RESUMEN
Genomic studies with Salmonella enterica serovar Typhimurium reveal a crucial role of horizontal gene transfer (HGT) in the acquisition of accessory cellular functions involved in host-interaction. Many virulence genes are located in genomic islands, plasmids and prophages. GreA and GreB proteins, Gre factors, interact transiently with the RNA polymerase alleviating backtracked complexes during transcription elongation. The overall effect of Gre factors depletion in Salmonella expression profile was studied. Both proteins are functionally redundant since only when both Gre factors were depleted a major effect in gene expression was detected. Remarkably, the accessory gene pool is particularly sensitive to the lack of Gre factors, with 18.6% of accessory genes stimulated by the Gre factors versus 4.4% of core genome genes. Gre factors involvement is particularly relevant for the expression of genes located in genomic islands. Our data reveal that Gre factors are required for the expression of accessory genes.
Asunto(s)
Proteínas Bacterianas , Salmonella typhimurium , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Plásmidos , Virulencia/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismoRESUMEN
ppGpp is an intracellular sensor that, in response to different types of stress, coordinates the rearrangement of the gene expression pattern of bacteria to promote adaptation and survival to new environmental conditions. First described to modulate metabolic adaptive responses, ppGpp modulates the expression of genes belonging to very diverse functional categories. In Escherichia coli, ppGpp regulates the expression of cellular factors that are important during urinary tract infections. Here, we characterize the role of this alarmone in the regulation of the hlyCABDII operon of the UPEC isolate J96, encoding the toxin α-hemolysin that induces cytotoxicity during infection of bladder epithelial cells. ppGpp is required for the expression of the α-hemolysin encoded in hlyCABDII by stimulating its transcriptional expression. Prototrophy suppressor mutations in a ppGpp-deficient strain restore the α-hemolysin expression from this operon to wild-type levels, confirming the requirement of ppGpp for its expression. ppGpp stimulates hlyCABDII expression independently of RpoS, RfaH, Zur, and H-NS. The expression of hlyCABDII is promoted at 37 °C and at low osmolarity. ppGpp is required for the thermoregulation but not for the osmoregulation of the hlyCABDII operon. Studies in both commensal and UPEC isolates demonstrate that no UPEC specific factor is strictly required for the ppGpp-mediated regulation described. Our data further support the role of ppGpp participating in the coordinated regulation of the expression of bacterial factors required during infection.
Asunto(s)
Infecciones por Escherichia coli , Proteínas de Escherichia coli , Escherichia coli Uropatógena , Humanos , Escherichia coli Uropatógena/genética , Escherichia coli Uropatógena/metabolismo , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Guanosina Tetrafosfato/metabolismo , Guanosina Pentafosfato/metabolismo , Regulación Bacteriana de la Expresión Génica , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Infecciones por Escherichia coli/microbiología , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Factores de Elongación de Péptidos/metabolismo , Transactivadores/metabolismoRESUMEN
The cAMP-CRP regulon coordinates transcription regulation of several energy-related genes, the lac operon among them. Lactose, or IPTG, induces the lac operon expression by binding to the LacI repressor, and releasing it from the promoter sequence. At the same time, the expression of the lac operon requires the presence of the CRP-cAMP complex, which promotes the binding of the RNA polymerase to the promoter region. The modified nucleotide cAMP accumulates in the absence of glucose and binds to the CRP protein, but its ability to bind to DNA can be impaired by lysine-acetylation of CRP. Here we add another layer of control, as acetylation of CRP seems to be modified by ppGpp. In cells grown in glycerol minimal media, ppGpp seems to repress the expression of lacZ, where ΔrelA mutants show higher expression of lacZ than in WT. These differences between the WT and ΔrelA strains seem to depend on the levels of acetylated CRP. During the growth in minimal media supplemented with glycerol, ppGpp promotes the acetylation of CRP by the Nε-lysine acetyltransferases YfiQ. Moreover, the expression of the different genes involved in the production and degradation of Acetyl-phosphate (ackA-pta) and the enzymatic acetylation of proteins (yfiQ) are stimulated by the presence of ppGpp, depending on the growth conditions.
