Diagrama de equilibrio Fe–Fe 3 C

The liquid culture assay allowed quantification of the amount of P released by Pseudomonas sp. Ha200 and its derivative mini-Tn5Km1 transposon mutant gcd::Tn5(B8). This liquid culture medium facilitated the use of HPLC enabling the quantification of organic acids released by the isolates (Chapter 2). Pseudomonas sp. Ha200 and gcd::Tn5(B8) that contained a mutation in the gcd gene encoding for membrane bound quinoprotein glucose dehydrogenase were cultured in the HSU HydroxP liquid culture assay (Appendix A.2.4). Culture filtrates were collected at 24, 36, 48 and 72 h of incubation and soluble P released was determined by colourimetry. Wildtype Pseudomonas sp. Ha200 increasingly released organic acid throughout the incubation period. The amount of organic acid released also correlated with the amount of P released into the liquid culture. Wildtype

Pseudomonas sp. Ha200 released a high amount of 2-keto-D-gluconic acid (2-KGA) with the highest concentration of 23.50 ± 2.83 mM at 48 h (Table 3.3.5). The wildtype strain also released D-Gluconic Acid (GA) and pyruvic acid (PyrA) at basal levels with 5.90 ± 0.54 mM and 9.85 ± 1.24 mM respectively (48h). It is interesting to note that the amount of each organic acid in the culture was reduced at 72 h; in particular GA was not detected at this time point. This suggests carbon recycling after cell density had reached a certain level. For the mutant strain gcd::Tn5(B8), both GA and 2-KGA were not detected at any time point, but PyrA levels were not significantly different from the wildtype at 72 h (4.81 ± 1.24 and 4.99 ± 0.50 mM respectively, p > 0.05). The amount of P released into the culture was also significantly reduced compared to the wildtype at each of the time points assessed (p < 0.001; Table 3.3.5).

Figure 3.3.6 Schematic diagram of the Pqq open reading frame of Pseudomonas fluorescens Pf0-1 which consists of pqqF, pqqA – E, followed by a putative Peptidase S9. The blue arrow indicates the location of mini-transposon Tn5km1 in mutant B50 which is predicted from the alignment of this region in Pseudomonas sp. Ha200 with P. fluorescens Pf0-1 to be inserted at 2552 bp from the pqqF initiation codon.

HPLC analysis revealed the presence of a yet to be defined organic acid named “Unknown2”, which had a retention time of 10.98 min using a Rezex ROA-Organic Acid HPLC ion-exclusion column (Table 3.3.5). The amount of the Unknown2 presented here is an arbitrary number taken from the HPLC because the molecular mass of this organic acid is unknown. Therefore, direct comparison between Unknown2 and other organic acids such as 2-KGA cannot be made. However, the result shows

gcd::Tn5(B8) also had significantly reduced production of Unknown2 compared to the wildtype at 72 h (17.85 ± 2.27 and 3.91 ± 0.19 respectively, p < 0.001). This suggests that “Unknown2” also plays a role in P solubilisation.

Table 3.3.5 Soluble phosphate and organic acids released from HSU HydroxP liquid culture assay by wildtype Pseudomonas sp. Ha200 and gcd::Tn5(B8) over 72 h.

+ _{Value represents mean ± standard error of the mean (n=3); nd, none detected}

ⱡ _{Phosphate concentration determined by Murphy and Riley’s colorimetric method}

§ Unknown organic acid presented in arbitrary units (AU)

*** Significantly different from the control Pseudomonas sp. Ha200 at corresponding time point (p < 0.001).

In an attempt to identify “Unknown2”, a mixture of 2-KGA (peak A, 200 ppm) and 5-keto-D-gluconic acid (5-KGA, peak B2, 200 ppm) was made and analysed by HPLC (Figure 3.1.12). The chromatogram was compared with the supernatant of wildtype Pseudomonas sp. Ha200 incubated for 24 h in HSU HydroxP medium. The resultant chromatograph revealed that Unknown2 (peak B1) was different from 5-KGA (peak B2) with a retention time slightly earlier than 5-KGA (10.98 min and 11.02 min, respectively). Therefore “Unknown2” is not likely to be 5-KGA. However, mutant strain gcd::Tn5(B8) with its mutation in gcd also produced less “Unknown2”. Furthermore, the retention time of Unknown 2 is between 2-KGA (peak A), 5-KGA (peak B2) and GA (peak C), indicating the organic acid

