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The evolution of gene regulation is a major source of evolutionary adaptation and innovation, particularly when organisms encounter new or changing environments. Central to this process is the emergence of new transcription factor binding sites (TFBSs). Adaptive landscapes provide a powerful framework to study such emergence by linking regulatory DNA sequences to their transcriptional outputs. Although several landscapes have been characterized for DNA, RNA, and proteins, large-scale in vivo adaptive landscapes for bacterial TFBSs remain scarce. Here, we address this gap by experimentally mapping the first comprehensive in vivo regulatory landscapes for three global transcription factors in : cAMP receptor protein, Fis, and IHF. Using a massively parallel reporter assay, we quantify the regulation strength of more than 30,000 TFBS variants for each factor, and reconstruct their adaptive landscapes. All three landscapes are highly rugged and exhibit pervasive epistasis, with thousands of local peaks distributed broadly across sequence space. This ruggedness contrasts sharply with the much smoother TFBS landscapes of eukaryotes. It suggests greater constraints on the evolution of prokaryotic gene regulation. Nonetheless, evolutionary simulations show that ~10% of evolving populations can reach a peak of strong regulation, a proportion that is significantly greater than in comparable random landscapes. Adaptive evolution starting from the same DNA sequence can attain different high peaks, and some peaks are reached more frequently than others. Together, our results show that de novo adaptive evolution of new gene regulation in bacteria is feasible, but subject to a blend of chance, historical contingency, and evolutionary biases.

The nucleoid-associated protein FIS is a global regulator of gene expression and chromosome structure in Escherichia coli and Salmonella enterica. Despite the importance of FIS for infection and intracellular invasion, very little is known about the regulation of S. enterica fis expression. Under standard laboratory growth conditions, fis is highly expressed during rapid growth but is then silenced as growth slows. However, if cells are cultured in non-aerated conditions, fis expression is sustained during stationary phase. This led us to test whether the redox-sensing transcription factors ArcA and FNR regulate S. enterica fis. Deletion of FNR had no detectable effect, whereas deletion of ArcA had the unexpected effect of further elevating fis expression in stationary phase. ArcA required RpoS for induction of fis expression, suggesting that ArcA indirectly affects fis expression. Other putative regulators were found to play diverse roles: FIS acted directly as an auto-repressor (as expected), whereas CRP had little direct effect on fis expression. Deleting regions of the fis promoter led to the discovery of a novel anaerobically-induced transcription start site (Pfis-2) upstream of the primary transcription start site (Pfis-1). Promoter truncation also revealed that the shortest functional fis promoter was incapable of sustained expression. Moreover, fis expression was observed to correlate directly with DNA supercoiling in non-aerated conditions. Thus, the full-length S. enterica fis promoter region may act as a topological switch that is sensitive to stress-induced duplex destabilisation and up-regulates expression in non-aerated conditions.

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 Δ fis,  Δ arcA  and double deletion mutant, Δ arcA Δ fis  under acetate using 13 C-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. 13 C-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 CO 2 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.

The model prokaryote Escherichia coli contains seven copies of the rRNA operon in the genome. The presence of multiple rRNA operons is an advantage for increasing the level of ribosome, the key apparatus of translation, in response to environmental conditions. The complete sequence of E. coli genome, however, indicated the micro heterogeneity between seven rRNA operons, raising the possibility in functional heterogeneity and/or differential mode of expression. The aim of this research is to determine the strength and regulation of the promoter of each rRNA operon in E. coli. For this purpose, we used the double-fluorescent protein reporter pBRP system that was developed for accurate and precise determination of the promoter strength of protein-coding genes. For application of this promoter assay vector for measurement of the rRNA operon promoters devoid of the signal for translation, a synthetic SD sequence was added at the initiation codon of the reporter GFP gene, and then approximately 500 bp-sequence upstream each 16S rRNA was inserted in front of this SD sequence. Using this modified pGRS system, the promoter activity of each rrn operon was determined by measuring the rrn promoter-directed GFP and the reference promoter-directed RFP fluorescence, both encoded by a single and the same vector. Results indicated that: the promoter activity was the highest for the rrnE promoter under all growth conditions analyzed, including different growth phases of wild-type E. coli grown in various media; but the promoter strength of other six rrn promoters was various depending on the culture conditions. These findings altogether indicate that seven rRNA operons are different with respect to the regulation mode of expression, conferring an advantage to E. coli through a more fine-tuned control of ribosome formation in a wide range of environmental situations. Possible difference in the functional role of each rRNA operon is also discussed.