Asunto(s)
Proteína Receptora de AMP Cíclico , Proteínas de Escherichia coli/genética , Escherichia coli , Regulación Bacteriana de la Expresión Génica , Operón Lac , Acetilación , Proteína Receptora de AMP Cíclico/genética , Proteína Receptora de AMP Cíclico/metabolismo , ADN Bacteriano , Escherichia coli/genética , Escherichia coli/metabolismo , Genes Bacterianos , Regiones Promotoras GenéticasRESUMEN
Two (p)ppGpp nucleotide analogs, sometimes abbreviated simply as ppGpp, are widespread in bacteria and plants. Their name alarmone reflects a view of their function as intracellular hormone-like protective alarms that can increase a 100-fold when sensing any of an array of physical or nutritional dangers, such as abrupt starvation, that trigger lifesaving adjustments of global gene expression and physiology. The diversity of mechanisms for stress-specific adjustments of this sort is large and further compounded by almost infinite microbial diversity. The central question raised by this review is whether the small basal levels of (p)ppGpp functioning during balanced growth serve very different roles than alarmone-like functions. Recent discoveries that abrupt amino acid starvation of Escherichia coli, accompanied by very high levels of ppGpp, occasion surprising instabilities of transfer RNA (tRNA), ribosomal RNA (rRNA), and ribosomes raises new questions. Is this destabilization, a mode of regulation linearly related to (p)ppGpp over the entire continuum of (p)ppGpp levels, including balanced growth? Are regulatory mechanisms exerted by basal (p)ppGpp levels fundamentally different than for high levels? There is evidence from studies of other organisms suggesting special regulatory features of basal levels compared to burst of (p)ppGpp. Those differences seem to be important even during bacterial infection, suggesting that unbalancing the basal levels of (p)ppGpp may become a future antibacterial treatment. A simile for this possible functional duality is that (p)ppGpp acts like a car's brake, able to stop to avoid crashes as well as to slow down to drive safely.
RESUMEN
There is a growing appreciation for the diverse regulatory consequences of the family of proteins that bind to the secondary channel of E. coli RNA polymerase (RNAP), such as GreA, GreB or DksA. Similar binding sites could suggest a competition between them. GreA is characterised to rescue stalled RNAP complexes due to its antipause activity, but also it is involved in transcription fidelity and proofreading. Here, overexpression of GreA is noted to be lethal independent of its antipause activity. A library of random GreA variants has been used to isolate lethality suppressors to assess important residues for GreA functionality and its interaction with the RNA polymerase. Some mutant defects are inferred to be associated with altered binding competition with DksA, while other variants seem to have antipause activity defects that cannot reverse a GreA-sensitive pause site in a fliC::lacZ reporter system. Surprisingly, apparent binding and cleavage defects are found scattered throughout both the coiled-coil and globular domains. Thus, the coiled-coil of GreA is not just a measuring stick ensuring placement of acidic residues precisely at the catalytic centre but also seems to have binding functions. These lethality suppressor mutants may provide valuable tools for future structural and functional studies.
Asunto(s)
Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Unión Competitiva , Análisis Mutacional de ADN , ARN Polimerasas Dirigidas por ADN/metabolismo , Proteínas de Escherichia coli/química , Eliminación de Gen , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Genes Letales , Variación Genética , Modelos Moleculares , Mutagénesis , Mutación , Regiones Promotoras Genéticas , Factores de Transcripción/química , Factores de Elongación Transcripcional/química , Factores de Elongación Transcripcional/genética , Factores de Elongación Transcripcional/metabolismo , Regulación hacia ArribaRESUMEN
Antigen 43 (Ag43) is a self-recognizing outer membrane protein of Escherichia coli expressed during intracellular growth and biofilm formation, suggesting a role in infection. The expression of agn43 is under phase variation control, meaning that there are regulatory mechanisms adjusting the percentage of agn43-expressing cells in the population, in addition to mechanisms modulating the transcriptional expression level in each expressing cell. Phenotypic and transcriptional studies indicate that Ag43 expression is induced upon entry into the stationary phase in a ppGpp-dependent and RpoS-independent manner. The use of single-cell approaches and phase variation deficient strains let to conclude that ppGpp stimulates agn43 promoter activity, rather than affecting the percentage of agn43-expressing cells. The data highlight the relevance that promoter activity regulation may have, without any involvement of the phase variation state, in the final Ag43 expression output. The agn43 promoter of the MG1655 strain carries an AT-rich discriminator between positions -10 and +1, which is highly conserved among the agn43 genes present in the different pathotypes of E. coli. Remarkably, the AT-rich discriminator is required for the positive transcriptional control mediated by ppGpp.
