Distinct heterochromatin-like domains promote transcriptional memory and silence parasitic genetic elements in bacteria.

There is increasing evidence that prokaryotes maintain chromosome structure, which in turn impacts gene expression. We recently characterized densely occupied, multi-kilobase regions in the E. coli genome that are transcriptionally silent, similar to eukaryotic heterochromatin. These extended protein occupancy domains (EPODs) span genomic regions containing genes encoding metabolic pathways as well as parasitic elements such as prophages. Here, we investigate the contributions of nucleoid-associated proteins (NAPs) to the structuring of these domains, by examining the impacts of deleting NAPs on EPODs genome-wide in E. coli and B. subtilis. We identify key NAPs contributing to the silencing of specific EPODs, whose deletion opens a chromosomal region for RNA polymerase binding at genes contained within that region. We show that changes in E. coli EPODs facilitate an extra layer of transcriptional regulation, which prepares cells for exposure to exotic carbon sources. Furthermore, we distinguish novel xenogeneic silencing roles for the NAPs Fis and Hfq, with the presence of at least one being essential for cell viability in the presence of domesticated prophages. Our findings reveal previously unrecognized mechanisms through which genomic architecture primes bacteria for changing metabolic environments and silences harmful genomic elements.

Negative feedback for DARS2-Fis complex by ATP-DnaA supports the cell cycle-coordinated regulation for chromosome replication.

In Escherichia coli, the replication initiator DnaA oscillates between an ATP- and an ADP-bound state in a cell cycle-dependent manner, supporting regulation for chromosome replication. ATP-DnaA cooperatively assembles on the replication origin using clusters of low-affinity DnaA-binding sites. After initiation, DnaA-bound ATP is hydrolyzed, producing initiation-inactive ADP-DnaA. For the next round of initiation, ADP-DnaA binds to the chromosomal locus DARS2, which promotes the release of ADP, yielding the apo-DnaA to regain the initiation activity through ATP binding. This DnaA reactivation by DARS2 depends on site-specific binding of IHF (integration host factor) and Fis proteins and IHF binding to DARS2 occurs specifically during pre-initiation. Here, we reveal that Fis binds to an essential region in DARS2 specifically during pre-initiation. Further analyses demonstrate that ATP-DnaA, but not ADP-DnaA, oligomerizes on a cluster of low-affinity DnaA-binding sites overlapping the Fis-binding region, which competitively inhibits Fis binding and hence the DARS2 activity. DiaA (DnaA initiator-associating protein) stimulating ATP-DnaA assembly enhances the dissociation of Fis. These observations lead to a negative feedback model where the activity of DARS2 is repressed around the time of initiation by the elevated ATP-DnaA level and is stimulated following initiation when the ATP-DnaA level is reduced.

IHF and Fis as Escherichia coli Cell Cycle Regulators: Activation of the Replication Origin oriC and the Regulatory Cycle of the DnaA Initiator.

This review summarizes current knowledge about the mechanisms of timely binding and dissociation of two nucleoid proteins, IHF and Fis, which play fundamental roles in the initiation of chromosomal DNA replication in Escherichia coli. Replication is initiated from a unique replication origin called oriC and is tightly regulated so that it occurs only once per cell cycle. The timing of replication initiation at oriC is rigidly controlled by the timely binding of the initiator protein DnaA and IHF to oriC. The first part of this review presents up-to-date knowledge about the timely stabilization of oriC-IHF binding at oriC during replication initiation. Recent advances in our understanding of the genome-wide profile of cell cycle-coordinated IHF binding have revealed the oriC-specific stabilization of IHF binding by ATP-DnaA oligomers at oriC and by an initiation-specific IHF binding consensus sequence at oriC. The second part of this review summarizes the mechanism of the timely regulation of DnaA activity via the chromosomal loci DARS2 (DnaA-reactivating sequence 2) and datA. The timing of replication initiation at oriC is controlled predominantly by the phosphorylated form of the adenosine nucleotide bound to DnaA, i.e., ATP-DnaA, but not ADP-ADP, is competent for initiation. Before initiation, DARS2 increases the level of ATP-DnaA by stimulating the exchange of ADP for ATP on DnaA. This DARS2 function is activated by the site-specific and timely binding of both IHF and Fis within DARS2. After initiation, another chromosomal locus, datA, which inactivates ATP-DnaA by stimulating ATP hydrolysis, is activated by the timely binding of IHF. A recent study has shown that ATP-DnaA oligomers formed at DARS2-Fis binding sites competitively dissociate Fis via negative feedback, whereas IHF regulation at DARS2 and datA still remains to be investigated. This review summarizes the current knowledge about the specific role of IHF and Fis in the regulation of replication initiation and proposes a mechanism for the regulation of timely IHF binding and dissociation at DARS2 and datA.

