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NEW EMBO MEMBERS' REVIEW: The black cat/white cat principle of signal integration in bacterial promoters

2001; Springer Nature; Volume: 20; Issue: 1 Linguagem: Inglês

10.1093/emboj/20.1.1

1460-2075

Ildefonso Cases,

Transgenic Plants and Applications

New EMBO Member's Review15 January 2001free access The black cat/white cat principle of signal integration in bacterial promoters Ildefonso Cases Ildefonso Cases Department of Microbial Biotechnology, Centro Nacional de Biotecnología CSIC, Campus de Cantoblanco, 28049 Madrid, Spain Search for more papers by this author Víctor de Lorenzo Corresponding Author Víctor de Lorenzo Department of Microbial Biotechnology, Centro Nacional de Biotecnología CSIC, Campus de Cantoblanco, 28049 Madrid, Spain Search for more papers by this author Ildefonso Cases Ildefonso Cases Department of Microbial Biotechnology, Centro Nacional de Biotecnología CSIC, Campus de Cantoblanco, 28049 Madrid, Spain Search for more papers by this author Víctor de Lorenzo Corresponding Author Víctor de Lorenzo Department of Microbial Biotechnology, Centro Nacional de Biotecnología CSIC, Campus de Cantoblanco, 28049 Madrid, Spain Search for more papers by this author Author Information Ildefonso Cases1 and Víctor de Lorenzo 1 1Department of Microbial Biotechnology, Centro Nacional de Biotecnología CSIC, Campus de Cantoblanco, 28049 Madrid, Spain *Corresponding author. E-mail: [email protected] The EMBO Journal (2001)20:1-11https://doi.org/10.1093/emboj/20.1.1 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Introduction ‘Black cat, white cat: whatever catches the mouse is a good cat’ (Chinese proverb). Throughout the 3000 million years that bacteria have been on planet Earth, they have evolved amazing mechanisms for rapid adaptation to every imaginable type of environmental change. To give one extreme example, as little as 50 years after the introduction of the first antibiotics, very few of these drugs remain effective today at combating microbial infections (Davies, 1997). When bacteria are faced with antimicrobials, the only challenge they must overcome is to defeat the toxic effect of the compound through modification, cleavage or pumping the drug out of the cells; these processes generally involve one or just a few proteins. A far more complex environmental threat is that posed by organic chemicals, which have been discharged into many ecosystems through industrial and urban activities. Many of these chemicals are xenobiotic compounds (literally, alien to life), which include types of chemical ligatures (typically covalent C–Cl bonds) that have never been present in significant amounts in the biosphere and against which the housekeeping metabolic pathways of most microbes are generally useless (van der Meer et al., 1992). Unlike antibiotics, the challenge in this case is the construction of entire biodegradation pathways that endow bacteria with the ability to grow on these otherwise unpalatable chemicals. Such an outcome involves not just one protein, but sometimes dozens, which must adapt to entirely new substrates and intermediates. However, difficult as this might be, the successful assembly of a degradation pathway does not guarantee per se the survival of a particular strain. Bacteria that colonize polluted sites are subject to extremely tough competition from other microbial residents of the same niche. Transcriptional regulation of biodegradative genes and operons thus becomes a critical asset for the success of a newly assembled pathway to ensure its expression at only the right moment with a minimal waste of energy (de Lorenzo and Pérez-Martín, 1996). But how do bacteria learn to respond optimally to novel environmental signals and substrates? Most of the known functional characteristics of prokaryotic promoters come from studies that use Escherichia coli as the test organism. Although far more complex than Buchnera (one of the simplest bacteria known so far; Shigenobu et al., 2000), the life cycle and the natural niches of E.coli can be relatively simple compared with those of not-so-distant relatives, such as pseudomonads, which thrive in soils polluted with toxic chemicals. Such rapidly adapting bacteria have become the experimental systems of choice in understanding how genes and pathways end up with regulated expression. In fact, since bacteria have been exposed to some such pollutants for only a few decades, it is possible, as discussed below, to find intermediate steps in the evolution process. In this review we summarize the features of the regulation of catabolic pathways for recalcitrant aromatic compounds that can help us to understand such a process. The conclusion is what we refer to as the ‘black cat/white cat principle’, which states that any regulatory mechanism is equally efficient provided that it ensures both a responsiveness to the new substrate and a suitable connection with the physiological