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ABSTRACTEnteroaggregative Escherichia coli(EAEC) is a common diarrhoeagenic human pathogen, isolated from patients in both developing and industrialized countries, that is becoming increasingly resistant to many frontline antibiotics. In this study, we screened 50 E. colistrains from children presenting with diarrhea at the outpatients clinic of Assiut University Children’s Hospital, Egypt. We show that all of these isolates were resistant to multiple classes of antibiotics and identified two as being typical EAEC strains. Using whole genome sequencing, we determined that both isolates carried, amongst others, blaCTX-Mand blaTEMantibiotic resistance genes, as well as many classical EAEC virulence determinants, including the transcriptional regulator, AggR. We demonstrate that the expression of these virulence determinants is dependent on AggR, including aar, which encodes for a repressor of AggR, Aar. Since biofilm formation is the hallmark of EAEC infection, we examined the effect of Aar overexpression on both biofilm formation and AggR-dependent gene expression. We show that whilst Aar has a minimal effect on AggR-dependent transcription it is able to completely disrupt biofilm formation, suggesting that Aar affects these two processes differently. Taken together, our results suggest a model for the induction of virulence gene expression in EAEC that may explain the ubiquity of EAEC in both sick and healthy individuals.

Despite enormous progress in understanding the fundamentals of bacterial gene regulation, our knowledge remains limited when compared with the number of bacterial genomes and regulatory systems to be discovered. Derived from a small number of initial studies, classic definitions for concepts of gene regulation have evolved as the number of characterized promoters has increased. Together with discoveries made using new technologies, this knowledge has led to revised generalizations and principles. In this Expert Recommendation, we suggest precise, updated definitions that support a logical, consistent conceptual framework of bacterial gene regulation, focusing on transcription initiation. The resulting concepts can be formalized by ontologies for computational modelling, laying the foundation for improved bioinformatics tools, knowledge-based resources and scientific communication. Thus, this work will help researchers construct better predictive models, with different formalisms, that will be useful in engineering, synthetic biology, microbiology and genetics.

Gene expression in bacteria relies on promoter recognition by the DNA-dependent RNA polymerase and subsequent transcription initiation. Bacterial cells are able to tune their transcriptional programmes to changing environments, through numerous mechanisms that regulate the activity of RNA polymerase, or change the set of promoters to which the RNA polymerase can bind. In this Review, we outline our current understanding of the different factors that direct the regulation of transcription initiation in bacteria, whether by interacting with promoters, with RNA polymerase or with both, and we discuss the diverse molecular mechanisms that are used by these factors to regulate gene expression.

ABSTRACTRamA is a transcription factor involved in regulating multidrug resistance in Salmonella entericaserovar Typhimurium SL1344. Green fluorescent protein (GFP) reporter fusions were exploited to investigate the regulation of RamA expression by RamR. We show that RamR represses the ramApromoter by binding to a palindromic sequence and describe a superrepressor RamR mutant that binds to the ramApromoter sequence more efficiently, thus exhibiting a ramAinactivated phenotype.

Bacteria use their genetic material with great effectiveness to make the right products in the correct amounts at the appropriate time. Studying bacterial transcription initiation in Escherichia coli has served as a model for understanding transcriptional control throughout all kingdoms of life. Every step in the pathway between gene and function is exploited to exercise this control, but for reasons of economy, it is plain that the key step to regulate is the initiation of RNA-transcript formation.

Transcription activation by the Escherichia coli cyclic AMP receptor protein (CRP) at different promoters has been studied using RNA polymerase holoenzyme derivatives containing two full-length alpha subunits, or containing one full-length alpha subunit and one truncated alpha subunit lacking the alpha C-terminal domain (alpha CTD). At a promoter having a single DNA site for CRP, activation requires only one full-length alpha subunit. Likewise, at a promoter having a single DNA site for CRP and one adjacent UP-element subsite (high-affinity DNA site for alpha CTD), activation requires only one full-length alpha subunit. In contrast, at promoters having two DNA sites for CRP, or one DNA site for CRP and two UP-element subsites, activation requires two full-length alpha subunits. We conclude that a single copy of alpha CTD is sufficient to interact with one CRP molecule and one adjacent UP-element subsite, but two copies of alpha CTD are required to interact with two CRP molecules or with one CRP molecule and two UP-element subsites.

