GENERALIZED MUTAGENESIS AND TAIL-PCR FOR THE IDENTIFICATION OF COMPONENTS INVOLVED IN THE INVERSE REGULATION BETWEEN T3SS AND MOTILITY IN MESORHIZOBIUM LOTI

GONZÁLEZ AC; BASILE, LA; LEPEK, VC

Buenos Aires

Congreso; Reunión Conjunta de Sociedades de Biociencias; 2017

Sociedad Argentina de Investigación Bioquímica y Biología Molecular

Mesorhizobium loti presents a type 3 secretion system (T3SS) that is involved in the determination of bacterial competitiveness for the nodulation of Lotus spp. It was found that TtsI, the transcriptional factor that induces T3SS expression, inhibits bacterial motility in soft agar plates. This effect is accompanied by a down regulation of flagellar genes and flagella production. Besides, it was found that T3SS must be localized in membrane for that inhibition to occur. The objective proposed was to determine which mechanisms are involved in the motility inhibition. A generalized mutagenesis was performed by using mini-Tn5 transposon in order to isolate mutants that do not show this inhibition under T3SS induction conditions. A bacterial suspension was inoculated in soft agar plates containing naringenin (for T3SS induction) and those mutants that presented a higher motility were transferred to a new motility plate. After 5-6 passages, isolated colonies were obtained. In order to identify the gene in which the transposon was inserted, a TAIL-PCR method was performed for each mutant. We used a combination of random primers and transposon specific primers for the amplification of the region flanking the Tn5 sequence. By the evaluation of the presence or absence of the pili protein in culture supernatants, as indicative of T3SS functionality, mutants are being classified in two groups, those which the mutation affected a component of the T3SS and those in which a different gene was affected.