state of the bacteria. A number of well studied cases to substantiate this notion are discussed below. Transcriptional noise: promoters learning to respond to novel chemicals The regulation of pathways for biodegradation of recalcitrant compounds by Gram-negative soil bacteria (mostly Pseudomonas, Alcaligenes, Bulkholderia and Acinetobacter) reveal some interesting mechanistic features by which operons acquire conditional promoters (Díaz and Prieto, 2000). The functioning of a new route depends on two major requirements that bacteria must attain to utilize the evolutionary advantage offered by the presence of fresh chemical species as potential carbon sources. One is the complement of genes encoding the whole suite of enzymes that build a pathway of reactions leading to metabolism of the compound to cardon dioxide and water. Operons destined for work in polluted sites need, in addition, an efficient transcriptional control system (de Lorenzo and Pérez-Martín, 1996). Regulated promoters are the key element that permit catabolic operons to be transcribed only when required and at levels adequate to guarantee a satisfactory metabolic return from the substrate. These two steps (assembly of a catabolic operon and acquisition of a substrate-responsive promoter) seem to be independent events, governed by different rules that operate at various times. In general, isoenzymes that catalyse similar steps within a pathway tend to be alike at the sequence (DNA, protein) level also, even in cases where the initial substrates of the pathway are very different. This allows us to trace the origin of novel pathways to the patchwork assembly of pre-existing DNA segments bearing gene variants active on the novel substrates (van der Meer et al., 1992). Excellent examples of this include the adaptation of the entire set of catabolic genes of Pseudomonas putida for degradation of benzoate (involving the ben and cat genes) [Cl-benzoates (Parsek et al., 1994) or methyl-benzoates (van der Meer et al., 1992; Ramos et al., 1997)] or the recruitment of at least three catabolic segments for degradation of the very recalcitrant compound pentachlorophenol (Copley, 2000). There are no rules, however, to anticipate the type of regulator that may appear to respond to a novel chemical structure. New pathways generally start with low-level constitutive expression, on top of which increasingly specific promoters might develop. In contrast to the assembly of the enzymatic pathways, there is no specific requirement for a given type of regulator; on the contrary, it is common to find nearly identical catabolic operons preceeded by entirely different regulatory devices and proteins (de Lorenzo and Pérez-Martín, 1996). The bottom line is that a novel specificity may evolve through the leakiness of an earlier transcriptional control scheme (i.e. transcriptional noise). Any new control system should therefore start by recruiting the residual responsiveness of an already existing promoter/regulator to another signal, and then evolve by selecting changes such that residual responsiveness becomes predominant. As a consequence, the process from constitutive expression to response to a single inducer involves intermediate steps with various degrees of specificity, which can be found as remnants of the process in many catabolic pathways. Table I shows a number of cases that have been examined in detail. They include gratuitous induction (responsiveness to non-metabolizable compounds), cross-talk between structurally, but not functionally, related regulators, full replacement of one factor by another, or promiscuous activation among regulators of the same family. Pathways found in bacteria able to degrade substrates only very recently found in the environment (e.g. pentachlorophenol, polychlorobiphenyls, hexachlorocyclohexane, nitroaromatics, etc.) are often poorly regulated (Mouz et al., 1999; Copley, 2000; Watanabe et al., 2000), probably reflecting only an early step in the optimization of the corresponding metabolic route. Table 1. Levels of regulatory noise in catabolic promoters Phenomenon Examples Reference Minor/major gratuitous induction Induction of the upper TOL pathway by o-xylene Abril et al. (1989) Induction of the lower NAH pathway by anthranylic acid Cebolla et al. (1997) Induction of the alk pathway by DCPK van Beilen et al. (1994) Induction of the lower TOL pathway by 2Br-benzoate Ramos et al. (1986) Residual induction of σ54-dependent promoters by largecollections of non-substrates Abril et al. (1989);Garmendia and de Lorenzo (2000);Kahng et al. (2000);Jaspers et al. (2000) Cross-talk between akin regulators Benzoate-responsive activation of the Pm promoter of thelower TOL pathway in the absence of XylS Kessler et al. (1994) Activation of the tfd gene cluster of Ralstonia eutrophaJMP134 by TfdR in the absence of TfdT Leveau and van der Meer (1996) Activation of clc genes by CatR in the absence of cognateregulator ClcR in P.putida AC27 