Direct and indirect control of Rho-dependent transcription termination by the Escherichia coli lysC riboswitch.

Bacterial riboswitches are molecular structures that play a crucial role in controlling gene expression to maintain cellular balance. The Escherichia coli lysC riboswitch has been previously shown to regulate gene expression through translation initiation and mRNA decay. Recent research suggests that lysC gene expression is also influenced by Rho-dependent transcription termination. Through a series of in silico, in vitro, and in vivo experiments, we provide experimental evidence that the lysC riboswitch directly and indirectly modulates Rho transcription termination. Our study demonstrates that Rho-dependent transcription termination plays a significant role in the cotranscriptional regulation of lysC expression. Together with previous studies, our work suggests that lysC expression is governed by a lysine-sensing riboswitch that regulates translation initiation, transcription termination, and mRNA degradation. Notably, both Rho and RNase E target the same region of the RNA molecule, implying that RNase E may degrade Rho-terminated transcripts, providing a means to selectively eliminate these incomplete messenger RNAs. Overall, this study sheds light on the complex regulatory mechanisms used by bacterial riboswitches, emphasizing the role of transcription termination in the control of gene expression and mRNA stability.

Promoter selectivity of the RhlR quorum-sensing transcription factor receptor in Pseudomonas aeruginosa is coordinated by distinct and overlapping dependencies on C4-homoserine lactone and PqsE.

Quorum sensing is a mechanism of bacterial cell-cell communication that relies on the production and detection of small molecule autoinducers, which facilitate the synchronous expression of genes involved in group behaviors, such as virulence factor production and biofilm formation. The Pseudomonas aeruginosa quorum sensing network consists of multiple interconnected transcriptional regulators, with the transcription factor, RhlR, acting as one of the main drivers of quorum sensing behaviors. RhlR is a LuxR-type transcription factor that regulates its target genes when bound to its cognate autoinducer, C4-homoserine lactone, which is synthesized by RhlI. RhlR function is also regulated by the metallo-ß-hydrolase enzyme, PqsE. We recently showed that PqsE binds RhlR to alter its affinity for promoter DNA, a new mechanism of quorum-sensing receptor activation. Here, we perform ChIP-seq analyses of RhlR to map the binding of RhlR across the P. aeruginosa genome, and to determine the impact of C4-homoserine lactone and PqsE on RhlR binding to different sites across the P. aeruginosa genome. We identify 40 RhlR binding sites, all but three of which are associated with genes known to be regulated by RhlR. C4-homoserine lactone is required for maximal binding of RhlR to many of its DNA sites. Moreover, C4-homoserine lactone is required for maximal RhlR-dependent transcription activation from all sites, regardless of whether it impacts RhlR binding to DNA. PqsE is required for maximal binding of RhlR to many DNA sites, with similar effects on RhlR-dependent transcription activation from those sites. However, the effects of PqsE on RhlR specificity are distinct from those of C4-homoserine lactone, and PqsE is sufficient for RhlR binding to some DNA sites in the absence of C4-homoserine lactone. Together, C4-homoserine lactone and PqsE are required for RhlR binding at the large majority of its DNA sites. Thus, our work reveals three distinct modes of activation by RhlR: i) when RhlR is unbound by autoinducer but bound by PqsE, ii) when RhlR is bound by autoinducer but not bound by PqsE, and iii) when RhlR is bound by both autoinducer and PqsE, establishing a stepwise mechanism for the progression of the RhlR-RhlI-PqsE quorum sensing pathway in P. aeruginosa.

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.

Genome-Wide Mapping of the Escherichia coli PhoB Regulon Reveals Many Transcriptionally Inert, Intragenic Binding Sites.

Genome-scale analyses have revealed many transcription factor binding sites within, rather than upstream of, genes, raising questions as to the function of these binding sites. Here, we use complementary approaches to map the regulon of the Escherichia coli transcription factor PhoB, a response regulator that controls transcription of genes involved in phosphate homeostasis. Strikingly, the majority of PhoB binding sites are located within genes, but these intragenic sites are not associated with detectable transcription regulation and are not evolutionarily conserved. Many intragenic PhoB sites are located in regions bound by H-NS, likely due to shared sequence preferences of PhoB and H-NS. However, these PhoB binding sites are not associated with transcription regulation even in the absence of H-NS. We propose that for many transcription factors, including PhoB, binding sites not associated with promoter sequences are transcriptionally inert and hence are tolerated as genomic "noise." IMPORTANCE Recent studies have revealed large numbers of transcription factor binding sites within the genes of bacteria. The function, if any, of the vast majority of these binding sites has not been investigated. Here, we map the binding of the transcription factor PhoB across the Escherichia coli genome, revealing that the majority of PhoB binding sites are within genes. We show that PhoB binding sites within genes are not associated with regulation of the overlapping genes. Indeed, our data suggest that bacteria tolerate the presence of large numbers of nonregulatory, intragenic binding sites for transcription factors and that these binding sites are not under selective pressure.

