Reduction of cellular stress by TolC-dependent efflux pumps in Escherichia coli indicated by BaeSR and CpxARP activation of spy in efflux mutants.

Escherichia coli has nine inner membrane efflux pumps which complex with the outer membrane protein TolC and cognate membrane fusion proteins to form tripartite transperiplasmic pumps with diverse functions, including the expulsion of antibiotics. We recently observed that tolC mutants have elevated activities for three stress response regulators, MarA, SoxS, and Rob, and we suggested that TolC-dependent efflux is required to prevent the accumulation of stressful cellular metabolites. Here, we used spy::lacZ fusions to show that two systems for sensing/repairing extracytoplasmic stress, BaeRS and CpxARP, are activated in the absence of TolC-dependent efflux. In either tolC mutants or bacteria with mutations in the genes for four TolC-dependent efflux pumps, spy expression was increased 6- to 8-fold. spy encodes a periplasmic chaperone regulated by the BaeRS and CpxARP stress response systems. The overexpression of spy in tolC or multiple efflux pump mutants also depended on these systems. spy overexpression was not due to acetate, ethanol, or indole accumulation, since external acetate had only a minor effect on wild-type cells, ethanol had a large effect that was not CpxA dependent, and a tolC tnaA mutant which cannot accumulate internal indole overexpressed spy. We propose that, unless TolC-dependent pumps excrete certain metabolites, the metabolites accumulate and activate at least five different stress response systems.

SoxS-dependent coregulation of ompN and ydbK in a multidrug-resistant Escherichia coli strain.

SoxS, MarA, and Rob are homologous transcriptional activators of numerous superoxide- and antibiotic resistance genes but many of the regulated genes are yet to be characterized. In this study, microarrays and RT-PCR analysis were used to show the overexpression of the ompN porin and its upstream gene, ydbK, in an Escherichia coli multidrug-resistant mutant and in a strain constitutive for SoxS. However, transcriptional fusions revealed that SoxS (not MarA or Rob) only activated the ydbK promoter but not the ompN upstream region. RT-PCR experiments showed the overexpression of a combined ydbK - ompN transcript in the SoxS-overexpressing strain. Surprisingly, a bioinformatic approach revealed no soxbox upstream of the ydbK promoter. Thus, the ydbK and ompN genes are coexpressed in an operon and are likely activated by SoxS indirectly. It is known that YdbK is involved in superoxide resistance. Thus, individual ompN and ydbK mutants were tested for superoxide susceptibility. Nonetheless, only the ydbK mutant was susceptible to paraquat, a superoxide generator. These mutants, as well as an OmpN-overproducing strain, were further tested for antibiotic resistance. No significant decreased susceptibility was observed. Thus, ydbK plays a role in superoxide resistance but no role for either gene is found in resistance to the antibiotics tested.

Promoter discrimination at class I MarA regulon promoters mediated by glutamic acid 89 of the MarA transcriptional activator of Escherichia coli.

Three paralogous transcriptional activators MarA, SoxS, and Rob, activate > 40 Escherichia coli promoters. To understand why MarA does not activate certain promoters as strongly as SoxS, we compared MarA, MarA mutants, and SoxS for their abilities to activate 16 promoters and to bind their cognate marbox binding sites. Replacement of the MarA glutamic acid residue 89 with alanine greatly increased the marbox binding and activation of many class I promoters. Like cells constitutive for SoxS, cells expressing the MarA with the E89A mutation were more resistant to superoxides than those harboring WT MarA. The activities of several other E89 substitutions ranked as follows: E89A > E89G > E89V > WT > E89D. Increased binding and activation occurred only at class I promoters when the 12th base of the promoter's marbox (a position at which there is no known interaction between the marbox and MarA) was not a T residue. Furthermore, WT MarA binding to a synthetic marbox in vitro was enhanced when the phosphate group between positions 12 and 13 was eliminated on one strand. The results demonstrate that relatively minor changes in a single amino acid side chain (e.g., alanine to valine or glutamic acid to aspartic acid) can strongly influence activity despite any evidence that the side chain is involved in positive interactions with either DNA or RNA polymerase. We present a model which attributes the differences in binding and activation to the interference between the ß- and γ-carbons of the amino acid at position 89 and the phosphate group between positions 12 and 13.

