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Pseudomonas fluorescens ATCC 13525 Containing an Artificial Oxalate Operon and Vitreoscilla Hemoglobin Secretes Oxalic Acid and Solubilizes Rock Phosphate in Acidic Alfisols

Pseudomonas fluorescens ATCC 13525 Containing an Artificial Oxalate Operon and Vitreoscilla... Oxalate secretion was achieved in Pseudomonas fluorescens ATCC 13525 by incorporation of genes encoding Aspergillus niger oxaloacetate acetyl hydrolase (oah), Fomitopsis plaustris oxalate transporter (FpOAR) and Vitreoscilla hemoglobin (vgb) in various combinations. Pf (pKCN2) transformant containing oah alone accumulated 19 mM oxalic acid intracellularly but secreted 1.2 mM. However, in the presence of an artificial oxalate operon containing oah and FpOAR genes in plasmid pKCN4, Pf (pKCN4) secreted 13.6 mM oxalate in the medium while 3.6 mM remained inside. This transformant solubilized 509 mM of phosphorus from rock phosphate in alfisol which is 4.5 fold higher than the Pf (pKCN2) transformant. Genomic integrants of P. fluorescens (Pf int1 and Pf int2) containing artificial oxalate operon (plac-FpOAR-oah) and artificial oxalate gene cluster (plac-FpOAR-oah, vgb, egfp) secreted 4.8 mM and 5.4 mM oxalic acid, released 329 mM and 351 mMP, respectively, in alfisol. The integrants showed enhanced root colonization, improved growth and increased P content of Vigna radiata plants. This study demonstrates oxalic acid secretion in P. fluorescens by incorporation of an artificial operon constituted of genes for oxalate synthesis and transport, which imparts mineral phosphate solubilizing ability to the organism leading to enhanced growth and P content of V. radiata in alfisol soil. Citation: Yadav K, Kumar C, Archana G, Naresh Kumar G (2014) Pseudomonas fluorescens ATCC 13525 Containing an Artificial Oxalate Operon and Vitreoscilla Hemoglobin Secretes Oxalic Acid and Solubilizes Rock Phosphate in Acidic Alfisols. PLoS ONE 9(4): e92400. doi:10.1371/journal.pone.0092400 Editor: Adam Driks, Loyola University Medical Center, United States of America Received September 8, 2013; Accepted February 21, 2014; Published April 4, 2014 Copyright:  2014 Yadav et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors have no support or funding to report. Competing Interests: The authors have declared that no competing interests exist. * E-mail: gnaresh_k@yahoo.co.in . These authors contributed equally to this work. efficient. An alternative approach is to use organic acid secreting Introduction microorganisms to solubilize RP in alfisols [11,12]. Low molecular Acidic soils occupy about 30% of world’s ice-free land area and weight organic acids are known to be most effective in chelation of are considered important for future agricultural development, as Fe and Al and thus solubilization of P [13–15]. Plants secreting presently only 24.2% of the total land area of the world is piscidic, citric and oxalic acid in root exudates show increased potentially arable [1,2]. Phosphorous (P) is the second major growth and shoot P content [15–17]. Improved growth of pigeon nutrient in the soil limiting plant growth after nitrogen (N). In pea plants as compared to other crops in alfisols is attributed to acidic alfisols, plant growth and crop yields are limited by low P piscidic acid mediated FeP solubilization [7]. availability combined with high refixation of applied P [3–5]. Due Although many microorganisms are known to solubilize mineral to the high reactivity of soluble inorganic phosphate (Pi) with Al, phosphates [6], majority of them solubilize Ca-P and very few Fe and Ca, most soil P exists in the bound form and very low (, microorganisms are known to solubilize Fe-P and Al-P [18]. As a 10 mM) amount of free P is available for plant growth in soil result most phosphate solubilizing microorganisms are ineffective solution [6]. In acidic alfisols, P is mainly complexed with Fe and in supplying P to plants grown in alfisols. For instance, Enterobacter Al [7,8] which are difficult to dissolve by simple acidification. asburiae PSI3, a gluconic acid secreting bacterium, solubilizes P However, organic acids have a demonstrated chelation capacity, from alkaline vertisols [19] but does not release free P from alfisols making them potentially ideal for releasing P from alfisols [5]. supplemented with RP [5]. This has been attributed to the nature Application of rock phosphate (RP) as a P fertilizer to acidic soils and amount of the organic acid secreted by the microorganism. It is considered as an important strategy for enhancing plant P has been shown that P is released efficiently from alfisol amended nutrition [9]. RP is rich in mineral phosphate complexes which with RP when treated with organic acids such as oxalic and citric could be solubilized in acidic conditions in alfisols. However, as compared to gluconate, succinate and malate which are more partially acidulated rock phosphate (PARP) obtained by mild acid effective in vertisol or alkaline soils [5]. About 5–10 mM oxalic treatment of RP, renders it more easily available for plants [10], acid solubilizes P from RP in acidic alfisol which could be indicating that mere addition of untreated RP to alfisols is not accredited to the excellent chelating properties of this acid that PLOS ONE | www.plosone.org 1 April 2014 | Volume 9 | Issue 4 | e92400 Oxalate Secretion in Pseudomonas fluorescens plausibly hinders refixation of P by chelation of Fe and Al ions Construction of artificial oxalate operon [5,15,20]. Addition of oxalate to different phosphate rocks and RNA was isolated from A. niger by TRizol method (Sigma soils resulted in efficient mineral phosphate solubilization [5,15]. Aldrich, India) and oah gene was amplified from mRNA using gene Only a few bacterial strains belonging to Bacillus subtilis, specific primers (Integrated DNA Technology, USA) (Table S2). Pseudomonas fluorescens, Arthrobacter spp. and Micrococcus spp. are Amplicon was digested with BamHI/PstI and cloned in BamHI/ known to secrete oxalic acid that too in very low amounts (, PstI digested plasmid pUCPM18Gm under lac promoter (Figure 2 mM) [21,22]. High levels of oxalic acid secretion are reported in S1). The resultant construct was designated as pKCN2. The RNA fungi such as Aspergillus niger, A. fumigatus, Botrytis cinerea, Fomitopsis isolated from F. plaustris was amplified using gene specific primers plaustris and Penicillium spp. [21]. In fungi, oxalic acid synthesis is of FpOAR gene (Table S2). Amplicon digested with SacI/BamHI mediated by the cytosolic enzyme, oxaloacetate acetyl hydrolase was cloned upstream of oah gene, in SacI/BamHI digested plasmid (OAH), which breaks down oxaloacetate into oxalic and acetic pKCN2 under lac promoter to construct artificial oxalate operon, acids [23–26]. A. niger OAH is a pH inducible enzyme belonging to designated as pKCN4 (Figure S1). Amplicon of 2.8 kb containing PEP mutase/isocitrate lyase super family and requires divalent lac-FpOAR-oah was amplified with forward lac primer (Table S2) metal ions for catalysis [23,25–27]. In bacteria such as Oxalobacter and oah reverse primer from pKCN4, using XT-20 polymerase formigens oxalate specific transporter (OxlT) is responsible for (Merck Genei, India) and was cloned in SmaI digested integration oxalate uptake and helps in ATP generation [28,29]. On the other vector pUC18T-mini-Tn7T-Gm-eyfp. hand, a high amount of oxalate secretion in fungi is mediated by efficient oxalate transporter [30]. F. plaustris is a wood rotting