Extreme Spore UV Resistance of <Emphasis Type="Italic">Bacillus pumilus</Emphasis> Isolates Obtained from an Ultraclean Spacecraft Assembly Facility

Recent environmental microbial sampling of the ultraclean Spacecraft Assembly Facility at NASA Jet Propulsion Laboratory (JPL-SAF) identified spores of Bacillus pumilus as major culturable bacterial contaminants found on and around spacecraft. As part of an effort to assess the efficacy of various spacecraft sterilants, purified spores of 10 JPL-SAF B. pumilus isolates were subjected to 254-nm UV and their UV resistance was compared to spores of standard B. subtilis biodosimetry strains. Spores of six of the 10 JPL-SAF isolates were significantly more resistant to UV than the B. subtilis biodosimetry strain, and one of the JPL-SAF isolates, B. pumilus SAFR-032, exhibited the highest degree of spore UV resistance observed by any Bacillus spp. encountered to date.     

枯草芽孢杆菌表面展示技术用于黏膜疫苗的研究进展

枯草杆菌全名枯草芽孢杆菌(Bacillus subtilis),因其优秀的益生特性及芽孢良好的抗逆性而备受研究者青睐,由于芽孢的特殊结构及独特的生理特性,是酶和免疫原等外源蛋白的理想锚定点。采用枯草杆菌进行芽孢表面展示被认为是表达高活性和高稳定性的外源蛋白的方法之一。本文主要对枯草杆菌芽孢表面展示抗原蛋白以生产黏膜疫苗的策略和应用前景进行综述。     

Immunomodulatory effects of Bacillus subtilis (natto) B4 spores on murine macrophages

To investigate the immunomodulatory effects of Bacillus subtilis (B. subtilis) (natto) B4 spores on murine macrophage, RAW 264.7 cells were cultured alone or with B subtilis (natto) B4 spores at 37°C for 12 hrs, then both cells and culture supernatants were collected for analyses. Exposure of RAW 264.7 cells to B. subtilis (natto) B4 spores had no significant effects on macrophage viability and amounts of extracellular lactate dehydrogenase (LDH). However, it remarkably increased the activities of acid phosphatase (ACP), lactate dehydrogenase (LDH) and inducible nitric oxide synthase (iNOS) in cells and the amounts of nitric oxide (NO) and cytokines (tumor necrosis factor‐alpha, interferon‐gamma, interleukin [IL]‐1 beta, IL‐6, IL‐12, IL‐10 and macrophage inflammatory protein‐2) in culture supernatants. These results demonstrate that B. subtilis (natto) B4 spores are harmless to murine macrophages and can stimulate their activation through up‐regulation of ACP and LDH activities and enhance their immune function by increasing iNOS activity and stimulating NO and cytokine production. The above findings suggest that B. subtilis (natto) B4 spores have immunomodulatory effects on macrophages.     

Formulation and Delivery of the Bacterial Antagonist Bacillus subtilis for Management of Lentil Vascular Wilt Caused by Fusarium oxysporum f. sp. lentis

Different formulations of Bacillus subtilis were prepared using standard laboratory protocols. Bacillus subtilis survived in glucose and talc powders at 8.6 and 7.8 log₁₀ CFU/g, respectively, for 1 year of storage at room temperature compared with 3.5 log₁₀ CFU/g on a peat formulation. Glasshouse experiments using soil and seed treatments were conducted to test the efficacy of B. subtilis for protecting lentil against the wilt disease caused by Fusariumoxysporum f. sp. lentis. Seed treatments with formulations of B. subtilis on glucose, talc and peat significantly enhanced its biocontrol activity against Fusarium compared with a treatment in which spores were applied directly to seed. The formulations decreased disease severity by reducing colonization of plants by the pathogen, promoting their growth and increased the dry weight of lentil plants. Of these treatments the glucose and talc‐based powder formulations were more effective than the peat formulation and the spore application without a carrier. It was shown that the B. subtilis spores applied with glucose were viable for longer than those applied with other carriers. Seed treatment with these formulated spores is an effective delivery system that can provide a conducive environment for B. subtilis to suppress vascular wilt disease on lentil and has the potential for utilization in commercial field application.     

