Regulation of phosphate starvation-specific responses in Escherichia coli.

Toxic agents added into the medium of rapidly growing Escherichia coli induce specific stress responses through the activation of specialized transcription factors. Each transcription factor and downstream regulon (e.g. SoxR) are linked to a unique stress (e.g. superoxide stress). Cells starved of phosphate induce several specific stress regulons during the transition to stationary phase when the growth rate is steadily declining. Whereas the regulatory cascades leading to the expression of specific stress regulons are well known in rapidly growing cells stressed by toxic products, they are poorly understood in cells starved of phosphate. The intent of this review is to both describe the unique mechanisms of activation of specialized transcription factors and discuss signalling cascades leading to the induction of specific stress regulons in phosphate-starved cells. Finally, I discuss unique defence mechanisms that could be induced in cells starved of ammonium and glucose.

Rapid evolution of acetic acid-detoxifying Escherichia coli under phosphate starvation conditions requires activation of the cryptic PhnE permease and induction of translesion synthesis DNA polymerases.

Escherichia coli incubated in phosphate-limiting minimal medium dies during prolonged incubation as a result of the production of acetic acid. Variants that consume acetic acid generally sweep through the population after three serial cultures. Evolvability may primarily result from induction of the potentially mutagenic LexA DNA damage response or from growth of preexisting mutants. Cells starved of phosphate induce the LexA regulon through a unique mechanism based on an increase in the internal pH at the approach of the stationary phase. Evolved cells resume growth on phosphorylated products as a result of the activation of the cryptic PhnE permease. Here, it is shown that first PhnE-expressing revertants swept through starved populations independently of the expression of the LexA regulon. Induction of the LexA regulon and especially of the translesion synthesis DNA polymerases Pol IV and Pol V was, however, absolutely required for the ultimate evolution of acetic acid-detoxifying mutant strains. Both growth under selection and induction of translesion synthesis DNA polymerases are therefore required for adaptive evolution under phosphate starvation conditions.

Characterization of acetic acid-detoxifying Escherichia coli evolved under phosphate starvation conditions.

BACKGROUND: During prolonged incubation of Escherichia coli K-12 in batch culture under aerobic, phosphate (Pi) starvation conditions, excess glucose is converted into acetic acid, which may trigger cell death. Following serial cultures, we isolated five evolved strains in two populations that survived prolonged incubation. METHODS: We sequenced the genomes of the ancestral and evolved strains, and determined the effects of the genetic changes, tested alone and in combination, on characteristic phenotypes in pure and in mixed cultures. RESULTS: Evolved strains used two main strategies: (1) the constitutive expression of the Trk- and Kdp-dependent K(+) transport systems, and (2) the inactivation of the ArcA global regulator. Both processes helped to maintain a residual activity of the tricarboxylic acid cycle, which decreased the production of acetic acid and eventually allowed its re-consumption. Evolved strains acquired a few additional genetic changes besides the trkH, kdpD and arcA mutations, which might increase the scavenging of organophosphates (phnE (+), lapB, and rseP) and the resistance to oxidative (rsxC) and acetic acid stresses (e14(-)/icd (+)). CONCLUSIONS: Evolved strains rapidly acquired mutations (phnE (+) lapB rpoS trkH and phnE (+) rseP kdpD) that were globally beneficial to growth on glucose and organophosphates, but detrimental to long-term viability. The spread of these mutant strains might give the ancestral strain time to accumulate up to five genetic changes (phnE (+) arcA rsxC crfC e14(-)/icd (+)), which allowed growth on glucose and organophosphates, and provided a long-term survival. The latter strain, which expressed several mechanisms of protection against endogenous and exogenous stresses, might provide a platform for producing toxic recombinant proteins and chemicals during prolonged incubation under aerobic, Pi starvation conditions.

Protective role of the RpoE (σE) and Cpx envelope stress responses against gentamicin killing of nongrowing Escherichia coli incubated under aerobic, phosphate starvation conditions.

