H-NS is a bacterial transposon capture protein.
Published: 20th August 2024
Authors: Cooper C, Legood S, Wheat RL, Forrest D, Sharma P, Haycocks JRJ, Grainger DC.
Journal: Nature Communications
Abstract:
The histone-like nucleoid structuring (H-NS) protein is a DNA binding factor, found in gammaproteobacteria, with functional equivalents in diverse microbes. Universally, such proteins are understood to silence transcription of horizontally acquired genes. Here, we identify transposon capture as a major overlooked function of H-NS. Using genome-scale approaches, we show that H-NS bound regions are transposition “hotspots”. Since H-NS often interacts with pathogenicity islands, such targeting creates clinically relevant phenotypic diversity. For example, in Acinetobacter baumannii, we identify altered motility, biofilm formation, and interactions with the human immune system. Transposon capture is mediated by the DNA bridging activity of H-NS and, if absent, more ubiquitous transposition results. Consequently, transcribed and essential genes are disrupted. Hence, H-NS directs transposition to favour evolutionary outcomes useful for the host cell.
The environmentally-regulated interplay between local three-dimensional chromatin organisation and transcription of proVWX in E. coli.
Published: 17th November 2023
Authors: Rashid FM, Crémazy FGE, Hofmann A, Forrest D, Grainger DC, Heermann DW, Dame RT.
Journal: Nature Communications
Abstract:
Nucleoid associated proteins (NAPs) maintain the architecture of bacterial chromosomes and regulate gene expression. Thus, their role as transcription factors may involve three-dimensional chromosome re-organisation. While this model is supported by in vitro studies, direct in vivo evidence is lacking. Here, we use RT-qPCR and 3C-qPCR to study the transcriptional and architectural profiles of the H-NS (histone-like nucleoid structuring protein)-regulated, osmoresponsive proVWX operon of Escherichia coli at different osmolarities and provide in vivo evidence for transcription regulation by NAP-mediated chromosome re-modelling in bacteria. By consolidating our in vivo investigations with earlier in vitro and in silico studies that provide mechanistic details of how H-NS re-models DNA in response to osmolarity, we report that activation of proVWX in response to a hyperosmotic shock involves the destabilization of H-NS-mediated bridges anchored between the proVWX downstream and upstream regulatory elements (DRE and URE), and between the DRE and ygaY that lies immediately downstream of proVWX. The re-establishment of these bridges upon adaptation to hyperosmolarity represses the operon. Our results also reveal additional structural features associated with changes in proVWX transcript levels such as the decompaction of local chromatin upstream of the operon, highlighting that further complexity underlies the regulation of this model operon. H-NS and H-NS-like proteins are wide-spread amongst bacteria, suggesting that chromosome re-modelling may be a typical feature of transcriptional control in bacteria.
A simple mechanism for integration of quorum sensing and cAMP signalling in Vibrio cholerae
Published: 6th July 2023
Authors: Walker LM, Haycocks JRJ, Van Kessel JC, Dalia TN, Dalia AB, Grainger DC.
Journal: Elife
Abstract:
Many bacteria use quorum sensing to control changes in lifestyle. The process is regulated by microbially derived ‘autoinducer’ signalling molecules, that accumulate in the local environment. Individual cells sense autoinducer abundance, to infer population density, and alter their behaviour accordingly. In Vibrio cholerae, quorum-sensing signals are transduced by phosphorelay to the transcription factor LuxO. Unphosphorylated LuxO permits expression of HapR, which alters global gene expression patterns. In this work, we have mapped the genome-wide distribution of LuxO and HapR in V. cholerae. Whilst LuxO has a small regulon, HapR targets 32 loci. Many HapR targets coincide with sites for the cAMP receptor protein (CRP) that regulates the transcriptional response to carbon starvation. This overlap, also evident in other Vibrio species, results from similarities in the DNA sequence bound by each factor. At shared sites, HapR and CRP simultaneously contact the double helix and binding is stabilised by direct interaction of the two factors. Importantly, this involves a CRP surface that usually contacts RNA polymerase to stimulate transcription. As a result, HapR can block transcription activation by CRP. Thus, by interacting at shared sites, HapR and CRP integrate information from quorum sensing and cAMP signalling to control gene expression. This likely allows V. cholerae to regulate subsets of genes during the transition between aquatic environments and the human host.
An unexpected abundance of bidirectional promoters within Salmonella Typhimurium plasmids
Published: 19th May 2023
Authors: Middlemiss AD, Warman EA, Forrest D, Haycocks JRJ, Grainger DC.
Journal: Microbiology
Abstract:
Transcription of the DNA template, to generate an RNA message, is the first step in gene expression. The process initiates at DNA sequences called promoters. Conventionally, promoters have been considered to drive transcription in a specific direction. However, in recent work, we showed that many prokaryotic promoters can drive divergent transcription. This is a consequence of key DNA sequences for transcription initiation being inherently symmetrical. Here, we used global transcription start site mapping to determine the prevalence of such bidirectional promoters in Salmonella Typhimurium. Surprisingly, bidirectional promoters occur three times more frequently in plasmid components of the genome compared to chromosomal DNA. Implications for the evolution of promoter sequences are discussed.
Mapping direct and indirect MarA/SoxS/Rob/RamA regulons in Salmonella Typhimurium reveals repression of csgD and biofilm formation
Published: 19th May 2023
Authors: Middlemiss AD, Haycocks JRJ, Stringer AM, Piddock LJV, Wade JT, Grainger DC.
Journal: Microbiology
Abstract:
The closely related transcription factors MarA, SoxS, Rob and RamA control overlapping stress responses in many enteric bacteria. Furthermore, constitutive expression of such regulators is linked to clinical antibiotic resistance. In this work we have mapped the binding of MarA, SoxS, Rob and RamA across the Salmonella Typhimurium genome. In parallel, we have monitored changes in transcription start site use resulting from expression of the regulators. Together, these data allow direct and indirect gene regulatory effects to be disentangled. Promoter architecture across the regulon can also be deduced. At a phylogenetic scale, around one third of regulatory targets are conserved in most organisms encoding MarA, SoxS, Rob or RamA. We focused our attention on the control of csgD, which encodes a transcriptional activator responsible for stimulating production of curli fibres during biofilm formation. We show that expression of csgD is particularly sensitive to SoxS that binds upstream to repress transcription. This differs to the situation in Escherichia coli, where MarA regulates csgD indirectly.
Homologs of the Escherichia coli F Element Protein TraR, Including Phage Lambda Orf73, Directly Reprogram Host Transcription
Published: 18th May 2022
Authors: Gopalkrishnan S, Ross W, Akbari MS, Li X, Haycocks JRJ, Grainger DC, Court DL, Gourse RL.
Journal: mBio
Abstract:
Bacterial cells and their associated plasmids and bacteriophages encode numerous small proteins of unknown function. One example, the 73-amino-acid protein TraR, is encoded by the transfer operon of the conjugative F plasmid of Escherichia coli. TraR is a distant homolog of DksA, a protein found in almost all proteobacterial species that is required for ppGpp to regulate transcription during the stringent response. TraR and DksA increase or decrease transcription initiation depending on the kinetic features of the promoter by binding directly to RNA polymerase without binding to DNA. Unlike DksA, whose full activity requires ppGpp as a cofactor, TraR is fully active by itself and unaffected by ppGpp. TraR belongs to a family of divergent proteins encoded by proteobacterial bacteriophages and other mobile elements. Here, we experimentally addressed whether other members of the TraR family function like the F element-encoded TraR. Purified TraR and all 5 homologs that were examined bound to RNA polymerase, functioned at lower concentrations than DksA, and complemented a dksA-null strain for growth on minimal medium. One of the homologs, λ Orf73, encoded by bacteriophage lambda, was examined in greater detail. λ Orf73 slowed host growth and increased phage burst size. Mutational analysis suggested that λ Orf73 and TraR have a similar mechanism for inhibiting rRNA and r-protein promoters. We suggest that TraR and its homologs regulate host transcription to divert cellular resources to phage propagation or conjugation without induction of ppGpp and a stringent response.
