Anne M. Stringer

Genome-Scale Analyses of Escherichia coli and Salmonella enterica AraC Reveal Noncanonical Targets and an Expanded Core Regulon

Escherichia coli AraC is a well-described transcription activator of genes involved in arabinose metabolism. Using complementary genomic approaches, chromatin immunoprecipitation (ChIP)-chip, and transcription profiling, we identify direct regulatory targets of AraC, including five novel target genes: ytfQ, ydeN, ydeM, ygeA, and polB. Strikingly, only ytfQ has an established connection to arabinose metabolism, suggesting that AraC has a broader function than previously described. We demonstrate arabinose-dependent repression of ydeNM by AraC, in contrast to the well-described arabinose-dependent activation of other target genes. We also demonstrate unexpected read-through of transcription at the Rho-independent terminators downstream of araD and araE, leading to significant increases in the expression of polB and ygeA, respectively. AraC is highly conserved in the related species Salmonella enterica. We use ChIP sequencing (ChIP-seq) and RNA sequencing (RNA-seq) to map the AraC regulon in S. enterica. A comparison of the E. coli and S. enterica AraC regulons, coupled with a bioinformatic analysis of other related species, reveals a conserved regulatory network across the family Enterobacteriaceae comprised of 10 genes associated with arabinose transport and metabolism.

FRUIT, a Scar-Free System for Targeted Chromosomal Mutagenesis, Epitope Tagging, and Promoter Replacement in Escherichia coli and Salmonella enterica

Recombineering is a widely-used approach to delete genes, introduce insertions and point mutations, and introduce epitope tags into bacterial chromosomes. Many recombineering methods have been described, for a wide range of bacterial species. These methods are often limited by (i) low efficiency, and/or (ii) introduction of “scar” DNA into the chromosome. Here, we describe a rapid, efficient, PCR-based recombineering method, FRUIT, that can be used to introduce scar-free point mutations, deletions, epitope tags, and promoters into the genomes of enteric bacteria. The efficiency of FRUIT is far higher than that of the most widely-used recombineering method for Escherichia coli. We have used FRUIT to introduce point mutations and epitope tags into the chromosomes of E. coli K-12, Enterotoxigenic E. coli, and Salmonella enterica. We have also used FRUIT to introduce constitutive and inducible promoters into the chromosome of E. coli K-12. Thus, FRUIT is a versatile, efficient recombineering approach that can be applied in multiple species of enteric bacteria.

Identification of HilD-Regulated Genes in Salmonella enterica Serovar Typhimurium

Salmonella enterica serovar Typhimurium (S. Typhimurium) pathogenicity island 1 (SPI-1) encodes a type III secretion system required for invasion of host gut epithelial cells. Expression of SPI-1 virulence genes is controlled by a complex hierarchy of transcription factors encoded within and outside SPI-1. The master regulator of SPI-1, HilA, is itself regulated by three homologous transcription factors, HilD, HilC, and RtsA. HilD activates transcription of hilA and other target genes in response to environmental conditions associated with the intestinal microenvironment of the host. We have mapped the binding of HilD across the S. Typhimurium genome using chromatin immunoprecipitation-sequencing (ChIP-seq). Thus, we have identified 17 regions bound by HilD, including 11 novel targets. The majority of HilD targets are located outside SPI-1. We demonstrate transcription activation of 8 genes by HilD; four of these genes have not been previously described as being regulated by HilD, including lpxR, which encodes a lipid A deacylase important for immune evasion. We also show that HilD-activated genes are frequently activated by HilC and RtsA, indicating extensive overlap of the HilD, HilC, and RtsA regulons.

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.