A horizontally acquired Legionella genomic island encoding a LuxR type regulator and effector proteins displays variation in gene content and regulation.

The intracellular pathogen Legionella pneumophila translocates >300 effector proteins into host cells, many of which are regulated at the transcriptional level. Here, we describe a novel L. pneumophila genomic island, which undergoes horizontal gene transfer within the Legionella genus. This island encodes two Icm/Dot effectors: LegK3 and a previously uncharacterized effector which we named CegK3, as well as a LuxR type regulator, which we named RegK3. Analysis of this island in different Legionella species revealed a conserved regulatory element located upstream to the effector-encoding genes in the island. Further analyses, including gene expression analysis, mutagenesis of the RegK3 regulatory element, controlled expression studies, and gel-mobility shift assays, all demonstrate that RegK3 directly activates the expression levels of legK3 and cegK3 effector-encoding genes. Additionally, the expression of all the components of the island is silenced by the Fis repressors. Comparison of expression profiles of these three genes among different Legionella species revealed variability in the activation levels mediated by RegK3, which were positively correlated with the Fis-mediated repression. Furthermore, LegK3 and CegK3 effectors moderately inhibit yeast growth, and importantly, they have a strong synergistic inhibitory effect on yeast growth, suggesting these two effectors are not only co-regulated but also might function together.

Mutants lacking global regulators, fis and arcA, in Escherichia coli enhanced growth fitness under acetate metabolism by pathway reprogramming.

Global regulatory transcription factors play a significant role in controlling microbial metabolism under genetic and environmental perturbations. A system-level effect of carbon sources such as acetate on microbial metabolism under disrupted global regulators has not been well established. Acetate is one of the major substrates available in various nutrient niches such as the mammalian gut and a keto diet. A substantial amount of acetate gets secreted in aerobic metabolism. Therefore, investigating the study on acetate metabolism is highly significant. It is known that the global regulators fis and arcA regulate acetate uptake genes in E. coli under glucose conditions. This study deciphered the growth and flux distribution of E. coli transcription regulatory knockouts Δfis, ΔarcA and double deletion mutant, ΔarcAΔfis under acetate using 13C-metabolic flux analysis (MFA), which has not been investigated before. We observed that the mutants exhibited an expeditious growth rate (~ 1.2-1.6-fold) with a proportionate increase in acetate uptake rates compared to the wild type. 13C-MFA displayed the distinct metabolic reprogramming of intracellular fluxes via the TCA cycle, anaplerotic pathway and gluconeogenesis, which conferred an advantage of a faster growth rate with better carbon usage in all the mutants. This resulted in higher metabolic fluxes through the TCA cycle (~ 18-90%), lower gluconeogenesis (~ 15-35%) and higher CO2 and ATP production with the proportional increase in growth rate. The study reveals a novel insight by stating the sub-optimality of the wild-type strain grown under acetate substrate aerobically. These mutant strains efficiently oxidize acetate, thus acting as potential candidates for the biosynthesis of isoprenoids, biofuels, vitamins and various pharmaceutical products.Key Points• Mutants exhibited a better balance between energy and precursor synthesis than WT.• Leveraged in the unravelling of regulatory control under various nutrient shifts.• Metabolic readjustment resulted in optimal biomass requirement and faster growth.

A bacteriophage mimic of the bacterial nucleoid-associated protein Fis.

We report the identification and characterization of a bacteriophage λ-encoded protein, NinH. Sequence homology suggests similarity between NinH and Fis, a bacterial nucleoid-associated protein (NAP) involved in numerous DNA topology manipulations, including chromosome condensation, transcriptional regulation and phage site-specific recombination. We find that NinH functions as a homodimer and is able to bind and bend double-stranded DNA in vitro. Furthermore, NinH shows a preference for a 15 bp signature sequence related to the degenerate consensus favored by Fis. Structural studies reinforced the proposed similarity to Fis and supported the identification of residues involved in DNA binding which were demonstrated experimentally. Overexpression of NinH proved toxic and this correlated with its capacity to associate with DNA. NinH is the first example of a phage-encoded Fis-like NAP that likely influences phage excision-integration reactions or bacterial gene expression.