Parsek et al. (1994) Cross-regulation of toluene monooxygenases by TbmR andTbuT in Ralstonia pickettii PKO1 Leahy et al. (1997) Regulatory takeover between relatedregulators Phenol-dependent activation of upperTOL operon by DmpRand toluene-dependent activation of the dmp operon by XylR Fernández et al. (1994) Activation of the pheBA promoter of P.putida PaW85 by CatR Parsek et al. (1995) Promiscuous activation Activation from solution of σ54-dependent promoters byvarious regulators of the NtrC family Pérez-Martín and de Lorenzo (1995) Physiological control of transcription: the phenomenon Bacterial regulation of catabolic pathways in the environment implies not just the ability to respond to a substrate, but also whether or not expression of the whole complement of enzymes is beneficial or detrimental to ecological performance. Bacteria thriving in a polluted niche receive a range of physical and chemical signals, other than just the presence of a substrate, which need to be processed to achieve a positive or negative outcome for each specific promoter (Cases and de Lorenzo, 1998). Such signals include nutrient availability, but also osmolarity, temperature, chaotropic agents, contact with surfaces, and interactions with other microorganisms. Under such tough conditions, it is of essence that expression of biodegradative operons becomes tightly coupled to the physiological and metabolic state of the cells. Figure 1 shows the various types of response found in promoters that drive expression of catabolic promoters. We would predict that biodegradative operons evolve from constitutive expression to substrate-responsive and metabolically controlled transcription. In this respect, pathways found in bacteria able to degrade man-made xenobiotic compounds, either totally or partially, frequently display a range of suboptimal, non-regulated expression profiles. In contrast, biodegradation of compounds that, despite being recalcitrant, have been available to bacteria for a long time is controlled through promoters endowed with sophisticated facets to ensure the processing of substrate-specific and general physiological signals. Typical promoters of this type are those that drive biodegradation of BTEX components of petroleum (benzene, toluene, ethylbenzene, xylenes), styrene, n -alkanes, or side-products of wood decay such as phenylacetate, phenols and benzoates (Díaz and Prieto, 2000). Some chloro-aromatic compounds (Cl-benzoates, Cl-catechols) may also be of natural origin, thus the cognate catabolic pathways frequently show responsiveness to both the substrate and the metabolic state. Figure 1.Expression profiles and development of promoters responding to novel environmental signals (e.g. latest carbon sources). Expression of genes and gene clusters encoding new catabolic abilities towards an evolutionarily recent substrate may be simply achieved through constitutive expression (type 1). This may evolve further into inducible expression (type 2). The presence of other nutrients in the medium that are easier to metabolize can later influence such induction and cause a C-source inhibition (type 3). Finally, both the presence of other nutrients, growth rate, and other environmental and physiological signals can be integrated for downregulation of the promoter during rapid growth, causing the so-called exponential silencing (type 4). Download figure Download PowerPoint Table II shows a number of examples where the presence of a physiological control of biodegradative pathways has been observed as something superimposed on the substrate-dependent expression of the catabolic genes. Some of these will be examined in detail below. One typical environmental factor is the presence in the same niche of alternative carbon sources, the preferential consumption of which must be decided. This may or may not be related to the overall growth rate and growth phase, a major origin of signals that can be exploited for adjustment of promoter output. The most frequent induction pattern of highly evolved catabolic promoters is that referred to as type 4 in Figure 1, which exhibits a phenomenon termed ‘post-exponential induction’ or ‘exponential silencing’. Regardless of the mechanisms involved (discussed below), such conduct consists of a lack of transcriptional activity while bacteria grow rapidly on a nutrient-rich media, irrespective of the presence of the effector. This is then followed by rapid induction of the promoter once the growth rate of the bacteria decreases, they enter stationary phase, or cease to grow altogether. Effector-triggered post-exponential induction requires both the presence of a given effector and that cells reach a specific physiological stage. Such a stage might be defined not only by the depletion of a limiting nutrient or growth factor, but also by a particular overall energy state. To detect such a metabolic and physiological condition, and to connect it to the transcription of specific catabolic