Isolates P Conc (mM)ⱡ _{Organic acid concentration (mM)}+

Time post

inoculation (h) Unknown2§ 2-Keto-D- _{Gluconic Acid} D-Gluconic _{Acid Pyruvic Acid}

Pseudomonas sp. Ha200 24 4.34 ± 0.09 1.80 ± 0.24 5.37 ± 0.33 1.40 ± 0.12 0.16 ± 0.25 36 11.23 ± 0.11 13.27 ± 2.52 8.96 ± 1.50 2.01 ± 0.57 7.51± 1.22 48 19.00 ± 0.09 24.87 ± 2.97 23.50 ± 2.83 5.90 ± 0.54 9.85 ± 1.24 72 20.95 ± 0.38 17.85 ± 2.27 20.60 ± 2.38 nd 4.99 ± 0.50 gcd::Tn5(B8) 24 0.14 ± 0.00*** _{nd nd nd nd} 36 1.24 ± 0.01*** _{nd nd nd 0.03 ± 0.01}*** 48 2.12 ± 0.07*** _{1.63 ± 0.19}*** _{nd nd 2.02 ± 0.25}*** 72 5.31 ± 0.04*** _{3.91 ± 0.19}*** _{nd nd 4.81 ± 1.24}

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exhibited similar charges and the structure of this molecule may be similar. One possible organic acid might be the 2, 5-diketo-gluconic acid (2, 5-KGA) which is produced by a different pathway.

3.4

Discussion

Three strains—Enterobacter sp. Wi28, Pseudomonas sp. Ha200, and Burkholderia sp. Ha185—were selected for this study (discussion in Chapter 2). Two non-auxotrophic mutants with mutations affecting their ability to solubilise P were identified. These mutants were found to contain transposon insertions in genes involved in the well characterised direct oxidation pathway of glucose metabolism. In the Pseudomonas sp. Ha200 strain gcd::Tn5(B8), the insertion occurred in the gcd

gene, encoding enzyme quinoprotein glucose dehydrogenase (Gcd), while the insertion in the mutant B50 was located upstream of pqqA encoding pyrroloquinoline quinone PqqA. Mutations in these genes have been well characterised in Pseudomonas spp. for their role in P solubilisation where they are involved in the production of GA by Gcd facilitated by the co-factor PQQ (de Werra et al., 2009) (Figure 3.1.13). A C B1 D B2 E 10.0 10.5 11.0 11.5 12.0 12.5 13.0 13.5 14.0 14.5 15.0 min 0 2500 5000 7500 10000 12500 15000 17500 20000uV

Figure 3.3.7 Chromatogram of organic acids released by wildtype Pseudomonas sp. Ha200 in HSU HydroxP liquid culture medium at 24 h (black) and standard solution of 2-keto-D-gluconic acid (200 ppm) and 5-keto-D-gluconic acid (200 ppm) resuspend in HSU medium control (pink). Supernatants were analysed for organic acid by high performance liquid chromatography (HPLC) (n = 3). Peak A denotes 2-Keto-D-Gluconic acid (2-KGA, 10.64 min), B1 denotes peak unknown2 (Un2, 10.98 min), B2 denotes 5-Keto-D-Gluconic acid (5-KGA, 11.02 min), C denotes D-Gluconic acid (GA, 11.55 min), D denotes pyruvic acid (PyrA, 12.11 min) and E is an unknown peak.

Current literature has implicated gluconic acid (GA) released by PSB as the key organic acid responsible for P solubilisation. This was validated through this study where the production of 2-KGA was identified as the major organic acid released by Pseudomonas sp. Ha200, and the amount of P solubilised is correlated with the amount of 2-KGA released. Previous research has failed to identify the key organic acid produced by bacteria, possibly due to the similar structure of GA and 2-KGA making them difficult to separate them in a column using HPLC. This study has shown that GA and 2- KGA co-eluted when analysed using the Prevail TM organic acid column for HPLC, but can be separated using the Rezex ROA-Organic Acid H+ (8%) column (See discussion from Chapter 2). Moreover, many researchers have not included 2-KGA analysis in their study. For example, Farhat et al., (2013) determined the amount of GA produced by a recombinant E. coli strain that carries both

gcd and pqqABCDE genes from Serratia marcescens CTM 50650. Castagno et al. (2011) determined the amount of GA produced by plant growth promoting P solubilising bacteria reside in different

