNA suggest an ordered series of events in which changes in core to final sigma(54) interactions and final sigma(54)-DNA interactions occur in response to activator to allow final sigma(54) isomerization and the holoenzyme to progress from the closed complex to the open complex.

Single amino acid substitution mutants of Klebsiella pneumoniae sigma(54) defective in transcription.

Transcription initiation by the sigma(54) RNA polymerase requires specialised activators and their associated nucleoside triphosphate hydrolysis. To explore the roles of sigma(54) in initiation we used random mutagenesis of rpoN and an in vivo activity screen to isolate functionally altered sigma(54) proteins. Five defective mutants, each with a different single amino acid substitution, were obtained. Three failed in transcription after forming a closed complex. One such mutant mapped to regulatory Region I of sigma(54), the other two to Region III. The Region I mutant allowed transcription independently of activator and showed reduced activator-dependent sigma(54) isomerisation. The two Region III mutants displayed altered behaviour in a sigma(54) isomerisation assay and one failed to stably bind early melted DNA as the holoenzyme; they may contribute to a communication pathway linking changes in sigma to open complex formation. Two further Region III mutants showed gross defects in overall DNA binding. For one, sufficient residual DNA binding activity remained to allow us to demonstrate that other activities were largely unaffected. Changes in DNA binding preferences and core polymerase-dependent properties were evident amongst the mutants.

Interaction of sigma factor sigmaN with Escherichia coli RNA polymerase core enzyme.

The equilibrium binding and kinetics of assembly of the DNA-dependent RNA polymerase (RNAP) sigma(N)-holoenzyme has been investigated using biosynthetically labelled 7-azatryptophyl- (7AW)sigma(N). The spectroscopic properties of such 7AW proteins allows their absorbance and fluorescence to be monitored selectively, even in the presence of high concentrations of other tryptophan-containing proteins. The 7AWsigma(N) retained its biological activity in stimulating transcription from sigma(N)-specific promoters, and in in vitro gel electrophoresis assays of binding to core RNAP from Escherichia coli. Furthermore, five Trp-->Ala single mutants of sigma(N) were shown to support growth under conditions of nitrogen limitation, and showed comparable efficiency in activating the sigma(N)-dependent nifH promoter in vivo, indicating that none of the tryptophan residues were essential for activity. The equilibrium binding of 7AWsigma(N) to core RNAP was examined by analytical ultracentrifugation. In sedimentation equilibrium experiments, absorbance data at 315 nm (which reports selectively on the distribution of free and bound 7AWsigma(N)) established that a 1:1 complex was formed, with a dissociation constant lower than 2 microM. The kinetics of the interaction between 7AWsigma(N) and core RNAP was investigated using stopped-flow spectrofluorimetry. A biphasic decrease in fluorescence intensity was observed when samples were excited at 280 nm, whereas only the slower of the two phases was observed at 315 nm. The kinetic data were analysed in terms of a mechanism in which a fast bimolecular association of sigma(N) with core RNAP is followed by a relatively slow isomerization step. The consequences of these findings on the competition between sigma(N) and the major sigma factor, sigma(70), in Escherichia coli are discussed.

Isomerization of a binary sigma-promoter DNA complex by transcription activators.

Multisubunit RNA polymerases are targets of sophisticated signal transduction pathways that link environmental or temporal cues to changes in gene expression. Here we show that the sigma 54 protein (sigma54), responsible for promoter specific binding by bacterial RNA polymerase, undergoes a nucleotide hydrolysis dependent isomerization on DNA. Changes in protein structure are evident. The isomerization has all the known requirements of sigma 54-dependent transcription, including a dependence on enhancer binding activator proteins and occurs independently of the core RNA polymerase. We suggest that activator driven changes in sigma54 conformation trigger the conversion of a transcriptionally silent RNA polymerase conformation to one able to interact productively with template DNA. Our results illustrate the types of changes that must occur for multisubunit complexes to manipulate DNA, and show that transcription activators can remodel key nucleoprotein structures to achieve direct activation of transcription.

