Display of Bacterial Exochitanase on Bacillus subtilis Spores Improved Enzyme Stability and Recyclability.

Chitin is the second most prevalent polysaccharide found in nature, following cellulose. Amino-oligosaccharides, the byproducts of chitin degradation, exhibit favorable biological properties and potential for various uses. Chitinases play a crucial function in the breakdown of chitin, and their exceptionally effective production has garnered significant interest. Here, in this study, the exochitinase PbChiA, obtained from Paenibacillus barengoltzii, was recombinantly produced and immobilized using the CotG surface protein of Bacillus subtilis WB800N. The resulting strain Bacillus subtilis WB800N pHS-CotG-Chi exhibited exceptional heat stability and efficacy across various pH levels. The chitinolytic activity of the enzyme, which had been isolated and immobilized on the spore surface, was measured to be approximately 16.06 U/mL. Including Ni2+, Zn+2, and K+, and EDTA at various concentration levels in the reaction system, has significantly enhanced the activity of the immobilized enzyme. The immobilized exochitinase demonstrated a notable rate of recycling, as the recombinant spores sustained a relative enzyme activity of more than 70% after three cycles and 62.7% after four cycles. These findings established a basis for additional investigation into the role and practical use of the immobilized bacterial exochitinase in industry.

Differential effects of 'resurrecting' Csp pseudoproteases during Clostridioides difficile spore germination.

Clostridioides difficile is a spore-forming bacterial pathogen that is the leading cause of hospital-acquired gastroenteritis. C. difficile infections begin when its spore form germinates in the gut upon sensing bile acids. These germinants induce a proteolytic signaling cascade controlled by three members of the subtilisin-like serine protease family, CspA, CspB, and CspC. Notably, even though CspC and CspA are both pseudoproteases, they are nevertheless required to sense germinants and activate the protease, CspB. Thus, CspC and CspA are part of a growing list of pseudoenzymes that play important roles in regulating cellular processes. However, despite their importance, the structural properties of pseudoenzymes that allow them to function as regulators remain poorly understood. Our recently solved crystal structure of CspC revealed that its pseudoactive site residues align closely with the catalytic triad of CspB, suggesting that it might be possible to 'resurrect' the ancestral protease activity of the CspC and CspA pseudoproteases. Here, we demonstrate that restoring the catalytic triad to these pseudoproteases fails to resurrect their protease activity. We further show that the pseudoactive site substitutions differentially affect the stability and function of the CspC and CspA pseudoproteases: the substitutions destabilized CspC and impaired spore germination without affecting CspA stability or function. Thus, our results surprisingly reveal that the presence of a catalytic triad does not necessarily predict protease activity. Since homologs of C. difficile CspA occasionally carry an intact catalytic triad, our results indicate that bioinformatic predictions of enzyme activity may underestimate pseudoenzymes in rare cases.

Characterization of an Endolysin Targeting Clostridioides difficile That Affects Spore Outgrowth.

Clostridioides difficile is a spore-forming enteric pathogen causing life-threatening diarrhoea and colitis. Microbial disruption caused by antibiotics has been linked with susceptibility to, and transmission and relapse of, C. difficile infection. Therefore, there is an urgent need for novel therapeutics that are effective in preventing C. difficile growth, spore germination, and outgrowth. In recent years bacteriophage-derived endolysins and their derivatives show promise as a novel class of antibacterial agents. In this study, we recombinantly expressed and characterized a cell wall hydrolase (CWH) lysin from C. difficile phage, phiMMP01. The full-length CWH displayed lytic activity against selected C. difficile strains. However, removing the N-terminal cell wall binding domain, creating CWH351-656, resulted in increased and/or an expanded lytic spectrum of activity. C. difficile specificity was retained versus commensal clostridia and other bacterial species. As expected, the putative cell wall binding domain, CWH1-350, was completely inactive. We also observe the effect of CWH351-656 on preventing C. difficile spore outgrowth. Our results suggest that CWH351-656 has therapeutic potential as an antimicrobial agent against C. difficile infection.

Optimization of spore laccase production by Bacillus amyloliquefaciens isolated from wastewater and its potential in green biodecolorization of synthetic textile dyes.

