Analysis of mutants that are affected in phosphate solubilisation activity

LUDUEÑA LILIANA, ANGELINI JORGE, FABRA ADRIANA, TAURIAN TANIA

Congreso; Congreso Argentino de Microbiologia General; 2011

Phosphorus is a limiting factor for plant growth. Low levels are due to the fact that soluble soil phosphorus reacts with ions that cause the precipitation, reducing their availability to plants. Many soil microorganisms are involved in processes that affect the transformation of phosphorus from soil being an integral part of its cycle. The main mechanism by which microbial phosphate compounds are mobilized is the release of organic acids of low molecular weight with a significant decrease of pH values. Understanding the basic mechanisms related to the solubilization of these compounds will allow the application of microorganisms with an effective and high capacity to increase the available levels of phosphorus for plants. Objective: To identify genes involved in phosphate solubilising mechanism. Getting transpositional insertion mutants: The plasmid pUTminiTn5Km was transferred to the strain Serratia sp S119 by triparental conjugation using Escherichia coli CC118 λ pir (donor) and E. coli DH5α (helper) strains. Selection of mutants: in NBRIP-BPB containing tricalcium phosphate supplemented with appropriate antibiotics and determination of phosphate solubilization halo in NBRIP-BPB. Isolation of genomic DNA. ERIC-fingerprints. MiniTn5 detection by PCR. Viability of mutants in NBRIP broth determining CFU/ml. Quantification of soluble phosphorus released in NBRIP containing glucose or glycerol as carbon source and analysis of pH from 2 to 48 hs of growth. There were selected 3 colonies (4A, 5A and 6A) that showed a significant reduction in their phosphate solubilization halo on NBRIP plates. These strains showed identical ERIC profiles being thus isogenic mutants of Serratia sp S119. In all of them it was possible to confirm the presence of the insert. Viability of mutants 4A and 5A was similar to that of wt while mutant 6A showed a significant decrease in CFU/ml. The mutant 4A released significantly less amounts of soluble phosphorus compared to the wt after 8 and 10 hs of growth, while in the mutant 5A this diminution was only observed after 8 hs of growth. The quantity of soluble phosphorus released was negatively correlated with pH values of the medium for all strains. The presence of a non-fermentative carbon source such as glycerol significantly decreased the solubilisation of phosphate in both the wt and the mutant 4A strains after 8 and 10 hs of growth. The pH values of the supernatants from wt or mutant 4A growing in presence of glycerol also differed respect to values obtained when glucose was used, showing a statistically significant increase at 8, 10 and 12 h of growth for the former whereas in the mutant 4A, difference was found only at 10 hs. The results obtained suggest that the mutant 4A is affected in the phosphate solubilisation mechanism. Further studies are needed to characterize the affected gene and its role in this mechanism.