CONTRUCTION AND STUDY OF MUTANT BACTERIA OF Pseudomonas putida KT2440 MUTANTS, WITH REDUCED SALT TOLERANCE.

COSTA GUTIERREZ SB; LAMI MJ,; CARAM DI SANTO C.; ,ZENOFF AM,; ADLER C.; DE CRISTOBAL, R.E.; VINCENT PA

San Miguel de Tucuman

Congreso; SAMIGE 2017; 2017

Asociacion Civil de Microbiologia General

CONTRUCTION AND STUDY OF MUTANT BACTERIA OF Pseudomonas putida KT2440 MUTANTS, WITH REDUCED SALT TOLERANCE.CONSTRUCCIÓN Y ESTUDIO DE MUTANTES DE LA BACTERIA Pseudomonas putida KT2440 CON REDUCIDA TOLERANCIA A SALINIDAD.Costa Gutierrez SB1, Lami MJ1, Caram di Santo MC1, Zenoff AM1, Vincent PA1, Espinosa Urgel M2 & de Cristóbal RE11INSIBIO (CONICET-UNT), Tucumán, Argentina. 2Estación Experimental del Zaidín, Granada-España.The aim of this work is to delve into stress salt tolerante mechanisms of Pseudomonas putida KT2440, which is a well known PGPR (Plant Growth Promoting Rhizobacteria). In order to evaluate this, transposon mutagenesis with mini-Tn5 (Km) was performed by triparental mating, with KT2440 as the recipient and both Escherichia coli CC118λpir (pUT-Km) and HB101 (RK600) as donor and helper strains, respectively; the mutants obtained were screened for their reduced tolerance to salinity in solid media.After evaluating 1500 transposon mutants, only four showed less growth under saline conditions in solid media, in the preliminary screening: Mut10, Mut11, Mut50 and Mut59. Growth curves in different liquid culture media and spots assay, with and without saline conditions, were carried out. The curves highlighted that mutants Mut11 and Mut59, grew markedly less with respect to the wild type, in saline conditions.Given these results, transposon insertion sites were determined by arbitrary PCR, followed by sequencing; obtained sequences were analyzed and compared with genome databases. After analyzing the sequences for the Mut11 mutant, it was determined that the transposon was inserted into the gene PP_0024, coding for a membrane-associated metal-dependent hydrolase, involved in the synthesis of lipopolysaccharides; whereas for the Mut59 mutant, the transposon was inserted into the gene PP_0003, which encodes a 16S RNA methyltransferase, whereby its growth is affected, not only in saline conditions.For a better understanding of the importance of the correct synthesis of lipopolysaccharides in the stress salt tolerance, we studied the behavior of mutant bacteria (mus-40).This mutant is affected in galU, encoding UDP-glucose pyrophosphorylase and presents deficiencies in the synthesis of intact lipopolysaccharide. Growth curves and spots assay, in different media and in saline and non saline conditions, were carried out. As expected both mutants, Mut11 and mus-40 showed reduce stress salt tolerance, compared to the wild type.Also Congo red binding assay in saline and non saline conditions was performed, and the results showed rough and less red colonies for Mut11 and darker colonies for mus-40, compared to the wild type. Congo red is a dye with cellulose fibers affinity, therefore the more links of this type there are, the more red the colony will be. But it is not a specific technique for exopolysaccharide. To evaluate EPS, calcofluor assay with and without saline stress was carried out, then calcofluor stainable EPS were visualized under UV light; mus-40 colonies were more fluorescent and Mut11 less fluorescent compared with the wild type colonies; the more fluorescence is an indicative for more EPS presence.Finally LPS quantification was performed using SDS-PAGE and visualized after silver stain; in the LPS profile the incomplete lipopolyshaccharide structure can be observed, especially under saline conditions.The results presented in this work give an idea of the great importance of the study of polysaccharides to improve bacteria tolerance to saline stress.