Enhanced Tolerance to Naphthalene and Enhanced Rhizoremediation Performance for Pseudomonas putida KT2440 via the NAH7 Catabolic Plasmid

Abstract

In this work, we explore the potential use of the Pseudomonas putida KT2440 strain for bioremediation of naphthalene-polluted soils. Pseudomonas putida strain KT2440 thrives in naphthalene-saturated medium, establishing a complex response that activates genes coding for extrusion pumps and cellular damage repair enzymes, as well as genes involved in the oxidative stress response. The transfer of the NAH7 plasmid enables naphthalene degradation by P. putida KT2440 while alleviating the cellular stress brought about by this toxic compound, without affecting key functions necessary for survival and colonization of the rhizosphere. Pseudomonas putida KT2440(NAH7) efficiently expresses the Nah catabolic pathway in vitro and in situ, leading to the complete mineralization of [¹⁴C]naphthalene, measured as the evolution of ¹⁴CO₂, while the rate of mineralization was at least 2-fold higher in the rhizosphere than in bulk soil.

INTRODUCTION

Bioremediation refers to the degradation of environmental pollutants by free-living and symbiotic microbes and plants (12, 30). The removal of pollutants via biological systems is safer and cheaper than conventional physicochemical treatments (e.g., incineration or landfill for cleaning up contaminated sites), and it is considered environmentally friendly because it often results in complete mineralization of the pollutants without significant alteration of the sites being treated. The main hurdles to achieving effective bioremediation are that the processes are often slow and, in certain cases, the presence of the toxic compound inhibits the proliferation of microbes and prevents the metabolic removal of the target chemicals (30, 44, 52). Therefore, methodologies that enhance the action of microbes in the environment are needed. When pollutant-degrading microorganisms are associated with the rhizosphere of plants and the plant-microbe association is used to remove pollutants, the process is called rhizoremediation, and it often results in enhanced removal of contaminants (5, 30, 44, 53). Rhizoremediation is considered an effective way to increase biodegradation, although the microbes used in rhizoremediation should encode the appropriate degradative routes, express their catabolic potential in situ, and be able to colonize the root system of plants proliferating in the polluted site. In addition, microorganisms for rhizoremediation use must be safe from the clinical, veterinary, and ecological points of view.

Naphthalene is the simplest member of the polycyclic aromatic hydrocarbons (PAHs), a well-known family of widespread environmental pollutants. Naphthalene is toxic for eukaryotic and prokaryotic cells (26, 40, 48) and even for naphthalene-metabolizing microorganisms (2, 23, 37, 39, 41). However, a number of bacteria belonging to different genera have been shown to use naphthalene as a source of carbon and energy (45). Naphthalene is often first oxidized to salicylate, which is further channeled to Krebs cycle intermediates via catechol or gentisate catabolic pathways. The metabolism of naphthalene via catechol has been analyzed in detail in Pseudomonas organisms bearing the catabolic plasmids NAH7 (in Pseudomonas putida G7) and pDTG1 (in P. putida NCIB 9816-4) (6, 9, 47, 54). The gentisate pathway has been found in microorganisms, including in Ralstonia sp. strain U2 (56) and Polaromonas naphthalenivorans CJ2 (49). Bacterial tolerance to naphthalene has been linked to the ability to degrade this toxic compound (37, 49); however, other potential cellular mechanisms are also implicated in naphthalene tolerance, and the tolerance mechanisms used by microorganisms that do not degrade naphthalene remain poorly studied.

Pseudomonas putida KT2440 is an efficient root-colonizing microorganism (36) that does not bear the set of genes needed for naphthalene degradation, but it is amenable to genetic manipulation and it has been declared a biologically safe microorganism by the Recombinant DNA Advisory Committee (19). For these reasons, it is considered a model microorganism to study bioremediation, plant-microbe interactions, chemotaxis, and other relevant processes with the aim of understanding the biology of soil microorganisms (17, 33, 34, 49).

Previous studies reported the potential use of P. putida strains in naphthalene rhizoremediation and focused on bacterium-mediated naphthalene phytoprotection (24, 31, 32). In this study, we have explored the potential of P. putida KT2440 both with and without the NAH7 plasmid and in the context of rhizoremediation of naphthalene-polluted sites. Our broader perspective is based on the transcriptional response to the toxic compound and on its potential in efficient rhizoremediation.

MATERIALS AND METHODS

Bacterial strains, growth conditions, and plasmid transference.

