Insight into the Mechanism of Lysogeny Control of phiCDKH01 Bacteriophage Infecting Clinical Isolate of Clostridioides difficile.

Clostridioides difficile is a causative agent of antibiotic-associated diarrhea as well as pseudomembranous colitis. So far, all known bacteriophages infecting these bacteria are temperate, which means that instead of prompt lysis of host cells, they can integrate into the host genome or replicate episomally. While C. difficile phages are capable of spontaneous induction and entering the lytic pathway, very little is known about the regulation of their maintenance in the state of lysogeny. In this study, we investigated the properties of a putative major repressor of the recently characterized C. difficile phiCDKH01 bacteriophage. A candidate protein belongs to the XRE family and controls the transcription of genes encoding putative phage antirepressors, known to be involved in the regulation of lytic development. Hence, the putative major phage repressor is likely to be responsible for maintenance of the lysogeny.

High-Quality Complete Genome Resource of Tomato Rhizosphere Strain Pseudomonas donghuensis P482, a Representative of a Species with Biocontrol Activity Against Plant Pathogens.

Strain P482 was isolated from a tomato rhizosphere and classified as Pseudomonas donghuensis. The P. donghuensis species was first established in 2015 and currently consists of only four strains: P482, HYST, SVBP6, and 22G5. P. donghuensis strains antagonize plant pathogens, including bacteria, fungi, and oomycetes, and, therefore, are of high interest regarding their biological control potential to combat plant diseases. The antimicrobial activity of P. donghuensis P482 is based on the production of iron-scavenging compound 7-hydroxytropolone, antifungal volatile organic compounds, and as-yet-unidentified secondary metabolites. Here, we report a complete genome resource for P. donghuensis strain P482. The genome consists of a single chromosome (5,656,185 bp) with 5,258 open reading frames (5,158 protein-coding genes, 74 transfer RNAs, 22 ribosomal RNAs, 3 noncoding RNAs, and 1 transfer-messenger RNA) and no plasmid. We believe that information on the first high-quality, complete genome of P. donghuensis will provide resources for analyses targeting the biological control potential of this species and understanding the traits essential for plant-microbe interaction.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Complete genome sequence of the newly discovered temperate Clostridioides difficile bacteriophage phiCDKH01 of the family Siphoviridae.

A temperate siphovirus, phiCDKH01, was obtained from a clinical isolate of Clostridioides difficile. The phage genome is a 45,089-bp linear double-stranded DNA molecule with an average G+C content of 28.7%. It shows low similarity to known phage genomes, except for phiCD24-1. Genomic and phylogenetic analysis revealed that phiCDKH01 is a newly discovered phage. Sixty-six putative ORFs were predicted in the genome, 37 of which code for proteins with predicted functions. The phiCDKH01 prophage was localized in the host genome. The results of this study increase our knowledge about the genetic diversity of tailed phages.

The combination of recombinant and non-recombinant Bacillus subtilis spore display technology for presentation of antigen and adjuvant on single spore.

BACKGROUND: Bacillus subtilis spores can be used for presentation of heterologous proteins. Two main approaches have been developed, the recombinant one, requiring modification of bacterial genome to express a protein of interest as a fusion with spore-coat protein, and non-recombinant, based on the adsorption of a heterologous protein onto the spore. So far only single proteins have been displayed on the spore surface. RESULTS: We have used a combined approach to adsorb and display FliD protein of Clostridium difficile on the surface of recombinant IL-2-presenting spores. Such spores presented FliD protein with efficiency comparable to FliD-adsorbed spores produced by wild-type 168 strain and elicited FliD-specific immune response in intranasally immunized mice. CONCLUSIONS: Our results indicate that such dual display technology may be useful in creation of spores simultaneously presenting adjuvant and antigen molecules. Regarding the characteristics of elicited immune response it seems plausible that such recombinant IL-2-presenting spores with adsorbed FliD protein might be an interesting candidate for vaccine against infections with Clostridium difficile.

A system of vectors for Bacillus subtilis spore surface display.