UV Resistance of Bacillus anthracis Spores Revisited: Validation of Bacillus subtilis Spores as UV Surrogates for Spores of B. anthracis Sterne

Recent bioterrorism concerns have prompted renewed efforts towards understanding the biology of bacterial spore resistance to radiation with a special emphasis on the spores of Bacillus anthracis. A review of the literature revealed that B. anthracis Sterne spores may be three to four times more resistant to 254-nm-wavelength UV than are spores of commonly used indicator strains of Bacillus subtilis. To test this notion, B. anthracis Sterne spores were purified and their UV inactivation kinetics were determined in parallel with those of the spores of two indicator strains of B. subtilis, strains WN624 and ATCC 6633. When prepared and assayed under identical conditions, the spores of all three strains exhibited essentially identical UV inactivation kinetics. The data indicate that standard UV treatments that are effective against B. subtilis spores are likely also sufficient to inactivate B. anthracis spores and that the spores of standard B. subtilis strains could reliably be used as a biodosimetry model for the UV inactivation of B. anthracis spores.     

Surface-displayed VP28 on Bacillus subtilis spores induce protection against white spot syndrome virus in crayfish by oral administration

Aim: Surface‐displayed heterologous antigens on Bacillus subtilis spores can induce the vertebrate animals tested to generate local and systematic immune response through oral immunization. Here, the protection potential of the recombinant spores displaying the VP28 protein of white spot syndrome virus (WSSV) was investigated in the invertebrate crayfish (Cambarus clarkii). Methods and Results: The VP28 protein was successfully displayed on the surfaces of B. subtilis spores using CotB or CotC as a fusion partner. Crayfish were administrated orally by feeding the feed pellets coated with B. subtilis spores for 7 days and immediately followed by WSSV challenge. Oral administration of either spores expressing CotB‐VP28 or CotC‐VP28 resulted in significantly higher relative survival rates of 37·9 and 44·8% compared with the crayfish orally administrated with the spores nonexpressing VP28 (10·3% relative survival rate). When challenges were separately conducted at 7 and 21 days after oral administration, the relative survival rates increased to 46·4 and 50% at 7 days post‐oral administration, but decreased to 30 and 33·3% at 21 days after oral administration. Conclusion: These evidences indicate that the surface‐displayed VP28 on B. subtilis spore could induce protection of crayfish against WSSV via oral administration. Significance and Impact of the Study: This is the first report to use the spore surface display system to deliver orally a heterologous antigen in an aquatic invertebrate animal, crayfish. The results presented here suggest that the spore‐displayed VP28 might be suitable for an oral booster vaccine on prevention of WSSV infection in shrimp farming.     

Peptidoglycan transformations during Bacillus subtilis sporulation

While vegetative Bacillus subtilis cells and mature spores are both surrounded by a thick layer of peptidoglycan (PG, a polymer of glycan strands cross‐linked by peptide bridges), it has remained unclear whether PG surrounds prespores during engulfment. To clarify this issue, we generated a slender ΔponA mutant that enabled high‐resolution electron cryotomographic imaging. Three‐dimensional reconstructions of whole cells in near‐native states revealed a thin PG‐like layer extending from the lateral cell wall around the prespore throughout engulfment. Cryotomography of purified sacculi and fluorescent labelling of PG in live cells confirmed that PG surrounds the prespore. The presence of PG throughout engulfment suggests new roles for PG in sporulation, including a new model for how PG synthesis might drive engulfment, and obviates the need to synthesize a PG layer de novo during cortex formation. In addition, it reveals that B. subtilis can synthesize thin, Gram‐negative‐like PG layers as well as its thick, archetypal Gram‐positive cell wall. The continuous transformations from thick to thin and back to thick during sporulation suggest that both forms of PG have the same basic architecture (circumferential). Endopeptidase activity may be the main switch that governs whether a thin or a thick PG layer is assembled.     