Asunto(s)
Adhesinas de Escherichia coli/metabolismo , Escherichia coli/crecimiento & desarrollo , Escherichia coli/metabolismo , Guanosina Tetrafosfato/metabolismo , Regiones Promotoras Genéticas , Adhesinas de Escherichia coli/genética , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Transcripción GenéticaRESUMEN
The initiation of Escherichia coli chromosomal DNA replication starts with the oligomerization of the DnaA protein at repeat sequences within the origin (ori) region. The amount of ori DNA per cell directly correlates with the growth rate. During fast growth, the cell generation time is shorter than the time required for complete DNA replication; therefore, overlapping rounds of chromosome replication are required. Under these circumstances, the ori region DNA abundance exceeds the DNA abundance in the termination (ter) region. Here, high ori/ter ratios are found to persist in (p)ppGpp-deficient [(p)ppGpp0] cells over a wide range of balanced exponential growth rates determined by medium composition. Evidently, (p)ppGpp is necessary to maintain the usual correlation of slow DNA replication initiation with a low growth rate. Conversely, ori/ter ratios are lowered when cell growth is slowed by incrementally increasing even low constitutive basal levels of (p)ppGpp without stress, as if (p)ppGpp alone is sufficient for this response. There are several previous reports of (p)ppGpp inhibition of chromosomal DNA synthesis initiation that occurs with very high levels of (p)ppGpp that stop growth, as during the stringent starvation response or during serine hydroxamate treatment. This work suggests that low physiological levels of (p)ppGpp have significant functions in growing cells without stress through a mechanism involving negative supercoiling, which is likely mediated by (p)ppGpp regulation of DNA gyrase.IMPORTANCE Bacterial cells regulate their own chromosomal DNA synthesis and cell division depending on the growth conditions, producing more DNA when growing in nutritionally rich media than in poor media (i.e., human gut versus water reservoir). The accumulation of the nucleotide analog (p)ppGpp is usually viewed as serving to warn cells of impending peril due to otherwise lethal sources of stress, which stops growth and inhibits DNA, RNA, and protein synthesis. This work importantly finds that small physiological changes in (p)ppGpp basal levels associated with slow balanced exponential growth incrementally inhibit the intricate process of initiation of chromosomal DNA synthesis. Without (p)ppGpp, initiations mimic the high rates present during fast growth. Here, we report that the effect of (p)ppGpp may be due to the regulation of the expression of gyrase, an important enzyme for the replication of DNA that is a current target of several antibiotics.
Asunto(s)
Cromosomas Bacterianos/genética , Replicación del ADN , ADN Bacteriano/biosíntesis , Escherichia coli/crecimiento & desarrollo , Escherichia coli/genética , Guanosina Pentafosfato/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Girasa de ADN/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Escherichia coli/enzimología , Biosíntesis de ProteínasRESUMEN
GreA is a well-characterized transcriptional factor that acts primarily by rescuing stalled RNA polymerase complexes, but has also been shown to be the major transcriptional fidelity and proofreading factor, while it inhibits DNA break repair. Regulation of greA gene expression itself is still not well understood. So far, it has been shown that its expression is driven by two overlapping promoters and that greA leader encodes a small RNA (GraL) that is acting in trans on nudE mRNA. It has been also shown that GreA autoinhibits its own expression in vivo. Here, we decided to investigate the inner workings of this autoregulatory loop. Transcriptional fusions with lacZ reporter carrying different modifications (made both to the greA promoter and leader regions) were made to pinpoint the sequences responsible for this autoregulation, while GraL levels were also monitored. Our data indicate that GreA mediated regulation of its own gene expression is dependent on GraL acting in cis (a rare example of dual-action sRNA), rather than on the promoter region. However, a yet unidentified, additional factor seems to participate in this regulation as well. Overall, the GreA/GraL regulatory loop seems to have unique but hard to classify properties.
