Requirement of Polyphosphate by Pseudomonas fluorescens Pf0-1 for Competitive Fitness and Heat Tolerance in Laboratory Media and Sterile Soil

Bacterial strains, plasmids, and culture conditions.All bacterial strains and plasmids used in this study are listed in Table 1. Pseudomonas fluorescens strains were grown at 30°C, and Escherichia coli strains were grown at 37°C. E. coli strains were routinely grown in LB medium (35), while P. fluorescens strains were cultured in LB or in Pseudomonas minimal medium (PMM) (17). For defined phosphate medium, MOPS (morpholinepropanesulfonic acid) minimal medium (26, 31) was used. MOPS medium was amended with either 2 mM (high-phosphate) or 0.14 mM (low-phosphate [25]) K₂HPO₄, creating media called MOPS-H and MOPS-L, respectively. For solid medium, agar was added to a final concentration of 1.5%. For motility assays, the agar concentration was 0.3%. The following antibiotics were used at the concentrations indicated: ampicillin, 50 μg/ml; streptomycin (Sm), 50 μg/ml; kanamycin (Km), 25 μg/ml; tetracycline, 15 μg/ml (for strains carrying replicating plasmids) or 7.5 μg/ml (for strains with plasmids integrated into the genome); gentamicin, 10 μg/ml. Plasmids were introduced into P. fluorescens strains by conjugation from E. coli S17-1. Briefly, donor and recipient strains were grown for 16 h. Cells were collected by centrifugation, washed twice in LB broth, and finally suspended in 200 μl of LB. Cells were mixed in a 1:4 donor-to-recipient ratio, and a 100-μl drop was placed onto the surface of an LB agar plate and incubated at 30°C for 8 h, after which the cells were scraped from the plate and plated onto appropriate medium to select the desired transconjugants.

DNA manipulations and sequencing.Recombinant DNA techniques were performed as described previously (35) or according to the supplier's instructions. Restriction enzymes and DNA-modifying enzymes were purchased from Invitrogen (Carlsbad, CA), New England Biolabs (Ipswich, MA), and Promega (Madison, WI). Plasmid DNA was extracted using a QIAprep Spin miniprep kit (Qiagen, Valencia, CA). DNA fragments were recovered from agarose gel slices using a QIAquick gel extraction kit (Qiagen). The PCR was carried out in a GeneAmp PCR System 2700 (Applied Biosystems, Foster City, CA), using Taq or Platinum Taq High Fidelity DNA polymerase (Invitrogen). PCRs to amplify DNA for cloning were all carried out using purified genomic DNA for the template (Wizard DNA isolation kit; Promega). Screening of mutants was carried out by colony PCR. When required, PCR products were cloned with pGEM-T Easy (Promega). Oligonucleotides were synthesized by IDT (Coralville, IA). All custom primers are shown in Table 2. DNA sequences were determined by the Tufts University Core Facility (Boston, MA).

Construction of a ppk iiv8 mutant.A ppk-iiv8 deletion mutant was constructed by splicing-by-overlap extension-PCR (SOE-PCR) (11) and allele exchange between the suicide plasmid pSRΔiiv8 and the Pf0-1 genome to replace the wild-type sequence with a sequence in which nucleotides 6131357 to 6133054 were deleted. The suicide plasmid was introduced into Pf0-1 by conjugation, and single-crossover integration was selected by Km resistance. Sensitivity to sucrose, because of the plasmid-carried sacB gene, confirmed the presence of the plasmid. This strain was then grown for 24 h in LB broth in the absence of selection to allow a second recombination and loss of the suicide plasmid, and those strains were selected by plating dilutions from the second recombination culture on sucrose-containing medium. After plasmid loss was confirmed by sensitivity to Km, colonies possessing the desired deletion were identified by PCR. Primers for the SOE-PCR are listed in Table 2. This deletion spanned the transcribed region of iiv8 identified by IVET (40), a possible open reading frame in which the transcribed sequence was found (bases 6131357 to 6133057 in the Pf0-1 genome), and the first 1,665 bases of ppk (the full length is 2,226 bases). The resulting strain was called Pf0-1Δ8-ppk. To provide a selectable marker to allow Pf0-1Δ8-ppk to be distinguished in competition experiments, mini-Tn7Km (24) was allowed to insert into the unique Tn7 integration site downstream of glmS in Pf0-1Δ8-ppk, producing strain Pf0-1Δ8-ppk-Km.