Xenogeneic silencing strategies in bacteria are dictated by RNA polymerase promiscuity
Published: 3rd March 2022
Authors: Forrest D, Warman EA, Erkelens AM, Dame RT and Grainger DC.
Journal: Nature Communications
Abstract:
Horizontal gene transfer facilitates dissemination of favourable traits among bacteria. However, foreign DNA can also reduce host fitness: incoming sequences with a higher AT content than the host genome can misdirect transcription. Xenogeneic silencing proteins counteract this by modulating RNA polymerase binding. In this work, we compare xenogeneic silencing strategies of two distantly related model organisms: Escherichia coli and Bacillus subtilis. In E. coli, silencing is mediated by the H-NS protein that binds extensively across horizontally acquired genes. This prevents spurious non-coding transcription, mostly intragenic in origin. By contrast, binding of the B. subtilis Rok protein is more targeted and mostly silences expression of functional mRNAs. The difference reflects contrasting transcriptional promiscuity in E. coli and B. subtilis, largely attributable to housekeeping RNA polymerase σ factors. Thus, whilst RNA polymerase specificity is key to the xenogeneic silencing strategy of B. subtilis, transcriptional promiscuity must be overcome to silence horizontally acquired DNA in E. coli.
Genome-wide mapping of Vibrio cholerae VpsT binding identifies a mechanism for c-di-GMP homeostasis
Published: 11th January 2022
Authors: Guest T, Haycocks JRJ, Warren GZL and Grainger DC.
Journal: Nucleic Acids Research
Abstract:
Many bacteria use cyclic dimeric guanosine monophosphate (c-di-GMP) to control changes in lifestyle. The molecule, synthesized by proteins having diguanylate cyclase activity, is often a signal to transition from motile to sedentary behaviour. In Vibrio cholerae, c-di-GMP can exert its effects via the transcription factors VpsT and VpsR. Together, these proteins activate genes needed for V. cholerae to form biofilms. In this work, we have mapped the genome-wide distribution of VpsT in a search for further regulatory roles. We show that VpsT binds 23 loci and recognises a degenerate DNA palindrome having the consensus 5′-W-5R-4[CG]-3Y-2W-1W+1R+2[GC]+3Y+4W+5-3′. Most genes targeted by VpsT encode functions related to motility, biofilm formation, or c-di-GMP metabolism. Most notably, VpsT activates expression of the vpvABC operon that encodes a diguanylate cyclase. This creates a positive feedback loop needed to maintain intracellular levels of c-di-GMP. Mutation of the key VpsT binding site, upstream of vpvABC, severs the loop and c-di-GMP levels fall accordingly. Hence, as well as relaying the c-di-GMP signal, VpsT impacts c-di-GMP homeostasis.
A dual-function RNA balances carbon uptake and central metabolism in Vibrio cholerae
Published: 6th October 2021
Authors: Venkat K, Hoyos M, Haycocks JR, Cassidy L, Engelmann B, Rolle-Kampczyk U, von Bergen M, Tholey A, Grainger DC and Papenfort K.
Journal: EMBO Journal
Abstract:
Bacterial small RNAs (sRNAs) are well known to modulate gene expression by base pairing with trans-encoded transcripts and are typically non-coding. However, several sRNAs have been reported to also contain an open reading frame and thus are considered dual-function RNAs. In this study, we discovered a dual-function RNA from Vibrio cholerae, called VcdRP, harboring a 29 amino acid small protein (VcdP), as well as a base-pairing sequence. Using a forward genetic screen, we identified VcdRP as a repressor of cholera toxin production and link this phenotype to the inhibition of carbon transport by the base-pairing segment of the regulator. By contrast, we demonstrate that the VcdP small protein acts downstream of carbon transport by binding to citrate synthase (GltA), the first enzyme of the citric acid cycle. Interaction of VcdP with GltA results in increased enzyme activity and together VcdR and VcdP reroute carbon metabolism. We further show that transcription of vcdRP is repressed by CRP allowing us to provide a model in which VcdRP employs two different molecular mechanisms to synchronize central metabolism in V. cholerae.
Widespread divergent transcription from bacterial and archaeal promoters is a consequence of DNA-sequence symmetry
Published: 6th May 2021
Authors: Warman EA, Forrest D, Guest T, Haycocks JRJ, Wade JT and Grainger DC
Journal: Nature Microbiology
Abstract:
Transcription initiates at promoters, DNA regions recognized by a DNA-dependent RNA polymerase. We previously identified horizontally acquired Escherichia coli promoters from which the direction of transcription was unclear. In the present study, we show that more than half of these promoters are bidirectional and drive divergent transcription. Using genome-scale approaches, we demonstrate that 19% of all transcription start sites detected in E. coli are associated with a bidirectional promoter. Bidirectional promoters are similarly common in diverse bacteria and archaea, and have inherent symmetry: specific bases required for transcription initiation are reciprocally co-located on opposite DNA strands. Bidirectional promoters enable co-regulation of divergent genes and are enriched in both intergenic and horizontally acquired regions. Divergent transcription is conserved among bacteria, archaea and eukaryotes, but the underlying mechanisms for bidirectionality are different.
A non-canonical promoter element drives spurious transcription of horizontally acquired bacterial genes
Published: 21st May 2020
Authors: Warman EA, Singh SS, Gubieda AG, Grainger DC.
Journal: Nucleic Acids Research
Abstract:
RNA polymerases initiate transcription at DNA sequences called promoters. In bacteria, the best conserved promoter feature is the AT-rich -10 element; a sequence essential for DNA unwinding. Further elements, and gene regulatory proteins, are needed to recruit RNA polymerase to the -10 sequence. Hence, -10 elements cannot function in isolation. Many horizontally acquired genes also have a high AT-content. Consequently, sequences that resemble the -10 element occur frequently. As a result, foreign genes are predisposed to spurious transcription. However, it is not clear how RNA polymerase initially recognizes such sequences. Here, we identify a non-canonical promoter element that plays a key role. The sequence, itself a short AT-tract, resides 5 base pairs upstream of otherwise cryptic -10 elements. The AT-tract alters DNA conformation and enhances contacts between the DNA backbone and RNA polymerase.
Chromosome organization in bacteria: mechanistic insights into genome structure and function
Published: 25th November 2019
Authors: Dame RT, Rashid FM, Grainger DC.
Journal: Nature Reviews Genetics
Abstract:
Bacterial chromosomes are folded to compact DNA and facilitate cellular processes. Studying model bacteria has revealed aspects of chromosome folding that are applicable to many species. Primarily controlled by nucleoid-associated proteins, chromosome folding is hierarchical, from large-scale macrodomains to smaller-scale structures that influence DNA transactions, including replication and transcription. Here we review the environmentally regulated, architectural and regulatory roles of nucleoid-associated proteins and the implications for bacterial cell biology. We also highlight similarities and differences in the chromosome folding mechanisms of bacteria and eukaryotes.
The quorum sensing transcription factor AphA directly regulates natural competence in Vibrio cholerae
Published: 28th October 2019
Authors: Haycocks JRJ, Warren GZL, Walker LM, Chlebek JL, Dalia TN, Dalia AB, Grainger DC.