Gene expression is controlled by the joint effect of (i) the global physiological state of the cell, in particular the activity of the gene expression machinery, and (ii) DNA‐binding transcription factors and other specific regulators. We present a model‐based approach to distinguish between these two effects using time‐resolved measurements of promoter activities. We demonstrate the strength of the approach by analyzing a circuit involved in the regulation of carbon metabolism in E. coli . Our results show that the transcriptional response of the network is controlled by the physiological state of the cell and the signaling metabolite cyclic AMP (cAMP). The absence of a strong regulatory effect of transcription factors suggests that they are not the main coordinators of gene expression changes during growth transitions, but rather that they complement the effect of global physiological control mechanisms. This change of perspective has important consequences for the interpretation of transcriptome data and the design of biological networks in biotechnology and synthetic biology. A simple, parameterless mathematical model, in combination with real‐time monitoring of promoter activities, shows how control of gene expression in bacteria is shared between transcription factors and global physiological effects. Synopsis A simple, parameterless mathematical model, in combination with real‐time monitoring of promoter activities, shows how control of gene expression in bacteria is shared between transcription factors and global physiological effects. We present an approach based on a simple, paramaterless mathematical model to analyze the control of gene expression by transcription factors and the global physiological state of the cell. We illustrate the strength of this approach by means of time‐resolved measurements of the transcriptional activities of genes in a central regulatory circuit in Escherichia coli . We conclude that global physiological effects rather than transcription factors dominate the control of gene expression during a growth transition. Our results call for a reappraisal of the role of transcription factors, which may be most appropriately viewed as complementing and finetuning global control exerted by the physiological state of the cell.

Pasteurella multocida ( P. multocida ) is an important pathogen causing various diseases in both domestic and wild animals. The factor for inversion stimulation (Fis) is a nucleoid-associated protein with diverse functions in various bacteria, which positively regulate the transcription of capsular glycosaminoglycan genes in P. multocida . However, the precise mechanistic understanding of how the fis regulate virulence of P. multocida remains largely unknown. In this study, we discovered that fis transcript levels of P. multocida CQ2, serotype A (PmCQ2) were significantly increased in vivo, and showed a positive correlation with the capsule and virulence of P. multocida . To further understand how the fis regulated P. multocida pathogenesis, a homologous recombination strategy was used to generate fis -deleted strain. Then, the growth velocity, virulence characteristics, immune/inflammatory responses, and the survival rates of challenged mice were determined. The findings revealed that the presence of fis promoted the growth, regulated synthesis of capsule and biofilm of PmCQ2, and helped to resist phagocytosis by macrophages. Notably, we firstly demonstrated that Fis determined whether P. multocida can use bound iron ion for its survival. Consequently, the loss of fis greatly restricted P. multocida pathogenicity, as evidenced by reducing tissue bacterial loads as well as inflammatory factors levels. Moreover, the fis deletion strain was endowed with strong cross immunoprotected properties against P. multocida serotype A and B. Thus, these results suggested the pathogenic role of fis in P. multocida and proposed that fis deletion strain could be used as an attenuated vaccine candidate against P. multocida of serotype A and B.

Key Points Nucleoid-associated proteins in bacteria perform roles that influence all of the major DNA transactions. In Escherichia coli and other Gram-negative bacteria, the H-NS nucleoid-associated protein has a global and negative influence on gene expression. Detailed information is emerging about the mechanisms by which H-NS exerts its negative effects. Knowledge of the domain structure of the H-NS protein is central to the understanding of its biological role. It is becoming clear that the ability of H-NS to oligomerize is of great biological significance. The formation of heteromeric oligomers between related but distinct proteins is a growing theme in studies of H-NS. It is also becoming apparent that H-NS can interact with unrelated proteins. The finding that genes encoding proteins that are capable of interacting with H-NS can be located on mobile genetic elements has potentially important implications for the development and evolution of global regulatory circuits in bacteria. The effect of the bacterial heat-stable nucleoid-structuring (H-NS) protein on gene expression is overwhelmingly negative and extends throughout the genome, pointing to an almost universal role for this nucleoid-associated protein as a transcriptional repressor. Its ability to exert widespread effects on gene expression probably reflects the fact that it binds to curved DNA, which is commonly found at promoters. H-NS and related proteins can engage in both homologous and heterologous protein–protein interactions. Recent data show that the genes that encode H-NS-like proteins can be carried on mobile genetic elements. This raises the possibility that horizontal gene transfer expands the repertoire of protein–protein interactions that nucleoid-associated proteins can engage in, with potentially profound consequences for the global gene-expression profile of the cell.

