P released by (a) SM6 and SM11, and (b) SM6Δ and SM11Δ in Pikovskaya broth with glucose (G) and glucose+succinate (GS).

<p>P released by (a) SM6 and SM11, and (b) SM6Δ and SM11Δ in Pikovskaya broth with glucose (G) and glucose+succinate (GS).</p

MPS phenotype of wildtype SM6 and SM11 on (a) TRP agar with glucose (b) TRP agar with glucose+succinate (c) Pikovskaya agar with glucose (d) Pikovskaya agar with glucose+succinate, and MPS phenotype of SM6Δ and SM11Δ on (e) TRP agar with glucose (f) TRP agar with glucose+succinate (g) Pikovskaya agar with glucose (h) Pikovskaya agar with glucose+succinate.

<p>MPS phenotype of wildtype SM6 and SM11 on (a) TRP agar with glucose (b) TRP agar with glucose+succinate (c) Pikovskaya agar with glucose (d) Pikovskaya agar with glucose+succinate, and MPS phenotype of SM6Δ and SM11Δ on (e) TRP agar with glucose (f) TRP agar with glucose+succinate (g) Pikovskaya agar with glucose (h) Pikovskaya agar with glucose+succinate.</p

Monoauxic growth profile of (a) SM6Δ and (b) SM11Δ, and diauxic growth profile of (c) SM6Δ and (d) SM11Δ.

<p>Monoauxic growth profile of (a) SM6Δ and (b) SM11Δ, and diauxic growth profile of (c) SM6Δ and (d) SM11Δ.</p

Derepression of Mineral Phosphate Solubilization Phenotype by Insertional Inactivation of <i>iclR</i> in <i>Klebsiella pneumoniae</i>

<div><p>The mode of succinate mediated repression of mineral phosphate solubilization and the role of repressor in suppressing phosphate solubilization phenotype of two free-living nitrogen fixing <i>Klebsiella pneumoniae</i> strains was studied. Organic acid mediated mineral phosphate solubilization phenotype of oxalic acid producing <i>Klebsiella pneumoniae</i> SM6 and SM11 were transcriptionally repressed by IclR in presence of succinate as carbon source. Oxalic acid production and expression of genes of the glyoxylate shunt (<i>aceBAK</i>) was found only in glucose but not in succinate- and glucose+succinate-grown cells. IclR, repressor of <i>aceBAK</i> operon, was inactivated using an allelic exchange system resulting in derepressed mineral phosphate solubilization phenotype through constitutive expression of the glyoxylate shunt. Insertional inactivation of <i>iclR</i> resulted in increased activity of the glyoxylate shunt enzymes even in succinate-grown cells. An augmented phosphate solubilization up to 54 and 59% soluble phosphate release was attained in glucose+succinate-grown SM6Δ and SM11Δ strains respectively, compared to glucose-grown cells, whereas phosphate solubilization was absent or negligible in wildtype cells grown in glucose+succinate. Both wildtype and <i>iclR</i> deletion strains showed similar indole-3-acetic acid production. Wheat seeds inoculated with wildtype SM6 and SM11 improved both root and shoot length by 1.2 fold. However, <i>iclR</i> deletion SM6Δ and SM11Δ strains increased root and shoot length by 1.5 and 1.4 folds, respectively, compared to uninoculated controls. The repressor inactivated phosphate solubilizers better served the purpose of constitutive phosphate solubilization in pot experiments, where presence of other carbon sources (e.g., succinate) might repress mineral phosphate solubilization phenotype of wildtype strains.</p></div

Primers used in the study.

<p>Restriction sites added at the 5’ end of primer are underlined.</p><p>Primers used in the study.</p

Enzyme activity of glucose- and succinate-grown SM6, SM11, SM6Δ and SM11Δ: (a) ICL (b) GO.

<p>Enzyme activity of glucose- and succinate-grown SM6, SM11, SM6Δ and SM11Δ: (a) ICL (b) GO.</p

Expression profile of <i>aceBAK</i> operon genes and <i>iclR</i> in glucose-, glucose+succinate-and succinate-grown <i>K</i>. <i>pneumoniae</i> strains (a) SM6, (b) SM11, (c) SM6Δ and (d) SM11Δ.

<p>Expression profile of <i>aceBAK</i> operon genes and <i>iclR</i> in glucose-, glucose+succinate-and succinate-grown <i>K</i>. <i>pneumoniae</i> strains (a) SM6, (b) SM11, (c) SM6Δ and (d) SM11Δ.</p

Bacterial strains and plasmids used in the study.

<p>Amp^R ampicillin resistance, Kan^R kanamycin resistance, Ery^R erythromycin resistance, <i>oriTS</i> temperature sensitive origin of replication, <i>pldhL Lactobacillus</i> lactate dehydrogenase promoter, <i>gfp</i>_<i>uv</i> UV excitable green fluorescent protein.</p><p>Bacterial strains and plasmids used in the study.</p