Emerging Applications of Bacterial Spores in Nanobiotechnology

Bacterial spores are robust and dormant life forms with formidable resistance properties, in part, attributable to the multiple layers of protein that encase the spore in a protective and flexible shield. The coat has a number of features pertinent to the emerging field of nanobiotechnology including self-assembling protomers and the capacity for engineering and delivery of foreign molecules. This review gives an account of recent progress describing the use of the spore, and specifically, the spore coat as a vehicle for heterologous antigen presentation and protective immunization (vaccination). As interest in the spore coat increases it seems likely that they will be exploited further for drug and enzyme delivery as well as a source of novel self-assembling proteins

DEVELOPMENT OF PROTEIN DISPLAY SYSTEMS AND GENETIC TOOLS FOR SPORE-FORMING BACTERIA

One major area of synthetic biology is to engineer microbial cells and subcellular systems for diverse applications including biosynthesis, biocatalysis, therapeutics, drug delivery, and bioremediation. For most applications, robust cellular systems are preferred for longer activity half-life and resistance to harsh environments. Two projects related to robust cellular systems involving Gram-positive bacteria are presented in this work. One is to develop thermostable genetic reporters for Geobacilli species and the other is to display an enzyme on the Bacillus subtilis spore surface to enhance its robustness and present an alternative to purified enzymes for industrial applications. Bacillus subtilis and Geobacillus thermoglucosidans are gram-positive, spore-forming bacteria. They secrete many proteins used industrially for the production of paper, food, textiles, chemicals, medicine, and cosmetics. Since G. thermoglucosidans is thermostable with an optimal growth temperature of 60ºC, its secreted proteins are also thermostable which proves advantageous for a variety of industrial applications. Additionally, a strain of G. thermoglucosidans has been used for the production of ethanol from biomass. Unfortunately the inner workings of G. thermoglucosidans are still poorly understood and a genetic toolkit is necessary to better discover how to improve them via genetic engineering for industrial use. Important components of this toolkit are genetic reporters which allow for the analysis of gene expression in G. thermoglucosidans. Fluorescent proteins are commonly used reporters for other bacterial species due to their easily observed and readily measured signal, however no thermostable fluorescent proteins have been shown to be functional in Geobacillus. Seven different fluorescent proteins including mCherry, Venus, GFP, sfGFP, GFPmut3, mCherry (Gt), and Venus (Gt) were tested for stability and functionality in Geobacillus thermoglucosidans. Venus (Gt) and mCherry (Gt) were codon optimized for this bacterium with the goal of increasing expression level and thus improving the fluorescence signal. The fluorescence intensity of each fluorescent protein expressed in G. thermoglucosidans was measured after several hours of bacterial growth at 50ºC and 60ºC. Venus, mCherry, Venus (Gt), mCherry (Gt), and sfGFP all had signal when expressed in G. thermoglucosidans at 50ºC and sfGFP had signal at 60ºC. Therefore, fluorescent reporter proteins in three different colors were found to be functional in G. thermoglucosidans. This will further genetic engineering of the species for thermostable protein production, bioremediation, and biofuel production. Bacillus subtilis is Generally Regarded as Safe (GRAS) by the FDA and amenable toward genetic manipulation. Thus it has been engineered for the production of many heterologous proteins. Oftentimes, proteins secreted by bacteria are purified for industrial use. However, protein purification is expensive and time-consuming and long-term storage of purified proteins requires extremely low temperatures (-20ºC). B. subtilis spores have been used to immobilize a variety of proteins for vaccines, biosensors, and bioremediation applications. Spore surface display eliminates the need for purification and provides a way to easily separate proteins from the final product if necessary. A novel and thermostable laccase, a copper-containing oxidase, was isolated and purified from G. thermoglucosidans. It can be used to degrade lignin and a variety of phenolic compounds and thus has applications for the production of paper, textiles, food, and biofuel. This laccase was isolated, characterized, and immobilized on the surface of B. subtilis spores. The purified and spore displayed laccase were tested for heat stability and catalytic function. The purified laccase showed high activity toward 2,6-dimethoxyphenol (2,6-DMP) and moderate activity toward veratryl alcohol and 2,2′-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) while the spore displayed laccase showed high activity toward 2,6-DMP. The purified laccase was considerably more heat stable than a commonly used fungal laccase. The spore displayed laccase was also found to be heat stable with a half-life of about 6 hours at 80ºC. The binding affinity of the immobilized laccase for the substrate 2,6-DMP was virtually the same as that of the purified laccase, plus the immobilized laccase showed solid activity. These results show that spore surface display of proteins is a promising, more inexpensive alternative to purifying proteins for industrial use

