Preliminary indication of the role of AHL-dependent quorum sensing systems in calcium carbonate precipitation in Gram-negative bacteria

Numerous microbial species participate in precipitation of carbonates in various natural environments, including soils, geological formations, freshwater biofilms and oceans. Despite the geochemical interest of such a biomineralization process, its molecular mechanisms and adaptive aspects remain poorly known. Many Gram-negative bacteria use cell-to-cell communication systems relying on N-acylhomoserine lactone (AHLs) signal molecules to express certain phenotypic traits in a density-dependent manner, a phenomenon referred as to quorum-sensing (QS). In this work, bacterial isolates collected from cave and rhizosphere soil were analyzed to study the occurrence of the AHL-mediated QS in bacterial calcium carbonate (CaCO3) precipitation. To test the production of AHLs signal molecules, we cross-streaked Gram-negative calcifying strains, selected among the environmental strains studied, with the AHL-negative mutant Chromobacterium subtsugae strain CV026. Only Burkholderia ambifaria LMG 11351 was able to restore violacein production in CV026 among the tested strains. The constructed AHL-negative mutant of B. ambifaria LMG 11351 could not precipitate CaCO3 on B-4 agar. Scanning Electron Microscopy (SEM) analysis on CaCO3 crystals obtained in vitro shows crystals of different morphologies, calcified biofilms and bacteria in close contact with the precipitated crystals. In the inner layers of the bioliths deposited by B. ambifaria LMG 11351, a stream-like organization of the Burkholderia imprints was not detected by SEM. Our data provide preliminary evidence that the activation of AHL-regulated genes may be a prerequisite for in vitro bacterial carbonatogenesis, in some cases, confirming the specific role of bacteria as CaCO3 precipitating agents. We enhance the understanding of bacterial CaCO3 biomineralization and has potential biotechnology implications for QS-based strategies to enhance or decrease CaCO3 precipitation through specific bacterial processes. The AHL-negative mutant of B. ambifaria LMG 11351 (a well-known plant growth-promoting bacterium) could also be used to study plant-bacteria interactions. The adaptive role of bacterial CaCO3 biomineralization was also discussed.