Variable DNA topology is an epigenetic generator of physiological heterogeneity in bacterial populations.

Transcription is a noisy and stochastic process that produces sibling-to-sibling variations in physiology across a population of genetically identical cells. This pattern of diversity reflects, in part, the burst-like nature of transcription. Transcription bursting has many causes and a failure to remove the supercoils that accumulate in DNA during transcription elongation is an important contributor. Positive supercoiling of the DNA ahead of the transcription elongation complex can result in RNA polymerase stalling if this DNA topological roadblock is not removed. The relaxation of these positive supercoils is performed by the ATP-dependent type II topoisomerases DNA gyrase and topoisomerase IV. Interference with the action of these topoisomerases involving, inter alia, topoisomerase poisons, fluctuations in the [ATP]/[ADP] ratio, and/or the intervention of nucleoid-associated proteins with GapR-like or YejK-like activities, may have consequences for the smooth operation of the transcriptional machinery. Antibiotic-tolerant (but not resistant) persister cells are among the phenotypic outliers that may emerge. However, interference with type II topoisomerase activity can have much broader consequences, making it an important epigenetic driver of physiological diversity in the bacterial population.

Mechanistic insights from molecular microbiology into the production of immunological and neuronal diversity.

Bacteria deal with an unpredictable, and often hostile, environment by being unpredictable themselves. This article will link some contributions made by variable DNA topology and nucleoid-associated proteins to the generation of stochasticity in bacterial gene expression and describe how the associated mechanistic insights can elucidate the means by which diversity in antibody and neuronal cell development might be produced in humans and other higher organisms. The focus here will not be on mutation; instead, the article will address epigenetic effects on gene expression brought about by the modulation of topoisomerase activity in both prokaryotes and eukaryotes.

Physiological Robustness of Model Gram-Negative Bacteria in Response to Genome Rewiring.

DNA supercoiling and nucleoid-associated proteins (NAPs) are two of the factors that govern the architecture of the bacterial genome, influencing the expression of the genetic information that it contains. Alterations to DNA topology, and to the numbers and types of NAPs, have pleiotropic effects on gene expression, suggesting that modifications to the production patterns of DNA topoisomerases and/or NAPs are likely to result in marked impacts on bacterial physiology. Knockout mutations in the genes encoding these proteins (where the mutants remain viable) result in clear physiological effects. However, genetic modifications that involve rewiring, or repositioning, of topoisomerase or NAP genes produce much more subtle outcomes. These findings demonstrate that the high-level regulatory circuitry of bacteria is robust in the face of genomic rearrangements that, a priori, might be expected to produce significant changes in bacterial lifestyle. Examples from genomic rewiring experiments, performed chiefly with the Gram-negative model bacteria Escherichia coli K-12 and Salmonella enterica serovar Typhimurium, will be used to illustrate these features. The results show not only the ability of naturally occurring bacteria to tolerate regulatory rewiring but also indicate the limits within which experiments in synthetic biology may be designed.

Reciprocally rewiring and repositioning the Integration Host Factor (IHF) subunit genes in Salmonella enterica serovar Typhimurium: impacts on physiology and virulence.

The Integration Host Factor (IHF) is a heterodimeric nucleoid-associated protein that plays roles in bacterial nucleoid architecture and genome-wide gene regulation. The ihfA and ihfB genes encode the subunits and are located 350 kbp apart, in the Right replichore of the Salmonella chromosome. IHF is composed of one IhfA and one IhfB subunit. Despite this 1 : 1 stoichiometry, MS revealed that IhfB is produced in 2-fold excess over IhfA. We re-engineered Salmonella to exchange reciprocally the protein-coding regions of ihfA and ihfB, such that each relocated protein-encoding region was driven by the expression signals of the other's gene. MS showed that in this 'rewired' strain, IhfA is produced in excess over IhfB, correlating with enhanced stability of the hybrid ihfB-ihfA mRNA that was expressed from the ihfB promoter. Nevertheless, the rewired strain grew at a similar rate to the wild-type and was similar in competitive fitness. However, compared to the wild-type, it was less motile, had growth-phase-specific reductions in SPI-1 and SPI-2 gene expression, and was engulfed at a higher rate by RAW macrophage. Our data show that while exchanging the physical locations of its ihf genes and the rewiring of their regulatory circuitry are well tolerated in Salmonella, genes involved in the production of type 3 secretion systems exhibit dysregulation accompanied by altered phenotypes.

