The DINGGG thermoprotein is membrane bound in the Crenarchaeon Sulfolobus solfataricus

Background Sulfolobus solfataricus N-terminus and other regions of the partial amino acid sequence of a thermoprotein exhibiting poly(ADP-ribose) polymerase activity suggest that it belongs to the DINGGG class of proteins that are often described as membrane bound. Our previous biochemical studies demonstrated that the thermoprotein is also strictly associated with DNA, and is only partially solubilized from cell homogenate. The present research is focused on the analysis of the sulfolobal DING thermozyme localization within the archaeal cell

Surface charge and hydrodynamic coefficient measurements of {\it Bacillus subtilis} spore by Optical Tweezers

In this work we report on the simultaneous measurement of the hydrodynamic coefficient and the electric charge of single {\it Bacillus subtilis} spores. The latter has great importance in protein binding to spores and in the adhesion of spores onto surfaces. The charge and the hydrodynamic coefficient were measured by an accurate procedure based on the analysis of the motion of single spores confined by an optical trap. The technique has been validated using charged spherical polystyrene beads. The excellent agreement of our results with the expected values demonstrates the quality of our procedure. We measured the charge of spores of {\it B. subtilis} purified from a wild type strain and from two isogenic mutants characterized by an altered spore surface. Our technique is able to discriminate the three spore types used, by their charge and by their hydrodynamic coefficient which is related to the hydrophobic properties of the spore surface.Comment: 21 pages 5 figure

CotG-Like modular proteins are common among spore-forming bacilli

CotG is an abundant protein initially identified as an outer component of the Bacillus subtilis spore coat. It has an unusual structure characterized by several repeats of positively charged amino acids that are probably the outcome of multiple rounds of gene elongation events in an ancestral minigene. CotG is not highly conserved, and its orthologues are present in only two Bacillus and two Geobacillus species. In B. subtilis, CotG is the target of extensive phosphorylation by a still unidentified enzyme and has a role in the assembly of some outer coat proteins. We report now that most spore-forming bacilli contain a protein not homologous to CotG of B. subtilis but sharing a central “modular” region defined by a pronounced positive charge and randomly coiled tandem repeats. Conservation of the structural features in most spore-forming bacilli suggests a relevant role for the CotG-like protein family in the structure and function of the bacterial endospore. To expand our knowledge on the role of CotG, we dissected the B. subtilis protein by constructing deletion mutants that express specific regions of the protein and observed that they have different roles in the assembly of other coat proteins and in spore germination. IMPORTANCE CotG of B. subtilis is not highly conserved in the Bacillus genus; however, a CotG-like protein with a modular structure and chemical features similar to those of CotG is common in spore-forming bacilli, at least when CotH is also present. The conservation of CotG-like features when CotH is present suggests that the two proteins act together and may have a relevant role in the structure and function of the bacterial endospore. Dissection of the modular composition of CotG of B. subtilis by constructing mutants that express only some of the modules has allowed a first characterization of CotG modules and will be the basis for a more detailed functional analysis

Alternative use of Bacillus subtilis spores: Protection against environmental oxidative stress in human normal keratinocytes

Inorganic trivalent arsenic is a major environmental pollutant and exposure to human results in many pathologies, including keratosis and carcinoma. Here, we analyzed the effects of B. subtilis spores on human normal keratinocytes in the presence of sodium arsenite oxidative stress. Pre-treatment of cells with spores before inducing oxidative stress was able to keep normal levels of intracellular ROS, GSH and lipid peroxidation, as well as to inhibit the activation of the MAPK cascade. Moreover, spores showed a positive effect on cell proliferation, probably due to their binding on the cell surface and the activation of intracellular catalases. We found that spores exert their protective effect by the nuclear translocation of Nrf-2, involved in the activation of stress response genes. This, in turn, resulted in a protective effect against sodium arsenite stress injury, as oxidative stress markers were reported to physiological levels when cells were stressed before incubating them with spores. Therefore, B. subtilis spores can be considered as a new agent to counteract oxidative stress on normal human keratinocytes

Conversion of xylan by recyclable spores of Bacillus subtilis displaying thermophilic enzymes