The spore laccase enzyme production by B. amyloliquefaciens was optimized. It was characterized and tested for its textile dye decolorization potential. LB medium was found to be the most promising growth medium with addition of glucose (1-2%), yeast extract (0.1%), FeCl3 (0.01 mM) and MnCl2 (0.001 mM). The optimum spore laccase production was at pH 8, 30 °C, 1:5 medium to air ratio, 2% inoculum size and 7 days incubation. The characterization study of the enzyme showed the maximum activity at 60 °C and pH 6-7.5. It was induced by Ca+2, Mg+2, Fe+3, Zn+2, Cu+2 and Na+ at 1 mM concentration. Also, it was stable in the presence of methanol, ethanol, acetone and chloroform. In addition, it enhanced about 34% by 5 mM H2O2 and it was nearly stable at 10-20 mM H2O2. Furthermore, mediators such as ABTS, syrengaldazine and 2, 6 dimethyl phenol enhanced the spore laccase activity. The spore laccase enzyme efficiently decolorized direct red 81 and acid black 24 after 24 h. Phytotoxicity of the direct red 81 solution after decolorization by tested spore laccase was lower than that of the untreated dye solution. Finally, this study added a promising spore laccase candidate for ecofriendly and cost-effective dye wastewater bio-decolorization.

Role of SpoIVA ATPase Motifs during Clostridioides difficile Sporulation.

The nosocomial pathogen Clostridioides difficile is a spore-forming obligate anaerobe that depends on its aerotolerant spore form to transmit infections. Functional spore formation depends on the assembly of a proteinaceous layer known as the coat around the developing spore. In C. difficile, coat assembly depends on the conserved spore protein SpoIVA and the clostridial-organism-specific spore protein SipL, which directly interact. Mutations that disrupt their interaction cause the coat to mislocalize and impair spore formation. In Bacillus subtilis, SpoIVA is an ATPase that uses ATP hydrolysis to drive its polymerization around the forespore. Loss of SpoIVA ATPase activity impairs B. subtilis SpoIVA encasement of the forespore and activates a quality control mechanism that eliminates these defective cells. Since this mechanism is lacking in C. difficile, we tested whether mutations in the C. difficile SpoIVA ATPase motifs impact functional spore formation. Disrupting C. difficile SpoIVA ATPase motifs resulted in phenotypes that were typically >104-fold less severe than the equivalent mutations in B. subtilis Interestingly, mutation of ATPase motif residues predicted to abrogate SpoIVA binding to ATP decreased the SpoIVA-SipL interaction, whereas mutation of ATPase motif residues predicted to disrupt ATP hydrolysis but maintain ATP binding enhanced the SpoIVA-SipL interaction. When a sipL mutation known to reduce binding to SpoIVA was combined with a spoIVA mutation predicted to prevent SpoIVA binding to ATP, spore formation was severely exacerbated. Since this phenotype is allele specific, our data imply that SipL recognizes the ATP-bound form of SpoIVA and highlight the importance of this interaction for functional C. difficile spore formation.IMPORTANCE The major pathogen Clostridioides difficile depends on its spore form to transmit disease. However, the mechanism by which C. difficile assembles spores remains poorly characterized. We previously showed that binding between the spore morphogenetic proteins SpoIVA and SipL regulates assembly of the protective coat layer around the forespore. In this study, we determined that mutations in the C. difficile SpoIVA ATPase motifs result in relatively minor defects in spore formation, in contrast with Bacillus subtilis Nevertheless, our data suggest that SipL preferentially recognizes the ATP-bound form of SpoIVA and identify a specific residue in the SipL C-terminal LysM domain that is critical for recognizing the ATP-bound form of SpoIVA. These findings advance our understanding of how SpoIVA-SipL interactions regulate C. difficile spore assembly.

Regulation of Sporangium Formation, Spore Dormancy, and Sporangium Dehiscence by a Hybrid Sensor Histidine Kinase in Actinoplanes missouriensis: Relationship with the Global Transcriptional Regulator TcrA.

