INTRODUCTION

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Upon nutrient starvation or entry into the stationary phase, a pleiotropic phenotype comprising cell morphology change, stress tolerance, and starvation survival develops in bacteria (18). In enterobacteria and Pseudomonas this transition is genetically controlled by the rpoS gene (14, 21, 26). This gene encodes a transcription factor that activates the expression of genes involved in cellular shape change and in cross protection against damaging agents, such as ethanol, H₂O₂, high temperature, or high salt concentration. There is strong evidence that guanosine tetraphosphate (ppGpp) induces expression of the rpoS gene (7).

Many bacterial species, including Pseudomonas oleovorans, synthesize polyhydroxyalkanoic acids (PHAs) under growth conditions characterized by an abundance of carbon sources with respect to other nutrients, such as nitrogen or phosphorus (1). Synthesis and degradation of PHAs are part of a cycle in which the acyl coenzyme A (acyl-CoA) precursors are converted by different metabolic pathways into PHAs. The depolymerization produces acyl-CoAs and acetyl-CoA, which is metabolized in the tricarboxylic acid cycle as a source of carbon and energy (13). PHA accumulation increases bacterial survival in nutrient-depleted cultures (17). Previous studies from our laboratory using wild-type strains and mutants deficient in the synthesis of PHA in natural waters have shown that the capability for synthesis of the polymer endows the bacteria with enhanced survival and competition abilities (15). The growth of bacteria in microcosms resembling natural environments where starvation conditions prevail is an alternative for the study of starvation-related phenomena, like those of growth in limiting conditions or the stationary phase achieved in laboratory cultures. The approach used in the above-mentioned microcosm experiments based on PHA-minus strains excluded the possibility of controlling PHA accumulation. Further experiments performed with a P. oleovorans mutant affected in the ability to degrade the accumulated polymer showed that it was a more appropriate system for studying the role of PHAs in bacterial survival (24). The influence of PHA has not been analyzed in bacterial stress resistance studies.

The mechanisms by which PHA favors bacterial survival are not yet fully understood (17). Early work suggested an association of PHA utilization with respiration and oxidative phosphorylation in Ralstonia eutropha (9).

In this work, we assessed the influence of PHA utilization in survival and tolerance of ethanol and heat challenge in starved cells of P. oleovorans by comparing wild-type and PHA depolymerase-minus (phaZ) strains in river water microcosms. Based on previous results (9, 15, 17), we investigated whether the polymer could be related to the synthesis of nucleotides, including ppGpp as well. This molecule was chosen as a compound involved in the enhanced survival and stress resistance specific to bacteria subjected to starvation conditions.

The accumulation of ppGpp depends on multiple factors, such as energy source availability, amino acid starvation, and growth inhibition. Therefore, the detection of an association between its synthesis and a single phenomenon requires a special experimental approach (4). In the case of the stringent response, the association with ppGpp accumulation is visualized either by amino acid starvation in an auxotroph or by the addition of serine hydroxamate (4). In our case, the study of a relationship between nucleotide synthesis and PHA depolymerization also required a special experimental approach, as the nucleotides could be synthesized from other precursors. The genetic approach chosen in this work in order to provide evidence for an association between the two phenomena relied on the utilization of a phaZ mutation, together with special culture conditions that allowed the observation of spontaneous polymer degradation. Thus, it was possible to observe a relationship between nucleotide accumulation and PHA degradation independently of the availability of other precursors.

	MATERIALS AND METHODS

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Bacterial strains, plasmids, and genetic manipulations. The strains used in this study were P. oleovorans GPo1 and its PHA depolymerase-minus mutant, P. oleovorans GPo500 phaZ (10). Plasmid pGEc422 carrying phaC1 and phaZ genes and their promoter region from P. oleovorans GPo1, which restores PHA degradation in the strain GPo500 (10), was digested with EcoRI and ClaI, and the fragment harboring the pha genes was subcloned in the mobilizable vector pBBR1MCS-2 (23), producing pmPoCZ. This plasmid was mobilized from Escherichia coli DH5 into P. oleovorans strains using the helper plasmid pRK24 (28). Transconjugants were isolated on E medium (8) plates containing sodium octanoate (5 g · liter¹) and kanamycin (50 µg · ml¹). Plasmids and DNA fragments were purified with purification kits (Concert; GIBCO BRL). DNA manipulations were performed according to the method of Sambrook et al. (25).