The viability of Escherichia coli starved of nitrogen (N) or phosphorus (P) decreased by up to seven orders of magnitude during prolonged incubation under aerobic conditions when exposed to high levels of the antibiotic gentamicin, whereas viability of cells starved of carbon (C) was barely affected. However, the initial rate of killing was lower for P-starved cells than for N-starved cells. The transient resistance of P-starved cells was partially dependent upon the expression of the phosphate (Pho) and Cpx responses. Constitutive activity of the Cpx and RpoE (σ(E)) envelope stress regulons increased the resistance of P- and N-starved cells. The level of expression of the RpoE regulon was fourfold higher in P-starved cells than in N-starved cell at the time gentamicin was added. Gentamicin killing of nongrowing cells may thus require ongoing aerobic glucose metabolism and faulty synthesis of structural membrane proteins. However, membrane protein damage induced by gentamicin can be eliminated or repaired by RpoE- and Cpx-dependent mechanisms pre-emptively induced in P-starved cells, which reveals a novel mechanism of resistance to gentamicin that is active in certain circumstances.

Transcriptional and preliminary functional analysis of the six genes located in divergence of phoR/phoP in Streptomyces lividans.

Streptomyces lividans senses and adjusts to a situation of Pi limitation via the expression of genes of the pho regulon controlled by the two-component system PhoR/PhoP. Interestingly, an in silico analysis of the proteins encoded by the six genes located in divergence of phoR/phoP revealed that the latter bear features often found in metalloproteins involved in the sensing/resistance to oxidative stress. We determined whether genes of this region were belonging to the pho regulon and whether the encoded proteins do play a role in the resistance to oxidative stress. For this purpose, a transcriptional analysis of these genes was carried out on the carbon and nitrogen rich medium R2YE and on a minimal medium (MM). On R2YE, the expression of the genes phoU to sco4225 was much higher than on MM and constant throughout growth. On this medium, the expression of phoU was totally PhoP-dependent whereas the expression of sco4227 and sco4226 was partially PhoP-dependent, taking place from the phoU promoter region. In contrast, on MM, the expression of sco4227 and sco4226 was PhoP-independent whereas that of phoU remained PhoP-dependent and showed, as phoR/phoP, a peak of expression at 48 h. sco4225, sco4224, and sco4223 were transcribed from their own promoter independently of PhoP in both media. The mutants of five out of six genes of the region (Δsco4226 mutant could not be obtained) grew poorly in the presence of exogenous oxidants, suggesting a role of the encoded proteins in the resistance to oxidative stress, especially on the rich medium R2YE.

Activation of the cryptic PhnE permease promotes rapid adaptive evolution in a population of Escherichia coli K-12 starved for phosphate.

Escherichia coli K-12 suffers acetic acid stress during prolonged incubation in glucose minimal medium containing a limiting concentration of inorganic phosphate (0.1 mM P(i)), which decreases the number of viable cells from 6 × 10(8) to ≤10 CFU/ml between days 6 and 14 of incubation. Here we show that following two serial transfers into P(i)-limiting medium, evolved mutants survived prolonged incubation (≈10(7) CFU/ml on day 14 of incubation). The evolved strains that overtook the populations were generally PhnE(+), whereas the ancestral K-12 strain carries an inactive phnE allele, which prevents the transport of phosphonates. The switching in phnE occurred with a high frequency as a result of the deletion of an 8-bp repeated sequence. In a mixed culture starved for P(i) that contained the K-12 ancestral strain in majority, evolved strains grew through PhnE-dependent scavenging of probably organic phosphate esters (not phosphonates or P(i)) released by E. coli K-12 between days 1 and 3, before acetic acid excreted by E. coli K-12 reached toxic levels. The growth yield of phnE(+) strains in mixed culture was dramatically enhanced by mutations that affect glucose metabolism, such as an rpoS mutation inactivating the alternative sigma factor RpoS. The long-term viability of evolved populations was generally higher when the ancestral strain carried an inactive rather than an active phnE allele, which indicates that cross-feeding of phosphorylated products as a result of the phnE polymorphism may be essential for the spread of mutants which eventually help populations to survive under P(i) starvation conditions.