Construction of artificial oxalate gene cluster (plac- fungus and degradation of wood is promoted by oxalic acid FpOAR-oah, vgb, egfp) secretion with the help of an oxalate transporter encoded by pUCVHb-egfp plasmid was digested with PvuII to obtain 3.2 kb FpOAR gene. insert containing vgb and egfp genes. Insert was cloned in pKCN5 Oxygen is present in limited amounts in the rhizosphere which digested with NheI and end filled using Klenow fragment (Thermo could limit the colonization and survival of rhizobacteria [31]. The Scientific, USA). The resultant construct (artificial oxalate gene obligate aerobic bacterium, Vitreoscilla, synthesizes elevated quan- cluster) containing F. plaustris FpOAR and A. niger oah genes under tities of homodimeric hemoglobin (VHb) under hypoxic growth lac promoter, vgb gene under its natural oxygen sensitive promoter conditions which allows improved growth under microaerobic and egfp under rrnB promoter in pUC18T-mini-Tn7T-Gm-eyfp conditions when dissolved oxygen is less than 2% of air saturation vector, was designated as pKCN7 (Figure S1). All the plasmids [32,33]. Expression of vgb gene encoding VHb protein in were transformed in Pf13525 using modified NaCl/CaCl method heterologous hosts often enhances growth and metabolism by [39] and integration in the genome of Pf13525 was done by facilitating oxygen transfer to the respiratory membranes [34]. transformation method [40]. Beneficial effect of vgb overexpression for improved bacterial growth has been demonstrated in plant associated bacteria [31]. Physiological and analytical experiments Fluorescent pseudomonads are well-known plant-growth pro- Bacterial inoculum was used to inoculate Tris rock phosphate moting rhizobacteria with root colonization and efficient biocon- (TRP) minimal medium [41] and alfisol soil medium (containing trol properties [35,36]. The present study deals with the genetic 0.5 g/ml alfisol in sterile medium containing 100 mM glucose, modification of Pseudomonas fluorescens ATCC 13525 for oxalic acid 10 mM KNO , micronutrient cocktail and 30 mg RP/g of soil) for secretion by the incorporation of A. niger oah and F. plaustris FpOAR batch studies in 150 ml conical flask containing 30 ml of genes and determination of its effect on its mineral phosphate inoculated medium. The culture supernatants collected at the solubilizing (MPS) ability and growth promotion of mung bean end were used for extracellular organic acid analysis by (Vigna radiata) plants in acidic alfisols. Additionally, vgb gene was Prominence UFLC (Shimadzu, Japan) and P estimation [42]. incorporated as a part of an artificial operon containing the oah Cell free extract was used for intracellular organic acid analysis. and FpOAR genes to enhance the survival and colonization of organism in the soil environment. Incorporation of the artificial MPS ability of Pf13525 transformants and integrants oxalate operon in P. fluorescens ATCC 13525 resulted in secretion MPS ability of Pf13525 transformants and integrants was of high amounts of oxalate which in turn released P from RP in determined on TRP minimal medium plates (containing 100 mM acidic alfisols. Incorporation of vgb gene along with artificial Tris buffer pH-8.0, 1% methyl red, 1.8% agar and 50 mM oxalate operon resulted in better colonization and improved plant glucose) with RP as the sole P source (1mg/ml), respectively. parameters in acidic alfisols. Saline washed bacterial inoculum (5 ml) was spot inoculated on plates and incubated at 30uC. Phosphate solubilization and acid Materials and Methods secretion was determined by monitoring the growth and red zone of acidification. Bacterial strains, plasmids and media The plasmids, bacterial and fungal strains used in this study are shown in Table S1. Routine DNA manipulations were done with OAH assay E. coli DH10B (Invitrogen, Carlsband, CA, USA) as a host using Cells grown in M9 minimal medium were used for cell free standard molecular biology protocols [37]. pUC18T-mini-Tn7T- extract preparation and OAH enzyme activity measurements were Gm-eyfp was generously gifted by Dr. H. P. Schweizer, Colorado done by the method described by Lenz et al (1976) [23]. OAH State University, USA (Table S1) [38]. P. fluorescens ATCC 13525 enzyme specific activity was expressed in nmole per minute per mg (Pf13525) and its plasmid derivatives were grown at 30uC and total protein. Total protein was estimated using a modified maintained on Pseudomonas agar (Hi Media, India) containing Lowry’s method [43]. One unit of enzyme activity was defined as 50 mg/ml ampicillin and 10 mg/ml gentamycin as and when the amount of protein required to convert 1 nmole of substrate per required. Fungal cultures were grown in minimal medium at 27uC minute. [25,30]. PLOS ONE | www.plosone.org 2 April 2014 | Volume 9 | Issue 4 | e92400 Oxalate Secretion in Pseudomonas fluorescens Figure 2. Effect of genetic manipulation on MPS phenotype, oxalic acid secretion and P release by Pf13525 strains. (A) Phenotype of Pf13525 strains on TRP-methyl red agar plates containing 50 mM Tris-HCl and 100 mM glucose. Red zone indicates acidification of the medium; (B) Oxalic acid secretion and (C) P released by Pf13525 integrants and transformants in TRP medium (with 50 mM Tris HCl) and alfisol soil containing 100 mM glucose as carbon source. Results are given as mean 6 SD of four to six independent observations where ***, P, 0.0001; ns, non-significant. doi:10.1371/journal.pone.0092400.g002 Figure 1. OAH activity, growth and pH profiles of Pf13525 wild type and genetically modified strains. (A) OAH activity in M9 minimal medium. Results are given as mean 6 SD of three independent observed for bacterial colonization by using Confocal laser observations. Values are compared with Pf(pKCN2), **, P , 0.0067; ns, scanning microscopy (LSM 700 Carl Zeiss, GmbH). non-significant; (B) Growth profile and (C) pH profile in TRP minimal medium containing 100 mM glucose as carbon source. O.D. and 600nm pH values at each time point are represented as the mean 6 SD of four Data analysis to six independent observations. Physiological experiments were done in three to four indepen- doi:10.1371/journal.pone.0092400.g001 dent replicates for batch culture study. Data are expressed in mean with standard deviation. In plant experiments, three independent Plant experiments triplicate studies were carried out. Differences in mean values were Plant studies were done in Murashige and Skoog medium as determined using general analysis of variance (ANOVA) and well as in alfisol soil (containing 10 mg/g of RP). Soil analysis was linear regression analysis. The statistical analysis of all the done from Pulse Research Station (Anand Agriculture University, parameters has been done using Graph Pad Prism (version 5.0) Vadodara) and was found to contain 0.085% organic carbon, software. 