Copper tolerance mediated by polyphosphate degradation and low-affinity inorganic phosphate transport system in Escherichia coli
© Grillo-Puertas et al.; licensee BioMed Central Ltd. 2014
Received: 26 November 2013
Accepted: 14 March 2014
Published: 19 March 2014
Metal tolerance in bacteria has been related to polyP in a model in which heavy metals stimulate the polymer hydrolysis, forming metal-phosphate complexes that are exported. As previously described in our laboratory, Escherichia coli cells grown in media containing a phosphate concentration >37 mM maintained an unusually high polyphosphate (polyP) level in stationary phase. The aim of the present work was to evaluate the influence of polyP levels as the involvement of low-affinity inorganic phosphate transport (Pit) system in E. coli copper tolerance.
PolyP levels were modulated by the media phosphate concentration and/or using mutants in polyP metabolism. Stationary phase wild-type cells grown in high phosphate medium were significantly more tolerant to copper than those grown in sufficient phosphate medium. Copper addition to tolerant cells induced polyP degradation by PPX (an exopolyphosphatase), phosphate efflux and membrane polarization. ppk − ppx − (unable to synthesize/degrade polyP), ppx − (unable to degrade polyP) and Pit system mutants were highly sensitive to metal even in high phosphate media. In exponential phase, CopA and polyP-Pit system would act simultaneously to detoxify the metal or one could be sufficient to safeguard the absence of the other.
Our results support a mechanism for copper detoxification in exponential and stationary phases of E. coli, involving Pit system and degradation of polyP. Data reflect the importance of the environmental phosphate concentration in the regulation of the microbial physiological state.
Inorganic polyphosphate (polyP) is a linear polymer of hundreds of orthophosphate residues linked by phosphoanhydride bonds. The main enzymes associated with polyP metabolism in bacteria are polyphosphate kinase (PPK, encoded by ppk) and exopolyphosphatase (PPX, encoded by ppx) [1, 2]. In most organisms, including bacteria, archaea and eukaryotes, metal tolerance was related to polyP levels . Rachlin et al.  have proposed that polyP, as a metal chelator, reduces intracellular heavy metals concentration in the Cyanophycean alga Plectonema boryanum. Similarly, resistance to cadmium in Anacystis nidulans R2 strain  and in Klebsiella aerogenes was related to high polyP levels. Keasling proposed a model where metals (Na+, Mg2+, Co2+ Cd2+) can be chelated by polyP and/or regulate the activity of PPX, which would in turn degrade polyP, allowing the removal of metal-phosphate complexes possibly via low-affinity inorganic phosphate transport (Pit) system . This model was supported in acidophilic bacteria  and archaea , where Cu2+ increases PPX activity and phosphate (Pi) efflux.
Pit system in Escherichia coli includes PitA (encoded by pitA) and PitB (encoded by pitB) . van Veen et al.  have shown that Pit can reversibly transport Ca2+, Co2+ or Mg2+ phosphates in E. coli and Acinetobacter johnsonii. The uptake of a neutral metal-phosphate (MeHPO) complex is mediated by an electrogenic proton symport mechanism. Conversely, the excretion of the metal-phosphate complex via Pit generates a proton motive force in A. johnsonii.
Copper is an essential nutrient required for many biochemical functions, acting as a cofactor for several enzymes . However, copper is also a toxic element able to catalyze free radicals formation, producing alteration of nucleic acids, lipids and proteins [14, 15]. Thus, cells ensure their viability by a tight regulation of copper levels, involving several homeostatic mechanisms. E. coli is equipped with multiple systems to ensure copper handling under varying environmental conditions. For instance, the Cu+-translocating P-type ATPase CopA is responsible for removing excess Cu+ from the cytoplasm. Multi-copper oxidase CueO and the multi-component copper transport system CusCFBA appears to safeguard the periplasmic space from copper-induced toxicity [16–18]. In aerobic conditions, E. coli usually tolerate copper concentrations in the μM range, although minimal inhibitory concentrations for metals depend on the growth media and the methodology used [17–20].