Serine recombinases are often tightly controlled by elaborate, topologically-defined, nucleoprotein complexes. Hin is a member of the DNA invertase subclass of serine recombinases that are regulated by a remote recombinational enhancer element containing two binding sites for the protein Fis. Two Hin dimers bound to specific recombination sites associate with the Fis-bound enhancer by DNA looping where they are remodeled into a synaptic tetramer competent for DNA chemistry and exchange. Here we show that the flexible beta-hairpin arms of the Fis dimers contact the DNA binding domain of one subunit of each Hin dimer. These contacts sandwich the Hin dimers to promote remodeling into the tetramer. A basic region on the Hin catalytic domain then contacts enhancer DNA to complete assembly of the active Hin tetramer. Our results reveal how the enhancer generates the recombination complex that specifies DNA inversion and regulates DNA exchange by the subunit rotation mechanism. Many processes in biology rely on enzymes that break both the strands in a DNA molecule, then rearrange the strands, and finally join them back together in a new configuration. These recombination reactions can, for example, change the positions of genetic elements such as enhancers and promoters within the DNA molecule and, therefore, influence how a given gene is expressed as a protein. Cells need to be able to control recombination reactions because they can lead to leukemia and lymphomas if they go wrong. The enzymes that catalyze these recombination reactions are called recombinases. One type of recombinase binds to specific sequences of DNA bases and uses an amino acid in the enzyme–usually serine or tyrosine–to break and rejoin the DNA strands. Recombination reactions require the assembly of complexes containing many proteins bound to DNA. Tyrosine recombinases form relatively simple protein-DNA complexes, and these have been studied in detail. Serine recombinases, on the other hand, form more elaborate protein-DNA complexes, and much less is known about these. Now McLean et al. have unraveled the mechanism that a serine recombinase called Hin uses to reverse the direction of a stretch of chromosomal DNA in the bacteria Salmonella enterica. Inverting this stretch of DNA–which contains about 1000 base pairs–changes the position of a gene promoter that is responsible for the production of flagellin, which is the protein that enables the bacterium to move. This is one of the tricks that Salmonella uses to evade the immune system of its host. Previous research has established that four Hin subunits and two copies of a protein called Fis are needed to invert this stretch of DNA: two Hin subunits bind to each of the two hix recombination sites, and the Fis proteins (which are dimers) bind to each end of an enhancer that is located between the hix sites. A protein called HU then causes the DNA to bend and form a loop, and the four Hin subunits and the two Fis dimers all come together at the enhancer to form a structure called the invertasome where the recombination reaction occurs. All four DNA strands at the crossover point are broken as a result of a near simultaneous attack by the catalytic serine amino acids in the Hin subunits. One pair of Hin subunits–and the two DNA strands attached to them–then rotate by 180 degrees around the other pair of Hin subunits. This means that the stretch of DNA between the hix sites is inverted when the DNA strands are rejoined at the end of the reaction. Enhancers often regulate transcription and other reactions from a distance. McLean et al. reveal how an enhancer of a DNA recombination reaction works. The pairs of Hin subunits that initially bind to the DNA are not catalytically active, but when they are brought together by the enhancer and form a tetramer, they become active. Two of the Hin subunits are clamped onto the enhancer by the Fis dimers and by directly interacting with the enhancer DNA, but the other two (and the DNA strands attached to them) are free to rotate within the tetramer. In the Salmonella chromosome the enhancer is located close to one of the hix sites (∼100 base pairs away from it), so the length of the DNA between the enhancer and hix site physically limits the number of Hin subunit rotations to just one.

LapA is the biggest protein in Pseudomonas putida and a key factor for biofilm formation. Its importance and posttranslational regulation is rather thoroughly studied but less is known about the transcriptional regulation. Here we give evidence that transcription of lapA in LB-grown bacteria is initiated from six promoters, three of which display moderate RpoS-dependence. The global transcription regulator Fis binds to the lapA promoter area at six positions in vitro, and Fis activates the transcription of lapA while overexpressed in cells. Two of the six Fis binding sites, Fis-A7 and Fis-A5, are necessary for the positive effect of Fis on the transcription of lapA in vivo. Our results indicate that Fis binding to the Fis-A7 site increases the level of transcription from the most distal promoter of lapA, whereas Fis binding to the Fis-A5 site could be important for modifying the promoter area topology.

Extra-intestinal pathogenic Escherichia coli (ExPEC) are responsible for diverse infections including meningitis, sepsis and urinary tract infections. The alarming rise in anti-microbial resistance amongst ExPEC complicates treatment and has highlighted the need for alternative preventive measures. SslE is a lipoprotein secreted by a dedicated type II secretion system in E. coli that was first identified as a potential vaccine candidate using reverse genetics. Although the function and protective efficacy of SslE has been studied, the molecular mechanisms that regulate SslE expression remain to be fully elucidated. Here, we show that while the expression of SslE can be detected in E. coli culture supernatants, different strains express and secrete different amounts of SslE when grown under the same conditions. While the histone-like transcriptional regulator H-NS strongly represses sslE at ambient temperatures, the variation in SslE expression at human physiological temperature suggested a more complex mode of regulation. Using a genetic screen to identify novel regulators of sslE in the high SslE-expressing strain UTI89, we defined a new role for the nucleoid-associated regulator Fis and the ribosome-binding GTPase TypA as positive regulators of sslE transcription. We also showed that Fis-mediated enhancement of sslE transcription is dependent on a putative Fis-binding sequence located upstream of the -35 sequence in the core promoter element, and provide evidence to suggest that Fis may work in complex with H-NS to control SslE expression. Overall, this study has defined a new mechanism for sslE regulation and increases our understanding of this broadly conserved E. coli vaccine antigen.