165.1 kg/ha total nitrogen, 262.4 kg/ha available K and 17.9 kg/ ha available P. V. radiata (mung bean) plant studies were done as Results described in [44] and plant parameters such as lengths of the main root and shoot, dry weight and P content were monitored. Effect of genetic modifications on OAH activity, growth Molybdate-blue method [45] was used to determine P content. and MPS phenotype of Pf13525 th Root colonization of Pf13525 integrant was observed on 5 and Pf (pKCN2) and Pf (pKCN4) (Table S1) transformants showed th 10 day after inoculation and different sections of root were around 230 U/mg of OAH activity while integrants Pf int1 and Pf PLOS ONE | www.plosone.org 3 April 2014 | Volume 9 | Issue 4 | e92400 Oxalate Secretion in Pseudomonas fluorescens Figure 3. Root colonization study of V. radiata inoculated with Pf int2. (A) and (B) colonization study in Murashige-Skoog (MS) medium th th th th (hydroponics study) on 5 and 10 day respectively; (C) and (D) in alfisol soil (pot experiment) on 5 and 10 day, respectively, by Confocal Laser Scanning Microscopy (CLSM). Left panel shows fluorescence imaging, middle panel shows bright field images and rightmost panel shows overlapped images. doi:10.1371/journal.pone.0092400.g003 int2 showed about 165 U/mg of OAH activity in M9 minimal Effect of genetic modifications on organic acid secretion medium containing 100 mM glucose (Figure 1A). On the other by Pf13525 hand, Pf13525 and the vector control did not show detectable To study the effect of increased OAH activity on organic acid OAH activity as the gene is absent in the organism. Growth of Pf secretion in genetically modified Pf13525 strains, the cell lysate (pKCN4) transformant was not significantly different than the and extracellular culture supernatants were analyzed for oxalic untransformed strain in 50 mM Tris-HCl (pH 8.0) medium acid levels using HPLC. Pf (pKCN2) transformants carrying the containing 100 mM glucose as carbon source and RP as the sole oah gene showed the highest intracellular accumulation of oxalic P source (Figure 1B). However, the Pf (pKCN2) transformant acid up to 19.1 mM, but secreted a relatively less amount and the integrants grew slowly and reached 0.28 O.D after 600 nm (1.2 mM) in the medium (Figure 2B). On the other hand, Pf 168 h. Pf (pKCN4) transformant was most effective in acidification (pKCN4) transformants possessing artificial oxalate operon, of the medium from pH 8.0 to 4.2 while Pf (pKCN2) did not consisting of oah gene along with the fungal oxalate transporter, decrease the pH to less than 7.0 (Figure 1C). Although growth of accumulated only 3.6 mM oxalic acid and secreted 13.6 mM in the integrants was slower as compared to the pKCN4 transfor- the medium. Genomic integrants of Pf13525, int1 and int2 mant, they were effective at reducing the pH of the medium secreted 4.1 and 4.7 mM of oxalic acid, respectively, while indicating organic acid secretion. Further, organic acid mediated intracellular levels were 2.6 and 3.1 mM, respectively (Figure 2B). acidification by genetically modified Pf13525 strains was observed In order to study the organic acid secretion in soil conditions, on TRP plates. Pf13525 and Pf (pKCN2) transformant did not alfisol soil supplemented with 100 mM glucose and 30 mg RP/g show red zone of acidification while Pf (pKCN4) transformant, Pf of soil was inoculated with genetically modified Pf13525 strains int1 and Pf int2 acidified TRP agar plate containing 100 mM and the oxalate secreted in the soil solution was estimated. As seen glucose as carbon source and 50 mM Tris HCl (pH 8.0) in Figure 2B, in agreement with the TRP medium studies, in (Figure 2A). alfisol Pf (pKCN4) secreted 15 mM of oxalate. Oxalate secretion PLOS ONE | www.plosone.org 4 April 2014 | Volume 9 | Issue 4 | e92400 Oxalate Secretion in Pseudomonas fluorescens in both media abundant colonization was seen on the root surfaces, root tips and at branching points and the colonization th decreased on 10 day (Figure 3). Effect of inoculation of genetically modified Pf13525 strains on growth parameters of V. radiata Inoculation of V. radiata with Pf int1 and Pf int2 in pot experiments with unsterilized alfisol soil supplemented with RP showed better growth (root and shoot length) and increased root and shoot dry weight. Pf int1 and Pf int2 inoculations showed 1.7 and 1.9 fold increase in root length, 1.3 and 1.4 fold increase in shoot length of V. radiata, respectively, as compared to wild type inoculations (Figure 4A). Pf int1 and Pf int2 inoculations resulted in 1.5 and 2 fold increase in root dry weight and 1.3 and 1.4 fold increase in shoot dry weight, respectively (Figure 4B). P content in Pf int1 and Pf int2 inoculations increased in shoot and root by 1.8 and 2.1 fold, respectively, as compared to plants inoculated with wild type strain (Figure 4C). The improvement in plant parameters is correlated with the amount of oxalic acid secreted by Pf int1 and Pf int2. Discussion Growth and yields of crops in alfisols are low due to acidic pH, aluminum toxicity and limited amount of available P, with high P refixation capacity of the soil [46]. P is strongly complexed with Fe and Al in alfisols, which are difficult to solubilize by mineral acids. Several lines of evidences have shown that certain low molecular weight organic acids can release P from Fe-P and Al-P complexes. Among several organic acids tested, oxalic and citric acid solubilized RP in alfisol [5]. Only few bacteria are known to naturally secrete oxalate but do so in very low amounts as compared to several fungi which secrete oxalate in molar amounts [21]. Thus, the present work was aimed at genetic modification of P. fluorescens so as to enable it to secrete high amounts of oxalic acid and render it proficient at solubilizing P from RP amended alfisol. This bacterium was chosen since fluorescent pseudomonads are recognized as plant growth promoting bacteria with efficient root Figure 4. Plant parameters and P content of V. radiata plants colonizing ability [35,36]. The strategy used was to express the key inoculated with Pf13525 wild type and genetically modified strains. (A) Plant length; (B) Dry weight and (C) P content of enzyme oxaloacetate acetyl hydrolase (OAH) for oxalate synthesis inoculated V. radiata plants in alfisol soil. Values are represented as from a fungal system. Presence of this enzyme for oxalate Mean 6 SD of n = 9 observations for plant length and weight while biosynthesis has not been reported so far from bacteria. In order n = 4 to 8 for P content analysis. *, P , 0.05; **, P,0.01; ***, P,0.001; ns, to enable the OAH transformants to secrete the oxalate in the non-significant as compared to the uninoculated control. extracellular milieu, an oxalate transporter from another fungal doi:10.1371/journal.pone.0092400.g004 system was deployed. by integrants in alfisol was also comparable to that on TRP P. fluorescens ATCC 13525 transformant harboring oah gene medium. Wild type Pf13525 secreted 2.0 mM gluconic acid under alone (without the heterologous transporter system) secreted low similar growth conditions but did not show either oxalate amount of oxalic acid (Figure 5). The ability to secrete the accumulation or secretion. All genetic modifications resulted in a organic acid in the cell free supernatant may be attributed to the decrease in gluconic acid secretion. resident dicarboxylate transporters (DctA and DctB) [47]. Secretion of oxalic acid by P. fluorescens ATCC 13525 has been shown in response to Al toxicity in presence of external citrate Effect of genetic modifications of Pf13525 on MPS ability [48], suggesting that the resident dicarboxylate transporters are In TRP minimal medium, Pf (pKCN2), Pf (pKCN4), Pf int1 and functional. Pf (pCNK4) transformant harboring artificial oxalate Pf int2 released 62 mM, 217 mM, 152 mM and 155 mM of P from operon containing oah and the fungal transporter FpOAR showed RP, respectively (Figure 2C). In alfisol soil medium containing enhanced oxalate secretion (Figure 5) indicating more efficient 100 mM glucose as the carbon source and supplemented with transport. The transformant harboring artificial oxalate operon 30 mg RP/g of soil, Pf (pKCN4), Pf