Stationary phase cells are particularly vulnerable to oxidative damage since they lack the energy and materials needed to repair or replace the damaged molecules. In our laboratory, it has been demonstrated that E. coli stationary cells presented high viability, low oxidative damage and elevated resistance to exogenous H2O2 when Pi concentration in the medium was above 37 mM . These events were related to the maintenance of high polyP level in late stationary phase .
According to the model proposed previously by Keasling , we examined here the involvement of polyP metabolism and Pit system components in E. coli copper tolerance in stationary or exponential phase cells. Our approach included the use of mutants in PPK, PPX, PitA and PitB encoding genes and the modulation of polyP levels by varying media phosphate concentration.
Cu2+tolerance of stationary phase cells grown in different phosphate concentration media
PolyP levels during growth in different Pi concentrations media
ppk − ppx −
pitA − pitB −
MT + P
MT + P
MT + P
MT + P
MT + P
MT + P
123650 ± 10540a
152951 ± 8120a
45541 ± 5563a
38254 ± 4521a
220152 ± 15120a
252651 ± 11120a
80524 ± 9452a
91523 ± 8563a
82536 ± 8652a
95623 ± 9563a
81524 ± 9452a
90523 ± 5563a
54000 ± 9500b
125420 ± 10245a
42564 ± 4521a
40251 ± 6523a
200536 ± 16245a
241536 ± 12155a
32564 ± 4152b
93056 ± 6652a
24563 ± 3254b
89654 ± 10254a
28564 ± 4152b
88056 ± 8652a
44652 ± 4556b
138456 ± 8486a
38563 ± 7521a
41251 ± 5125a
208456 ± 12486a
238456 ± 10286a
22563 ± 5634b
89862 ± 4128a
32564 ± 4635b
92365 ± 8365a
20563 ± 5634b
91862 ± 4658a
As a first step to elucidate the differential copper tolerance in cells grown in MT or MT + P for 48 h, assays using ppk − ppx − (unable to synthesize/degrade polyP [24, 25]) and ppx − (unable to degrade polyP) cells were performed in these conditions. Both mutants were highly sensitive to metal even in MT + P (Figure 1B and C). Note that, polyP levels in ppx − strain were always high, independently of the growth phase and the media used, while the ppkppx mutant exhibits greatly reduced synthesis of polyP, evidenced by low values of fluorescence emission (Table 1).
The implication of Pit system components in copper tolerance was also analyzed using E. coli strains lacking one or both transporter encoding genes (Figure 1D-F). pitA and pitB single mutants were unable to tolerate 0.5 mM Cu2+ in both media. This sensitivity was more pronounced in the pitApitB double mutant. It is worth noting that polyP levels in Pit system mutants depended on media Pi concentration, similarly to WT (Table 1).
Above results using different strains and culture media support the idea that stationary phase copper tolerance is mediated by a mechanism which involves both polyP metabolism and Pit system.
PolyP levels in cells exposed to Cu2+
Pi efflux in cells exposed to Cu2+
Membrane polarization in cells exposed to Cu2+
Cu2+tolerance of exponential phase cells
Cellular functions can be disrupted when Cu2+ concentration exceeds acceptable levels . In order to survive the adverse environment, several mechanisms of resistance are switched on in bacteria . In the present study, we demonstrated that polyP levels and Pit system are involved in E. coli copper tolerance.