Soil growth and competition assays.The soil used in these experiments was a gamma-irradiated fine loam from Sherborn, MA, as described previously (6). Bacterial strains were grown for 16 h in LB with appropriate antibiotics. Cells were diluted to approximately 1 × 10⁵ CFU/ml in sterile distilled H₂O. For survival experiments, 1 ml of diluted cell suspension was mixed with 5 g of soil, achieving a water-holding capacity of approximately 50%. For competition experiments, cultures were adjusted to equal values of optical density at 600 nm prior to dilution, and then 500 μl of each diluted competing strain was combined and mixed with soil as described for the survival experiments. Inoculated soil samples were transferred to a 15-ml conical polypropylene Falcon tube. The initial recoverable population was established by removal of 0.5 g of soil after 30 min and recovery and enumeration of bacteria from that sample. Briefly, 1 ml of sterile water was added to the 0.5-g soil sample, followed by vigorous vortexing for 30 s. After the soil particulates had settled, bacteria in suspension were recovered and enumerated by CFU determination. This method has been described elsewhere (39, 40). The initial populations of wild-type and mutant strains were approximately equal. Populations were monitored over time by extraction of bacteria from the soil and determination of numbers by counting colonies on the appropriate selective medium (39, 40). The wild-type strain in competition experiments was Pf0-1Sm^r. In wild-type versus wild-type control assays, Pf0-1Sm^r was competed with Pf0-1Km^r. The competitive index (CI) is the ratio of mutant to wild type at a given time point divided by the initial mutant/wild-type ratio.

Competition experiments in LB medium.Competition experiments were carried out in 12.5 ml of LB in 125-ml flasks, in a shaking water bath at 150 rpm. Strains for competition experiments were grown for 20 h in LB with appropriate selection and then washed in fresh LB. Competition cultures were established as 1:1,000 dilutions of the washed cells. A 100-μl sample was immediately removed, diluted, and plated on selective medium to allow enumeration of each competitor in the culture. Samples (100 μl) were then removed, diluted, and plated on selective medium each for 24 h for 5 days. The CI was calculated as described for soil competition experiments.

Construction of complementation plasmids.Plasmids bearing the genes for complementation were constructed in the vector pME6000. PCR was used to amplify bases 6130970 to 6133214 for iiv8 (primers 8compF and 8compR) and bases 6130717 to 6134285 for ppk (primers 8D5F2H and hemF-H). The genomic regions amplified were chosen so that the native promoters would be included. The PCR products were cloned into the HindIII site of pME6000 by using restriction enzyme sites incorporated into the primer sequences.

Restoration of ppk by allele exchange.Pf0-1Δ8-ppk was restored to the ppk⁺ genotype for soil experiments. DNA spanning the deletion in Pf0-1Δ8-ppk was amplified by PCR using primers 8D5F and 8D3R, cloned in pGEM-T Easy, and then cloned in the NotI restriction site of pSR47s. The resulting clone (pSR-8DFR) was transferred to Pf0-1Δ8-ppk by conjugation. Allele exchange was used to replace the Δiiv8-ppk region with the wild-type sequence, generating strains Pf0-1ppkRev-1 and Pf0-1ppkRev-2 (independently selected). The allele exchange was confirmed by PCR with primers 8D5F2 and 8D3R2 and by confirming restored poly P production.

Stress tolerance assays. (i) Heat tolerance.Heat tolerance during growth in culture medium was tested by starting 15-ml cultures in the appropriate medium with a 1:50 dilution of a 16-h culture grown in the same medium. The 15-ml cultures were grown for 24 h at 30°C and then transferred to 45°C. Samples (100 μl) were periodically taken, serially diluted in 10-fold increments, and plated to determine CFU/ml. To examine heat tolerance during growth in soil, the soil was inoculated with single strains as described above for soil growth assays, and the initial recoverable population was determined. After 2 days, 0.5 g of soil was removed for CFU determination and the remainder of the soil was subjected to the various temperatures used. The population was ascertained for each of the next 2 days.