Sequences in sigma(54) region I required for binding to early melted DNA and their involvement in sigma-DNA isomerisation.

The bacterial sigma(54) RNA polymerase functions in a transcription activation mechanism that fully relies upon nucleotide hydrolysis by an enhancer binding activator protein to stimulate open complex formation. Here, we describe results of DNA-binding assays used to probe the role of the sigma(54) amino terminal region I in activation. Of the 15 region I alanine substitution mutants assayed, several specifically failed to bind to a DNA structure representing an early conformation in DNA melting. The same mutants are defective in activated transcription and in forming an isomerised sigma-DNA complex on the early opened DNA. The mechanism of activation may therefore require tight binding of sigma(54) to particular early melted DNA structures. Where mutant sigma(54) binding to early melted DNA was detected, activator-dependent isomerisation generally occurred as efficiently as with the wild-type protein, suggesting that certain region I sequences are largely uninvolved in sigma isomerisation. DNA-binding, sigma isomerisation and transcription activation assays allow formulation of a functional map of region I.

Functionality of purified sigma(N) (sigma(54)) and a NifA-like protein from the hyperthermophile Aquifex aeolicus.

The genome sequence of the extremely thermophilic bacterium Aquifex aeolicus encodes alternative sigma factor sigma(N) (sigma(54), RpoN) and five potential sigma(N)-dependent transcriptional activators. Although A. aeolicus possesses no recognizable nitrogenase genes, two of the activators have a high degree of sequence similarity to NifA proteins from nitrogen-fixing proteobacteria. We identified five putative sigma(N)-dependent promoters upstream of operons implicated in functions including sulfur respiration, nitrogen assimilation, nitrate reductase, and nitrite reductase activity. We cloned, overexpressed (in Escherichia coli), and purified A. aeolicus sigma(N) and the NifA homologue, AQ_218. Purified A. aeolicus sigma(N) bound to E. coli core RNA polymerase and bound specifically to a DNA fragment containing E. coli promoter glnHp2 and to several A. aeolicus DNA fragments containing putative sigma(N)-dependent promoters. When combined with E. coli core RNA polymerase, A. aeolicus sigma(N) supported A. aeolicus NifA-dependent transcription from the glnHp2 promoter. The E. coli activator PspFDeltaHTH did not stimulate transcription. The NifA homologue, AQ_218, bound specifically to a DNA sequence centered about 100 bp upstream of the A. aeolicus glnBA operon and so is likely to be involved in the regulation of nitrogen assimilation in this organism. These results argue that the sigma(N) enhancer-dependent transcription system operates in at least one extreme environment, and that the activator and sigma(N) have coevolved.

Systematic analysis of sigma54 N-terminal sequences identifies regions involved in positive and negative regulation of transcription.

The conserved amino-terminal region of sigma 54 (Region I) contains sequences that allow response to activator proteins, and inhibit initiation in the absence of activator. Alanine-scanning mutagenesis has been used to systematically define Region I elements that contribute to each of these functions. Amino acid residues from 6 to 50 were substituted with alanine in groups of three consecutive residues, making a total of 15 mutants. Mutants were tested for their ability to mediate activation in vivo, and in vitro, and to support transcription in the absence of activator in vitro. Most mutations located between residues 15 and 47 altered sigma function, while mutations between residues 6 and 14, and 48-50 had little effect. The defective mutants ala 15-17, 42-44, and 45-47 define new amino acids required for normal sigma function. In general, there is an inverse correlation between the levels of activated and activator-independent transcription, suggesting that the two functions are linked. When activated, the defective sigma mutants, except for ala 24-26, formed heparin-resistant open complexes similar to wild-type sigma. Mutant ala 24-26 formed heparin-unstable open complexes, suggesting that this mutation interferes with a different step in the initiation pathway.