Consequences of producing DNA gyrase from a synthetic gyrBA operon in Salmonella enterica serovar Typhimurium.

DNA gyrase is an essential type II topoisomerase that is composed of two subunits, GyrA and GyrB, and has an A2 B2 structure. Although the A and B subunits are required in equal proportions to form DNA gyrase, the gyrA and gyrB genes that encode them in Salmonella (and in many other bacteria) are at separate locations on the chromosome, are under separate transcriptional control, and are present in different copy numbers in rapidly growing bacteria. In wild-type Salmonella, gyrA is near the chromosome's replication terminus, while gyrB is near the origin. We generated a synthetic gyrBA operon at the oriC-proximal location of gyrB to test the significance of the gyrase gene position for Salmonella physiology. Although the strain producing gyrase from an operon had a modest alteration to its DNA supercoiling set points, most housekeeping functions were unaffected. However, its SPI-2 virulence genes were expressed at a reduced level and its survival was reduced in macrophage. Our data reveal that the horizontally acquired SPI-2 genes have a greater sensitivity to disturbance of DNA topology than the core genome and we discuss its significance in the context of Salmonella genome evolution and the gyrA and gyrB gene arrangements found in other bacteria.

Human Health and Ocean Pollution.

Background: Pollution - unwanted waste released to air, water, and land by human activity - is the largest environmental cause of disease in the world today. It is responsible for an estimated nine million premature deaths per year, enormous economic losses, erosion of human capital, and degradation of ecosystems. Ocean pollution is an important, but insufficiently recognized and inadequately controlled component of global pollution. It poses serious threats to human health and well-being. The nature and magnitude of these impacts are only beginning to be understood. Goals: (1) Broadly examine the known and potential impacts of ocean pollution on human health. (2) Inform policy makers, government leaders, international organizations, civil society, and the global public of these threats. (3) Propose priorities for interventions to control and prevent pollution of the seas and safeguard human health. Methods: Topic-focused reviews that examine the effects of ocean pollution on human health, identify gaps in knowledge, project future trends, and offer evidence-based guidance for effective intervention. Environmental Findings: Pollution of the oceans is widespread, worsening, and in most countries poorly controlled. It is a complex mixture of toxic metals, plastics, manufactured chemicals, petroleum, urban and industrial wastes, pesticides, fertilizers, pharmaceutical chemicals, agricultural runoff, and sewage. More than 80% arises from land-based sources. It reaches the oceans through rivers, runoff, atmospheric deposition and direct discharges. It is often heaviest near the coasts and most highly concentrated along the coasts of low- and middle-income countries. Plastic is a rapidly increasing and highly visible component of ocean pollution, and an estimated 10 million metric tons of plastic waste enter the seas each year. Mercury is the metal pollutant of greatest concern in the oceans; it is released from two main sources - coal combustion and small-scale gold mining. Global spread of industrialized agriculture with increasing use of chemical fertilizer leads to extension of Harmful Algal Blooms (HABs) to previously unaffected regions. Chemical pollutants are ubiquitous and contaminate seas and marine organisms from the high Arctic to the abyssal depths. Ecosystem Findings: Ocean pollution has multiple negative impacts on marine ecosystems, and these impacts are exacerbated by global climate change. Petroleum-based pollutants reduce photosynthesis in marine microorganisms that generate oxygen. Increasing absorption of carbon dioxide into the seas causes ocean acidification, which destroys coral reefs, impairs shellfish development, dissolves calcium-containing microorganisms at the base of the marine food web, and increases the toxicity of some pollutants. Plastic pollution threatens marine mammals, fish, and seabirds and accumulates in large mid-ocean gyres. It breaks down into microplastic and nanoplastic particles containing multiple manufactured chemicals that can enter the tissues of marine organisms, including species consumed by humans. Industrial releases, runoff, and sewage increase frequency and severity of HABs, bacterial pollution, and anti-microbial resistance. Pollution and sea surface warming are triggering poleward migration of dangerous pathogens such as the Vibrio species. Industrial discharges, pharmaceutical wastes, pesticides, and sewage contribute to global declines in fish stocks. Human Health Findings: Methylmercury and PCBs are the ocean pollutants whose human health effects are best understood. Exposures of infants in utero to these pollutants through maternal consumption of contaminated seafood can damage developing brains, reduce IQ and increase children's risks for autism, ADHD and learning disorders. Adult exposures to methylmercury increase risks for cardiovascular disease and dementia. Manufactured chemicals - phthalates, bisphenol A, flame retardants, and perfluorinated chemicals, many of them released into the seas from plastic waste - can disrupt endocrine signaling, reduce male fertility, damage the nervous system, and increase risk of cancer. HABs produce potent toxins that accumulate in fish and shellfish. When ingested, these toxins can cause severe neurological impairment and rapid death. HAB toxins can also become airborne and cause respiratory disease. Pathogenic marine bacteria cause gastrointestinal diseases and deep wound infections. With climate change and increasing pollution, risk is high that Vibrio infections, including cholera, will increase in frequency and extend to new areas. All of the health impacts of ocean pollution fall disproportionately on vulnerable populations in the Global South - environmental injustice on a planetary scale. Conclusions: Ocean pollution is a global problem. It arises from multiple sources and crosses national boundaries. It is the consequence of reckless, shortsighted, and unsustainable exploitation of the earth's resources. It endangers marine ecosystems. It impedes the production of atmospheric oxygen. Its threats to human health are great and growing, but still incompletely understood. Its economic costs are only beginning to be counted.Ocean pollution can be prevented. Like all forms of pollution, ocean pollution can be controlled by deploying data-driven strategies based on law, policy, technology, and enforcement that target priority pollution sources. Many countries have used these tools to control air and water pollution and are now applying them to ocean pollution. Successes achieved to date demonstrate that broader control is feasible. Heavily polluted harbors have been cleaned, estuaries rejuvenated, and coral reefs restored.Prevention of ocean pollution creates many benefits. It boosts economies, increases tourism, helps restore fisheries, and improves human health and well-being. It advances the Sustainable Development Goals (SDG). These benefits will last for centuries. Recommendations: World leaders who recognize the gravity of ocean pollution, acknowledge its growing dangers, engage civil society and the global public, and take bold, evidence-based action to stop pollution at source will be critical to preventing ocean pollution and safeguarding human health.Prevention of pollution from land-based sources is key. Eliminating coal combustion and banning all uses of mercury will reduce mercury pollution. Bans on single-use plastic and better management of plastic waste reduce plastic pollution. Bans on persistent organic pollutants (POPs) have reduced pollution by PCBs and DDT. Control of industrial discharges, treatment of sewage, and reduced applications of fertilizers have mitigated coastal pollution and are reducing frequency of HABs. National, regional and international marine pollution control programs that are adequately funded and backed by strong enforcement have been shown to be effective. Robust monitoring is essential to track progress.Further interventions that hold great promise include wide-scale transition to renewable fuels; transition to a circular economy that creates little waste and focuses on equity rather than on endless growth; embracing the principles of green chemistry; and building scientific capacity in all countries.Designation of Marine Protected Areas (MPAs) will safeguard critical ecosystems, protect vulnerable fish stocks, and enhance human health and well-being. Creation of MPAs is an important manifestation of national and international commitment to protecting the health of the seas.

