David C. Grainger

Association of nucleoid proteins with coding and non-coding segments of the Escherichia coli genome

The Escherichia coli chromosome is condensed into an ill-defined structure known as the nucleoid. Nucleoid-associated DNA-binding proteins are involved in maintaining this structure and in mediating chromosome compaction. We have exploited chromatin immunoprecipitation and high-density microarrays to study the binding of three such proteins, FIS, H-NS and IHF, across the E.coli genome in vivo. Our results show that the distribution of these proteins is biased to intergenic parts of the genome, and that the binding profiles overlap. Hence some targets are associated with combinations of bound FIS, H-NS and IHF. In addition, many regions associated with FIS and H-NS are also associated with RNA polymerase.

Effects of nucleoid-associated proteins on bacterial chromosome structure and gene expression

Bacterial nucleoid-associated proteins play a key role in the organisation, replication, segregation, repair and expression of bacterial chromosomes. Here, we review some recent progress in our understanding of the effects of these proteins on DNA and their biological role, focussing mainly on Escherichia coli and its chromosome. Certain nucleoid-associated proteins also regulate transcription initiation at specific promoters, and work in concert with dedicated transcription factors to regulate gene expression in response to growth phase and environmental change. Some specific examples, involving the E. coli IHF and Fis proteins, that illustrate new principles, are described in detail.

Extensive functional overlap between σ factors in Escherichia coli

Bacterial core RNA polymerase (RNAP) must associate with a σ factor to recognize promoter sequences. Escherichia coli encodes seven σ factors, each believed to be specific for a largely distinct subset of promoters. Using microarrays representing the entire E. coli genome, we identify 87 in vivo targets of σ32, the heat-shock σ factor, and estimate that there are 120–150 σ32 promoters in total. Unexpectedly, 25% of these σ32 targets are located within coding regions, suggesting novel regulatory roles for σ32. The majority of σ32 promoter targets overlap with those of σ70, the housekeeping σ factor. Furthermore, their DNA sequence motifs are often interdigitated, with RNAPσ70 and RNAPσ32 initiating transcription in vitro with similar efficiency and from identical positions. σE-regulated promoters also overlap extensively with those for σ70. These results suggest that extensive functional overlap between σ factors is an important phenomenon.

The Escherichia coli RutR transcription factor binds at targets within genes as well as intergenic regions

The Escherichia coli RutR protein is the master regulator of genes involved in pyrimidine catabolism. Here we have used chromatin immunoprecipitation in combination with DNA microarrays to measure the binding of RutR across the chromosome of exponentially growing E. coli cells. Twenty RutR-binding targets were identified and analysis of these targets generated a DNA consensus logo for RutR binding. Complementary in vitro binding assays showed high-affinity RutR binding to 16 of the 20 targets, with the four low-affinity RutR targets lacking predicted key binding determinants. Surprisingly, most of the DNA targets for RutR are located within coding segments of the genome and appear to have little or no effect on transcript levels in the conditions tested. This contrasts sharply with other E. coli transcription factors whose binding sites are primarily located in intergenic regions. We suggest that either RutR has yet undiscovered function or that evolution has been slow to eliminate non-functional DNA sites for RutR because they do not have an adverse effect on cell fitness.

Transcription factor distribution in Escherichia coli: studies with FNR protein

Using chromatin immunoprecipitation (ChIP) and high-density microarrays, we have measured the distribution of the global transcription regulator protein, FNR, across the entire Escherichia coli chromosome in exponentially growing cells. Sixty-three binding targets, each located at the 5′ end of a gene, were identified. Some targets are adjacent to poorly transcribed genes where FNR has little impact on transcription. In stationary phase, the distribution of FNR was largely unchanged. Control experiments showed that, like FNR, the distribution of the nucleoid-associated protein, IHF, is little altered when cells enter stationary phase, whilst RNA polymerase undergoes a complete redistribution.

Selective repression by Fis and H‐NS at the Escherichia coli dps promoter

Dps is a nucleoid‐associated protein that plays a major role in condensation of the Escherichia coli chromosome in stationary phase. Here we show that two other nucleoid‐associated proteins, Fis and H‐NS, can bind at the dps gene promoter and downregulate its activity. Both Fis and H‐NS selectively repress the dps promoter, preventing transcription initiation by RNA polymerase containing σ70, the housekeeping σ factor, but not by RNA polymerase containing σ38, the stationary‐phase σ factor. Fis represses by trapping RNA polymerase containing σ70 at the promoter. In contrast, H‐NS functions by displacing RNA polymerase containing σ70, but not RNA polymerase containing σ38. Dps levels are known to be very low in exponentially growing cells and rise sharply as cells enter stationary phase. Conversely, Fis levels are high in growing cells but fall to nearly zero in stationary‐phase cells. Our data suggest a simple model to explain how the Dps‐dependent super‐compaction of the folded chromosome is triggered as cell growth ceases.

Genomic Studies with Escherichia coli MelR Protein: Applications of Chromatin Immunoprecipitation and Microarrays

Escherichia coli MelR protein is a transcription activator that is essential for melibiose-dependent expression of the melAB genes. We have used chromatin immunoprecipitation to study the binding of MelR and RNA polymerase to the melAB promoter in vivo. Our results show that MelR is associated with promoter DNA, both in the absence and presence of the inducer melibiose. In contrast, RNA polymerase is recruited to the melAB promoter only in the presence of inducer. The MelR DK261 positive control mutant binds to the melAB promoter but cannot recruit RNA polymerase. Further analysis of immunoprecipitated DNA, by using an Affymetrix GeneChip array, showed that the melAB promoter is the major, if not the sole, target in E. coli for MelR. This was confirmed by a transcriptomics experiment to analyze RNA in cells either with or without melR.

Genomic analysis of protein-DNA interactions in bacteria : insights into transcription and chromosome organization

Chromatin immunoprecipitation (ChIP) is a powerful method to measure protein–DNA interactions in vivo, and it can be applied on a genomic scale with microarray technology (ChIP‐chip). ChIP‐chip has been used extensively to map DNA–protein interactions across eukaryotic chromosomes. Here we review recent applications of ChIP‐chip to the study of bacteria, which provide important and unexpected insights into transcription and chromosome organization.

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.

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.