1.8 IMPERFECCIONES EN LA ESTRUCTURA CRISTALINA
1.8.3 Defectos de superficie
The ability of wildtype isolates and non-auxotrophic mutants to solubilize mineral P was measured using two different agar media to determine the reproducibility of using agar plates for testing P solubilisation. The TCaP medium contains additional nitrate, manganese, iron and trace elements (Appendix A.1.4) whereas the NBRIP medium lacks trace elements but is widely used for isolating and testing P solubilising bacteria (Nautiyal, 1999). Table 3.3.2 shows the solubilisation index (SI) of
Pseudomonas sp. Ha200, Enterobacter sp. Wi28, Burkholderia sp. Ha185 and their derivative mutants on NBRIP and TCaP plates. In general, there was no significant difference in terms of P solubilisation for wildtype Pseudomonas sp. Ha200 and Enterobacter sp. Wi28 on both TCaP and NBRIP plates, but the solubilisation by Burkholderia sp. Ha185 was reduced on TCaP plates (Table 3.3.2).
Several non-auxotrophic mutants that exhibited reduced MPS on both TCaP and NBRIP plates (Table 3.3.2) had insertions in genes involved in the direct oxidation pathway of Pseudomonas sp. Ha200 (Table 3.3.1). These mutants had the transposon inserted in either the genes encoding quinoprotein glucose dehydrogenase (B8), glucose-6-phosphate-1-dehydrogenase (B31) or 35 bp upstream of pyrroloquinoline quinone PqqA (B50). The Pseudomonas sp. Ha200 mutants B8 and B50 showed reduced SI on NBRIP plates with 0.00 ± 0.00 and 0.57 ± 0.14 respectively compared to wildtype control (1.26 ± 0.14; p < 0.05). In contrast, mutant B31, where the mutation occurred in glucose-6- phosphate-1-dehydrogenase, demonstrated no significant difference in ability to solubilise P on NBRIP plates but had a significantly reduced SI on TCaP plates (1.56 ± 0.10; p < 0.05), in comparison with the wildtype Pseudomonas sp. Ha200 (1.63 ± 0.10) (Table 3.3.2).
Table 3.3.1 Mutant library of Pseudomonas sp. Ha200 (B), Enterobacter sp. Wi28 (D) and Burkholderia sp. Ha185 (F).
Isolate Mutant MPS Phenotypea Accession Numberb Organismb BlastX closest match to mutated ORFc Biological process or pathway E-Value Identity
Pseudomonas sp. B8 - YP_350305.1 Pseudomonasfluorescens Pf0-1 Quinoprotein glucose dehydrogenase Direct oxidation pathway 3.00E-94 91%
Ha200 B31 ++ ZP_10650857.1 Pseudomonas sp. GM50 Glucose-6-phosphate 1-dehydrogenase Pentose phosphate pathway 5.00E-88 100% B50 + YP_002875094.1 Pseudomonasfluorescens SBW25 Pyrroloquinoline quinone PqqA Direct oxidation pathway 8.00E-91 94% B42 ++ ZP_10594719.1 Pseudomonas sp. GM102 Flagellar motor switch protein G Chemotaxis 4.00E-148 100% B34AUX - ZP_10663122.1 Pseudomonas sp. GM48 Histidinol dehydrogenase Histidine biosynthesis 0.00E+00 97%
B45AUX + YP_350846.1 P. fluorescens Pf0-1 Phosphoribosylformimino-5-aminoimidazole Histidine biosynthesis 1.00E-160 99%
Carboxamideribotideisomerase
B35AUX - ZP_10682030.1 Pseudomonas sp. GM30 Anthranilate synthase component I Tryptophan biosynthesis 5.00E-79 86%
B46AUX + ZP_10682026.1 Pseudomonas sp. GM30 Anthranilatephosphoribosyltransferase Tryptophan biosynthesis 7.00E-159 97%
B37AUX + ZP_10620673.1 Pseudomonas sp. GM78 Glutamate synthase small subunit Glutamate synthesis 1.00E-57 100%
Enterobacter sp. D22AUX - YP_001175330.1 Enterobacter sp. 638 Carbamoyl phosphate synthase large subunit Pyrimidine synthesis 2.00E-143 94%
Wi28 D5AUX - YP_003614144.1 EnterobactercloacaeATCC 13047 Amidophosphoribosyltransferase Purine synthesis 8.00E-133 93%
