Année académique 2017 - 2018
Promoteur : Pr. Laurence VAN MELDEREN
Laboratoire de Génétique et Physiologie Bactérienne
Département de Biologie Moléculaire
Faculté de Science
Université Libre de Bruxelles
Regulation of the expression of the
global regulator CsrA by the two
component system CpxR/A in
Escherichia coli
MASSON Clément
2
Remerciements
Ce manuscrit est l'aboutissement d'un travail long de 5 ans et des nombreuses expériences qui l'ont pavé. Il m'aurait été impossible d'achever ce travail sans toutes les personnes qui, au quotidien, m'ont apporté leur aide, leurs conseils, leurs soutien ou simplement leur présence. Tous, vous m'avez permis de tenir dans les moments les plus difficiles et de m'accrocher à ce doctorat. Pour tout cela et bien plus :
Merci
Je me dois de nommer certaine personnes pour tout ce qu'elle m'ont apporté en plus.
Laurence, qui m'a accordé sa confiance et m'a donné la chance de faire ce doctorat, qui s'est montrée être une Cheffe hors du commun au labo comme au…dehors.
Les membres de mon comité d'accompagnement, Anne-Marie Marini, Louis Droogmans et Xavier De Bolle, pour leurs conseils et discussions.
Eveline Peeters et les membres de son laboratoire pour leur aide sur une partie de ce travail.
Freddus dit 'the best' et Monsieur Dukas, qui ont été d'une aide sans équivalent pour mon travail, ma culture et mes connaissances dans l'aérodynamisme des élastiques.
Les inséparables Thibaut et Baptiste, grâce à qui je sais à quoi ressemblent tous les accents de Belgique et bien d'autres choses que je ne saurais/pourrais jamais placer dans une conversation. Nathan, pour nos échanges commerciaux cordiales. Hedvig, Katleen, Zhongshu, Nathalie, Ethel, Marie, Marie junior, Julien et Thomas pour les rires et les échanges plus ou moins constructifs au cours de ces années.
Laura, Elodie, Paula, Simon 'Kekette' et la ferme du hameau du roi, pour vos bons et loyaux services. Safia et Natacha pour m'avoir montré comment survivre dans le monde hostile de l'expérimentation. Mes vieux amis, que je connais depuis plus de la moitié de ma vie (par ordre alphabétique): Geoffrey, Adirne, Berno, Grégré, Soso, Laura, Emilie, Marion et Sandra.
Sara qui a été là tous les jours pour me soutenir et corriger mon français au détriment de ses yeux et de ses oreilles.
Et bien sur ma famille qui me soutient depuis toujours et me pousse vers mes rêves.
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INTRODUCTION
... 8GENERAL INTRODUCTION ... 10
THE TRANSCRIPTION IN ESCHERICHIA COLI ... 11
THE TRANSCRIPTION MECHANISM ... 11
STRUCTURE OF THE RNA POLYMERASE ... 11
REGULATIONS OF GENE TRANSCRIPTION ... 12
MOLECULAR MECHANISM OF CSRA ACTIVITY ... 14
STRUCTURE OF CSRA ... 14
TARGET MOTIF OF CSRA ... 14
POSITIVE AND NEGATIVE REGULATION ... 15
TARGETS OF CSRA... 15
CSRA REGULATES ITS OWN EXPRESSION ... 15
CSRA REGULATES THE CENTRAL CARBON METABOLISM ... 15
CSRA REGULATES THE STRINGENT RESPONSE ... 17
CSRA REGULATES BIOFILM FORMATION... 18
MOTILITY AND CHEMOTAXIS ... 19
CSRA REGULATES QUORUM SENSING ... 19
CSRA REGULATES VIRULENCE ... 20
CSRA AND DRUG RESISTANCE ... 20
CSRA AND OTHER GLOBAL REGULATORS ... 20
IDENTIFICATION OF THE CSRA REGULON BY HIGH THROUGHPUT ANALYSIS ... 21
REGULATION OF CSRA EXPRESSION ... 23
STRUCTURE OF CSRA PROMOTER REGION ... 23
REGULATION OF CSRA ACTIVITY BY SRNAS ... 24
REGULATION OF THE TWO SRNA CSRB AND CSRC ... 26
TWO-COMPONENT SYSTEMS ... 27
GENERAL INTRODUCTION ... 27
THE BARA/UVRY TWO-COMPONENT SYSTEM REGULATES CSRB AND CSRC EXPRESSION... 28
THE TWO-COMPONENT SYSTEM CPXA-CPXR IN ESCHERICHIA COLI ... 30
ENVELOPE ORGANIZATION AND ENVELOPE STRESS RESPONSE PATHWAYS ... 30
THE CPX SYSTEM ... 31
THE CPXA SENSOR-KINASE ... 31
THE CPXR RESPONSE REGULATOR ... 32
THE CPX REGULON ... 32
EXPRESSION AND ACTIVATION OF THE CPX SYSTEM ... 33
TRANSCRIPTIONAL AND POST-TRANSCRIPTIONAL REGULATION OF CPX GENES ... 33
ACTIVATION OF THE CPX STRESS RESPONSE THROUGH SIGNALS ... 35
ACTIVATION OF THE CPX PATHWAY INDEPENDENTLY OF THE CPXA SENSOR KINASE BY ACETYL-PHOSPHATE ... 36
THE ACETYL-PHOSPHATE PATHWAY AND ITS IMPLICATION IN TCS PHOSPHORYLATION ... 36
OBJECTIVES
... 40RESULTS
... 44PHENOTYPIC SCREEN ON TWO COMPONENT SYSTEMS ... 46
THE CSRA-ASSOCIATED PHENOTYPES ... 46
MUTATIONS OF THE TCS AFFECTS THE CELL MOTILITY ... 46
GLYCOGEN ACCUMULATION IS AFFECTED IN SOME TCS MUTANTS ... 47
5
CONCLUSIONS OF THE PHENOTYPIC SCREEN ... 49
BETA-GALACTOSIDASE ASSAYS REPORTING PROMOTER ACTIVITY OF CSRA, CSRB AND CSRC PARTIALLY CONFIRM THE SCREEN RESULTS ... 49
THE ROLE OF THE CPX SYSTEM ON CSRA TRANSCRIPTION IN RICH MEDIUM ... 53
CSRA EXPRESSION DURING GROWTH ... 53
CPXR POSITIVELY REGULATES CSRA PROMOTER ACTIVITY ... 53
THE PHOSPHATASE ACTIVITY OF CPXA REPRESSES CSRA EXPRESSION DURING THE LOG PHASE... 53
IMPLICATION OF THE ACETYL-PHOSPHATE IN THE CSRA TRANSCRIPTION LEVEL ... 56
TRANSCRIPTION LEVEL OF THE DIFFERENT CSRA PROMOTERS ARE AFFECTED BY CPXR ... 56
PROMOTER P3 ACTIVITY IS DEPENDENT ON CPXR IN RT-QPCR ASSAYS ... 61
THE AMOUNT OF CSRA SEEMS TO NOT BE AFFECTED BY CPXR ... 62
CONCLUSION ... 63
PROBLEM OF EXPERIMENTS REPRODUCIBILITY IN LB MEDIUM ... 64
CPXR DIRECTLY UP-REGULATES CSRA TRANSCRIPTION IN CHEMICALLY-DEFINED MEDIUM IN E. COLI ... 65
CSRA TRANSCRIPTION LEVEL DURING GROWTH IN MINIMAL MEDIUM ... 65
TRANSCRIPTIONAL REGULATION OF CSRA EXPRESSION BY CPXR ... 65
CPXA DOWN-REGULATES CSRA TRANSCRIPTION DURING THE EXPONENTIAL PHASE ... 68
IMPLICATION OF THE ACETYL-PHOSPHATE ON CSRA EXPRESSION IS CPXR DEPENDENT ... 68
CPXR BINDS TO THE CSRA PROMOTER REGION IN VITRO ... 72
ITC EXPERIMENTS INDICATE THE PRESENCE OF TWO CPXR~P BINDING SITES ON THE P3 PROMOTER REGION ... 76
CONCLUSIONS ... 76
DISCUSSION AND PERSPECTIVES
... 80LIMITATIONS OF THE PHENOTYPIC SCREEN APPROACH ... 82
PROBLEM OF REPRODUCIBILITY IN LB MEDIUM... 84
THE CPXR/ATCS REGULATES CSRA PROMOTER ACTIVITY ... 84
SHED THE ENVELOPE, GROWING IN OUR PERCEPTION OF CSRA ... 87
TO GROW OR NOT TO GROW, THAT IS CSRA ... 88
INVOLVEMENT OF CSRA IN SIGNALS INTEGRATION AND ADAPTIVE RESPONSE ... 89
MATERIALS AND METHODS
... 92REFERENCES
... 1048
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General introduction
Bacteria are confronted to a large variety of stresses due to ever changing environmental conditions. Cells have to quickly adapt and revert to the original state when the conditions return to steady state conditions. To achieve this short-term adaptation, bacteria possess global regulators able to regulate the expression of tens to hundreds targets belonging to specific pathways.