Cooperative DNA binding by proteins through DNA shape complementarity.

Localized arrays of proteins cooperatively assemble onto chromosomes to control DNA activity in many contexts. Binding cooperativity is often mediated by specific protein-protein interactions, but cooperativity through DNA structure is becoming increasingly recognized as an additional mechanism. During the site-specific DNA recombination reaction that excises phage λ from the chromosome, the bacterial DNA architectural protein Fis recruits multiple λ-encoded Xis proteins to the attR recombination site. Here, we report X-ray crystal structures of DNA complexes containing Fis + Xis, which show little, if any, contacts between the two proteins. Comparisons with structures of DNA complexes containing only Fis or Xis, together with mutant protein and DNA binding studies, support a mechanism for cooperative protein binding solely by DNA allostery. Fis binding both molds the minor groove to potentiate insertion of the Xis ß-hairpin wing motif and bends the DNA to facilitate Xis-DNA contacts within the major groove. The Fis-structured minor groove shape that is optimized for Xis binding requires a precisely positioned pyrimidine-purine base-pair step, whose location has been shown to modulate minor groove widths in Fis-bound complexes to different DNA targets.

Reconstruction of transcriptional regulatory networks of Fis and H-NS in Escherichia coli from genome-wide data analysis.

Fis (Factor for inversion stimulation) and H-NS (Histone-like nucleoid-structuring protein) are two well-known nucleoid-associated proteins (NAPs) in proteobacteria, which play crucial roles in genome organization and transcriptional regulation. We performed RNA-sequencing to identify genes regulated by these NAPs. Study reveals that Fis and H-NS affect expression of 462 and 88 genes respectively in Escherichia coli at mid-exponential growth phase. By integrating available ChIP-seq data, we identified direct and indirect regulons of Fis and H-NS proteins. Functional analysis reveals that Fis controls expression of genes involved in translation, oxidative phosphorylation, sugar metabolism and transport, amino acid metabolism, bacteriocin transport, cell division, two-component system, biofilm formation, pilus organization and lipopolysaccharide biosynthesis pathways. However, H-NS represses expression of genes in cell adhesion, recombination, biofilm formation and lipopolysaccharide biosynthesis pathways under mid-exponential growth condition. The current regulatory networks thus provide a global glimpse of coordinated regulatory roles for these two important NAPs.

Fis Contributes to Resistance of Pseudomonas aeruginosa to Ciprofloxacin by Regulating Pyocin Synthesis.

Factor for inversion stimulation (Fis) is a versatile DNA binding protein that plays an important role in coordinating bacterial global gene expression in response to growth phases and environmental stresses. Previously, we demonstrated that Fis regulates the type III secretion system (T3SS) in Pseudomonas aeruginosa In this study, we explored the role of Fis in the antibiotic resistance of P. aeruginosa and found that mutation of the fis gene increases the bacterial susceptibility to ciprofloxacin. We further demonstrated that genes related to pyocin biosynthesis are upregulated in the fis mutant. The pyocins are produced in response to genotoxic agents, including ciprofloxacin, and the release of pyocins results in lysis of the producer cell. Thus, pyocin biosynthesis genes sensitize P. aeruginosa to ciprofloxacin. We found that PrtN, the positive regulator of the pyocin biosynthesis genes, is upregulated in the fis mutant. Genetic experiments and electrophoretic mobility shift assays revealed that Fis directly binds to the promoter region of prtN and represses its expression. Therefore, our results revealed novel Fis-mediated regulation on pyocin production and bacterial resistance to ciprofloxacin in P. aeruginosaIMPORTANCEPseudomonas aeruginosa is an important opportunistic pathogenic bacterium that causes various acute and chronic infections in human, especially in patients with compromised immunity, cystic fibrosis (CF), and/or severe burn wounds. About 60% of cystic fibrosis patients have a chronic respiratory infection caused by P. aeruginosa The bacterium is intrinsically highly resistant to antibiotics, which greatly increases difficulties in clinical treatment. Therefore, it is critical to understand the mechanisms and the regulatory pathways that are involved in antibiotic resistance. In this study, we elucidated a novel regulatory pathway that controls the bacterial resistance to fluoroquinolone antibiotics, which enhances our understanding of how P. aeruginosa responds to ciprofloxacin.

