CONSTRUCTION AND STUDY OF Pseudomonas putida KT2440 MUTANTS, WITH REDUCED SALT TOLERANCE

LAMI, MARÍA JESÚS; ADLER, CONRADO; DE CRITÓBAL, RICARDO EZEQUIEL; CARAM DI SANTO, CAROLINA M; COSTA GUTIERREZ, STEFANIE BERNARDETTE; VINCENT, PAULA ANDREA; ZENOFF, ANA M; ESPINOSA-URGEL, MANUEL

San Miguel de Tucumán

Congreso; XII Congreso Argentino de Microbiología General; 2017

Asociación Civil de Microbiología General

The aim of this work is to delve into stress salt tolerant 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. 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 showed that the 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. The obtained sequences were analyzed and compared with genome databases. After analyzing the sequences for the Mut11, 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. For the Mut59, 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. 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 extraction with phenol-chloroform technique, running in SDS-PAGE and visualized after silver stain; was performed. In the LPS profile the incomplete lipopolysaccharide 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.