CotG controls spore surface formation in response to the temperature of growth in Bacillus subtilis.

Bacterial spores of the Bacillus genus are ubiquitous in nature and are commonly isolated from a variety of diverse environments. Such wide distribution mainly reflects the spore resistance properties but some Bacillus species can grow/sporulate in at least some of the environments where they have been originally isolated. Growing and sporulating at different conditions is known to affect the structure and the resistance properties of the produced spore. In B. subtilis the temperature of growth and sporulation has been shown to influence the structure of the spore surface throughout the action of a sporulation-specific and heat-labile kinase CotH. Here we report that CotG, an abundant component of the B. subtilis spore surface and a substrate of the CotH kinase, assembles around the forming spore but also accumulates in the mother cell cytoplasm where it forms aggregates with at least two other coat components. Our data suggest that the thermo-regulator CotH contributes to the switch between the coat of 25°C and that of 42°C spores by controlling the phosphorylation levels of CotG that, in turn, regulates the assembly of at least two other coat components.

Occurrence of Capnophilic Lactic Fermentation in the Hyperthermophilic Anaerobic Bacterium Thermotoga sp. Strain RQ7.

Capnophilic lactic fermentation (CLF) is an anaplerotic pathway exclusively identified in the anaerobic hyperthermophilic bacterium Thermotoga neapolitana, a member of the order Thermotogales. The CO2-activated pathway enables non-competitive synthesis of hydrogen and L-lactic acid at high yields, making it an economically attractive process for bioenergy production. In this work, we discovered and characterized CLF in Thermotoga sp. strain RQ7, a naturally competent strain, opening a new avenue for molecular investigation of the pathway. Evaluation of the fermentation products and expression analyses of key CLF-genes by RT-PCR revealed similar CLF-phenotypes between T. neapolitana and T. sp. strain RQ7, which were absent in the non-CLF-performing strain T. maritima. Key CLF enzymes, such as PFOR, HYD, LDH, RNF, and NFN, are up-regulated in the two CLF strains. Another important finding is the up-regulation of V-ATPase, which couples ATP hydrolysis to proton transport across the membranes, in the two CLF-performing strains. The fact that V-ATPase is absent in T. maritima suggested that this enzyme plays a key role in maintaining the necessary proton gradient to support high demand of reducing equivalents for simultaneous hydrogen and lactic acid synthesis in CLF.

Bacillus subtilis builds structurally and functionally different spores in response to the temperature of growth.

Bacterial spores are commonly isolated from a variety of different environments, including extreme habitats. Although it is well established that such ubiquitous distribution reflects the spore resistance properties, it is not clear whether the growing conditions affect the spore structure and function. We used Bacillus subtilis spores of similar age but produced at 25, 37, or 42°C to compare their surface structures and functional properties. Spores produced at the 25°C were more hydrophobic while those produced at 42°C contained more dipicolinic acid, and were more resistant to heat or lysozyme treatments. Electron microscopy analysis showed that while 25°C spores had a coat with a compact outer coat, not tightly attached to the inner coat, 42°C spores had a granular, not compact outer coat, reminiscent of the coat produced at 37°C by mutant spores lacking the protein CotG. Indeed, CotH and a series of CotH-dependent coat proteins including CotG were more abundantly extracted from the coat of 25 or 37°C than 42°C spores. Our data indicated that CotH is a heat-labile protein with a major regulatory role on coat formation when sporulation occurs at low temperatures, suggesting that B. subtilis builds structurally and functionally different spores in response to the external conditions.

The temperature of growth and sporulation modulates the efficiency of spore-display in Bacillus subtilis.