Non-canonical protein-DNA interactions identified by ChIP are not artifacts

BackgroundChIP-chip and ChIP-seq are widely used methods to map protein-DNA interactions on a genomic scale in vivo. Waldminghaus and Skarstad recently reported, in this journal, a modified method for ChIP-chip. Based on a comparison of our previously-published ChIP-chip data for Escherichia coli σ32 with their own data, Waldminghaus and Skarstad concluded that many of the σ32 targets identified in our earlier work are false positives. In particular, we identified many non-canonical σ32 targets that are located inside genes or are associated with genes that show no detectable regulation by σ32. Waldminghaus and Skarstad propose that such non-canonical sites are artifacts, identified due to flaws in the standard ChIP methodology. Waldminghaus and Skarstad suggest specific changes to the standard ChIP procedure that reportedly eliminate the claimed artifacts.ResultsWe reanalyzed our published ChIP-chip datasets for σ32 and the datasets generated by Waldminghaus and Skarstad to assess data quality and reproducibility. We also performed targeted ChIP/qPCR for σ32 and an unrelated transcription factor, AraC, using the standard ChIP method and the modified ChIP method proposed by Waldminghaus and Skarstad. Furthermore, we determined the association of core RNA polymerase with disputed σ32 promoters, with and without overexpression of σ32. We show that (i) our published σ32 ChIP-chip datasets have a consistently higher dynamic range than those of Waldminghaus and Skarstad, (ii) our published σ32 ChIP-chip datasets are highly reproducible, whereas those of Waldminghaus and Skarstad are not, (iii) non-canonical σ32 target regions are enriched in a σ32 ChIP in a heat shock-dependent manner, regardless of the ChIP method used, (iv) association of core RNA polymerase with some disputed σ32 target genes is induced by overexpression of σ32, (v) σ32 targets disputed by Waldminghaus and Skarstad are predominantly those that are most weakly bound, and (vi) the modifications to the ChIP method proposed by Waldminghaus and Skarstad reduce enrichment of all protein-bound genomic regions.ConclusionsThe modifications to the ChIP-chip method suggested by Waldminghaus and Skarstad reduce rather than increase the quality of ChIP data. Hence, the non-canonical σ32 targets identified in our previous study are likely to be genuine. We propose that the failure of Waldminghaus and Skarstad to identify many of these σ32 targets is due predominantly to the lower data quality in their study. We conclude that surprising ChIP-chip results are not artifacts to be ignored, but rather indications that our understanding of DNA-binding proteins is incomplete.

Horizontally acquired AT-rich genes in Escherichia coli cause toxicity by sequestering RNA polymerase

Horizontal gene transfer permits rapid dissemination of genetic elements between individuals in bacterial populations. Transmitted DNA sequences may encode favourable traits. However, if the acquired DNA has an atypical base composition, it can reduce host fitness. Consequently, bacteria have evolved strategies to minimize the harmful effects of foreign genes. Most notably, xenogeneic silencing proteins bind incoming DNA that has a higher AT content than the host genome. An enduring question has been why such sequences are deleterious. Here, we showed that the toxicity of AT-rich DNA in Escherichia coli frequently results from constitutive transcription initiation within the coding regions of genes. Left unchecked, this causes titration of RNA polymerase and a global downshift in host gene expression. Accordingly, a mutation in RNA polymerase that diminished the impact of AT-rich DNA on host fitness reduced transcription from constitutive, but not activator-dependent, promoters.

Mapping the Regulatory Network for Salmonella enterica Serovar Typhimurium Invasion

ABSTRACT Salmonella enterica pathogenicity island 1 (SPI-1) encodes proteins required for invasion of gut epithelial cells. The timing of invasion is tightly controlled by a complex regulatory network. The transcription factor (TF) HilD is the master regulator of this process and senses environmental signals associated with invasion. HilD activates transcription of genes within and outside SPI-1, including six other TFs. Thus, the transcriptional program associated with host cell invasion is controlled by at least 7 TFs. However, very few of the regulatory targets are known for these TFs, and the extent of the regulatory network is unclear. In this study, we used complementary genomic approaches to map the direct regulatory targets of all 7 TFs. Our data reveal a highly complex and interconnected network that includes many previously undescribed regulatory targets. Moreover, the network extends well beyond the 7 TFs, due to the inclusion of many additional TFs and noncoding RNAs. By comparing gene expression profiles of regulatory targets for the 7 TFs, we identified many uncharacterized genes that are likely to play direct roles in invasion. We also uncovered cross talk between SPI-1 regulation and other regulatory pathways, which, in turn, identified gene clusters that likely share related functions. Our data are freely available through an intuitive online browser and represent a valuable resource for the bacterial research community. IMPORTANCE Invasion of epithelial cells is an early step during infection by Salmonella enterica and requires secretion of specific proteins into host cells via a type III secretion system (T3SS). Most T3SS-associated proteins required for invasion are encoded in a horizontally acquired genomic locus known as Salmonella pathogenicity island 1 (SPI-1). Multiple regulators respond to environmental signals to ensure appropriate timing of SPI-1 gene expression. In particular, there are seven transcription regulators that are known to be involved in coordinating expression of SPI-1 genes. We have used complementary genome-scale approaches to map the gene targets of these seven regulators. Our data reveal a highly complex and interconnected regulatory network that includes many previously undescribed target genes. Moreover, our data functionally implicate many uncharacterized genes in the invasion process and reveal cross talk between SPI-1 regulation and other regulatory pathways. All datasets are freely available through an intuitive online browser. Invasion of epithelial cells is an early step during infection by Salmonella enterica and requires secretion of specific proteins into host cells via a type III secretion system (T3SS). Most T3SS-associated proteins required for invasion are encoded in a horizontally acquired genomic locus known as Salmonella pathogenicity island 1 (SPI-1). Multiple regulators respond to environmental signals to ensure appropriate timing of SPI-1 gene expression. In particular, there are seven transcription regulators that are known to be involved in coordinating expression of SPI-1 genes. We have used complementary genome-scale approaches to map the gene targets of these seven regulators. Our data reveal a highly complex and interconnected regulatory network that includes many previously undescribed target genes. Moreover, our data functionally implicate many uncharacterized genes in the invasion process and reveal cross talk between SPI-1 regulation and other regulatory pathways. All datasets are freely available through an intuitive online browser.