The rare actinomycete Actinoplanes missouriensis forms terminal sporangia containing a few hundred flagellated spores. In response to water, the sporangia open and release the spores into external environments. The orphan response regulator TcrA functions as a global transcriptional activator during sporangium formation and dehiscence. Here, we report the characterization of an orphan hybrid histidine kinase, HhkA. Sporangia of an hhkA deletion mutant contained many distorted or ectopically germinated spores and scarcely opened to release the spores under sporangium dehiscence-inducing conditions. These phenotypic changes are quite similar to those observed in a tcrA deletion mutant. Comparative RNA sequencing analysis showed that genes controlled by HhkA mostly overlap TcrA-regulated genes. The direct interaction between HhkA and TcrA was suggested by a bacterial two-hybrid assay, but this was not conclusive. The phosphorylation of TcrA using acetyl phosphate as a phosphate donor markedly enhanced its affinity for the TcrA box sequences in the electrophoretic mobility shift assay. Taking these observations together with other results, we proposed that HhkA and TcrA compose a cognate two-component regulatory system, which controls the transcription of the genes involved in many aspects of morphological development, including sporangium formation, spore dormancy, and sporangium dehiscence in A. missouriensisIMPORTANCEActinoplanes missouriensis goes through complex morphological differentiation, including formation of flagellated spore-containing sporangia, sporangium dehiscence, swimming of zoospores, and germination of zoospores to filamentous growth. Although the orphan response regulator TcrA globally activates many genes required for sporangium formation, spore dormancy, and sporangium dehiscence, its partner histidine kinase remained unknown. Here, we analyzed the function of an orphan hybrid histidine kinase, HhkA, and proposed that HhkA constitutes a cognate two-component regulatory system with TcrA. That HhkA and TcrA homologues are highly conserved among the genus Actinoplanes and several closely related rare actinomycetes indicates that this possible two-component regulatory system is employed for complex morphological development in sporangium- and/or zoospore-forming rare actinomycetes.

The serine proteases CspA and CspC are essential for germination of spores of Clostridium perfringens SM101 through activating SleC and cortex hydrolysis.

Clostridium perfringens SM101 genome encodes three serine proteases (CspA, CspB, and CspC), and genetic evidence indicates that CspB is required for processing of pro-SleC into active SleC, an enzyme essential for degradation of the peptidoglycan cortex during spore germination. In this study, the expression of cspA and cspC, as well as the germination and colony formation by spores of cspAC and cspC mutants of strain SM101, were assessed. We demonstrated that 1) the cspA and cspC genes were expressed as a bicistronic operon only during sporulation in the mother cell compartment of SM101; 2) both cspAC and cspC mutant spores were unable to germinate significantly with either KCl, l-glutamine, brain heart infusion (BHI) broth, or a 1:1 chelate of Ca2+ and dipicolinic acid (DPA); 3) consistent with germination results, both cspAC and cspC mutant spores were defective in normal DPA release; 4) the colony formation by cspAC and cspC mutant spores was ~106-fold lower than that of wild-type spores, although decoated mutant spores yielded wild-type level colony formation on plates containing lysozyme; 5) no processing of inactive pro-SleC into active SleC was observed in cspAC and cspC mutant spores during germination; and finally, 6) the defects in germination, DPA release, colony formation and SleC processing in cspAC and cspC mutant spores were complemented by the wild-type cspA-cspC operon. Collectively, these results indicate that both CspA and CspC are essential for C. perfringens spore germination through activating SleC and inducing cortex hydrolysis.

A two-step transport pathway allows the mother cell to nurture the developing spore in Bacillus subtilis.

One of the hallmarks of bacterial endospore formation is the accumulation of high concentrations of pyridine-2,6-dicarboxylic acid (dipicolinic acid or DPA) in the developing spore. This small molecule comprises 5-15% of the dry weight of dormant spores and plays a central role in resistance to both wet heat and desiccation. DPA is synthesized in the mother cell at a late stage in sporulation and must be translocated across two membranes (the inner and outer forespore membranes) that separate the mother cell and forespore. The enzymes that synthesize DPA and the proteins required to translocate it across the inner forespore membrane were identified over two decades ago but the factors that transport DPA across the outer forespore membrane have remained mysterious. Here, we report that SpoVV (formerly YlbJ) is the missing DPA transporter. SpoVV is produced in the mother cell during the morphological process of engulfment and specifically localizes in the outer forespore membrane. Sporulating cells lacking SpoVV produce spores with low levels of DPA and cells engineered to express SpoVV and the DPA synthase during vegetative growth accumulate high levels of DPA in the culture medium. SpoVV resembles concentrative nucleoside transporters and mutagenesis of residues predicted to form the substrate-binding pocket supports the idea that SpoVV has a similar structure and could therefore function similarly. These findings provide a simple two-step transport mechanism by which the mother cell nurtures the developing spore. DPA produced in the mother cell is first translocated into the intermembrane space by SpoVV and is then imported into the forespore by the SpoVA complex. This pathway is likely to be broadly conserved as DPA synthase, SpoVV, and SpoVA proteins can be found in virtually all endospore forming bacteria.