As an example (for review see [157]), Escherichia coli is able to respond to a depletion in amino acids by synthesizing (p)ppGpp, an alarmone which redirects transcription towards biosynthetic operons and indirectly leads to degradation of free ribosomal proteins. During heat-shock, E. coli induces the production of chaperones and proteases that will help to refold or degrade damaged proteins. Similarly, cell responds to oxidative stress by inducing expression of antioxidant enzymes. Cell can also detect and use preferred carbon sources among a mixture of carbon sources. Adaptation to these environmental changes reflects gene expression reprogramming caused by global regulators. Usually, those regulators act at the level of transcription. However, global post-transcriptional regulators have been characterized (for review see [68]). Global transcriptional regulators are themselves regulated by post-transcriptional regulators. Therefore, global regulation implies a cascade of regulations from the integration of extracellular signal to the phenotypic traits resulting from differential gene expression.
11 itself negatively regulated by CsrA [15]. The stringent response, central carbon metabolism as well as the two-component system BarA/UvrY are involved in CsrA activity regulation mainly through feedback loops. The csrA gene is transcribed thanks to σ70 (housekeeping sigma factor) and RpoS (σ38, stationary phase and general stress response sigma factor), via five promoters. CsrA is expressed at the end of exponential phase and during the stationary phase [16].
The transcription in Escherichia coli
The transcription mechanism
Promoter sequences are located upstream of genes and contain all the elements for the transcription machinery recognition and transcription initiation as well as regulatory sites [160]. The E. coli RNA-polymerase is composed of 5 subunits (core enzyme) and of a sigma factor (figure 1) [160]. The core enzyme is responsible for the synthesis of RNA while sigma factors specifically recognize the promoter. Sigma factors recognize 2 specific sequences, the -10 box and -35 box, named after their approximate location from the transcription start site [160]. Upon binding of the sigma factor to the promoter, the core enzyme opens up the double strand DNA helix and synthesises RNA using the coding strand as template (figure 1A) [161]. The transcription stops when the polymerase reaches a terminator sequence. The terminator sequences are composed of repeated sequences that form a hairpin structure followed by a stretch of adenines. The hairpin loop blocks the polymerase and slows down the elongation. The polymerase is released due to the weak interaction between the DNA adenine-rich sequence and the uracil rich-sequence of the neo-synthesised RNA. Specific genes possess a Rho-dependent terminator sequence. In this case, the protein Rho recognizes a specific sequence and separates the DNA and the RNA to release RNA-polymerase [160].
Structure of the RNA polymerase
12 The σ subunit is part of the RNA-polymerase and allows the recognition of the promoter by the core polymerase [160]. It is released from the core polymerase core to allow RNA synthesis. In E. coli, 7 alternative sigma factors have been described [for review 162]. Each sigma factor recognizes a different consensus sequence of the -10 and -35 boxes, leading to the transcription of specific genes. Variations of these sequences affect the strength of the promoter and reduce its transcription level. Thereby, except for few highly expressed genes, the consensus sequences are rarely found [162].
σ70 (RpoD) is the house keeping sigma factor, it is expressed all along the growth and
recognizes most of the genes [162]. σ38 (RpoS) is the general stress response and stationary
phase sigma factor. It recognizes the same -10 and -35 boxes as σ70 but with a lower affinity.
RpoS is favoured over RpoD during the stationary phase thanks to an anti-sigma factor that prevents recruitment of RpoD by the core polymerase. Furthermore, RpoS recognizes -10 and -35 boxes more degenerated than RpoD does, allowing regulation of genes ignored by this latter [162]. σ19 (FecI) is activated upon intracellular iron deprivation and regulates
expression of genes involved in the detection and import of iron [162]. σ28 (FliA) regulates
genes involved in the flagella synthesis. It is antagonised by the anti-sigma factor FlgM that is secreted to allow FliA activity [162]. σ32 (RpoH) is the sigma factor of heat-shock response,
it is activated by the presence of unfolded proteins in the cytoplasm and recognises genes encoding proteases and chaperones [162]. σ24 (RpoE) is activated by outer membrane
stresses and especially by misfolded proteins in the outer membrane. It regulates expression of genes involved in the envelope stress response [162]. Finally, σ54 (RpoN) responds to
limiting nitrogen conditions and recognises genes involved in nitrogen stress response. Interestingly, RpoN recognises -12 and -24 boxes instead of the -10 and -35 boxes and requires ATP binding to allow initiation of the transcription. Furthermore, RpoN can bind DNA in absence of the core polymerase [162].
Regulations of gene transcription
13 thereby favour transcription initiation. For example, the CpxR transcriptional regulator (see below) positively regulates the transcription of the cpxP gene by binding on 2 sites, one overlapping the -35 box and helping RNA polymerase recognition while the second site overlaps the transcription starting site favouring the DNA opening [116].
In the case of negative regulation, the transcription factor can bind in the transcribed sequence and prevent transcription elongation by blocking the RNA polymerase progression. On the other hand, repressors binding usually overlap the recognition site of the RNA polymerase and prevent recruitment of this latter. For example, the expression of the csgD gene, encoding a regulator of curli and cellulose synthesis, is repressed by the CpxR regulator. CpxR binds on 2 sites within the csgD promoter. The first site is located between the -10 and -35 boxes and prevent RNA polymerase binding and the second site is located few nucleotides downstream in the transcribed region and prevent RNA elongation [163]. Some transcription factors can bind upstream of the promoter, up to hundreds of nucleotides. These regulators regulate transcription via DNA bending. In other cases, regulators are dependent on the presence of other regulators, such as HU and IHF [164]. These histone-like proteins are involved in recombination, inversion and integration events as well as DNA bending. They bind and bend DNA and in this way bring DNA-bound regulators to close proximity of the promoter allowing them to regulate transcription [164].