Network Rewiring: Physiological Consequences of Reciprocally Exchanging the Physical Locations and Growth-Phase-Dependent Expression Patterns of the Salmonella fis and dps Genes.

The Fis nucleoid-associated protein controls the expression of a large and diverse regulon of genes in Gram-negative bacteria. Fis production is normally maximal in bacteria during the early exponential phase of batch culture growth, becoming almost undetectable by the onset of stationary phase. We tested the effect on the Fis regulatory network in Salmonella of moving the complete fis gene from its usual location near the origin of chromosomal replication to the position normally occupied by the dps gene in the right macrodomain of the chromosome, and vice versa, creating the gene exchange (GX) strain. In a parallel experiment, we tested the effect of rewiring the Fis regulatory network by placing the fis open reading frame under the control of the stationary-phase-activated dps promoter at the dps genetic location within the right macrodomain, and vice versa, creating the open reading frame exchange (OX) strain. Chromatin immunoprecipitation sequencing (ChIP-seq) was used to measure global Fis protein binding levels and to determine gene expression patterns. Strain GX showed few changes compared with the wild type, although we did detect increased Fis binding at Ter, accompanied by reduced binding at Ori. Strain OX displayed a more pronounced version of this distorted Fis protein-binding pattern together with numerous alterations in the expression of genes in the Fis regulon. OX, but not GX, had a reduced ability to infect cultured mammalian cells. These findings illustrate the inherent robustness of the Fis regulatory network with respect to the effects of rewiring based on gene repositioning alone and emphasize the importance of fis expression signals in phenotypic determination.IMPORTANCE We assessed the impact on Salmonella physiology of reciprocally translocating the genes encoding the Fis and Dps nucleoid-associated proteins (NAPs) and of inverting their growth-phase production patterns such that Fis was produced in stationary phase (like Dps) and Dps was produced in exponential phase (like Fis). Changes to peak binding of Fis were detected by ChIP-seq on the chromosome, as were widespread impacts on the transcriptome, especially when Fis production mimicked Dps production. Virulence gene expression and the expression of a virulence phenotype were altered. Overall, these radical changes to NAP gene expression were well tolerated, revealing the robust and well-buffered nature of global gene regulation networks in the bacterium.

