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2.
Methods Enzymol ; 616: 117-132, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30691640

RESUMO

The specificity of CRISPR-Cas systems for nucleic acid targets is determined by a combination of binding and cleavage. Understanding the mechanisms by which Cas proteins specifically select their targets is critical for the development of CRISPR-Cas systems for biotechnology applications. Moreover, the specificity of CRISPR-Cas systems plays an important role in prokaryote evolution due to its role in distinguishing self from nonself. Here, I describe Library-ChIP, a high-throughput method for measuring Cas protein occupancy at many DNA sequence variants in a native prokaryotic host. Library-ChIP can be used to identify the determinants of specificity for Cas protein binding to nucleic acid targets.

4.
Infect Immun ; 86(10)2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30082479

RESUMO

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.

5.
Proc Natl Acad Sci U S A ; 115(32): 8191-8196, 2018 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-30038002

RESUMO

Bacteria respond to zinc starvation by replacing ribosomal proteins that have the zinc-binding CXXC motif (C+) with their zinc-free (C-) paralogues. Consequences of this process beyond zinc homeostasis are unknown. Here, we show that the C- ribosome in Mycobacterium smegmatis is the exclusive target of a bacterial protein Y homolog, referred to as mycobacterial-specific protein Y (MPY), which binds to the decoding region of the 30S subunit, thereby inactivating the ribosome. MPY binding is dependent on another mycobacterial protein, MPY recruitment factor (MRF), which is induced on zinc depletion, and interacts with C- ribosomes. MPY binding confers structural stability to C- ribosomes, promoting survival of growth-arrested cells under zinc-limiting conditions. Binding of MPY also has direct influence on the dynamics of aminoglycoside-binding pockets of the C- ribosome to inhibit binding of these antibiotics. Together, our data suggest that zinc limitation leads to ribosome hibernation and aminoglycoside resistance in mycobacteria. Furthermore, our observation of the expression of the proteins of C- ribosomes in Mycobacterium tuberculosis in a mouse model of infection suggests that ribosome hibernation could be relevant in our understanding of persistence and drug tolerance of the pathogen encountered during chemotherapy of TB.


Assuntos
Antibióticos Antituberculose/farmacologia , Proteínas de Bactérias/metabolismo , Mycobacterium tuberculosis/fisiologia , Proteínas Ribossômicas/metabolismo , Tuberculose/tratamento farmacológico , Zinco/deficiência , Aminoglicosídeos/farmacologia , Animais , Microscopia Crioeletrônica , Modelos Animais de Doenças , Farmacorresistência Bacteriana , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Testes de Sensibilidade Microbiana , Modelos Moleculares , Mycobacterium smegmatis/efeitos dos fármacos , Mycobacterium smegmatis/fisiologia , Mycobacterium tuberculosis/efeitos dos fármacos , Biossíntese de Proteínas/fisiologia , Ribossomos/metabolismo , Ribossomos/ultraestrutura , Tuberculose/microbiologia , Tuberculose/patologia
6.
MBio ; 9(2)2018 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-29666291

RESUMO

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.

7.
RNA Biol ; 15(6): 679-682, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29537923

RESUMO

Riboswitches are RNA regulators that control gene expression by modulating their structure in response to metabolite binding. The study of mechanisms by which riboswitches modulate gene expression is crucial to understand how riboswitches are involved in maintaining cellular homeostasis. Previous reports indicate that riboswitches can control gene expression at the level of translation, transcription or mRNA decay. However, there are very few described examples where riboswitches regulate multiple steps in gene expression. Recent studies of a translation-regulating, TPP-dependent riboswitch have revealed that ligand binding is also involved in the control of mRNA levels. In this model, TPP binding to the riboswitch leads to the inhibition of translation, which in turn allows for Rho-dependent transcription termination. Thus, mRNA levels are indirectly controlled through ribosome occupancy. This is in contrast to other riboswitches that directly control mRNA levels by modulating the access of regulatory sequences involved in either Rho-dependent transcription termination or RNase E cleavage activity. Together, these findings indicate that riboswitches modulate both translation initiation and mRNA levels using multiple strategies that direct the outcome of gene expression.


Assuntos
Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Biossíntese de Proteínas/fisiologia , Estabilidade de RNA/fisiologia , Riboswitch/fisiologia , Transcrição Genética/fisiologia , Bactérias/genética
8.
Mol Microbiol ; 108(3): 221-225, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29473964

RESUMO

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.