(ii) Osmotic stress.Cultures (15 ml) were started in MOPS-H and MOPS-L by 1:50 dilution of cells (washed in MOPS-L) that had grown for 16 h in MOPS-H. After 20 h of growth, the bacteria from 15-ml cultures were recovered by centrifugation and then suspended in the same MOPS medium amended with NaCl at concentrations ranging up to 2.5 M. Controls were suspended in medium lacking the NaCl supplement. Bacterial populations were monitored over 3 h by plating to determine the CFU/ml.

(iii) Oxidative stress.Cultures (15 ml) in MOPS-H and MOPS-L were started as described above for osmotic stress. After 20 h of growth, H₂O₂ was added to final concentrations ranging up to 150 mM, and populations were monitored over 3 h by plating to determine the CFU/ml.

(iv) Acid stress.Acid stress experiments were carried out following the same method as that for the osmotic stress experiments, except that medium was adjusted to pH 4.5 instead of being amended with NaCl. Populations were monitored over 3 h by plating to determine the CFU/ml.

Extraction and quantification of poly P.poly P was extracted and measured essentially as described previously (4). A standard poly P curve was made by serially diluting (10-fold increments) a solution of a known quantity of sodium poly P (Sigma-Aldrich, catalog no. 305553) and adding 100-μl aliquots to 900 μl of 6-mg/liter toluidine blue O dye (Sigma-Aldrich) in 40 mM acetic acid. After incubation at room temperature for 15 min, A₅₃₀ and A₆₃₀ levels were measured, and the amount of poly P was expressed as the ratio of A₅₃₀ to A₆₃₀.

For poly P quantification in cells, bacteria were grown for 16 h in LB, 1 ml of culture was washed with MOPS-L, and the appropriate media were inoculated to achieve a 1:50 dilution of bacteria. At required time points, 1 ml of culture was removed for poly P extraction, and a 100-μl sample was taken for CFU determination. Cells from the 1-ml aliquot were collected by centrifugation, suspended by being vortexed in prewarmed (95°C) guanidine thiocyanate lysis buffer (4 M guanidine thiocyanate in 500 mM Tris-HCl [pH 7]), and incubated at 95°C for 5 min, after which 30 μl of 10% sodium dodecyl sulfate, 500 μl of 95% ethanol, and 10 μl of Glassmilk (Qbiogene catalog no. 1001-404) were added. Samples were mixed by vortexing and then briefly centrifuged in a bench top centrifuge (15 s, maximum speed) to pellet the Glassmilk. Pellets were suspended in New Wash buffer (5 mM Tris-HCl [pH 7.5], 50 mM NaCl, 5 mM EDTA, 50% ethanol) and pelleted again. The New Wash buffer treatment was repeated twice more. The pellet was then suspended in 100 μl of 50 mM Tris-HCl (pH 7.4), 10 mM MgCl₂, and 1 mg/ml of DNase I and RNase A. Samples were incubated at 37°C for 30 min, and then the Glassmilk was pelleted and suspended in 300 μl of a 1:1 mix of guanidine thiocyanate buffer and 95% ethanol. The Glassmilk was again pelleted and then suspended in New Wash buffer. This wash was repeated once. poly P was eluted from the Glassmilk by suspending the pellet in 50 μl of 50 mM Tris-HCl (pH 8) and incubating it at 95°C for 2 min. After the Glassmilk was pelleted, the supernatant was collected. The elution was repeated twice more, and the three 50-μl eluates were pooled. poly P was quantified by adding 10-, 25-, or 50-μl samples to 900 μl of toluidine blue O dye and bringing the final volume to 1 ml. After 15 min at room temperature, the A₅₃₀/A₆₃₀ was determined and compared to the standard curve.

RNA isolation, RT-PCR, and rapid amplification of cDNA ends (RACE).RNA was extracted from P. fluorescens Pf0-1 using an RNeasy minikit and on-column DNase I digestion (Qiagen). RNA so recovered was treated with RQ1 DNase (37°C, 1 h) (Promega) and subsequently purified using an RNeasy minikit column. First-strand synthesis for reverse transcription-PCR (RT-PCR) was carried out using Superscript III (Invitrogen) and gene-specific primers (8RT-F for ppk and 8RT-R for iiv8) (Table 2), at 52°C for 1 h. The cDNAs for both iiv8 and ppk were amplified by PCR using primers 8RT-F and 8RT-R. A negative control consisting of a reverse transcriptase-free reaction mixture was used in each experiment.

