Isolation and characterization of a Mn(II)-oxidizing Bacillus strain from the demosponge Suberites domuncula.

In this study we demonstrate that the demosponge Suberites domuncula harbors a Mn(II)-oxidizing bacterium, a Bacillus strain, termed BAC-SubDo-03. Our studies showed that Mn(II) stimulates bacterial growth and induces sporulation. Moreover, we show that these bacteria immobilize manganese on their cell surface. Comparison of the 16S rDNA sequence allowed the grouping of BAC-SubDo-03 to the Mn-precipitating bacteria. Analysis of the spore cell wall revealed that it contains an Mn(II)-oxidizing enzyme. Co-incubation studies of BAC-SubDo-03 with 100 µM MnCl(2) and >1 µM of CuCl(2) showed an increase in their Mn(II)-oxidizing capacity. In order to prove that a multicopper oxidase-like enzyme(s) (MCO) exists in the cell wall of the S. domuncula-associated BAC-SubDo-03 Bacillus strain, the gene encoding this enzyme was cloned (mnxG-SubDo-03). Sequence alignment of the deduced MCO protein (MnxG-SubDo-03) revealed that the sponge bacterium clusters together with known Mn(II)-oxidizing bacteria. The expression of the mnxG-SubDo-03 gene is under strong control of extracellular Mn(II). Based on these findings, we assume that BAC-SubDo-03 might serve as a Mn reserve in the sponge providing the animal with the capacity to detoxify Mn in the environment. Applying the in vitro primmorph cell culture system we could demonstrate that sponge cells, that were co-incubated with BAC-SubDo-03 in the presence of Mn(II), show an increased proliferation potential.

Bioencapsulation of living bacteria (Escherichia coli) with poly(silicate) after transformation with silicatein-alpha gene.

Bioencapsulation is an intriguing way to immobilize biological materials, including cells, in silica, metal-oxides or hybrid sol-gel polymers. Until now only the sol-gel precursor technology was utilized to immobilize bacteria or yeast cells in silica. With the discovery of silicatein, an enzyme from demosponges that catalyzes the formation of poly(silicate), it became possible to synthesize poly(silicate) under physiological (ambient) conditions. Here we show that Escherichia coli can be transformed with the silicatein gene, its expression level in the presence of isopropyl beta-D-thiogalactopyranoside (IPTG) can be efficiently intensified by co-incubation with silicic acid. This effect could be demonstrated on the level of recombinant protein synthesis as well as by immunostaining analysis. The heterologously produced silicatein is enzymatically active, as confirmed by staining with Rhodamine 123 (formation for poly[silicate] from silicic acid) and by reacting free silicic acid with the beta-silicomolybdato color system. Electron microscopic analysis revealed that the bacteria that express silicatein form a viscous cover around them when growing in the presence of silicic acid. Finally, we demonstrate that the growth kinetics of E. coli remains unaffected whether or not the bacteria had been transformed with silicatein or grown in medium, supplemented with silicic acid. It is concluded that silicatein-mediated encapsulation of bacteria with silica might improve, extend and optimize the range of application of bacteria for the production of recombinant protein.