CONJUGATIVE PROPERTIES OF PLASMIDS BELONGING TO GROUP I-C OF RHIZOBIA

CABRERA, MARÍA DELFINA; CASTELLANI, LUCAS G.; LUCHETTI, ABRIL; PISTORIO, MARIANO; TORRES TEJERIZO, GONZALO A.

Chapadmalal

Congreso; XVIII Congreso de la Sociedad Argentina de Microbiología General (SAMIGE 2023); 2023

Sociedad Argentina de Microbiología General

Rhizobia are gram-negative bacteria capable of interacting symbiotically with leguminous plants. These bacteria usually have plasmids that can be transferred by conjugation. Two types of plasmid transfer regulation systems have been deeply described in rhizobia: Quorum Sensing (QS) and rctA/rctB system. In the QS regulation mechanism, a signal molecule is produced by traI (luxI-like) gene and it is accumulated in the environment. When this molecule reaches a certain concentration, it enters the cell and binds with the TraR regulator, allowing the expression of conjugative genes. In the rctA/rctB system, the product of rctA gene inhibits conjugative genes expression. This inhibitory function is reduced by RctB, allowing conjugation. Nevertheless, conditions for RctB expression are not known yet.Rhizobial plasmids are classified in group I, II, III and IV. Group I includes plasmids regulated, principally, by QS. Among plasmids belonging to group I, four subgroups were described: I-A, I-B, I-C and I-D. Plasmid pLPU83a from Rhizobium favelukesii LPU83 is the model of group I-C. The regulatory network involved in its transfer has been studied recently. pLPU83a harbors a traR gene in the conjugation locus, but there is no traI within the conjugative region, implying a new regulatory system of rhizobial plasmid transfer. In view of this, in this work we used molecular biology and bioinformatic tools to study the conjugative transfer (CT) of other rhizobial plasmids of group I-C: pRL8 from Rhizobium leguminosarum bv. viciae 3841, pSmeSM11b from Sinorhizobium meliloti SM11 and pDD12c from Shinella sp. DD12. Plasmids were tagged and the conjugative phenotype was observed from the parental strain and from a plasmid-free Agrobacterium strain UBAPF2.pLPU83a is conjugative from its parental strain and showed a 3-fold lower CT frequency from plasmid-free Agrobacterium strain UBAPF2. pRL8 has a CT frequency higher than pLPU83a in both genomic backgrounds. Regarding pSmeSM11b, it showed an undetectable CT frequency from its parental strain. Bioinformatic analysis showed that it has a frameshift in trbE gene. It was shown that TrbE is required for type 4 secretion system machinery. Thus, we complemented pSmeSM11b with the complete TrbE from pLPU83a and that lead to detectable CT frequencies, similar to pLPU83a from its parental strain and higher than pLPU83a from UBAPF2. Last of all, pDD12 has a CT frequency lower than pLPU83a from the parental strain.The differences in CT frequencies between pRL8, pDD12c and pLPU83a indicates that the conjugal transfer regulation may be partially different, either by the presence or the possible differential expression of certain genes in the plasmid. Furthermore, we demonstrated that the frameshift in trbE gene is responsible for the undetectable CT frequency of pSmeSM11b. Future efforts will aim to unravel the molecular system that regulates CT of plasmids of group I-C.