Water samples. Experiments were performed with surface water samples collected from the top 10 cm of water from the Rio de la Plata (Buenos Aires, Argentina) using a plastic container with a lid, previously rinsed three times with the same water. The water was processed in the laboratory within a few hours of collection. The river water is poor in carbon and nutrient (15).

Microcosms. Microcosms were constructed as previously described (15). Bacteria were grown with shaking up to the early stationary phase (optical density at 600 nm, 1 to 1.1) in nutrient broth supplemented with 5 g · of sodium octanoate liter¹ at 30°C. The cells were harvested by centrifugation and rinsed twice with saline solution (9 g of NaCl liter¹). The pellets were then resuspended in the microcosms, consisting of 100 ml of filtered river water in 500-ml Erlenmeyer flasks, and incubated with shaking at 30°C. The bacteria were counted immediately after the inocula were added (day zero) and then at appropriate times over 25 days by serial dilutions onto triplicate nutrient agar plates. The variation coefficient for replicate platings was 20%. Microcosm samples were microscopically checked for PHA content periodically by staining them with Nile blue (19).

Challenge protocol. One-milliliter water samples of the microcosms were diluted in saline solution according to cell numbers and then, at time zero, were diluted 10 times in saline solution supplemented with 20% ethanol or preheated at 47°C. The ethanol challenge was performed at 25°C. The challenge conditions (concentration, time, and temperature) were chosen so that a rapid decline in the survival of a growing culture was obtained. At different time intervals, aliquots (0.1 ml) were spread onto nutrient agar plates and incubated overnight at 30°C. Each challenge experiment was performed at least twice. Variations in replicate platings were 20%. Aliquots from an exponentially growing culture were challenged by the same protocol to compare exponential-phase cells with cells residing in microcosms. Stress resistance was expressed as the percentage of survival compared to the situation shortly before stress application, which was set at 100%.

Culture conditions. PHA accumulation was achieved as previously described (11) with slight modifications. Cells were precultured at 30°C for 24 h in 250-ml Erlenmeyer flasks containing 25 ml of E medium supplemented with 5 g of sodium octanoate liter¹. The resulting cultures were used to inoculate 1-liter Erlenmeyer flasks containing 250 ml of 0.5 N E₂P medium. This medium contains 0.5 g of K₂HPO₄ · 3H₂O, 0.24 g of KH₂PO₄, 0.45 g of NH₄Cl, 0.5 g of NaCl, and 1 g of yeast extract per liter, supplemented with either 5 or 2.5 g of sodium octanoate liter¹ and 2 mM MgSO₄. Phosphate concentrations in 0.5 N E₂P medium are appropriate for performing ppGpp accumulation studies. When necessary, kanamycin was added at 10 µg · ml¹.

Analytical determinations. PHA was measured chromatographically (3). The PHA content was expressed as the percentage of cellular dry weight. Proteins were determined by the method of Lowry et al. (16). Intracellular levels of ATP and ppGpp were measured chromatographically as described previously (5) in cultures uniformly labeled with [³²P]Na₂HPO₄. One-milliliter aliquots of the parent culture were taken at the indicated time intervals and incubated further in the presence of 0.1 mCi of [³²P]Na₂HPO₄/ml for 2 h. Then extraction was performed in accordance with the method of Bochner and Ames (2). Cultures of Salmonella enterica serovar Typhimurium argC 95 grown with and without arginine were used as controls. In the absence of arginine, ppGpp synthesis is strongly induced in this strain. Extracts were analyzed by thin-layer chromatography in polyethyleneimine-cellulose plaques using 1.5 M KH₂PO₄ (pH 3.5) as the mobile phase. The nucleotide spots were identified as previously described (2). Those spots corresponding to ATP and ppGpp were scraped, and radioactivity was quantified in a liquid scintillation counter.