BACKGROUND: Bacterial spores displaying heterologous antigens or enzymes have long been proposed as mucosal vaccines, functionalized probiotics or biocatalysts. Two main strategies have been developed to display heterologous molecules on the surface of Bacillus subtilis spores: (i) a recombinant approach, based on the construction of a gene fusion between a gene coding for a coat protein (carrier) and DNA coding for the protein to be displayed, and (ii) a non-recombinant approach, based on the spontaneous and stable adsorption of heterologous molecules on the spore surface. Both systems have advantages and drawbacks and the selection of one or the other depends on the protein to be displayed and on the final use of the activated spore. It has been recently shown that B. subtilis builds structurally and functionally different spores when grown at different temperatures; based on this finding B. subtilis spores prepared at 25, 37 or 42 °C were compared for their efficiency in displaying various model proteins by either the recombinant or the non-recombinant approach. RESULTS: Immune- and fluorescence-based assays were used to analyze the display of several model proteins on spores prepared at 25, 37 or 42 °C. Recombinant spores displayed different amounts of the same fusion protein in response to the temperature of spore production. In spores simultaneously displaying two fusion proteins, each of them was differentially displayed at the various temperatures. The display by the non-recombinant approach was only modestly affected by the temperature of spore production, with spores prepared at 37 or 42 °C slightly more efficient than 25 °C spores in adsorbing at least some of the model proteins tested. CONCLUSION: Our results indicate that the temperature of spore production allows control of the display of heterologous proteins on spores and, therefore, that the spore-display strategy can be optimized for the specific final use of the activated spores by selecting the display approach, the carrier protein and the temperature of spore production.

Effect of Cultivation Parameters on Fermentation and Hydrogen Production in the Phylum Thermotogae.

The phylum Thermotogae is composed of a single class (Thermotogae), 4 orders (Thermotogales, Kosmotogales, Petrotogales, Mesoaciditogales), 5 families (Thermatogaceae, Fervidobacteriaceae, Kosmotogaceae, Petrotogaceae, Mesoaciditogaceae), and 13 genera. They have been isolated from extremely hot environments whose characteristics are reflected in the metabolic and phenotypic properties of the Thermotogae species. The metabolic versatility of Thermotogae members leads to a pool of high value-added products with application potentials in many industry fields. The low risk of contamination associated with their extreme culture conditions has made most species of the phylum attractive candidates in biotechnological processes. Almost all members of the phylum, especially those in the order Thermotogales, can produce bio-hydrogen from a variety of simple and complex sugars with yields close to the theoretical Thauer limit of 4 mol H2/mol consumed glucose. Acetate, lactate, and L-alanine are the major organic end products. Thermotagae fermentation processes are influenced by various factors, such as hydrogen partial pressure, agitation, gas sparging, culture/headspace ratio, inoculum, pH, temperature, nitrogen sources, sulfur sources, inorganic compounds, metal ions, etc. Optimization of these parameters will help to fully unleash the biotechnological potentials of Thermotogae and promote their applications in industry. This article gives an overview of how these operational parameters could impact Thermotogae fermentation in terms of sugar consumption, hydrogen yields, and organic acids production.

Localization of a red fluorescence protein adsorbed on wild type and mutant spores of Bacillus subtilis.

BACKGROUND: Bacterial spores have been proposed as vehicles to display heterologous proteins for the development of mucosal vaccines, biocatalysts, bioremediation and diagnostic tools. Two approaches have been developed to display proteins on the spore surface: a recombinant approach, based on the construction of gene fusions between DNA molecules coding for a spore surface protein (carrier) and for the heterologous protein to be displayed (passenger); and a non-recombinant approach based on spore adsorption, a spontaneous interaction between negatively charged, hydrophobic spores and purified proteins. The molecular details of spore adsorption have not been fully clarified yet. RESULTS: We used the monomeric Red Fluorescent Protein (mRFP) of the coral Discosoma sp. and Bacillus subtilis spores of a wild type and an isogenic mutant strain lacking the CotH protein to clarify the adsorption process. Mutant spores, characterized by a strongly altered coat, were more efficient than wild type spores in adsorbing mRFP but the interaction was less stable and mRFP could be in part released by raising the pH of the spore suspension. A collection of isogenic strains carrying GFP fused to proteins restricted in different compartments of the B. subtilis spore was used to localize adsorbed mRFP molecules. In wild type spores mRFP infiltrated through crust and outer coat, localized in the inner coat and was not surface exposed. In mutant spores mRFP was present in all surface layers, inner, outer coat and crust and was exposed on the spore surface. CONCLUSIONS: Our results indicate that different spores can be selected for different applications. Wild type spores are preferable when a very tight protein-spore interaction is needed, for example to develop reusable biocatalysts or bioremediation systems for field applications. cotH mutant spores are instead preferable when the heterologous protein has to be displayed on the spore surface or has to be released, as could be the case in mucosal delivery systems for antigens and drugs, respectively.