Laccase mediator system obtained from a marine spore exhibits decolorization potential in harsh environmental conditions.

Laccases play a significant role in remedying dye pollutants. Most of these enzymes are originated from terrestrial fungi and bacteria, thus they are not proper to be used in the environments with neutral/alkaline pH, or they may require laborious extraction/purification steps. These limitations can be solved using marine spore laccases through high stability and easy to use application. In the current study, laccase activity of the marine spore -forming Bacillus sp. KC2 was measured according to the guaiacol and syringaldazine oxidation. Abiotic stresses like pH of 6, temperature of 37 °C and 0.3 mM CuSO4 (in comparison with optimal sporulation conditions: pH of 8, temperature of 20 °C and 0.0 mM CuSO4) enhanced laccase formation in sporal coat. Maximum activity of enzyme was observed at 50 °C and pH 7, which did not change in the alkaline pH and temperature range of 20-70 °C. Results indicated ions, inhibitors and solvent stability of the enzyme and its activity were stimulated by Co2+, Mn2+, PMSF, acetone, acetonitrile, ethanol, and methanol. The spore laccase could decolorize synthetic dyes from various chemical groups including azo (acid orange, amaranth, trypan blue, congo red, and amido black), indigo (indigo carmine), thiazine (methylene blue, and toluidine blue), and triarylmethane (malachite green) with ABTS/syringaldazine mediators after 5 h. Degradation products were not toxic against Sorghum vulgare and Artemia salina model organisms. The enzyme mediator system showed high potentials for dye bioremediation over a wide range of harsh conditions.

Economical production of isomaltulose from agricultural residues in a system with sucrose isomerase displayed on Bacillus subtilis spores.

A safe, efficient, environmentally friendly process for producing isomaltulose is needed. Here, the biocatalyst, sucrose isomerase (SIase) from Erwinia rhapontici NX-5, displayed on the surface of Bacillus subtilis 168 spores (food-grade strain) was applied for isomaltulose production. The anchored SIase showed relatively high bioactivity, suggesting that the surface display system using CotX as the anchoring protein was successful. The stability of the anchored SIase was also significantly better. Thermal stability analysis showed that 80% of relative activity was retained after incubation at 40 °C and 45 °C for 60 min. To develop an economical industrial fermentation medium, untreated beet molasses (30 g/L) and cold-pressed soybean powder (50 g/L) were utilised as the main broth components for SIase pilot-scale production. Under the optimal conditions, the productive spores converted 92% of sucrose after 6 h and the conversion rate was 45% after six cycles. Isomaltulose production with this system using the agricultural residues, untreated beet molasses and soybean powder, as substrates is cost-effective and environmentally friendly and can help to overcome issues due to the genetic background.

The early stage peptidoglycan biosynthesis Mur enzymes are antibacterial and antisporulation drug targets for recurrent Clostridioides difficile infection.

Sporulation during Clostridioides difficile infection (CDI) contributes to recurrent disease. Cell division and sporulation both require peptidoglycan biosynthesis. We show C. difficile growth and sporulation is attenuated by antisenses to murA and murC or the MurA inhibitor fosfomycin. Thus, targeting the early steps of peptidoglycan biosynthesis might reduce the onset of recurrent CDI.

A LysM Domain Intervenes in Sequential Protein-Protein and Protein-Peptidoglycan Interactions Important for Spore Coat Assembly in Bacillus subtilis.