Figure 1: Structure of the prokaryotic RNA polymerase and transcription mechanism
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Molecular mechanism of CsrA activity
Structure of CsrA
CsrA is a conserved protein composed of 61 amino acids in E. coli (figure2A) [1]. CsrA is composed of five β-strands and, at the C-terminal region, by one α-helix followed by an unstructured ten amino acids region (figure 2B) [18]. In physiological conditions, CsrA forms homo-dimers [19] in which the β-strand 1 and 2 interacts with the β-strand 4 and 5 from the other monomer, respectively, forming an anti-parallel sheet (figure 2C). The binding of CsrA to RNA occurs via the β-strand 1 and 5 forming a positively charged region on each side of
the dimer [20, 21] allowing the binding of two DNA sites per dimer.
Figure 2: Sequence and structure of CrsA
(A) Amino acids sequence of CsrA from E. coli K-12 aligned with CsrA sequence from other bacteria. Conserved acidic, basic, polar, and hydrophobic residues are indicated in red, blue, green, and gray, respectively. Secondary structure elements are shown on top of the sequence alignment. (B) Structure of the CsrA homodimer from residue 1 to 55 (pink and blue). Superposition of 10 conformers. Secondary structures are annotated. (C) Depiction of the CsrA homodimer structure. Adapted from [18].
Target motif of CsrA
15 sequence, however binding sites can be found as well in the coding region (see below). Using the SELEX method, Dubey and al [22] identified the consensus motif recognized by CsrA as being RUACARGGAUGU (where R is a U or C) with the underlined GGA highly conserved. Secondary structure of the mRNA appears to be an important feature as well. The binding motif of CsrA is in general located in a hairpin with the GGA sequence always located in the single strand loop of the hairpin [22]. As described above, a dimer of CsrA binds two sites on the mRNA. The optimal distance between both sites is 18 nucleotides, although distance may vary from 10 to 63 nucleotides [23].
Positive and negative regulation
CsrA regulates either positively or negatively the expression of its gene targets. In the case of negative regulation, CsrA prevents translation initiation usually by masking the Shine-Dalgarno sequence (RBS) and in specific cases, by leading to degradation of the mRNA [8]. CsrA also prevents translation by binding to the early coding region [26], therefore preventing translation elongation instead of initiation.
Examples of positive regulation are less common. CsrA bind on the 5'UTR, and thereby preventing mRNAs degradation by RNAse E [9]. Alternative mechanisms have been described in which CsrA binding site overlaps a repressor binding site [27] or CsrA binding disrupts a inhibitory binding secondary structure [28, 29].
Targets of CsrA
CsrA regulates its own expression
It was shown that CsrA negatively regulates its own expression. It binds its own mRNA on 4 binding sites (figure 3A) [16]. This feedback loop also goes through negative regulation of csrD expression, which is an indirect activator of csrA expression (see below), emphasizing the negative control of CsrA on itself.
CsrA regulates the central carbon metabolism
16 glgC expression prevents glycogen synthesis and eventually redirects glucose into the glycolysis flux.
CsrA up-regulates the expression of the acs gene (figure 3A) [30]. The Acs enzyme converts the glycolysis by-product acetate into pyruvate, a metabolite produced by the glycolysis last step. During growth on glucose, glycolysis leads to pyruvate, which constitutes the entry
Figure 3: The CsrA regulon
17 metabolite for the Krebs cycle. An excessive production of pyruvate leads to its conversion into acetate and secretion out of the cell. Upon glucose exhaustion, acetate is up taken and consumed. Up-regulation of acs expression emphasizes the role of CsrA in glucose consumption.
CsrA is also involved in anaerobic fermentation. Sugar fermentation is activated by CsrA via positive regulation of the ldhA gene expression encoding lactate dehydrogenase (IdhA) which converts pyruvate into lactate, a substrate for anaerobic respiration (figure 3A) [31]. Expression of the moaA gene, involved in the synthesis of the anaerobic metabolism cofactor MOCO, is activated by CsrA (figure 3A). Indeed, CsrA binds on the repressive MOCO-responsive riboswitch and prevents the binding of the MOCO cofactor itself. Thus, it prevents the feedback repression by MOCO and therefore activates moaA expression [27]. In addition CsrA, represses the expression of nrf gene (figure 3A) [32]. NrfA is a periplasmic nitrite reductase converting nitrite into ammonium ions that support growth during anaerobic conditions. The regulation of nrf expression by CsrA appears to be direct although no strong evidences have been provided. Interestingly, nrf expression is also repressed by CsrA in Salmonella enterica [32]. In addition, different metabolisms are affected by CsrA in Salmonella strains, notably maltose transport [33]. Alongside, in other bacterial species, CsrA regulates central carbon metabolism, such as in Legionella [34], Campylobacter jejuni [35], or in the more distant Borelia burgdoferi [36] and Clostridium acetobutylicum [37]. To sum up, the role of CsrA in carbon metabolism is well conserved among bacterial species. CsrA appears to favor the consumption of energy against its storage, by activating glycolysis and repressing glycogen biosynthesis in E. coli. The role of CsrA in the anaerobic metabolism remains unclear as CsrA both activates and represses activators of the anaerobic metabolism. CsrA could increase specific types of anaerobic growth (fermentation on sugar) and repress others (nitrate respiration).
csrA regulates the stringent response
18 Expression of the cstA gene is activated by amino acids starvation thus providing a way to increase amino acids uptake in starvation conditions. Expression of cstA is also positively regulated by the catabolic repressor CRP. CRP is a global regulator activated by cyclic AMP, which is synthesized by the adenylate cyclase CyaA. CRP regulates expression of about 180 genes. CRP is responsible for the catabolic repression, it hierarchies the carbon sources consumption. Interestingly, crp and cyaA expression is also regulated by CsrA [40].
Moreover, expression of two other putative peptide transporters, yjiY and yhjX, appears to be regulated by CsrA [41]. Expression of these 2 genes is also under the control of 2 two-component systems, YehU/T and YpdA/B, that sense and respond to presence of amino acids and pyruvate, respectively. Altogether, these information clearly indicate that CsrA negatively regulates the import of amino acids by repressing expression of transporters (CstA and YjiY) and (p)ppGpp synthesis (RelA/SpoT). CsrA also regulates the import of pyruvate (CstA and YhjX). Repression of the stringent response is consistent with the positive role of CsrA on glycolysis and growth state of the cell. Interestingly, in Legionella, CsrA is described as an essential activator of DNA replication [42], an activity consistent with the proposal that CsrA favors cell growth. Moreover, deletion of the csrA gene in E. coli leads to reduction of growth rate and cell size [Hallaert, Seyll and Van Melderen unpublished].
CsrA regulates biofilm formation
19 CsrA prevents biofilm formation not only in E. coli but also in Pseudomonas putida [50] through decrease of the synthesis of (p)ppGpp. Unexpectedly, CsrA was shown to increase biofilm formation in Campylobacter jejuni [35]. Moreover, deletion of csrA leads to a loss of the colony organization [Hallaert in preparation].
Motility and chemotaxis
Coherent with the observation that CsrA presses biofilm formation, CsrA activates motility (figure 3C). This regulation occurs trough post-transcriptional activation of flhDC expression [51]. The flhDC operon encodes the transcription factor required for the synthesis of the flagellum and chemotaxis [for review 52]. CsrA binds to two sites located in the 5'UTR of the flhDC mRNA [51] and stabilizes it by masking the recognition site for RNAse E, thus preventing its degradation [9]. Motility is controlled by the csr system in different bacterial species, such as Legionella, Salmonella, Yersinia and Borrelia [53, 54, 55 and 56]. Interestingly, in Borrelia burgdoferi, CsrA represses the expression of flaB, a major element of the flagellum [36]. These data indicate that CsrA positively regulates the motility, except in the case of Borrelia burgdoferi. The difference of regulation depending on the bacterial species (as observed for biofilm formation in Campylobacter jejuni) might reflect adaption to ecological niches. In most of the cases, activation of the motility in a CsrA-dependent manner is consistent with the activation of the growth state and the consumption of nutrients. Indeed, motility and chemotaxis are important mechanisms for cells to detect and reach new nutrient sources in order to colonize favorable niches.