5′- and 3′-RACE experiments were carried out using Invitrogen 5′- and 3′-RACE system protocols, except that prior to 3′ RACE, total RNA was treated with E. coli poly(A) polymerase (Ambion). The gene-specific primers used were 8RT-R, 8GSP2, and 8GSP3 (for 5′ RACE) and 8RT-F and 8GSP5 (for 3′ RACE) (Table 2).

Measuring ppk transcription by β-galactosidase assay.A ppk-lacZ transcriptional fusion was constructed to measure expression of ppk. Bases 6132852 to 6134419 of the Pf0-1 genome, which include the hemB gene and the first 170 bases of ppk, were amplified by PCR (primers ppk-UIC-FWD and ppk-UIC-REV; Table 2). The product was cloned into the BglII and SpeI sites of plasmid pUIC3, which carries a promoterless lacZ gene. The resulting plasmid, pUICppk-lac, cloned in E. coli S17-1, was transferred to Pseudomonas strains by conjugation. Transconjugants were selected on tetracycline-containing PMM agar. Since pUIC3 and its derivatives do not replicate in Pseudomonas (28), transconjugants have a single copy of the plasmid integrated in the genome of the recipient by a single homologous recombination event, resulting in strains carrying ppk-lacZ in a near-native genetic context, with ppk transcription from its native promoter. Strains for the assay were grown for 16 h in MOPS-H medium, before 1 ml of culture was removed and washed twice in MOPS-L medium. The appropriate fresh medium was then inoculated with washed cells using a 1:50 dilution. β-Galactosidase assays were carried out essentially as described by Miller (23), using 20 μl of chloroform and 10 μl of 0.1% sodium dodecyl sulfate to permeabilize the cells. Data were expressed as pmol of orthonitrophenol produced per 10³ CFU per min of reaction.

Statistical analysis.Data were analyzed with a t test using Graph Pad Prism version 4.

The iiv8-ppk locus is important for competitive fitness in soil and laboratory culture.DNA sequence analysis revealed that iiv8 is located opposite the ppk gene in P. fluorescens Pf0-1. Since iiv8 was upregulated in a soil environment (40), we reasoned that the iiv8-ppk locus may be important for fitness in soil. Moreover, ppk has been linked to a number of stress response phenotypes including survival in the stationary phase during laboratory culture (15, 30) and survival of Mycobacterium tuberculosis in macrophages (43). We therefore created a Pf0-1 derivative (Pf0-1Δ8-ppk) with this locus deleted (see Materials and Methods) and added a Km^r marker to facilitate selection after competition experiments with the Pf0-1 wild type bearing Sm^r. As shown in Fig. 1A, loss of the iiv8-ppk locus resulted in reduced competitive fitness in soil. After 9 days of competition, the CI for the Km^riiv8-ppk mutant dropped to less than 0.05 compared to a value of approximately 1.0 for a Km^r wild-type strain in competition with the Sm^r Pf0-1 (Fig. 1A).

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FIG. 1.

Competition between Pf0-1 wild-type and ppk mutant strains. (A) Competition in sterile loam soil. Soil was inoculated with equal numbers of each competing bacterium. Bacteria were recovered from 0.5 g of soil 1, 2, and 9 days after inoculation and enumerated on media containing Sm or Km. A mock CI was derived from soil experiments in which Pf0-1 and the ppk mutant were grown separately, to show the absence of growth defects in monoculture (inverted triangles). The CI of Pf0-1Δ8-ppk was significantly different from those of Pf0-1, Pf0-1ppkRev-Km, and the mock experiment (P < 0.05). Note that the x axis is not continuous. (B) Competition in LB culture. LB (15 ml) was inoculated with equal numbers of each competing strain. Bacteria were enumerated by diluting a 100-μl sample and plating it on medium containing Sm or Km. All strains carried the plasmid vector pME6000, except where the complementation plasmid is indicated. The CI of Pf0-1Δ8-ppk was significantly different from that of Pf0-1 and the complemented strain. For both panels, the CI was calculated by dividing the ratio of mutant to wild type on a particular day by the initial ratio. The y axis on both panels is log plotted. Means and standard errors of the means of at least three replicate experiments are shown. Note the different scales of the two panels.

