Analysis of Serratia sp. PQQE- mutants affected in their phosphate solubilization activity

LUDUEÑA, L., ANGELINI, J., FABRA, A., TAURIAN, T.

Mar del Plata

Congreso; VIII Congreso de Microbiologia General; 2012

Introduction. Phosphorus (P) is one of the major essential macronutrients for biological growth and development. The concentration of soluble P in soil is usually very low due to the fact that it reacts with ions that cause its precipitation. Phosphate solubilizing bacteria play an important role in P mobilization in soil. The main mechanism of inorganic P mineralization is the production of organic acids, being gluconic and ketogluconic acid the major compounds released by these bacteria. These acids are produced by Gram-negative bacteria through a direct glucose oxidation pathway involving the glucose dehydrogenase and gluconate dehydrogenase enzymes. These enzymes require the cofactor pyrroloquinoline quinone (PQQ) which has been described to be essential for phosphate solubilizing activity in many bacterial genera. In several bacteria, the PQQ biosynthetic genes are organized in a single pqqABCDEF cluster. The aim of this work was to analyze the role of PQQ bacterial protein in phosphate solubilization activity in the native peanut bacteria Serratia sp S119. Methods. Amplification and sequencing of pqqE gene: A 700 bp fragment of pqqE gene from Serratia sp. S119 was amplified by PCR using primers 1019R-317F. The PCR product was purified and its sequence was determined. Site-directed mutagenesis: the PCR product of partial pqqE gene was cleaved with PstI enzyme and a 540 pb fragment was cloned into the PstI site of plasmid pKnock-Gm. The plasmid pKnock-Gm-pqqE538 obtained was used to transform Escherichia coli SM10 ?É pir cells and it was transferred to the strain Serratia sp S119 by biparental conjugation. Transformants clones were identified in NBRIP-BPB medium containing tricalcium phosphate supplemented with appropriate antibiotics determining the diameter of phosphate solubilization halo. Isolation of genomic bacterial DNA was performed using Genomic DNA extraction kit (Real genomics). Characterization of mutants: ERIC-fingerprints, pKnock insert detection by PCR, viability and quantification of soluble P released in NBRIP broth from 2 to 24 hs. Results. The 3 selected pqqE- mutants (6, 11 and L) showed a significant reduction in their phosphate solubilization halo on NBRIP plates (