Genome-wide mapping of the Escherichia coli PhoB regulon reveals many transcriptionally inert, intragenic binding sites.

Genome-scale analyses have revealed many transcription factor binding sites within, rather than upstream of genes, raising questions as to the function of these binding sites. Here, we use complementary approaches to map the regulon of the Escherichia coli transcription factor PhoB, a response regulator that controls transcription of genes involved in phosphate homeostasis. Strikingly, the majority of PhoB binding sites are located within genes, but these intragenic sites are not associated with detectable transcription regulation and are not evolutionarily conserved. Many intragenic PhoB sites are located in regions bound by H-NS, likely due to shared sequence preferences of PhoB and H-NS. However, these PhoB binding sites are not associated with transcription regulation even in the absence of H-NS. We propose that for many transcription factors, including PhoB, binding sites not associated with promoter sequences are transcriptionally inert, and hence are tolerated as genomic "noise". IMPORTANCE: Recent studies have revealed large numbers of transcription factor binding sites within the genes of bacteria. The function, if any, of the vast majority of these binding sites has not been investigated. Here, we map the binding of the transcription factor PhoB across the Escherichia coli genome, revealing that the majority of PhoB binding sites are within genes. We show that PhoB binding sites within genes are not associated with regulation of the overlapping genes. Indeed, our data suggest that bacteria tolerate the presence of large numbers of non-regulatory, intragenic binding sites for transcription factors, and that these binding sites are not under selective pressure.

Identification of novel translated small ORFs in Escherichia coli using complementary ribosome profiling approaches.

Small proteins of <51 amino acids are abundant across all domains of life but are often overlooked because their small size makes them difficult to predict computationally, and they are refractory to standard proteomic approaches. Ribosome profiling has been used to infer the existence of small proteins by detecting the translation of the corresponding open reading frames (ORFs). Detection of translated short ORFs by ribosome profiling can be improved by treating cells with drugs that stall ribosomes at specific codons. Here, we combine the analysis of ribosome profiling data for Escherichia coli cells treated with antibiotics that stall ribosomes at either start or stop codons. Thus, we identify ribosome-occupied start and stop codons with high sensitivity for ∼400 novel putative ORFs. The newly discovered ORFs are mostly short, with 365 encoding proteins of <51 amino acids. We validate translation of several selected short ORFs, and show that many likely encode unstable proteins. Moreover, we present evidence that most of the newly identified short ORFs are not under purifying selection, suggesting they do not impact cell fitness, although a small subset have the hallmarks of functional ORFs. IMPORTANCE Small proteins of <51 amino acids are abundant across all domains of life but are often overlooked because their small size makes them difficult to predict computationally, and they are refractory to standard proteomic approaches. Recent studies have discovered small proteins by mapping the location of translating ribosomes on RNA using a technique known as ribosome profiling. Discovery of translated sORFs using ribosome profiling can be improved by treating cells with drugs that trap initiating ribosomes. Here, we show that combining these data with equivalent data for cells treated with a drug that stalls terminating ribosomes facilitates the discovery of small proteins. We use this approach to discover 365 putative genes that encode small proteins in Escherichia coli.

Regulatory Cross Talk between Motility and Interbacterial Communication in Salmonella enterica Serovar Typhimurium.

FliA is a broadly conserved σ factor that directs transcription of genes involved in flagellar motility. We previously identified FliA-transcribed genes in Escherichia coli and Salmonella enterica serovar Typhimurium, and we showed that E. coli FliA transcribes many unstable, noncoding RNAs from intragenic promoters. Here, we show that FliA in S Typhimurium also directs the transcription of large numbers of unstable, noncoding RNAs from intragenic promoters, and we identify two previously unreported FliA-transcribed protein-coding genes. One of these genes, sdiA, encodes a transcription factor that responds to quorum-sensing signals produced by other bacteria. We show that FliA-dependent transcription of sdiA is required for SdiA activity, highlighting a regulatory link between flagellar motility and intercellular communication.IMPORTANCE Initiation of bacterial transcription requires association of a σ factor with the core RNA polymerase to facilitate sequence-specific recognition of promoter elements. FliA is a widely conserved σ factor that directs transcription of genes involved in flagellar motility. We previously showed that Escherichia coli FliA transcribes many unstable, noncoding RNAs from promoters within genes. Here, we demonstrate the same phenomenon in Salmonella Typhimurium. We also show that S Typhimurium FliA directs transcription of the sdiA gene, which encodes a transcription factor that responds to quorum-sensing signals produced by other bacteria. FliA-dependent transcription of sdiA is required for transcriptional control of SdiA target genes, highlighting a regulatory link between flagellar motility and intercellular communication.

Transcription termination and antitermination 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 that each include a single spacer, and direct Cas proteins to complementary sequences 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 can prematurely terminate transcription 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 transcription termination and antitermination in the evolution of bacterial CRISPR-Cas systems.

MrpJ Directly Regulates Proteus mirabilis Virulence Factors, Including Fimbriae and Type VI Secretion, during Urinary Tract Infection.