int1 and Pf int2 released also exhibited MPS phenotype in minimal medium containing RP 509 mM, 329 mM and 352 mM levels of P, respectively as the P source and in alfisol. Attaining MPS phenotype in alfisol (Figure 2C). by oxalate secretion is supported by the fact that 5–10 mM oxalate solubilizes significant amount of RP in alfisols [5]. The oxalic acid Root colonization study secreted by the transformant is also expected to be effective in Root colonization ability of Pf int2 was observed in V. radiata releasing P from alkaline vertisols which contain high amount of th plants in Murashige-Skoog’s medium and alfisol soil. On 5 day, Ca-P [41]. PLOS ONE | www.plosone.org 5 April 2014 | Volume 9 | Issue 4 | e92400 Oxalate Secretion in Pseudomonas fluorescens Figure 5. Schematic representation of the genetic modifications of Pf13525 and their effect on oxalic acid accumulation and secretion. doi:10.1371/journal.pone.0092400.g005 Integration of genes in to the genome is a preferred method for levels of oxalate accumulation and secretion in the genomic genetic manipulation of bacteria for environmental applications as integrants of artificial oxalate operon as compared to the plasmid compared to plasmid transformation, due to increased stability of transformants (Figure 5). However, the amount of oxalate the integrant, absence of antibiotic resistance genes, lack of secreted in the integrants was sufficient to solubilize RP in horizontal transfer and reduced metabolic load [49]. However, buffered medium as well as in alfisols. genomic integrants have the limitation of single copy expression Hydroponic and alfisol soil experiments of V. radiata inoculated leading to weak phenotype. This was reflected in the decrease in with genomic integrants demonstrated enhanced root colonization PLOS ONE | www.plosone.org 6 April 2014 | Volume 9 | Issue 4 | e92400 Oxalate Secretion in Pseudomonas fluorescens and plant growth as compared to the vector control suggesting that improved plant growth and P content. The present work genetic manipulation supported colonization and survival in the demonstrates the potential of oxalic acid secretion in mineral rhizosphere. On the other hand, E. asburiae PSI3, which is efficient phosphate solubilization by rhizobacteria. in releasing P from alkaline vertisols mediated by secretion of high levels of gluconic acid, did not improve the growth of V. radiata in Supporting Information alfisol [5]. Significant enhancement in plant growth parameters Figure S1 Schematic representation of arrangement of and P content in Pf int2 inoculations as compared to Pf int1 could genes in plasmid constructs used in this study. (A) be attributed to the presence of VHb which is known to improve Expression plasmid and (B) Integration plasmid constructs. the metabolism of bacteria under microaerobic conditions. Squares denote the genes, operon and gene clusters cloned in Similarly, presence of VHb in Rhizobium etli increased nitrogenase the vector backbone. activity and N content in bean plants [31]. This suggests that (TIF) metabolism of bacteria in the rhizosphere corresponds to that under microoxic conditions. Since oxalate is also implicated in Table S1 Plasmids, bacterial and fungal strains used in alleviation of Al toxicity to plants [50] and organic acid secreting this study. Amp = ampicillin; Gm = gentamycin; r = resistance. rhizobacteria are known to promote plant growth by multiple (DOCX) processes [51], it may be hypothesized that the genetically modified strain could be beneficial to plants in diverse soil Table S2 List of primers used in this study. Underlined sequences represent restriction enzyme sites used for cloning and conditions. To summarize, in this study we report the genetic manipulation sequences in italics indicate universal ribosome binding site (RBS). of rhizosphere colonizing bacterium for the secretion of oxalic acid (DOCX) with the aim of imparting it the ability to carry out mineral phosphate solubilization (MPS) from acidic alfisol to enhance P Acknowledgments availability to plants. Pf13525 harboring oah gene resulted in CK was provided with a fellowship from the Council for Scientific and intracellular accumulation of high amounts of oxalic acid while Industrial Research, (CSIR) New Delhi, Government of India. incorporation of an artificial oxalate operon, containing addition- ally an oxalate transporter, lead to the secretion of oxalate in the Author Contributions medium, which in turn resulted in MPS ability in the organism. A genomic integrant of artificial oxalate operon showed improved Conceived and designed the experiments: KY CK GA GNK. Performed growth and increased P content of V. radiata in alfisol soil. the experiments: KY CK. Analyzed the data: KY CK GA GNK. Furthermore, presence of VHb contributed to improved root Contributed reagents/materials/analysis tools: KY CK GNK. Wrote the paper: KY GA GNK. colonization and better survival of Pf13525 integrant in soil, thus, References 1. El-Swaify SA, Pathak P, Rego TJ, Singh S (1985) Soil management for 16. Jones DL, Darrah PR (1994) Role of root derived organic acids in the optimized productivity under rainfed conditions in the semi-arid tropics. mobilization of nutrients from the rhizosphere. Plant Soil 166: 247–257. Springer New York, pp 1–64. 17. Khademi Z, Jones DL, Malakouti MJ, Asadi F (2010) Organic acids differ in 2. 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Plant Soil 237: 173–195. PLOS ONE | www.plosone.org 7 April 2014 | Volume 9 | Issue 4 | e92400 Oxalate Secretion in Pseudomonas fluorescens 29. Ye L, Jia Z, Jung T, Maloney PC (2001) Topology of OxlT the oxalate 40. Choi KH, Schweizer HP (2006) Mini-Tn7 insertion in bacteria with single attTn7 sites: example Pseudomonas aeruginosa. Nat Protoc 1: 153–161. transporter of Oxalobacter formigenes, determined by site-directed fluorescence 41. Gyaneshwar P, Naresh Kumar G, Parekh LJ (1998) Effect of buffering on the labeling. J Biol Chem 183: 2490–2496. phosphate solubilizing ability of microorganisms. World J Microbiol Biotechnol 30. Watanabe T, Shitan N, Suzuki S, Umezawa T, Shimada M, et al. (2010) 14: 669–673. Oxalate efflux transporter from the brown rot fungus Fomitopsis palustris. Appl 42. Ames BN (1964) Assay of inorganic phosphate, total phosphate and Environ Microbiol 76: 7683–7690. phosphatases. Methods Enzymol 8: 115–118. 31. 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Rodrıguez H, Fraga R (1999) Phosphate solubilizing bacteria and their role in Adaptation of Pseudomonas fluorescens to Al-citrate: involvement of tricarboxylic plant growth promotion. Biotechnol Adv 17: 319–339. acid and glyoxylate cycle enzymes and the influence of phosphate. Curr 36. Hass D, De ´fago G (2005) Biological control of soil-borne pathogens by Microbiol 47: 521–527. fluorescent pseudomonads. Nat Rev Microbiol 3: 307–319. 49. Buch AD, Archana G, Naresh Kumar G (2010) Broad-host-range plasmid- 37. Sambrook J, Russell DW (2001) Molecular Cloning: a Laboratory Manual, Cold mediated metabolic perturbations in Pseudomonas fluorescens 13525. Appl Spring Harbor, NY: Cold Spring Harbor Laboratory. Microbiol Biotechnol 88: 209–218. 38. Choi KH, Gaynor JB, White KG, Lopez C, Bosio CM, et al. (2005) A Tn7- 50. Ma JF, Hiradate S, Matsumoto H (1998) High aluminum resistance in based broad-range bacterial cloning and expression system. Nat Methods 2: buckwheat II. Oxalic acid detoxifies aluminum internally. Plant Physiol 117: 443–448. 753–759. 39. Cohen S, Chang ACY, Leslie HSU (1972) Nonchromosomal antibiotic 51. Archana G, Buch A, Naresh Kumar G (2012) Pivotal role of organic acid resistance in bacteria: Genetic transformation of Escherichia coli by R-factor secretion by rhizobacteria in plant growth promotion. In: Satayanarayana T, DNA (CaCl /extrachromosomal DNA/plasmid). Proc Nat Acad Sci USA 69: Johri BN, Prakash AA, editors. Microorganisms in Sustainable Agriculture and 2110–2114. Biotechnology. Springer, Heidelberg, New York pp. 35–53. PLOS ONE | www.plosone.org 8 April 2014 | Volume 9 | Issue 4 | e92400 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png PLoS ONE Unpaywall