Lack of Arabidopsis chloroplastic glucose-6-phosphate dehydrogenase 1 (G6PD1) affects lipid synthesis during cold stress response.

Cold stress represents one of the major constraints for agricultural systems and crops productivity, inducing a wide range of negative effects. Particularly, long-term cold stress affects lipid metabolism, modifying the lipids/proteins ratio, the levels of phospholipids and glycolipids, and increasing lipids' unsaturation in bio-membranes. Glucose-6-phosphate dehydrogenase (G6PDH) reported prominent roles as NADPH suppliers in response to oxidative perturbations. Cytosolic G6PDH was suggested as the main isoform involved in cold stress response, while a down-regulation of the chloroplastic P1-G6PDH was observed. We thus investigated an Arabidopsis mutant defective for the P1-G6PDH (KO-P1) using integrated approaches to verify a possible role of this isoform in low temperature tolerance. KO-P1 genotype showed an improved tolerance to cold stress, highlighting a better photosynthetic efficiency, a reduction in stress markers content and a different regulation of genes involved in stress response. Intriguingly, the lack of P1-G6PDH induced modification in the levels of the main fatty acid and lipid species affecting the morphology of chloroplasts and mitochondria, which was restored under cold. Globally, these results indicate a priming effect induced by the absence of P1-G6PDH able to improve the tolerance to abiotic stress. Our results suggest novel and specific abilities of P1-G6PDH, highlighting its central role in different aspects of plant physiology and metabolism.

LINC00958 as new diagnostic and prognostic biomarker of childhood acute lymphoblastic leukaemia of B cells.

Background: Paediatric acute B-cell lymphoblastic leukaemia is the most common cancer of the paediatric age. Although the advancement of scientific and technological knowledge has ensured a huge step forward in the management of this disease, there are 15%-20% cases of recurrence leading to serious complications for the patient and sometimes even death. It is therefore necessary to identify new and increasingly personalised biomarkers capable of predicting the degree of risk of B-ALL in order to allow the correct management of paediatric leukaemia patients. Methods: Starting from our previously published results, we validate the expression level of LINC00958 in a cohort of 33 B-ALL and 9 T-ALL childhood patients, using in-silico public datasets as support. Expression levels of LINC00958 in B-ALL patients stratified by risk (high risk vs. standard/medium risk) and who relapsed 3 years after the first leukaemia diagnosis were also evaluated. Results: We identified the lncRNA LINC00958 as a biomarker of B-ALL, capable of discriminating B-ALL from T-ALL and healthy subjects. Furthermore, we associated LINC00958 expression levels with the disease risk classification (high risk and standard risk). Finally, we show that LINC00958 can be used as a predictor of relapses in patients who are usually stratified as standard risk and thus not always targeted for marrow transplantation. Conclusions: Our results open the way to new diagnostic perspectives that can be directly used in clinical practice for a better management of B-ALL paediatric patients.

Potential of Lipid Biosynthesis under Heterotrophy in the Marine Diatom Cyclotella cryptica.