When is a transcription factor a NAP?

Proteins that regulate transcription often also play an architectural role in the genome. Thus, it has been difficult to define with precision the distinctions between transcription factors and nucleoid-associated proteins (NAPs). Anachronistic descriptions of NAPs as 'histone-like' implied an organizational function in a bacterial chromatin-like complex. Definitions based on protein abundance, regulatory mechanisms, target gene number, or the features of their DNA-binding sites are insufficient as marks of distinction, and trying to distinguish transcription factors and NAPs based on their ranking within regulatory hierarchies or positions in gene-control networks is also unsatisfactory. The terms 'transcription factor' and 'NAP' are ad hoc operational definitions with each protein lying along a spectrum of structural and functional features extending from highly specific actors with few gene targets to those with a pervasive influence on the transcriptome. The Streptomyces BldC protein is used to illustrate these issues.

CRISPR-Cas, DNA Supercoiling, and Nucleoid-Associated Proteins.

In this opinion article we highlight links between the H-NS nucleoid-associated protein, variable DNA topology, the regulation of CRISPR-cas locus expression, CRISPR-Cas activity, and the recruitment of novel genetic information by the CRISPR array. We propose that the requirement that the invading mobile genetic element be negatively supercoiled limits effective CRISPR action to a window in the bacterial growth cycle when DNA topology is optimal, and that this same window is used for the efficient integration of new spacer sequences at the CRISPR array. H-NS silences CRISPR promoters, and we propose that antagonists of H-NS, such as the LeuO transcription factor, provide a basis for a stochastic genetic switch that acts at random in each cell in the bacterial population. In addition, we wish to propose a mechanism by which mobile genetic elements can suppress CRISPR-cas transcription using H-NS homologues. Although the individual components of this network are known, we propose a new model in which they are integrated and linked to the physiological state of the bacterium. The model provides a basis for cell-to-cell variation in the expression and performance of CRISPR systems in bacterial populations.

DNA supercoiling and transcription in bacteria: a two-way street.

BACKGROUND: The processes of DNA supercoiling and transcription are interdependent because the movement of a transcription elongation complex simultaneously induces under- and overwinding of the DNA duplex and because the initiation, elongation and termination steps of transcription are all sensitive to the topological state of the DNA. RESULTS: Policing of the local and global supercoiling of DNA by topoisomerases helps to sustain the major DNA-based transactions by eliminating barriers to the movement of transcription complexes and replisomes. Recent data from whole-genome and single-molecule studies have provided new insights into how interactions between transcription and the supercoiling of DNA influence the architecture of the chromosome and how they create cell-to-cell diversity at the level of gene expression through transcription bursting. CONCLUSIONS: These insights into fundamental molecular processes reveal mechanisms by which bacteria can prevail in unpredictable and often hostile environments by becoming unpredictable themselves.

Regulatory Hierarchies Controlling Virulence Gene Expression in Shigella flexneri and Vibrio cholerae.