promoters, bacteria have evolved a diverse array of molecular artifices. Table 2. Biodegradative pathways subject to physiological control in pseudomonads Pathway/operon and phenotype Substrate Reference Degradation of the hydrocarbon by P.putida CA3 is inhibited by glutamate and citrate,not by glucose. styrene O'Connor et al. (1995) Degradation by P.fluorescensST subjected to inhibition by glucose, acetate andglutamate, and down-regulated by succinate and lactate. Santos et al. (2000) Metabolic integration of 3 operons for catabolism of the aromatic substrate in afunctional unit (catabolon) of P.putida U. phenyl acetate Olivera et al. (1998) Succinate inhibits consuption of the aromatic hydrocarbon in P.putida ML2. benzene Mason (1994) Benzoate inhibits catabolism of phenol and acetate by Ralstonia eutropha. Succinateimpairs benzoate consumption by blocking one key enzyme for its metabolism. phenol, benzoate Ampe and Lindley (1995);Ampe et al. (1997, 1998) Alk pathway of the OCT plasmid of P.oleovorans is inhibited in rich media and rapidgrowth. Repressed by C-sources. n-alkanes Yuste et al. (1998);Staijen et al. (1999) TOL pathway of plasmid pWW0 of P.putida mt2. Repressed by rich media, rapidgrowth and some carbohydrates (i.e. glucose). toluene, m-xylene,p-xylene Caseset al. (1996); Marqués et al. (1994); Duetz et al. (1994, 1996, 1997);Du et al. (1996) Activity of the PnahG promoter of P.fluorescens HK44 is down-regulated by richmedia, glucose and toluene. naphthalene, salicylate Heitzer et al. (1994) Catabolism of the aromatic substrate by P.fluorescens CA4 is inhibited by glutamate,but not by glucose or citrate. ethyl benzene Corkery et al. (1994) clc pathway of P.putida AC27 down regulated by fumarate through inhibition of theregulator ClcR. 3Cl-benzoate McFall et al. (1997, 1998) Downregulation of the metabolism of the xenobiotic compound by glutamate, glucoseand cellobiose in a Flavobacterium strain. pentachlorophenol Topp et al. (1988); Topp and Hanson (1990) DMP pathway of plasmid pVI150 of Pseudomonas sp. CF600. Inhibited by rich mediaand C substrates allowing fast growth. phenol, methyl-phenols Sze et al. (1996) Assets for physiological control of catabolic operons Unlike their eukaryotic counterparts, which seem to have a large number of transcription factors and controllable steps available to them, prokaryotic promoters have a very limited number of potential targets to integrate transcriptional co-regulation elements, namely specific regulators, sigma factors and promoter DNA. Such a paucity of molecular instruments has, however, been maximally combined and exploited with remarkable success. Of the many examples known (Table II) in which biodegradative pathways are subject to physiological regulation, the mechanisms involved have been studied to a significant degree in only a few instances. The diversity of regulatory strategies resulting in the same eventual phenotype is demonstrated in the cases that follow. Metabolites that inhibit regulators A remarkable example of how the transcriptional regulators of catabolic promoters can be subdued to the overall carbon metabolic and energetic status of cells is provided by the control of 3Cl-benzoate degradation in some strains of P.putida. The ortho-cleavage pathways of catechol and 3Cl-catechol are central catabolic pathways of P.putida that convert aromatic and chloroaromatic compounds (such as benzoate and 3Cl-benzoate) to tricarboxylic acid (TCA) cycle intermediates (McFall et al., 1997, 1998). They are encoded by the evolutionarily related catBCA and clcABD operons, respectively. Expression of the cat and clc operons requires the LysR-type transcriptional activators CatR and ClcR, and the inducer molecules cis,cis-muconate and 2Cl-cis,cis-muconate. Although the core transcriptional activation mechanisms of CatR and ClcR have been conserved in response to the presence of inducer, nature has provided some flexibility to respond to physiological signals. Transcriptional fusion studies demonstrated that the expression from the clc promoter is repressed when the cells are grown on succinate, citrate or fumarate, and that this repression is ClcR-dependent and occurs at the transcriptional level. The presence of these organic acids did not affect the expression from the cat promoter. In vitro transcription assays demonstrate that the TCA cycle intermediate, fumarate, directly and specifically inhibits the formation of the clcA transcript. No such inhibition was observed when CatR was used as an activator on either the cat or the clc template. Since both the catechol and the Cl-catechol pathways feed into the TCA cycle, but only the Cl-catechol pathway is inhibited by fumarate, there is a subtle difference in the regulation of these two pathways, where intracellular sensing of a TCA cycle intermediate leads to a reduction of chloroaromatic degradation. Titration studies of fumarate and 2-chloromuconate in vitro transcription assays show