Despite the theoretical high productivity, microalgae-based oil production is not economically sustainable due to the high cost of photoautotrophic cultures. Heterotrophic growth is a suitable economic alternative to overcoming light dependence and climatic/geographic fluctuations. Here we report data about growth performance, biomass production, and lipid composition of the marine diatom Cyclotella cryptica, chosen as a model strain for biodiesel production in heterothrophy. A repeated-batch process of heterotrophic cultivation has also been investigated to assess the robustness and phenotypic stability. The process consisting of six constant cycle repetitions was carried out for 42 days and led to an average dry biomass production of 1.5 ± 0.1 g L-1 of which 20% lipids composed of 60% triglycerides, 20% phospholipids. and 20% glycolipids. The major fatty acids were C16:0 (∼26%), C16:1 ω-7 (∼57%), and C20:5 ω-3 (∼12%), with a significant reduction in the unsaturated fatty acids in comparison to other microalgae grown in heterotrophy. Fatty acids were differently distributed among the glycerolipid classes, and the lipid composition was used to compare the potential properties of C. cryptica oil with traditional vegetable biofuels.

Genomic and Physiological Characterization of Bacilli Isolated From Salt-Pans With Plant Growth Promoting Features.

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.

Improvement of CO2 and Acetate Coupling into Lactic Acid by Genetic Manipulation of the Hyperthermophilic Bacterium Thermotoga neapolitana.

Capnophilic lactic fermentation (CLF) represents an attractive biotechnological process for biohydrogen production and synthesis of L-lactic acid from acetate and CO2. The present study focuses on a genetic manipulation approach of the Thermotoga neapolitana DSM33003 strain to enhance lactic acid synthesis by the heterologous expression of a thermostable acetyl-CoA synthetase that catalyses the irreversible acetate assimilation. Because of the scarcity of available genetic tools, each transformation step was optimized for T. neapolitana DSM33003 to cope with the specific needs of the host strain. Batch fermentations with and without an external source of acetate revealed a strongly increased lactate production (up to 2.5 g/L) for the recombinant strain compared to wild type. In the engineered bacterium, the assimilation of CO2 into lactic acid was increased 1.7 times but the hydrogen yield was impaired in comparison to the wild type strain. Analysis of fermentation yields revealed an impaired metabolism of hydrogen in the recombinant strain that should be addressed in future studies. These results offer an important prospective for the development of a sustainable approach that combines carbon capture, energy production from renewable source, and the synthesis of high value-added products, which will be addressed in future studies.

Hyperthermiphile biofilms of Thermotoga neapolitana on different materials and electrostimulated: SEM micrographs and chemical data of the glucose fermentation in electrochemical reactors.

Hyperthermophile bacteria were seldom investigated in bioelectrochemical systems although they allow more effective control of the inoculum in comparison with mesophilic bacteria. Biofilm formed in hyperthermophilic conditions (>60 °C) also rarely was documented (d'Ippolito et al., 2020; Belkin et al., 1986, Pysz et al., 2004). Scanning Electron Microscopy (SEM) micrographs documenting biofilms formed by the Hyperthermophile bacterium Thermotoga neapolitana on different solid materials (ceramic carrier, stainless steel mesh, carbon felt, carbon paper, expanse graphite, and carbon cloth) are shown in this report. Also, micrographs of the biofilm formed on electrodes of carbon cloth under a dynamic polarization oscillating around ±1 V (±0.8 V and ±1.2 V) are reported. Two procedures of sample preparation for SEM analyses are described and used: 1) a fast drying of samples, which is enough to underline the biofilm shape that covers solids, and 2) a chemical treating of the samples with glutaraldehyde, which better preserves the shape of bacterial cell components in the biofilm, although this treatment might cause the detachment of pieces of the biofilm. The different effect of potentiostatic and potentiodynamic polarizations on the glucose metabolism of T. neapolitana has been screened and discussed in the associated article [1]. Here, data of Optical Densities (O.D.) of culture media are provided, indicating the presence or absence of bacteria growth in the bulk of the media. Data have been collected every 24 h from the differently polarized bioreactors. The electrodes set-up of small bioreactors is also illustrated. Chemical data, optical data and SEM images, accordingly, document a retard in the glucose fermentation process due to a settlement of T. Neapolitana in a stationary phase. The polarization of electrodes can modify the stationary condition, inducing a possible change of the bacteria metabolism.