Journal: PLoS Genetics
Abstract:
Many bacteria use population density to control gene expression via quorum sensing. In Vibrio cholerae, quorum sensing coordinates virulence, biofilm formation, and DNA uptake by natural competence. The transcription factors AphA and HapR, expressed at low and high cell density respectively, play a key role. In particular, AphA triggers the entire virulence cascade upon host colonisation. In this work we have mapped genome-wide DNA binding by AphA. We show that AphA is versatile, exhibiting distinct modes of DNA binding and promoter regulation. Unexpectedly, whilst HapR is known to induce natural competence, we demonstrate that AphA also intervenes. Most notably, AphA is a direct repressor of tfoX, the master activator of competence. Hence, production of AphA markedly suppressed DNA uptake; an effect largely circumvented by ectopic expression of tfoX. Our observations suggest dual regulation of competence. At low cell density AphA is a master repressor whilst HapR activates the process at high cell density. Thus, we provide deep mechanistic insight into the role of AphA and highlight how V. cholerae utilises this regulator for diverse purposes.
The Escherichia coli MarA protein regulates the ycgZ–ymgABC operon to inhibit biofilm formation
Published: 30th September 2019
Authors: Kettles RA, Tschowri N, Lyons KJ, Sharma P, Hengge R, Webber MA, Grainger DC.
Journal: Molecular Microbiology
Abstract:
The Escherichia coli marRAB operon is a paradigm for chromosomally encoded antibiotic resistance. The operon exerts its effect via an encoded transcription factor called MarA that modulates efflux pump and porin expression. In this work we show that MarA is also a regulator of biofilm formation. Control is mediated by binding of MarA to the intergenic region upstream of the ycgZ–ymgABC operon. The operon, known to influence the formation of curli fibres and colanic acid, is usually expressed during periods of starvation. Hence, the ycgZ–ymgABC promoter is recognised by s38 (RpoS) associated RNA polymerase. We show that MarA does not influence s38 dependent transcription. Instead, MarA drives transcription by the housekeeping s70 associated RNA polymerase. The effects of MarA on ycgZ–ymgABC expression are coupled to biofilm formation by the rcsCDB phosphorelay system, with YcgZ, YmgA and YmgB forming a complex that directly interacts with the histidine kinase domain of RcsC.
The Escherichia coli multiple antibiotic resistance activator protein represses transcription of the lac operon
Published: March 8th 2019
Authors: Lankester A, Ahmed S, Lamberte LE, Kettles RA, Grainger DC.
Journal: Biochemical Society Transactions
Abstract:
In Escherichia coli, the marRAB operon is a determinant for antibiotic resistance. Such phenotypes require the encoded transcription factor MarA that activates efflux pump expression. To better understand all genes controlled by MarA, we recently mapped binding of the regulator across the E. coli genome. As expected, many MarA targets were adjacent to genes encoding stress response systems. Surprisingly, one MarA-binding site overlapped the lac operon regulatory region. Here, we show that MarA specifically targets this locus and can block transcription of the lac genes. Repression is mediated by binding of MarA to a site overlapping the lacP1 promoter -35 element. Control of the lac operon by MarA does not impact antibiotic resistance.
Determination of the 3D Genome Organization of Bacteria Using Hi-C
Published: 10th July 2018
Authors: Crémazy FG, Rashid FM, Haycocks JR, Lamberte LE, Grainger DC, Dame RT.
Journal: Methods in Molecular Biology
Abstract: 15 August 2018
The spatial organization of genomes is based on their hierarchical compartmentalization in topological domains. There is growing evidence that bacterial genomes are organized into insulated domains similar to the Topologically Associating Domains (TADs) detected in eukaryotic cells. Chromosome conformation capture (3C) technologies are used to analyze in vivo DNA proximity based on ligation of distal DNA segments crossed-linked by bridging proteins. By combining 3C and high-throughput sequencing, the Hi-C method reveals genome-wide interactions within topological domains and global genome structure as a whole. This chapter provides detailed guidelines for the preparation of Hi-C sequencing libraries for bacteria.
cAMP Receptor Protein Controls Vibrio cholerae Gene Expression in Response to Host Colonization
Published: 10th July 2018
Authors: Manneh-Roussela J, Haycocks JRJ, Magán A, Perez-Soto N, Voelz K, Camilli A, Krachler A-M, Grainger DC.
Journal: mBio
Abstract:
The bacterium Vibrio cholerae is native to aquatic environments and can switch lifestyles to cause disease in humans. Lifestyle switching requires modulation of genetic systems for quorum sensing, intestinal colonization, and toxin production. Much of this regulation occurs at the level of gene expression and is controlled by transcription factors. In this work, we have mapped the binding of cAMP receptor protein (CRP) and RNA polymerase across the V. cholerae genome. We show that CRP is an integral component of the regulatory network that controls lifestyle switching. Focusing on a locus necessary for toxin transport, we demonstrate CRP-dependent regulation of gene expression in response to host colonization. Examination of further CRP-targeted genes reveals that this behavior is commonplace. Hence, CRP is a key regulator of many V. cholerae genes in response to lifestyle changes.
Waking the neighbours: disruption of H-NS repression by overlapping transcription
Published: 23rd March 2018
Authors: Wade JT, Grainger DC.
Journal: Molecular Microbiology
Abstract:
The histone-like nucleoid structuring (H-NS) protein and its analogues bind large stretches of horizontally acquired AT-rich DNA in a broad range of bacterial species. Binding by H-NS silences the promoters within such DNA that would otherwise deplete the cellular pool of RNA polymerase. Selective de-repression can occur when sequence-specific DNA-binding proteins locally disrupt H-NS function; this mechanism is important for the regulation of many virulence genes. In this issue of Molecular Microbiology, Rangarajan and Schnetz show that when transcription from a neighbouring region invades an H-NS-bound locus, it can disrupt local H-NS repression. Moreover, they show that de-repression occurs in a dose-dependent manner, and they demonstrate a natural example of this in Escherichia coli. This finding has important implications for H-NS function and its impact on genome evolution.
The multiple antibiotic resistance operon of enteric bacteria controls DNA repair and outer membrane integrity
Published: 13th November 2017
Authors: Sharma P, Haycocks JRJ, Middlemiss AD, Kettles RA, Sellars LE, Ricci V, Piddock LJV, Grainger DC.
Journal: Nature Communications
Abstract:
The multiple antibiotic resistance (mar) operon of Escherichia coli is a paradigm for chromosomally encoded antibiotic resistance in enteric bacteria. The locus is recognised for its ability to modulate efflux pump and porin expression via two encoded transcription factors, MarR and MarA. Here we map binding of these regulators across the E. coli genome and identify an extensive mar regulon. Most notably, MarA activates expression of genes required for DNA repair and lipid trafficking. Consequently, the mar locus reduces quinolone-induced DNA damage and the ability of tetracyclines to traverse the outer membrane. These previously unrecognised mar pathways reside within a core regulon, shared by most enteric bacteria. Hence, we provide a framework for understanding multidrug resistance, mediated by analogous systems, across the Enterobacteriaceae.
CsrA maximizes expression of the AcrAB multidrug resistance transporter
Published: 13th October 2017
Authors: Ricci V, Attah V, Overton T, Grainger DC, Piddock LJV.