Pseudomonas fluorescens ATCC 13525 Containing an Artificial Oxalate Operon and Vitreoscilla Hemoglobin Secretes Oxalic Acid and Solubilizes Rock Phosphate in Acidic Alfisols

Pseudomonas fluorescens ATCC 13525 Containing an Artificial Oxalate Operon and Vitreoscilla Hemoglobin Secretes Oxalic Acid and Solubilizes Rock Phosphate in Acidic Alfisols

Abstract

Oxalate secretion was achieved in Pseudomonas fluorescens ATCC 13525 by incorporation of genes encoding Aspergillus niger oxaloacetate acetyl hydrolase (oah), Fomitopsis plaustris oxalate transporter (FpOAR) and Vitreoscilla hemoglobin (vgb) in various combinations. Pf (pKCN2) transformant containing oah alone accumulated 19 mM oxalic acid intracellularly but secreted 1.2 mM. However, in the presence of an artificial oxalate operon containing oah and FpOAR genes in plasmid pKCN4, Pf (pKCN4) secreted 13.6 mM oxalate in the medium while 3.6 mM remained inside. This transformant solubilized 509 mM of phosphorus from rock phosphate in alfisol which is 4.5 fold higher than the Pf (pKCN2) transformant. Genomic integrants of P. fluorescens (Pf int1 and Pf int2) containing artificial oxalate operon (plac-FpOAR-oah) and artificial oxalate gene cluster (plac-FpOAR-oah, vgb, egfp) secreted 4.8 mM and 5.4 mM oxalic acid, released 329 mM and 351 mMP, respectively, in alfisol. The integrants showed enhanced root colonization, improved growth and increased P content of Vigna radiata plants. This study demonstrates oxalic acid secretion in P. fluorescens by incorporation of an artificial operon constituted of genes for oxalate synthesis and transport, which imparts mineral phosphate solubilizing ability to the organism leading to enhanced growth and P content of V. radiata

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Abstract

Oxalate secretion was achieved in Pseudomonas fluorescens ATCC 13525 by incorporation of genes encoding Aspergillus niger oxaloacetate acetyl hydrolase (oah), Fomitopsis plaustris oxalate transporter (FpOAR) and Vitreoscilla hemoglobin (vgb) in various combinations. Pf (pKCN2) transformant containing oah alone accumulated 19 mM oxalic acid intracellularly but secreted 1.2 mM. However, in the presence of an artificial oxalate operon containing oah and FpOAR genes in plasmid pKCN4, Pf (pKCN4) secreted 13.6 mM oxalate in the medium while 3.6 mM remained inside. This transformant solubilized 509 mM of phosphorus from rock phosphate in alfisol which is 4.5 fold higher than the Pf (pKCN2) transformant. Genomic integrants of P. fluorescens (Pf int1 and Pf int2) containing artificial oxalate operon (plac-FpOAR-oah) and artificial oxalate gene cluster (plac-FpOAR-oah, vgb, egfp) secreted 4.8 mM and 5.4 mM oxalic acid, released 329 mM and 351 mMP, respectively, in alfisol. The integrants showed enhanced root colonization, improved growth and increased P content of Vigna radiata plants. This study demonstrates oxalic acid secretion in P. fluorescens by incorporation of an artificial operon constituted of genes for oxalate synthesis and transport, which imparts mineral phosphate solubilizing ability to the organism leading to enhanced growth and P content of V. radiata in alfisol soil. Citation: Yadav K, Kumar C, Archana G, Naresh Kumar G (2014) Pseudomonas fluorescens ATCC 13525 Containing an Artificial Oxalate Operon and Vitreoscilla Hemoglobin Secretes Oxalic Acid and Solubilizes Rock Phosphate in Acidic Alfisols. PLoS ONE 9(4): e92400. doi:10.1371/journal.pone.0092400 Editor: Adam Driks, Loyola University Medical Center, United States of America Received September 8, 2013; Accepted February 21, 2014; Published April 4, 2014 Copyright:  2014 Yadav et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors have no support or funding to report. Competing Interests: The authors have declared that no competing interests exist. * E-mail: gnaresh_k@yahoo.co.in . These authors contributed equally to this work. efficient. An alternative approach is to use organic acid secreting Introduction microorganisms to solubilize RP in alfisols [11,12]. Low molecular Acidic soils occupy about 30% of world’s ice-free land area and weight organic acids are known to be most effective in chelation of are considered important for future agricultural development, as Fe and Al and thus solubilization of P [13–15]. Plants secreting presently only 24.2% of the total land area of the world is piscidic, citric and oxalic acid in root exudates show increased potentially arable [1,2]. Phosphorous (P) is the second major growth and shoot P content [15–17]. Improved growth of pigeon nutrient in the soil limiting plant growth after nitrogen (N). In pea plants as compared to other crops in alfisols is attributed to acidic alfisols, plant growth and crop yields are limited by low P piscidic acid mediated FeP solubilization [7]. availability combined with high refixation of applied P [3–5]. Due Although many microorganisms are known to solubilize mineral to the high reactivity of soluble inorganic phosphate (Pi) with Al, phosphates [6], majority of them solubilize Ca-P and very few Fe and Ca, most soil P exists in the bound form and very low (, microorganisms are known to solubilize Fe-P and Al-P [18]. As a 10 mM) amount of free P is available for plant growth in soil result most phosphate solubilizing microorganisms are ineffective solution [6]. In acidic alfisols, P is mainly complexed with Fe and in supplying P to plants grown in alfisols. For instance, Enterobacter Al [7,8] which are difficult to dissolve by simple acidification. asburiae PSI3, a gluconic acid secreting bacterium, solubilizes P However, organic acids have a demonstrated chelation capacity, from alkaline vertisols [19] but does not release free P from alfisols making them potentially ideal for releasing P from alfisols [5]. supplemented with RP [5]. This has been attributed to the nature Application of rock phosphate (RP) as a P fertilizer to acidic soils and amount of the organic acid secreted by the microorganism. It is considered as an important strategy for enhancing plant P has been shown that P is released efficiently from alfisol amended nutrition [9]. RP is rich in mineral phosphate complexes which with RP when treated with organic acids such as oxalic and citric could be solubilized in acidic conditions in alfisols. However, as compared to gluconate, succinate and malate which are more partially acidulated rock phosphate (PARP) obtained by mild acid effective in vertisol or alkaline soils [5]. About 5–10 mM oxalic treatment of RP, renders it more easily available for plants [10], acid solubilizes P from RP in acidic alfisol which could be indicating that mere addition of untreated RP to alfisols is not accredited to the excellent chelating properties of this acid that PLOS ONE | www.plosone.org 1 April 2014 | Volume 9 | Issue 4 | e92400 Oxalate Secretion in Pseudomonas fluorescens plausibly hinders refixation of P by