Figure 3.4.1 Glucose dehydrogenase (Gcd) and gluconate dehydrogenase (Gad) involved in periplasmic glucose metabolism via direct oxidation pathway involved with pyrroloquinoline quinone (PQQ) in Pseudomonas spp.. Glk, glucokinase; Zwf, glucose-6- phosphate 1-dehydrogenase; GnuK, gluconokinase; KguK, 2-ketogluconate kinase; and KguD, 2-ketogluconate 6-phosphate reductase. (Modified from de Werra et al., 2009) (detailed mechanism outlined in Chapter 1).

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genera (Pantoea, Erwinia, Pseudomonas, Rhizobium and Enterobacter). However, the production of 2-KGA was not investigated in either study.

Results from this study show production of 2-KGA by Pseudomonas sp. Ha200 is the key organic acid responsible for HydroxP solubilisation in liquid culture, whereas GA was produced at basal levels from 24 h to 48 h, and was below the detection limit at 72 h. Interestingly, mutation in the gcd gene (gcd::Tn5(B8)), abolished the production of GA, and 2-KGA was not detected. This also correlated with the reduced soluble P found in the culture filtrate. This result indicates production of 2-KGA by

Pseudomonas sp. Ha200 is dependent on the production of GA, which corresponds to the direct oxidation pathway as described by de Werra et al. (2009). This also further indicates glucose was first converted to GA by the membrane bound PQQ-dependent Gcd, then oxidised to 2-KGA possibly by gluconate dehydrogenase (Gad) in the periplasmic space of Pseudomonas sp. Ha200 cell, followed by transportation of 2-KGA out of the cell.

One of the important plant growth promoting traits is phytase production by rhizobacteria (Chapter 1). It was found that organic acids such as citric acid improved phytate solubilisation before phytase hydrolysis takes place, this organic acid-driven solubilisation is theorised to be the preliminary step in the hydrolysis of the phytate salt (Tang et al., 2006). Giles et al. (2013) found that organic acid producing Pseudomonas sp. CCAR59 in the rhizosphere of tobacco plants improved calcium phytate solubilisation and subsequently increased the availability of P to plants increasing plant growth. The

Pseudomonas sp. Ha200 strain and its mutant derivative gcd::Tn5(B8) that is unable to produce GA and subsequently 2-KGA from this study were provided to Dr. Courtney Giles (University of Vermont, Burlington, USA). The strains were used to enable comparison between the wildtype and its gcd

mutant derivative that is unable to produce GA and subsequently 2-KGA for their role in phytate solubilisation (Giles et al., 2014).

Furthermore, gcd::Tn5(B8) mutant was shown to only have a partial reduction in MPS in the HSU liquid culture assay. This indicates apart from P solubilisation by 2-KGA derived from GA, another organic acid also contributed significantly to the MPS phenotype, the “Unknown 2” organic acid (Un2). It was also found Un2 was released during HydroxP solubilisation by all the effective P solubilising Pseudomonas strains previously described in Chapter 2, EN101, EE131, EE132, EE127, and Wh15. This may indicate the release of Un2 is common within the genus Pseudomonas and is related to MPS. It was shown that the amount of Un2 decreased to a near basal level in the gcd::Tn5(B8) mutant. Therefore, the production of Un2 is dependent, but not limited to, the direct oxidative pathway (GA and 2-KGA production), and is possibly involved in the tricarboxylic acid cycle (TCA). The chemical composition of this organic acid molecule is uncertain, but is postulated to be 2, 5-KGA that

possesses similar chemical composition to 2-KGA and 5-KGA. To find out the composition of this molecule, Liquid chromatography–mass spectrometry (Tandem MS, LC-MS/MS) could be employed in the future to determine the mass-to-charge ratio by multiple steps mass spectrometry analysis. Biosynthesis of cofactor PQQ is pre-requisite for P solubilisation by Gcd, as shown by Kim et al. (2003). The authors cloned pqqA-F from Enterobacter intermedium (60-2G) in different fragments and expressed them in E. coli DH5α that has Gcd without the pqq gene cluster, confirming the importance of pqqA-E for P solubilisation via the direct oxidative pathway. However, it has also been shown that biosynthesis of PQQ does not require pqqA in Methylobacterium extorquens AM1 (Toyama & Lidstrom, 1998), and the significance of the pqqA gene within the cluster for PQQ production remains debatable (Ge et al., 2013). In this study, the results show the Pseudomonas sp. Ha200 B50 mutant, with the transposon insertion 35 bp upstream of the pqqA initiation codon, had partial reduction in MPS on both TCaP and NBRIP plates. Therefore, it was postulated the mutation occurred within the promoter region of the pqq gene cluster. This result highlights the importance of