Involvement of the sigmaN DNA-binding domain in open complex formation.

sigmaN (sigma54) RNA polymerase holoenzyme closed complexes isomerize to open complexes in a reaction requiring nucleoside triphosphate hydrolysis by enhancer binding activator proteins. Here, we characterize Klebsiella pneumoniae sigmaN mutants, altered in the carboxy DNA-binding domain (F354A/F355A, F402A, F403A and F402A/F403A), that fail in activator-dependent transcription. The mutant holoenzymes have altered activator-dependent interactions with promoter sequences that normally become melted. Activator-dependent stable complexes accumulated slowly in vitro (F402A) and to a reduced final level (F403A, F402A/F403A, F354A/F355A). Similar results were obtained in an assay of activator-independent stable complex formation. Premelted templates did not rescue the mutants for stable preinitiation complex formation but did for deleted region I sigmaN, suggesting different defects. The DNA-binding domain substitutions are within sigmaN sequences previously shown to be buried upon formation of the wild-type holoenzyme or closed complex, suggesting that, in the mutants, alteration of the sigmaN-core and sigmaN-DNA interfaces has occurred to change holoenzyme activity. Core-binding assays with the mutant sigmas support this view. Interestingly, an internal deletion form of sigmaN lacking the major core binding determinant was able to assemble into holoenzyme and, although unable to support activator-dependent transcription, formed a stable activator-independent holoenzyme promoter complex on premelted DNA templates.

Functions of the sigma(54) region I in trans and implications for transcription activation.

Control of transcription frequently involves the direct interaction of activators with RNA polymerase. In bacteria, the formation of stable open promoter complexes by the sigma(54) RNA polymerase is critically dependent on sigma(54) amino Region I sequences. Their presence correlates with activator dependence, and removal allows the holoenzyme to engage productively with melted DNA independently of the activator. Using purified Region I sequences and holoenzymes containing full-length or Region I-deleted sigma(54), we have explored the involvement of Region I in transcription activation. Results show that Region I in trans inhibits a reversible conformational change in the holoenzyme believed to be polymerase isomerization. Evidence is presented indicating that the holoenzyme (and not the promoter DNA per se) is one interacting target used by Region I in preventing polymerase isomerization. Activator overcomes this inhibition in a reaction requiring nucleotide hydrolysis. Region I in trans is able to inhibit activated transcription by the holoenzyme containing full-length sigma(54). Inhibition appeared to be noncompetitive with respect to the activator, suggesting that a direct activator interaction occurs with parts of the holoenzyme outside Region I. Stabilization of isomerized holoenzyme bound to melted DNA by Region I in trans occurs largely independently of the initiating nucleotide, suggesting a role for Region I in maintaining the open complex.

Sequences in sigmaN determining holoenzyme formation and properties.

Sigma subunits of bacterial RNA polymerases are closely involved in many steps of promoter-specific transcription initiation. Holoenzyme formed with the specialised minor sigma-N (sigmaN) protein binds rare promoters in a transcriptionally inactive state and functions in enhancer-dependent transcription. Using competition and dissociation assays, we show that sigmaN-holoenzyme has a stability comparable to the major sigma70-holoenzyme. Purified partial sequences of sigmaN were prepared and assayed for retention of core RNA polymerase binding activity. Two discrete fragments of sigmaN which both bind the core but with significantly different affinities were identified, demonstrating that the sigmaN interface with core RNA polymerase is extensive. The low affinity segment of sigmaN included region I sequences, an amino terminal domain which mediates activator responsiveness and formation of open promoter complexes. The higher affinity site lies within a 95 residue fragment of region III. We propose that the core to region I contact mediates properties of the sigmaN-holoenzyme important for enhancer responsiveness. Heparin is shown to dissociate sigmaN and core, indicating that disruption of the holoenzyme is involved in the heparin sensitivity of the sigmaN closed complex.

The XylS-dependent Pm promoter is transcribed in vivo by RNA polymerase with sigma 32 or sigma 38 depending on the growth phase.