BACKGROUND: Bacterial spores have been utilized as platforms for protein display. The best studied display systems are based on Bacillus subtilis spores in which several coat proteins have successfully been used as anchors for heterologous protein. Increasing knowledge about spore coat structure enables selection of new anchor proteins such as CotZ and CgeA. Here we describe a system of vectors for display of proteins on the surface of B. subtilis spores. RESULTS: We have designed and constructed a set of 16 vectors for ectopic integration which can be used for spore surface display of heterologous proteins. There is a selection of five coat proteins: CotB, CotC, CotG, CotZ and CgeA which can be used for construction of fusions. Three of these (CotB, CotC and CotG) enable obtaining N-terminal and C-terminal fusions and other two (CotZ and CgeA) are designed to produce C-terminal fusions only. All the vectors enable introduction of an additional peptide linker between anchor and displayed protein to enhance surface display. As a selection marker trophic genes are used. Additionally we describe an example application of presented vector system to display CagA protein of Helicobacter pylori in fusion with CgeA spore coat protein. CONCLUSIONS: Described system of vectors is a versatile tool for display of heterologous proteins on the surface of B. subtilis spores. Such recombinant spores can be further used as for example biocatalysts or antigen-carriers in vaccine formulations. The lack of antibiotic resistance genes in the system makes such spores an interesting option for applications in which a possible release to the environment can occur.

A genome-wide transcriptional profiling of sporulating Bacillus subtilis strain lacking PrpE protein phosphatase.

The sporulation process is a complex genetic developmental program leading to profound changes in global gene expression profile. In this work, we have applied genome-wide microarray approach for transcriptional profiling of Bacillus subtilis strain lacking a gene coding for PrpE protein phosphatase. This protein was previously shown to be involved in the regulation of germination of B. subtilis spores. Moreover, the deletion of prpE gene resulted in changing the resistance properties of spores. Our results provide genome-wide insight into the influence of this protein phosphatase on the physiology of B. subtilis cells. Although the precise role of PrpE in shaping the observed phenotype of ΔprpE mutant strain still remains beyond the understanding, our experiments brought observations of possible indirect implication of this protein in the regulation of cell motility and chemotaxis, as well as the development of competence. Surprisingly, prpE-deleted cells showed elevated level of general stress response, which turned out to be growth medium specific.

Investigation of spore coat display of Bacillus subtilis ß-galactosidase for developing of whole cell biocatalyst.

The production of highly efficient, recyclable and cost-effective enzymes is one of the most important goals in industrial biotechnology. Bacterial spores are highly resistant to harsh environmental conditions, easy to produce and are suitable for manipulation of genetic materials. These features make them a very efficient tool for biotechnology. Here, we show the use bacterial spores for presentation of functional enzyme. Spore coat display was used to produce a biocatalyst, which expresses ß-galactiosidase (LacA). This enzyme is commonly used to produce lactose-free milk for lactose intolerant individuals. The lacA gene from Bacillus subtilis strain 168 was expressed on the surface of B. subtilis RH101(ΔcotC) spores using CotC as protein carrier. Presence of LacA protein is verified by western blotting. Results of ß-galactiosidase assay show that the expressed enzyme retained its activity in condition of freezing and drying, as well as after recovery from the reaction's mixture.

New stable anchor protein and peptide linker suitable for successful spore surface display in B. subtilis.

BACKGROUND: In last decade spores have been successfully used as a surface display platform. Various peptides or proteins were displayed this way as functional enzymes or antigens. Nearly all attempts involved use of three coat proteins: CotB, CotC or CotG. Increasing knowledge of the structure of the spore coat allowed us to propose the use of other proteins whose localization in the spore envelope has been determined. We also propose the application of a new linker suitable for building fusion proteins. RESULTS: We show that a member of the outer coat, CotZ, is a good candidate as a new anchor protein useful in spore surface display. This protein allows use of relatively large passenger proteins and their efficient display on the spore surface. Analysis by Western- and dot-blotting, combined with immunofluorescence microscopy, allowed us to estimate the number of displayed fusion proteins molecules as 1.4 × 10(2) per spore. In addition, we present data indicating that the use of a peptide linker, which forms a stable α-helix, may greatly improve the display of anchored proteins on the spore surface. CONCLUSION: CotZ can be used as an efficient anchor protein in the outer spore coat. Its localisation in the coat crust layer should guarantee surface display of passenger proteins. Moreover, a CotZ based fusion can tolerate relatively large passenger proteins for efficient spore surface display. In addition, to the properties of both the anchor and passenger proteins, an important issue is the nature of the linker. Here we present evidence that the linker, which forms a stable α-helix, may be crucial for successful display.

Influence of glucose on swarming and quorum sensing of Dickeya solani.

