Analysis of a bac operon-silenced strain suggests pleiotropic effects of bacilysin in Bacillus subtilis.

Bacilysin, as the simplest peptide antibiotic made up of only L-alanine and L-anticapsin, is produced and excreted by Bacillus subtilis under the control of quorum sensing. We analyzed bacilysin-nonproducing strain OGU1 which was obtained by bacA-targeted pMutin T3 insertion into the parental strain genome resulting in a genomic organization (bacA'::lacZ::erm::bacABCDEF) to form an IPTG-inducible bac operon. Although IPTG induction provided 3- to 5-fold increment in the transcription of bac operon genes, no bacilysin activity was detectable in bioassays and inability of the OGU1 to form bacilysin was confirmed by UPLC-mass spectrometry analysis. Phenotypic analyses revealed the deficiencies in OGU1 with respect to colony pigmentation, spore coat proteins, spore resistance and germination, which could be rescued by external addition of bacilysin concentrate into its cultures. 2DE MALDI-TOF/MS and nanoLC-MS/MS were used as complementary approaches to compare cytosolic proteomes of OGU1. 2-DE identified 159 differentially expressed proteins corresponding to 121 distinct ORFs. In nanoLC-MS/MS, 76 proteins were differentially expressed in OGU1. Quantitative transcript analyses of selected genes validated the proteomic findings. Overall, the results pointed to the impact of bacilysin on expression of certain proteins of sporulation and morphogenesis; the members of mother cell compartment-specific σE and σK regulons in particular, quorum sensing and two component-global regulatory systems, peptide transport, stress response as well as CodY- and ScoC-regulated proteins.

In vitro labeling of hydroxyapatite minerals by an engineered protein.

Biological and biomimetic synthesis of inorganics have been a major focus in hard tissue engineering as well as in green processing of advanced materials. Among the minerals formed by organisms, calcium phosphate mineralization is studied extensively to understand the formation of mineral-rich tissues. Herein, we report an engineered fusion protein that not only targets calcium phosphate minerals but also allows monitoring of biomineralization. To produce the bi-functional fusion protein, nucleotide sequence encoding combinatorially selected hydroxyapatite-binding peptides (HABP) was genetically linked to the 3' end of the open reading frame of green fluorescence protein (GFPuv) and successfully expressed in Escherichia coli. The fluorescence and binding activities of the bi-functional proteins were characterized by, respectively, using fluorescence microscopy and quartz crystal microbalance spectroscopy. The utility of GFPuv-HABP fusion protein was assessed for both time-wise monitoring of mineralization and the visualization of the mineralized tissues. We used an alkaline phosphatase-based reaction to control phosphate release, thereby mimicking biological processes, to monitor calcium phosphate mineralization. The increase in mineral amount was observed using the fusion protein at different time points. GFPuv-HABP1 was also used for efficient fluorescence labeling of mineralized regions on the extracted human incisors. Our results demonstrate a simple and versatile application of inorganic-binding peptides conjugated with bioluminescence proteins as bi-functional bioimaging molecular probes that target mineralization, and which can be employed to a wide range of biomimetic processing and cell-free tissue engineering.

The effects of insertional mutations in comQ, comP, srfA, spo0H, spo0A and abrB genes on bacilysin biosynthesis in Bacillus subtilis.

In Bacillus subtilis, two extracellular signaling peptides, ComX pheromone and CSF (competence and sporulation factor), stimulate the development of genetic competence and surfaction biosynthesis in response to high cell density (quorum sensing) by regulating the activity of transcription factor ComA. We recently showed that biosynthesis of dipeptide antibiotic bacilysin is linked to ComA and PhrC(CSF) in a Spo0K(Opp)-dependent manner by constructing phrC-, comA- and oppA-disrupted mutants of B. subtilis. In the present study, another pathway of quorum-sensing global regulation, namely, ComQ/ComX was found to be essential for bacilysin biosynthesis. ComP function in this chain was dispensable, most probably because of the existence of an alternative mean of ComA activation. The disruption of srfA operon in the bacilysin producer resulted with the bacilysin-negative phenotype; thus, our study verified that the srfA operon functions directly in the production of bacilysin. The abrB mutation suppressed the bacilysin-negative phenotype of a spo0A mutant, whereas the same mutation in the wild-type strain resulted in a significant increase in the production of bacilysin. This indicated that abrB gene product negatively controls the transcription of the gene(s) involved in bacilysin formation.