The Pm promoter of the TOL plasmid of Pseudomonas putida is expressed at high level along the growth curve. This transcription is dependent on the positive regulator XylS activated by 3-methylbenzoate. The sigma factor sigma 38 is required for expression in early stationary phase and thereafter. To test whether sigma 70 was involved in Pm transcription in exponential phase, we have followed mRNA synthesis in a rpoD thermosensitive strain. No difference in Pm transcription was found between the wild type and the thermosensitive strain at the restrictive temperature of 42 degrees C, indicating that transcription was independent of the sigma factor sigma 70. However, basal levels of mRNA expression from Pm in this strain in exponential phase were more than twofold higher at 42 degrees C, suggesting involvement of sigma 32 in Pm transcription. In a rpoH background, no expression of Pm took place in the exponential phase, whereas it increased during stationary phase, and in a rpoH rpoS double mutant no activity from the Pm promoter was detected along the growth curve. We have shown that the increase in the amount of sigma 32 factor necessary for transcription in exponential phase is provided through induction of the heat shock response by the presence of the effector 3-methylbenzoate, which is also required for activation of the positive regulator XylS. We conclude that activation of Pm transcription is achieved through a switch between two stress-responsive factors, sigma 32 in exponential phase and sigma 38 in stationary phase. In both cases, transcription is dependent on the activator XylS and presents the same transcription start point.

Amino-terminal sequences of sigmaN (sigma54) inhibit RNA polymerase isomerization.

In bacteria, association of the specialized sigmaN protein with the core RNA polymerase subunits forms a holoenzyme able to bind promoter DNA, but unable to melt DNA and initiate transcription unless acted on by an activator protein. The conserved amino-terminal 50 amino acids of sigmaN (Region I) are required for the response to activators. We have used pre-melted DNA templates, in which the template strand is unpaired and accessible for transcription initiation, to mimic a naturally melted promoter and explore the function of Region I. Our results indicate that one activity of Region I sequences is to inhibit productive interaction of holoenzyme with pre-melted DNA. On pre-melted DNA targets, either activation of sigmaN-holoenzyme or removal of Region I allowed efficient formation of complexes in which melted DNA was sequestered by RNA polymerase. Like natural pre-initiation complexes formed on conventional DNA templates through the action of activator, such complexes were heparin-resistant and transcriptionally active. The inhibitory sigmaN Region I domain functioned in trans to confer heparin sensitivity to complexes between Region I-deleted holoenzyme and pre-melted promoter DNA. Evidence that Region I senses the conformation of the promoter was obtained from protein footprint experiments. We suggest that one function for Region I is to mask a single-strand DNA-binding activity of the holoenzyme. On the basis of extended DNA footprints of Region I-deleted holoenzyme, we also propose that Region I prevents RNA polymerase isomerization, a conformational change necessary for access to and the subsequent stable association of holoenzyme with melted DNA.

Critical nucleotides in the upstream region of the XylS-dependent TOL meta-cleavage pathway operon promoter as deduced from analysis of mutants.

The Pm promoter, dependent on TOL plasmid XylS regulator, which is activated by benzoate effectors, drives transcription of the meta-cleavage pathway for the metabolism of alkylbenzoates. This promoter is unique in that in vivo transcription is mediated by RNA-polymerase with different sigma factors. In vivo footprinting analysis shows that XylS interacted with nucleotides in the -40 to -70 region. In vivo and in vitro methylation of Pm shows extensive methylation of T at position -42 in the bottom strand, suggesting that it represents a key distortion point that may favor XylS/RNA polymerase interactions. Methylation of T-42 was highest in cells bearing XylS and in the presence of an effector. Gs in the -47 to -61 region appeared to be more protected in cells harboring XylS in the presence than in the absence of the effector. Almost 100 mutants in the Pm region between -41 and -78 were generated; transcriptional analysis of these mutants defined the XylS target as two direct repeats with the sequence TGCAN6GGNCA. These motifs cover the -70 to -56 and the -49 to -35 regions. Single point mutations revealed that nucleotides located at -49 to -46 and at -59, -60, -62, and -70 are the most critical for appropriate XylS-Pm interactions.

