Transposon search and identification of genes involved in biofilm formation regulated by c-di-GMP in Bordetella bronchiseptica.

C. SEBASTIÁN GIMÉNEZ; JULIETA FERNÁNDEZ; FEDERICO SISTI

Mar del Plata

Congreso; VII Reunión de la SAMIGE (Sociedad Argentina de Microbiología General); 2012

Sociedad Argentina de Microbiología General

Bordetella bronchiseptica isa pathogenic bacterium that causes respiratory infections in a wide variety of host. In a previous work we determined that high cyclic diguanylate (c-di-GMP) enhances biofilm formation in B. bronchiseptica. C-di-GMP is produced from two molecules of GTP by diguanylate cyclase (DGC). Activity is conferred with the GGDEF functional domain whereas c-di-GMP-specific phosphodiesterase activity is carried out by unrelated EAL or HD-GYP domains. However is established GGDEF, EAL and HD-GYP proteins regulates c-di-GMP intracellular concentration, little is known about how this second messenger interacts with other proteins to regulate phenotypes. In order to find proteins involved in biofilm c-di-GMP mediated formation we constructed a transposon library. We transformed B. bronchiseptica overexpressing a DGC (Bb-DGC) with transposon Tn5 by biparental conjugation. Transcojugants were selected with antibiotics and stocked at -20° C. We present here results after obtained 723 clones. If a transposon insertion interrupted a gen that codifies for a protein involved in c-di-GMP signaling and biofilm formation we expected to observe differences in adhesion to abiotic surfaces. Approximately 300 clones were screened for adhesion capacity to polycarbonate with the cristal violet (CV) technique. Bb-DGC presented a hyperbiofilm formation as described previously by us when growth media was supplemented with nicotinic acid (1.0 mM or 4.0 mM). Hence, these conditions were selected for screening. Those clones that presented CV values significantly lower than presented by Bb-DGC but over from wild type B. bronchiseptica were selected. Approximately 30 clones were re-evaluated in different nicotinic acid concentrations (0 to 4.0 mM) to confirm phenotype. With this method we were able to differentiate 20 clones whose capacity to adhesion to abiotic surface was impaired. Interestingly 6 clones presented significantly higher capacity to adhere than Bb-DGC. In order to determinate any anomaly in planktonic growth due to transposon insertion all clones were evaluated in batch cultures. All of them presented none differences with wild type or Bb-DGC strains. To identify interrupted open reading frames, transposon insertion were rescued and cloned in pLow2 plasmid. Insertion were sequenced and compared to B. bronchiseptica RB50 sequenced genome. We were able to identify to the moment the ORF interrupted in clone 12. The BB2515 gen presents homology to a LysR regulator and belongs to a big family of regulators in bacteria. The work presented here is expected to contribute to knowledge of c-di-GMP network, particularly in B. bronchiseptica. Comprehension of pathogenesis mechanism may be extrapolated to other Bordetella species and help to develop rationalized strategies to control the diseases that they cause.