Multiple Binding Configurations of Fis Protein Pairs on DNA: Facilitated Dissociation versus Cooperative Dissociation.

As a master transcription regulator, the Fis protein influences over two hundred genes of E. coli. The Fis protein's nonspecific binding to DNA is widely acknowledged, and its kinetics of dissociation from DNA is strongly influenced by its surroundings: the dissociation rate increases as the concentration of the Fis protein in the solution phase increases. In this study, we use computational methods to explore the global binding energy landscape of the Fis1:Fis2:DNA ternary complex. The complex contains a binary-Fis molecular dyad whose formation relies on complex structural rearrangements. The simulations allow us to distinguish several different pathways for the dissociation of the protein from DNA with different functional outcomes and involving different protein stoichiometries: (1) simple exchange of proteins and (2) cooperative unbinding of two Fis proteins to yield bare DNA. In the case of exchange, the protein on the DNA is replaced by the solution-phase protein through competition for DNA binding sites. This process seen in fluorescence imaging experiments has been called facilitated dissociation. In the latter case of cooperative unbinding of pairs, two neighboring Fis proteins on DNA form a unique binary-Fis configuration via protein-protein interactions, which in turn leads to the codissociation of both molecules simultaneously, a process akin to the "molecular stripping" seen in the NFκB/IκB genetic broadcasting system. This simulation shows that the existence of multiple binding configurations of transcription factors can have a significant impact on the kinetics and outcome of transcription factor dissociation from DNA, with important implications for the systems biology of gene regulation by Fis.

ThreaDNA: predicting DNA mechanics' contribution to sequence selectivity of proteins along whole genomes.

Motivation: Many DNA-binding proteins recognize their target sequences indirectly, by sensing DNA's response to mechanical distortion. ThreaDNA estimates this response based on high-resolution structures of the protein-DNA complex of interest. Implementing an efficient nanoscale modeling of DNA deformations involving essentially no adjustable parameters, it returns the profile of deformation energy along whole genomes, at base-pair resolution, within minutes on usual laptop/desktop computers. Our predictions can also be easily combined with estimations of direct selectivity through a generalized form of position-weight-matrices. The formalism of ThreaDNA is accessible to a wide audience. Results: We demonstrate the importance of indirect readout for the nucleosome as well as the bacterial regulators Fis and CRP. Combined with the direct contribution provided by usual sequence motifs, it significantly improves the prediction of sequence selectivity, and allows quantifying the two distinct physical mechanisms underlying it. Availability and implementation: Python software available at bioinfo.insa-lyon.fr, natively executable on Linux/MacOS systems with a user-friendly graphical interface. Galaxy webserver version available. Contact: sam.meyer@insa-lyon.fr. Supplementary information: Supplementary data are available at Bioinformatics online.

Genome-wide prediction of minor-groove electrostatic potential enables biophysical modeling of protein-DNA binding.

Protein-DNA binding is a fundamental component of gene regulatory processes, but it is still not completely understood how proteins recognize their target sites in the genome. Besides hydrogen bonding in the major groove (base readout), proteins recognize minor-groove geometry using positively charged amino acids (shape readout). The underlying mechanism of DNA shape readout involves the correlation between minor-groove width and electrostatic potential (EP). To probe this biophysical effect directly, rather than using minor-groove width as an indirect measure for shape readout, we developed a methodology, DNAphi, for predicting EP in the minor groove and confirmed the direct role of EP in protein-DNA binding using massive sequencing data. The DNAphi method uses a sliding-window approach to mine results from non-linear Poisson-Boltzmann (NLPB) calculations on DNA structures derived from all-atom Monte Carlo simulations. We validated this approach, which only requires nucleotide sequence as input, based on direct comparison with NLPB calculations for available crystal structures. Using statistical machine-learning approaches, we showed that adding EP as a biophysical feature can improve the predictive power of quantitative binding specificity models across 27 transcription factor families. High-throughput prediction of EP offers a novel way to integrate biophysical and genomic studies of protein-DNA binding.

The shape of the DNA minor groove directs binding by the DNA-bending protein Fis.