Dickeya solani is a pathogen most frequently responsible for infecting potato plants in Europe. As in the case of most plant pathogens, its ability to colonize and invade the host depends on chemotaxis and motility. The coordinated movement of Dickeya over solid surfaces is governed by a quorum sensing mechanism. In D. solani motility is regulated by ExpI-ExpR proteins, homologous to luxI-luxR system from Vibrio fisheri, in which N-acyl-homoserine lactones (AHLs) serve as signaling molecules. Moreover, in many Gram-negative bacteria motility is coupled with central metabolism via carbon catabolite repression. This enables them to reach more nutrient-efficient niches. The aim of this study was to analyze the swarming motility of D. solani depending on the volume of the medium in the cultivation plate and glucose content. We show that the ability of this bacterium to move is strictly dependent on both these factors. Moreover, we analyze the production of AHLs and show that the quorum sensing mechanism in D. solani is also influenced by the availability of glucose in the medium and that the distribution of these signaling molecules are different depending on the volume of the medium in the plate.

Expression and display of UreA of Helicobacter acinonychis on the surface of Bacillus subtilis spores.

BACKGROUND: The bacterial endospore (spore) has recently been proposed as a new surface display system. Antigens and enzymes have been successfully exposed on the surface layers of the Bacillus subtilis spore, but only in a few cases the efficiency of expression and the effective surface display and have been determined. We used this heterologous expression system to produce the A subunit of the urease of the animal pathogen Helicobater acinonychis. Ureases are multi-subunit enzymes with a central role in the virulence of various bacterial pathogens and necessary for colonization of the gastric mucosa by the human pathogen H. pylori. The urease subunit UreA has been recognized as a major antigen, able to induce high levels of protection against challenge infections. RESULTS: We expressed UreA from H. acinonychis on the B. subtilis spore coat by using three different spore coat proteins as carriers and compared the efficiency of surface expression and surface display obtained with the three carriers. A combination of western-, dot-blot and immunofluorescence microscopy allowed us to conclude that, when fused to CotB, UreA is displayed on the spore surface (ca. 1 x 10(3) recombinant molecules per spore), whereas when fused to CotC, although most efficiently expressed (7-15 x 10(3) recombinant molecules per spore) and located in the coat layer, it is not displayed on the surface. Experiments with CotG gave results similar to those with CotC, but the CotG-UreA recombinant protein appeared to be partially processed. CONCLUSION: UreA was efficiently expressed on the spore coat of B. subtilis when fused to CotB, CotC or CotG. Of these three coat proteins CotC allows the highest efficiency of expression, whereas CotB is the most appropriate for the display of heterologous proteins on the spore surface.

IL-1 Fragment Modulates Immune Response Elicited by Recombinant Bacillus subtilis Spores Presenting an Antigen/Adjuvant Chimeric Protein.

Mucosal immunizations are convenient ways of vaccination, which do not require any trained personnel for administration. One of the major challenges for developing an effective mucosal vaccine is finding appropriate adjuvant. Bacillus subtilis endospores have been shown to help solving these obstacles while serving as a platform for presentation of both, antigens and adjuvants. In this study, we have successfully designed and constructed recombinant spores displaying an antigen/adjuvant chimeric protein. We have used a fragment of Clostridium difficile flagellar cap FliD protein as antigen and VQGEESNDK peptide, a fragment of human IL-1ß, as adjuvant. Recombinant spores presenting FliD were able to elicit immune response in orally immunized mice which could be evaluated by detection of FliD-specific IgA antibodies in feces of immunized animals. Moreover, the presence of IL-1ß fragment significantly changed characteristics of elicited immune response. Obtained results show that recombinant spores presenting an antigen/adjuvant chimeric protein exhibit both properties in mucosal immunization of mice. Moreover, IL-1ß fragment could serve as valuable adjuvant in B. subtilis spore-based mucosal vaccines.

Positions 299 and 302 of the GerAA subunit are important for function of the GerA spore germination receptor in Bacillus subtilis.