Characterization of new <Emphasis Type="SmallCaps">l,d</Emphasis>-endopeptidase gene product CwlK (previous YcdD) that hydrolyzes peptidoglycan in <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Bacillus subtilis has various cell wall hydrolases, however, the functions and hydrolase activities of some enzymes are still unknown. B. subtilis CwlK (YcdD) exhibits high sequence similarity with the peptidoglycan hydrolytic l,d-endopeptidase (PLY500) of Listeria monocytogenes phage and CwlK has the VanY motif which is a d-alanyl-d-alanine carboxypeptidase (Pfam: http://www.sanger.ac.uk/Software/Pfam/). The β-galactosidase activity observed on cwlK-lacZ fusion indicated that the cwlK gene was expressed during the vegetative growth phase, and Western blotting suggested that CwlK seems to be localized in the membrane. Truncated CwlK fused with a histidine-tag (h-ΔCwlK) was produced in Escherichia coli and purified on a nickel column. The h-ΔCwlK protein hydrolyzed the peptidoglycan of B. subtilis, and the optimal pH, temperature and NaCl concentration for h-ΔCwlK were pH 6.5, 37°C, and 0 M, respectively. Interestingly, h-ΔCwlK could hydrolyze the linkage of l-alanine-d-glutamic acid in the stem of the peptidoglycan, however, this enzyme could not hydrolyze the linkage of d-alanine-d-alanine, suggesting that CwlK is an l,d-endopeptidase not a d,d-carboxypeptidase. CwlK could not hydrolyze polyglutamate from B. natto or peptidoglycan of Staphylococcus aureus. This is the first report describing the characterization of an l,d-endopeptidase in B. subtilis and also the first report in bacteria of the characterization of a PLY500 family protein encoded in chromosomal DNA. Tatsuya Fukushima and Yang Yao contributed equally to this work.     

Probiotic Potential of the Farmed Olive Flounder,Paralichthys olivaceus,Autochthonous Gut Microbiota

In recent years, considerable and growing attention has been given to the application of host-associated microorganisms as a more suitable source of probiotics in aquaculture sector. Herein, we isolated and screened the olive flounder gut microbiota for beneficial bacterial strains that might serve as potential probiotics in a low fishmeal extruded aquafeed. Among the ten identified isolates, Bacillus amyloliquefaciens SK4079 and B. subtilis SK4082 were screened out based on their heat-resistant ability as well as enzymatic and non-hemolytic activities. Although both strains were well able to utilize carboxymethyl cellulose (CMC), xylan, and soybean meal (SBM) as a single carbon source in the minimal nutrient M9 medium, B. subtilis exhibited significantly higher cellulase, xylanase, and protease activities than B. amyloliquefaciens. The two selected strains were well able to degrade the undesirable anti-nutritional component of the SBM, which would limit its utilization as protein source in aquafeed industry. Significantly higher biofilm formation capacity and notably stronger adhesive interactions with the flounder’s skin mucus were detected in B. subtilis than B. amyloliquefaciens. Immobilization of the spores from the selected strains, in a SBM complex carrier, remarkably enhances their thermal resistance at 120 °C for 5 min and different drying conditions. It was also interesting to learn that the B. subtilis spores could survive and remain viable after being sprayed onto extruded low-fish meal feed pellets for as long as 6 months. Overall, the findings of the present study could help the food/feed industries achieve their goal of developing cost-effective yet efficient products.

     

Effects of Mn levels on resistance of Bacillus megaterium spores to heat, radiation and hydrogen peroxide

Aims: To determine the effects of Mn levels in Bacillus megaterium sporulation and spores on spore resistance. Methods and Results: Bacillus megaterium was sporulated with no added MnCl₂ and up to 1 mmol l^?1 MnCl₂. The resultant spores were purified and loosely bound Mn removed, and spore Mn levels were found to vary c. 100‐fold. The Mn level had no effect on spore γ‐radiation resistance, but B. megaterium spores with elevated Mn levels had higher resistance to UVC radiation (as did Bacillus subtilis spores), wet and dry heat and H₂O₂. However, levels of dipicolinic acid and the DNA‐protective α/β‐type small, acid‐soluble spore proteins were the same in spores with high and low Mn levels. Conclusions: Mn levels either in sporulation or in spores are important factors in determining levels of B. megaterium spore resistance to many agents, with the exception of γ‐radiation. Significance and Impact of the Study: The Mn level in sporulation is an important factor to consider when resistance properties of B. megaterium spores are examined, and will influence the UV resistance of B. subtilis spores, some of which are used as biological dosimeters.     