A “mock” CI for monocultures was calculated by combining data from Pf0-1Δ8-ppk-Km and Pf0-1Sm^r monocultures. This mock CI demonstrated that when Pf0-1Δ8-ppk-Km was grown alone in sterile soil, no growth defect was detected, compared to Km^r Pf0-1 grown under the same conditions (Fig. 1A). Since stationary-phase defects have been seen in ppk mutants of other species (e.g., reference 15), we asked whether the competitive defect was confined to soil or was a more general phenotype associated with Pf0-1Δ8-ppk-Km. Pf0-1Δ8-ppk-Km was cocultured with Sm^r Pf0-1 in LB broth for 5 days. As seen in the soil experiments, Pf0-1Δ8-ppk-Km was unable to compete with the wild type (Fig. 1B). Notably, the mutant population was comparable to that of the wild type for 2 days, after which the iiv8 ppk mutant population declined dramatically, resulting in very low CI values of about 0.0005 (Fig. 1B). These data show that the competitive defect is not limited to soil and not associated with a failure to reach a high population at the start of the experiment or the result of a lower initial growth rate than that of Pf0-1. The competitive defect of Pf0-1Δ8-ppk-Km in culture and in sterile soil was reversed when the ppk-iiv8 deletion was reverted to wild type by allele replacement (strain Pf0-1ppkRev; see Materials and Methods), thus confirming the importance of the ppk-iiv8 locus in competitive fitness (Fig. 1). As shown in Fig. 2, the ppk deletion also results in removal of the last 15 bases of the upstream gene, hemB. Experiments with a different ppk-null mutant (Pf0-1Δppk-full) confirmed that the hemB 3′ deletion was not responsible for the fitness defects (data not shown).

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FIG. 2.

Organization of the ppk and iiv8 locus in Pf0-1. The ppk (Pfl01_5464), hemB (Pfl01_5465), and iiv8 genes as well as the positions of flanking genes (Pfl01_5463 and Pfl01_5466) are shown. In the arrow representing iiv8, the embedded small dark arrow shows the longest open reading frame (orf) in the transcribed region. The region deleted in Pf0-1Δ8-ppk (bases 6131357 to 6133054) is indicated, as are the regions cloned in plasmids pUICppk-lac and pMEppk. Parallel diagonal lines through the Pfl01_5463 and Pfl01_5466 arrows indicate that the full length is not shown. Nucleotide positions in the Pf0-1 genome are shown for the genes, deleted region, and cloned regions.

The iiv8-ppk locus produces overlapping transcripts.We tested whether production of both iiv8 and ppk transcripts could be detected after growth in PMM, using strand-specific RT-PCR (data not shown; Table 2 shows primer sequences). Products were readily detected, indicating that genes are located on both strands of this single stretch of DNA. While ppk is well characterized in other organisms and generally well conserved, the iiv8 gene is novel. Using 5′ and 3′ RACE, we found that the transcribed iiv8 gene spans bases 6131885 to 6132542, producing a transcript of 658 bases (Fig. 2). It is unlikely that the transcript is translated into a protein since the longest open reading frame that starts and finishes within this region is only 180 bases long (bases 6132309 to 6132488 of the Pf0-1 genome). If translated, it would produce a small 59-amino-acid protein, and the mRNA would also have a 424-base 5′ untranslated sequence (Fig. 2). These data suggest that iiv8, the gene opposite ppk, probably specifies a noncoding RNA molecule, although the possibility that a small protein is also produced has not been excluded.

Loss of ppk is responsible for the competitive defect in laboratory culture.To distinguish between loss of ppk and loss of iiv8 as the cause of the competitive defect shown by Pf0-1Δ8-ppk-Km, we carried out complementation experiments in LB culture, where the maintenance of complementing plasmids could be easily controlled and verified. Two different constructs were tested, one encoding both iiv8 and ppk (pMEppk) and the other encoding just iiv8 (pME8comp). Pf0-1Δ8-ppk-Km carrying each plasmid was competed against the Sm^r Pf0-1 carrying the parent plasmid pME6000. While carriage of pME8comp did not improve the fate of Pf0-1Δ8-ppk-Km (data not shown), possession of pMEppk resulted in a more-than-1,000-fold improvement relative to Pf0-1Δ8-ppk-Km (Fig. 1B). The plasmid pMEppk did not completely restore the competitive fitness of the mutant because of a growth defect associated with cells carrying pMEppk but did confer a significant improvement in the CI of Pf0-1Δ8-ppk-Km, relative to Pf0-1Δ8-ppk-Km carrying the plasmid vector (Fig. 1B). A truncated version of pMEppk which included the complete hemB sequence (pME-hemB) failed to complement the competitive fitness defect (data not shown). We conclude from these data that loss of ppk, not iiv8, in Pf0-1 results in reduced competitive fitness.