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.

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.

Model of transcriptional activation by MarA in Escherichia coli.

The AraC family transcription factor MarA activates approximately 40 genes (the marA/soxS/rob regulon) of the Escherichia coli chromosome resulting in different levels of resistance to a wide array of antibiotics and to superoxides. Activation of marA/soxS/rob regulon promoters occurs in a well-defined order with respect to the level of MarA; however, the order of activation does not parallel the strength of MarA binding to promoter sequences. To understand this lack of correspondence, we developed a computational model of transcriptional activation in which a transcription factor either increases or decreases RNA polymerase binding, and either accelerates or retards post-binding events associated with transcription initiation. We used the model to analyze data characterizing MarA regulation of promoter activity. The model clearly explains the lack of correspondence between the order of activation and the MarA-DNA affinity and indicates that the order of activation can only be predicted using information about the strength of the full MarA-polymerase-DNA interaction. The analysis further suggests that MarA can activate without increasing polymerase binding and that activation can even involve a decrease in polymerase binding, which is opposite to the textbook model of activation by recruitment. These findings are consistent with published chromatin immunoprecipitation assays of interactions between polymerase and the E. coli chromosome. We find that activation involving decreased polymerase binding yields lower latency in gene regulation and therefore might confer a competitive advantage to cells. Our model yields insights into requirements for predicting the order of activation of a regulon and enables us to suggest that activation might involve a decrease in polymerase binding which we expect to be an important theme of gene regulation in E. coli and beyond.

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.

Transcriptional activation by MarA, SoxS and Rob of two tolC promoters using one binding site: a complex promoter configuration for tolC in Escherichia coli.

SUMMARY: The Escherichia coli tolC encodes a major outer membrane protein with multiple functions in export (e.g. diverse xenobiotics, haemolysin) and as an attachment site for phage and colicins. tolC is regulated in part by MarA, SoxS and Rob, three paralogous transcriptional activators which bind a sequence called the marbox and which activate multiple antibiotic and superoxide resistance functions. Two previously identified tolC promoters, p1 and p2, are not regulated by MarA, SoxS or Rob but p2 is activated by EvgAS and PhoPQ which also regulate other functions. Using transcriptional fusions and primer extension assays, we show here that tolC has two additional strong overlapping promoters, p3 and p4, which are downstream of p1, p2 and the marbox and are activated by MarA, SoxS and Rob. p3 and p4 are configured so that a single marbox suffices to activate transcription from both promoters. At the p3 promoter, the marbox is separated by 20 bp from the -10 hexamer for RNA polymerase but at the p4 promoter, the same marbox is separated by 30 bp from the -10 hexamer. The multiple tolC promoters may allow the cell to respond to diverse environments by co-ordinating tolC transcription with other appropriate functions.

Activation of the Escherichia coli marA/soxS/rob regulon in response to transcriptional activator concentration.

The paralogous transcriptional activators MarA, SoxS, and Rob activate a common set of promoters, the marA/soxS/rob regulon of Escherichia coli, by binding a cognate site (marbox) upstream of each promoter. The extent of activation varies from one promoter to another and is only poorly correlated with the in vitro affinity of the activator for the specific marbox. Here, we examine the dependence of promoter activation on the level of activator in vivo by manipulating the steady-state concentrations of MarA and SoxS in Lon protease mutants and by measuring promoter activation using lacZ transcriptional fusions. We found that: (i) the MarA concentrations needed for half-maximal stimulation varied by at least 19-fold among the 10 promoters tested; (ii) most marboxes were not saturated when there were 24,000 molecules of MarA per cell; (iii) the correlation between the MarA concentration needed for half-maximal promoter activity in vivo and marbox binding affinity in vitro was poor; and (iv) the two activators differed in their promoter activation profiles. The marRAB and sodA promoters could both be saturated by MarA and SoxS in vivo. However, saturation by MarA resulted in greater marRAB and lesser sodA transcription than did saturation by SoxS, implying that the two activators interact with RNA polymerase in different ways at the different promoters. Thus, the concentration and nature of activator determine which regulon promoters are activated, as well as the extent of their activation.

