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Novel method to achieve crystallinity of calcite by Bacillus subtilis in coupled and non-coupled calcium-carbon sources.

ABSTRACT: Bacteria mineralization is a promising biotechnological approach to apply in biomaterials development. In this investigation, we demonstrate that Bacillus subtilis 168 induces and influences CaCO₃ composites precipitation. Crystals were formed in calcium-carbon non-coupled (glycerol?+?CaCl₂, GLY; or glucose?+?CaCl₂, GLC) and coupled (calcium lactate, LAC; or calcium acetate, ACE) agar-sources, only maintaining the same Ca²⁺ concentration. The mineralized colonies showed variations in morphology, size, and crystallinity form properties. The crystals presented spherulitic growth in all conditions, and botryoidal shapes in GLC one. Birefringence and diffraction patterns confirmed that all biogenic carbonate crystals (BCC) were organized as calcite. The CaCO₃ in BCC was organized as calcite, amorphous calcium carbon (ACC) and organic matter (OM) of biofilm; all of them with relative abundance related to bacteria growth condition. BCC-GLY presented greatest OM composition, while BCC-ACE highest CaCO₃ content. Nucleation mechanism and OM content impacted in BCC crystallinity.

SUBMITTER: Ferral-Perez H

PROVIDER: S-EPMC7524977 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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Novel method to achieve crystallinity of calcite by Bacillus subtilis in coupled and non-coupled calcium-carbon sources.

Ferral-Pérez Héctor H Galicia-García Mónica M Alvarado-Tenorio Bonifacio B Izaguirre-Pompa Aldo A Aguirre-Ramírez Marisela M

AMB Express 20200929 1

Bacteria mineralization is a promising biotechnological approach to apply in biomaterials development. In this investigation, we demonstrate that Bacillus subtilis 168 induces and influences CaCO3 composites precipitation. Crystals were formed in calcium-carbon non-coupled (glycerol + CaCl2, GLY; or glucose + CaCl2, GLC) and coupled (calcium lactate, LAC; or calcium acetate, ACE) agar-sources, only maintaining the same Ca2+ concentration. The mineraliz ...[more]

PMID: 32990816

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Project description:BackgroundMicrobially induced calcite precipitation (MICP) is an ancient property of bacteria, which has recently gained considerable attention for biotechnological applications. It occurs as a by-product of bacterial metabolism and involves a combination of chemical changes in the extracellular environment, e.g. pH increase, and presence of nucleation sites on the cell surface or extracellular substances produced by the bacteria. However, the molecular mechanisms underpinning MICP and the interplay between the contributing factors remain poorly understood, thus placing barriers to the full biotechnological and synthetic biology exploitation of bacterial biomineralisation.ResultsIn this study, we adopted a bottom-up approach of systematically engineering Bacillus subtilis, which has no detectable intrinsic MICP activity, for biomineralisation. We showed that heterologous production of urease can induce MICP by local increases in extracellular pH, and this can be enhanced by co-expression of urease accessory genes for urea and nickel uptake, depending on environmental conditions. MICP can be strongly enhanced by biofilm-promoting conditions, which appeared to be mainly driven by production of exopolysaccharide, while the protein component of the biofilm matrix was dispensable. Attempts to modulate the cell surface charge of B. subtilis had surprisingly minor effects, and our results suggest this organism may intrinsically have a very negative cell surface, potentially predisposing it for MICP activity.ConclusionsOur findings give insights into the molecular mechanisms driving MICP in an application-relevant chassis organism and the genetic elements that can be used to engineer de novo or enhanced biomineralisation. This study also highlights mutual influences between the genetic drivers and the chemical composition of the surrounding environment in determining the speed, spatial distribution and resulting mineral crystals of MICP. Taken together, these data pave the way for future rational design of synthetic precipitator strains optimised for specific applications.

Project description:The clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system from Streptococcus pyogenes has been widely deployed as a tool for bacterial strain construction. Conventional CRISPR-Cas9 editing strategies require design and molecular cloning of an appropriate guide RNA (gRNA) to target genome cleavage and a repair template for introduction of the desired site-specific genome modification. Here, we present a streamlined method that leverages the existing collection of nearly 4,000 Bacillus subtilis strains (the BKE collection) with individual genes replaced by an integrated erythromycin (erm) resistance cassette. A single plasmid (pAJS23) with a gRNA targeted to erm allows cleavage of the genome at any nonessential gene and at sites nearby to many essential genes. This plasmid can be engineered to include a repair template, or the repair template can be cotransformed with the plasmid as either a PCR product or genomic DNA. We demonstrate the utility of this system for generating gene replacements, site-specific mutations, modification of intergenic regions, and introduction of gene-reporter fusions. In sum, this strategy bypasses the need for gRNA design and allows the facile transfer of mutations and genetic constructions with no requirement for intermediate cloning steps. IMPORTANCE Bacillus subtilis is a well-characterized Gram-positive model organism and a popular platform for biotechnology. Although many different CRISPR-based genome editing strategies have been developed for B. subtilis, they generally involve the design and cloning of a specific guide RNA (gRNA) and repair template for each application. By targeting the erm resistance cassette with an anti-erm gRNA, genome editing can be directed to any of nearly 4,000 gene disruptants within the existing BKE collection of strains. Repair templates can be engineered as PCR products, or specific alleles and constructions can be transformed as chromosomal DNA, thereby bypassing the need for plasmid construction. The described method is rapid and facilitates a wide range of genome manipulations.

Dataset Information

Novel method to achieve crystallinity of calcite by Bacillus subtilis in coupled and non-coupled calcium-carbon sources.

Publications

Novel method to achieve crystallinity of calcite by Bacillus subtilis in coupled and non-coupled calcium-carbon sources.

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