CsrA regulates quorum sensing
20
CsrA regulates virulence
CsrA is involved in virulence and host invasion regulation (figure 3C). It was shown that overexpression of CsrA inhibits translation of the glrA mRNA [60]. GlrA is an activator of the type 3 secretion system (T3SS), a system that is responsible for eukaryote cells invasion. Single-copy csrA also potentially stabilizes the transcripts of sepL and espADB operon, which products are involved in the assembly the T3SS needle [60]. Moreover, expression of the nleA gene is negatively regulated by CsrA. NleA is involved in the translocation of proteins via the T3SS [61]. Therefore, it is proposed that CsrA might modulate the T3SS synthesis/activity depending on specific conditions.
Other virulence/pathogenic pathways regulated by CsrA have been identified. Firstly, expression of the tnaA gene is positively regulated by CsrA [62]. TnaA is a tryptophanase that catalyzes the hydrolysis of tryptophan into indole, leading to the synthesis of exotoxin capable of killing the worm Caenorhabditis elegans [62]. Secondly, the expression of the colE7 operon appears to be down-regulated by CsrA [63]. The colE7 operon encodes three proteins, ColE7, ImmE7 and LyzE7 allowing the synthesis of colicin (ColE7). ColE7 is a colicin, the LyzE7 protein increases permeability to allow colicin secretion and finally ImmE7 immunizes the cell against the toxin.
The regulation of virulence and host invasion pathways are described in many other bacteria to be under the control of the csr pathway [for review 64], e.g. host invasion and macrophage intracellular survival of Salmonella, the intracellular growth of Legionella, and the switch between acute and chronic infection in Pseudomonas species [for review 64].
CsrA and drug resistance
CsrA is also involved in stress responses, like drug-resistance through the stabilization of the acrAB mRNA (figure 3C). AcrA and AcrB are, with TolC, part of a tripartite efflux pump conferring resistance to a variety of drugs [65]. CsrA bind to acrAB mRNA and increase its stability by supposedly preventing the formation of a secondary structure that blocks ribosome binding [28].
CsrA and other global regulators
21 targets RpoS to degradation. iraD expression is regulated by (p)ppGpp and is activated by the stringent response. The negative regulation of iraD translation by CsrA leads to RssB activation and thus a decrease in RpoS expression [29]. The effect of CsrA on the iraD expression is double, first directly has mentioned above but also through the repression of (p)ppGpp synthesis pathway (see above 'stringent response'). It appears that CsrA represses the pathways responding to stress and stationary phase, consistent with its role in cell growth.
As mentioned in the previous chapter on stringent response, CsrA positively regulates expression of crp and cyaA. CRP regulates the expression of up to 180 genes in E. coli, mainly involved in catabolism and secondary carbons sources [for review 69] but also in stringent response, biofilm formation, virulence and metabolism.
Finally, the expression of the 3' to 5' exo-nuclease PNP (polynucleotides phosphorylase) was recently described to be down-regulated by CsrA [70]. PNP is part of the degradosome, a protein complex that degrades RNA. CsrA is unable to bind pnp mRNA unless the double strands exonuclease RNAse III digests a portion of the 5'UTR of the pnp mRNA. Park et al. showed that one of the two binding sites of CsrA on pnp mRNA is blocked by a mRNA secondary structure. The degradation of the upstream region of the mRNA by RNAse III then by PNP itself releases the binding site allowing CsrA to repress the expression of PNP. Aside this unusual mechanism of regulation, the regulation of an important member of the degradosome allows CsrA to indirectly regulate the stability of a large number of mRNAs, expanding therefore the csr regulon.
Identification of the csrA regulon by high throughput analysis
So far, it has been shown that CsrA directly regulates the expression 32 targets genes. High throughput analyses suggest hundreds of potential targets. The first high throughput analysis [39] was based on co-purification of recombinant CsrA protein and interacting mRNAs. The co-purified mRNAs were then reverse-transcribed into cDNAs and sequenced. To confirm these high throughput data, Edwards and al. compared the proteomes of a wild-type strain and csrA mutant. Interestingly amongst all the transcripts that interact with CsrA, the authors estimated that about 40% of them respond to ppGpp and/or DksA. As mentioned above, ppGpp and DksA modulate expression of genes during the stringent response. Also, up to 67% of the identified transcripts are related to energy production or consumption. As shown above, CsrA is involved in energy storage (glycogen accumulation), consumption (glycolysis, motility), as well as in the stringent response itself (DksA and synthesis of ppGpp).
22 potential binding sites located directly upstream of the translation start site. They used a degenerate consensus sequence to predict potential biding sites. Cross-analysis of the results highlights targets in the usual pathways (glycolysis/neoglucogenesis, glycogen biosynthesis and biofilm formation) as well as genes involved in the Krebs cycle and pentose phosphate pathway. All these putative target pathways correlate with the known roles of CsrA and suggest a wider/stronger control on metabolism.
Additionally, this analysis identified potential CsrA targets in a variety of unrelated pathways such as sulfate transport, export and efflux pumps as well in peptidoglycan synthesis.
A high throughput analysis by Esquerré et al. [72] focused on the mRNA stability and mRNA level in csrA51 mutant strain and ΔcsrD strain. The csrA51 mutant encodes a truncated version of CsrA that retains partial activity. CsrD is an indirect activator of the CsrA activity (see below and [15]). Esquerré and al. first observed a general lower stability of total mRNAs in the csrA51 strain compared to the wild-type strain. Indeed, up to 1600 mRNAs out of the 4377 genes (4290 encode for proteins) in E. coli present a differential stability in the mutant strain as compared to the wild-type strain. Deletion of csrD did not lead to a significant increase in mRNAs stability as it was expected. A feedback loop appears to prevent variations in the level of csrB and csrC mRNA and thereby buffering the effect on CsrA activity. On the other hand, the level of mRNAs (and not stability) is strongly affected in both strains compared to the wild-type strain. CsrA appears to affect the transcription level of 500 mRNAs. Results indicate that this change in the transcription level is, at least partially, CsrD-dependent. The mechanism remains unclear, but a hypothesis could be that CsrA and/or CsrD regulate the expression of transcriptional factors. Indeed, Esquerré et al. observed that within the list of genes down-regulated by CsrA, they detected the RNA polymerase genes rpoB and rpoC (coding for β and β' subunits), 6 sigma factors genes (including the general stress response rpoS, and the housekeeping rpoD), as well as transcription factors involved in carbon flow (the catabolic repressors crp and cyaA). The general idea behind this analysis is that CsrA not only regulates a large range of targets, directly or through other global regulators, but that CsrA is an important mRNA stabilizer element.
