Physiological requirements for carbonate precipitation during biofilm development ofBacillus subtilis etfAmutant

Abstract

Although the implications of calcium carbonate (CaCO(3)) precipitation by microorganisms in natural environments are quite relevant, the physiology and genetics of this phenomenon are poorly understood. We have chosen Bacillus subtilis 168 as our model to study which physiological aspects are associated with CaCO(3) (calcite) formation during biofilm development when grown on precipitation medium. A B. subtilis eftA mutant named FBC5 impaired in calcite precipitation was used for comparative studies. Our results demonstrate that inactivation of etfA causes a decrease in the pH of the precipitation medium during biofilm development. Further analysis demonstrated that eftA extrudes an excess of 0.7 mol H(+) L(-1) with respect to B. subtilis 168 strain. Using media buffered at different pH values, we were able to control calcite formation. Because etfA encodes the alpha-subunit of a putative flavoprotein involved in fatty acid metabolism, we compared the intracellular levels of NADH among strains. Our physiological assay showed that FBC5 accumulated up to 32 times more NADH than the wild-type strain. We propose that the accumulation of NADH causes a deregulation in the generation of the proton motive force (DeltamicroH(+)) in FBC5 producing the acidification.

Importance of B4 Medium in Determining Organomineralization Potential of Bacterial Environmental Isolates

“…Based on our light microscopic observations, this matrix could consist of extracellular polymeric substances (Marvasi et al 2010), dead cells, and spores (of those spore-forming bacteria). A drop of 0.1 N HCl was used to completely dissolve the CaCO 3 and reveal the presence of an intact amorphous matrix (Fig.…”

“…Our previous studies with a non-precipitating laboratory strain Bacillus subtilis etfA mutant revealed that a decrease in the pH during biofilm growth prevented CaCO 3 precipitation, which was always observed in wild-type B. subtilis 168 strain (Barabesi et al 2007;Marvasi et al 2010). Further analysis demonstrated that when compared to the 168 strain, the etfA mutant extruded an excess of 0.7 mol H + L −1 .…”

“…PR stock solution (20 mg/ml in NaOH 0.1N) was added to B4 medium before autoclaving at a final concentration of 0.0025% (v/v (Marvasi et al 2010). The pH of each plate was maintained by buffering the medium with TRIS 1.2% (w/v) and HCl 2N.…”

“…Modified versions of B4 medium have also been used; for example, calcium acetate was replaced by calcium citrate to study vaterite precipitation , and by barium acetate to study barite precipitation (SanchezMoral et al 2003). Dextrose levels were decreased to 0.5% to study carbonate precipitation in Bacillus (Barabesi et al 2007;Marvasi et al 2010).…”

“…Further analysis demonstrated that when compared to the 168 strain, the etfA mutant extruded an excess of 0.7 mol H + L −1 . This excessive proton extrusion prevented the precipitation of CaCO 3 (Marvasi et al 2010). Using media buffered at different pH values, we were able to control CaCO 3 precipitation by the two strains (Marvasi et al 2010).…”