Activation and repression of transcription at the double tandem divergent promoters for the xylR and xylS genes of the TOL plasmid of Pseudomonas putida.

The xylR and xylS genes are divergent and control transcription of the TOL plasmid catabolic pathways for toluene metabolism. Four promoters are found in the 300-bp intergenic region: Pr1 and Pr2 are constitutive sigma70-dependent tandem promoters that drive expression of xylR, while expression of the xylS gene is driven from Ps2, a constitutive sigma70-dependent promoter, and by the regulatable sigma54 class Ps1 promoter. In Ps1 the XylR targets (upstream activator sequences [UASs]) overlap the Pr promoters, and two sites for integration host factor (IHF) binding are located at the region from positions -2 to -30 (-2/-30 region) and the -137/-156 region, the latter overlapping the Pr promoters. When the XylR protein binds to the UASs in the absence of effector, it represses expression from Pr promoters. In the XylR-plus background and in the absence of an effector, the level of expression from Ps1 is low, although detectable, whereas Ps2 is active. In this background and in the presence of an effector, XylR increases autorepression. In a sigma54-deficient Pseudomonas putida background, no expression occurred from Ps1 regardless of the presence of an effector. However, in the presence of an effector, the amount of RNA produced from Pr promoters was almost undetectable. This finding suggests that when no transcription occurred at the Ps1 promoter, clearance of XylR from the UASs was almost negligible. In this background, expression from Ps2 was very high regardless of the presence of an effector; this finding suggests that RNA polymerase containing sigma54 modulates expression from the downstream Ps2 sigma70-dependent promoter. In a P. putida IHF-minus background and in the presence of effector, Ps1 expression was the highest found; in contrast, the basal levels of this promoter were the lowest observed. This finding suggests that IHF acts in vivo as a repressor of the sigma54-dependent Ps1 promoter. In an IHF-deficient host background, expression from Ps2 in the presence of effector was negligible. Thus, binding of RNA polymerase containing sigma54 at the upstream promoter may modulate expression from the Ps2 promoter.

Arac/XylS family of transcriptional regulators.

The ArC/XylS family of prokaryotic positive transcriptional regulators includes more than 100 proteins and polypeptides derived from open reading frames translated from DNA sequences. Members of this family are widely distributed and have been found in the gamma subgroup of the proteobacteria, low- and high-G + C-content gram-positive bacteria, and cyanobacteria. These proteins are defined by a profile that can be accessed from PROSITE PS01124. Members of the family are about 300 amino acids long and have three main regulatory functions in common: carbon metabolism, stress response, and pathogenesis. Multiple alignments of the proteins of the family define a conserved stretch of 99 amino acids usually located at the C-terminal region of the regulator and connected to a nonconserved region via a linker. The conserved stretch contains all the elements required to bind DNA target sequences and to activate transcription from cognate promoters. Secondary analysis of the conserved region suggests that it contains two potential alpha-helix-turn-alpha-helix DNA binding motifs. The first, and better-fitting motif is supported by biochemical data, whereas existing biochemical data neither support nor refute the proposal that the second region possesses this structure. The phylogenetic relationship suggests that members of the family have recruited the nonconserved domain(s) into a series of existing domains involved in DNA recognition and transcription stimulation and that this recruited domain governs the role that the regulator carries out. For some regulators, it has been demonstrated that the nonconserved region contains the dimerization domain. For the regulators involved in carbon metabolism, the effector binding determinants are also in this region. Most regulators belonging to the AraC/XylS family recognize multiple binding sites in the regulated promoters. One of the motifs usually overlaps or is adjacent to the -35 region of the cognate promoters. Footprinting assays have suggested that these regulators protect a stretch of up to 20 bp in the target promoters, and multiple alignments of binding sites for a number of regulators have shown that the proteins recognize short motifs within the protected region.