pqqA transcription regulation and correlates with the result from Gómez et al., (2010) who found mutation of pqqA in Enterobacter intermedium 60-2G reduced MPS on NBRIP, while in the complemented strain the MPS phenotype was restored. It is known that there might be multiple copies of pqqA within a bacterial genome. For example, there are five copies of pqqA in the

Methylovorus sp. MP688 genome, each of which possess different promoter activity and are transcribed independently from the pqqBCDE operon (Ge et al., 2013). Furthermore, it was also found there is a second copy of a pqqA-like gene present in both Pseudomonas fluorescens F113 and

P. fluorescens Pf-5 (Miller et al., 2010), which is likely to increase PQQ production and consequently Gcd activity. Therefore, it is also likely that multiple pqqA exist in Pseudomonas sp. Ha200. Transcription of the second pqqA likely complements the B50 mutation, resulting in only partial reduction in MPS. However, further investigation of the presence of multiple pqqA genes present in

Pseudomonas sp. Ha200 is required. Together, transcription of pqqA is pre-requisite to the production of PQQ and the PqqA peptide is a precursor of the PQQ cofactor.

Random mutagenesis of Enterobacter sp. Wi28 using the mini-Tn5Km1 generated 2208 mutants, but most retained the ability to solubilise P. It was found that this isolate produced Un2, PyrA, and DL- lactic acid during HydroxP solubilisation, but no GA and 2-KGA was detected (Chapter 2). Furthermore, it was found that this isolate is able to utilise variable sugar substrates for P solubilisation such as fructose, inositol and mannitol (Dr. Carolyn Mander, personal communication). This suggests Enterobacter sp. Wi28 solubilises P by secondary metabolites (organic acids) produced via the TCA cycle because it is postulated that carbon substrates other than glucose are converted to

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fructose-6-P within the cytosol via the Embden-Meyerhof-Parnas pathway and then subjected to fructolysis before entering the TCA cycle (Wisselink et al., 2002). The results suggest that P solubilisation by Enterobacter sp. Wi28 does not occur by the direct oxidation pathway at the cellular membrane, and the bacteria possibly lacks the pqq gene cluster similar to E. coli MC1061 (Rodrı ́guez

et al., 2000). This would account for the inability to obtain non-auxotrophic mutants from

Enterobacter sp. Wi28 with impaired MPS but able to maintain cellular growth. The only

Enterobacter sp. Wi28 non-auxotrophic mutant with impaired MPS ability that was obtained in this study was D23, which contained an insert in the gltD gene encoding a glutamate synthase. However, this mutant derivative was significantly impaired in growth and was therefore not a suitable candidate for further investigation.

The relationship between Gcd, the pqqA-E operon and P solubilisation of Pseudomonas spp. is well understood (Intorne et al., 2009, Farhat et al., 2013), and no novel genes implicated in MPS from the

Enterobacter sp. Wi28 isolate were identified. Therefore, study of these isolates was not pursued. Instead, the bacterium Burkholderia sp. Ha185 was chosen for further study of novel genetic determinants of P solubilisation. Sequencing of the transposon insertion points generated from random mutagenesis, revealed two non-auxotrophic mutants in genes that had not previously been implicated in P solubilisation, F13 and F18. Mutant F13 had a mutation in a gene encoding for a hypothetical protein that is closely related to Burkholderia sp. CCGE1003 (YP_003907489.1) and F18 had a mutation in a gene encoding bifunctional uroporphyrinogen-III synthetase/uroporphyrin-III C- methyltransferase which is closely related to Burkholderia xenovorans LB400 (YP_557627.1) (Table 3.3.1). Mutation in these novel genes independently resulted in reduced P solubilisation compared to the wildtype Burkholderia sp. Ha185. Detail of these two novel genes and the relationship to P solubilisation are investigated and discussed in Chapter 4.