Genetic inactivation of acrAB or inhibition of efflux induces expression of ramA.

OBJECTIVES: The transcriptional activator RamA regulates production of the multidrug resistance efflux AcrAB-TolC system in several Enterobacteriaceae. This study investigated factors that lead to increased expression of ramA. METHODS: In order to monitor changes in ramA expression, the promoter region of ramA was fused to a gfp gene encoding an unstable green fluorescence protein (GFP) on the reporter plasmid, pMW82. The ramA reporter plasmid was transformed into Salmonella Typhimurium SL1344 and a ΔacrB mutant. The response of the reporter to subinhibitory concentrations of antibiotics, dyes, biocides, psychotropic agents and efflux inhibitors was measured during growth over a 5 h time period. RESULTS: Our data revealed that the expression of ramA was increased in a ΔacrB mutant and also in the presence of the efflux inhibitors phenylalanine-arginine-ß-naphthylamide, carbonyl cyanide m-chlorophenylhydrazone and 1-(1-naphthylmethyl)-piperazine. The phenothiazines chlorpromazine and thioridazine also increased ramA expression, triggering the greatest increase in GFP expression. However, inducers of Escherichia coli marA and soxS and 12 of 17 tested antibiotic substrates of AcrAB-TolC did not induce ramA expression. CONCLUSIONS: This study shows that expression of ramA is not induced by most substrates of the AcrAB-TolC efflux system, but is increased by mutational inactivation of acrB or when efflux is inhibited.

An evaluation of the potential to use tumor-associated antigens as targets for antitumor T cell therapy using transgenic mice expressing a retroviral tumor antigen in normal lymphoid tissues.

A major obstacle to the development of T cell therapy for the treatment of human tumors has been the difficulty generating T cells specifically reactive with the tumor. Most of the characterized human tumor antigens have been classified as tumor associated, because of demonstrable expression at low levels in some normal cells, and thus have not been extensively studied as potential targets of a therapeutic immune response. However, the quantitative difference in expression of such antigens between the tumor and normal cells might permit the generation of antigen-specific T cells capable of selective antitumor and not autoimmune activity. To address this issue, transgenic (TG) mice were generated that expressed low levels of Friend murine leukemia virus (FMuLV) envelope protein in lymphoid cells under the control of an immunoglobulin promoter. This protein is expressed at high levels by a Friend virus-induced erythroleukemia of C57BL/6 (B6) origin, FBL, and has been shown to serve as an efficient tumor-specific rejection antigen in B6 mice. The env-TG mice were tolerant to envelope, as reflected by the failure to detect an envelope-specific response after in vivo priming and in vitro stimulation with preparations of FMuLV envelope. However, adoptively transferred envelope-specific T cells from immunized non-TG B6 mice mediated complete eradication of FBL tumor cells in TG mice, and did not induce detectable autoimmune damage to TG lymphoid tissues. The transferred immune cells were not permanently inactivated in the TG mice, since donor T cells responded to envelope after removal from the TG mice. The lack of autoimmune injury did not reflect inadequate expression of envelope by TG lymphocytes for recognition by T cells, since TG lymphocytes functioned effectively in vitro as stimulators for envelope-specific T cells. The results suggest that this and analogous strains of TG mice may prove useful for elucidating principles for the generation and therapeutic use of tumor-reactive T cells specific for tumor-associated antigens.

The bacterial LexA transcriptional repressor.

Bacteria respond to DNA damage by mounting a coordinated cellular response, governed by the RecA and LexA proteins. In Escherichia coli, RecA stimulates cleavage of the LexA repressor, inducing more than 40 genes that comprise the SOS global regulatory network. The SOS response is widespread among bacteria and exhibits considerable variation in its composition and regulation. In some well-characterised pathogens, induction of the SOS response modulates the evolution and dissemination of drug resistance, as well as synthesis, secretion and dissemination of the virulence. In this review, we discuss the structure of LexA protein, particularly with respect to distinct conformations that enable repression of SOS genes via specific DNA binding or repressor cleavage during the response to DNA damage. These may provide new starting points in the battle against the emergence of bacterial pathogens and the spread of drug resistance among them.

Cyclic AMP receptor protein: role in transcription activation.