Bacillus subtilis, as a model spore-forming Gram-positive bacterium, has been extensively used for spore germination research. Within this field, nutrient-dependent germination with specific germinant receptors (GerA, responding to L-alanine or L-valine; GerB and GerK, acting together to start spore germination process in response to AGFK) has been the most studied. There are three different variants of the GerAA subunit (299T/302S, 299A/302P, 299A/302S) of the GerA germination receptor present in B. subtilis subs. subtilis laboratory strains. According to our research, the 299A/302P one, unlike the others, interferes with the spore's ability to germinate in L-alanine as assessed by the measurement of DPA release upon stimulation with the germinant. Multiple genetic manipulations described in this work followed by spore germination tests, together with secondary structure predictions led us to the following conclusions. First, position 302 of GerAA protein is crucial in terms of GerA germination receptor functionality; a proline residue at this position renders the GerA receptor non-functional, most probably due to a change in the protein secondary structure. Second, the 302P GerAA variant has most probably an impaired affinity to other components of GerA receptor. Together, these may explain the loss of GerA receptor's function. Analysis of the GerAA protein should get us closer to understanding the mechanism of GerA receptor function.

Protective effects of voltage-gated calcium channel antagonists against zinc toxicity in SN56 neuroblastoma cholinergic cells.

One of the pathological site effects in excitotoxic activation is Zn2+ overload to postsynaptic neurons. Such an effect is considered to be equivalent to the glutamate component of excitotoxicity. Excessive uptake of Zn2+ by active voltage-dependent transport systems in these neurons may lead to significant neurotoxicity. The aim of this study was to investigate whether and which antagonists of the voltage gated calcium channels (VGCC) might modify this Zn2+-induced neurotoxicity in neuronal cells. Our data demonstrates that depolarized SN56 neuronal cells may take up large amounts of Zn2+ and store these in cytoplasmic and mitochondrial sub-fractions. The mitochondrial Zn2+ excess suppressed pyruvate uptake and oxidation. Such suppression was caused by inhibition of pyruvate dehydrogenase complex, aconitase and NADP-isocitrate dehydrogenase activities, resulting in the yielding of acetyl-CoA and ATP shortages. Moreover, incoming Zn2+ increased both oxidized glutathione and malondialdehyde levels, known parameters of oxidative stress. In depolarized SN56 cells, nifedipine treatment (L-type VGCC antagonist) reduced Zn2+ uptake and oxidative stress. The treatment applied prevented the activities of PDHC, aconitase and NADP-IDH enzymes, and also yielded the maintenance of acetyl-CoA and ATP levels. Apart from suppression of oxidative stress, N- and P/Q-type VGCCs presented a similar, but weaker protective influence. In conclusion, our data shows that in the course of excitotoxity, impairment to calcium homeostasis is tightly linked with an excessive neuronal Zn2+ uptake. Hence, the VGCCs types L, N and P/Q share responsibility for neuronal Zn2+ overload followed by significant energy-dependent neurotoxicity. Moreover, Zn2+ affects the target tricarboxylic acid cycle enzymes, yields acetyl-CoA and energy deficits as well.

The choice of the anchoring protein influences the interaction of recombinant Bacillus spores with the immune system.

The technology of display of heterologous proteins on the surface of Bacillus subtilis spores enables use of these structures as carriers of antigens for mucosal vaccination. Currently, there are no technical possibilities to predict whether a designed fusion will be efficiently displayed on the spore surface and how such recombinant spores will interact with cells of the immune system. In this study, we compared four variants of B. subtilis spores presenting a fragment of a FliD protein from Clostridium difficile in fusion with CotB, CotC, CotG or CotZ spore coat proteins. We show that these spores promote their own phagocytosis and activate both, the J774 macrophages and JAWSII dendritic cells of murine cell lines. Moreover, we used these spores for mucosal immunization of mice. We conclude that the observed effects vary with the type of displayed FliD-spore coat protein fusion and seem to be mostly independent of its abundance and localization in the spore coat structure.

Mapping of a transcription promoter located inside the priA gene of the Bacillus subtilis chromosome.

The genome sequence of the Gram-positive soil bacterium Bacillus subtilis was completed in 1997 (Kunst et al., 1998) and the results included the identification of a putative transcription unit encompassing the yloI to yloS genes. Within this region of the B. subtilis chromosome 11 putative open reading frames were found with a wide diversity of probable functions. In this work we have analyzed transcription in the region of the priA-cpgA genes and we have mapped a promoter which is located inside the priA gene and its activity directs transcription of the def-yloM genes. Moreover, this transcript can be extended at low level to the prpC-priK-cpgA genes. Analysis of the sequence in proximity of the transcription start site revealed a sequence suitable for the housekeeping sigma(A) subunit of RNA polymerase. Analysis of the beta-glactosidase activity of transcription fusions revealed that the identified promoter is active at low level and its activity is increased during late exponential phase of growth.