Journal: Nucleic Acids Research
Abstract:
Carbon Storage Regulator A (CsrA) is an RNA binding protein that acts as a global regulator of diverse genes. Using a combination of genetics and biochemistry we show that CsrA binds directly to the 5′ end of the transcript encoding AcrAB. Deletion of csrA or mutagenesis of the CsrA binding sites reduced production of both AcrA and AcrB. Nucleotide substitutions at the 5′ UTR of acrA mRNA that could potentially weaken the inhibitory RNA secondary structure, allow for more efficient translation of the AcrAB proteins. Given the role of AcrAB-TolC in multi-drug efflux we suggest that CsrA is a potential drug target.
Spurious transcription and its impact on cell function
Published: 5th October 2017
Authors: Wade JT, Grainger DC.
Journal: Transcription
Abstract:
Most RNA polymerases can initiate transcription from diverse DNA template sequences with relatively few outright sequence restraints. Recent reports have demonstrated that failure to subdue the promiscuity of RNA polymerase in vivo can severely impede cell function. This phenomenon appears common to all cell types with undesirable effects ranging from growth inhibition in prokaryotes to cancer in higher organisms. Here we discuss similarities and differences in strategies employed by cells to minimise spurious transcription across life’s domains.
Horizontally acquired AT-rich genes in Escherichia coli cause toxicity by sequestering RNA polymerase
Published: 9th January 2017
Authors: Lamberte LE, Baniulyte G, Singh SS, Stringer AM, Bonocora RP, Stracy M, Kapanidis AN, Wade JT, Grainger DC.
Journal: Nature Microbiology
Abstract:
Horizontal gene transfer permits rapid dissemination of genetic elements between individuals in bacterial populations. Transmitted DNA sequences may encode favourable traits. However, if the acquired DNA has an atypical base composition, it can reduce host fitness. Consequently, bacteria have evolved strategies to minimize the harmful effects of foreign genes. Most notably, xenogeneic silencing proteins bind incoming DNA that has a higher AT content than the host genome. An enduring question has been why such sequences are deleterious. Here, we showed that the toxicity of AT-rich DNA in Escherichia coli frequently results from constitutive transcription initiation within the coding regions of genes. Left unchecked, this causes titration of RNA polymerase and a global downshift in host gene expression. Accordingly, a mutation in RNA polymerase that diminished the impact of AT-rich DNA on host fitness reduced transcription from constitutive, but not activator-dependent, promoters.
The unexpected complexity of bacterial genomes
Published: 16th July 2016
Authors: Grainger DC.
Journal: Microbiology
Abstract:
Gene organization and control are described by models conceived in the 1960s. These models explain basic gene regulatory mechanisms and underpin current genome annotation. However, such models struggle to explain recent genome-scale observations. For example, accounts of RNA synthesis initiating within genes, widespread antisense transcription and non-canonical DNA binding by gene regulatory proteins are difficult to reconcile with traditional thinking. As a result, unexpected observations have often been dismissed and downstream consequences ignored. In this paper I will argue that, to fully understand the biology of bacterial chromosomes, we must embrace their hidden layers of complexity.
Unusually Situated Binding Sites for Bacterial Transcription Factors Can Have Hidden Functionality
Published: 3 June 2016
Authors: Haycocks JR, Grainger DC.
Journal: PLoS One
Abstract:
A commonly accepted paradigm of molecular biology is that transcription factors control gene expression by binding sites at the 5′ end of a gene. However, there is growing evidence that transcription factor targets can occur within genes or between convergent genes. In this work, we have investigated one such target for the cyclic AMP receptor protein (CRP) of enterotoxigenic Escherichia coli. We show that CRP binds between two convergent genes. When bound, CRP regulates transcription of a small open reading frame, which we term aatS, embedded within one of the adjacent genes. Our work demonstrates that non-canonical sites of transcription factor binding can have hidden functionality.
DNA recognition by Escherichia coli CbpA protein requires a conserved arginine-minor-groove interaction
Authors: Chintakayala K, Sellars LE, Singh SS, Shahapure R, Westerlaken I, Meyer AS, Dame RT, Grainger DC.
Journal: Nucleic Acids Research
Abstract:
Curved DNA binding protein A (CbpA) is a co-chaperone and nucleoid associated DNA binding protein conserved in most ?-proteobacteria. Best studied in Escherichia coli, CbpA accumulates to >2500 copies per cell during periods of starvation and forms aggregates with DNA. However, the molecular basis for DNA binding is unknown; CbpA lacks motifs found in other bacterial DNA binding proteins. Here, we have used a combination of genetics and biochemistry to elucidate the mechanism of DNA recognition by CbpA. We show that CbpA interacts with the DNA minor groove. This interaction requires a highly conserved arginine side chain. Substitution of this residue, R116, with alanine, specifically disrupts DNA binding by CbpA, and its homologues from other bacteria, whilst not affecting other CbpA activities. The intracellular distribution of CbpA alters dramatically when DNA binding is negated. Hence, we provide a direct link between DNA binding and the behaviour of CbpA in cells.
H-NS and RNA polymerase: a love-hate relationship?
Published: 29 January 2015
Authors: Landick R, Wade JT, Grainger DC.
Journal: Current Opinion in Microbiology
Abstract:
Histone-like nucleoid structuring (H-NS) protein is a component of bacterial chromatin and influences gene expression both locally and on a global scale. Although H-NS is broadly considered a silencer of transcription, the mechanisms by which H-NS inhibits gene expression remain poorly understood. Here we discuss recent advances in the context of a ‘love-hate’ relationship between H-NS and RNA polymerase, in which these factors recognise similar DNA sequences but interfere with each other’s activity. Understanding the complex relationship between H-NS and RNA polymerase may unite the multiple models that have been proposed to describe gene silencing by H-NS.
The Molecular Basis for Control of ETEC Enterotoxin Expression in Response to Environment and Host
Published: 8 January 2015
Authors: Haycocks JR, Sharma P, Stringer AM, Wade JT, Grainger DC.
Journal: PLoS Pathogens
Abstract:
Enterotoxigenic Escherichia coli (ETEC) cause severe diarrhoea in humans and neonatal farm animals. Annually, 380,000 human deaths, and multi-million dollar losses in the farming industry, can be attributed to ETEC infections. Illness results from the action of enterotoxins, which disrupt signalling pathways that manage water and electrolyte homeostasis in the mammalian gut. The resulting fluid loss is treated by oral rehydration. Hence, aqueous solutions of glucose and salt are ingested by the patient. Given the central role of enterotoxins in disease, we have characterised the regulatory trigger that controls toxin production. We show that, at the molecular level, the trigger is comprised of two gene regulatory proteins, CRP and H-NS. Strikingly, this renders toxin expression sensitive to both conditions encountered on host cell attachment and the components of oral rehydration therapy. For example, enterotoxin expression is induced by salt in an H-NS dependent manner. Furthermore, depending on the toxin gene, expression is activated or repressed by glucose. The precise sensitivity of the regulatory trigger to glucose differs because of variations in the regulatory setup for each toxin encoding gene.
Pervasive transcription: illuminating the dark matter of bacterial transcriptomes
Published: September 2014
Authors: Wade JT, Grainger DC.
Journal: Nature Reviews Microbiology
Abstract:
The conventional view of transcription posits that mRNAs are generated from the coding DNA strand and are delineated by gene boundaries; however, recent reports have mapped transcription start sites to unexpected locations in bacterial genomes, including the non-coding strand. The resultant RNAs were previously dismissed as artefacts, but models that describe such events as ‘pervasive transcription’ are now gaining support. In this Opinion article, we discuss our current understanding of pervasive transcription, its genetic origin and its regulation. On the basis of existing observations, we propose that RNAs that result from pervasive transcription are more than ‘transcriptional noise’ and have important functions in gene regulation and genome evolution.