Gram-negative enteropathogenic bacteria use a variety of strategies to cause disease in the human host and gene regulation in some form is typically a part of the strategy. This article will compare the toxin-based infection strategy used by the non-invasive pathogen Vibrio cholerae, the etiological agent in human cholera, with the invasive approach used by Shigella flexneri, the cause of bacillary dysentery. Despite the differences in the mechanisms by which the two pathogens cause disease, they use environmentally-responsive regulatory hierarchies to control the expression of genes that have some features, and even some components, in common. The involvement of AraC-like transcription factors, the integration host factor, the Factor for inversion stimulation, small regulatory RNAs, the RNA chaperone Hfq, horizontal gene transfer, variable DNA topology and the need to overcome the pervasive silencing of transcription by H-NS of horizontally acquired genes are all shared features. A comparison of the regulatory hierarchies in these two pathogens illustrates some striking cross-species similarities and differences among mechanisms coordinating virulence gene expression. S. flexneri, with its low infectious dose, appears to use a strategy that is centered on the individual bacterial cell, whereas V. cholerae, with a community-based, quorum-dependent approach and an infectious dose that is several orders of magnitude higher, seems to rely more on the actions of a bacterial collective.

Negative supercoiling of DNA by gyrase is inhibited in Salmonella enterica serovar Typhimurium during adaptation to acid stress.

DNA in intracellular Salmonella enterica serovar Typhimurium relaxes during growth in the acidified (pH 4-5) macrophage vacuole and DNA relaxation correlates with the upregulation of Salmonella genes involved in adaptation to the macrophage environment. Bacterial ATP levels did not increase during adaptation to acid pH unless the bacterium was deficient in MgtC, a cytoplasmic-membrane-located inhibitor of proton-driven F1 F0 ATP synthase activity. Inhibiting ATP binding by DNA gyrase and topo IV with novobiocin enhanced the effect of low pH on DNA relaxation. Bacteria expressing novobiocin-resistant (NovR ) derivatives of gyrase or topo IV also exhibited DNA relaxation at acid pH, although further relaxation with novobiocin was not seen in the strain with NovR gyrase. Thus, inhibition of the negative supercoiling activity of gyrase was the primary cause of enhanced DNA relaxation in drug-treated bacteria. The Salmonella cytosol reaches pH 5-6 in response to an external pH of 4-5: the ATP-dependent DNA supercoiling activity of purified gyrase was progressively inhibited by lowering the pH in this range, as was the ATP-dependent DNA relaxation activity of topo IV. We propose that DNA relaxation in Salmonella within macrophage is due to acid-mediated impairment of the negative supercoiling activity of gyrase.

Broad-scale redistribution of mRNA abundance and transcriptional machinery in response to growth rate in Salmonella enterica serovar Typhimurium.

We have investigated the connection between the four-dimensional architecture of the bacterial nucleoid and the organism's global gene expression programme. By localizing the transcription machinery and the transcriptional outputs across the genome of the model bacterium Salmonella enterica serovar Typhimurium at different stages of the growth cycle, a surprising disconnection between gene dosage and transcriptional output was revealed. During exponential growth, gene output occurred chiefly in the Ori (origin), Ter (terminus) and NSL (non-structured left) domains, whereas the Left macrodomain remained transcriptionally quiescent at all stages of growth. The apparently high transcriptional output in Ter was correlated with an enhanced stability of the RNA expressed there during exponential growth, suggesting that longer mRNA half-lives compensate for low gene dosage. During exponential growth, RNA polymerase (RNAP) was detected everywhere, whereas in stationary phase cells, RNAP was concentrated in the Ter macrodomain. The alternative sigma factors RpoE, RpoH and RpoN were not required to drive transcription in these growth conditions, consistent with their observed binding to regions away from RNAP and regions of active transcription. Specifically, these alternative sigma factors were found in the Ter macrodomain during exponential growth, whereas they were localized at the Ori macrodomain in stationary phase.

Control of virulence gene transcription by indirect readout in Vibrio cholerae and Salmonella enterica serovar Typhimurium.

Indirect readout mechanisms of transcription control rely on the recognition of DNA shape by transcription factors (TFs). TFs may also employ a direct readout mechanism that involves the reading of the base sequence in the DNA major groove at the binding site. TFs with winged helix-turn-helix (wHTH) motifs use an alpha helix to read the base sequence in the major groove while inserting a beta sheet 'wing' into the adjacent minor groove. Such wHTH proteins are important regulators of virulence gene transcription in many pathogens; they also control housekeeping genes. This article considers the cases of the non-invasive Gram-negative pathogen Vibrio cholerae and the invasive pathogen Salmonella enterica serovar Typhimurium. Both possess clusters of A + T-rich horizontally acquired virulence genes that are silenced by the nucleoid-associated protein H-NS and regulated positively or negatively by wHTH TFs: for example, ToxR and LeuO in V. cholerae; HilA, LeuO, SlyA and OmpR in S. Typhimurium. Because of their relatively relaxed base sequence requirements for target recognition, indirect readout mechanisms have the potential to engage regulatory proteins with many more targets than might be the case using direct readout, making indirect readout an important, yet often ignored, contributor to the expression of pathogenic phenotypes.

