QUORUM SENSING INTERCELLULAR COMUNICATION SYSTEM PARTICIPATES IN Yersinia enterocolitica BIOFILM FORMATION

CECILIA LUCERO ESTRADA; NATALIA DI MARCO; JOHANA WEIRICH; ERWIN BOHN

Cordoba

Congreso; XI Congreso Argentino de Microbiologia General (SAMIGE); 2015

Sociedad Argentina de Microbiología General

Yersinia enterocolitica (Ye) causes acute gastroenteritis which may result in severe post-infectiouscomplications as reactive arthritis. It is able to form biofilms onto biotic and abiotic surfaces but till themoment it is not clear the function of biofilm formation in its pathogenesis. Every species of the genera Yersinia is able to produce and release N-acyl-homoserin lactones (AHLs), diffusible molecules thatare part of a complex intercellular system known as Quorum Sensing (QS). In Ye the YenI/R system,which presents significant analogy to the LuxI/R family, participates in synthesis and recognition ofAHLs. The aim of this work was to obtain mutant strains with yenI/R deletion in order to know if QSparticipate in Ye biofilm formation. The reference strain Y. enterocolitica WA 1B/O:8 (bio/serotype),was used to obtain mutant strains. The Gibson assembling reaction was used to obtain a suicideplasmid that was cloned into Escherichia coli pir+ by electroporation. After obtaining multiples copiesof the plasmid, the E. coli b2168Dnic35 strain was transformed. The latter strain mated with Ye andthen suicide plasmid was integrated into Yersinia chromosome via homologous recombination creatingpartial diploids (merodiploid) with tetracycline resistance. The suicide plasmid also contains thecounter selection marker sacB. The SacB enzyme metabolites yield the cytotoxic product sucrose. Ye merodiploids were cultured on sucrose plates therefore clones that lost the suicide plasmid wereselected in this medium. The looping out of the suicide plasmid can either restore the original wild typeallele or yield the desired mutant allele. The result of the looping out reaction was tested by colonyPCR and also, mutants were validated by sequencing the mutated genes and analyzing the mRNAexpression. The crystal violet technique in a 96-well polystyrene plate (PE) was used to observe thebiofilm formed after 24 h of incubation at 25°C without shaking in trypticase soy broth added with0.25% glucose (TSBG). Laser Scanning Confocal Microscopy (LSCM) was used to observe biofilmsfixed on glass and stained with DAPI. With the Gibson cloning technique it was possible to obtain the yenI/R mutants without antibiotic resistance. The biofilm observed with mutant strains was significantlythinner than with wild type (WT) strain; the mean values were Ye Δ yenI 0.54±0.09, Ye Δ yenR0.64±0.08 andYe WT1.70±0.23 (p