Widespread suppression of intragenic transcription initiation by H-NS
Published: 1 February 2014
Authors: Singh SS, Singh N, Bonocora RP, Fitzgerald DM, Wade JT, Grainger DC.
Journal: Genes and Development
Abstract:
Widespread intragenic transcription initiation has been observed in many species. Here we show that the Escherichia coli ehxCABD operon contains numerous intragenic promoters in both sense and antisense orientations. Transcription from these promoters is silenced by the histone-like nucleoid structuring (H-NS) protein. On a genome-wide scale, we show that 46% of H-NS-suppressed transcripts in E. coli are intragenic in origin. Furthermore, many intergenic promoters repressed by H-NS are for noncoding RNAs (ncRNAs). Thus, a major overlooked function of H-NS is to prevent transcription of spurious RNA. Our data provide a molecular description for the toxicity of horizontally acquired DNA and explain how this is counteracted by H-NS.
H-NS can facilitate specific DNA-binding by RNA polymerase in AT-rich gene regulatory regions
Published: 9 June 2013
Authors: Singh SS, Grainger DC.
Journal: PLoS Genetics
Abstract:
Extremely AT-rich DNA sequences present a challenging template for specific recognition by RNA polymerase. In bacteria, this is because the promoter -10 hexamer, the major DNA element recognised by RNA polymerase, is itself AT-rich. We show that Histone-like Nucleoid Structuring (H-NS) protein can facilitate correct recognition of a promoter by RNA polymerase in AT-rich gene regulatory regions. Thus, at the Escherichia coli ehxCABD operon, RNA polymerase is unable to distinguish between the promoter -10 element and similar overlapping sequences. This problem is resolved in native nucleoprotein because the overlapping sequences are masked by H-NS. Our work provides mechanistic insight into nucleoprotein structure and its effect on protein-DNA interactions in prokaryotic cells.
The Escherichia coli Nucleoid in Stationary Phase
Published: 2013
Authors: Meyer AS, Grainger DC.
Journal: Advances in Applied Microbiology
Abstract:
Compaction of DNA is an essential phenomenon that affects all facets of cellular biology. Surprisingly, given the abundance and apparent simplicity of bacteria, our understanding of chromosome organization in these ancient organisms is inadequate. In this chapter we will focus on arguably the best understood aspect of DNA folding in the model bacterium Escherichia coli: the supercondensation of the chromosome that occurs during periods of starvation and stress.
E. coli Fis protein insulates the cbpA gene from uncontrolled transcription.
Published: January 2013
Authors: Chintakayala K, Singh SS, Rossiter AE, Shahapure R, Dame RT, Grainger DC.
Journal: PLoS Genetics
Abstract:
The Escherichia coli curved DNA binding protein A (CbpA) is a poorly characterised nucleoid associated factor and co-chaperone. It is expressed at high levels as cells enter stationary phase. Using genetics, biochemistry, and genomics, we have examined regulation of, and DNA binding by, CbpA. We show that Fis, the dominant growth-phase nucleoid protein, prevents CbpA expression in growing cells. Regulation by Fis involves an unusual “insulation” mechanism. Thus, Fis protects cbpA from the effects of a distal promoter, located in an adjacent gene. In stationary phase, when Fis levels are low, CbpA binds the E. coli chromosome with a preference for the intrinsically curved Ter macrodomain. Disruption of the cbpA gene prompts dramatic changes in DNA topology. Thus, our work identifies a novel role for Fis and incorporates CbpA into the growing network of factors that mediate bacterial chromosome structure.
Multidisciplinary perspectives on bacterial genome organization and dynamics.
Published: December 2012
Authors: Dame RT, Espéli O, Grainger DC, Wiggins PA.
Journal: Molecular Microbiology
Abstract:
Bacterial genomes are organized by a plethora of chromatin proteins and physical mechanisms. This organization appears to be hierarchical with DNA folding events at the nm scale influencing higher levels of chromosome organization. Besides acting in shaping the genome these factors also play important regulatory roles in numerous DNA transactions. While DNA folding mechanisms operating at the nm scale are fairly well understood, it has been hard to translate this knowledge into accurate models that describe the complete dynamics of the genome. In recent years new techniques have evolved that are key to filling the current gaps in understanding. Particularly insightful in this light appear techniques that probe architectural properties of chromatin proteins on single molecules, techniques that map the binding of protein components and spatial structure on a genome-wide basis and improved imaging techniques that provide resolutions capable of resolving substructures/heterogeneities in the nucleoid. Moreover, bioinformatic and polymer physics approaches are starting to provide novel insights. In our opinion, an important aim in the field is to generate an accurate and complete description of the nucleoid and its dynamics at all scales. A first step towards this aim has now been set by bringing together people from diverse disciplinary backgrounds at the Lorentz centre workshop ‘Biology and Physics of Bacterial Genome Organization’ in Leiden, the Netherlands from 18 to 22 June 2012.
A conserved acidic amino acid mediates the interaction between modulators and co-chaperones in enterobacteria
Published: 12 August 2011
Authors: Chintakayala K, Grainger DC.
Journal: Journal of Molecular Biology
Abstract:
Hsp40-like co-chaperones are ubiquitous enzymes that stimulate the protein refolding activity of Hsp70 family chaperones. They are widespread in prokaryotic and eukaryotic systems. In bacteria, the best characterized co-chaperone is the Escherichia coli DnaJ protein. Many ?-proteobacteria encode a functional homologue of DnaJ, known as CbpA, which is expressed in response to starvation and environmental stress. The activity of CbpA is regulated by the “modulator” protein CbpM. Here, we have used a combination of genetics and biochemistry to identify the co-chaperone contact determinant of CbpM. We show that the nature of the interaction is conserved in enterobacteria.
Escherichia coli δ70 senses sequence and conformation of the promoter spacer region
Published: July 2011
Authors: Singh SS, Typas A, Hengge R, Grainger DC.
Journal: Nucleic Acids Research
Abstract:
In bacteria, promoter identification by RNA polymerase is mediated by a dissociable ? factor. The housekeeping ?(70) factor of Escherichia coli recognizes two well characterized DNA sequence elements, known as the ‘-10’ and ‘-35’ hexamers. These elements are separated by ‘spacer’ DNA, the sequence of which is generally considered unimportant. Here, we use a combination of bioinformatics, genetics and biochemistry to show that ?(70) can sense the sequence and conformation of the promoter spacer region. Our data illustrate how alterations in spacer region sequence can increase promoter activity. This stimulatory effect requires ?(70) side chain R451, which is located in close proximity to the non-template strand at promoter position -18. Conversely, R451 is not required to mediate transcriptional stimulation by improvement of the -10 element. Mutation of ?(70) residue R451, which is highly conserved, results in reduced growth rate, consistent with a central role in promoter recognition.
Chromosomal macrodomains and associated proteins: implications for DNA organization and replication in gram negative bacteria
Published: June 2011
Authors: Dame RT, Kalmykowa OJ, Grainger DC.
Journal: PLoS Genetics
Abstract:
The Escherichia coli chromosome is organized into four macrodomains, the function and organisation of which are poorly understood. In this review we focus on the MatP, SeqA, and SlmA proteins that have recently been identified as the first examples of factors with macrodomain-specific DNA-binding properties. In particular, we review the evidence that these factors contribute towards the control of chromosome replication and segregation by specifically targeting subregions of the genome and contributing towards their unique properties. Genome sequence analysis of multiple related bacteria, including pathogenic species, reveals that macrodomain-specific distribution of SeqA, SlmA, and MatP is conserved, suggesting common principles of chromosome organisation in these organisms. This discovery of proteins with macrodomain-specific binding properties hints that there are other proteins with similar specificity yet to be unveiled. We discuss the roles of the proteins identified to date as well as strategies that may be employed to discover new factors.
