Judah L. Rosner

The AraC transcriptional activators

The AraC family of bacterial transcriptional activators regulate diverse genetic systems. Recent X-ray diffraction studies show that the monomeric MarA and Rob activators bind to their asymmetric degenerate DNA sites via two different helix-turn-helix elements. Activation by MarA, SoxS or Rob requires a particular orientation of the asymmetric binding sequence (and hence the activator), depending on its distance from the -10 RNAP signal. Genetic studies are beginning to clarify how the activators interact with RNAP. Growing evidence suggests that for the sugar metabolism activators, multiple binding sites upstream of the promoter anchor the activator in a repressing or nonactivating configuration. By interaction with the sugar and/or CRP, the activator is allosterically altered so it can bind a new set of sites that enable it to activate the promoter. Surprisingly, the virulence activator, Rns, must bind to both upstream and downstream sites in order to activate the rns promoter.

Structural requirements for marbox function in transcriptional activation of mar/sox/rob regulon promoters in Escherichia coli: sequence, orientation and spatial relationship to the core promoter

The promoters of the mar/sox/rob regulon of Escherichia coli contain a binding site (marbox) for the homologous transcriptional activators MarA, SoxS and Rob. In spite of data from footprinting studies, the marbox has not been precisely defined because of its degeneracy and asymmetry and seemingly variable location with respect to the −10 and −35 hexamers for RNA polymerase (RNP) binding. Here, we use DNA retardation studies and hybrid promoters to identify optimally binding 20u2003bp minimal marboxes from a number of promoters. This has yielded a more defined marbox consensus sequence (AYnGCACnnWnnRYYAAAYn) and has led to the demonstration that some marboxes are inverted relative to others. Using transcriptional fusions to lacZ, we have found that only one marbox orientation is functional at a given location. Moreover, the functional orientation is determined by marbox location: marboxes that are 15 or more basepairs upstream of the −35 hexamer are oriented opposite those closer to the −35 hexamer. Marbox orientation and the spacing between marbox and signals for RNP binding are critical for transcriptional activation, presumably to align MarA with RNP.

Promoter discrimination by the related transcriptional activators MarA and SoxS: differential regulation by differential binding

MarA and SoxS are closely related proteins (≈45% identical) that transcriptionally activate a common set of unlinked genes, resulting in multiple antibiotic and superoxide resistance in Escherichia coli. Both proteins bind as monomers to a 20u2003bp degenerate asymmetric recognition sequence, the ‘marbox’, located upstream of the promoter. However, the proteins differ widely in the extents to which they activate particular promoters, with the consequence that overexpression of SoxS leads to greater superoxide resistance than does overexpression of MarA. This ‘discrimination’ between activators by promoters was demonstrated in vivo, using promoters fused to lacZ, and in vitro, using purified RNA polymerase, promoter DNA and MarA or SoxS. The marbox was found to be a critical element in discrimination by in vivo and in vitro assays of hybrid promoters containing the marbox from one gene and the core promoter from another. Furthermore, by sequential mutation of its marbox, a promoter that discriminated 35‐fold in favour of SoxS was converted into one that did not discriminate. The relative activation of a promoter by MarA or SoxS was paralleled by the relative binding of the two activators to the promoters marbox as assayed by band shift experiments. Thus, differential recognition of closely related marbox sequences by the closely related activators is the primary basis for promoter discrimination. Discrimination enables the cell to customize its response to the stresses that trigger synthesis of the activators.

Posttranscriptional activation of the transcriptional activator Rob by dipyridyl in Escherichia coli

