SCREENING, IDENTIFICATION AND CHARACTERIZATION OF GLUCONACETOBACTER DIAZOTROPHICUS PAL5 MUTANTS POTTENTIALLY RELATED WITH BIOFILM FORMATION

Marcio Loureiro (IFRJ); Vania Padua (UEZO); Juan Santos (IFRJ); Larissa Almeida (IFRJ); Rafael Ventura (UFRJ); Claudia Costa (UEZO); Bianca Oliveira (UFRJ); Jose Baldani (Embrapa-Agrobiologia); Adriana Hemerly (UFRJ); Ana Coelho (UFRJ)

Several bacterial strains are very creative in their regulatory processes and adaptation to sudden changes in nutrient availability and response to host secondary defenses. A particularly important example of bacterial adaptation is the ability to grow as a biofilm, forming an optimized niche embedded in a gel-like extracellular polymer network of microbial origin. The bacterium Gluconacetobacter diazotrophicus is found mainly in the intercellular spaces and vascular tissues of plants from different families, especially sugarcane, resulting in substantial benefits for vegetal development. The complete genomic sequences of G. diazotrophicus PAL5 was completely decoded (RioGene Project) and as a follow up of this work, a library containing random transposon mutations was generated at Embrapa-Agrobiologia – Seropédica – RJ. This work is based on the identification of two G. diazotrophicus mutants with reduced biofilm formation capacity, using glasswool as a test surface. Selection was based on growth in LGIP medium in 96 well polystyrene plates containing glasswool fibers. The culture was stained with crystal violet, washed with ethanol and was measured by OD 600nm of the solution to estimate the amount of biofilm formation. Knockout genes were identified through direct bi-directional sequencing from genomic DNA, using terminal transposon-specific primers. In one case, the insertion occurred in a gene responsible by production of a putative transcriptional regulator with a LacI-like domain. In the other mutant the insertion was detected in a coding gene of the diguanylate cyclase protein (GGDEF) that is often linked to regulatory domains, synthesizing cyclic di-GMP, which is used as an intracellular signalizing molecule.