The Escherichia coli K-12 MntR miniregulon includes dps, which encodes the major stationary-phase DNA-binding protein.
Published: March 2011
Authors: Yamamoto K, Ishihama A, Busby SJ, Grainger DC.
Journal: Journal of Bacteriology
Abstract:
Escherichia coli MntR protein is the Mn(2+)-responsive transcriptional repressor of the MntH manganese transporter. We have used chromatin immunoprecipitation to determine the distribution of Mn(2+)-MntR across the entire E. coli chromosome in vivo, and we report that MntR binds to only four targets, adjacent to the mntH, mntR, yebN, and dps genes. Unexpectedly, we found that dps expression is directly repressed by Mn(2+)-MntR.
Effects of nucleoid-associated proteins on bacterial chromosome structure and gene expression
Published: December 2010
Authors: Browning DF, Grainger DC, Busby SJ.
Journal: Current Opinion in Microbiology
Abstract:
Bacterial nucleoid-associated proteins play a key role in the organisation, replication, segregation, repair and expression of bacterial chromosomes. Here, we review some recent progress in our understanding of the effects of these proteins on DNA and their biological role, focussing mainly on Escherichia coli and its chromosome. Certain nucleoid-associated proteins also regulate transcription initiation at specific promoters, and work in concert with dedicated transcription factors to regulate gene expression in response to growth phase and environmental change. Some specific examples, involving the E. coli IHF and Fis proteins, that illustrate new principles, are described in detail.
Dimerization and DNA-dependent aggregation of the Escherichia coli nucleoid protein and chaperone CbpA
Published: September 2010
Authors: Cosgriff S, Chintakayala K, Chim YT, Chen X, Allen S, Lovering AL, Grainger DC.
Journal: Molecular Microbiology
Abstract:
The Escherichia coli curved DNA-binding protein A (CbpA) is a nucleoid-associated DNA-binding factor and chaperone that is expressed at high levels as cells enter stationary phase. Using a combination of genetics, biochemistry, structural modelling and single-molecule atomic force microscopy we have examined dimerization of, and DNA binding by, CbpA. Our data show that CbpA dimerization is driven by a hydrophobic surface comprising amino acid side chains W287 and L290 located on the same side of an ? helix close to the C-terminus of CbpA. Derivatives of CbpA that are unable to dimerize are also unable to bind DNA. Free in solution, CbpA can exist as either a monomer or dimer. However, when bound to DNA, CbpA forms large aggregates that can protect DNA from degradation by nucleases. These CbpA-DNA aggregates are similar in morphology to protein-DNA complexes formed by the DNA-binding protein from starved cells (Dps), the only other stationary phase-specific nucleoid protein. Conversely, protein-DNA complexes formed by Fis, the major growth phase nucleoid protein, have a markedly different appearance.
Dynamic distribution of SeqA protein across the chromosome of Escherichia coli K-12
Published: 18 May 2010
Authors: Sánchez-Romero MA, Busby SJ, Dyer NP, Ott S, Millard AD, Grainger DC.
Journal: mBio
Abstract:
The bacterial SeqA protein binds to hemi-methylated GATC sequences that arise in newly synthesized DNA upon passage of the replication machinery. In Escherichia coli K-12, the single replication origin oriC is 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 coli K-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 ter macrodomain. 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.
Direct methods for studying transcription regulatory proteins and RNA polymerase in bacteria
Published: October 2010
Authors: Grainger DC, Lee DJ, Busby SJ.
Journal: Current Opinion in Microbiology
Abstract:
Transcription factors and sigma factors play a major role in bacterial gene regulation by guiding the distribution of RNA polymerase between the promoters of different transcription units in response to changes in the environment. For 40 years Escherichia coli K-12 has been the paradigm for investigating this regulation and most studies have focused on small numbers of promoters studied by a combination of genetics and biochemistry. Since the first complete sequence for a bacterial genome was reported, the emphasis has switched to studying transcription on a global scale, with transcriptomics and bioinformatics becoming the methods of choice. Here we discuss two complementary direct experimental methods for studying transcription factors and sigma factors and we outline their potential use in rapidly establishing the regulatory networks in newly sequenced bacteria.
Dissecting regulatory networks in host-pathogen interaction using ChIP-on-chip technology
Published: 8 May 2009
Authors: Sala C, Grainger DC, Cole ST.
Journal: Cell Host and Microbe
Abstract:
Understanding host-microbe interactions has been greatly enhanced by our broadening knowledge of the regulatory mechanisms at the heart of pathogenesis. The “transcriptomics” approach of measuring global gene expression has identified genes involved in bacterial pathogenesis. More recently, chromatin immunoprecipitation (ChIP) and hybridization to microarrays (chIP-on-chip) has emerged as a complementary tool that permits protein-DNA interactions to be studied in vivo. Thus, chIP-on-chip can be used to map the binding sites of transcription factors, thereby teasing apart gene regulatory networks. In this Review, we discuss the ChIP-on-chip technique and focus on its application to the study of host-pathogen interactions.
Global regulators of transcription in Escherichia coli: mechanisms of action and methods for study.
Published: 2008
Authors: Grainger DC, Busby SJ.
Journal: Advances in Applied Microbiology
Abstract:
None available.
Methods for studying global patterns of DNA binding by bacterial transcription factors and RNA polymerase
Published: August 2008
Authors: Grainger DC, Busby SJ.
Journal: Biochemical Society Transactions
Abstract:
A major goal in the study of gene regulation is to untangle the transcription-regulatory networks of Escherichia coli and other ‘simple’ organisms. To do this we must catalogue the binding sites of all transcription factors. ChIP (chromatin immunoprecipitation), combined with DNA microarray analysis, is a powerful tool that permits global patterns of DNA binding to be measured. Here, we discuss the benefits of this approach and the application of this technique to bacterial systems.
The Escherichia coli RutR transcription factor binds at targets within genes as well as intergenic regions
Published: July 2008
Authors: Shimada T, Ishihama A, Busby SJ, Grainger DC.
Journal: Nucleic Acids Research
Abstract:
The Escherichia coli RutR protein is the master regulator of genes involved in pyrimidine catabolism. Here we have used chromatin immunoprecipitation in combination with DNA microarrays to measure the binding of RutR across the chromosome of exponentially growing E. coli cells. Twenty RutR-binding targets were identified and analysis of these targets generated a DNA consensus logo for RutR binding. Complementary in vitro binding assays showed high-affinity RutR binding to 16 of the 20 targets, with the four low-affinity RutR targets lacking predicted key binding determinants. Surprisingly, most of the DNA targets for RutR are located within coding segments of the genome and appear to have little or no effect on transcript levels in the conditions tested. This contrasts sharply with other E. coli transcription factors whose binding sites are primarily located in intergenic regions. We suggest that either RutR has yet undiscovered function or that evolution has been slow to eliminate non-functional DNA sites for RutR because they do not have an adverse effect on cell fitness.
Selective repression by Fis and H-NS at the Escherichia coli dps promoter
Published: June 2008
Authors: Grainger DC, Goldberg MD, Lee DJ, Busby SJ.