23 Interestingly, the binding of CsrA in the coding sequence of target mRNAs was mostly associated with positive regulation. Binding art the 5'UTR or the early coding sequence was correlated with negative regulation. Potts et al. propose that the binding of CsrA in the targets coding sequence could modify the mRNA structure and prevent action of nucleases. Potts and al. integrated the results of previous high throughput analyses with their own results to identify the most relevant information. First of all, their results were consistent with the already identified pathways, namely CsrA up-regulates carbon consumption, motility, and represses biofilm formation. Results also indicate a CsrA-dependent regulation of alternative carbon sources pathways. Potts et al. are the first to discuss the importance of CsrA in the envelope homeostasis [10]. Unlike McKee et al. they did not observe changes in mRNA levels for genes related to peptidoglycan synthesis, however they identified potential regulation of the RpoE-dependent envelope stress response. CsrA appears to regulate the envelope stress response by regulating regulators expression, notably RpoE, and not by regulating effectors expression. Potts et al. identified up to 87 transcription factors, 11 sensor-regulators systems (see below 'two component systems' and figure 5A), sigma factors and potentially post-transcriptional regulators and base pairing sRNA. These results are consistent with those obtained by Esquerré et al. [72]. This also echoes the down-regulation of the degradosome (via modulation of pnp expression) by CsrA. The high number of targets identified by the different methods presented in this chapter is most likely indirect and relies on the lack or at least decrease of RNA degradation induced by the decrease of degradosome expression/activity.
Regulation of CsrA expression
Structure of CsrA promoter region
24 Expression of csrA is low during the exponential growth phase and increases at the late exponential growth/early stationary phase. This increase is mainly due to P3 and a to a less extent to P4-5 promoters. Activity of P1-2 remains stable along the growth curve [16]. During stationary phase, the transcription is mainly sustained by P3, responsible for about 75% of the total transcription, whereas P4-5 accounts for about 25%. P1-2 shows only a residual level of activity (≈5%). It was shown that CsrA regulates its own transcription from the promoter P3 [16] in a DksA-dependent manner (see below) [39].
Regulation of CsrA activity by sRNAs
The main regulation of CsrA activity occurs at the post-translational level through 3 sRNAs: csrB, csrC and mcaS (figure 4). These 3 sRNAs contain hairpins exposing single strand GGA sequences (figure 4A, B and C). Thanks to this structure, they bind and sequester CsrA thereby down-regulate CsrA activity. The csrB and csrC sRNAs present 18 and 9 CsrA binding sites, respectively while mcaS present only 2 sites [7, 13, 14]. The 3 sRNAs show a very similar structure despite their overall differences in sequence and length (366 nt for csrB, 242 nt for csrC and 95 nt for mcaS) as well as in the number of CsrA binding sites (18, 9 and 2 for csrB, csrC and mcaS respectively). The csrB and csrC expression is similar to that of the csrA gene, it increases along the growth curve and reaches a plateau at the beginning of stationary phase [17]. Their expression is similar to that of CsrA. The transcription level of csrB is about ten 10 to fifteen15-fold higher than that of csrC in rich medium [17]. csrB expression is strongly dependent on the transcription factor UvrY (see below 'the TCS BarA/UvrY') and on CsrA. csrC transcription is also regulated by UvrY and CsrA but to a lesser extent than that of csrB. The effect of CsrA is dependent on UvrY. More specifically, CsrA acts through the activation of UvrY in a manner both dependent and independent of the cognate activation pathway of UvrY (see below) [12]. On the other hand, deletion of csrB, csrC or both leads to an increase of csrC transcription, an effect not observed in the case of csrB [13]. This indicates a negative feedback loop on csrC mediated by the two sRNAs. Catabolic repression also represses csrB and csrC transcription, via the regulator CRP. CRP binds 2 sites in the csrC promoter, 2 potential binding sites have been predicted in the csrB promoter region, however no specific binding was observed (figure 4D and E)
The regulation of CsrA activity by csrB and csrC appears to be part of a complex feedback loops system. Indeed, csrB and csrC are indirectly regulated by CsrA, via UvrY (positive feedback loop), and via CRP (negative feedback loop), itself under the positive control of CsrA (see above 'Stringent response').
25 csrB. In minimum medium supplemented with casamino acids as sole carbon source, the levels of csrB and csrC strongly decrease during late stationary phase while the level of mcaS remains elevated, indicating a differential regulation for the 3 regulators. mcaS is also capable of regulating flhDC and curli genes expression by acting has a ‘true’ sRNA by base-pairing on their mRNA and preventing their translation [76]. Thereby, mcaS regulates CsrA
Figure 4: Structure of the sRNAs and regulation of the Csr network
26 activity as well as expression of CsrA target genes. Expression of mcaS is under the control of σ70 and activated by CRP in condition of nutrient limitations. In late stationary phase, expression of mcaS is down-regulated at the post transcriptional level by an unknown element but in an RpoS-dependent manner.
Altogether, these information provide evidences that the three regulatory sRNAs are differentially expressed, indicating that their action on CsrA activity might rely on different signals, reflecting the tight regulation of CsrA activity.
In other bacterial species, studies characterizing the csr network highlighted a similar regulation by sRNAs [for review 68]. Between one and three sRNAs have been identified, depending on the bacterial species. These sRNAs are homologs to csrB and csrC and use the same sequestration mechanism. However, no homolog of mcaS sRNA has been identified in other species yet.
Regulation of the two sRNA csrB and csrC
Expression of the sRNAs csrB and csrC is regulated at the post-transcriptional level by the CsrD protein (figure 4D) [15]. This latest is involved in degradation of csrB and csrC mRNA through the activity of the exonuclease RNAse E. CsrD is a protein described to possess an oligomerisation domain (HAMP) and non-functional GGDEF and EAL domains, usually c-di-GMP synthesis and hydrolase domains, respectively [15]. Although they are non functional in terms of c-di-GMP synthesis and degradation, these two domains are essential for CsrD activity. RNAse E activity on csrB is counteracted by the binding of CsrA on the 3' end of the sRNA [77]. Thus, the binding of CsrA protects csrB from degradation and increases its stability. CsrD is also involved in the degradation of csrC through a similar mechanism. However, the interaction between CsrA and csrC is not affected by CsrD.
27 As mentioned above, CsrA down-regulates the expression of relA and spoT and up-regulates that of DksA (see above 'stringent response'). DksA in turn activates csrB and csrC transcription in RpoE- and UvrYdependent manner [39].
The csrB and csrC homologs in other bacterial species are also regulated by the UvrY homologues, which are also part of two-component systems as in E. coli [for review 68].
Two-component systems
General introduction
Two-component systems (TCS) are widespread in the bacterial kingdom. TCS are typically composed of two proteins, a sensor kinase and a specific response regulator (figure 5B). The sensor is a histidine-kinase bound to the inner membrane. The N-terminal domain of the protein is sensitive to specific signals (which remain unknown in general for many different systems). In general, the sensing domain is located in the periplasm or at the external surface of the outer membrane. Upon signal detection, the sensor kinase is activated. The signal is transmitted to the enzymatic domain on the C-terminus of the sensor by conformational changes of the protein. The catalytic domain contains a conserved histidine that is phosphorylated, an ATP catalytic site and a dimerization motif. Two activated sensors form then a homodimer thanks to the dimerization motif and cross-phosphorylate each other on the conserved histidine in an ATP-dependent manner [80 and 81]. The phosphate group is then transferred to the response regulator on a specific aspartate residue [82]. It induces conformational changes and modulation of regulatory domain activity. The response regulator returns to the initial conformation through dephosphorylation by discrete phosphatases or by the phosphatase activity of the cognate histidine kinase. Most of the sensor kinases appear to possess an intrinsic phosphatase activity. It is assumed that it prevents activation of the response regulator by cross-activation by a non cognate sensor [83]. Another hypothesis proposes that the phosphatase activity allows a rapid turn-off of the TCS when conditions return to steady-state [for review 81].