Pseudomonas putida KT2440R is a rifampin-resistant spontaneous variant of KT2440 (17). Isogenic insertional mutants of this strain were obtained from the Pseudomonas Reference Culture Collection (PRCC) (14) and are listed in Table S1 in the supplemental material.

Pseudomonas putida strains were grown in LB medium at 30°C with shaking or in modified M9 minimal medium with glucose (0.5% [wt/vol]), citrate (15 mM), or naphthalene as a carbon source (1, 14). When required, antibiotics were added to reach the following final concentrations: chloramphenicol, 30 μg/ml; kanamycin, 50 μg/ml; and rifampin, 10 μg/ml. Naphthalene was purchased from Sigma-Aldrich and dissolved in methanol to produce a stock solution of 230 mM.

The NAH7 plasmid was transferred via conjugation from P. putida G7 (13) to KT2440R and two isogenic knockout mutants deficient in pqqC (PP0378) or pqqB (PP0379). Transconjugants appeared at a rate of 10⁻³ per recipient and were selected in M9 minimal medium with rifampin and naphthalene as the sole carbon source (NAH7). Transconjugants bearing the NSH7 plasmid were confirmed by fingerprinting after PCR amplification of a 35-mer primer designed based on the KT2440-specific REP sequence (3, 50). More than 99.9% of cells of P. putida KT2440R(NAH7) kept the plasmid upon cultivation for 50 generations in minimal medium without naphthalene and with citrate as a carbon source.

Bacterial growth was monitored by turbidity at 660 nm in a UV-1700 Pharma Spec spectrophotometer (Shimadzu, Kyoto, Japan). Biomass production is expressed as milligrams of cell dry weight per milliliter of culture medium (51).

In rhizosphere assays, bacterial densities are expressed as numbers of CFU per gram of soil and were determined by serial dilution and spreading on plates of selective medium (M9 minimal medium with citrate and rifampin).

Chemical shock assays in liquid culture medium.

Cells were grown in LB medium until cultures reached the mid-exponential growth phase (turbidity between 0.7 and 0.85 at 660 nm). Then, cultures were divided into different aliquots; one was kept as a control, and the other was dosed with increasing concentrations of naphthalene dissolved in methanol to reach a final concentration of 230 μM (note that the same amount of methanol was added to the control cultures). The numbers of viable cells were determined as CFU/ml before the stressor was added and 10, 20, and 40 min after. Assays were run in duplicate and repeated at least three times.

DNA microarrays.

For all assays, cells were grown overnight in M9 minimal medium with glucose and then diluted in the appropriate culture medium without or with naphthalene to reach an initial turbidity of 0.05 at 660 nm. When cultures were in the exponential phase, cells were harvested and processed for RNA extraction, and RNA was used for preparation of fluorescently labeled cDNA (55).

The Pseudomonas putida array (Progenika, Spain) contains 5,539 gene-specific oligonucleotides (50-mer) spotted in duplicate onto γ-amino silane-treated 25- by 75-mm microscope slides and bound to the slide with UV light and heat (55). Hybridization conditions, slide scanning, and data processing were carried out as described previously (15, 43, 55).

Each experiment was repeated 3 to 5 times; therefore, as every gene is represented twice on each slide, the results reported come from at least 6 hybridizations. Data were normalized and filtered to identify genes with statistically significant changes in expression levels and significant P values. An open reading frame (ORF) was considered differentially expressed when the fold change was at least 2, the P value was ≤0.05, and the average intensity of the signal was >64.

Seed attachment and rhizosphere colonization assays.

Corn seeds (Zea mays) were sterilized and inoculated as described previously (18). After 2 days of rehydration, for which seeds were wrapped in a wet filter paper, seeds were planted in pots that contained 1 kg of sterilized soil, either naphthalene-free or supplemented with different amounts of naphthalene.

Cypress (Cupressus sempervirens) rhizosphere colonization assays were performed using young trees whose heights were 30 ± 3 cm (mean ± standard deviation). Inoculation was done by soaking the root mass system in a 1/10-diluted overnight bacterial culture, and then plants were transferred to a pot containing 1 kg of either sterile pristine soil or soil containing increasing concentrations of naphthalene. In all cases, pots were kept under greenhouse conditions for 2 months (16 h light, 25°C ± 1°C, 40% humidity). Samples were taken periodically, cells were separated from roots by vortexing with glass beads, and the numbers of viable cells were determined by plate counting on selective medium as described previously (17).