At a late stage in spore development in Bacillus subtilis, the mother cell directs synthesis of a layer of peptidoglycan known as the cortex between the two forespore membranes, as well as the assembly of a protective protein coat at the surface of the forespore outer membrane. SafA, the key determinant of inner coat assembly, is first recruited to the surface of the developing spore and then encases the spore under the control of the morphogenetic protein SpoVID. SafA has a LysM peptidoglycan-binding domain, SafALysM, and localizes to the cortex-coat interface in mature spores. SafALysM is followed by a region, A, required for an interaction with SpoVID and encasement. We now show that residues D10 and N30 in SafALysM, while involved in the interaction with peptidoglycan, are also required for the interaction with SpoVID and encasement. We further show that single alanine substitutions on residues S11, L12, and I39 of SafALysM that strongly impair binding to purified cortex peptidoglycan affect a later stage in the localization of SafA that is also dependent on the activity of SpoVE, a transglycosylase required for cortex formation. The assembly of SafA thus involves sequential protein-protein and protein-peptidoglycan interactions, mediated by the LysM domain, which are required first for encasement then for the final localization of the protein in mature spores.IMPORTANCEBacillus subtilis spores are encased in a multiprotein coat that surrounds an underlying peptidoglycan layer, the cortex. How the connection between the two layers is enforced is not well established. Here, we elucidate the role of the peptidoglycan-binding LysM domain, present in two proteins, SafA and SpoVID, that govern the localization of additional proteins to the coat. We found that SafALysM is a protein-protein interaction module during the early stages of coat assembly and a cortex-binding module at late stages in morphogenesis, with the cortex-binding function promoting a tight connection between the cortex and the coat. In contrast, SpoVIDLysM functions only as a protein-protein interaction domain that targets SpoVID to the spore surface at the onset of coat assembly.

Activity of Spore-Specific Respiratory Nitrate Reductase 1 of Streptomyces coelicolor A3(2) Requires a Functional Cytochrome bcc-aa3 Oxidase Supercomplex.

Spores have strongly reduced metabolic activity and are produced during the complex developmental cycle of the actinobacterium Streptomyces coelicolor Resting spores can remain viable for decades, yet little is known about how they conserve energy. It is known, however, that they can reduce either oxygen or nitrate using endogenous electron sources. S. coelicolor uses either a cytochrome bd oxidase or a cytochrome bcc-aa3 oxidase supercomplex to reduce oxygen, while nitrate is reduced by Nar-type nitrate reductases, which typically oxidize quinol directly. Here, we show that in resting spores the Nar1 nitrate reductase requires a functional bcc-aa3 supercomplex to reduce nitrate. Mutants lacking the complete qcr-cta genetic locus encoding the bcc-aa3 supercomplex showed no Nar1-dependent nitrate reduction. Recovery of Nar1 activity was achieved by genetic complementation but only when the complete qcr-cta locus was reintroduced to the mutant strain. We could exclude that the dependence on the supercomplex for nitrate reduction was via regulation of nitrate transport. Moreover, the catalytic subunit, NarG1, of Nar1 was synthesized in the qcr-cta mutant, ruling out transcriptional control. Constitutive synthesis of Nar1 in mycelium revealed that the enzyme was poorly active in this compartment, suggesting that the Nar1 enzyme cannot act as a typical quinol oxidase. Notably, nitrate reduction by the Nar2 enzyme, which is active in growing mycelium, was not wholly dependent on the bcc-aa3 supercomplex for activity. Together, our data suggest that Nar1 functions together with the proton-translocating bcc-aa3 supercomplex to increase the efficiency of energy conservation in resting spores.IMPORTANCEStreptomyces coelicolor forms spores that respire with either oxygen or nitrate, using only endogenous electron donors. This helps maintain a membrane potential and, thus, viability. Respiratory nitrate reductase (Nar) usually receives electrons directly from reduced quinone species; however, we show that nitrate respiration in spores requires a respiratory supercomplex comprising cytochrome bcc oxidoreductase and aa3 oxidase. Our findings suggest that the Nar1 enzyme in the S. coelicolor spore functions together with the proton-translocating bcc-aa3 supercomplex to help maintain the membrane potential more efficiently. Dissecting the mechanisms underlying this survival strategy is important for our general understanding of bacterial persistence during infection processes and of how bacteria might deal with nutrient limitation in the natural environment.

N-Deacetylases required for muramic-δ-lactam production are involved in Clostridium difficile sporulation, germination, and heat resistance.