9.
Mol Microbiol ; 108(4): 361-378, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29476659

RESUMO

In Escherichia coli, one sigma factor recognizes the majority of promoters, and six 'alternative' sigma factors recognize specific subsets of promoters. The alternative sigma factor FliA (σ28 ) recognizes promoters upstream of many flagellar genes. We previously showed that most E. coli FliA binding sites are located inside genes. However, it was unclear whether these intragenic binding sites represent active promoters. Here, we construct and assay transcriptional promoter-lacZ fusions for all 52 putative FliA promoters previously identified by ChIP-seq. These experiments, coupled with integrative analysis of published genome-scale transcriptional datasets, strongly suggest that most intragenic FliA binding sites are active promoters that transcribe highly unstable RNAs. Additionally, we show that widespread intragenic FliA-dependent transcription may be a conserved phenomenon, but that specific promoters are not themselves conserved. We conclude that intragenic FliA-dependent promoters and the resulting RNAs are unlikely to have important regulatory functions. Nonetheless, one intragenic FliA promoter is broadly conserved and constrains evolution of the overlapping protein-coding gene. Thus, our data indicate that intragenic regulatory elements can influence bacterial protein evolution and suggest that the impact of intragenic regulatory sequences on genome evolution should be considered more broadly.

10.
Transcription ; 9(3): 182-189, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-28980880

RESUMO

Most RNA polymerases can initiate transcription from diverse DNA template sequences with relatively few outright sequence restraints. Recent reports have demonstrated that failure to subdue the promiscuity of RNA polymerase in vivo can severely impede cell function. This phenomenon appears common to all cell types with undesirable effects ranging from growth inhibition in prokaryotes to cancer in higher organisms. Here we discuss similarities and differences in strategies employed by cells to minimise spurious transcription across life's domains.


Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Regiões Promotoras Genéticas , Transcrição Genética , Animais , Escherichia coli/enzimologia , Escherichia coli/genética , Humanos , Neoplasias/enzimologia , Neoplasias/genética , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Schizosaccharomyces/enzimologia , Schizosaccharomyces/genética , Especificidade por Substrato , Iniciação da Transcrição Genética
11.
Nat Commun ; 8(1): 2027, 2017 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-29229908

RESUMO

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.


Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Dobramento de RNA , RNA Ribossômico/química , Sequência de Bases , Sítios de Ligação/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Fatores de Alongamento de Peptídeos/genética , Fatores de Alongamento de Peptídeos/metabolismo , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Ligação Proteica , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Homologia de Sequência do Ácido Nucleico , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/metabolismo
12.
MBio ; 8(5)2017 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-29018122

RESUMO

Nearly all virulence factors in Bordetella pertussis are activated by a master two-component system, BvgAS, composed of the sensor kinase BvgS and the response regulator BvgA. When BvgS is active, BvgA is phosphorylated (BvgA~P), and virulence-activated genes (vags) are expressed [Bvg(+) mode]. When BvgS is inactive and BvgA is not phosphorylated, virulence-repressed genes (vrgs) are induced [Bvg(-) mode]. Here, we have used transcriptome sequencing (RNA-seq) and reverse transcription-quantitative PCR (RT-qPCR) to define the BvgAS-dependent regulon of B. pertussis Tohama I. Our analyses reveal more than 550 BvgA-regulated genes, of which 353 are newly identified. BvgA-activated genes include those encoding two-component systems (such as kdpED), multiple other transcriptional regulators, and the extracytoplasmic function (ECF) sigma factor brpL, which is needed for type 3 secretion system (T3SS) expression, further establishing the importance of BvgA~P as an apex regulator of transcriptional networks promoting virulence. Using in vitro transcription, we demonstrate that the promoter for brpL is directly activated by BvgA~P. BvgA-FeBABE cleavage reactions identify BvgA~P binding sites centered at positions -41.5 and -63.5 in bprL Most importantly, we show for the first time that genes for multiple and varied metabolic pathways are significantly upregulated in the B. pertussis Bvg(-) mode. These include genes for fatty acid and lipid metabolism, sugar and amino acid transporters, pyruvate dehydrogenase, phenylacetic acid degradation, and the glycolate/glyoxylate utilization pathway. Our results suggest that metabolic changes in the Bvg(-) mode may be participating in bacterial survival, transmission, and/or persistence and identify over 200 new vrgs that can be tested for function.IMPORTANCE Within the past 20 years, outbreaks of whooping cough, caused by Bordetella pertussis, have led to respiratory disease and infant mortalities, despite good vaccination coverage. This is due, at least in part, to the introduction of a less effective acellular vaccine in the 1990s. It is crucial, then, to understand the molecular basis of B. pertussis growth and infection. The two-component system BvgA (response regulator)/BvgS (histidine kinase) is the master regulator of B. pertussis virulence genes. We report here the first RNA-seq analysis of the BvgAS regulon in B. pertussis, revealing that more than 550 genes are regulated by BvgAS. We show that genes for multiple and varied metabolic pathways are highly regulated in the Bvg(-) mode (absence of BvgA phosphorylation). Our results suggest that metabolic changes in the Bvg(-) mode may be participating in bacterial survival, transmission, and/or persistence.


