Chromosome organization in bacteria: mechanistic insights into genome structure and function.

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.

A dual-function RNA balances carbon uptake and central metabolism in Vibrio cholerae.

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.

Genome-wide mapping of Vibrio cholerae VpsT binding identifies a mechanism for c-di-GMP homeostasis.

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.

An unexpected abundance of bidirectional promoters within Salmonella Typhimurium plasmids.

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.

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.

A non-canonical promoter element drives spurious transcription of horizontally acquired bacterial genes.

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.

The quorum sensing transcription factor AphA directly regulates natural competence in Vibrio cholerae.

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.

Widespread suppression of intragenic transcription initiation by H-NS.

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.

The Escherichia coli MarA protein regulates the ycgZ-ymgABC operon to inhibit biofilm formation.

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 σ38 (RpoS)-associated RNA polymerase (RNAP). Surprisingly, MarA does not influence σ38 -dependent transcription. Instead, MarA drives transcription by the housekeeping σ70 -associated RNAP. The effects of MarA on ycgZ-ymgABC expression are coupled with 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.

Waking the neighbours: disruption of H-NS repression by overlapping transcription.

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 Escherichia coli multiple antibiotic resistance activator protein represses transcription of the lac operon.

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.

CsrA maximizes expression of the AcrAB multidrug resistance transporter.

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.

The molecular basis for control of ETEC enterotoxin expression in response to environment and host.

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.

DNA recognition by Escherichia coli CbpA protein requires a conserved arginine-minor-groove interaction.

Curved DNA binding protein A (CbpA) is a co-chaperone and nucleoid associated DNA binding protein conserved in most γ-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.

The unexpected complexity of bacterial genomes.

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.

Structure and function of bacterial H-NS protein.

The histone-like nucleoid structuring (H-NS) protein is a major component of the folded chromosome in Escherichia coli and related bacteria. Functions attributed to H-NS include management of genome evolution, DNA condensation, and transcription. The wide-ranging influence of H-NS is remarkable given the simplicity of the protein, a small peptide, possessing rudimentary determinants for self-association, hetero-oligomerisation and DNA binding. In this review, I will discuss our understanding of H-NS with a focus on these structural elements. In particular, I will consider how these interaction surfaces allow H-NS to exert its different effects.

H-NS can facilitate specific DNA-binding by RNA polymerase in AT-rich gene regulatory regions.

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.

E. coli Fis protein insulates the cbpA gene from uncontrolled transcription.

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.

Chromosomal macrodomains and associated proteins: implications for DNA organization and replication in gram negative bacteria.

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.

Multidisciplinary perspectives on bacterial genome organization and dynamics.

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.