The bacterial nucleoid-associated protein Fis regulates diverse reactions by bending DNA and through DNA-dependent interactions with other control proteins and enzymes. In addition to dynamic nonspecific binding to DNA, Fis forms stable complexes with DNA segments that share little sequence conservation. Here we report the first crystal structures of Fis bound to high- and low-affinity 27-base-pair DNA sites. These 11 structures reveal that Fis selects targets primarily through indirect recognition mechanisms involving the shape of the minor groove and sequence-dependent induced fits over adjacent major groove interfaces. The DNA shows an overall curvature of approximately 65 degrees , and the unprecedented close spacing between helix-turn-helix motifs present in the apodimer is accommodated by severe compression of the central minor groove. In silico DNA structure models show that only the roll, twist, and slide parameters are sufficient to reproduce the changes in minor groove widths and recreate the curved Fis-bound DNA structure. Models based on naked DNA structures suggest that Fis initially selects DNA targets with intrinsically narrow minor grooves using the separation between helix-turn-helix motifs in the Fis dimer as a ruler. Then Fis further compresses the minor groove and bends the DNA to generate the bound structure.

Characterization of Two Novel Lipopolysaccharide Phosphoethanolamine Transferases in Pasteurella multocida and Their Role in Resistance to Cathelicidin-2.

The lipopolysaccharide (LPS) produced by the Gram-negative bacterial pathogen Pasteurella multocida has phosphoethanolamine (PEtn) residues attached to lipid A, 3-deoxy-d-manno-octulosonic acid (Kdo), heptose, and galactose. In this report, we show that PEtn is transferred to lipid A by the P. multocida EptA homologue, PetL, and is transferred to galactose by a novel PEtn transferase that is unique to P. multocida called PetG. Transcriptomic analyses indicated that petL expression was positively regulated by the global regulator Fis and negatively regulated by an Hfq-dependent small RNA. Importantly, we have identified a novel PEtn transferase called PetK that is responsible for PEtn addition to the single Kdo molecule (Kdo1), directly linked to lipid A in the P. multocida glycoform A LPS. In vitro assays showed that the presence of a functional petL and petK, and therefore the presence of PEtn on lipid A and Kdo1, was essential for resistance to the cationic, antimicrobial peptide cathelicidin-2. The importance of PEtn on Kdo1 and the identification of the transferase responsible for this addition have not previously been shown. Phylogenetic analysis revealed that PetK is the first representative of a new family of predicted PEtn transferases. The PetK family consists of uncharacterized proteins from a range of Gram-negative bacteria that produce LPS glycoforms with only one Kdo molecule, including pathogenic species within the genera Vibrio, Bordetella, and Haemophilus We predict that many of these bacteria will require the addition of PEtn to Kdo for maximum protection against host antimicrobial peptides.

Mechanical constraints on Hin subunit rotation imposed by the Fis/enhancer system and DNA supercoiling during site-specific recombination.

Hin, a member of the serine family of site-specific recombinases, regulates gene expression by inverting a DNA segment. DNA inversion requires assembly of an invertasome complex in which a recombinational enhancer DNA segment bound by the Fis protein associates with the Hin synaptic complex at the base of a supercoiled DNA branch. Each of the four Hin subunits becomes covalently joined to the cleaved DNA ends, and DNA exchange occurs by translocation of a Hin subunit pair within the tetramer. We show here that, although the Hin tetramer forms a bidirectional molecular swivel, the Fis/enhancer system determines both the direction and number of subunit rotations. The chirality of supercoiling directs rotational direction, and the short DNA loop stabilized by Fis-Hin contacts limit rotational processivity, thereby ensuring that the DNA strands religate in the recombinant configuration. We identify multiple rotational conformers that are formed under different supercoiling and solution conditions.

Chromosomal position shift of a regulatory gene alters the bacterial phenotype.

Recent studies strongly suggest that in bacterial cells the order of genes along the chromosomal origin-to-terminus axis is determinative for regulation of the growth phase-dependent gene expression. The prediction from this observation is that positional displacement of pleiotropic genes will affect the genetic regulation and hence, the cellular phenotype. To test this prediction we inserted the origin-proximal dusB-fis operon encoding the global regulator FIS in the vicinity of replication terminus on both arms of the Escherichia coli chromosome. We found that the lower fis gene dosage in the strains with terminus-proximal dusB-fis operons was compensated by increased fis expression such that the intracellular concentration of FIS was homeostatically adjusted. Nevertheless, despite unchanged FIS levels the positional displacement of dusB-fis impaired the competitive growth fitness of cells and altered the state of the overarching network regulating DNA topology, as well as the cellular response to environmental stress, hazardous substances and antibiotics. Our finding that the chromosomal repositioning of a regulatory gene can determine the cellular phenotype unveils an important yet unexplored facet of the genetic control mechanisms and paves the way for novel approaches to manipulate bacterial physiology.