Fe-S clusters, fragile sentinels of the cell.

Iron-sulfur (Fe-S) clusters are ubiquitous cofactors present in a myriad of proteins controlling processes as diverse as DNA replication, photosynthesis, respiration and gene regulation. Their assembly and delivery into apo-proteins are catalysed by different multi-protein systems conserved throughout prokaryotes and eukaryotes. Because so many cellular processes are dependent upon Fe-S proteins, alteration of the Fe-S clusters or of the systems that make them has profound impact on cellular physiology. The present review aims at covering and discussing those situations wherein these highly efficient redox sensitive cofactors turn from faithful sentinels into enfeebled assistants or, worse, into dangerous insiders.

Fur-dependent detoxification of organic acids by rpoS mutants during prolonged incubation under aerobic, phosphate starvation conditions.

The activity of amino acid-dependent acid resistance systems allows Escherichia coli to survive during prolonged incubation under phosphate (P(i)) starvation conditions. We show in this work that rpoS-null mutants incubated in the absence of any amino acid survived during prolonged incubation under aerobic, P(i) starvation conditions. Whereas rpoS(+) cells incubated with glutamate excreted high levels of acetate, rpoS mutants grew on acetic acid. The characteristic metabolism of rpoS mutants required the activity of Fur (ferric uptake regulator) in order to decrease the synthesis of the small RNA RyhB that might otherwise inhibit the synthesis of iron-rich proteins. We propose that RpoS (sigma(S)) and the small RNA RyhB contribute to decrease the synthesis of iron-rich proteins required for the activity of the tricarboxylic acid (TCA) cycle, which redirects the metabolic flux toward the production of acetic acid at the onset of stationary phase in rpoS(+) cells. In contrast, Fur activity, which represses ryhB, and the lack of RpoS activity allow a substantial activity of the TCA cycle to continue in stationary phase in rpoS mutants, which decreases the production of acetic acid and, eventually, allows growth on acetic acid and P(i) excreted into the medium. These data may help explain the fact that a high frequency of E. coli rpoS mutants is found in nature.

The lysine decarboxylase CadA protects Escherichia coli starved of phosphate against fermentation acids.

Conflicting results have been reported for the rate and extent of cell death during a prolonged stationary phase. It is shown here that the viability of wild-type cells (MG1655) could decrease >or=10(8)-fold between days 1 and 14 and between days 1 and 6 of incubation under aerobic and anaerobic phosphate (P(i)) starvation conditions, respectively, whereas the cell viability decreased moderately under ammonium and glucose starvation conditions. Several lines of evidence indicated that the loss of viability of P(i)-starved cells resulted primarily from the catabolism of glucose into organic acids through pyruvate oxidase (PoxB) and pyruvate-formate lyase (PflB) under aerobic and anaerobic conditions, respectively. Weak organic acids that are excreted into the medium can reenter the cell and dissociate into protons and anions, thereby triggering cell death. However, P(i)-starved cells were efficiently protected by the activity of the inducible GadABC glutamate-dependent acid resistance system. Glutamate decarboxylation consumes one proton, which contributes to the internal pH homeostasis, and removes one intracellular negative charge, which might compensate for the accumulated weak acid anions. Unexpectedly, the tolerance of P(i)-starved cells to fermentation acids was markedly increased as a result of the activity of the inducible CadBA lysine-dependent acid resistance system that consumes one proton and produces the diamine cadaverine. CadA plays a key role in the defense of Salmonella at pH 3 but was thought to be ineffective in Escherichia coli since the protection of E. coli challenged at pH 2.5 by lysine is much weaker than the protection by glutamate. CadA activity was favored in P(i)-starved cells probably because weak organic acids slowly reenter cells fermenting glucose. Since the environmental conditions that trigger the death of P(i)-starved cells are strikingly similar to the conditions that are thought to prevail in the human colon (i.e., a combination of low levels of P(i) and oxygen and high levels of carbohydrates, inducing the microbiota to excrete high levels of organic acids), it is tempting to speculate that E. coli can survive in the gut because of the activity of the GadABC and CadBA glutamate- and lysine-dependent acid resistance systems.