KT2440R(NAH7) naphthalene in situ mineralization assays.

The NAH7 plasmid-conferred catabolic potential in KT2440R was determined by measuring the mineralization of ¹⁴C-labeled naphthalene in sealed flasks as indicated by the evolution of ¹⁴CO₂, which was trapped in a solution of 1 M NaOH in a beaker placed in the flasks. Microcosms were prepared in tightly sealed Erlenmeyer flasks with 20 g of sterile soil supplemented with 12.5 μCi of [¹⁴C]naphthalene (American Radiolabeled Chemicals, Inc., Saint Louis, MO) and inoculated with 1.5% (vol/wt) of an overnight culture of KT2440R(NAH7). To test different conditions, the study combined two variants: (i) soil with or without 0.05% (wt/wt) unlabeled naphthalene, to guarantee that the concentration of naphthalene was high enough to induce the catabolic pathways, and (ii) flasks with soil in which four sterilized corn seeds, pregerminated for 3 days, were sown to analyze the role of plants in naphthalene degradation. Each flask contained a trap made of 5 ml of 1 M NaOH. Flasks were kept under greenhouse conditions. Samples were taken periodically, and radioactivity measured in an LS6500 multipurpose scintillation counter (Beckman Coulter, Fullerton, CA).

Microarray data accession numbers.

The raw microarray data were deposited in the ArrayExpress Archive database, www.ebi.ac.uk/arrayexpress/ (38), under accession numbers E-MEXP-2823, E-MEXP-3411, and E-MEXP-3412.

RESULTS

Growth and survival of Pseudomonas putida KT2440R in the presence of naphthalene, and the effect of the NAH7 plasmid on naphthalene tolerance.

The growth of P. putida KT2440R was tested in liquid medium (M9 with glucose as a carbon source) with different concentrations of naphthalene, whose maximal solubility in water is 230 μM (29.5 μg/ml). Increasing concentrations of naphthalene were added to the culture medium until saturation. The addition of naphthalene to KT2440R cultures had no bactericidal effect at any of the concentrations tested (data not shown). However, the doubling time of KT2440R cultures was influenced by the presence of naphthalene such that the rapid growth of the strain in the absence of naphthalene (64 ± 2 min) was progressively delayed with the increase in naphthalene concentration and reached a doubling time of 103 ± 4 min when the aromatic compound was supplied at saturation (Fig. 1A). Furthermore, the KT2440R biomass yield was reduced by about half in the naphthalene-saturated medium, from 1.3 to 0.6 mg/ml (Fig. 1B).

Fig 1.

Effects of naphthalene on the growth of P. putida KT2440R and several isogenic knockout mutants, with and without the NAH7 plasmid. (A) Doubling time in M9 minimal medium in the absence of naphthalene or in medium saturated with naphthalene. (B) Biomass production in naphthalene-free medium or naphthalene-saturated medium. Assays were run in triplicate, and a minimum of three independent experiments were carried out. Error bars show the standard error.

When we carried out these experiments with P. putida KT2440R harboring NAH7, we only found a slight difference in growth rates, with doubling times of 75 ± 1 min in the absence of the stressor compound and 96 ± 2 min at a saturating naphthalene concentration. Nevertheless, the presence of the NAH7 plasmid clearly had a positive effect on bacterial biomass production in the presence of naphthalene (Fig. 1B): the biomass of KT2440R(NAH7) cultures was 1.5 mg/ml, and that of the parental strain was only 0.63 mg/ml, a 50% higher yield when the strain bore the plasmid. This effect was not detected in naphthalene-free medium, where KT2440R produced approximately 1.3 mg/ml of biomass regardless of the presence of the plasmid (Fig. 1B).

P. putida KT2440R and P. putida KT2440R(NAH7) transcriptional analysis. (i) Cellular response to naphthalene: transcriptional profiles of P. putida KT2440R grown with and without naphthalene and phenotypic analysis of knockout mutants in naphthalene-upregulated genes.