that the fumarate effect is concentration-dependent and reversible, indicating that fumarate and 2-chloromuconate most probably compete for the same binding site on ClcR (McFall et al., 1997, 1998). This is an interesting example of the transcriptional regulation of a biodegradative pathway through the sensing of the levels of one key metabolite of the TCA cycle. Unsophisticated as it may appear, this type of metabolic downregulation of a xenobiotic-degrading pathway (clc) caused by a side-metabolite from a substrate that is easier to consume is probably very frequent, since just a few mutations in the targeted protein makes it amenable to a degree of physiological control. Parasitizing sigma factors The general transcription machinery can also be used by degradation pathways to couple expression of biodegradative operons with different physiological signals. One remarkable example is the TOL plasmid of P.putida mt-2 for degradation of toluene, in which the interplay of two promoters, two regulators and four sigma factors provide a very efficient control mechanism. Pseudomonas putida cells harbouring the TOL plasmid pWW0 are able to grow on toluene and m-/p-xylene as the only carbon source, owing to expression of a two-step pathway for the complete mineralization of these hydrocarbons (Ramos et al., 1997). The first step (Figure 2A) involves the biotransformation of toluene/xylenes to their corresponding carboxylic acids through oxidation of one methyl group of the aromatic substrate. The second stage channels the benzoate (or toluate) into the Krebs' cycle. This follows a complex pathway summarized in Figure 2A. The biochemical steps are reflected in two separate transcriptional units, the so-called upper operon (encoding enzymes for oxidation of the methyl group of toluene) and the lower (or meta) operon (responsible for the aromatic ring fission leading to pyruvate and acetaldehyde). Expression of the xyl genes is tightly regulated through a complex cascade of transcriptional controls (Ramos et al., 1997) that involve two regulators, the XylR and XylS proteins. These are responsible for the activation of the upper and meta operons, respectively, thus ensuring optimal expression of the degradative activities only in the presence of pathway substrates (Figure 2A). Figure 2.Organization of the TOL and dmp biodegradation pathways and their cognate Pu and Po promoters. (A) The regulatory cascade of the xyl genes in the TOL plasmid pWW0 of P.putida mt-2. In the presence of upper pathway substrates like m-xylene, the upper-operon promoter Pu and the xylS promoter Ps are activated by XylR in combination with the σ54-containing RNA polymerase (σ54-RNAP). Subsequently, an excess of XylS product or XylS bound to its effectors (i.e. substrates of the meta pathway) activate Pm. There is no physical continuity between the upper and the meta operons. Below the scheme of the pathway, the Pu promoter region is expanded, showing the boundaries of relevant DNA sequences: upstream binding sites (UAS) for XylR, the −12/−24 sequences recognized by σ54-RNAP, and a single IHF binding site located within the intervening region. (B) Regulation of the pVI150-encoded dmp-operon of Pseudomonas sp. CF600. The dmpR gene product that is responsive to phenol and cresols activates transcription of the divergently transcribed dmp-operon from the Po promoter. A subset of the dmp genes are involved in phenol hydroxylation, while the rest encode enzymatic activities of the meta-cleavage pathway for dissimilation of the catechol intermediate. The Po promoter region is expanded below the scheme of the dmp pathway. Relevant portions of the sequence are pinpointed. Download figure Download PowerPoint The Pm promoter (also called OP2), which drives expression of the lower operon for metabolism of benzoate and toluates all the way to the TCA cycle intermediates, is expressed at a high level throughout the growth curve (Marqués et al., 1995). This transcription is dependent on the positive activator XylS (of the AraC family of prokaryotic regulators) activated by 3-methyl benzoate. Although from just inspecting the DNA sequence this promoter would be predicted to be dependent on the housekeeping sigma factor σ70, recent observations (Marqués et al., 1995, 1999; Miura et al., 1998) have revealed a most intricate sigma succession mechanism that ensures continuous Pm activity throughout all stages of growth. First, it appears that thermosensitive rpoD mutants that transiently lack σ70 are still able to support Pm activity at the non-permissive temperature, thereby suggesting that other sigmas may actually drive promoter activity. In fact, it could be shown that it is the heat shock factor σH, rather than σ70, that is required for Pm output following induction with 3-methyl benzoate. The surprising finding is that σH levels are generally very low unless a signal triggering the heat shock response occurs. When cells are challenged with the aromatic