The response regulators are mostly transcription factors, seldom enzymatic regulators, and more rarely RNA binding or ligand binding proteins. In the same way as the activated sensors, the response regulators generally form homodimers upon phosphorylation, allowing DNA binding to two repeated specific half-sites [81].
28 phosphorylated elements allows more checkpoints in the regulation of the TCS activity [81]. On the other hand, proteins can interfere with the TCS activation by influencing the activity of the response regulators [for review 84] or the sensor activity [for review 85]. These proteins are called connectors and can be produced in response to a signal different from the one sensed by the TCS. It allows connections between different signals and pathways. Also TCS appear to not act as completely independent elements, but more like combinatorial sensors that modulate the response in a coordinated manner depending on the signals combination perceived [86].
The BarA/UvrY two-component system regulates csrB and csrC
expression
The BarA/UvrY system is a major element in the control of csrB and csrC transcription (figure 4D and E) [12, 13]. This TCS is a non-conventional TCS in which the sensor-kinase BarA is a tripartite kinase [for review 84, 87]. (figure 5B). Upon activation, the phosphate group is transferred from the conserved histidine to an aspartate residue and to a second histidine residue within the BarA protein. The phosphate group is in turn transferred to the cognate transcription factor UvrY [88]. It has been shown that BarA [89] perceives acetate or short carboxylic-acid chain molecules in general [90].
As mentioned above, it has been shown that the BarA/UvrY TCS regulates expression of csrB and csrC. Deletion of UvrY drastically reduces the level of csrB [12] and to a lesser extent that of csrC (60%) (figure 4D) [13]. The regulation by UvrY does not take place in a csrA::kan mutant strain indicating that CsrA has an important role in the transcription and translation of uvrY through an unknown pathway (figure 4E) [91] [12]. Moreover, the activation of BarA is dependent on acetate-like molecules but also requires CsrA, again the intermediary elements have not been identified [91]. CsrA does not modify the transcription or translation of barA gene but modify the activity of BarA by regulating the switch from the BarA phosphatase to the kinase activity. In absence of CsrA, BarA only dephosphorylates UvrY. Based on these data it is clear that CsrA impacts the expression of the complete csr system through activation of BarA and expression of UvrY.
29
Figure 5: TCS potentially regulated by CsrA and TCS organization.
(A) List and description of the TCS potentially regulated by CsrA identified by Potts et al. [11]. (B)
30 The regulation of the csrB and csrC sRNA is poorly characterized in other bacterial species [for review 68]. Expression of homolgs of csrB and csrC is also up-regulated by the hortologs of BarA/UvrY. In Pseudomonas aeruginosa and P. fluorescens, two hybrid sensor-kinases are involved in sRNAs regulation. The sensors LadS and RetS contain both sensor kinase and response regulator activities. They cross-regulate the sensor GacS (hortolog of BarA) activity, and thus affect the sRNAs expression. RetS and LadS are involved in pathogenesis, promoting acute and chronic infection, respectively [for review 68]. This cross-regulation allows integration of multiple signals and fine-tuning of the csr network activity. Interestingly, in S. typhimurium, SirA (UvrY hortolog) activity is positively regulated by catabolic repression and acetyl-phosphate (by-product of acetate metabolism). Finally, the motility regulator FlhDC positively regulates the expression of GacA in P. carotovorum, activating the csrB hortolog (rsmB) expression. This regulation also occurs on rsmB expression via the HexA regulator, a regulator involved in exoenzymes production [for review 68]. The regulator of the flagella assembly FliW in Bacillus subtilis also regulates the activity of CsrA by direct binding [93]. The same mechanism applies in Campylobacter Jejuni [94].
The two-component system CpxA-CpxR in Escherichia coli
Envelope organization and envelope stress response pathways
The envelope of gram-negative bacteria is composed of an outer membrane and an inner membrane, defining the periplasm compartment, which contains the peptidoglycan and numerous proteins. The outer-member is a lipid bilayer composed of phospholipids in the inner leaflet and glycolipids and lipopolysaccharides (LPS) in the outer leaflet. Outer membrane contains mainly lipoproteins and β-barrel porins. The latter ones allow a controlled diffusion of hydrophilic small molecules across the outer membrane. The LPS molecules bind to each other, creating a dense barrier against hydrophobic molecules. The lipoproteins bridge the outer membrane to the peptidoglycan, a rigid exoskeleton composed of repeated disaccharides and short peptides. The peptidoglycan defines the shape of the cell and protects it against osmotic stresses. The periplasm is dense in proteins, particularly transport, chemotaxis, and chaperone proteins among others. The inner membrane gathers the energetic production, lipid synthesis, transport and secretion systems [For review 95]. To maintain the homeostasis of such a complex organelle, E. coli has evolved 5 stress response systems [96]. These pathways sense specific stresses or perturbations of the envelope and regulate both different and common pathways [97].
31 signals by inducing expression of efflux pumps to reduce the level of toxic compounds to sub-lethal levels [96]. The σE pathway detects outer-membrane and LPS perturbations by sensing outer membrane misfolded proteins. Activation of σE leads to the induction of chaperons, proteases and adaptive proteins expression to counteract the stress and reduce the number of unfolded proteins in the periplasm. The RcsA/B/C/D phosphorelay is characterized for its role in the peptidoglycan stress sensing. Activation of the Rcs pathway notably alters formation of biofilms, motility, virulence and capsule synthesis as well as expression of envelope proteins [96]. The PSP pathway senses perturbations of the inner membrane and the loss of the proton motive force. PSP appears to decrease energy-consuming processes like motility, and favors synthesis of phospholipids to restore the inner membrane integrity and proton motive force [96, 97].
All these pathways, together with the Cpx two-component system (see below), partially overlap both for the detection signals and for the rescue mechanisms. However, they differentially respond to perturbations of the envelope homeostasis, indicating intricate and interconnected responses [102].
The Cpx system
CpxA was discovered in 1980 in a screen for mutants affecting F plasmid conjugation in E. coli [151]. It was later shown that mutation of cpxA reduces the abundance of outer membrane proteins and alters the membrane protein composition [152 and 153]. In 1986, CpxA proposed to be a potential inner membrane histidine kinase [154]. CpxR was discovered in 1993 by primary sequence comparison with other TCS response regulators [155]. The two-component system CpxA/R [98] monitors envelope homeostasis and peptidoglycan perturbations [99]. CpxA/R senses misfolded proteins in the periplasm [100]. Upon activation, the Cpx system negatively regulates the expression of numerous proteins located in the envelope to decrease the envelope burden. CpxA/R also positively regulates the expression of proteases and efflux pumps to clear unfolded proteins or toxic compounds [101].
The CpxA sensor-kinase
32 transmitting domain that appears to induce the conformational changes required for the phosphorylation of CpxA (figure 5C) [100]. HAMP is a cytoplasmic domain and is composed of two amphipathic helices linked by a loop. The catalytic domain of CpxA forms an ATP-binding pocket of 4 α-helix and 5 β-strands [103]. The last domain is the dimerization domain, it includes the conserved histidine (His 248), target of the phosphorylation (figure 5C) [100, 103]. CpxA possesses a phosphatase activity that relies on the dimerization domain and/or the catalytic domain of CpxA. The phosphatase and the phosphorylation activities depend on different amino acids. Indeed, mutations can affect the phosphatase activity without effect on phosphorylation activity [81 and personal communication from T. Silhavy].