To study the cellular strategies used by KT2440 to thrive in the presence of naphthalene, we decided to investigate the changes in the transcriptional expression profiles of KT2440R when cells were grown with and without naphthalene (230 μM). We found that in cells growing with naphthalene, 49 genes showed significant changes in expression level (fold change of ≥2 or ≤−2, P < 0.05, signal intensity average, >64) in comparison to their levels in cells growing in the same medium without naphthalene (the complete list of genes is provided in Table S1 in the supplemental material); 42 of these genes were upregulated, and only 7 were downregulated. Functional analysis of the upregulated genes revealed that the corresponding gene products include stress response proteins, transporters, regulators, and proteins involved in metabolism and energy production, as well as hypothetical proteins of unknown function (Fig. 2). Among the downregulated genes, we distinguished four functional subgroups: metabolism, bacterial motility, protein biosynthesis, and hypothetical or unknown function (Fig. 2; also see Table S1). Bibliographic searches revealed that 74% of the upregulated genes and 42% of the downregulated genes had been previously correlated with transcriptional changes in response to other chemicals by Pseudomonas putida (see Table S1).

Fig 2.

Functional distribution of genes differentially expressed in P. putida KT2440R grown in naphthalene-saturated medium. Genes induced in response to naphthalene are represented by hatched bars and downregulated genes by dotted bars. Genes were considered differentially expressed when their transcription level was changed 2-fold or more, with a P value of ≤0.05 and an average signal of >64.

A bank of mini-Tn5 mutants of P. putida KT2440 is available at the Pseudomonas Reference Culture Collection (PRCC). The collection consists of independent mutants with mutations in almost 50% of all ORFs (14). Mutants with mutations in 11 of the upregulated genes were available (see Table S1 in the supplemental material), and we subsequently tested their growth characteristics in naphthalene-free and naphthalene-saturated medium. Two of the mutants, those that harbored a mini-Tn5 insertion in the PQQ coenzyme biosynthesis genes, namely, pqqC (PP0378) and pqqB (PP0379), grew more slowly than the parental strain (Fig. 1A and B). These mutants had doubling times of around 5 h in medium with 230 μM naphthalene versus the doubling time of around 1.7 h for the parental strain under the same conditions (Fig. 1A). When grown with naphthalene, the biomass production was reduced by approximately 80% for both mutants compared to the production with growth in naphthalene-free conditions (Fig. 1B). The deleterious effect of naphthalene for these mutants was overcome when cells carried the NAH7 plasmid, which allowed them to grow faster (doubling time of 2 h approximately) and exhibit biomass production at levels similar to those in naphthalene-free medium conditions (Fig. 1). The remaining mutant strains from the PRCC showed growth rates and biomass production similar to those of the parental KT2440 strain in the presence of naphthalene (data not shown).

(ii) Effect of the NAH7 plasmid on cellular response to naphthalene: transcriptional profile of KT2440R versus that of KT2440R(NAH7) in naphthalene-containing medium.

To explore the putative protective effect of the NAH7 plasmid, we performed a transcriptional analysis of KT2440R(NAH7) and compared it to the parental strain growing in naphthalene-saturated medium. The results revealed no upregulated genes and 22 downregulated genes. Of these downregulated genes, most were involved in central metabolism or signal transduction or encoded outer membrane proteins (see Table S2 in the supplemental material).

By cross-referencing these results with those obtained in microarrays performed with the parental KT2440R grown in naphthalene-free versus naphthalene-saturated medium (see above), we identified a set of 13 naphthalene-upregulated genes whose expression levels were not increased when cells harbored NAH7 (Table 1). Most of these genes (12 out of 13) had been previously shown to be involved in the Pseudomonas stress response at the transcriptional level.

Table 1.