A comparison of higher order aberrations following implantation of four foldable intraocular lens designs.

PURPOSE: To determine the higher order aberrations following implantation of four foldable intraocular lens (IOL) designs. METHODS: One hundred twenty-two eyes were randomly assigned to undergo implantation with one of four foldable IOL designs--Collamer (STAAR Surgical, Monrovia, Calif), plate haptic silicone (STAAR Surgical), AcrySof (Alcon, Ft Worth, Tex), or the Sensar (Advanced Medical Optics, Santa Ana, Calif). All enrolled eyes underwent wavefront assessment using the Tracey Visual Function Analyzer (Tracey Technologies, Houston, Tex). RESULTS: Preoperatively, no significant differences were noted in higher order aberrations between the four lens groups. No significant differences were noted among the plate haptic silicone, Sensar, or AcrySof lenses at 1 week or 1 month postoperatively, or among the Collamer, plate haptic silicone, or Sensar lenses, with regard to total higher order aberrations, although differences were observed in individual aberration terms. At 1 week and 1 month postoperatively, the Collamer IOL had significantly less higher order aberrations than the AcrySof lens (P < .01). Significantly less third and fourth order aberrations, coma, trefoil, spherical aberration, and tetrafoil were observed postoperatively with the Collamer than the AcrySof IOL. There were no parameters where the AcrySof had significantly less optical aberrations than the Collamer. CONCLUSIONS: The Collamer IOL design is associated with fewer higher order aberrations than the AcrySof lens.

Detection of low-level promoter activity within open reading frame sequences of Escherichia coli.

The search for promoters has largely been confined to sequences upstream of open reading frames (ORFs) or stable RNA genes. Here we used a cloning approach to discover other potential promoters in Escherichia coli. Chromosomal fragments of approximately 160 bp were fused to a promoterless lacZ reporter gene on a multi-copy plasmid. Eight clones were deliberately selected for high activity and 105 clones were selected at random. All eight of the high-activity clones carried promoters that were located upstream of an ORF. Among the randomly-selected clones, 56 had significantly elevated activity. Of these, 7 had inserts which also mapped upstream of an ORF, while 49 mapped within or downstream of ORFs. Surprisingly, the eight promoters selected for high activity matched the canonical sigma70 -35 and -10 sequences no better than sequences from the randomly-selected clones. For six of the nine most active sequences with orientations opposite to that of the ORF, chromosomal expression was detected by RT-PCR, but defined transcripts were not detected by northern analysis. Our results indicate that the E.coli chromosome carries numerous -35 and -10 sequences with weak promoter activity but that most are not productively expressed because other features needed to enhance promoter activity and transcript stability are absent.

Versatility of the carboxy-terminal domain of the alpha subunit of RNA polymerase in transcriptional activation: use of the DNA contact site as a protein contact site for MarA.

The transcriptional activator, MarA, interacts with RNA polymerase (RNAP) to activate promoters of the mar regulon. Here, we identify the interacting surfaces of MarA and of the carboxy-terminal domain of the alpha subunit of RNAP (alpha-CTD) by NMR-based chemical shift mapping. Spectral changes were monitored for a MarA-DNA complex upon titration with alpha-CTD, and for alpha-CTD upon titration with MarA-DNA. The mapping results were confirmed by mutational studies and retention chromatography. A model of the ternary complex shows that alpha-CTD uses a '265-like determinant' to contact MarA at a surface distant from the DNA. This is unlike the interaction of alpha-CTD with the CRP or Fis activators where the '265 determinant' contacts DNA while another surface of the same alpha-CTD molecule contacts the activator. These results reveal a new versatility for alpha-CTD in transcriptional activation.