Journal: Molecular Microbiology
Abstract:
Dps is a nucleoid-associated protein that plays a major role in condensation of the Escherichia coli chromosome in stationary phase. Here we show that two other nucleoid-associated proteins, Fis and H-NS, can bind at the dps gene promoter and downregulate its activity. Both Fis and H-NS selectively repress the dps promoter, preventing transcription initiation by RNA polymerase containing sigma(70), the housekeeping sigma factor, but not by RNA polymerase containing sigma(38), the stationary-phase sigma factor. Fis represses by trapping RNA polymerase containing sigma(70) at the promoter. In contrast, H-NS functions by displacing RNA polymerase containing sigma(70), but not RNA polymerase containing sigma(38). Dps levels are known to be very low in exponentially growing cells and rise sharply as cells enter stationary phase. Conversely, Fis levels are high in growing cells but fall to nearly zero in stationary-phase cells. Our data suggest a simple model to explain how the Dps-dependent super-compaction of the folded chromosome is triggered as cell growth ceases.
Autoregulation of the Escherichia coli melR promoter: repression involves four molecules of MelR
Published: May 2008
Authors: Samarasinghe S, El-Robh MS, Grainger DC, Zhang W, Soultanas P, Busby SJ.
Journal: Nucleic Acids Research
Abstract:
The Escherichia coli MelR protein is a transcription activator
that autoregulates its own promoter by repressing transcription
initiation. Optimal repression requires MelR binding to a site that
overlaps the melR transcription start point and to upstream
sites. In this work, we have investigated the different determinants
needed for optimal repression and their spatial requirements. We show
that repression requires a complex involving four DNA-bound MelR
molecules, and that the global CRP regulator plays little or no role.
Genomic analysis of protein-DNA interactions in bacteria: insights into transcription and chromosome organization
Published: July 2007
Authors: Wade JT, Struhl K, Busby SJ, Grainger DC.
Journal: Molecular Microbiology
Abstract:
Chromatin immunoprecipitation (ChIP) is a powerful method to measure protein-DNA interactions in vivo, and it can be applied on a genomic scale with microarray technology (ChIP-chip). ChIP-chip has been used extensively to map DNA-protein interactions across eukaryotic chromosomes. Here we review recent applications of ChIP-chip to the study of bacteria, which provide important and unexpected insights into transcription and chromosome organization.
Transcription factor distribution in Escherichia coli: studies with FNR protein
Published: 2007
Authors: Grainger DC, Aiba H, Hurd D, Browning DF, Busby SJ.
Journal: Nucleic Acids Research
Abstract:
Using chromatin immunoprecipitation (ChIP) and high-density microarrays, we have measured the distribution of the global transcription regulator protein, FNR, across the entire Escherichia coli chromosome in exponentially growing cells. Sixty-three binding targets, each located at the 5′ end of a gene, were identified. Some targets are adjacent to poorly transcribed genes where FNR has little impact on transcription. In stationary phase, the distribution of FNR was largely unchanged. Control experiments showed that, like FNR, the distribution of the nucleoid-associated protein, IHF, is little altered when cells enter stationary phase, whilst RNA polymerase undergoes a complete redistribution.
Association of nucleoid proteins with coding and non-coding segments of the Escherichia coli genome
Published: 2006
Authors: Grainger DC, Hurd D, Goldberg MD, Busby SJ.
Journal: Nucleic Acids Research
Abstract:
The Escherichia coli chromosome is condensed into an ill-defined structure known as the nucleoid. Nucleoid-associated DNA-binding proteins are involved in maintaining this structure and in mediating chromosome compaction. We have exploited chromatin immunoprecipitation and high-density microarrays to study the binding of three such proteins, FIS, H-NS and IHF, across the E. coli genome in vivo. Our results show that the distribution of these proteins is biased to intergenic parts of the genome, and that the binding profiles overlap. Hence some targets are associated with combinations of bound FIS, H-NS and IHF. In addition, many regions associated with FIS and H-NS are also associated with RNA polymerase.
Extensive functional overlap between sigma factors in Escherichia coli
Published: September 2006
Authors: Wade JT, Castro Roa D, Grainger DC, Hurd D, Busby SJ, Struhl K, Nudler E.
Journal: Nature Structural and Molecular Biology
Abstract:
Bacterial core RNA polymerase (RNAP) must associate with a sigma factor to recognize promoter sequences. Escherichia coli encodes seven sigma factors, each believed to be specific for a largely distinct subset of promoters. Using microarrays representing the entire E. coli genome, we identify 87 in vivo targets of sigma32, the heat-shock sigma factor, and estimate that there are 120-150 sigma32 promoters in total. Unexpectedly, 25% of these sigma32 targets are located within coding regions, suggesting novel regulatory roles for sigma32. The majority of sigma32 promoter targets overlap with those of sigma70, the housekeeping sigma factor. Furthermore, their DNA sequence motifs are often interdigitated, with RNAPsigma70 and RNAPsigma32 initiating transcription in vitro with similar efficiency and from identical positions. SigmaE-regulated promoters also overlap extensively with those for sigma70. These results suggest that extensive functional overlap between sigma factors is an important phenomenon.
Studies of the distribution of Escherichia coli cAMP-receptor protein and RNA polymerase along the E. coli chromosome.
Published: 6 December 2005
Authors: Grainger DC, Hurd D, Harrison M, Holdstock J, Busby SJ.
Journal: Proceedings of the National Academy of Sciences
Abstract:
Chromatin immunoprecipitation and high-density microarrays have been used to monitor the distribution of the global transcription regulator Escherichia coli cAMP-receptor protein (CRP) and RNA polymerase along the E. coli chromosome. Our results identify targets occupied by CRP and genes transcribed by RNA polymerase in vivo. Many of the loci of CRP binding are at known CRP regulated promoters. However, our results show that CRP also interacts with thousands of weaker sites across the whole chromosome and that this “background” binding can be used as a probe for organization within the E. coli folded chromosome. In rapidly growing cells, we show that the major sites of RNA polymerase binding are approximately 90 transcription units that include genes needed for protein synthesis. Upon the addition of rifampicin, RNA polymerase is distributed among >500 functional promoters. We show that the chromatin immunoprecipitation and high-density-microarrays methodology can be used to study the redistribution of RNA polymerase induced by environmental stress, revealing previously uncharacterized aspects of RNA polymerase behavior and providing an alternative to the “transcriptomics” approach for studying global transcription patterns.
Integration of three signals at the Escherichia coli nrf promoter: a role for Fis protein in catabolite repression
Published: July 2005
Authors: Browning DF, Grainger DC, Beatty CM, Wolfe AJ, Cole JA, Busby SJ.
Journal: Molecular Microbiology
Abstract:
Expression from the Escherichia coli nrf operon promoter is activated by the anaerobically triggered transcription factor, FNR, and by the nitrate/nitrite ion-controlled response regulators, NarL or NarP, but is repressed by the IHF and Fis proteins. Here, we present in vitro studies on the nrf promoter, using permanganate footprinting to measure open complex formation, and DNase I footprinting to monitor binding of the different regulators and the interactions between them. Our results show that open complex formation is completely dependent on FNR and is enhanced by NarL, but is repressed by IHF or Fis. NarL counteracts repression by IHF but is unable to alter repression by Fis. These results suggest mechanisms by which nrf promoter activity is modulated by the different factors. Expression from the nrf promoter is known to be repressed in rich media, especially in the presence of glucose, but the molecular basis of this is not understood. Here, we show that this catabolite repression is relieved by mutations that weaken the DNA site for Fis, improve the DNA site for FNR or improve the promoter -10 or -35 elements. Hence, Fis protein is a major factor responsible for catabolite repression at the nrf promoter, and Fis can override activation by FNR and NarL or NarP.
Genomic studies with Escherichia coli MelR protein: applications of chromatin immunoprecipitation and microarrays
Published: October 2004
Authors: Grainger DC, Overton TW, Reppas N, Wade JT, Tamai E, Hobman JL, Constantinidou C, Struhl K, Church G, Busby SJ.