D23 + YP_003940046.1 Enterobactercloacae SCF1 Glutamate synthase, small subunit Glutamate synthesis 6.00E-39 75%
Burkholderia sp. F65AUX + ZP_06465278.1 Burkholderia sp. CCGE1003 2-dehydro-3-deoxyphosphogluconate aldolase Phosphorylative pathway 6.00E-30 74%
Ha185 F59AUX - YP_560625.1 Burkholderiaxenovorans LB400 3-dehydroquinate synthase Chorismate biosynthesis 3.00E-161 95%
F55AUX - YP_004229710.1 Burkholderia sp. CCGE1001 Shikimate kinase Chorismate biosynthesis 1.00E-26 81%
F68AUX + ZP_06297177.1 Burkholderia sp. CCGE1001 Chorismate synthase Chorismate biosynthesis 1.00E-77 98%
F66AUX - YP_001897167.1 Burkholderiaphytofirmans PsJN
Phosphoribosyl-AMP cyclohydrolase Histidine biosynthesis 1.00E-93 96% F78AUX - YP_003908394.1 Burkholderia sp. CCGE1003 Imidazole glycerol phosphate synthase
(subunit HisF) Histidine biosynthesis 3.00E-55 98%
F57AUX - ZP_02882233.1 Burkholderiagraminis C4D1M Tryptophan synthase, beta subunit Tryptophan biosynthesis 5.00E-81 95%
F62AUX - YP_001896485.1 B. phytofirmans PsJN Carbamoyl phosphate synthase large subunit Pyrimidine synthesis 1.00E-154 99%
F77AUX - YP_001896485.1 B. phytofirmans PsJN Carbamoyl phosphate synthase large subunit Pyrimidine synthesis 1.00E-154 98%
F69AUX - YP_003908510.1 Burkholderia sp. CCGE1003 Orotate phosphoribosyltransferase Pyrimidine biosynthesis 1.00E-110 99%
F76AUX - YP_001890498.1 B. phytofirmans PsJN Amido phosphoribosyltransferase Purine synthesis 5.00E-70 87%
75
F72AUX - ZP_06297057.1
Burkholderia sp. CCGE1001 Adenylylsulfatereductase, thioredoxin dependent Cysteine biosynthetic process 4.00E-71 91%
F81AUX ++ YP_001894616.1 B. phytofirmans PsJN Sulfate adenylyltransferase subunit 2 Hydrogen sulfide biosynthesis 7.00E-82 98%
F13 + YP_003907489.1 Burkholderia sp. CCGE1003 Hypothetical protein Unknown 3.00E-38 84% F18† + YP_557627.1 B. xenovorans LB400 Bifunctional uroporphyrinogen-III
synthetase/uroporphyrin-III C-methyltransferase Haem biosynthesis 1.00E-111 93% F80 + ZP_02886362.1 Burkholderiagraminis C4D1M Penicillin-binding protein, 1A family Peptidoglycan biosynthesis 2.00E-105 92% F85 ++ YP_004228782.1 Burkholderia sp. CCGE1001 Multi-sensor signal transduction, histidine kinase Peptidyl-histidine phosphorylation 9.00E-146 98% F86† +++ YP_559465.1 B. xenovorans LB400 Dihydrolipoamide acetyltransferase Glucose metabolism 0.00E+00 99%
a; Phenotype of the mutant on tricalcium phosphate agar plate (TCaP); + indicates reduced solubilisation, ++ denotes similar ratio to the wide type, +++ denotes enhanced solubilisation; - no solubilisation activity b; Bacteria species and GenBank accession number searched using BlastX against the reference proteins database at NCBI in November 2012
c; DNA sequence flanking the transposon insertion site obtained by touchdown PCR
d; Colours represent similar biological pathways identified by IntoPro and KEGG pathway database where highlighted; dark blue, pentose phosphate pathway and direct oxidation pathway; green, aromatic amino acid synthesis; orange, glutamate synthesis; purple, pyrimidine and purine synthesis; light blue, others.
†; Transposon insertion site identified by cloning strategy with pUC19 vector
The Enterobacter sp. Wi28 D23 mutant, where the transposon had inserted into the gltD gene encoding the small subunit of glutamate synthase, had significantly reduced ability to solubilise TCaP with a SI of 0.33 ± 0.24 (D23) compared to the wildtype 1.31 ± 0.24 on NBRIP plate, but there was no significant difference between the mutant or wildtype strain on the TCaP plates (1.53 ± 0.17).