The structure of this pleiotropic activator of gene transcription in bacteria and its interaction sites at promoter DNA's as well as the role of this protein in the RNA polymerase-promoter interactions are reviewed.

Characterization of multiple promoters and transcript stability in the sacB-sacC gene cluster in Zymomonas mobilis.

In Zymomonas mobilis, the extracellular levansucrase (SacB) and extracellular sucrase (SacC) are involved in sucrose hydrolysis. Genes coding for these two enzymes (sacB and sacC) are arranged in a cluster in the genome and separated by a short intervening sequence. The level of sacC transcript was 12-fold higher than that of sacB transcript. On the other hand, transcript stability analysis in sucrose grown cultures revealed that the half-life of the sacB transcripts (153 s) was more than twofold higher than that of sacC transcript (66 s). The decay curves of sacB and sacC transcripts analyzed by the semi-quantitative RT-PCR correlated well with the decay curves of the respective enzyme activities. In the sacB promoter disruption mutant, Z. moblis BT2, the extracellular sucrase activity decreased from 2.6 to 2.0 U mg(-1) in sucrose medium due to the loss of SacB expression. The expression of sacC in the absence of the sacB promoter suggested that these two genes could be transcribed as different mRNAs. The promoter-lacZ fusion studies in Escherichia coli proved that the short intervening region acts as a strong promoter for the sacC gene.

The Escherichia coli RNA polymerase alpha subunit: structure and function.

Recent work has established that the Escherichia coli RNA polymerase alpha subunit consists of an amino-terminal domain containing determinants for interaction with the remainder of RNA polymerase, a carboxy-terminal domain containing determinants for interaction with DNA and interaction with transcriptional activator proteins, and a 13-36 amino acid unstructured and/or flexible linker. These findings suggest a simple, integrated model for the mechanism of involvement of alpha in promoter recognition and transcriptional activation.

The Deuterator: software for the determination of backbone amide deuterium levels from H/D exchange MS data.

BACKGROUND: The combination of mass spectrometry and solution phase amide hydrogen/deuterium exchange (H/D exchange) experiments is an effective method for characterizing protein dynamics, and protein-protein or protein-ligand interactions. Despite methodological advancements and improvements in instrumentation and automation, data analysis and display remains a tedious process. The factors that contribute to this bottleneck are the large number of data points produced in a typical experiment, each requiring manual curation and validation, and then calculation of the level of backbone amide exchange. Tools have become available that address some of these issues, but lack sufficient integration, functionality, and accessibility required to address the needs of the H/D exchange community. To date there is no software for the analysis of H/D exchange data that comprehensively addresses these issues. RESULTS: We have developed an integrated software system for the automated analysis and representation of H/D exchange data that has been titled "The Deuterator". Novel approaches have been implemented that enable high throughput analysis, automated determination of deuterium incorporation, and deconvolution of overlapping peptides. This has been achieved by using methods involving iterative theoretical envelope fitting, and consideration of peak data within expected m/z ranges. Existing common file formats have been leveraged to allow compatibility with the output from the myriad of MS instrument platforms and peptide sequence database search engines.A web-based interface is used to integrate the components of The Deuterator that are able to analyze and present mass spectral data from instruments with varying resolving powers. The results, if necessary, can then be confirmed, adjusted, re-calculated and saved. Additional tools synchronize the curated calculation parameters with replicate time points, increasing throughput. Saved results can then be used to plot deuterium buildup curves and 3D structural overlays. The system has been used successfully in a production environment for over one year and is freely available as a web tool at the project home page http://deuterator.florida.scripps.edu. CONCLUSION: The automated calculation and presentation of H/D exchange data in a user interface enables scientists to organize and analyze data efficiently. Integration of the different components of The Deuterator coupled with the flexibility of common data file formats allow this system to be accessible to the broadening H/D exchange community.

Catecholaminergic neurons in medullary nuclei are among the post-synaptic targets of descending projections from infralimbic area 25 of the rat medial prefrontal cortex.