Protein engineering of cota laccase by using bacillus subtilis spore display

Spore display offers advantages over more commonly utilized microbe cell-surface display systems. For instance, protein-folding problems associated with the expressed recombinant polypeptide crossing membranes are avoided. Hence, a different region of protein space can be explored that previously was not accessible. In addition, spores tolerate many physical/chemical extremes. The aim is to improve pH stability using spore display. The maximum activity of CotA is between pH 4 and 5 for the substrate ABTS (ABTS = diammonium 2, 2’-azino-bis(3-ethylbenzothiazoline-6-sulfonate)). However, the activity dramatically decreases at pH 4. The activity is not significantly altered at pH 5. CotA is used as a model to prove that enzymes could be improved for pH resistance by using Bacillus subtilis spore display. First, CotA is evolved for increased half-life (t1/2) at pH 4. Next, a double mutant is constructed. This variant combines the amino acid substitutions from the improved t1/2 variant (E498G) and organic solvent tolerant mutant (T480A). The t1/2 and kinetic parameters are evaluated for the double mutant. Consequently, T480A/E498G-CotA is constructed and the t1/2 is 62.1 times greater than wt-CotA. Finally, T480A/E498G-CotA yields 5.3-fold more product than does wt-CotA after recycling the biocatalyst seven times over 42 h. Also, the mutant and wild-type are overexpressed in E. coli and purified. The enzymes immobilized in the spore coat are compared with the purified free protein. The t1/2 and catalytic efficiency follow the same trends for spore or E. coli expressed wt-CotA and E498G-CotA, although the kinetic parameters are different. In a previous investigation, a laccase (CotA), which is found on the spore coat of Bacillus subtilis, was engineered by directed evolution for improved activity in organic solvents. A CotA variant was identified with a Thr480Ala (T480A-CotA) amino acid substitution after only one round of evolution. The screen was performed at 60 % DMSO and it was 2.38-fold more active than the wild-type CotA (wt-CotA) with substrate ABTS. T480A-CotA was more active from a range of 0 - 70 % DMSO. In addition, the variant was more active in ethanol, methanol and acetonitrile. In this study, the catalysis of T480A-CotA and wt-CotA in the spore coat is determined with natural phenolic compounds, such as (+)-catechin, (-)-epicatechin and sinapic acid in aqueous-organic media. In general, the catalytic efficiency (Vmax/Km (δA/OD580)/mM) of T480A-CotA is higher than wt-CotA for all the substrates. Then, the Vmax for T480A-CotA is greater than the wt-CotA in all organic solvents used in this study. The Vmax for T480A-CotA is up to 3.4-fold, 7.9-fold and 6.4-fold greater than wt-CotA for substrate (+)-catechin, (-)-epicatechin and sinapic acid, respectively. In addition, the catalyst can be easily removed from the reaction solution and reused. This allows for simpler recovery of the product from the enzyme. This investigation indicates that enzymes expressed on the spore coat can be utilized for industrial applications

GENE REGULATION PATHWAYS AFFECT TOXIN GENE EXPRESSION, SPORULATION AND PIGMENT GENERATION IN BACILLUS ANTHRACIS AND

B. anthracis alters its virulence gene expression profile in response to a number of environmental signals, including levels of bicarbonate and CO2. Virulence plasmid pXO1 is important to Bacillus anthracis pathogenicity as it carries the genes encoding the anthrax toxin and virulence regulatory factors. Induction of toxin and other virulence genes requires the pXO1-encoded AtxA regulatory protein. The cytochrome c maturation system influences the expression of virulence factors in Bacillus anthracis. B. anthracis carries two copies of the ccdA gene, encoding predicted thiol-disulfide oxidoreductases that contribute to cytochrome c maturation. Loss of both ccdA genes results in a reduction of cytochrome c production, an increase in virulence factor expression, and a reduction in sporulation efficiency. pXO1 also carries a gene encoding an Hfq-like protein, pXO1-137. Loss of pXO1-137 results in significant growth defects and reductions in toxin gene expression only when grown under toxin inducing conditions. Similarly, loss of a small RNA on pXO1, sRNA-1, results in similar growth defects and reductions in toxin gene production. Both increased and decreased expression of pXO1-137 and sRNA-1 result in growth defects suggesting narrow functional set points for Hfq and sRNA levels