Spores are produced by many organisms as a survival mechanism activated in response to several environmental stresses. Bacterial spores are multilayered structures, one of which is a peptidoglycan layer called the cortex, containing muramic-δ-lactams that are synthesized by at least two bacterial enzymes, the muramoyl-l-alanine amidase CwlD and the N-deacetylase PdaA. This study focused on the spore cortex of Clostridium difficile, a Gram-positive, toxin-producing anaerobic bacterial pathogen that can colonize the human intestinal tract and is a leading cause of antibiotic-associated diarrhea. Using ultra-HPLC coupled with high-resolution MS, here we found that the spore cortex of the C. difficile 630Δerm strain differs from that of Bacillus subtilis Among these differences, the muramic-δ-lactams represented only 24% in C. difficile, compared with 50% in B. subtilis CD630_14300 and CD630_27190 were identified as genes encoding the C. difficile N-deacetylases PdaA1 and PdaA2, required for muramic-δ-lactam synthesis. In a pdaA1 mutant, only 0.4% of all muropeptides carried a muramic-δ-lactam modification, and muramic-δ-lactams were absent in the cortex of a pdaA1-pdaA2 double mutant. Of note, the pdaA1 mutant exhibited decreased sporulation, altered germination, decreased heat resistance, and delayed virulence in a hamster infection model. These results suggest a much greater role for muramic-δ-lactams in C. difficile than in other bacteria, including B. subtilis In summary, the spore cortex of C. difficile contains lower levels of muramic-δ-lactams than that of B. subtilis, and PdaA1 is the major N-deacetylase for muramic-δ-lactam biosynthesis in C. difficile, contributing to sporulation, heat resistance, and virulence.

Differential requirements for conserved peptidoglycan remodeling enzymes during Clostridioides difficile spore formation.

Spore formation is essential for the bacterial pathogen and obligate anaerobe, Clostridioides (Clostridium) difficile, to transmit disease. Completion of this process depends on the mother cell engulfing the developing forespore, but little is known about how engulfment occurs in C. difficile. In Bacillus subtilis, engulfment is mediated by a peptidoglycan degradation complex consisting of SpoIID, SpoIIP and SpoIIM, which are all individually required for spore formation. Using genetic analyses, we determined the functions of these engulfment-related proteins along with the putative endopeptidase, SpoIIQ, during C. difficile sporulation. While SpoIID, SpoIIP and SpoIIQ were critical for engulfment, loss of SpoIIM minimally impacted C. difficile spore formation. Interestingly, a small percentage of ∆spoIID and ∆spoIIQ cells generated heat-resistant spores through the actions of SpoIIQ and SpoIID, respectively. Loss of SpoIID and SpoIIQ also led to unique morphological phenotypes: asymmetric engulfment and forespore distortions, respectively. Catalytic mutant complementation analyses revealed that these phenotypes depend on the enzymatic activities of SpoIIP and SpoIID, respectively. Lastly, engulfment mutants mislocalized polymerized coat even though the basement layer coat proteins, SpoIVA and SipL, remained associated with the forespore. Collectively, these findings advance our understanding of several stages during infectious C. difficile spore assembly.

Peptidoglycan degradation machinery in Clostridium difficile forespore engulfment.

Clostridium difficile remains the leading cause of antibiotic-associated diarrhoea in hospitals worldwide, linked to significant morbidity and mortality. As a strict anaerobe, it produces dormant cell forms - spores - which allow it to survive in the aerobic environment. Importantly, spores are the transmission agent of C. difficile infections. A key aspect of sporulation is the engulfment of the future spore by the mother cell and several proteins have been proposed to be involved. Here, we investigated the role of the SpoIID, SpoIIM and SpoIIP (DMP) machinery and its interplay with the SpoIIQ:SpoIIIAH (Q:AH) complex in C. difficile. We show that, surprisingly, SpoIIM, the proposed machinery anchor, is not required for efficient engulfment and sporulation. We demonstrate the requirement of DP for engulfment due to their sequential peptidoglycan degradation activity, both in vitro and in vivo. Finally, new interactions within DMP and between DMP and Q:AH suggest that both systems form a single engulfment machinery to keep the mother cell and forespore membranes together throughout engulfment. This work sheds new light upon the engulfment process and on how different sporeformers might use the same components in different ways to drive spore formation.

Surface display of organophosphorus-degrading enzymes on the recombinant spore of Bacillus subtilis.

Organophosphorus-degrading enzymes show high hydrolysis efficiency and provide an environmentally friendly solution to the pollution of organophosphorus compound. However, poor enzyme stability and tedious purification process have limited practical applications. Spore-based display system can provide many advantages, such as safety, low cost, easy preparation and high resistance to harsh conditions. Recently, we have constituted the recombinant spore displaying organophosphorus hydrolase and organophosphorus acid anhydrolase. In the spore display systems, recombinant spores could be reliably produced and normal sporulation was not affected; the activities of recombinant spores were 15.81 and 10.67 U/mg spores (dry weight) respectively; furthermore, the recombinant spores exhibited significantly enhanced resistance to various harsh conditions compared to free-form enzymes. These results indicated that the spore display could contribute to the practical application of organophosphorus-degrading enzymes and provide a promising solution to bioremediation of organophosphorus compounds.