The non-auxotrophic Burkholderia sp. Ha185 mutant F13, had an insertion in a gene encoding an hypothetical protein (Table 3.3.1). The SI of this strain showed no significant difference compared to the wildtype control on both NBRIP and TCaP plates (Table 3.3.2). The mutation in the bifunctional Uroporphyrinogen III synthase gene (hemX)in Burkholderia sp. Ha185 (F18) resulted in a significantly reduced SI on NBRIP plates (2.23 ± 0.32, p < 0.05) when compared with the wildtype but there was no significant difference in SI when F18 and the wildtype were compared on TCaP plates (2.11 ± 0.26 and 1.83 ± 0.26 respectively).
Table 3.3.2 Solubilisation Index of wildtype Pseudomonas sp. Ha200, Enterobacter sp. Wi28, Burkholderia sp. Ha185 and their derivative mutants on TCaP and NBRIP plates.
Average SI ± SEM
TCaP Plate Average SI ± SEM NBRIP Pseudomonas sp. Ha200 1.63 ± 0.10 1.26 ± 0.14 B8 0.00 ± 0.00 * 0.00 ± 0.00 * B31 1.56 ± 0.10 * 1.22 ± 0.14 B50 1.46 ± 0.10 * 0.57 ± 0.14 * B42 1.39 ± 0.10 * 1.29 ± 0.14 Enterobacter sp. Wi28 1.34 ± 0.17 1.31 ± 0.24 D23 1.53 ± 0.17 0.33 ± 0.24 * Burkholderia sp. Ha185 2.11 ± 0.26 3.30 ± 0.32 F13 1.49 ± 0.26 3.11 ± 0.32 F18 1.83 ± 0.26 2.23 ± 0.32 * F80 1.28 ± 0.26 * 2.18 ± 0.32 * F85 1.83 ± 0.26 3.27 ± 0.32 F86 2.03 ± 0.26 3.50 ± 0.32
* indicates a statistically significant difference between means at a 5% level compared to the appropriate wildtype strain
The Burkholderia sp. Ha185 mutant F80, containing a mutation in the gene encoding the penicillin- binding protein (1A family) involved in cell wall formation, exhibited a significant reduction in SI on both NBRIP and TCaP plates relative to the wildtype Burkholderia sp. Ha185 (2.18 ± 0.32 and 1.28 ± 0.26 respectively; p < 0.05). Independent Burkholderia sp. Ha185 mutants in the gene encoding for histidine kinase (F85) or dihydrolipoamide cetyltransferase (F86), showed no significant differences on either the NBRIP or the TCaP plate compared to the wildtype strain.
Overall, the results show there are discrepancies in the SI when measured on either TCaP or NBRIP plates, although TCaP was the only source of insoluble P in both media (Figure 3.3.2). The inconsistencies of the SI between these two different media and for the comparison of the wildtype strain and its mutant derivatives on the same media indicated using an agar plate based assay for P solubilisation was an unreliable method. This is in an agreement with Bash et al. (2013) who suggested liquid culture testing should replace the traditional plate assays in determining P solubilisation. Therefore, a liquid culture assay was used that enabled the determination of MPS in a homogenous solution. Liquid culture also allows the surface contact of bacterial cells to TCaP. Only a couple of non-auxotophic mutants from Pseudomonas sp. Ha200, Enterobacter sp. Wi28 and
Burkholderia sp. Ha185 were selected for the liquid culture assay due to a limited amount of incubator space available at the time of this study. The non-auxotrophic mutant B8 that had a mutation on the membrane bound quinoprotein glucose dehydrogenase of Pseudomonas sp. Ha200, was an ideal mutant for confirming the direct oxidation pathway involved in P solubilisation as outlined in Chapter 1.3.3. The only non-auxotrophic mutant from Enterobacter sp. Wi28 (D23) was also selected for liquid culture assay. Although there were discrepancies in the SI on TCaP and NBRIP plates, the DNA sequence analysis of the genes involved in non-auxotrophic mutant F13 and F18 revealed that they encoded a hypothetical protein and a bifunctional uroporphyrinogen III synthase protein respectively. As these genes have not previously been implicated in P solubilisation, these mutants were also chosen for further analysis.