Asunto(s)
Proteínas de Escherichia coli/genética , Retroalimentación Fisiológica , Regulación Bacteriana de la Expresión Génica , ARN Pequeño no Traducido/metabolismo , Factores de Transcripción/genética , Escherichia coli , Proteínas de Escherichia coli/metabolismo , Regiones Promotoras Genéticas , ARN Pequeño no Traducido/genética , Factores de Transcripción/metabolismoRESUMEN
The (p)ppGpp nucleotide functions as a global regulator in bacteria in response to a variety of physical and nutritional stress. It has a rapid onset, in seconds, which leads to accumulation of levels that approach or exceed those of GTP pools. Stress reversal occasions a rapid disappearance of (p)ppGpp, often with a half-life of less than a minute. The presence of (p)ppGpp results in alterations of cellular gene expression and metabolism that counter the damaging effects of stress. Gram-negative and Gram-positive bacteria have different response mechanisms, but both depend on (p)ppGpp concentration. In any event, there is a need to simultaneously monitor many radiolabeled bacterial cultures at time intervals that may vary from 10 seconds to hours during critical stress transition periods. This protocol addresses this technical challenge. The method takes advantage of temperature- and shaker-controlled microtiter dish incubators that allow parallel monitoring of growth (absorbance) and rapid sampling of uniformly phosphate-radiolabeled cultures to resolve and quantitate nucleotide pools by thin-layer chromatography on PEI-cellulose. Small amounts of sample are needed for multiple technical and biological replicates of analyses. Complex growth transitions, such as diauxic growth and rapid (p)ppGpp turnover rates can be quantitatively assessed by this method.
Asunto(s)
Cromatografía en Capa Delgada , Escherichia coli/metabolismo , Guanosina Pentafosfato/metabolismo , Radioisótopos de Fósforo , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Guanosina Pentafosfato/química , Estrés Fisiológico/genéticaRESUMEN
During the diauxic shift, Escherichia coli exhausts glucose and adjusts its expression pattern to grow on a secondary carbon source. Transcriptional profiling studies of glucose-lactose diauxic transitions reveal a key role for ppGpp. The amount of ppGpp depends on RelA synthetase and the balance between a strong SpoT hydrolase and its weak synthetase. In this study, mutants are used to search for synthetase or hydrolase specific regulation. Diauxic shifts experiments were performed with strains containing SpoT hydrolase and either RelA or SpoT synthetase as the sole source of ppGpp. Here, the length of the diauxic lag times is determined by the presence of ppGpp, showing contributions of both ppGpp synthetases (RelA and SpoT) as well as its hydrolase (SpoT). A balanced ppGpp response is key for a proper adaptation during diauxic shift. The effects of one or the other ppGpp synthetase on diauxic shifts are abolished by addition of amino acids or succinate, although by different mechanisms. While amino acids control the RelA response, succinate blocks the uptake of the excreted acetate via SatP. Acetate is converted to Acetyl-CoA through the ackA-pta pathway, producing Ac-P as intermediate. Evidence of control of the ackA-pta operon as well as a correlation between ppGpp and Ac-P is shown. Finally, acetylation of proteins is shown to occur during a diauxic glucose-lactose shift.
RESUMEN
Bacteriophage T4 relies on host RNA polymerase to transcribe three promoter classes: early (Pe, requires no viral factors), middle (Pm, requires early proteins MotA and AsiA), and late (Pl, requires middle proteins gp55, gp33, and gp45). Using primer extension, RNA-seq, RT-qPCR, single bursts, and a semi-automated method to document plaque size, we investigated how deletion of DksA or ppGpp, two E. coli global transcription regulators, affects T4 infection. Both ppGpp° and ΔdksA increase T4 wild type (wt) plaque size. However, ppGpp° does not significantly alter burst size or latent period, and only modestly affects T4 transcript abundance, while ΔdksA increases burst size (2-fold) without affecting latent period and increases the levels of several Pe transcripts at 5 min post-infection. In a T4motAam infection, ΔdksA increases plaque size and shortens latent period, and the levels of specific middle RNAs increase due to more transcription from Pe’s that extend into these middle genes. We conclude that DksA lowers T4 early gene expression. Consequently, ΔdksA results in a more productive wt infection and ameliorates the poor expression of middle genes in a T4motAam infection. As DksA does not inhibit Pe transcription in vitro, regulation may be indirect or perhaps requires additional factors.