The Escherichia coli cAMP receptor protein (CRP) is a factor that activates transcription at over 100 target promoters. At Class I CRP-dependent promoters, CRP binds immediately upstream of RNA polymerase and activates transcription by making direct contacts with the C-terminal domain of the RNA polymerase α subunit (αCTD). Since αCTD is also known to interact with DNA sequence elements (known as UP elements), we have constructed a series of semi-synthetic Class I CRP-dependent promoters, carrying both a consensus DNA-binding site for CRP and a UP element at different positions. We previously showed that, at these promoters, the CRP–αCTD interaction and the CRP–UP element interaction contribute independently and additively to transcription initiation. In this study, we show that the two halves of the UP element can function independently, and that, in the presence of the UP element, the best location for the DNA site for CRP is position -69.5. This suggests that, at Class I CRP-dependent promoters where the DNA site for CRP is located at position -61.5, the two αCTDs of RNA polymerase are not optimally positioned. Two experiments to test this hypothesis are presented.

We have studied the formation of open complexes between purified RNA polymerase from Escherichia coli and DNA fragments carrying the galP1 promoter, a promoter with an extended -10 region. Unusually, these complexes are formed readily at low temperatures. This low-temperature opening is unaffected by deletions of either upstream or downstream promoter sequences. We conclude that low-temperature open-complex formation is due to specific base sequences in and just upstream of the extended -10 region. In contrast, open complexes are not formed at low temperatures with DNA fragments carrying the E. coli cysG promoter, which also has an extended -10 region. This demonstrates that an extended -10 sequence alone is not sufficient for low-temperature opening. Additionally, we report the temperature dependence of a hybrid galP1–cysG promoter, the related galP2 and galP3 promoters and a derivative of galP1 with an improved -10 hexamer sequence.

ABSTRACTIn a previous study of promoters dependent on the Escherichia colicyclic AMP receptor protein (CRP), carrying tandem DNA sites for CRP, we found that the upstream-bound CRP could either enhance or repress transcription, depending on its location. Here, we have analyzed the interactions between CRP and the C-terminal domains of the RNA polymerase α subunits at some of these promoters. We report that the upstream-bound CRP interacts with these domains irrespective of whether it up- or downregulates promoter activity. Hence, disruption of this interaction can lead to either down- or upregulation, depending on its location.IMPORTANCEMany bacterial promoters carry multiple DNA sites for transcription factors. While most factors that downregulate promoter activity bind to targets that overlap or are downstream of the transcription start and −10 element, very few cases of repression from upstream locations have been reported. Since more Escherichia colipromoters are regulated by cyclic AMP receptor protein (CRP) than by any other transcription factor, and since multiple DNA sites for CRP are commonplace at promoters, our results suggest that promoter downregulation by transcription factors may be more prevalent than hitherto thought, and this will have implications for the annotation of promoters from new bacterial genome sequences.

ABSTRACTThe bacterial SeqA protein binds to hemi-methylated GATC sequences that arise in newly synthesized DNA upon passage of the replication machinery. In Escherichia coliK-12, the single replication origin oriCis a well-characterized target for SeqA, which binds to multiple hemi-methylated GATC sequences immediately after replication has initiated. This sequesters oriC, thereby preventing reinitiation of replication. However, the genome-wide DNA binding properties of SeqA are unknown, and hence, here, we describe a study of the binding of SeqA across the entire Escherichia coliK-12 chromosome, using chromatin immunoprecipitation in combination with DNA microarrays. Our data show that SeqA binding correlates with the frequency and spacing of GATC sequences across the entire genome. Less SeqA is found in highly transcribed regions, as well as in the termacrodomain. Using synchronized cultures, we show that SeqA distribution differs with the cell cycle. SeqA remains bound to some targets after replication has ceased, and these targets locate to genes encoding factors involved in nucleotide metabolism, chromosome replication, and methyl transfer.IMPORTANCEDNA replication in bacteria is a highly regulated process. In many bacteria, a protein called SeqA plays a key role by binding to newly replicated DNA. Thus, at the origin of DNA replication, SeqA binding blocks premature reinitiation of replication rounds. Although most investigators have focused on the role of SeqA at replication origins, it has long been suspected that SeqA has a more pervasive role. In this study, we describe how we have been able to identify scores of targets, across the entire Escherichia colichromosome, to which SeqA binds. Using synchronously growing cells, we show that the distribution of SeqA between these targets alters as replication of the chromosome progresses. This suggests that sequential changes in SeqA distribution orchestrate a program of gene expression that ensures coordinated DNA replication and cell division.