The transcriptional activator Rob consists of an N-terminal domain (NTD) of 120 amino acids responsible for DNA binding and promoter activation and a C-terminal domain (CTD) of 169 amino acids of unknown function. Although several thousand molecules of Rob are normally present per Escherichia coli cell, they activate promoters of the rob regulon poorly. We report here that in cells treated with either 2,2- or 4,4-dipyridyl (the latter is not a metal chelator), Rob-mediated transcription of various rob regulon promoters was increased substantially. A small, growth-phase-dependent effect of dipyridyl on the rob promoter was observed. However, dipyridyl enhanced Robs activity even when rob was regulated by a heterologous (lac) promoter showing that the action of dipyridyl is mainly posttranscriptional. Mutants lacking from 30 to 166 of the C-terminal amino acids of Rob had basal levels of activity similar to that of wild-type cells, but dipyridyl treatment did not enhance this activity. Thus, the CTD is not an inhibitor of Rob but is required for activation of Rob by dipyridyl. In contrast to its relatively low activity in vivo, Rob binding to cognate DNA and activation of transcription in vitro is similar to that of MarA, which has a homologous NTD but no CTD. In vitro nuclear magnetic resonance studies demonstrated that 2,2-dipyridyl binds to Rob but not to the CTD-truncated Rob or to MarA, suggesting that the effect of dipyridyl on Rob is direct. Thus, it appears that Rob can be converted from a low activity state to a high-activity state by a CTD-mediated mechanism in vivo or by purification in vitro.

An Excretory Function for the Escherichia coli Outer Membrane Pore TolC: Upregulation of marA and soxS Transcription and Rob Activity Due to Metabolites Accumulated in tolC Mutants

Efflux pumps function to rid bacteria of xenobiotics, including antibiotics, bile salts, and organic solvents. TolC, which forms an outer membrane channel, is an essential component of several efflux pumps in Escherichia coli. We asked whether TolC has a role during growth in the absence of xenobiotics. Because tolC transcription is activated by three paralogous activators, MarA, SoxS, and Rob, we examined the regulation of these activators in tolC mutants. Using transcriptional fusions, we detected significant upregulation of marRAB and soxS transcription and Rob protein activity in tolC mutants. Three mechanisms could be distinguished: (i) activation of marRAB transcription was independent of marRAB, soxR, and rob functions; (ii) activation of soxS transcription required SoxR, a sensor of oxidants; and (iii) Rob protein was activated posttranscriptionally. This mechanism is similar to the mechanisms of upregulation of marRAB, soxS, and Rob by treatment with certain phenolics, superoxides, and bile salts, respectively. The transcription of other marA/soxS/rob regulon promoters, including tolC itself, was also elevated in tolC mutants. We propose that TolC is involved in the efflux of certain cellular metabolites, not only xenobiotics. As these metabolites accumulate during growth, they trigger the upregulation of MarA, SoxS, and Rob, which in turn upregulate tolC and help rid the bacteria of these metabolites, thereby restoring homeostasis.

Identification of the Escherichia coli K-12 ybhE Gene as pgl, Encoding 6-Phosphogluconolactonase

We report identification of the Escherichia coli ybhE gene as the pgl gene that encodes 6-phosphogluconolactonase. A tentative identification was first made based on the known approximate location of the pgl gene and the similarity of the presumptive ybhE-encoded protein sequence to a known Pgl enzyme. To test this notion, the ybhE gene was deleted and replaced with a drug marker. Like previously characterized pgl mutants, the ybhE deletion mutant had a Blu- phenotype (dark-blue staining with iodine due to accumulation of starch after growth on minimal maltose) and demonstrated impaired growth on minimal glucose medium when combined with a pgi mutation. Biochemical assay of crude extracts for 6-phosphogluconolactonase enzymatic activity showed that ybhE encodes this activity. The ybhE gene was transferred from the E. coli chromosome to an expression vector. This ybhE clone complemented both the precise deletion of the ybhE gene and a larger deletion, pglDelta8, for the Blu- phenotype and for phosphogluconolactonase activity, confirming that ybhE is the pgl gene. A newly observed phenotype of pgl strains is a lowered frequency of appearance of Bgl+ mutants that can utilize the beta-glucoside salicin. This is likely due to poor growth of Bgl+ pgl strains on salicin due to the accumulation of 6-phosphogluconolactone.