The infralimbic (IL) 'visceromotor' area of the rat medial prefrontal cortex projects to strategic subcortical nuclei involved in autonomic functions. Central among these targets are the nucleus tractus solitarius (NTS) and the rostral ventrolateral medulla (rVLM). By combining tract-tracing using the anterograde tracer biotinylated dextran amine (BDA) with immunolabeling for tyrosine hydroxylase (TH; an enzyme marker of catecholaminergic neurons), a limited proportion of BDA-labeled IL axonal boutons in the NTS and rVLM was found to be closely associated with TH immunopositive (+) target structures. Such structural appositions were mainly located proximally over the labeled dendritic arbors of identified TH+ neurons. Quantitative ultrastructural examination revealed that in NTS, TH+ dendritic shafts comprised 7.0% of the overall post-synaptic target population innervated by BDA-labeled IL boutons, whereas TH+ dendritic spines represented 1.25% of targets. In rVLM, TH+ shafts represented 9.0% and TH+ spines 2.5% of IL targets. Labeled IL boutons established exclusively asymmetric Gray Type 1 (presumed excitatory) synaptic junctions. The results indicate that subpopulations of catecholaminergic neurons in the NTS and rVLM are among the spectrum of post-synaptic neurons monosynaptically innervated by descending 'excitatory' input from IL cortex. Such connectivity, albeit restricted, identifies the potential direct influence of IL cortex on the processing and distribution of cardiovascular, respiratory and related autonomic information by catecholaminergic neurons in the NTS and VLM of the rat.

A Multinational Analysis of Mutations and Heterogeneity in PZase, RpsA, and PanD Associated with Pyrazinamide Resistance in M/XDR Mycobacterium tuberculosis.

Pyrazinamide (PZA) is an important first-line drug in all existing and new tuberculosis (TB) treatment regimens. PZA-resistance in M. tuberculosis is increasing, especially among M/XDR cases. Noted issues with PZA Drug Susceptibility Testing (DST) have driven the search for alternative tests. This study provides a comprehensive assessment of PZA molecular diagnostics in M/XDR TB cases. A set of 296, mostly XDR, clinical M. tuberculosis isolates from four countries were subjected to DST for eight drugs, confirmatory Wayne's assay, and whole-genome sequencing. Three genes implicated in PZA resistance, pncA, rpsA, and panD were investigated. Assuming all non-synonymous mutations cause resistance, we report 90% sensitivity and 65% specificity for a pncA-based molecular test. The addition of rpsA and panD potentially provides 2% increase in sensitivity. Molecular heterogeneity in pncA was associated with resistance and should be evaluated as a diagnostic tool. Mutations near the N-terminus and C-terminus of PZase were associated with East-Asian and Euro-American lineages, respectively. Finally, Euro-American isolates are most likely to have a wild-type PZase and escape molecular detection. Overall, the 8-10% resistance without markers may point to alternative mechanisms of resistance. Confirmatory mutagenesis may improve the disconcertingly low specificity but reduce sensitivity since not all mutations may cause resistance.

Amygdala input monosynaptically innervates parvalbumin immunoreactive local circuit neurons in rat medial prefrontal cortex.

The projection from the basolateral nucleus of the amygdala (BLA) conveys information about the affective significance of sensory stimuli to the medial prefrontal cortex (mPFC). By using an anterograde tract-tracing procedure combined with immunocytochemistry and correlated light/electron microscopical examination, labeled BLA afferents to layers 2-6 of the rat mPFC are shown to establish asymmetrical synaptic contacts, not only with dendritic spines (approximately 95.7% of targets innervated), but also with the aspiny dendritic shafts and somata of multipolar parvalbumin immunopositive (PV+) neurons. A population of PV- dendritic shafts was also innervated. Labeled BLA synaptic input to identified PV+ structures occurred in layers 2-6 of mPFC. The results indicate that labeled BLA afferents predominantly contact the spiny processes of presumed pyramidal cells and also provide a direct and specific innervation to a sub-population of local circuit neurons in mPFC containing PV. Since PV+ cells include two significant classes of fast-spiking GABAergic inhibitory interneuron (basket and axo-axonic cells), these novel observations indicate that the amygdalocortical pathway in the rat has the ability to directly influence functionally strategic 'feed-forward' inhibitory mechanisms at the first stage of processing amygdalocortical information.

Positive activation of gene expression.