	RESULTS

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Different cellular traits were used in order to determine the sampling periods for the challenge experiments. As bacterial cells grown under starvation conditions acquire a round shape (14), this easily detectable trait, along with survival and PHA content, was used to analyze starved P. oleovorans GPo1 and GPo500 cells in river water microcosms. Challenge experiments were performed on bacteria residing for 3, 10, and 21 days in river water microcosms. These days were selected on the basis of changes in cell shape and survival.

Effect of starvation on survival, cell shape, and PHA content. Survival of the wild-type strain was higher than that of the PHA depolymerase-minus strain. Cell numbers increased during the first days of microcosm residence for both strains. This increase has also been observed in other species (8, 15, 20) and reflects residual cell division. At day 10, the mutant strain showed a large decrease. At day 21, survival differences between the strains were at 1 order of magnitude (Fig. 1). This experiment was repeated twice, and the same survival pattern was displayed under these conditions (data not shown).

View larger version (21K): [in this window] [in a new window]   FIG. 1.   Survival of P. oleovorans GPo1 and GPo500 in sterile river water microcosms. Bacterial counts were expressed as CFU per milliliter. The values represent means ± 1 standard deviation of three replicated plate counts.

View larger version (73K): [in this window] [in a new window]   FIG. 2.   Microscopic observations of the change in cell shape of P. oleovorans GPo1 (A to D) and GPo500 (E to H). Growing cells before microcosm inoculation (A and E) and cells after 3 (B and F), 10 (C and G), and 21 (D and H) days of microcosm residence are shown. Magnification, ×1,000.

Ethanol challenge. Exponentially growing cells and cells residing in the microcosms were challenged with 20% ethanol (Fig. 3). The wild-type strain developed enhanced resistance with increasing residence time in microcosms (Fig. 3A). Cells of the mutant strain were sensitive to ethanol on day 3, and resistance was observed on day 10 (Fig. 3B). On day 21, both strains showed resistance; however, the percentages of survival after 25 min of exposure were 23 and 9% for the wild-type and mutant strains, respectively.

View larger version (20K): [in this window] [in a new window]   FIG. 3.   Challenge of P. oleovorans GPo1 (A) and P. oleovorans GPo500 (B) residing in microcosms with 20% ethanol. An exponential culture and cells residing in the microcosms for 3, 10, and 21 days were challenged with 20% ethanol at 25°C. Percent survival was determined as the number of viable cells at each time divided by the number of viable cells before exposure to ethanol. The data are means of two independent experiments.

Heat challenge. Exponentially growing cells and cells residing in microcosms were challenged with heat shock at 47°C (Fig. 4). The wild-type strain showed a gradual increase in heat shock resistance with increasing residence time in microcosms in agreement with the ethanol challenge results (Fig. 4A). Resistance was at a maximum on day 21 (13% survival at the end of the exposure). The mutant strain did not show stress resistance on days 3 and 10 of the experiment. By day 21, the mutant strain had slight resistance, as demonstrated by the low percentage of survival (2%) after 30 min of exposure to 47°C (Fig. 4B).

View larger version (21K): [in this window] [in a new window]   FIG. 4.   Challenge of P. oleovorans GPo1 (A) and P. oleovorans GPo500 (B) residing in microcosms at 47°C. An exponential culture and cells residing in the microcosms for 3, 10, and 21 days were challenged at 47°C. Percent survival was determined as the number of viable cells at each time divided by the number of viable cells before exposure to heat. The data are means of two independent experiments.

Nucleotide accumulation in P. oleovorans wild type and depolymerase-minus mutant. These results and those previously reported (15) clearly show that PHA enhances survival and stress resistance in bacteria in natural environments. As this phenomenon is also observed in nutrient-limited cultures (17), and as PHA is a reservoir of carbon and energy, it can be hypothesized that the products of polymer degradation are used for the synthesis of nucleotides and other compounds.

View larger version (32K): [in this window] [in a new window]   FIG. 5.   Intracellular levels of ppGpp and PHA content of P. oleovorans GPo1 (squares) and GPo500 (triangles). Cells were grown in 0.5 N E2P supplemented with 5 g of sodium octanoate per liter. Culture samples were analyzed for ppGpp and PHA content at the start of PHA degradation. Each value is the result of two determinations.