Comparison of gene expression levels under differing conditions

Stress response trigger(s)a	TIGR identifier	Gene product	Fold change under indicated conditionsb
			A	B	C
	PP2422	Alkylhydroperoxidase-like protein	3.6	−2.6
Chloramphenicol	PP2663	FIST N sensory domain-containing protein	8.5	−2.3
Chloramphenicol	PP2664	Sensory box histidine kinase/response regulator	5.4	−2.2
Chloramphenicol	PP2667	ABC efflux transporter permease protein	5.7	−1.7
Chloramphenicol	PP2675	Cytochrome c-type protein	5.8	−2.1
Chloramphenicol	PP2676	Putative periplasmic binding protein	4.5	−1.8
Chloramphenicol	PP2679	Putative quinoprotein ethanol dehydrogenase	2.8	−1.9
Chloramphenicol	PP2680	Aldehyde dehydrogenase family protein	6.4	−1.8
Formaldehyde	PP2695	Transcriptional regulator of the LysR family	2.3	−2.2
O-xylene, toluene, and TNT	PP4250	ccoN-1-cytochrome c oxidase cbb3-type subunit I	3.1	−2.5
O-xylene, toluene, and TNT	PP4251	ccoO-1-cytochrome c oxidase cbb3-type subunit II	2.7	−2
O-xylene, toluene, and TNT	PP4252	ccoQ-1-cytochrome c oxidase cbb3-type CcoQ subunit	3.8	−2.3
O-xylene, toluene, and TNT	PP4253	ccoP-1-cytochrome c oxidase cbb3-type subunit III	2.9	−2.4
	PP0031	Hypothetical protein		−2	−1.6
	PP0056	Oxidoreductase of the GMC family		−2	−2.6
	PP0186	Porphobilinogen deaminase		−2	−1.7
	PP0192	fkl-peptidyl-prolyl cis-trans isomerase FklB		−2	−1.7
	PP0541	Acetyltransferase of the GNAT family		−2	−3
	PP0680	Putative ATP-dependent protease		−2	−2.5
	PP1075	glpK-glycerol kinase		−1.8	−3
	PP2120	Methyl-accepting chemotaxis transducer		−2	−1.8
	PP2292	Hypothetical protein		−2.2	−1.8
	PP2643	Methyl-accepting chemotaxis transducer		−1.7	−2.3
	PP4038	Dihydropyrimidine dehydrogenase		−1.8	−2

^aGene was previously reported as involved in transcriptional stress response to O-xylene (11), toluene (15), formaldehyde (43), TNT (21), or chloramphenicol (21).

^bA, KT2440R grown in minimal medium saturated with naphthalene versus KT2440R grown in naphthalene-free medium; B, KT2440R(NAH7) versus parental KT2440R, both in minimal medium saturated with naphthalene; and C, KT2440R(NAH7) versus parental KT2440R, both grown in medium without naphthalene. For this comparison, we considered only those genes with fold change of ≤−1.8 or ≥1.8, P value of <0.09, and average intensity signal of >64. Full microarray results are available in Tables S1, S2, and S3 in the supplemental material.

(iii) Plasmid NAH7 load effect: transcriptional profile of KT2440R versus that of KT2440R(NAH7) in naphthalene-free medium.

To analyze changes in the gene expression profile of KT2440R due to the presence of the NAH7 plasmid, the transcriptional profile of KT2440R(NAH7) was compared with that of the plasmid-free strain when grown in M9 minimal medium without naphthalene. Data analysis showed 2 upregulated genes and 17 downregulated genes when the plasmid was present (see Table S3 in the supplemental material). The two upregulated genes (PP0038 and PP0354) encode proteins of unknown function, whereas the downregulated genes encode proteins involved in metabolism, cellular transport, and gene regulation (see Table S3). This indicates that the NAH7 plasmid exerts only a modest effect on global gene expression, since only 0.4% of genes were altered.

Finally, we compared all the differentially expressed genes found on the transcriptional profiles of KT2440R(NAH7) and the plasmid-free strain both in the presence of naphthalene and in its absence (see above); the results showed a set of 11 genes, which lack any commonality in regard to function, that are downregulated in KT2440R(NAH7) due to the presence of the plasmid and regardless of whether naphthalene is present in the culture medium.

In situ phenotypic analysis of KT2440(NAH7).

To test how the carriage of NAH7 affects phenotypical bacterial traits of interest, such as seed colonization, survival in the rhizosphere, and specifically, in situ naphthalene catabolic route expression, KT2440R(NAH7) was subjected to a battery of assays.

(i) Seed colonization.

Maintenance of the desired KT2440 ability to colonize seed was tested for KT2440R(NAH7) using corn seeds and compared with the colonization ability of the plasmid-free strain. The results revealed that the number of viable bacteria adhering per seed (about 1 to 3 × 10⁵) was similar in both cases and that their persistence and proliferation during corn germination were almost identical (data not shown).

(ii) Survival in the rhizosphere.

KT2440R(NAH7) was subjected to a series of experiments in the rhizosphere of corn and cypress farmed in soil containing different concentrations of naphthalene (0.05% naphthalene [wt/wt] in the assays with corn and 0.05, 0.1, and 0.15% [wt/wt] naphthalene with cypress) or in pristine soil and compared with those obtained for the plasmid-free strain. The pots were maintained under greenhouse conditions as described in Materials and Methods. The results showed similar survival ratios and bacterial population evolution in all cases regardless of the presence of naphthalene in the soil or plasmid carriage (not shown).