Constitutive SoxS Expression in a Fluoroquinolone-Resistant Strain with a Truncated SoxR Protein and Identification of a New Member of the marA-soxS-rob Regulon, mdtG

ABSTRACT Elevated levels of fluoroquinolone resistance are frequently found among Escherichia coli clinical isolates. This study investigated the antibiotic resistance mechanisms of strain NorE5, derived in vitro by exposing an E. coli clinical isolate, PS5, to two selection steps with increasing concentrations of norfloxacin. In addition to the amino acid substitution in GyrA (S83L) present in PS5, NorE5 has an amino acid change in ParC (S80R). Furthermore, we now find by Western blotting that NorE5 has a multidrug resistance phenotype resulting from the overexpression of the antibiotic resistance efflux pump AcrAB-TolC. Microarray and gene fusion analyses revealed significantly increased expression in NorE5 of soxS, a transcriptional activator of acrAB and tolC. The high soxS activity is attributable to a frameshift mutation that truncates SoxR, rendering it a constitutive transcriptional activator of soxS. Furthermore, microarray and reverse transcription-PCR analyses showed that mdtG (yceE), encoding a putative efflux pump, is overexpressed in the resistant strain. SoxS, MarA, and Rob activated an mdtG::lacZ fusion, and SoxS was shown to bind to the mdtG promoter, showing that mdtG is a member of the marA-soxS-rob regulon. The mdtG marbox sequence is in the backward or class I orientation within the promoter, and its disruption resulted in a loss of inducibility by MarA, SoxS, and Rob. Thus, chromosomal mutations in parC and soxR are responsible for the increased antibiotic resistance of NorE5.

Detection of chemicals that stimulate Tn9 transposition in Escherichia coli K12.

SummaryA spot test has been developed for detecting substances that enhance the transposition of Tn9 in Escherichia coli. Phage λ:: Tn9-infected cells were plated on chloramphenicol media and a drop of the test substance was placed at the center of the plate. Following incubation, chloramphenicol-resistant colonies appeared due to the transposition of Tn9 to the bacterial chromosome. By comparing the test plate and a control plate with respect to the number and distribution of colonies, the effect of the test compound can be evaluated.Out of over 100 compounds tested, acetate, two detergents (Brij 58 and Nonidet P40) and dimethylsulfoxide were found to enhance transposition 3–20 fold. Acetate was also found to enhance the transposition of Tn5 and Tn10. The stimulating effect of Brij 58 was lost when palmitic acid was added with the Brij 58. The nature of these substances, which we refer to as “transposagens”, suggests an involvement of lipid or membrane in the transposition process.

Transposition of IS1-?BIO-IS1 from a bacteriophage ? derivative carrying the IS1-cat-IS1 transposon (Tn9)

SummaryTn9 is a transposable element in which a gene (cat) determining chloramphenicol resistance is flanked by directly repeated sequences that are homologous to the insertion sequence IS1. We show here that infection of Escherichia coli K12 (under Rec- Red- Int- conditions) with a λbio transducing phage carrying Tn9 results in the formation of λbio transductants as frequently as cat transductants (about 1 per 106 to 107 infected cells). Most of the λbio transductants do not carry cat, just as most of the cat transductants do not carry λbio. In spite of the absence of cat, the λbio prophage can transpose a second time, from the E. coli chromosome to different sites on an F′ gal plasmid. Analysis of the structure of the transposed λbio element, by restriction nuclease digestion and by electron microscopy, demonstrates that the integrated λbio prophage is flanked by directly repeated IS1 elements. We conclude that there is no genetic information for the ability to transpose encoded in the non-repeated portion of Tn9, i.e. that the directly repeated IS1 elements alone are responsible for Tn9 transposition.

Analysis of Microarray Data for the marA, soxS, and rob Regulons of Escherichia coli

Publisher Summary This chapter explores microarray technology and its potential to identify a large number of the genes/promoters affected by the environmental stimulus or the regulatory protein, or RNA. It discusses the new experimental, statistical, and computational methods that are expected to improve the technique, but may not resolve all issues. Ultimately, each candidate gene or promoter has to be confirmed directly. Study of the marA, soxS, and rob regulons of Escherichia coli illustrates the approach for using microarray results. These regulons are highly overlapping because the transcriptional activators that directly control them—MarA, SoxS, and Rob—are highly homologous and are bound with roughly similar affinities to a 20-bp asymmetric degenerate sequence. The chapter reviews the data that was pooled from three microarray conditions which used arrays from the same manufacturer, and followed similar protocols.