Proteus mirabilis is a leading cause of catheter-associated urinary tract infections (CAUTIs) and urolithiasis. The transcriptional regulator MrpJ inversely modulates two critical aspects of P. mirabilis UTI progression: fimbria-mediated attachment and flagellum-mediated motility. Transcriptome data indicated a network of virulence-associated genes under MrpJ's control. Here, we identify the direct gene regulon of MrpJ and its contribution to P. mirabilis pathogenesis, leading to the discovery of novel virulence targets. Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) was used for the first time in a CAUTI pathogen to probe for in vivo direct targets of MrpJ. Selected MrpJ-regulated genes were mutated and assessed for their contribution to UTI using a mouse model. ChIP-seq revealed a palindromic MrpJ binding sequence and 78 MrpJ-bound regions, including binding sites upstream of genes involved in motility, fimbriae, and a type VI secretion system (T6SS). A combinatorial mutation approach established the contribution of three fimbriae (fim8A, fim14A, and pmpA) to UTI and a new pathogenic role for the T6SS in UTI progression. In conclusion, this study (i) establishes the direct gene regulon and an MrpJ consensus binding site and (ii) led to the discovery of new virulence genes in P. mirabilis UTI, which could be targeted for therapeutic intervention of CAUTI.

Determining the Specificity of Cascade Binding, Interference, and Primed Adaptation In Vivo in the Escherichia coli Type I-E CRISPR-Cas System.

In clustered regularly interspaced short palindromic repeat (CRISPR)-Cas (CRISPR-associated) immunity systems, short CRISPR RNAs (crRNAs) are bound by Cas proteins, and these complexes target invading nucleic acid molecules for degradation in a process known as interference. In type I CRISPR-Cas systems, the Cas protein complex that binds DNA is known as Cascade. Association of Cascade with target DNA can also lead to acquisition of new immunity elements in a process known as primed adaptation. Here, we assess the specificity determinants for Cascade-DNA interaction, interference, and primed adaptation in vivo, for the type I-E system of Escherichia coli Remarkably, as few as 5 bp of crRNA-DNA are sufficient for association of Cascade with a DNA target. Consequently, a single crRNA promotes Cascade association with numerous off-target sites, and the endogenous E. coli crRNAs direct Cascade binding to >100 chromosomal sites. In contrast to the low specificity of Cascade-DNA interactions, >18 bp are required for both interference and primed adaptation. Hence, Cascade binding to suboptimal, off-target sites is inert. Our data support a model in which the initial Cascade association with DNA targets requires only limited sequence complementarity at the crRNA 5' end whereas recruitment and/or activation of the Cas3 nuclease, a prerequisite for interference and primed adaptation, requires extensive base pairing.IMPORTANCE Many bacterial and archaeal species encode CRISPR-Cas immunity systems that protect against invasion by foreign DNA. In the Escherichia coli CRISPR-Cas system, a protein complex, Cascade, binds 61-nucleotide (nt) CRISPR RNAs (crRNAs). The Cascade complex is directed to invading DNA molecules through base pairing between the crRNA and target DNA. This leads to recruitment of the Cas3 nuclease, which destroys the invading DNA molecule and promotes acquisition of new immunity elements. We made the first in vivo measurements of Cascade binding to DNA targets. Thus, we show that Cascade binding to DNA is highly promiscuous; endogenous E. coli crRNAs can direct Cascade binding to >100 chromosomal locations. In contrast, we show that targeted degradation and acquisition of new immunity elements require highly specific association of Cascade with DNA, limiting CRISPR-Cas function to the appropriate targets.

Identification of regulatory targets for the bacterial Nus factor complex.

Nus factors are broadly conserved across bacterial species, and are often essential for viability. A complex of five Nus factors (NusB, NusE, NusA, NusG and SuhB) is considered to be a dedicated regulator of ribosomal RNA folding, and has been shown to prevent Rho-dependent transcription termination. Here, we identify an additional cellular function for the Nus factor complex in Escherichia coli: repression of the Nus factor-encoding gene, suhB. This repression occurs primarily by translation inhibition, followed by Rho-dependent transcription termination. Thus, the Nus factor complex can prevent or promote Rho activity depending on the gene context. Conservation of putative NusB/E binding sites upstream of Nus factor genes suggests that Nus factor autoregulation occurs in many bacterial species. Additionally, many putative NusB/E binding sites are also found upstream of other genes in diverse species, and we demonstrate Nus factor regulation of one such gene in Citrobacter koseri. We conclude that Nus factors have an evolutionarily widespread regulatory function beyond ribosomal RNA, and that they are often autoregulatory.

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.

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.

Mapping the Regulatory Network for Salmonella enterica Serovar Typhimurium Invasion.

UNLABELLED: 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.

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

UNLABELLED: 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.

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.

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.

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.

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

BACKGROUND: ChIP-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. RESULTS: We 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. CONCLUSIONS: The 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.

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.