Most bacterial transcription activators function by making direct contact with RNA polymerase at target promoters. Some activators contact the carboxy-terminal domain of the RNA polymerase alpha subunit, some contact region 4 of the sigma70 subunit, whilst others interact with other contact sites. A number of activators are ambidextrous and can, apparently simultaneously, contact more than one target site on RNA polymerase. Expression from many promoters is co-dependent on two or more activators. There are several different mechanisms for coupling promoter activity to more than one activator: in one such mechanism, the different activators make independent contacts with different target sites on RNA polymerase.

DNA sequence elements located immediately upstream of the -10 hexamer in Escherichia coli promoters: a systematic study.

We have made a systematic study of how the activity of an Escherichia coli promoter is affected by the base sequence immediately upstream of the -10 hexamer. Starting with an activator-independent promoter, with a 17 bp spacing between the -10 and -35 hexamer elements, we constructed derivatives with all possible combinations of bases at positions -15 and -14. Promoter activity is greatest when the 'non-template' strand carries T and G at positions -15 and -14, respectively. Promoter activity can be further enhanced by a second T and G at positions -17 and -16, respectively, immediately upstream of the first 'TG motif'. Our results show that the base sequence of the DNA segment upstream of the -10 hexamer can make a significant contribution to promoter strength. Using published collections of characterised E.coli promoters, we have studied the frequency of occurrence of 'TG motifs' upstream of the promoters' -10 elements. We conclude that correctly placed 'TG motifs' are found at over 20% of E.coli promoters.

UP element-dependent transcription at the Escherichia coli rrnB P1 promoter: positional requirements and role of the RNA polymerase alpha subunit linker.

The UP element stimulates transcription from the rrnB P1 promoter through a direct interaction with the C-terminal domain of the RNA polymerase alpha subunit (alphaCTD). We investigated the effect on transcription from rrnB P1 of varying both the location of the UP element and the length of the alpha subunit interdomain linker, separately and in combination. Displacement of the UP element by a single turn of the DNA helix resulted in a large decrease in transcription from rrnB P1, while displacement by half a turn or two turns totally abolished UP element-dependent transcription. Deletions of six or more amino acids from within the alpha subunit linker resulted in a decrease in UP element-dependent stimulation, which correlated with decreased binding of alphaCTD to the UP element. Increasing the alpha linker length was less deleterious to RNA polymerase function at rrnB P1 but did not compensate for the decrease in activation that resulted from displacing the UP element. Our results suggest that the location of the UP element at rrnB P1 is crucial to its function and that the natural length of the alpha subunit linker is optimal for utilisation of the UP element at this promoter.

Point mutations that affect translation initiation in the Escherichia coli gal E gene.

This paper describes the selection and characterization of several mutations in the Escherichia coli galactose operon that affect translation initiation of the galE gene but are located outside of the Shine-Dalgarno sequence and the initiator codon. One mutation lies in the gal promoter region and shifts transcription initiation from the galP1 to the galP2 promoter. This results in a gal messenger that is five nucleotides longer and that is translated threefold more efficiently in vivo. This accords with previous observations from in vitro experiments which showed that the longer gal messenger was better translated (Queen & Rosenberg, 1981). The other mutations that affect galE translation are located in the coding sequence immediately downstream from the initiator codon. In contrast to the promoter mutation, these cause alterations in galE expression only when the gene carries a mutated initiator codon or Shine-Dalgarno sequence and have no effect on the wild-type galE gene. These findings are discussed with respect to our present knowledge of translation initiation mechanisms.

Transcription activation by catabolite activator protein (CAP).

