Mutational analysis of Escherichia coli GreA protein reveals new functional activity independent of antipause and lethal when overexpressed

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

ppGpp, the general stress response alarmone, is required for the expression of the alpha-hemolysin toxin in the uropathogenic EScherichia coli isolate, J96.

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

Secondary channel of the RNA polymerase, a target for transcriptional regulation in bacteria

[cat] El control de l’expressió gènica en bacteris recau principalment sobre un complex enzimàtic anomenat ARN polimerasa (ARNpol). A procariotes, la seva unitat bàsica (core) està formada per 5 subunitats proteiques (a2bb’w). S’han determinat dos canals entre les diferents subunitats de l’ARNpol: el canal primari, on es desenvolupa la transcripció, i el canal secundari, que comunica el medi exterior amb el centre catalític de l’ARNpol. Tot i així, aquest holoenzim necessita la unió d’una subunitat σ per ser capaç de reconèixer una seqüència promotora i iniciar la transcripció. S’han descrit diferents factors, tant proteics com no proteics, que poden interaccionar amb el canal secundari de l’ARNpol i causar alteracions a l’expressió gènica. En aquesta tesi ens hem centrat en la possible competència entre els diferents factors que poden interaccionar amb el canal secundari de l’ARNpol. Estudis anterior duts a terme en el nostre grup d’investigació, ens van permetre postular una possible competència entre els diferents factors que interaccionen amb el canal secundari de l’ARNpol, més concretament entre les proteïnes GreA i DksA. Aquesta competència provocaria alteracions en el patró d’expressió gènica d’Escherichia coli. En aquest treball s’han dut a terme estudis funcionals, estructurals i filogenètics de la proteïna GreA que ens han permès determinar quins aminoàcids, i com a conseqüència quins dominis, podrien ser importants per la funcionalitat de la proteïna, la seva capacitat d’unir-se a l’ARNpol i la seva capacitat de competir amb altres factors. A més, hem estudiat quin efecte té la competència entre els diferents factors que interaccionen amb el canal secundari sobre l’expressió d’un gen diana. Canvis en els nivells de la proteïna GreA, poden afectar la competència pel canal secundari de l’ARNpol Per això hem determinat el patró d’expressió del gen greA, així com l’existència d’una regulació creuada entre les diferents proteïnes que interaccionen amb el canal secundari. Finalment, hem dut a terme un estudi transcriptòmic en Salmonella enterica serovar Typhimurium, amb l’objectiu de determinar quin és l’efecte d’aquesta competència en l’expressió de factors de virulència.[eng] Gene expression begins by an enzymatic complex known as RNA polymerase (RNApol). The basic unit (core) of RNApol in bacteria is formed by 5 protein subunits (α2ββ’ω). The three-dimensional structure of the RNApol defines two spaces that play a relevant role during transcription, defined as primary and secondary channel. The holoenzyme needs the binding of a σ subunit to recognise promoter sequences and initiate the transcription process. Transcription is a dynamic process controlled at different steps. Genetic regulation during transcription initiation has been highly studied, and several mechanisms of regulation exist. However, the aim of this project is to study some aspects of the regulation during transcription elongation. It has been described that the alamone ppGpp, as well as several proteins, such as GreA, GreB or DksA, enter within the secondary channel and interact directly with the catalytic centre of the RNApol. The swap between the different factors that bind to the secondary channel of the RNApol may cause changes in the expression pattern. It has been postulated that DksA and ppGpp act as cofactors, however, a previous study performed in our research group, indicated that the phenotype of ppGpp and DksA deficiencies were not always identical, letting us suggest that the occupancy degree of the secondary channel of the RNApol may have significant impact in the expression pattern in E. coli. The data obtained clearly indicate that upregulation of some genes, such as fliC, that occurs in absence of DksA, was the result of the vacancy of the secondary channel generated in a dksA strain rather than being the result of DksA having a direct repressor effect. We suggested that in the absence of DksA, the interactions of other proteins, such as GreA, are promoted and responsible of the upregulation observed. In this project, functional, structural and phylogenetical studies of the protein GreA were performed to determine which amino acids are important for i) the functionality of GreA, ii) the ability to bind to the secondary channel of the RNApol or iii) the capacity to compete with other factors, such as DksA. We have determined that greA overexpression produces a negative effect of the bacterial growth. Moreover, this negative effect is enhanced in absence of DksA, highlighting the hypothesis of a competition between factors that bind into the secondary channel. The effect of this competition between GreA and DksA was also determined studying the expression of the fliC gene. Our data showed that both, GreA and DksAare required for fliC expression but act at different levels in the regulatory cascade of flagella expression regulation. GreA may control fliC expression during transcription elongation whereas DksA may act during transcription initiation. Changes in the amount of GreA, could affect the competition between factors that bind to the secondary channel of the RNApol. Therefore, we have determined the expression pattern of greA. Transcriptional studies showed a crosstalk between the different factors that bind into the secondary channel of the RNApol exists. Finally, transcriptomic studies were performed to determine the effect of ppGpp and DksA on the expression pattern of Salmonella enterica serovar Typhimurium. The results obtained indicate : i) the effect of the possible competence between the factors that interact into the secondary channel of the RNApol and ii) the effect of ppGpp and DksA on the expression of several virulence factors as well as different mobile elements present in Salmonella

Image_2_Contributions of SpoT Hydrolase, SpoT Synthetase, and RelA Synthetase to Carbon Source Diauxic Growth Transitions in Escherichia coli.PDF

<p>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.</p

Table_1_Contributions of SpoT Hydrolase, SpoT Synthetase, and RelA Synthetase to Carbon Source Diauxic Growth Transitions in Escherichia coli.pdf

<p>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.</p

Image_1_Contributions of SpoT Hydrolase, SpoT Synthetase, and RelA Synthetase to Carbon Source Diauxic Growth Transitions in Escherichia coli.pdf

<p>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.</p

Image_3_Contributions of SpoT Hydrolase, SpoT Synthetase, and RelA Synthetase to Carbon Source Diauxic Growth Transitions in Escherichia coli.pdf

<p>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.</p

Table_3_Contributions of SpoT Hydrolase, SpoT Synthetase, and RelA Synthetase to Carbon Source Diauxic Growth Transitions in Escherichia coli.XLSX

<p>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.</p

Autoregulation of greA expression relies on GraL rather than on greA promoter region

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