chelation of Fe and Al ions Construction of artificial oxalate operon [5,15,20]. Addition of oxalate to different phosphate rocks and RNA was isolated from A. niger by TRizol method (Sigma soils resulted in efficient mineral phosphate solubilization [5,15]. Aldrich, India) and oah gene was amplified from mRNA using gene Only a few bacterial strains belonging to Bacillus subtilis, specific primers (Integrated DNA Technology, USA) (Table S2). Pseudomonas fluorescens, Arthrobacter spp. and Micrococcus spp. are Amplicon was digested with BamHI/PstI and cloned in BamHI/ known to secrete oxalic acid that too in very low amounts (, PstI digested plasmid pUCPM18Gm under lac promoter (Figure 2 mM) [21,22]. High levels of oxalic acid secretion are reported in S1). The resultant construct was designated as pKCN2. The RNA fungi such as Aspergillus niger, A. fumigatus, Botrytis cinerea, Fomitopsis isolated from F. plaustris was amplified using gene specific primers plaustris and Penicillium spp. [21]. In fungi, oxalic acid synthesis is of FpOAR gene (Table S2). Amplicon digested with SacI/BamHI mediated by the cytosolic enzyme, oxaloacetate acetyl hydrolase was cloned upstream of oah gene, in SacI/BamHI digested plasmid (OAH), which breaks down oxaloacetate into oxalic and acetic pKCN2 under lac promoter to construct artificial oxalate operon, acids [23–26]. A. niger OAH is a pH inducible enzyme belonging to designated as pKCN4 (Figure S1). Amplicon of 2.8 kb containing PEP mutase/isocitrate lyase super family and requires divalent lac-FpOAR-oah was amplified with forward lac primer (Table S2) metal ions for catalysis [23,25–27]. In bacteria such as Oxalobacter and oah reverse primer from pKCN4, using XT-20 polymerase formigens oxalate specific transporter (OxlT) is responsible for (Merck Genei, India) and was cloned in SmaI digested integration oxalate uptake and helps in ATP generation [28,29]. On the other vector pUC18T-mini-Tn7T-Gm-eyfp. hand, a high amount of oxalate secretion in fungi is mediated by efficient oxalate transporter [30]. F. plaustris is a wood rotting Construction of artificial oxalate gene cluster (plac- fungus and degradation of wood is promoted by oxalic acid FpOAR-oah, vgb, egfp) secretion with the help of an oxalate transporter encoded by pUCVHb-egfp plasmid was digested with PvuII to obtain 3.2 kb FpOAR gene. insert containing vgb and egfp genes. Insert was cloned in pKCN5 Oxygen is present in limited amounts in the rhizosphere which digested with NheI and end filled using Klenow fragment (Thermo could limit the colonization and survival of rhizobacteria [31]. The Scientific, USA). The resultant construct (artificial oxalate gene obligate aerobic bacterium, Vitreoscilla, synthesizes elevated quan- cluster) containing F. plaustris FpOAR and A. niger oah genes under tities of homodimeric hemoglobin (VHb) under hypoxic growth lac promoter, vgb gene under its natural oxygen sensitive promoter conditions which allows improved growth under microaerobic and egfp under rrnB promoter in pUC18T-mini-Tn7T-Gm-eyfp conditions when dissolved oxygen is less than 2% of air saturation vector, was designated as pKCN7 (Figure S1). All the plasmids [32,33]. Expression of vgb gene encoding VHb protein in were transformed in Pf13525 using modified NaCl/CaCl method heterologous hosts often enhances growth and metabolism by [39] and integration in the genome of Pf13525 was done by facilitating oxygen transfer to the respiratory membranes [34]. transformation method [40]. Beneficial effect of vgb overexpression for improved bacterial growth has been demonstrated in plant associated bacteria [31]. Physiological and analytical experiments Fluorescent pseudomonads are well-known plant-growth pro- Bacterial inoculum was used to inoculate Tris rock phosphate moting rhizobacteria with root colonization and efficient biocon- (TRP) minimal medium [41] and alfisol soil medium (containing trol properties [35,36]. The present study deals with the genetic 0.5 g/ml alfisol in sterile medium containing 100 mM glucose, modification of Pseudomonas fluorescens ATCC 13525 for oxalic acid 10 mM KNO , micronutrient cocktail and 30 mg RP/g of soil) for secretion by the incorporation of A. niger oah and F. plaustris FpOAR batch studies in 150 ml conical flask containing 30 ml of genes and determination of its effect on its mineral phosphate inoculated medium. The culture supernatants collected at the solubilizing (MPS) ability and growth promotion of mung bean end were used for extracellular organic acid analysis by (Vigna radiata) plants in acidic alfisols. Additionally, vgb gene was Prominence UFLC (Shimadzu, Japan) and P estimation [42]. incorporated as a part of an artificial operon containing the oah Cell free extract was used for intracellular organic acid analysis. and FpOAR genes to enhance the survival and colonization of organism in the soil environment. Incorporation of the artificial MPS ability of Pf13525 transformants and integrants oxalate operon in P. fluorescens ATCC 13525 resulted in secretion MPS ability of Pf13525 transformants and integrants was of high amounts of oxalate which in turn released P from RP in determined on TRP minimal medium plates (containing 100 mM acidic alfisols. Incorporation of vgb gene along with artificial Tris buffer pH-8.0, 1% methyl red, 1.8% agar and 50 mM oxalate operon resulted in better colonization and improved plant glucose) with RP as the sole P source (1mg/ml), respectively. parameters in acidic alfisols. Saline washed bacterial inoculum (5 ml) was spot inoculated on plates and incubated at 30uC. Phosphate solubilization and acid Materials and Methods secretion was determined by monitoring the growth and red zone of acidification. Bacterial strains, plasmids and media The plasmids, bacterial and fungal strains used in this study are shown in Table S1. Routine DNA manipulations were done with OAH assay E. coli DH10B (Invitrogen, Carlsband, CA, USA) as a host using Cells grown in M9 minimal medium were used for cell free standard molecular biology protocols [37]. pUC18T-mini-Tn7T- extract preparation and OAH enzyme activity measurements were Gm-eyfp was generously gifted by Dr. H. P. Schweizer, Colorado done by the method described by Lenz et al (1976) [23]. OAH State University, USA (Table S1) [38]. P. fluorescens ATCC 13525 enzyme specific activity was expressed in nmole per minute per mg (Pf13525) and its plasmid derivatives were grown at 30uC and total protein. Total protein was estimated using a modified maintained on Pseudomonas agar (Hi Media, India) containing Lowry’s method [43]. One unit of enzyme activity was defined as 50 mg/ml ampicillin and 10 mg/ml gentamycin as and when the amount of protein required to convert 1 nmole of substrate per required. Fungal cultures were grown in minimal medium at 27uC minute. [25,30]. PLOS ONE | www.plosone.org 2 April 2014 | Volume 9 | Issue 4 | e92400 Oxalate Secretion in Pseudomonas fluorescens Figure 2. Effect of genetic manipulation on MPS phenotype, oxalic acid secretion and P release by Pf13525 strains. (A) Phenotype of Pf13525 strains on TRP-methyl red agar plates containing 50 mM Tris-HCl and 100 mM glucose. Red zone indicates acidification of the medium; (B) Oxalic acid secretion and (C) P released by Pf13525 integrants and transformants in TRP medium (with 50 mM Tris HCl) and alfisol soil containing 100 mM glucose as carbon source. Results are given as mean 6 SD of four to six independent observations where ***, P, 0.0001; ns, non-significant. doi:10.1371/journal.pone.0092400.g002 