(iii) In situ naphthalene mineralization through rhizoremediation.

This set of assays was designed to test (i) in situ expression of the Nah pathway and (ii) the effect of the rhizosphere on bacterial catabolic activity. With these aims, we conducted the following experiment: sterilized bulk soil was supplemented with 0.05% (wt/wt) naphthalene and inoculated with KT2440R(NAH7), and corn seedlings were either sown or not sown. The respective negative controls were set up in naphthalene-free soil, and in all cases, 0.625 μCi of [¹⁴C]naphthalene was added per gram of soil. Data obtained after 7 days of incubation under greenhouse conditions (Fig. 3) showed ¹⁴CO₂ evolution as a readout of naphthalene mineralization. In both cases, we found that the total amount of naphthalene mineralized per gram of soil after 1 week was doubled when bacteria were present in the corn rhizosphere versus the amount mineralized in the vegetation-free soil.

Fig 3.

In situ naphthalene mineralization. Assays were performed in microcosms using [¹⁴C]naphthalene and CO₂ traps to test naphthalene mineralization by P. putida KT2440R(NAH7) in bulk soil and in the corn rhizosphere, both with naphthalene added at 0.05% (wt/wt). Naphthalene-free controls were run in parallel (data not shown). Assays were run in duplicate, and a minimum of three independent experiments were carried out. Error bars show the standard error.

DISCUSSION

P. putida KT2440 tolerance to naphthalene.

The toxic effect of naphthalene on Pseudomonas putida KT2440R growth was noticeable in vitro, where saturating concentrations (230 μM) of this compound caused a modest growth rate retardation and lower biomass production, although no bactericidal effect was detected. The presence of naphthalene in the rhizosphere or bulk soil did not alter the behavior of Pseudomonas putida KT2440R. The tolerance of KT2440 for naphthalene was somewhat surprising considering that even microorganisms able to degrade naphthalene are more sensitive to it, like Polaromonas naphthalenivorans, whose growth was completely inhibited in medium containing only 78 μM naphthalene, whereas for a nondegrading variant of this bacterium, the naphthalene tolerance limit dropped to 23 μM (41). The ability to degrade naphthalene was previously considered an essential mechanism for conferring naphthalene tolerance (37, 41); however, Kang and colleagues (28) reported a direct correlation between antioxidant enzyme levels and naphthalene biodegradation in the Pseudomonas sp. strain As1. We have found that P. putida KT2440R activates a broad stress response program in the presence of naphthalene, a response which resembles that reported for other toxic compounds in this bacterium (see below).

When KT2440R bore the NAH7 plasmid, the deleterious effect of naphthalene on biomass production disappeared (Fig. 1B) and the transcriptomic response to naphthalene was notably softened, since a number of the genes activated in the presence of naphthalene did not require such activation when cells harbored NAH7. Also, pqqC and pqqB mutants overcame their hypersensitivity to naphthalene when the catabolic plasmid NAH7 was carried. These findings agree with the hypothesis that naphthalene degradation itself has a role in cellular protection against this compound (37, 41), but they do not exclude other defensive actions or explain naphthalene resistance in bacteria that do not degrade naphthalene.

P. putida KT2440R stress response induced by naphthalene.

Of the 42 genes upregulated when cells were grown in the presence of naphthalene, 31 were previously found to be involved in the transcriptional response of P. putida to one or more toxic chemical compounds, such as chloramphenicol (21), toluene (11, 15), formaldehyde (43), and o-xylene (11), and chemicals that produced oxidative stress (our unpublished results). Therefore, it appears that naphthalene tolerance is also a multifactorial response involving several bacterial transcriptional regulators and that several response strategies are employed, including the active extrusion of the toxic drug to reduce the effective intracellular concentration, as suggested by the robust activation of transporters encoding genes such as PP2667 (fold change, 5.7) and PP5066 (fold change, 6.3), both of which were also involved in the cellular response to chloramphenicol in KT2440 (21). Nevertheless, we did not find a clear reduction in growth rate or biomass production when these mutants were analyzed under growth conditions that included naphthalene. This result could be explained by functional redundancy between the two transporters, similar to what has been reported previously in Pseudomonas (20, 25, 43).