Journal: Journal of Bacteriology
Abstract:
Escherichia coli MelR protein is a transcription activator that is essential for melibiose-dependent expression of the melAB genes. We have used chromatin immunoprecipitation to study the binding of MelR and RNA polymerase to the melAB promoter in vivo. Our results show that MelR is associated with promoter DNA, both in the absence and presence of the inducer melibiose. In contrast, RNA polymerase is recruited to the melAB promoter only in the presence of inducer. The MelR DK261 positive control mutant binds to the melAB promoter but cannot recruit RNA polymerase. Further analysis of immunoprecipitated DNA, by using an Affymetrix GeneChip array, showed that the melAB promoter is the major, if not the sole, target in E. coli for MelR. This was confirmed by a transcriptomics experiment to analyze RNA in cells either with or without melR.
Transcription activation at the Escherichia coli melAB promoter: interactions of MelR with the C-terminal domain of the RNA polymerase alpha subunit.
Published: March 2004
Authors: Grainger DC, Belyaeva TA, Lee DJ, Hyde EI, Busby SJ.
Journal: Molecular Microbiology
Abstract:
We have investigated the role of the RNA polymerase alpha subunit during MelR-dependent activation of transcription at the Escherichia coli melAB promoter. To do this, we used a simplified melAB promoter derivative that is dependent on MelR binding at two 18 bp sites, located from position -34 to -51 and from position -54 to -71, upstream of the transcription start site. Results from experiments with hydroxyl radical footprinting, and with RNA polymerase, carrying alpha subunits that were tagged with a chemical nuclease, show that the C-terminal domains of the RNA polymerase alpha subunits are located near position -52 and near position -72 during transcription activation. We demonstrate that the C-terminal domain of the RNA polymerase alpha subunit is needed for open complex formation, and we describe two experiments showing that the RNA polymerase alpha subunit can interact with MelR. Finally, we used alanine scanning to identify determinants in the C-terminal domain of the RNA polymerase alpha subunit that are important for MelR-dependent activation of the melAB promoter.
Transcription activation at the Escherichia coli melAB promoter: interactions of MelR with its DNA target site and with domain 4 of the RNA polymerase sigma subunit.
Published: March 2004
Authors: Grainger DC, Webster CL, Belyaeva TA, Hyde EI, Busby SJ.
Journal: Molecular Microbiology
Abstract:
Activation of transcription initiation at the Escherichia coli melAB promoter is dependent on MelR, a transcription factor belonging to the AraC family. MelR binds to 18 bp target sites using two helix-turn-helix (HTH) motifs that are both located in its C-terminal domain. The melAB promoter contains four target sites for MelR. Previously, we showed that occupation of two of these sites, centred at positions -42.5 and -62.5 upstream of the melAB transcription start point, is sufficient for activation. We showed that MelR binds as a direct repeat to these sites, and we proposed a model to describe how the two HTH motifs are positioned. Here, we have used suppression genetics to confirm this model and to show that MelR residue 273, which is in HTH 2, interacts with basepair 13 of each target site. As our model for DNA-bound MelR suggests that HTH 2 must be adjacent to the melAB promoter -35 element, we searched this part of MelR for amino acid side-chains that might be able to interact with sigma. We describe genetic evidence to show that MelR residue 261 is close to residues 596 and 599 of the RNA polymerase sigma(70) subunit, and that they can interact. Similarly, MelR residue 265 is shown to be able to interact with residue 596 of sigma(70). In the final part of the work, we describe experiments in which the MelR binding site at position -42.5 was improved. We show that this is detrimental to MelR-dependent transcription activation because bound MelR is mispositioned so that it is unable to make ‘correct’ interactions with sigma.
Binding of the Escherichia coli MelR protein to the melAB promoter: orientation of MelR subunits and investigation of MelR-DNA contacts.
Published: April 2003
Authors: Grainger DC, Belyaeva TA, Lee DJ, Hyde EI, Busby SJ.
Journal: Molecular Microbiology
Abstract:
The Escherichia coli MelR protein is a melibiose-triggered transcription factor, belonging to the AraC family, that activates transcription initiation at the melAB promoter. Activation is dependent on the binding of MelR to four 18 bp sites, centred at position -42.5 (site 2′), position -62.5 (site 2), position -100.5 (site 1) and position -120.5 (site 1′) relative to the melAB transcription start point. Activation also depends on the binding of CRP to a single site located between MelR binding site 1 and site 2. All members of the AraC family contain two helix-turn-helix (HTH) motifs that contact two segments of the DNA major groove at target sites on the same DNA face. In this work, we have studied the binding of MelR to different sites at the melAB promoter, focusing on the orientation of binding of the two MelR HTH motifs, and the juxtaposition of the different bound MelR subunits with respect to each other. To do this, MelR was engineered to contain a single cysteine residue adjacent to either one or the other HTH motif. The MelR derivatives were purified, and the cysteine residues were tagged with p-bromoacetamidobenzyl-EDTA-Fe, an inorganic DNA cleavage reagent. Patterns of DNA cleavage after MelR binding were then used to determine the positions of the two HTH motifs at target sites. In order to simplify our analysis, we exploited an engineered derivative of the melAB promoter in which MelR binding to site 2 and site 2′, in the absence of CRP, is sufficient for transcription activation. To assist in the interpretation of our results, we also used a shortened derivative of MelR, MelR173, that is able to bind to site 2 but not to site 2′. Our results show that MelR binds as a direct repeat to site 2 and site 2′ with the C-terminal HTH located towards the promoter-proximal end of each site. The orientation in which MelR binds to site 2′ appears to be determined by MelR-MelR interactions rather than by MelR-DNA interactions. In complementary experiments, we used genetic analysis to investigate the importance of different residues in the two HTH motifs of MelR. Epistasis experiments provided evidence that supports the proposed orientation of binding of MelR at its target site.
Homologs of the Escherichia coli F Element Protein TraR, Including Phage Lambda Orf73, Directly Reprogram Host Transcription
Published: 18th May 2022
Authors: Gopalkrishnan S, Ross W, Akbari MS, Li X, Haycocks JRJ, Grainger DC, Court DL, Gourse RL.
Journal: mBio
Abstract:
Bacterial cells and their associated plasmids and bacteriophages encode numerous small proteins of unknown function. One example, the 73-amino-acid protein TraR, is encoded by the transfer operon of the conjugative F plasmid of Escherichia coli. TraR is a distant homolog of DksA, a protein found in almost all proteobacterial species that is required for ppGpp to regulate transcription during the stringent response. TraR and DksA increase or decrease transcription initiation depending on the kinetic features of the promoter by binding directly to RNA polymerase without binding to DNA. Unlike DksA, whose full activity requires ppGpp as a cofactor, TraR is fully active by itself and unaffected by ppGpp. TraR belongs to a family of divergent proteins encoded by proteobacterial bacteriophages and other mobile elements. Here, we experimentally addressed whether other members of the TraR family function like the F element-encoded TraR. Purified TraR and all 5 homologs that were examined bound to RNA polymerase, functioned at lower concentrations than DksA, and complemented a dksA-null strain for growth on minimal medium. One of the homologs, λ Orf73, encoded by bacteriophage lambda, was examined in greater detail. λ Orf73 slowed host growth and increased phage burst size. Mutational analysis suggested that λ Orf73 and TraR have a similar mechanism for inhibiting rRNA and r-protein promoters. We suggest that TraR and its homologs regulate host transcription to divert cellular resources to phage propagation or conjugation without induction of ppGpp and a stringent response.