Bacterial pathogen gene regulation: a DNA-structure-centred view of a protein-dominated domain.

The mechanisms used by bacterial pathogens to regulate the expression of their genes, especially their virulence genes, have been the subject of intense investigation for several decades. Whole genome sequencing projects, together with more targeted studies, have identified hundreds of DNA-binding proteins that contribute to the patterns of gene expression observed during infection as well as providing important insights into the nature of the gene products whose expression is being controlled by these proteins. Themes that have emerged include the importance of horizontal gene transfer to the evolution of pathogens, the need to impose regulatory discipline upon these imported genes and the important roles played by factors normally associated with the organization of genome architecture as regulatory principles in the control of virulence gene expression. Among these architectural elements is the structure of DNA itself, its variable nature at a topological rather than just at a base-sequence level and its ability to play an active (as well as a passive) part in the gene regulation process.

The interplay between DNA topology and accessory factors in site-specific recombination in bacteria and their bacteriophages.

Site-specific recombination is employed widely in bacteria and bacteriophage as a basis for genetic switching events that control phenotypic variation. It plays a vital role in the life cycles of phages and in the replication cycles of chromosomes and plasmids in bacteria. Site-specific recombinases drive these processes using very short segments of identical (or nearly identical) DNA sequences. In some cases, the efficiencies of the recombination reactions are modulated by the topological state of the participating DNA sequences and by the availability of accessory proteins that shape the DNA. These dependencies link the molecular machines that conduct the recombination reactions to the physiological state of the cell. This is because the topological state of bacterial DNA varies constantly during the growth cycle and so does the availability of the accessory factors. In addition, some accessory factors are under allosteric control by metabolic products or second messengers that report the physiological status of the cell. The interplay between DNA topology, accessory factors and site-specific recombination provides a powerful illustration of the connectedness and integration of molecular events in bacterial cells and in viruses that parasitise bacterial cells.

DNA supercoiling is a fundamental regulatory principle in the control of bacterial gene expression.

Although it has become routine to consider DNA in terms of its role as a carrier of genetic information, it is also an important contributor to the control of gene expression. This regulatory principle arises from its structural properties. DNA is maintained in an underwound state in most bacterial cells and this has important implications both for DNA storage in the nucleoid and for the expression of genetic information. Underwinding of the DNA through reduction in its linking number potentially imparts energy to the duplex that is available to drive DNA transactions, such as transcription, replication and recombination. The topological state of DNA also influences its affinity for some DNA binding proteins, especially in DNA sequences that have a high A + T base content. The underwinding of DNA by the ATP-dependent topoisomerase DNA gyrase creates a continuum between metabolic flux, DNA topology and gene expression that underpins the global response of the genome to changes in the intracellular and external environments. These connections describe a fundamental and generalised mechanism affecting global gene expression that underlies the specific control of transcription operating through conventional transcription factors. This mechanism also provides a basal level of control for genes acquired by horizontal DNA transfer, assisting microbial evolution, including the evolution of pathogenic bacteria.

DNA supercoiling is a fundamental regulatory principle in the control of bacterial gene expression.

Although it has become routine to consider DNA in terms of its role as a carrier of genetic information, it is also an important contributor to the control of gene expression. This regulatory principle arises from its structural properties. DNA is maintained in an underwound state in most bacterial cells and this has important implications both for DNA storage in the nucleoid and for the expression of genetic information. Underwinding of the DNA through reduction in its linking number potentially imparts energy to the duplex that is available to drive DNA transactions, such as transcription, replication and recombination. The topological state of DNA also influences its affinity for some DNA binding proteins, especially in DNA sequences that have a high A + T base content. The underwinding of DNA by the ATP-dependent topoisomerase DNA gyrase creates a continuum between metabolic flux, DNA topology and gene expression that underpins the global response of the genome to changes in the intracellular and external environments. These connections describe a fundamental and generalised mechanism affecting global gene expression that underlies the specific control of transcription operating through conventional transcription factors. This mechanism also provides a basal level of control for genes acquired by horizontal DNA transfer, assisting microbial evolution, including the evolution of pathogenic bacteria.