In stationary phase, the significant metal resistance of WT cells grown in high phosphate medium could be attributed to the high polyP level in this condition , which could also account for enhancement in stationary-phase fitness . The copper sensitivity of ppk − ppx − is in agreement with previous work showing that this double mutant is deficient in stationary phase functions and lacks stress resistance [22, 24, 25]. On the other hand, considering ppx single mutant sensitive phenotype, not only polyP presence but also its degradation is relevant for Cu2+ resistance in our conditions, discarding the role of polymer merely as a metal chelator. The chelating effect constitutes one line of thought linking the metal tolerance and the polymer; however, abundance of polyP in exopolyphosphatase deficient strain may be damaging for the cell. Note that polymer molecules with high capacity to bind metal ions represent a source of potentially toxic species in equilibrium with the intracellular medium. Degradation of preformed polyP and Pi-copper complex formation that can be exported from the cells represent another alternative way to detoxify metals. In fact, our results provided lines of evidence that copper-induced polyP degradation through PPX in few minutes of exposure. In agreement, Acidithiobacillus ferrooxidans and Sulfolobus metallicus cells underwent to an increase of exopolyphosphatase activity with a concomitant decrease in polyP levels with increasing copper concentrations [8, 9]. In addition, viability assays with Pit system mutants indicate, for the first time, the direct involvement of PitA and PitB in E. coli copper tolerance, as it was previously suggested for other metals  and copper [8, 9]. Levels of pitA gene expression were invariant due to copper addition in each of our experimental conditions (data not shown). Expression was high in exponential phase either in MT and MT + P cells and moderate in stationary MT + P cells, coincidentally with copper tolerance conditions. In sufficient Pi medium MT, expression decay during stationary phase, where viability was impaired and polyP was minimal.
We consider that copper tolerance is a consequence of changes in polyP levels exerted by the metal. Even when copper efflux or formation of intracellular copper–phosphate complexes were not determined in this work, high Pi release and elevated membrane polarization in MT + P WT stationary phase cells, evidence that high polyP levels and its metal-induced degradation would lead to Cu2+-phosphate complexes formation and their subsequent efflux. Low changes in membrane polarization generated after copper addition in other strains and conditions may be due to differential diffusion of ions that induces complex movement of buffer and other ions.
According to present data and our previous results [21–23, 29], the salt composition of the culture media should be carefully considered in the experimental design, especially when stationary-phase events are studied. Note that commonly used minimal media, as M63  and M9 , contain Pi concentrations higher than 40 mM. Our strategy using differential Pi concentration media, allowed us to find the first copper detoxification mechanism acting in E. coli stationary phase, which only involves polyP-Pit system and is functional in high phosphate media. It should be noted that no copper induction of copA gene expression was observed in stationary phase in all the tested media (data not shown).
Our data show that polyP-Pit system is involved in copper tolerance also in exponential phase. Actually, CopA absence could be counteracted by a functional polyP-Pit system and, conversely, CopA would be responsible for metal tolerance in a polyP or Pit deficient background. Even we could not discard the participation of other copper detoxification mechanisms already described to be functional during this phase [17, 19, 28], CopA or polyP-Pit systems seem to be necessary to safeguard cells against copper toxicity, according to sensitive phenotypes of copA − ppk − ppx − and copA − ppx − strains. As it was previously described for E. coli, Pseudomonas fluorescensCorynebacterium glutamicum, Bacillus cereus and a wide range of microorganisms , high polyP levels were reached in the early exponential growth phase. Thus, polyP-Pit system would be a very important aspect to consider as an additional copper tolerance mechanism in bacterial exponential phase.
In conclusion, this work shed light on the previously proposed polyP-dependent mechanism for metal resistance in microorganisms. PolyP degradation and functionality of Pit, postulated as a metal-phosphate transporter system, mediates copper tolerance in E. coli both in exponential and stationary cells. Data represent the first experimental evidence of the involvement of Pit system components in this detoxification mechanism. Our study may also help to understand the importance of the environmental salt composition to regulate the microbial physiological state.
The present research does not involve human subjects, human material, human data, or animals.