The sporulation-specific small regulatory RNAs of Bacillus subtilis

Constantly changing environments in nature have led to bacteria evolving regulatory strategies that result in differential gene expression. A novel and understudied aspect of these networks are regulatory RNAs. The Gram-positive model organism Bacillus subtilis not only modulates gene expression to survive a variety of stresses, but also can form endospores to ensure its survival. Sporulation is an essential survival mechanism for many species, allowing them to enter a state of dormancy with resistance to various harsh conditions. This, in turn, ensures survival of not only the population, but also the species. The process of sporulation requires the controlled expression of approximately a quarter of the genes encoded by B. subtilis. Previous large-scale studies have identified that many transcripts do not encode proteins, but exhibited expression profiles similar to genes already known to be part of the sporulation network. Many of these transcripts were selected to likely function as small regulatory RNAs (sRNAs). This study has shown that many putative sRNAs are active during sporulation, three of which show specific phenotypes that alter germination capabilities in the presence of specific germinants. Cells lacking the necessary components to reverse this process are at a strong disadvantage. Detection of favorable growth conditions is key, but how is this conveyed during metabolic inactivity? Initial selection of putative sRNAs was done by in silico characterization. Prediction of transcriptional control and regulatory regions combined with tiling array profiling was used to select putative sRNAs for confirmation in vivo. Transcriptional fusion constructs were generated to confirm compartmental specific expression during sporulation. Spore specific sRNAs were further characterized with phenotypic studies, which suggested a role in endospore formation. This study explored some of the global analysis methods to identify sRNA targets. Whilst no targets for the four chosen sRNAs could be identified, this study produced the most comprehensive data set of proteins to be identified from a B. subtilis endospore

Bacterial Spore: analysis of surface proteins and biotechnological applications

In my PhD project, I focused on two bacteria of the same genus, Bacillus subtilis and Bacillus megaterium. These organisms are Gram-positive, aerobic and spore formers. This work includes studies on the sporulation temperature consequences on the ultrastructure and the protein composition of the spore surface layers and on the functional properties of spores produced at growth temperature 25 and 42°C, in comparison to an optimal growth temperature (37°C). In addition, I focused on the possibility to use the bacterial spore as a platform to deliver Bio-Drugs to gastro-intestinal as they are able to stabilize and protect the adsorbed molecules by degradation and to ensure its activity. Moreover, the antioxidant properties of B.subtilis and B.megaterium spores have been also investigated analyzing the mechanism of action and the effects in vitro and in vivo.The results have showed that the spores are able to exert a preventive and therapeutic effect on the cells through the nuclear translocation of the transcriptional factor NRF-2, involved in the activation of responsive genes stress

Determinants for the subcellular localization of the inner and outer spore coat hubs in Bacillus subtilis

Endospores, or spores for simplicity, are a highly resistant cell type produced by some bacterial species under adverse conditions. Two main protective layers contribute to the resilience of spores: the cortex, composed of peptidoglycan, and the outermost proteinaceous coat. In Bacillus subtilis, the coat comprises up to 80 different proteins, organized into four sublayers: the basement layer, the inner coat, the outer coat and the crust. These proteins are synthesized at different times during sporulation and deposited at the spore surface in multiple coordinated waves. Central to coat formation is a group of morphogenetic proteins that guide the assembly of the coat components. Targeting of the coat proteins to the surface of the developing spore is mainly controlled by the SpoIVA morphogenetic ATPase. In a second stage, the coat proteins fully encircle the spore, a process termed encasement that requires the morphogenetic protein SpoVID. Assembly of the inner coat requires SafA, whereas formation of the outer coat and the crust requires CotE. SafA interacts directly with the N terminus of SpoVID. (...)Fundação Luso-American