Figure 1. OAH activity, growth and pH profiles of Pf13525 wild type and genetically modified strains. (A) OAH activity in M9 minimal medium. Results are given as mean 6 SD of three independent observed for bacterial colonization by using Confocal laser observations. Values are compared with Pf(pKCN2), **, P , 0.0067; ns, scanning microscopy (LSM 700 Carl Zeiss, GmbH). non-significant; (B) Growth profile and (C) pH profile in TRP minimal medium containing 100 mM glucose as carbon source. O.D. and 600nm pH values at each time point are represented as the mean 6 SD of four Data analysis to six independent observations. Physiological experiments were done in three to four indepen- doi:10.1371/journal.pone.0092400.g001 dent replicates for batch culture study. Data are expressed in mean with standard deviation. In plant experiments, three independent Plant experiments triplicate studies were carried out. Differences in mean values were Plant studies were done in Murashige and Skoog medium as determined using general analysis of variance (ANOVA) and well as in alfisol soil (containing 10 mg/g of RP). Soil analysis was linear regression analysis. The statistical analysis of all the done from Pulse Research Station (Anand Agriculture University, parameters has been done using Graph Pad Prism (version 5.0) Vadodara) and was found to contain 0.085% organic carbon, software. 165.1 kg/ha total nitrogen, 262.4 kg/ha available K and 17.9 kg/ ha available P. V. radiata (mung bean) plant studies were done as Results described in [44] and plant parameters such as lengths of the main root and shoot, dry weight and P content were monitored. Effect of genetic modifications on OAH activity, growth Molybdate-blue method [45] was used to determine P content. and MPS phenotype of Pf13525 th Root colonization of Pf13525 integrant was observed on 5 and Pf (pKCN2) and Pf (pKCN4) (Table S1) transformants showed th 10 day after inoculation and different sections of root were around 230 U/mg of OAH activity while integrants Pf int1 and Pf PLOS ONE | www.plosone.org 3 April 2014 | Volume 9 | Issue 4 | e92400 Oxalate Secretion in Pseudomonas fluorescens Figure 3. Root colonization study of V. radiata inoculated with Pf int2. (A) and (B) colonization study in Murashige-Skoog (MS) medium th th th th (hydroponics study) on 5 and 10 day respectively; (C) and (D) in alfisol soil (pot experiment) on 5 and 10 day, respectively, by Confocal Laser Scanning Microscopy (CLSM). Left panel shows fluorescence imaging, middle panel shows bright field images and rightmost panel shows overlapped images. doi:10.1371/journal.pone.0092400.g003 int2 showed about 165 U/mg of OAH activity in M9 minimal Effect of genetic modifications on organic acid secretion medium containing 100 mM glucose (Figure 1A). On the other by Pf13525 hand, Pf13525 and the vector control did not show detectable To study the effect of increased OAH activity on organic acid OAH activity as the gene is absent in the organism. Growth of Pf secretion in genetically modified Pf13525 strains, the cell lysate (pKCN4) transformant was not significantly different than the and extracellular culture supernatants were analyzed for oxalic untransformed strain in 50 mM Tris-HCl (pH 8.0) medium acid levels using HPLC. Pf (pKCN2) transformants carrying the containing 100 mM glucose as carbon source and RP as the sole oah gene showed the highest intracellular accumulation of oxalic P source (Figure 1B). However, the Pf (pKCN2) transformant acid up to 19.1 mM, but secreted a relatively less amount and the integrants grew slowly and reached 0.28 O.D after 600 nm (1.2 mM) in the medium (Figure 2B). On the other hand, Pf 168 h. Pf (pKCN4) transformant was most effective in acidification (pKCN4) transformants possessing artificial oxalate operon, of the medium from pH 8.0 to 4.2 while Pf (pKCN2) did not consisting of oah gene along with the fungal oxalate transporter, decrease the pH to less than 7.0 (Figure 1C). Although growth of accumulated only 3.6 mM oxalic acid and secreted 13.6 mM in the integrants was slower as compared to the pKCN4 transfor- the medium. Genomic integrants of Pf13525, int1 and int2 mant, they were effective at reducing the pH of the medium secreted 4.1 and 4.7 mM of oxalic acid, respectively, while indicating organic acid secretion. Further, organic acid mediated intracellular levels were 2.6 and 3.1 mM, respectively (Figure 2B). acidification by genetically modified Pf13525 strains was observed In order to study the organic acid secretion in soil conditions, on TRP plates. Pf13525 and Pf (pKCN2) transformant did not alfisol soil supplemented with 100 mM glucose and 30 mg RP/g show red zone of acidification while Pf (pKCN4) transformant, Pf of soil was inoculated with genetically modified Pf13525 strains int1 and Pf int2 acidified TRP agar plate containing 100 mM and the oxalate secreted in the soil solution was estimated. As seen glucose as carbon source and 50 mM Tris HCl (pH 8.0) in Figure 2B, in agreement with the TRP medium studies, in (Figure 2A). alfisol Pf (pKCN4) secreted 15 mM of oxalate. Oxalate secretion PLOS ONE | www.plosone.org 4 April 2014 | Volume 9 | Issue 4 | e92400 Oxalate Secretion in Pseudomonas fluorescens in both media abundant colonization was seen on the root surfaces, root tips and at branching points and the colonization th decreased on 10 day (Figure 3). Effect of inoculation of genetically modified Pf13525 strains on growth parameters of V. radiata Inoculation of V. radiata with Pf int1 and Pf int2 in pot experiments with unsterilized alfisol soil supplemented with RP showed better growth (root and shoot length) and increased root and shoot dry weight. Pf int1 and Pf int2 inoculations showed 1.7 and 1.9 fold increase in root length, 1.3 and 1.4 fold increase in shoot length of V. radiata, respectively, as compared to wild type inoculations (Figure 4A). Pf int1 and Pf int2 inoculations resulted in 1.5 and 2 fold increase in root dry weight and 1.3 and 1.4 fold increase in shoot dry weight, respectively (Figure 4B). P content in Pf int1 and Pf int2 inoculations increased in shoot and root by 1.8 and 2.1 fold, respectively, as compared to plants inoculated with wild type strain (Figure 4C). The improvement in plant parameters is correlated with the amount of oxalic acid secreted by Pf int1 and Pf int2. Discussion Growth and yields of crops in alfisols are low due to acidic pH, aluminum toxicity and limited amount of available P, with high P refixation capacity of the soil [46]. P is strongly complexed with Fe and Al in alfisols, which are difficult to solubilize by mineral acids. Several lines of evidences have shown that certain low molecular weight organic acids can release P from Fe-P and Al-P complexes. Among several organic acids tested, oxalic and citric acid solubilized RP in alfisol [5]. Only few bacteria are known to naturally secrete oxalate but do so in very low amounts as compared to several fungi which secrete oxalate in molar amounts [21]. Thus, the present work was aimed at genetic modification of P. fluorescens so as to enable it to secrete high amounts of oxalic acid and render it proficient at solubilizing P from RP amended alfisol. This bacterium was chosen since fluorescent pseudomonads are recognized as plant growth promoting bacteria with efficient root Figure 4. Plant parameters and P content of V. radiata plants colonizing ability [35,36]. The strategy used was to express the key inoculated with Pf13525 wild type and