ppk is required for poly P accumulation.To verify that poly P is produced by Ppk in Pf0-1 and is thus important for fitness, we measured accumulation of poly P by the wild type and Pf0-1Δ8-ppk. MOPS-L and MOPS-H minimal media were used for the initial poly P accumulation assays, since those media have limited (MOPS-L) and sufficient (MOPS-H) concentrations of P_i for derepression and repression of the Pho regulon, respectively. These media have previously been used for poly P studies (26, 31). When the strain was grown in MOPS-H, accumulation of poly P by Pf0-1 was easily measured, whereas poly P accumulation during growth in MOPS-L medium was close to the lower limit of detection (data not shown). In contrast, Pf0-1Δ8-ppk failed to accumulate detectable poly P in either medium. The poly P defect was reversed by replacing the region carrying the ppk deletion with a wild-type sequence, by allele exchange (Fig. 3A). The presence of plasmid pMEppk impaired growth of Pf0-1Δ8-ppk relative to strains carrying the plasmid vector alone (data not shown) but led to greater poly P accumulation than that observed in Pf0-1, probably because of expression of ppk from a multicopy plasmid (Fig. 3B). The parent plasmid pME6000 did not complement the ppk mutation (Fig. 3B), and plasmid pME8comp bearing iiv8 also failed to restore poly P accumulation to Pf0-1Δ8-ppk (data not shown), demonstrating that ppk, not iiv8, was required for poly P accumulation.

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FIG. 3.

Accumulation of poly P under high-P_i conditions. (A) poly P accumulation by Pf0-1, Pf0-1Δ8-ppk, and two independent isolates of Pf0-1Δ8-ppk reverted to wild type by allele replacement (strains Pf0-1ppkRev-1 and Pf0-1ppkRev-2) after 8 and 24 h of growth. Loss of ppk results in a greater-than-10-fold reduction in measurable poly P. Replacing the ppk deletion with wild-type sequence (Pf0-1ppkRev strains) completely restores poly P accumulation. (B) Complementation of ppk by the plasmid pMEppk. Pf0-1 and Pf0-1Δ8-ppk carrying the plasmid vector pME6000 accumulate poly P to approximately the same level as do plasmid-free strains (A). Pf0-1Δ8-ppk carrying pMEppk accumulates almost 10 times more poly P than does Pf0-1. Plasmids used in the complementation experiments were maintained by including tetracycline in the medium. Means and standard errors of the means of at least two replicates are shown. Data are expressed as the quantity of poly P (in ng) per 10⁷ CFU.

A role for poly P in stress tolerance of Pf0-1.The ppk mutant of Pf0-1 did not show reduced tolerance to osmotic, oxidative, or acid stress conditions in either MOPS-L or MOPS-H medium (data not shown). In MOPS-L medium, loss of ppk led to increased sensitivity to elevated temperature (Fig. 4A), an effect that was apparent but much less profound in the P_i-sufficient MOPS-H medium (Fig. 4B). Since temperature fluctuations are likely to be experienced in natural soil environments, we examined the effect of increased temperature on wild-type and ppk mutant Pf0-1 during monoculture growth in sterile soil (Fig. 5). As was seen in the culture-based experiments, the absence of ppk was associated with decreased tolerance to elevated temperature. After 1 day of exposure to the increased temperature, the population of Pf0-1Δ8-ppk was only ∼10% of that of a Pf0-1 monoculture in soil. The Pf0-1 population had declined by 1 order of magnitude, compared with 2 orders of magnitude for Pf0-1Δ8-ppk. Although sustained exposure to 42°C caused a decline in both wild-type and mutant populations, this decline was more rapid in Pf0-1Δ8-ppk, implicating production of poly P in tolerance to heat stress.

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FIG. 4.