The CpxR response regulator
The cognate response regulator of CpxA is the cytoplasmic transcription factor CpxR (figure 5B). CpxR is a classical member of the TCS response regulator (from the OmpR subfamily) [105]. CpxR is composed of two domains connected by a flexible linker: the N-terminal receiver domain containing the conserved phosphorylation receiver aspartate (D51) and the C-terminal effector domain that binds DNA through an helix-turn-helix motif [106]. Upon phosphorylation, CpxR forms homodimers [107] and binds to the consensus sequence 5'-GTAAA(5N)GTAAA-3' [108] (also described as the reverse 5'-TTAC(6N)TTAC-3' shorter version, see http://regulondb.ccg.unam.mx). As the majority of TCS response regulators, CpxR has no specific activity when not phosphorylated. However, a weak activity has been observed in vivo is Yersinia pseudotuberculosis strain expressing a non-phosphorylatable CpxR allele [109]. As mentioned above, phosphorylated CpxR can be deactivated by the phosphatase activity of CpxA but also by PrpA, a heat-shock phosphatase [110].
The phosphatase activity of the sensor (or of PrpA) allows a rapid turn-off of the Cpx response when conditions return to steady state. This allows fine-tuning of CpxR activity to maintain envelope homeostasis.
The Cpx regulon
33 expression of the DegP periplasmic protease leading to degradation of misfolded proteins [98]. On the other hand, CpxR activates transcription of the amiA and amiC genes, encoding proteins involved in peptidoglycan homeostasis and structure and the impermeability of the outer-membrane. CpxR is also involved in drug resistance by enhancing expression of several efflux pumps [101].
Interestingly, CpxR/A regulates bacterial social behaviors. Notably, CpxR represses expression of the flhDC flagella regulator and motor flagella genes as well chemotaxis genes [116]. CpxR negatively regulates virulence by inhibiting pili formation. In the same line of idea, the expression of the curli operons csgBA (structural genes) and csgDEFG (regulation and curli assembly machinery) are down regulated by CpxR [117]. Biofilm formation is also positively regulated by CpxR/A via up-regulation of ydeH expression, encoding for a diguanylate cyclase.
Interestingly, CpxRA pathway is also responsible for modulating the other envelope stress response pathways. First, CpxR directly represses the σE pathway by negatively regulating the expression of rpoE gene. [111]. Second, the expression of the two TCS BaeS/R and EnvZ/OmpR responsible for antimicrobial compound response and osmotic shock response respectively, is up-regulated by CpxR [101]. BaeS/R induces synthesis of efflux pumps and drug resistance. CpxRA and BaeS/R regulate expression of shared targets and act in synergy in presence of specific stresses [101]. Meanwhile, the EnvZ/OmpR TCS works in synergy with CpxR in presence of commonly perceived stresses. Finally, CpxR regulates the activation of its own pathway via a feed-back loop involving the CpxP protein which is a negative regulator of CpxA activity (see below) [118].
This short overview of the Cpx regulon (figure 6A) shows that CpxR/A has an important role in periplasm and peptidoglycan homeostasis. This role is achieved by favoring degradation of unfolded proteins, and limiting the presence of non-essential proteins in membranes. It is assumed that repression of motility, virulence and biofilm formation allows the cell to maintain resources that can be limiting upon stress.
Expression and activation of the Cpx system
Transcriptional and post-transcriptional regulation of Cpx genes
34
Figure 6: Inducers and regulon of CpxR/A
35 are under the negative control of two sRNAs, rprA and micF (figure 6A). rprA expression is up-regulated by both Cpx signaling pathway and Rcs phosphorelay. rprA generates a negative feedback loop on the cpx gene expression [121]. Interestingly, rprA negatively regulates curli synthesis by pairing to the csgBA and csgDEFG mRNA expression [for review 122]. The second sRNA micF down-regulates the Cpx pathway by potentially binding to the cpxRA mRNA [123]. Interestingly, micF is indirectly controlled by CpxR since expression of micF is under the control of the EnvZ/OmpR TCS which is itself under the control of CpxR creating a negative feedback loop. These three retro-control loops (including the CpxP repressing loop) participate to negative regulation of CpxR/A expression and/or activity [101].
Activation of the Cpx stress response through signals
The signal generally accepted as sensed by CpxA is misfolded proteins in the periplasm [for review 100]. Many conditions can cause protein misfolding, notably environmental conditions such as alkaline pH, high osmolarity, presence of copper or zinc, indole, alcohols or acetone (figure 6B). In addition, problems arising with the machinery assembly of outer-membrane proteins or with protein translocation through the inner membrane lead to activation of CpxA [124, 125]. Stimuli can also derive from hydrophobic surface adhesion, P-pili and curli formation [100]. The lack of disulfide bonds in periplasmic proteins also trigger CpxA activation [126] as well as the depletion of phosphatidylethanolamine (phospholipids) in the inner membrane [100]. Indeed, changes in lipids length and charges can alter the phosphatase activity of CpxA [127]. Surprisingly, signals from the cytosol can activate CpxA phosphorylation as well, notably the level of acetyl-coA and of cAMP [for review 100].
36 and allows signal transmission to CpxA [131]. All these structural modifications and the underlying mechanisms are hypothetical and have not been tested yet [101].
A third protein (CacA) appears to be involved in Cpx activation but has been characterized only in Salmonella [132].
Activation of the Cpx pathway independently of the CpxA sensor kinase
by acetyl-phosphate
Finally, the last element that affects the activity of the Cpx stress response pathway can stimulate CpxR phosphorylation independently of CpxA (figure 6B). Indeed, acetyl-phosphate (acetyl-P) is a very reactive molecule that can directly phosphorylate CpxR thereby increasing the Cpx response [133]. During the exponential phase the acetyl-P is required for a normal CpxR response on CpxP expression. During the stationary phase, the lack of both CpxA and acetyl-P leads to a reduced response similar to the response of a strain lacking CpxR indicating that they are the sole phosphate donors of CpxR [133].
The
acetyl-phosphate
pathway
and
its
implication
in
TCS
phosphorylation
Acetyl-P is synthesized and degraded by the enzymes AckA (acetate kinase) and Pta (phosphotransacetylase). It is an intermediary compound of the acetate synthesis from acetyl-coenzyme A (acetyl-coA) or the reverse reaction. Acetate is a by-product of the glycolysis produced when the carbon flux into the cell exceeds the Krebs cycle capacity. In this condition, excess of acetyl-coA from the Krebs cycle is recycled by Pta into coenzyme A synthesizing acetyl-phosphate. AckA converts the acetyl-phosphate into acetate and ATP that are both secreted [for review 134]. In E. coli, the ackA and pta genes constitutes an operon that produces two transcripts, one that encodes both ackA and pta and a second that encodes only pta. A warm, alkaline, anaerobic, or a nutrient-rich environment increase ackA and pta expression and/or activity while a cool, acidic, aerobic, or acetate-rich environment does not [for review 134]. Acetyl-phosphate level reflects the nutritional status of the cell. A high concentration reflects either an excess carbon or a lack of oxygen, whereas a small concentration reflects limited carbon in the presence of oxygen.
40
42 The activity of CsrA is regulated by the csrB and csrC sRNAs. Expression of csrB and csrC is up-regulated by the BarA/UvrY two-component systems that responds to the presence of acetate-like molecules. Furthermore, the stringent response via (p)ppGpp and DksA up-regulates the expression of csrB and csrC while catabolic repression and glycolysis induce their degradation. Finally, the envelope stress response sigma factor RpoE positively regulates csrB and csrC expression.