Recombinant Bacillus subtilis Spores Elicit Th1/Th17-Polarized Immune Response in a Murine Model of Helicobacter pylori Vaccination.

Current progress in research on vaccines against Helicobacter pylori emphasizes the significance of eliciting the Th1/Th17-polarized immune response. Such polarization can be achieved by selection of appropriate antigen and adjuvant. In this study, we wanted to check the polarization of the immune response elicited by UreB protein of Helicobacter acinonychis delivered by recombinant Bacillus subtilis spores upon oral immunization. B. subtilis spores presenting fragment of UreB protein and able to express entire UreB in vegetative cells after germination were orally administered to mice along with aluminum hydroxide or recombinant spores presenting IL-2 as an adjuvant. The pattern of cytokines secreted by sensitized splenocytes assessed by the cytometric bead array clearly indicated polarization of the immune response toward both Th1 and Th17 in mice immunized with the use of above-mentioned adjuvants. Obtained result is promising regarding the usage of recombinant spores in formulations of vaccines against H. pylori and line up with the current state of research emphasizing the key role of appropriate adjuvants.

Mucosal adjuvant activity of IL-2 presenting spores of bacillus subtilis in a murine model of Helicobacter pylori vaccination.

The endospores of Bacillus subtilis are now widely used as a platform for presentation of heterologous proteins and due to their safety record and high resistance to harsh environmental conditions can be considered as potential vehicles for oral vaccination. In this research we show that recombinant B. subtilis spores presenting a fragment of the Helicobacter acinonychis UreB protein and expressing the ureB gene under vegetative promoter elicit a strong cellular immune response in orally immunized mice when co-administered with spores presenting IL-2. We show for the first time the successful application of two types of recombinant spores, one carrying an antigen and the other an adjuvant, in a single oral immunization.

Expression and display of Clostridium difficile protein FliD on the surface of Bacillus subtilis spores.

The endospores of Bacillus subtilis can serve as a tool for surface presentation of heterologous proteins. The unique properties of the spore protective layers make them perfect vehicles for orally administered vaccines. In this study, we successfully displayed a fragment of Clostridium difficile FliD protein on the surface of B. subtilis spores using the CotB, CotC, CotG and CotZ spore coat proteins. The presence of the fusion proteins in the spore coat was verified by Western blotting and immunofluorescence microscopy. The amount of recombinant proteins was assessed by a dot-blot technique. C. difficile is one of the most common infectious agents in nosocomial infections and is especially associated with antibiotic therapies. FliD is a flagellar cap protein of C. difficile and is known to be one of the immunogenic surface antigens of this bacterium. Therefore, its use in vaccine formulations gives a good perspective for successful immunization with a FliD-based vaccine. The recombinant spores presented here may be good candidates for C. difficile oral vaccines.

Genome Sequence of Bacillus subtilis MB73/2, a Soil Isolate Inhibiting the Growth of Plant Pathogens Dickeya spp. and Rhizoctonia solani.

Bacillus subilis MB73/2 is a Gram-positive bacterium isolated in Poland from a meadow soil sample. When tested in vitro, the strain shows strong antagonism toward plant pathogens-the soft rot-causing bacteria Dickeya spp. and the crown rot fungus Rhizoctonia solani. Here, we present the genome sequence of MB73/2.

Expression of genes coding for GerA and GerK spore germination receptors is dependent on the protein phosphatase PrpE.

The ability of Bacillus subtilis to form spores is a strategy for survival under unfavorable environmental conditions. It is equally crucial to break spore dormancy and return to vegetative growth at the appropriate time. Here we present data showing that the PrpE phosphatase is involved in the control of expression of genes coding for GerA receptors, which are necessary for L-alanine-induced spore germination. Moreover, PrpE is also involved in aspartic acid, glucose, fructose, and potassium (AGFK)-induced spore germination by controlling expression of genes coding for GerK receptors. In the absence of PrpE, the production of spores was essentially normal. However, L-alanine-induced spore germination and, to a lesser extent, the AGFK-induced pathway were abolished. In contrast, the germination pathway dependent on Ca2+-dipicolinate or dodecylamine remained intact. A protein phosphatase PrpE-green fluorescent protein fusion was localized to the prespore and to the dormant spore, consistent with a role in controlling expression of genes coding for GerA receptors. We propose that PrpE is an important element in a signal transduction pathway in Bacillus subtilis that controls the expression of genes coding for germination receptors.