Production of trehalose with trehalose synthase expressed and displayed on the surface of Bacillus subtilis spores.

BACKGROUND: Bacillus subtilis spores have been commonly used for the surface display of various food-related or human antigens or enzymes. For successful display, the target protein needs to be fused with an anchor protein. The preferred anchored proteins are the outer-coat proteins of spores; outer-coat proteins G (CotG) and C (CotC) are commonly used. In this study, mutant trehalose synthase (V407M/K490L/R680E TreS) was displayed on the surface of B. subtilis WB800n spores using CotG and CotC individually or in combination as an anchoring protein. RESULTS: Western blotting, immunofluorescence, dot blot, and enzymatic-activity assays detected TreS on the spore surface. The TreS activity with CotC and CotG together as the anchor protein was greater than the sum of the enzymatic activities with CotC or CotG alone. The TreS displayed on the spore surface with CotC and CotG together as the anchoring protein showed elevated and stable specific activity. To ensure spore stability and prevent spore germination in the trehalose preparation system, two germination-specific lytic genes, sleB and cwlJ, were deleted from the B. subtilis WB800n genome. It was demonstrated that this deletion did not affect the growth and spore formation of B. subtilis WB800n but strongly inhibited germination of the spores during transformation. The conversion rate of trehalose from 300 g/L maltose by B. subtilis strain WB800n(ΔsleB, ΔcwlJ)/cotC-treS-cotG-treS was 74.1% at 12 h (350 U/[g maltose]), and its enzymatic activity was largely retained, with a conversion rate of 73% after four cycles. CONCLUSIONS: The spore surface display system based on food-grade B. subtilis with CotC and CotG as a combined carrier appears to be a powerful technology for TreS expression, which may be used for the biotransformation of D-maltose into D-trehalose.

A disclosure gel for visual detection of live Bacillus anthracis spores.

AIMS: To develop a gel formulation to trigger a visual signal for rapid disclosure of the location and extent of surface contamination with viable Bacillus anthracis spores. METHODS AND RESULTS: Methylumbelliferyl-α-d-glucopyranoside was combined with hyaluronic acid to produce a gel that could be applied to a surface as a coating. It remained hydrated for a sufficient time for α-glucosidase activity present in intact B. anthracis spores to cleave the substrate and release the fluorescent product, methylumbelliferone. The presence of B. anthracis spores could be disclosed at 5 × 104 CFU per reaction test well (0·32 cm2 ) both visually and using fluorescence detection equipment. CONCLUSIONS: The disclosure gel provides a rapid, visual response to the presence of B. anthracis spores on a surface. SIGNIFICANCE AND IMPACT OF THE STUDY: The disclosure gel demonstrates the first steps towards the development of a formulation that can provide nonspecialist users with a visual alert to the presence of B. anthracis spores on a surface. It is envisioned that such a formulation would be beneficial in scenarios where exposure to spore release is a risk, and could be used in the initial assessment of equipment to aid prioritization and localized execution of a decontamination strategy.

Bacillus subtilis Spore Surface Display of Haloalkane Dehalogenase DhaA.

The haloalkane dehalogenase DhaA can degrade sulfur mustard (2,2'-dichlorethyl sulfide; also known by its military designation HD) in a rapid and environmentally safe manner. However, DhaA is sensitive to temperature and pH, which limits its applications in natural or harsh environments. Spore surface display technology using resistant spores as a carrier to ensure enzymatic activity can reduce production costs and extend the range of applications of DhaA. To this end, we cloned recombinant Bacillus subtilis spores pHY300PLK-cotg-dhaa-6his/DB104(FH01) for the delivery of DhaA from Rhodococcus rhodochrous NCIMB 13064. A dot blotting showed that the fusion protein CotG-linker-DhaA accounted for 0.41% ± 0.03% (P < 0.01) of total spore coat proteins. Immunofluorescence analyses confirmed that DhaA was displayed on the spore surface. The hydrolyzing activity of DhaA displayed on spores towards the HD analog 2-chloroethyl ethylsulfide was 1.74 ± 0.06 U/mL (P < 0.01), with a specific activity was 0.34 ± 0.04 U/mg (P < 0.01). This is the first demonstration that DhaA displayed on the surface of B. subtilis spores retains enzymatic activity, which suggests that it can be used effectively in real-world applications including bioremediation of contaminated environments.