Although regulation of CsrA activity has been extensively characterized, regulation of csrA expression at the transcriptional level is poorly characterized. The promoter region of the csrA gene is composed of 5 different promoters recognized by the general stress response sigma factor RpoS (σS) or the house keeping sigma factor RpoD (σ70). In addition, the DksA
transcription factor positively controls the activity of the main promoter of csrA. However, the presence of 5 promoters suggests the existence of differential regulations.
The roles of csrA in the metabolism as well as its implication in the envelope homeostasis suggest that csrA expression is responding to variations of the environmental conditions. In Escherichia coli and other bacteria, the detection of environmental signals is mainly due to TCS. TCS are composed of a sensor and of a response regulator. The sensor detects specific signals and activates the response regulator through its kinase activity. The phosphorylated response regulator controls the expression of its target genes and thus induces the adapted response to the stress.
44
46
Phenotypic screen on two component
systems
The CsrA-associated phenotypes
The mutation or deletion of csrA leads to the modification of different phenotypes (see 'introduction'). Motility, biofilm formation and glycogen accumulation are affected in a csrA::kan mutant and can be easily observed. The simultaneous modifications of the 3 phenotypes would be a strong indication of csrA-dependence and indicate a modification of csrA expression or CsrA activity. The TCS BarA/UvrY as well as the double mutant ΔcsrB ΔcsrC were used as controls as they are characterized regulators of CsrA activity. We choose TCS related to pathways regulated by CsrA, such as DcuS/R that regulates the fumarate anaerobic respiration, and EvgS/A that regulates virulence through the activation of PhoP/Q, as well as TCS related to envelope stresses (CpxR/A, BaeS/R, RcsCDAB and EnvZ/OmpR) (table 1).
Table 1: List of two component systems that will be screened
TCS sensors and regulators as well as induction signals and general response pathways are indicated.
Mutations of the TCS affects the cell motility
47 However, the deletion of barA or uvrY leads to an increase of the motility ring size. Interestingly, the ΔuvrY strain is less motile than the ΔbarA (figure 7). Similar result is observed with the ΔcpxR and ΔcpxA strains, both presenting a larger ring of motility as compared to the WT strain with the CpxA mutant being more motile than the CpxR mutant (figure 7). Similarly, the ΔphoP and ΔphoQ mutants present an increased motility but ΔphoP strain is more motile than the mutant of the cognate sensor-kinase. The ΔevgS and ΔevgA mutant strains show wide motility rings. The ΔenvZ, ΔdcuR and ΔbaeR mutants also present a wider motility ring than the WT strain. Interestingly, the ΔrcsA strain does not show any significant difference in motility compared to the WT strain (figure 7).
Figure 7: E. coli motility is affected by TCS
Motility was estimated via the extent of the motility ring. Pictures shown present representative results of at least three independent experiments.
Glycogen accumulation is affected in some TCS mutants
48 related to the envelope stresses detection, with the exceptions of the ΔcsrB ΔcsrC and ΔevgS strains that are also affected.
Figure 8 : Glycogen accumulation is affected by the TCS
The glycogen accumulation was observed thanks to a specific coloration. Photos show representative results of at least 3 independent experiments.
TCS affect biofilm formation
49
Figure 9: Biofilm formation is reduced by the deletion of TCS
Biofilm formation was measured as described in Materials and methods. Biofilm formation of the mutant strains was normalized to the WT biofilm formation. The graph represents the mean of at least 3 independent experiments. The error bars represent the standard deviation.
Conclusions of the phenotypic screen
Altogether, our results indicate that mutations of the TCS affect negatively biofilm formation, which is the opposite effect to what is known for the csrA::kan mutant (table 2). Similarly, the motility is increased in the TCS mutant strains inversely to a csrA::kan mutant (table 2). The glycogen accumulation is negatively affected in 5 mutant strains while it is positively affected in a csrA::kan mutant strain (table 2). Our results show that the double mutant ΔcsrB ΔcsrC and ΔbarA ΔuvrY affect 2 phenotypes out of the 3 tested. We thus used modification of at least 2 phenotypes as criteria for selecting potential candidates involved in regulation of csrA expression. The ΔrcsA mutant is behaving like the WT strain for the 3 different assays, indicating that the TCS RcsCDAB does not regulate csrA expression. DcuR/S only affects motility and thus is not a good candidate for csrA regulation. We decided to focus our work on the 4 mutants that affect all 3 phenotypes, ΔcpxR, ΔcpxA, ΔbaeR and ΔenvZ.
Beta-galactosidase assays reporting promoter activity of csrA, csrB and
csrC partially confirm the screen results
50 were cloned upstream of the lacZ gene in a medium-copy plasmid. For the pcsrA::lacZ fusion, a 500 bp fragment containing the described promoters of csrA was used. The natural RBS was replaced by a RBS consensus sequence to ensure detection of LacZ activity. For the pcsrB::lacZ and pcsrC::lacZ fusions, 500bp fragments containing the proposed promoter of csrB or csrC were used. As csrB and csrC are sRNAs, a RBS consensus sequence was added downstream (22nt and 25 nt respectively) of the transcription start site.
We measured LacZ activity of the 3 different fusions in the ΔcpxR, ΔcpxA, ΔbaeR, ΔenvZ mutants. We also measured the LacZ activity of the csrB and csrC fusions in strains deleted for barA or uvrY.
Table 2: Phenotypes affected by the different TCS deletions
Phenotypes not affected in the mutant strains as compared to the WT strain are indicated (0). For loss of motility, adhesion, and glycogen accumulation, phenotypes are indicated (---), increase of motility, adhesion and glycogen accumulation is indicted as (+++).
51 Figure 11A shows that the different strains are growing in a similar manner in LB medium. The LacZ activity of the pcsrB::lacZ promoter activity is strongly decreased in the ΔbarA and ΔuvrY strains as compared to the WT strain (figure 11B). In these mutant strains, the LacZ activity of the pcsrB::lacZ was mainly affected during stationary phase (figure 11C)., In our preliminary experiments, the LacZ activity of the pcsrB::lacZ was not affected in the ΔcpxR, ΔcpxA, ΔbaeR, ΔenvZ mutants (data not show).
The LacZ activity of the pcsrA::lacZ and pcsrB::lacZ fusions in the ΔbaeR strain was comparable to that in the WT strain (figures 11D and E). The LacZ activity of the pcsrA::lacZ and pcsrB::lacZ fusions in the ΔenvZ strain showed a decrease after 3 and 5 hours of growth respectively (figure 11B and F). Surprisingly, in our preliminary experiments, the LacZ activity of the pcsrA::lacZ and pcsrB::lacZ fusions in a strain deleted for ompR, the response regulator of envZ was similar to that of the WT strain (figure 11D and E).
In the ΔcpxR strain the LacZ activity of pcsrA::lacZ fusion show a decrease after 3 hours of growth (figure 13A). Moreover, a strain deleted for CpxA shows an increase in the LacZ activity of the pcsrA::lacZ fusion between 3 and 7 hours of growth (figure 13B).
As a conclusion, our data indicate that BaeR does not control the promoter activity of the csrA, csrB and csrC genes, indicating that the phenotype modifications observed in the previous section are csrA-independent.
52
Figure 11 : Implication of TCS in the expression of the csr genes
(A) The growth curve of the WT (square), ΔenvZ (diamond), ΔompR (plus), ΔbarA (cross) and ΔuvrY (circle), ΔbaeR (triangle) are presented as a function of time. LacZ activities are normalized to OD600nm
