INTERCELLULAR COMMUNICATION IN A POLY-EXTREMOPHILIC EXIGUOBACTERIUM STRAIN ISOLATED FROM MODERN STROMATOLITES

GALVAN F. S.; ALONSO REYES D.; FARIAS M.E.; ALBARRACÍN V. H.

Oxford

Congreso; MICROSCIENCE MICROSCOPY CONGRESS; 2021

Royal Microscopical Society

Exiguobacterium sp. S17 is a Gram-positive bacterium isolated from modern stromatolites that were formed at the shore of Lake Socompa (3570 m.a.s.l). This lake belongs to the group of high-altitude Andean lakes (HAAL)) located in the Puna Andean region. They are exposed to extreme environmental conditions such as high levels of UV radiation, temperature fluctuations between day and night, alkalinity, elevated salinity, and heavy metals presence. S17 strain is characterized by its resistance to these extreme environments highlighting its ability to survive critical arsenic concentrations, for the presence of cytoplasmic arsenite flow pumps as well as the development of a strong cellular aggregation (biofilm); and high levels of UV irradiation, linked to a complete DNA repair system called UV-resistome complex. To survive environmental conditions, gram-positive and gram-negative bacteria have developed complex communication systems that involving contact-independent and contact-dependent signaling mechanisms. Contact through tubular protrusions such as conjugating pili, tubular spine and nanotubes allow intercellular molecular exchange, highlighting the nanotube by producing extensive connecting networks. In this work, we combined genomics data of S17 strain with its ultrastructure and topology information, provided by electron microscopy technics, to identify their potential cell interactions. Cell culture was grown in Luria-Bertani broth (LB, Britania) at 30°C with shaking (180rpm), and bacteria were harvested in the mid-exponential phase by centrifugation (5000 rpm, 5 min). For scanning electron microscopy, the pellet was immediately fixed with Karnovsky´s fixative for 24h at 4°C. The sample fixed was placed onto a coverslip for electron microscopy and kept for three hours at room temperature for its adhesion. After that, it was dehydrated successively with ethanol (30%, 50%, 70%, 90%, and 100%) for 10 min each and finally maintained in acetone 100% for 40 min. The dehydration was completed with the critical drying point (Denton Vacuum model DCP-1). Then, samples were mounted on stubs and covered by gold (Ion Sputter Marca JEOL model JFC-1100) and observed under a Zeiss Supra 55VP (Carl Zeiss NTS GmbH, Germany). For transmission electron microscopy, we followed the negative staining technic. A drop of the culture without fixed was placed on a formvar supported copper grid and remained for 5 min. Then, the grid dried with blotting paper. After, the grid was covered with a contrast agent (Uranyl acetate) for 1 min. The remaining fluid was removed and the grid allowed to dry. Bacteria were examined using a Zeiss LIBRA 120 (Carl Zeiss AG, Germany), equipment both belonging to the Electron Microscopy Core Facility (CIME). The genomic study of strain S17 indicated the presence of coding genes for the biogenesis of the flagellum: the CORE complex (basal body of the flagellum) formed by five integral membrane proteins: FliP, FliQ, FliR, FlhB and FlhA, flagellar motor proteins (MotB, MotA, and others), and the flagellar hook (FlgK, FlgL, FlgE, and others); and the presence of type IV intercellular pili coding genes such as PilO, PilM, PilN, PilC, PilA, among others. Also, we found the presence of the gen ymdB which coding for YmdB protein, a component required for nanotube formation and the molecular exchange. These data were correlated with electron microscopy images, evidencing the presence of membranous structures like pili and nanotubes, formed extensive intercellular networks.