Bacillus megaterium SF185 spores exert protective effects against oxidative stress in vivo and in vitro.

Endogenous reactive oxygen species (ROS) are by-products of the aerobic metabolism of cells and have an important signalling role as secondary messengers in various physiological processes, including cell growth and development. However, the excessive production of ROS, as well as the exposure to exogenous ROS, can cause protein oxidation, lipid peroxidation and DNA damages leading to cell injuries. ROS accumulation has been associated to the development of health disorders such as neurodegenerative and cardiovascular diseases, inflammatory bowel disease and cancer. We report that spores of strain SF185, a human isolate of Bacillus megaterium, have antioxidant activity on Caco-2 cells exposed to hydrogen peroxide and on a murine model of dextran sodium sulfate-induced oxidative stress. In both model systems spores exert a protective state due to their scavenging action: on cells, spores reduce the amount of intracellular ROS, while in vivo the pre-treatment with spores protects mice from the chemically-induced damages. Overall, our results suggest that treatment with SF185 spores prevents or reduces the damages caused by oxidative stress. The human origin of SF185, its strong antioxidant activity, and its protective effects led to propose the spore of this strain as a new probiotic for gut health.

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.

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.

Fluorescent peptide dH3w: A sensor for environmental monitoring of mercury (II).

Heavy metals are hazardous environmental contaminants, often highly toxic even at extremely low concentrations. Monitoring their presence in environmental samples is an important but complex task that has attracted the attention of many research groups. We have previously developed a fluorescent peptidyl sensor, dH3w, for monitoring Zn2+ in living cells. This probe, designed on the base on the internal repeats of the human histidine rich glycoprotein, shows a turn on response to Zn2+ and a turn off response to Cu2+. Other heavy metals (Mn2+, Fe2+, Ni2+, Co2+, Pb2+ and Cd2+) do not interfere with the detection of Zn2+ and Cu2+. Here we report that dH3w has an affinity for Hg2+ considerably higher than that for Zn2+ or Cu2+, therefore the strong fluorescence of the Zn2+/dH3w complex is quenched when it is exposed to aqueous solutions of Hg2+, allowing the detection of sub-micromolar levels of Hg2+. Fluorescence of the Zn2+/dH3w complex is also quenched by Cu2+ whereas other heavy metals (Mn2+, Fe2+, Ni2+, Co2+, Cd2+, Pb2+, Sn2+ and Cr3+) have no effect. The high affinity and selectivity suggest that dH3w and the Zn2+/dH3w complex are suited as fluorescent sensor for the detection of Hg2+ and Cu2+ in environmental as well as biological samples.

The Exosporium of Bacillus megaterium QM B1551 Is Permeable to the Red Fluorescence Protein of the Coral Discosoma sp.

Bacterial spores spontaneously interact and tightly bind heterologous proteins. A variety of antigens and enzymes have been efficiently displayed on spores of Bacillus subtilis, the model system for spore formers. Adsorption on B. subtilis spores has then been proposed as a non-recombinant approach for the development of mucosal vaccine/drug delivery vehicles, biocatalysts, bioremediation, and diagnostic tools. We used spores of B. megaterium QM B1551 to evaluate their efficiency as an adsorption platform. Spores of B. megaterium are significantly larger than those of B. subtilis and of other Bacillus species and are surrounded by the exosporium, an outermost surface layer present only in some Bacillus species and lacking in B. subtilis. Strain QM B1551 of B. megaterium and a derivative strain totally lacking the exosporium were used to localize the adsorbed monomeric Red Fluorescent Protein (mRFP) of the coral Discosoma sp., used as a model heterologous protein. Our results indicate that spores of B. megaterium adsorb mRFP more efficiently than B. subtilis spores, that the exosporium is essential for mRFP adsorption, and that most of the adsorbed mRFP molecules are not exposed on the spore surface but rather localized in the space between the outer coat and the exosporium.