genetically modified strains. (A) Plant length; (B) Dry weight and (C) P content of enzyme oxaloacetate acetyl hydrolase (OAH) for oxalate synthesis inoculated V. radiata plants in alfisol soil. Values are represented as from a fungal system. Presence of this enzyme for oxalate Mean 6 SD of n = 9 observations for plant length and weight while biosynthesis has not been reported so far from bacteria. In order n = 4 to 8 for P content analysis. *, P , 0.05; **, P,0.01; ***, P,0.001; ns, to enable the OAH transformants to secrete the oxalate in the non-significant as compared to the uninoculated control. extracellular milieu, an oxalate transporter from another fungal doi:10.1371/journal.pone.0092400.g004 system was deployed. by integrants in alfisol was also comparable to that on TRP P. fluorescens ATCC 13525 transformant harboring oah gene medium. Wild type Pf13525 secreted 2.0 mM gluconic acid under alone (without the heterologous transporter system) secreted low similar growth conditions but did not show either oxalate amount of oxalic acid (Figure 5). The ability to secrete the accumulation or secretion. All genetic modifications resulted in a organic acid in the cell free supernatant may be attributed to the decrease in gluconic acid secretion. resident dicarboxylate transporters (DctA and DctB) [47]. Secretion of oxalic acid by P. fluorescens ATCC 13525 has been shown in response to Al toxicity in presence of external citrate Effect of genetic modifications of Pf13525 on MPS ability [48], suggesting that the resident dicarboxylate transporters are In TRP minimal medium, Pf (pKCN2), Pf (pKCN4), Pf int1 and functional. Pf (pCNK4) transformant harboring artificial oxalate Pf int2 released 62 mM, 217 mM, 152 mM and 155 mM of P from operon containing oah and the fungal transporter FpOAR showed RP, respectively (Figure 2C). In alfisol soil medium containing enhanced oxalate secretion (Figure 5) indicating more efficient 100 mM glucose as the carbon source and supplemented with transport. The transformant harboring artificial oxalate operon 30 mg RP/g of soil, Pf (pKCN4), Pf int1 and Pf int2 released also exhibited MPS phenotype in minimal medium containing RP 509 mM, 329 mM and 352 mM levels of P, respectively as the P source and in alfisol. Attaining MPS phenotype in alfisol (Figure 2C). by oxalate secretion is supported by the fact that 5–10 mM oxalate solubilizes significant amount of RP in alfisols [5]. The oxalic acid Root colonization study secreted by the transformant is also expected to be effective in Root colonization ability of Pf int2 was observed in V. radiata releasing P from alkaline vertisols which contain high amount of th plants in Murashige-Skoog’s medium and alfisol soil. On 5 day, Ca-P [41]. PLOS ONE | www.plosone.org 5 April 2014 | Volume 9 | Issue 4 | e92400 Oxalate Secretion in Pseudomonas fluorescens Figure 5. Schematic representation of the genetic modifications of Pf13525 and their effect on oxalic acid accumulation and secretion. doi:10.1371/journal.pone.0092400.g005 Integration of genes in to the genome is a preferred method for levels of oxalate accumulation and secretion in the genomic genetic manipulation of bacteria for environmental applications as integrants of artificial oxalate operon as compared to the plasmid compared to plasmid transformation, due to increased stability of transformants (Figure 5). However, the amount of oxalate the integrant, absence of antibiotic resistance genes, lack of secreted in the integrants was sufficient to solubilize RP in horizontal transfer and reduced metabolic load [49]. However, buffered medium as well as in alfisols. genomic integrants have the limitation of single copy expression Hydroponic and alfisol soil experiments of V. radiata inoculated leading to weak phenotype. This was reflected in the decrease in with genomic integrants demonstrated enhanced root colonization PLOS ONE | www.plosone.org 6 April 2014 | Volume 9 | Issue 4 | e92400 Oxalate Secretion in Pseudomonas fluorescens and plant growth as compared to the vector control suggesting that improved plant growth and P content. The present work genetic manipulation supported colonization and survival in the demonstrates the potential of oxalic acid secretion in mineral rhizosphere. On the other hand, E. asburiae PSI3, which is efficient phosphate solubilization by rhizobacteria. in releasing P from alkaline vertisols mediated by secretion of high levels of gluconic acid, did not improve the growth of V. radiata in Supporting Information alfisol [5]. Significant enhancement in plant growth parameters Figure S1 Schematic representation of arrangement of and P content in Pf int2 inoculations as compared to Pf int1 could genes in plasmid constructs used in this study. (A) be attributed to the presence of VHb which is known to improve Expression plasmid and (B) Integration plasmid constructs. the metabolism of bacteria under microaerobic conditions. Squares denote the genes, operon and gene clusters cloned in Similarly, presence of VHb in Rhizobium etli increased nitrogenase the vector backbone. activity and N content in bean plants [31]. This suggests that (TIF) metabolism of bacteria in the rhizosphere corresponds to that under microoxic conditions. Since oxalate is also implicated in Table S1 Plasmids, bacterial and fungal strains used in alleviation of Al toxicity to plants [50] and organic acid secreting this study. Amp = ampicillin; Gm = gentamycin; r = resistance. rhizobacteria are known to promote plant growth by multiple (DOCX) processes [51], it may be hypothesized that the genetically modified strain could be beneficial to plants in diverse soil Table S2 List of primers used in this study. Underlined sequences represent restriction enzyme sites used for cloning and conditions. To summarize, in this study we report the genetic manipulation sequences in italics indicate universal ribosome binding site (RBS). of rhizosphere colonizing bacterium for the secretion of oxalic acid (DOCX) with the aim of imparting it the ability to carry out mineral phosphate solubilization (MPS) from acidic alfisol to enhance P Acknowledgments availability to plants. Pf13525 harboring oah gene resulted in CK was provided with a fellowship from the Council for Scientific and intracellular accumulation of high amounts of oxalic acid while Industrial Research, (CSIR) New Delhi, Government of India. incorporation of an artificial oxalate operon, containing addition- ally an oxalate transporter, lead to the secretion of oxalate in the Author Contributions medium, which in turn resulted in MPS ability in the organism. A genomic integrant of artificial oxalate operon showed improved Conceived and designed the experiments: KY CK GA GNK. Performed growth and increased P content of V. radiata in alfisol soil. the experiments: KY CK. Analyzed the data: KY CK GA GNK. Furthermore, presence of VHb contributed to improved root Contributed reagents/materials/analysis tools: KY CK GNK. Wrote the paper: KY GA GNK. colonization and better survival of Pf13525 integrant in soil, thus, References 1. El-Swaify SA, Pathak P, Rego TJ, Singh S (1985) Soil management for 16. Jones DL, Darrah PR (1994) Role of root derived organic acids in the optimized productivity under rainfed conditions in the semi-arid tropics. mobilization of nutrients from the rhizosphere. Plant Soil 166: 247–257. Springer New York, pp 1–64. 17. Khademi Z, Jones DL, Malakouti MJ, Asadi F (2010) Organic acids differ in 2. 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