effector, however, it does trigger such a response, probably due to its effects on membrane properties. This is true mostly for cells that are exposed to 3-methyl benzoate (Marqués et al., 1999). When cells enter stationary phase, the starvation sigma σS seems to take over and replace σH as the factor that directs Pm activity. It thus seems that activation of Pm transcription is achieved through a switch between two stress-responsive factors: σH in exponential phase and σS in stationary phase. In both cases, Pm is dependent on the same activator, XylS, and starts transcription in the same point. The Pm/XylS system reveals a strategy of coupling transcription of a specific promoter to the cell physiology by ‘choosing’ general stress signals mediated by sigma factors. Since the whole TOL system is plasmid encoded, it is remarkable how expression of the biodegradation functions is the result of an interplay, if not a parasitism, of general host factors with system-specific, plasmid-encoded regulators. The σ54 promoter Pu of the TOL plasmid In 1986, a now classic paper (Dixon, 1986) reported that the Pu promoter of upper operon of the same TOL plasmid pWW0 of P.putida mt-2 mentioned above (Figure 2A) had features that made its expression dependent on the ntrA gene. This gene was later identified as the determinant of the sigma factor for nitrogen metabolism and thus called σN (or, more frequently, σ54; Merrick, 1993). Since then, the σ54-dependent promoter Pu has become a landmark for studies on the regulation of biodegradative pathways. Such extensive work has yielded detailed knowledge on both specific effector-mediated regulation and the devices that couple its performance in vivo to cell physiology. Pu is regulated by the XylR protein, which belongs to the family of prokaryotic enhancer-binding activators that act in concert with σ54 (Morett and Segovia, 1993; Shingler, 1996). In the presence of toluene, xylenes and other structural analogues, the XylR protein activates the Pu promoter of the upper TOL operon (Figure 2A), using a mechanism that is generally shared by other activators of the family. This involves the binding of the regulator to upstream activating sequences (UAS) and the looping-out of the complex into close proximity with the σ54-containing form or RNA polymerase bound to the −12/−24 region of the promoter (Morett and Segovia, 1993; North et al., 1993). This event is assisted by the presence of an integration host factor (IHF)-binding site at the intervening region between the UAS and the σ54-RNAP attachment site (Pérez-Martín and de Lorenzo, 1996a, b). Such an elaborate promoter architecture (Figure 2A) seems to be particularly well suited to integrating a repertoire of environmental signals. From the early studies on the regulation of this promoter, it became evident that expression of the upper TOL operon was inhibited when cells grew exponentially in rich medium (Hugouvieux-Cotte-Pattat et al., 1990; de Lorenzo et al., 1993). This effect seemed not to require the activity of the whole complement of TOL genes, since it could be faithfully reproduced with only the regulatory elements that control transcriptional activity of Pu. Pseudomonas putida cells devoid of the TOL plasmid carrying a chromosomal insertion of the xylR gene and a Pu–lacZ fusion were unable to accumulate β-galactosidase when growing exponentially in Luria–Bertani (LB) medium, regardless of the presence or absence of m-xylene (de Lorenzo et al., 1993). However, as soon as the cells leave the exponential growth phase and enter the stationary phase, the same Pu–lacZ fusion becomes extremely responsive to the aromatic inducer (de Lorenzo et al., 1993; Cases et al., 1996). The data of the reporter fusion match faithfully the quantitative S1 protection assays with mRNA from induced cells, so the effect certainly occurs at the transcriptional level (Marqués et al., 1994). Since Pu is functional in vitro simply by mixing purified and pre-activated XylR with σ54-containing RNAP and IHF (Pérez-Martín and de Lorenzo, 1996b), it is clear that additional elements, when induced, adjust transcription to the physiological state that governs the cells. These initial observations triggered a large number of studies on the mechanisms involved in such a physiological inhibition of Pu activity. Various reports from different perspectives have documented that Pu activity is downregulated in response to exponential growth in rich media, a phenomenon referred to as catabolite repression (Duetz et al., 1994, 1996, 1997; Holtel et al., 1994; Marqués et al., 1994), stationary-phase dependency (Hugouvieux-Cotte-Pattat et al., 1990) or, as we prefer to call it, exponential silencing (Cases et al., 1996) (Figure 1). At least in part, this effect can be traced to modulation of the activity of the sigma factor iself, because its overproduction shifts Pu derepression to an earlier growth stage (Cases et al., 1996). In addition, ftsH mutants of