Facilitated Dissociation of a Nucleoid Protein from the Bacterial Chromosome.

UNLABELLED: Off-rates of proteins from the DNA double helix are widely considered to be dependent only on the interactions inside the initially bound protein-DNA complex and not on the concentration of nearby molecules. However, a number of recent single-DNA experiments have shown off-rates that depend on solution protein concentration, or "facilitated dissociation." Here, we demonstrate that this effect occurs for the major Escherichia coli nucleoid protein Fis on isolated bacterial chromosomes. We isolated E. coli nucleoids and showed that dissociation of green fluorescent protein (GFP)-Fis is controlled by solution Fis concentration and exhibits an "exchange" rate constant (kexch) of ≈10(4) M(-1) s(-1), comparable to the rate observed in single-DNA experiments. We also show that this effect is strongly salt dependent. Our results establish that facilitated dissociation can be observed in vitro on chromosomes assembled in vivo IMPORTANCE: Bacteria are important model systems for the study of gene regulation and chromosome dynamics, both of which fundamentally depend on the kinetics of binding and unbinding of proteins to DNA. In experiments on isolated E. coli chromosomes, this study showed that the prolific transcription factor and chromosome packaging protein Fis displays a strong dependence of its off-rate from the bacterial chromosome on Fis concentration, similar to that observed in in vitro experiments. Therefore, the free cellular DNA-binding protein concentration can strongly affect lifetimes of proteins bound to the chromosome and must be taken into account in quantitative considerations of gene regulation. These results have particularly profound implications for transcription factors where DNA binding lifetimes can be a critical determinant of regulatory function.

Upstream binding of idling RNA polymerase modulates transcription initiation from a nearby promoter.

The bacterial gene regulatory regions often demonstrate distinctly organized arrays of RNA polymerase binding sites of ill-defined function. Previously we observed a module of closely spaced polymerase binding sites upstream of the canonical promoter of the Escherichia coli fis operon. FIS is an abundant nucleoid-associated protein involved in adjusting the chromosomal DNA topology to changing cellular physiology. Here we show that simultaneous binding of the polymerase at the canonical fis promoter and an upstream transcriptionally inactive site stabilizes a RNAP oligomeric complex in vitro. We further show that modulation of the upstream binding of RNA polymerase affects the fis promoter activity both in vivo and in vitro. The effect of the upstream RNA polymerase binding on the fis promoter activity depends on the spatial arrangement of polymerase binding sites and DNA supercoiling. Our data suggest that a specific DNA geometry of the nucleoprotein complex stabilized on concomitant binding of RNA polymerase molecules at the fis promoter and the upstream region acts as a topological device regulating the fis transcription. We propose that transcriptionally inactive RNA polymerase molecules can act as accessory factors regulating the transcription initiation from a nearby promoter.

Expression of different bacterial cytotoxins is controlled by two global transcription factors, CRP and Fis, that co-operate in a shared-recruitment mechanism.

Pet is a cytotoxic autotransporter protein secreted by the pathogenic enteroaggregative Escherichia coli strain 042. Expression of Pet is co-dependent on two global transcription regulators: CRP (cyclic AMP receptor protein) and Fis (factor for inversion stimulation). At the pet promoter CRP binds to a single site centred at position -40.5 upstream of the start site for transcription. Due to the suboptimal positioning of this site, CRP alone activates transcription poorly and requires Fis to bind upstream to promote full activation. Here, we show that CRP and Fis control the expression of other important autotransporter toxins, namely Sat from uropathogenic E. coli (UPEC) and SigA from Shigella sonnei, and that this regulation has been conserved in different pathogens. Furthermore, we investigate the mechanism of Fis-mediated co-activation, exploiting a series of semi-synthetic promoters, with similar architecture to the pet promoter. We show that, when bound at position -40.5, CRP recruits RNA polymerase inefficiently and that Fis compensates by aiding polymerase recruitment through a direct protein-protein interaction. We demonstrate that other suitably positioned upstream transcription factors, which directly recruit RNA polymerase, can also compensate for the inappropriate positioning of CRP. We propose that this is a simple 'shared-recruitment' mechanism, by which co-dependence of promoters on two transcription factors could evolve.