The IS6770 insertion sequence modifies the promoter region structure of the gene kup encoding for the potassium symporter in Enterococcus faecalis JH2-2

ACCIARRI, G; ALEMAN, M; GIZZI, F; BLANCATO, VS; MAGNI, C

Mendoza

Congreso; LVIII Reunión Anual de SAIB; 2022

SAIB

Enterococcus faecalis is a ubiquitous colonizer of the gastrointestinal tract of animals, however, this microorganism evolved in the last decade from an avirulent commensal to nosocomial multiresistant bacteria. The intrinsic robustness of E. faecalis, which allows it to tolerate different environmental stress conditions, is directly related to the pathogenic potential of this bacterium. In our laboratory demonstrated that Kup and KimA proteins, two members of the K+ transporter Kup family, catalyze the uptake of K+ inside the cells and are involved in the response to osmotic stress in E. faecalis. These genes are located in a chromosomal region of 16 kbp, including the opuABCD operon, which encodes for putative osmolyte transporters, and two putative cation transporters. Insertion Sequences (IS), which codify for transposases, are mediators of genetic diversity in prokaryotes. They could induce rearrangements including deletions, duplications, inversion and activation of genes. The IS6770 is composed of a unique Orf that codifies for a putative transposase (member of the IS30 element) and 30 nucleotides of the imperfect inverted repeat. Even though, IS6770 was identified in E. faecalis strain associated to clinical isolated, in the vancomycin resistant strain V583 is absent. In the laboratory strain JH2-2 we detected 12 copies of the IS6770, two of them located downstream of both the kup and the kimA gene forming a genetic structure that constitutes a putative compound transposon. The presence of the IS6770, located 79 bp upstream of the kup gene initiation, modified the structure of the promotor region. To analyze whether the presence of IS6770 in the JH2-2 strain modified the transcriptional expression of the kup gene, transcriptional fusions of kup upstream regions of different sizes with a fluorescent reporter gene were performed, and then compared with transcriptional fusions of the promoter region of the V583 strain (IS6770 deficient strain). It was observed that while the promoter region amplified from the V583 strain allowed the expression of the fluorescent protein, the upstream region amplified from JH2-2, which includes part of the IS6770, showed no activity. However, the region containing part of the kup promoting region and a full copy of IS6770 from JH2-2 did show activity. Thus, these experiments suggest that the promoter region required for kup gene expression was either displaced by the IS6770 insertion or replaced by an internal promoter region located in the IS. Finally, in silico analyses suggest a recent formation of this composite transposon with the potential to transfer genes involved in osmotic resistance, since transposition of this putative element was not found in other localization of the bacterial genome.