A second strategy involves the reparation of cellular damage caused by the toxic compound. At least two genes encoding universal stress proteins were upregulated when this bacterium was grown in naphthalene-containing medium: PP2187, whose expression level was also altered in the presence of o-xylene and toluene (11), and PP2648, which is involved in the toluene and formaldehyde transcriptional response (15, 43).

A new role for the pyrroloquinoline quinone (PQQ) coenzyme in naphthalene tolerance was revealed by the upregulation of several genes involved in its biosynthesis, localized at different positions in the bacterial chromosome, and confirmed by the slower growth and lower biomass production of pqqC and pqqB mutants in naphthalene-saturated medium. PQQ is also known to be involved in stress endurance in P. putida, which has been linked to the oxidative stress response (21). This seems also to be the case in other bacteria, such as Deinococcus radiodurans (35, 42) and Bradyrhizobium japonicum (7).

NAH7 affects the P. putida KT2440R transcriptomic profile but does not alter its relevant rhizosphere-colonizing abilities.

Plasmid acquisition by bacteria often imparts an important evolutionary advantage, such as antibiotic or heavy metal resistance or new metabolic abilities, but it is also well established that, especially in the absence of selective pressure, plasmid carriage represents a cost for the cells (4, 8, 10, 22, 46) and alters host chromosome gene expression through mechanisms not yet fully understood. Shintani and colleagues (46) carried out a transcriptional analysis of several species of Pseudomonas and found that dozens of genes were differentially transcribed as a mere consequence of pCAR1 carriage. We found that NAH7 carriage has only a modest impact on KT2440R chromosomal gene expression, altering the expression of only 0.4% of total genes. The difference in the costs imposed by the two plasmids may be due to their different sizes, 199 kb for pCAR1 versus 79 kb for NAH7, or the presence in pCAR1 of interference genes that are not present in NAH7. Whatever the molecular mechanism might be, the fact that KT2440(NAH7) retains its phenotypic characteristics considered “desirable” for seed attachment and rhizosphere colonization of different plants is of biotechnological interest in rhizoremediation.

Expression of the NAH7 plasmid-mediated catabolic naphthalene pathway in KT2440R.

In situ expression of nah genes in a heterologous host was reported in the endophytic strain P. putida VM1441 (24); however, the existence of multiple factors that can influence gene expression/enzyme activity in situ requires experimental verification for each strain and plasmid, as well as for each specific ecological niche. In this regard, Shintani and colleagues (46) described the finding that carbazole degradation by Pseudomonas carrying pCAR1 occurred in aquatic systems but was impaired in soil microcosms. In contrast, our study shows that naphthalene degradation by KT2440R bearing the NAH7 plasmid was efficient both in vitro in liquid cultures and in situ during bioremediation both in bulk soil and in the rhizosphere. We have found that naphthalene mineralization by KT2440R(NAH7) takes place in soil, an observation in line with the degradation of other aromatic compounds in the same matrix, e.g., polychlorinated biphenyls (5), PAHs (6, 27), p-methylbenzoate (16), and atrazine (29).

Rhizosphere effect on naphthalene degradation by KT2440R(NAH7).

This work clearly demonstrates how bioremediation can be greatly improved when a suitable microorganism is present in the rhizosphere. We found more than double the amount of naphthalene mineralized per gram of rhizosphere soil compared to that in bulk soil after 1 week of incubation. The effect of the rhizosphere on microbial biodegradation enhancement is usually explained by the contributions of root exudates to the number of microbes and to stimulation of bacterial metabolic enzymes together increasing bacterial dispersion mediated by the root system (30, 44). Kuiper and colleagues (31) found that grass roots acted as bioinjectors of bacteria, consequently favoring dissemination of microbes as a factor for degradation enhancement, and Molina and colleagues (36) reported a marked increase in bacteria in the rhizosphere because plant exudates stimulated microbial growth and metabolic activity. Our results support the idea that these combined effects promote rhizoremediation of naphthalene in situ.

In summary, we have explored the effectiveness of P. putida KT2440 for use in naphthalene bioremediation. We chose this strain because it readily colonizes the rhizosphere of a wide range of plants (36), is safe, and exhibits pollutant resistance. We have provided insight into the molecular mechanisms behind naphthalene tolerance in KT2440R(NAH7), which exhibits degradative potential both in vitro and in situ. Plasmid carriage had a low impact on the chromosomal gene expression of KT2440R and did not alter the traits desirable for rhizoremediation, such as survival in the rhizosphere; in fact, the NAH7 plasmid enhanced naphthalene mineralization.