Strains and growth conditions
E. coli strains and plasmids used in this work
Strains and plasmids
Relevant genotype or description
Construction or reference
araD, lac, rpsL, flbB, deoC, ptsF, rbsR, relA1
LSB022/pBC29((ppkppx::Km /ppk + , Ap)
RKP4353 [Φ(pitA–lacZ)] pitA::Cm
MC4100 pitA:: Cm
F-, araD-araB, lacZ, copA::km λ − , rph-1, rhaD-rhaB, hsdR
MGP003 ppkppx::Km, copA −
MGP004 /pBC29 ((ppkppx::Km, copA − /ppk + , Ap)
(ppk + , Ap)
Ap, Cm, cI857 lPR flp pSC101 oriTS
Cells grown in MT and MT + P during 6, 24 or 48 h were incubated with shaking at 37°C for 1 h with different CuSO4 concentrations in the same culture media. Identical aliquots of cells incubated without copper were used as controls. Then, metal tolerance was evaluated by qualitative viability assays, spotting 1/10 serial dilutions on LB-agar . Plates were incubated for 24 h at 37°C.
PolyP level measurement
Intracellular polyP was measured in cell suspensions by a fluorescence approach using 4′,6-diamidino-2-phenylindole (DAPI) . Briefly, cells were washed and resuspended in T buffer (100 mM Tris–HCl, pH 8). 17 μM DAPI (Sigma) was added to cuvettes containing cell suspensions (OD560 nm =0.02) in T buffer, with 0.075% SDS and chloroform for cell permeabilization . After 5 min at 37°C with agitation, the DAPI fluorescence spectra (excitation, 415 nm; emission, 445–650 nm) were recorded using an ISS PCI spectrofluorometer (ISS Inc., Champaign, IL). Fluorescence of the DAPI-polyP complex at 550 nm was used as a measurement of intracellular polyP, since emissions from free DAPI and DAPI-DNA are minimal at this wavelength .
Membrane electrical potential measurement
Changes in the transmembrane electrical potential (ΔΨ) were measured utilizing the potential sensitive fluorescent probe 3,3′-dipropylthiadicarbocyanine (DisC3 ) . Briefly, cells were harvested by centrifugation, washed twice with 100 mM T buffer and resuspended in 5 mM HEPES buffer pH 7.5 to an OD 560 nm = 0.1. Cell suspensions were incubated with shaking plus 0.4 μM DisC3  and 0.4% glucose. Fluorescence measurements were carried out at 37°C, adjusting the wavelengths of excitation and emission to 622 and 675 nm, respectively. When the dye uptake was maximal, as indicated by a decrease to a steady fluorescence value, (ΔΨi), 0.1 mM Cu2+ was added and fluorescence was followed for 5 min, achieving ΔΨf. The difference between ΔΨf and ΔΨi was defined as ΔΨCu. Measurements were repeated at least seven times under each condition. Distillated water was added instead of Cu2+ solutions in negative control.
Pi efflux determination
Cells were harvested and thoroughly washed by four steps of centrifugation and resuspension with T buffer to eliminate Pi present in the media. Then, cells were resuspended to the original volume in the same buffer (OD between 2.5 to 3, corresponding to ≈ 109 CFU mL−1) and incubated with agitation in the presence of 0.25 mM Cu2+ at 37°C for the indicated times. Phosphate was determinate in supernatants using ammonium molybdate and ascorbic acid as described by Chifflet et al. . T buffer incubated with copper for 60 min and cells without metal were used as negative controls.
Gene expression was evaluated by β-galactosidase activity and expressed in Miller Units (MU) .
Data were subjected to analysis of variance (ANOVA) followed by Tukey’s test with Statitix 9.0 Analytical Software 2008 for Windows (USA). Differences at p-value of 0.05 were considered significant.
We gratefully acknowledge Dr R. K. Poole for providing the strain RKP2935 and Dr S. Howitt for providing the strains AN3901 and AN4080. This research was supported by Argentinean grants of the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), the Agencia Nacional de Promoción Científica y Técnica (ANPCyT) and the Consejo de Investigaciones de la Universidad Nacional de Tucumán (CIUNT). M.G.P. thanks CONICET for doctoral fellowship.
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