Diversion of the metabolic flux from pyruvate dehydrogenase to pyruvate oxidase decreases oxidative stress during glucose metabolism in nongrowing Escherichia coli cells incubated under aerobic, phosphate starvation conditions.

Ongoing aerobic metabolism in nongrowing cells may generate oxidative stress. It is shown here that the levels of thiobarbituric acid-reactive substances (TBARSs), which measure fragmentation products of oxidized molecules, increased strongly at the onset of starvation for phosphate (P(i)). This increase in TBARS levels required the activity of the histone-like nucleoid-structuring (H-NS) protein. TBARS levels weakly increased further in DeltaahpCF mutants deficient in alkyl hydroperoxide reductase (AHP) activity during prolonged metabolism of glucose to acetate. Inactivation of pyruvate oxidase (PoxB) activity decreased the production of acetate by half and significantly increased the production of TBARS. Overall, these data suggest that during incubation under aerobic, P(i) starvation conditions, metabolic flux is diverted from the pyruvate dehydrogenase (PDH) complex (NAD dependent) to PoxB (NAD independent). This shift may decrease the production of NADH and in turn the adventitious production of H(2)O(2) by NADH dehydrogenase in the respiratory chain. The residual low levels of H(2)O(2) produced during prolonged incubation can be scavenged efficiently by AHP. However, high levels of H(2)O(2) may be reached transiently at the onset of stationary phase, primarily because H-NS may delay the metabolic shift from PDH to PoxB.

Role of Escherichia coli RpoS, LexA and H-NS global regulators in metabolism and survival under aerobic, phosphate-starvation conditions.

It has been suggested that Escherichia coli can resist aerobic, glucose-starvation conditions by switching rapidly from an aerobic to a fermentative metabolism, thereby preventing the production by the respiratory chain of reactive oxygen species (ROS) that can damage cellular constituents. In contrast, it has been reported that E. coli cannot resist aerobic, phosphate (Pi)-starvation conditions, probably because of the maintenance of an aerobic metabolism and the continuous production of ROS. This paper presents evidence that E. coli cells starved for Pi under aerobic conditions indeed maintain an active aerobic metabolism for about 3 d, which allows the complete degradation of exogenous nutrients such as arginine (metabolized probably to putrescine via the SpeA-initiated pathway) and glucose (metabolized notably to acetate), but cell viability is not significantly affected because of the protection afforded against ROS through the expression of the RpoS and LexA regulons. The involvement of the LexA-controlled RuvAB and RecA proteins with the RecG and RecBCD proteins in metabolism and cell viability implies that DNA double-strand breaks (DSB), and thus hydroxyl radicals that normally generate this type of damage, are produced in Pi-starved cells. It is shown that induction of the LexA regulon, which helps protect Pi-starved cells, is totally prevented by introduction of a recB mutation, which indicates that DSB are actually the main DNA lesion generated in Pi-starved cells. The requirement of RpoS for survival of cells starved for Pi may thus be explained by the role played by various RpoS-controlled gene products such as KatE, KatG and Dps in the protection of DNA against ROS. In the same light, the degradation of arginine and threonine may be accounted for by the synthesis of polyamines (putrescine and spermidine) that protect nucleic acids from ROS. Besides LexA and RpoS, a third global regulator, the nucleoid-associated protein H-NS, is also shown to play a key role in Pi-starved cells. Through a modulation of the metabolism during Pi starvation, H-NS may perform two complementary tasks: it helps maintain a rapid metabolism of glucose and arginine, probably by favouring the activity of aerobic enzymes such as the NAD-dependent pyruvate dehydrogenase complex, and it may enhance the cellular defences against ROS which are then produced by increasing RpoS activity via the synthesis of acetate and presumably homoserine lactone.