Effect of temperature stress on survival of Pf0-1Δ8-ppk in high- and low-P_i laboratory media. (A) Pf0-1 and Pf0-1Δ8-ppk were grown as monocultures in MOPS-L at 30°C prior to cultures being exposed to 45°C for 180 min. Pairs of bars at each time point show the ratios of surviving Pf0-1Δ8-ppk to Pf0-1 for control cultures left at 30°C compared to those shifted to 45°C. The ratio of surviving Pf0-1Δ8-ppk to Pf0-1 at 45°C decreases over time, showing the effect of the ppk deletion on temperature sensitivity. In contrast, in control experiments that remained at 30°C, no such decline was seen. The differences between the heat exposure and control data are significant (P < 0.05) at 120 and 180 min after heat exposure began. (B) As described for panel A except that cultures were grown in the high-P_i MOPS-H medium. After 180 min, there was a significant difference between the control and heat exposure experiments (P < 0.05), although the magnitude of the difference is far smaller in the high-P_i medium than in the low-P_i medium (A). In both panels, data shown are the means and standard errors from at least three independent experiments. Note that the y axis scales are not the same in panels A and B.

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FIG. 5.

Effect of temperature stress on survival of Pf0-1Δ8-ppk in sterile soil. Soil samples were inoculated with either Pf0-1 or Pf0-1Δ8-ppk. Bacteria were allowed to grow for 2 days at 20°C prior to a temperature shift. Data are shown as the ratio of surviving (CFU/0.5 g of soil) Pf0-1Δ8-ppk to Pf0-1. Bars indicate the ratios for experiments prior to temperature change (bars labeled “Pre”) and after 1 day at the new temperature (20 [control], 30, 37, or 42°C). When the ratios before and after temperature shifts are compared, the effect of 42°C on Pf0-1Δ8-ppk is significant (P < 0.05), whereas no other temperature caused a significant decline relative to Pf0-1. Data shown are the means and standard errors from at least three independent experiments.

Motility of P. fluorescens is not impaired in the ppk mutant.Motility and chemotaxis were found to be important for survival and spread of Pf0-1 in live soil (21). In P. aeruginosa, loss of poly P is associated with motility defects. We examined motility in Pf0-1Δ8-ppk as a potential explanation for the soil fitness defect. In contrast to P. aeruginosa, no motility difference was seen between Pf0-1 and Pf0-1Δ8-ppk on swimming agar containing 1% tryptone and 0.5% NaCl. These experiments, and those of Rashid and Kornberg (32), were carried out on the surface of rich medium in which the level of P_i was not determined but was likely to be high. Given the influence of P_i concentration on both accumulation of poly P and expression of the ppk gene (see below), the effect of P_i concentration on swimming motility of Pf0-1 was examined. On MOPS-H and MOPS-L swimming agar plates (0.3% agar), no substantial difference in motility was observed in comparison of wild-type and ppk mutant strains (data not shown).

ppk transcription is elevated under P_i-limiting conditions.poly P production in P. aeruginosa is induced in P_i-limiting conditions (7). To examine the impact of external P_i on ppk expression in Pf0-1, we constructed a ppk-lacZ fusion in plasmid pUIC3 and integrated the resulting construct (pUICppk-lac) into the native ppk locus in P. fluorescens Pf0-1. Transcription of ppk during growth in low- and high-P_i media was measured. During growth in high-P_i media, containing either high or limiting amino acid supplements, ppk-lacZ transcription remained reasonably constant at between 100 and 300 pmol o-nitrophenol/10³ CFU/min over a 24-h period (Fig. 6B). In contrast, in low-P_i media, ppk-lacZ transcription began to increase by 4 h of growth, reaching around 2,000 pmol o-nitrophenol/10³ CFU/min by 24 h (Fig. 6A). Thus, ppk is expressed at a relatively low level in high-P_i media, but this level is sufficient to allow accumulation of poly P in the cells (Fig. 3). In P_i-limiting media, ppk expression is elevated ∼10-fold, suggesting positive regulation in response to low phosphate (Fig. 6).

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FIG. 6.