View larger version (23K): [in this window] [in a new window]   FIG. 6.   Intracellular levels of ppGpp (A) or ATP (B) and PHA content of P. oleovorans GPo1/pmPoCZ (squares), GPo500/pmPoCZ (circles), and GPo500/pBBR1MCS-2 (triangles). Cells were grown in 0.5 N E2P supplemented with 2.5 g of sodium octanoate per liter and 50 µg of kanamycin per ml. Culture samples were analyzed for ppGpp (A) or ATP (B) and PHA content at the start of PHA degradation. Each value is the result of two determinations.

	DISCUSSION

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The experiments described in this work show that the phaZ mutation is a reliable system for studying the role of PHA in tolerance for starvation conditions and damaging agents in natural water microcosms and for suggesting a mechanism to explain this tolerance.

The wild-type strain showed increased survival compared to the PHA depolymerase-minus strain. This result had been previously observed in nonsterile microcosms (24). Survival was tested in association with changes in cell shape and PHA content. The change to a round cell shape favors the absorption of nutrients from the environment. Our results showed that morphological changes are delayed until 21 days of microcosm residence in the wild-type strain, while in the mutant strain these changes were observed on day 10. The wild-type strain could use PHA, a reservoir of carbon and energy, and hence this transformation was delayed. PHA granules were not detected in the wild-type strain the day after the start of the microcosm experiment; however, that does not mean that this carbon and energy source was exhausted at this stage of the experiment. Routinely, P. oleovorans cultures prepared as indicated in Materials and Methods contain 30% PHA relative to cellular dry weight. If this quantity of carbon is available for the cell, a delay in entrance into a resistant state is quite possible. Cells of both strains could have monitored transient C abundance that allowed them to accumulate the polymer and, in the case of GPo1, to degrade it in a dynamic way. The Nile blue staining method detects mainly extremely compact granules. The absence of granules does not mean that PHA is not available as a carbon source, as PHA synthesis is presumed to start with several polymer chains that coalesce in a hydrophilic cytoplasm (6) and polymer chains can also be originated by depolymerization (12).

P. oleovorans GPo1 showed enhanced resistance to ethanol and heat challenge compared to its corresponding PHA depolymerase-minus mutant, GPo500, in river water microcosms. For both strains of P. oleovorans tolerance to stress conditions was enhanced with the time of residence in the microcosms, as evidenced by comparing the resistance of bacteria residing for 3 days in river water with that of bacteria residing for 21 days (Fig. 3 and 4). However, the percent survival at the end of the challenges was always higher in the wild type than in the mutant strain (Fig. 3 and 4). The differences were more evident for heat challenge.

Our work presents clear genetic evidence, relying on phaZ mutation and complementation studies, that correlates PHA degradation with nucleotide accumulation. Depolymerization of a carbon and energy source like PHA could provide for their synthesis. The detection of ppGpp accumulation was a particular challenge, as it depends on multiple factors. The choice of approach in this work, to observe an association between ppGpp accumulation and PHA depolymerization, was based on previously well-established culture conditions that allow the observation of spontaneous polymer degradation. This degradation proceeds, as has been previously elucidated (27), under conditions of deficiency of reducing power (NADH₂) and acetyl-CoA and an excess of free CoA.

The cross protection from stress agents associated with the resistant state is less efficient in P. oleovorans GPo500 than in GPo1. We can speculate that as ppGpp is an activator of RpoS synthesis, low ppGpp levels would contribute to the reduced survival and stress resistance characteristic of the GPo500 strain. The phaZ genotype could be associated with alterations in the metabolism or uptake of phosphate or any step leading to nucleotide synthesis. Hippe studies (9), performed without the help of mutants in PHA-rich and-depleted cells, showed that the utilization of the polymer was not only associated with respiration and oxidative phosphorylation but also inhibited the degradation of nitrogenous cellular constituents in R. eutropha. Nevertheless, complementation of the phaZ mutation restores the wild-type phenotype and supports the hypothesis of the association between the phenomena.

Bacterial survival and stress resistance studies are important for many aspects of bioremediation, biocontrol, and plant growth promotion. Inorganic polyphosphate, another reserve polymer, has been shown to support stationary-phase survival and stress resistance in E. coli (22). These results, together with those presented in this work, indicate that an energy and carbon reservoir like polyhydroxyalkanoate should be taken into account while performing such studies.