Transcriptional and translational regulation of the marRAB multiple antibiotic resistance operon in Escherichia coli.

The marRAB multiple antibiotic resistance operon of Escherichia coli is autorepressed by MarR. MarR binds to two palindromic sequences in vitro: site I lies between and overlaps the -35 and -10 hexamers for RNA polymerase binding; site II lies between the transcription start site and the GTG initiation codon of marR. To assess the importance of these sites in vivo, the effects of mutant sites on transcription were analysed using fusions to lacZ in the presence and absence of wild-type MarR. When both sites were wild type, transcription in the derepressed marR-deleted strain was 19-fold that of the wild-type strain; when only site I or site II was wild type, this ratio was reduced to 4.3- and 2.6-fold, respectively, showing that full repression requires both sites, but some repression can occur at one site independently of the other. Translational fusions of the wild-type promoter to lacZ demonstrated that marR translation proceeds at only 4.5% of the transcription rate. Analysis of translational fusions with mutant leader sequences demonstrated that the principal reason for inefficient translation is a weak Shine-Dalgarno (SD) sequence, AGG(G). Although the SD sequence is located within the potential stem-loop structure of site II, no evidence for occlusion of the SD sequence was found in the wild-type strain. However, a single basepair mutation that strengthens the stem-loop structure drastically reduced the translational efficiency. Substitution of ATG for GTG as the initiation codon increased translational efficiency by 50%. Increasing the 5 bp spacing between the SD sequence and the GTG codon by one to four bases reduced the translational efficiency by 50-75%. Inefficient translation of marR may help to sensitize the cell to environmental signals.

Genomics of the marA/soxS/rob regulon of Escherichia coli: identification of directly activated promoters by application of molecular genetics and informatics to microarray data.

Microarray analyses are providing a plethora of data concerning transcriptional responses to specific gene regulators and their inducers but do not distinguish between direct and indirect responses. Here, we identify directly activated promoters of the overlapping marA, soxS and rob regulon(s) of Escherichia coli by applying informatics, genomics and molecular genetics to microarray data obtained by others. Those studies found that overexpression of marA, or the treatment of cells with salicylate to derepress marA, or treatment with paraquat to induce soxS, resulted in elevated transcription of 153 genes. However, only 27 out of the promoters showed increased transcription under at least two of the aforementioned conditions and eight of those were previously known to be directly activated. A computer algorithm was used to identify potential activator binding sites located upstream of the remaining 19 promoters of this subset, and conventional genetic and biochemical approaches were applied to test whether these sites are critical for activation by the homologous MarA, SoxS and Rob transcriptional activators. Only seven out of the 19 promoters were found to be activated when fused to lacZ and tested as single lysogens. All seven contained an essential activator binding site. The remaining promoters were insensitive to stimulation by the inducers suggesting that the great majority of elevated microarray transcripts either were misidentified or resulted from indirect effects requiring sequences outside of the promoter region. We estimate that the total number of directly activated promoters in the regulon is less than 40.

Complex formation between activator and RNA polymerase as the basis for transcriptional activation by MarA and SoxS in Escherichia coli.

Transcriptional activation in Escherichia coli is generally considered to proceed via the formation of an activator-DNA-RNA polymerase (RNP) ternary complex. Although the order of assembly of the three elements is thermodynamically irrelevant, a prevalent idea is that the activator-DNA complex is formed first, and recruitment of RNP to the binary complex occurs subsequently. We show here that the closely related activators, MarA, SoxS and Rob, which activate the same family of genes, are capable of forming complexes with RNP core or holoenzyme in the absence of DNA. In addition, we find that the ternary MarA-DNA-RNP and SoxS-DNA-RNP complexes are more stable than the corresponding Rob-DNA-RNP complex, although the binary Rob-DNA complex is often more stable than the corresponding MarA- or SoxS-DNA complexes. These results may help to explain certain puzzling aspects of the MarA/SoxS/Rob system. We suggest that activator-RNP complexes scan the chromosome and bind promoters of the regulon more efficiently than either RNP or the activators alone.

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 Rob's 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.