Transcription activation by Escherichia coli catabolite activator protein (CAP) at each of two classes of simple CAP-dependent promoters is understood in structural and mechanistic detail. At class I CAP-dependent promoters, CAP activates transcription from a DNA site located upstream of the DNA site for RNA polymerase holoenzyme (RNAP); at these promoters, transcription activation involves protein-protein interactions between CAP and the RNAP alpha subunit C-terminal domain that facilitate binding of RNAP to promoter DNA to form the RNAP-promoter closed complex. At class II CAP-dependent promoters, CAP activates transcription from a DNA site that overlaps the DNA site for RNAP; at these promoters, transcription activation involves both: (i) protein-protein interactions between CAP and RNAP alpha subunit C-terminal domain that facilitate binding of RNAP to promoter DNA to form the RNAP-promoter closed complex; and (ii) protein-protein interactions between CAP and RNAP alpha subunit N-terminal domain that facilitates isomerization of the RNAP-promoter closed complex to the RNAP-promoter open complex. Straightforward combination of the mechanisms for transcription activation at class I and class II CAP-dependent promoters permits synergistic transcription activation by multiple molecules of CAP, or by CAP and other activators. Interference with determinants of CAP or RNAP involved in transcription activation at class I and class II CAP-dependent promoters permits "anti-activation" by negative regulators. Basic features of transcription activation at class I and class II CAP-dependent promoters appear to be generalizable to other activators.

Deletion mutagenesis of the Escherichia coli galactose operon promoter region.

Using recombinant DNA technology we have created a series of progressively longer deletions both upstream and downstream from the Escherichia coli galactose operon regulatory region. The effects of these lesions on expression of the two overlapping galactose promoters have been quantitated after DNA fragments carrying these deletions were cloned in a plasmid vector, in which the beta-galactosidase gene could be expressed from the truncated galactose regulatory region. The results allow us to determine which sequences are necessary for the activity of the two promoters. Our results show that for the P1 promoter, which is controlled by the cyclic AMP-cyclic AMP receptor protein complex (cAMP-CRP), the sequence necessary for full activity starts 56 base-pairs upstream from the transcription initiation point. In contrast, for the P2 promoter, which functions in the absence of cAMP-CRP, the crucial sequence extends to only 39 base-pairs upstream from the transcription start. Deletions that cut into these sequences cause reductions in promoter strength, although some promoter activity is observed even when the "-35 region" of both P2 and P1 are deleted. Analysis of deletions originating downstream from the regulatory region shows that the elimination of the P1 and P2 Pribnow box sequences leads to loss of promoter activity. However, sequences downstream from the P1 start can be replaced without affecting the activity of either promoter. Finally examination of DNA fragments containing total deletions of both galactose promoters allows us to confirm that the flanking sequences contain no significant promoter activity and that the P1 and P2 promoters are principally responsible for galactose operon expression in vivo.

Binding of Escherichia coli RNA polymerase to a promoter carrying mutations that stop transcription initiation.

The gal P2 promoter can be inactivated by point mutations located in the -10 hexamer sequence or immediately upstream from it. Mutations at either site reduce expression in vivo and prevent the formation, in vitro, of tight complexes with RNA polymerase that give a strong footprint and can initiate transcription. However, with a mutation upstream from the -10 region, RNA polymerase could still make a specific contact with gal promoter DNA as judged by interference with cleavage by restriction enzyme SfaNI at a site within the promoter. In contrast, with a mutation in the -10 hexamer sequence, RNA polymerase could not make this contact and does not interfere with restriction by SfaNI.

Transcription activation by the Escherichia coli cyclic AMP receptor protein: determinants within activating region 3.

At Class II CRP-dependent promoters, the Escherichia coli cyclic AMP receptor protein (CRP) activates transcription by making multiple interactions with RNA polymerase (RNAP). Two discrete surfaces of CRP, known as Activating Region 1 (AR1) and Activating Region 2 (AR2), interact with the C-terminal and N-terminal domains, respectively, of the alpha subunit of RNAP. Activating Region 3 (AR3) is a third separate surface of CRP, which is thought to interact with a target in the C-terminal region of the RNAP sigma(70) subunit. We have used a CRP mutant that functions primarily via AR3, CRP HL159 KE101 KN52, as a tool to identify residues within AR3 that are important for activation. This was achieved by screening a random mutant library of the gene encoding CRP HL159 KE101 KN52 for positive control mutants at Class II CRP-dependent promoters, and also by performing alanine scanning mutagenesis. Using both in vivo reporter assays and in vitro transcription assays, we measured the effects of key substitutions within AR3 on transcription activation in both CRP HL159 KE101 KN52 and wild-type CRP. We show that a cluster of negatively charged surface-exposed residues at positions 53, 54, 55 and 58 is required for optimal activation at a Class II, but not at a Class I, CRP-dependent promoter. We conclude that these residues in AR3 of CRP form an activatory determinant for Class II transcription activation. Abortive initiation assays were used to show that this activatory determinant accelerates the rate of isomerisation from the closed to open complex at a Class II CRP-dependent promoter. AR3 of CRP also contains an inhibitory determinant: the lysine residue at position 52 of CRP is inhibitory to maximal levels of transcription activation from Class II promoters. We show that the negative effects of K52 are not simply due to "masking" of the negatively charged residues at positions 53, 54, 55 and 58. Our results suggest that, during activation by wild-type CRP, the activatory and inhibitory determinants of AR3 balance each other. Thus, activation is predominantly determined by AR1 and AR2.