Assuntos
Proteínas de Bactérias/genética , Bordetella pertussis/genética , Regulon , Fatores de Transcrição/genética , Bordetella pertussis/crescimento & desenvolvimento , Bordetella pertussis/metabolismo , Bordetella pertussis/patogenicidade , Regulação Bacteriana da Expressão Gênica , Genes Reguladores , Sequenciamento de Nucleotídeos em Larga Escala , Regiões Promotoras Genéticas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Transcriptoma , Virulência
13.
Nucleic Acids Res ; 45(12): 7474-7486, 2017 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-28520932

RESUMO

Riboswitches are regulatory elements that control gene expression by altering RNA structure upon the binding of specific metabolites. Although Bacillus subtilis riboswitches have been shown to control premature transcription termination, less is known about regulatory mechanisms employed by Escherichia coli riboswitches, which are predicted to regulate mostly at the level of translation initiation. Here, we present experimental evidence suggesting that the majority of known E. coli riboswitches control transcription termination by using the Rho transcription factor. In the case of the thiamin pyrophosphate-dependent thiM riboswitch, we find that Rho-dependent transcription termination is triggered as a consequence of translation repression. Using in vitro and in vivo assays, we show that the Rho-mediated regulation relies on RNA target elements located at the beginning of thiM coding region. Gene reporter assays indicate that relocating Rho target elements to a different gene induces transcription termination, demonstrating that such elements are modular domains controlling Rho. Our work provides strong evidence that translationally regulating riboswitches also regulate mRNA levels through an indirect control mechanism ensuring tight control of gene expression.


Assuntos
Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Biossíntese de Proteínas , Fator Rho/genética , Riboswitch , Terminação da Transcrição Genética , Sequência de Bases , Escherichia coli/metabolismo , Genes Reporter , Conformação de Ácido Nucleico , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fator Rho/metabolismo , Tiamina Pirofosfato/metabolismo
14.
Nat Microbiol ; 2: 16249, 2017 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-28067866

RESUMO

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.


Assuntos
Sequência Rica em At/genética , DNA Bacteriano/genética , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Proteínas de Fímbrias/metabolismo , Transferência Genética Horizontal , Composição de Bases , DNA Bacteriano/química , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação Bacteriana da Expressão Gênica , Inativação Gênica , Genes Bacterianos , Aptidão Genética , Genoma , Mutação , Regiões Promotoras Genéticas , Transcrição Genética
15.
J Bacteriol ; 199(7)2017 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-28115545

RESUMO

The alternative sigma factor RpoS is a central regulator of many stress responses in Escherichia coli The level of functional RpoS differs depending on the stress. The effect of these differing concentrations of RpoS on global transcriptional responses remains unclear. We investigated the effect of RpoS concentration on the transcriptome during stationary phase in rich media. We found that 23% of genes in the E. coli genome are regulated by RpoS, and we identified many RpoS-transcribed genes and promoters. We observed three distinct classes of response to RpoS by genes in the regulon: genes whose expression changes linearly with increasing RpoS level, genes whose expression changes dramatically with the production of only a little RpoS ("sensitive" genes), and genes whose expression changes very little with the production of a little RpoS ("insensitive"). We show that sequences outside the core promoter region determine whether an RpoS-regulated gene is sensitive or insensitive. Moreover, we show that sensitive and insensitive genes are enriched for specific functional classes and that the sensitivity of a gene to RpoS corresponds to the timing of induction as cells enter stationary phase. Thus, promoter sensitivity to RpoS is a mechanism to coordinate specific cellular processes with growth phase and may also contribute to the diversity of stress responses directed by RpoS.IMPORTANCE The sigma factor RpoS is a global regulator that controls the response to many stresses in Escherichia coli Different stresses result in different levels of RpoS production, but the consequences of this variation are unknown. We describe how changing the level of RpoS does not influence all RpoS-regulated genes equally. The cause of this variation is likely the action of transcription factors that bind the promoters of the genes. We show that the sensitivity of a gene to RpoS levels explains the timing of expression as cells enter stationary phase and that genes with different RpoS sensitivities are enriched for specific functional groups. Thus, promoter sensitivity to RpoS is a mechanism that coordinates specific cellular processes in response to stresses.


