Probing the regulatory effects of specific mutations in three major binding domains of the pleiotropic regulator CcpA of Bacillus subtilis

Carbon catabolite control is required for efficient use of available carbon sources to ensure rapid growth of bacteria. CcpA is a global regulator of carbon metabolism in Gram-positive bacteria like Bacillus subtilis. In this study the genome-wide gene regulation of a CcpA knockout and three specific CcpA mutants were studied by transcriptome analysis, to further elucidate the function of specific binding sites in CcpA. The following three amino acids were mutated to characterize their function: M17(R) which is involved in DNA binding, T62(H) which is important for the allosteric switch in CcpA upon HPr-Ser46-P binding, and R304(W) which is important for binding of the coeffectors HPr-Ser46-P and fructose-1,6-bisphosphate. The results confirm that CcpA was also involved in gene regulation in the absence of glucose. CcpA-M17R showed a small relief of Carbon Catabolite Control; the CcpA-M17R mutant regulates fewer genes than the CcpA-wt and the palindromicity of the cre site is less important for CcpA-M17R. CcpA-T62H was a stronger repressor than CcpA-wt and also acted as a strong repressor in the absence of glucose. CcpA-R304W was shown here to be less dependent on HPr-Ser46-P for its carbon catabolite control activities. The results presented here provide detailed information on alterations in gene regulation for each CcpA-mutant

Amphiphilic poly(ether ester amide) multiblock copolymers as biodegradable matrices for the controlled release of proteins

Amphiphilic poly(ether ester amide) (PEEA) multiblock copolymers were synthesized by polycondensation in the melt from hydrophilic poly(ethylene glycol) (PEG), 1,4-dihydroxybutane and short bisester-bisamide blocks. These amide blocks were prepared by reaction of 1,4-diaminobutane with dimethyl adipate in the melt. A range of multiblock copolymers were prepared, with PEG contents varying from 23-66 wt %. The intrinsic viscosity of the PEEA polymers varied from 0.58-0.78. Differential scanning calorimetry showed melting transitions for the PEG blocks and for the amide-ester blocks, suggesting a phase separated structure. Both the melting temperature and the crystallinity of the hard amide-ester segments decreased with increasing PEG content of the polymers. The equilibrium swelling ratio in phosphate buffered saline (PBS) increased with increasing amount of PEG in the polymers and varied from 1.7 to 3.7, whereas the polymer that contained 66 wt % PEG was soluble in PBS. During incubation of PEEA films in PBS, weight loss and a continuous decrease in the resulting inherent polymer viscosity was observed. The rate of degradation increased with increasing PEG content. The composition of the remaining matrices did not change during degradation. A preliminary investigation of the protein release characteristics of these PEEA copolymers showed that release of the model protein lysozyme was proportional to the square root of time. The release rate was found to increase with increasing degree of swelling of the polymers

Stress-induced activation of the proline biosynthetic pathway in Bacillus subtilis:A population-wide and single-cell study of the osmotically controlled proHJ promoter

Bacillus subtilis, in its natural habitat, is regularly exposed to rapid changes in the osmolarity of its surrounding. As its primary survival strategy, it accumulates large amounts of the compatible solute proline by activating the de novo proline biosynthesis pathway and exploiting the glutamate pools. This osmotically-induced biosynthesis requires activation of a SigA-type promoter that drives the expression of the proHJ operon. Population-wide studies have shown that the activity of the proHJ promoter correlates with the increased osmotic pressure of the environment. Therefore, the activation of the proHJ transcription should be an adequate measure of the adaptation to osmotic stress through proline synthesis in the absence of other osmoprotectants. In this study, we investigate the kinetics of the proHJ promoter activation and the early adaptation to mild osmotic upshift at the single-cell level. Under these conditions, we observed a switching point and heterogeneous proline biosynthesis gene expression, where the subpopulation of cells showing active proHJ transcription is able to continuously divide, and those unresponsive to osmotic stress remain dormant. Additionally, we demonstrate that bactericidal antibiotics significantly upregulate proHJ transcription in the absence of externally imposed osmotic pressure, suggesting that the osmotically-controlled proline biosynthesis pathway is also involved in the antibiotic-mediated stress response

Boosting heterologous protein production yield by adjusting global nitrogen and carbon metabolic regulatory networks in Bacillus subtilis

Bacillus subtilis is extensively applied as a microorganism for the high-level production of heterologous proteins. Traditional strategies for increasing the productivity of this microbial cell factory generally focused on the targeted modification of rate-limiting components or steps. However, the longstanding problems of limited productivity of the expression host, metabolic burden and non-optimal nutrient intake, have not yet been completely solved to achieve significant production-strain improvements. To tackle this problem, we systematically rewired the regulatory networks of the global nitrogen and carbon metabolism by random mutagenesis of the pleiotropic transcriptional regulators CodY and CcpA, to allow for optimal nutrient intake, translating into significantly higher heterologous protein production yields. Using a β-galactosidase expression and screening system and consecutive rounds of mutagenesis, we identified mutant variants of both CodY and CcpA that in conjunction increased production levels up to 290%. RNA-Seq and electrophoretic mobility shift assay (EMSA) showed that amino acid substitutions within the DNA-binding domains altered the overall binding specificity and regulatory activity of the two transcription factors. Consequently, fine-tuning of the central metabolic pathways allowed for enhanced protein production levels. The improved cell factory capacity was further demonstrated by the successfully increased overexpression of GFP, xylanase and a peptidase in the double mutant strain

Single Cell FRET Analysis for the Identification of Optimal FRET-Pairs in Bacillus subtilis Using a Prototype MEM-FLIM System

Protein-protein interactions can be studied in vitro, e.g. with bacterial or yeast two-hybrid systems or surface plasmon resonance. In contrast to in vitro techniques, in vivo studies of protein-protein interactions allow examination of spatial and temporal behavior of such interactions in their native environment. One approach to study protein-protein interactions in vivo is via Förster Resonance Energy Transfer (FRET). Here, FRET efficiency of selected FRET-pairs was studied at the single cell level using sensitized emission and Frequency Domain-Fluorescence Lifetime Imaging Microscopy (FD-FLIM). For FRET-FLIM, a prototype Modulated Electron-Multiplied FLIM system was used, which is, to the best of our knowledge, the first account of Frequency Domain FLIM to analyze FRET in single bacterial cells. To perform FRET-FLIM, we first determined and benchmarked the best fluorescent protein-pair for FRET in Bacillus subtilis using a novel BglBrick-compatible integration vector. We show that GFP-tagRFP is an excellent donor-acceptor pair for B. subtilis in vivo FRET studies. As a proof of concept, selected donor and acceptor fluorescent proteins were fused using a linker that contained a tobacco etch virus (TEV)-protease recognition sequence. Induction of TEV-protease results in loss of FRET efficiency and increase in fluorescence lifetime. The loss of FRET efficiency after TEV induction can be followed in time in single cells via time-lapse microscopy. This work will facilitate future studies of in vivo dynamics of protein complexes in single B. subtilis cells

The FRET efficiency Ea of the different FRET pairs, measured with FLIM.

<p>The fluorescence lifetime of GFP_only is 2.56 ns. The fluorescence lifetimes shown here are calculated with the LI-FLIM software from the average of all pixels in five regions of interest filled with a monolayer of cells.</p><p>The FRET efficiency Ea of the different FRET pairs, measured with FLIM.</p

The strains used in this study.

<p>The strains used in this study.</p