Pho regulon control of transcription of a ppk-lacZ fusion in low- and high-P_i media. Expression of a ppk-lacZ transcriptional fusion was monitored over time in wild-type and Δpst, ΔphoB, and Δpst ΔphoB mutants of Pf0-1. (A) Under low-P_i conditions (0.14 mM), ppk is induced in the wild type and the Δpst mutant but cannot be induced in strains lacking the Pho regulon regulator PhoB. (B) High-P_i conditions (2 mM) result in repression of ppk in Pf0-1, indicating Pho regulon control. Expression of ppk in the Δpst mutant is higher than that in the wild type, indicating induction of Pho regulon genes by PhoB. Over time, ppk expression is reduced in the pst mutant, indicating additional regulation of ppk. In both panels, means and standard errors of the means of at least three replicates are shown. The x axes are not continuous. Data are presented as the pmol of o-nitrophenol released from o-nitrophenyl-β-d-galactopyranoside per 10³ CFU, per min.

ppk is part of the Pho regulon in P. fluorescens Pf0-1.PhoB, the positive regulator of Pho regulon genes in P_i-limiting environments, is important for accumulation of poly P under certain stress conditions in E. coli (1) and for heterologous expression of Klebsiella aerogenes ppk in E. coli (13). The observations above show that ppk in Pf0-1 is regulated in response to P_i concentration, but definitive studies of regulation of ppk as part of the Pho regulon in Pseudomonas species are lacking. We sought to determine whether ppk was part of the Pho regulon in P. fluorescens as an initial step toward linking environmental sensing with fitness in soil, given the low concentration of phosphorus (10 ppm, wt/wt) in the soil used in this study (6).

We measured expression of the ppk-lacZ transcriptional fusion in pst and phoB deletion mutants of Pf0-1. Consistent with predictions, ppk transcription in Pf0-1Δpst was generally equivalent to that seen in the wild type under phosphate-limiting conditions (Fig. 6A), and ppk transcription was elevated in the Δpst strain during growth in high-P_i media (Fig. 6B). Transcription of ppk in the ΔphoB mutant remained at or below the level of that observed in the wild type under high-P_i medium conditions and was significantly lower in low-P_i conditions because of an inability to activate the Pho regulon. These data indicate that ppk is regulated by the Pho regulon system in Pf0-1. However, there are indications that ppk regulation is influenced by additional mechanisms. In high-P_i medium, ppk transcription in Pf0-1Δpst is elevated during exponential growth but declines over time. By the 8-h time point, ppk transcription in Pf0-1Δpst is less than threefold higher than in Pf0-1, and by 24 h, expression in Pf0-1Δpst is only a little more than double that in Pf0-1. These data reveal an additional mechanism that suppresses ppk transcription in stationary-phase cells growing in high-P_i medium, which is not dependent on Pst.

Overproduction of poly P reduces competitive fitness in soil.Since the ppk deletion mutant showed reduced competitive fitness in soil, we hypothesized that strains capable of overproducing poly P might have a competitive advantage. Pf0-1Δpst, which accumulates approximately sixfold more poly P than does Pf0-1 because of derepression of the Pho regulon, was competed against Pf0-1. Contrary to predictions, Pf0-1Δpst had a severe competitive defect relative to Pf0-1. One possible explanation for this finding is that derepression of the Pho regulon results in other, unknown changes that impair fitness. Alternatively, producing too much poly P could be deleterious. The CI of Pf0-1Δpst against Pf0-1 in soil was 10-fold lower than that of Pf0-1Δ8-ppk-Km (Fig. 7 and 1A). We predicted that if the latter possibility were true, deletion of ppk in Pf0-1Δpst would improve the competitive fitness to approximately the same level as that of Pf0-1Δ8-ppk-Km, whereas if the former were true, loss of ppk would have minimal impact. The prediction was tested by introducing the ppk mutation used to create Pf0-1Δ8-ppk-Km into Pf0-1Δpst (resulting in Pf0-1ΔpstΔppk) and carrying out soil competition experiments against Pf0-1. We observed a 10-fold improvement in the CI of Pf0-1ΔpstΔppk compared to that of Pf0-1Δpst (Fig. 7), indicating that an environmentally responsive control of poly P synthesis is important for competitive fitness in soil.

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FIG. 7.

Effect of deletion of ppk on Pf0-1Δpst competition with Pf0-1 in sterile loam soil. The average CI of Pf0-1Δpst was approximately 0.008, 0.003, 0.004, and 0.004 on days 1, 2, 4, and 9, respectively. When Δppk was introduced into Pf0-1Δpst, the CI of the resulting double mutant improved significantly, by approximately 10-fold at each time point (P < 0.05). Means and standard errors of the means of at least six replicates are shown. Note that the x axis is not continuous.