Interactions between activating region 3 of the Escherichia coli cyclic AMP receptor protein and region 4 of the RNA polymerase sigma(70) subunit: application of suppression genetics.

The Escherichia coli cyclic AMP receptor protein, CRP, induces transcription at Class II CRP-dependent promoters by making three different activatory contacts with different surfaces of holo RNA polymerase. One contact surface of CRP, known as Activating Region 3 (AR3), is functional in the downstream subunit of the CRP dimer and is predicted to interact with region 4 of the RNAP sigma(70) subunit. We have previously shown that a mutant CRP derivative that activates transcription primarily via AR3, CRP HL159 KE101 KN52, requires the positively charged residues K593, K597 and R599 in sigma(70) for activation. Here, we have used the positive control substitution, EK58, to disrupt AR3-dependent activation by CRP HL159 KE101 KN52. We then screened random mutant libraries and an alanine scan library of sigma(70) for candidates that restore activation by CRP HL159 KE101 KN52 EK58. We found that changes at R596 and R599 in sigma(70) can restore activation by CRP HL159 KE101 KN52 EK58. This suggests that the side-chains of both R596 and R599 in sigma(70) clash with K58 in CRP. Maximal activation by CRP HL159 KE101 KN52 EK58 is achieved with the substitutions RE596 or RD596 in sigma(70). We propose that there are specific charge-charge interactions between E596 or D596 in sigma(70) and K58 in AR3. Thus, no increase in activation is observed in the presence of another positive control substitution, EG58 (CRP HL159 KE101 KN52 EG58). Similarly, both sigma(70) RE596 and sigma(70) RD596 can restore activation by CRP EK58 but not CRP EG58, and they both decrease activation by wild-type CRP. We suggest that E596 and D596 in sigma(70) can positively interact with K58 in AR3, thereby enhancing activation, but negatively interact with E58, thereby decreasing activation. The substitution, KA52 in AR3 increases Class II CRP-dependent activation by removing an inhibitory lysine residue. However, this increase is not observed in the presence of either sigma(70) RE596 or sigma(70) RD596. We conclude that the inhibitory side-chain, K52 in AR3, clashes with R596 in sigma(70). Finally, we show that the sigma(70) RE596 and RD596 substitutions affect CRP-dependent activation from Class II, but not Class I, promoters.

Transcription activation by the Escherichia coli cyclic AMP receptor protein. Receptors bound in tandem at promoters can interact synergistically.

Starting with a semi-synthetic Escherichia coli promoter with a binding site for the cyclic AMP receptor protein (CRP) centred between base-pairs 41 and 42 upstream from the transcription start site, a second upstream CRP-binding site, centred between base-pairs 90 and 91, was introduced. CRP binding to this second upstream site results in a several-fold greater stimulation of CRP-dependent transcription initiation, compared to activation at the starting promoter with just one CRP-binding site. Activation of transcription by the upstream CRP molecule is blocked by the HL159 substitution, suggesting that the upstream-bound CRP makes a direct contact with RNA polymerase. Footprinting experiments suggest that RNA polymerase contacts the promoter DNA between the two CRP-binding sites, most likely due to interactions involving the C-terminal part of the alpha subunit. Synergy between tandem bound CRP molecules in transcription activation requires that the two CRP-binding sites be separated by around 40 or 50 base-pairs, but is not found at intermediate spacings. An experiment in which the upstream CRP-binding site is replaced by a site for the related transcription factor, FNR, shows that heterologous synergistic interactions between FNR and CRP are possible.