Background: The Bacillus subtilis spore has long been used to display antigens and enzymes. Spore display can be accomplished by a recombinant and a non-recombinant approach, with the latter proved more efficient than the recombinant one. We used the non-recombinant approach to independently adsorb two thermophilic enzymes, GH10-XA, an endo-1,4-β-xylanase (EC 3.2.1.8) from Alicyclobacillus acidocaldarius, and GH3-XT, a β-xylosidase (EC 3.2.1.37) from Thermotoga thermarum. These enzymes catalyze, respectively, the endohydrolysis of (1-4)-β-d-xylosidic linkages of xylans and the hydrolysis of (1-4)-β-d-xylans to remove successive d-xylose residues from the non-reducing termini. Results: We report that both purified enzymes were independently adsorbed on purified spores of B. subtilis. The adsorption was tight and both enzymes retained part of their specific activity. When spores displaying either GH10- XA or GH3-XT were mixed together, xylan was hydrolysed more efficiently than by a mixture of the two free, not spore-adsorbed, enzymes. The high total activity of the spore-bound enzymes is most likely due to a stabilization of the enzymes that, upon adsorption on the spore, remained active at the reaction conditions for longer than the free enzymes. Spore-adsorbed enzymes, collected after the two-step reaction and incubated with fresh substrate, were still active and able to continue xylan degradation. The recycling of the mixed spore-bound enzymes allowed a strong increase of xylan degradation. Conclusion: Our results indicate that the two-step degradation of xylans can be accomplished by mixing spores displaying either one of two required enzymes. The two-step process occurs more efficiently than with the two un-adsorbed, free enzymes and adsorbed spores can be reused for at least one other reaction round. The efficiency of the process, the reusability of the adsorbed enzymes, and the well documented robustness of spores of B. subtilis indicate the spore as a suitable platform to display enzymes for single as well as multi-step reactions

Display of the peroxiredoxin Bcp1 of Sulfolobus solfataricus on probiotic spores of Bacillus megaterium.

Bacterial spores displaying heterologous proteins have been proposed as a safe and efficient method for delivery of antigens and enzymes to animal mucosal surfaces. Initial studies have been performed using Bacillus subtilis spores, but other spore forming organisms have also been considered. B. megaterium spores have been shown capable of displaying large amounts of a model heterologous protein (Discosoma red fluorescent protein mRFP) that in part crossed the exosporium to localize in the space between the outer coat layer and the exosporium. Here, B. megaterium spores have been used to adsorb Bcp1 (bacterioferritin comigratory protein 1), a peroxiredoxin of the archaeon Sulfolobus solfataricus, known to have an antioxidant activity. The spores were highly efficient in adsorbing the heterologous enzyme which, once adsorbed, retained its activity. The adsorbed Bcp1 localized beneath the exosporium, filling the space between the outer coat and the exosporium. This unusual localization contributed to the stability of the enzyme-spore interaction and to the protection of the adsorbed enzyme in simulated intestinal or gastric conditions

Genomic and physiological characterization of Bacilli isolated from salt-pans with plant growth promoting features

© The Author(s), 2021. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Petrillo, C., Castaldi, S., Lanzilli, M., Selci, M., Cordone, A., Giovannelli, D., & Isticato, R. Genomic and physiological characterization of Bacilli isolated from salt-pans with plant growth promoting features. Frontiers in Microbiology, 12, (2021): 715678, https://doi.org/10.3389/fmicb.2021.715678.Massive application of chemical fertilizers and pesticides has been the main strategy used to cope with the rising crop demands in the last decades. The indiscriminate use of chemicals while providing a temporary solution to food demand has led to a decrease in crop productivity and an increase in the environmental impact of modern agriculture. A sustainable alternative to the use of agrochemicals is the use of microorganisms naturally capable of enhancing plant growth and protecting crops from pests known as Plant-Growth-Promoting Bacteria (PGPB). Aim of the present study was to isolate and characterize PGPB from salt-pans sand samples with activities associated to plant fitness increase. To survive high salinity, salt-tolerant microbes produce a broad range of compounds with heterogeneous biological activities that are potentially beneficial for plant growth. A total of 20 halophilic spore-forming bacteria have been screened in vitro for phyto-beneficial traits and compared with other two members of Bacillus genus recently isolated from the rhizosphere of the same collection site and characterized as potential biocontrol agents. Whole-genome analysis on seven selected strains confirmed the presence of numerous gene clusters with PGP and biocontrol functions and of novel secondary-metabolite biosynthetic genes, which could exert beneficial impacts on plant growth and protection. The predicted biocontrol potential was confirmed in dual culture assays against several phytopathogenic fungi and bacteria. Interestingly, the presence of predicted gene clusters with known biocontrol functions in some of the isolates was not predictive of the in vitro results, supporting the need of combining laboratory assays and genome mining in PGPB identification for future applications