Assuntos
Proteínas de Bactérias/metabolismo , Escherichia coli K12/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Estudo de Associação Genômica Ampla , Fator sigma/metabolismo , Proteínas de Bactérias/genética , Western Blotting , Mutação , Regiões Promotoras Genéticas , Fator sigma/genética , Transcriptoma
16.
Front Mol Biosci ; 3: 74, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27900321

RESUMO

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.

17.
MBio ; 7(5)2016 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-27601571

RESUMO

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.


Assuntos
Endocitose , Células Epiteliais/microbiologia , Redes Reguladoras de Genes , Salmonella typhimurium/genética , Salmonella typhimurium/fisiologia , Perfilação da Expressão Gênica , RNA não Traduzido , Regulon , Fatores de Transcrição/genética
18.
MBio ; 7(2): e00114, 2016 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-26980831

RESUMO

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.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Processamento Pós-Transcricional do RNA , RNA Ribossômico/metabolismo , Subunidades Ribossômicas Menores de Bactérias/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Ligação Proteica , Multimerização Proteica
19.
ACS Chem Biol ; 11(4): 1106-17, 2016 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-26785289

RESUMO

The opportunistic pathogen Pseudomonas aeruginosa utilizes multiple quorum sensing (QS) pathways to coordinate an arsenal of virulence factors. We previously identified several cysteine-based compounds inspired by natural products from the plant Petiveria alliacea which are capable of antagonizing multiple QS circuits as well as reducing P. aeruginosa biofilm formation. To understand the global effects of such compounds on virulence factor production and elucidate their mechanism of action, RNA-seq transcriptomic analysis was performed on P. aeruginosa PAO1 exposed to S-phenyl-l-cysteine sulfoxide, the most potent inhibitor from the prior study. Exposure to this inhibitor down-regulated expression of several QS-regulated virulence operons (e.g., phenazine biosynthesis, type VI secretion systems). Interestingly, many genes that were differentially regulated pertain to the related metabolic pathways that yield precursors of pyochelin, tricarboxylic acid cycle intermediates, phenazines, and Pseudomonas quinolone signal (PQS). Activation of the MexT-regulon was also indicated, including the multidrug efflux pump encoded by mexEF-oprN, which has previously been shown to inhibit QS and pathogenicity. Deeper investigation of the metabolites involved in these systems revealed that S-phenyl-l-cysteine sulfoxide has structural similarity to kynurenine, a precursor of anthranilate, which is critical for P. aeruginosa virulence. By supplementing exogenous anthranilate, the QS-inhibitory effect was reversed. Finally, it was shown that S-phenyl-l-cysteine sulfoxide competitively inhibits P. aeruginosa kynureninase (KynU) activity in vitro and reduces PQS production in vivo. The kynurenine pathway has been implicated in P. aeruginosa QS and virulence factor expression; however, this is the first study to show that targeted inhibition of KynU affects P. aeruginosa gene expression and QS, suggesting a potential antivirulence strategy.


Assuntos
Hidrolases/metabolismo , Pseudomonas aeruginosa/metabolismo , Percepção de Quorum , Pseudomonas aeruginosa/patogenicidade , Pseudomonas aeruginosa/fisiologia , Virulência
20.
Adv Exp Med Biol ; 883: 119-34, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26621465

RESUMO

Bacterial genomes encode numerous transcription factors, DNA-binding proteins that regulate transcription initiation. Identifying the regulatory targets of transcription factors is a major challenge of systems biology. Here I describe the use of two genome-scale approaches, ChIP-seq and RNA-seq, that are used to map transcription factor regulons. ChIP-seq maps the association of transcription factors with DNA, and RNA-seq determines changes in RNA levels associated with transcription factor perturbation. I discuss the strengths and weaknesses of these and related approaches, and I describe how ChIP-seq and RNA-seq can be combined to map individual transcription factor regulons and entire regulatory networks.


Assuntos
Imunoprecipitação da Cromatina/métodos , Redes Reguladoras de Genes , Análise de Sequência de RNA , Sequência de Bases , Código de Barras de DNA Taxonômico , Dados de Sequência Molecular
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