SuhB Associates with Nus Factors To Facilitate 30S Ribosome Biogenesis in Escherichia coli

ABSTRACT A complex of highly conserved proteins consisting of NusB, NusE, NusA, and NusG is required for robust expression of rRNA in Escherichia coli. This complex is proposed to prevent Rho-dependent transcription termination by a process known as “antitermination.” The mechanism of this antitermination in rRNA is poorly understood but requires association of NusB and NusE with a specific RNA sequence in rRNA known as BoxA. Here, we identify a novel member of the rRNA antitermination machinery: the inositol monophosphatase SuhB. We show that SuhB associates with elongating RNA polymerase (RNAP) at rRNA in a NusB-dependent manner. Although we show that SuhB is required for BoxA-mediated antitermination in a reporter system, our data indicate that the major function of the NusB/E/A/G/SuhB complex is not to prevent Rho-dependent termination of rRNA but rather to promote correct rRNA maturation. This occurs through formation of a SuhB-mediated loop between NusB/E/BoxA and RNAP/NusA/G. Thus, we have reassigned the function of these proteins at rRNA and identified another key player in this complex. IMPORTANCE As RNA polymerase transcribes the rRNA operons in E. coli, it complexes with a set of proteins called Nus that confer enhanced rates of transcription elongation, correct folding of rRNA, and rRNA assembly with ribosomal proteins to generate a fully functional ribosome. Four Nus proteins were previously known, NusA, NusB, NusE, and NusG; here, we discover and describe a fifth, SuhB, that is an essential component of this complex. We demonstrate that the main function of this SuhB-containing complex is not to prevent premature transcription termination within the rRNA operon, as had been long claimed, but to enable rRNA maturation and a functional ribosome fully competent for translation. As RNA polymerase transcribes the rRNA operons in E. coli, it complexes with a set of proteins called Nus that confer enhanced rates of transcription elongation, correct folding of rRNA, and rRNA assembly with ribosomal proteins to generate a fully functional ribosome. Four Nus proteins were previously known, NusA, NusB, NusE, and NusG; here, we discover and describe a fifth, SuhB, that is an essential component of this complex. We demonstrate that the main function of this SuhB-containing complex is not to prevent premature transcription termination within the rRNA operon, as had been long claimed, but to enable rRNA maturation and a functional ribosome fully competent for translation.

Genome-Wide Transcriptional Regulation and Chromosome Structural Arrangement by GalR in E. coli

The regulatory protein, GalR, is known for controlling transcription of genes related to D-galactose metabolism in Escherichia coli. Here, using a combination of experimental and bioinformatic approaches, we identify novel GalR binding sites upstream of several genes whose function is not directly related to D-galactose metabolism. Moreover, we do not observe regulation of these genes by GalR under standard growth conditions. Thus, our data indicate a broader regulatory role for GalR, and suggest that regulation by GalR is modulated by other factors. Surprisingly, we detect regulation of 158 transcripts by GalR, with few regulated genes being associated with a nearby GalR binding site. Based on our earlier observation of long-range interactions between distally bound GalR dimers, we propose that GalR indirectly regulates the transcription of many genes by inducing large-scale restructuring of the chromosome.

Nus Factors Prevent Premature Transcription Termination of Bacterial CRISPR Arrays

A hallmark of CRISPR-Cas immunity systems is the CRISPR array, a genomic locus consisting of short, repeated sequences (“repeats”) interspersed with short, variable sequences (“spacers”). CRISPR arrays are transcribed and processed into individual CRISPR RNAs (crRNAs) that each include a single spacer, and direct Cas proteins to complementary sequence in invading nucleic acid. Most bacterial CRISPR array transcripts are unusually long for untranslated RNA, suggesting the existence of mechanisms to prevent premature transcription termination by Rho, a conserved bacterial transcription termination factor that rapidly terminates untranslated RNA. We show that Rho termination functionally limits the length of bacterial CRISPR arrays, and we identify a widespread antitermination mechanism that antagonizes Rho to facilitate complete transcription of CRISPR arrays. Thus, our data highlight the importance of Rho termination in the evolution of bacterial CRISPR-Cas systems.