Transcription activation by the Escherichia coli cyclic AMP receptor protein (CRP) at Class II promoters is dependent on direct interactions between two surface-exposed activating regions (AR1 and AR2) and two contact sites in RNA polymerase. The effects on transcription activation of disrupting either AR1 or AR2 have been measured at different Class II promoters. AR2 but not AR1 is essential for activation at all the Class II promoters that were tested. The effects of single positive control substitutions in AR1 and AR2 vary from one promoter to another: the effects of the different substitutions are contingent on the −35 hexamer sequence. Abortive initiation assays have been used to quantify the effects of positive control substitutions in each activating region on the kinetics of transcription initiation at the Class II CRP-dependent promoter pmelRcon. At this promoter, the HL159 substitution in AR1 results in a defect in the initial binding of RNA polymerase whilst the KE101 substitution in AR2 reduces the rate of isomerization from the closed to the open complex.

Hydroxyl radical footprinting has been used to study different open complexes between Escherichia coli RNA polymerase and the galactose operon regulatory region, which contains two overlapping promoters, P1 and P2. Complexes at P1 were studied by exploiting a P2- mutant and complexes at P2 were studied with a P1- mutant. We have identified the precise location of a binding in both binary RNA polymerase-galP1 and RNA polymerase-P2 complexes from the effects of deletion of the C-terminal domain of the RNA polymerase α subunit: α binds to different sites at the upstream end of each complex. Transcription initiation at galP1 can be activated by the cyclic AMP receptor protein (CRP). Addition of CRP to the RNA polymerase-galP1 complex displaces the C-terminal domain of α, which then binds to a different site upstream of CRP in the ternary CRP-RNA polymerase-galP1 complex. Thus, the C-terminal domain of α can occupy three different sites at the gal operon regulatory region. We have also examined the effect of disrupting the Activating Region of CRP on interactions between CRP and the C-terminal domain of α in ternary CRP-RNA polymerase-galP1 complexes. Footprinting experiments show that these substitutions interfere with the contact between CRP and α but do not affect the position of α binding to its site upstream of bound CRP.

A library of random mutations in the Escherichia coli fnr gene has been screened to identify positive control mutants of FNR that are defective in transcription activation at Class I promoters. Single amino acid substitutions at D43, R72, S73, T118, M120, F181, F186, S187 and F191 identify a surface of FNR that is essential for activation which, presumably, makes contact with the C-terminal domain of the RNA polymerase α subunit. This surface is larger than the corresponding activating surface of the related transcription activator, CRP. To identify the contact surface in the C-terminal domain of the RNA polymerase α subunit, a library of mutations in the rpoA gene was screened for α mutants that interfered with transcription activation at Class I FNR-dependent promoters. Activation was reduced by deletions of the α C-terminal domain, by substitutions known to affect DNA binding by α, by substitutions at E261 and by substitutions at L300, E302, D305, A308, G315 and R317 that appear to identify contact surfaces of α that are likely to make contact with FNR at Class I promoters. Again, this surface differs from the surface used by CRP at Class I CRP-dependent promoters.

A set of nested deletions has been made In the Escherichia coll melR gene, encoding the MelR transcription activator protein. Expression of the resulting meIR derivatives led to the production of nine MeIR proteins with N-termlnal deletions of different lengths. The properties of the shortened proteins have been studied both In vivo and in vitro None of the truncated proteins activate transcription from the E.coll melAB promoter but three; MelR220, MelR183 and MelR173, Inhibit activation of the melAB promoter by chromoso-mally-encoded full-length MeIR. In gel retardation assays, both MeIR 183 and MelR173 clearly retard DNA fragments carrying the melAB promoter. MeIR 173 has been overproduced In a T7 expression system and shown to be stable In vivo for up to 2 h. DNAase I footprintlng assays of partially purified protein show that it binds to the melAB promoter, protecting the same sites as the full-length protein. This fragment may be suitable for further structure/function studies of this class of transcription activator.