Visualizing the production and arrangement of peptidoglycan in Gram‐positive cells

Decades of study have revealed the fine chemical structure of the bacterial peptidoglycan cell wall, but the arrangement of the peptidoglycan strands within the wall has been challenging to define. The application of electron cryotomography (ECT) and new methods for fluorescent labelling of peptidoglycan are allowing new insights into wall structure and synthesis. Two articles in this issue examine peptidoglycan structures in the model Gram‐positive species Bacillus subtilis. Beeby et al. combined visualization of peptidoglycan using ECT with molecular modelling of three proposed arrangements of peptidoglycan strands to identify the model most consistent with their data. They argue convincingly for a Gram‐positive wall containing multiple layers of peptidoglycan strands arranged circumferentially around the long axis of the rod‐shaped cell, an arrangement similar to the single layer of peptidoglycan in similarly shaped Gram‐negative cells. Tocheva et al. examined sporulating cells using ECT and fluorescence microscopy to demonstrate the continuous production of a thin layer of peptidoglycan around the developing spore as it is engulfed by the membrane of the adjacent mother cell. The presence of this peptidoglycan in the intermembrane space allows the refinement of a model for engulfment, which has been known to include peptidoglycan synthetic and lytic functions.     

Deposition of Bacillus subtilis spores using an airbrush-spray or spots to study surface decontamination by pulsed light

Microbial contamination on surfaces of food processing equipment is a major concern in industries. A new method to inoculate a single-cell layer (monolayer) of microorganisms onto polystyrene was developed, using a deposition with an airbrush. A homogeneous dispersion of Bacillus subtilis DSM 402 spores sprayed on the surface was observed using both plate count and scanning electron microscopy. No clusters were found, even with high spore concentrations (10⁷ spores/inoculated surface). A monolayer of microorganisms was also obtained after deposition of 10 μL droplets containing 3 × 10⁴ spores/spot on polystyrene disks, but not with a higher spore concentration. Pulsed light (PL) applied to monolayers of B. subtilis spores allowed log reductions higher than 6. As a consequence of clusters formation in spots of 10 μL containing more than 3 × 10⁵ spores, log reductions obtained by PL were significantly lower. The comparative advantages of spot and spray depositions were discussed.     

Mechanism of killing of spores of Bacillus cereus and Bacillus megaterium by wet heat

Aims: To determine the mechanism of wet heat killing of spores of Bacillus cereus and Bacillus megaterium. Methods and Results: Bacillus cereus and B. megaterium spores wet heat‐killed 82–99% gave two bands on equilibrium density gradient centrifugation. The lighter band was absent from spores that were not heat‐treated and increased in intensity upon increased heating times. These spores lacked dipicolinic acid (DPA) were not viable, germinated minimally and had much denatured protein. The spores in the denser band had viabilities as low as 2% of starting spores but retained normal DPA levels and most germinated, albeit slowly. However, these largely dead spores outgrew poorly if at all and synthesized little or no ATP following germination. Conclusions: Wet heat treatment appears to kill spores of B. cereus and B. megaterium by denaturing one or more key proteins, as has been suggested for wet heat killing of Bacillus subtilis spores. Significance and Impact of the Study: This work provides further information on the mechanisms of killing of spores of Bacillus species by wet heat, the most common method for spore inactivation.     

Differentiated roles for MreB‐actin isologues and autolytic enzymes in Bacillus subtilis morphogenesis

Cell morphogenesis in most bacteria is governed by spatiotemporal growth regulation of the peptidoglycan cell wall layer. Much is known about peptidoglycan synthesis but regulation of its turnover by hydrolytic enzymes is much less well understood. Bacillus subtilis has a multitude of such enzymes. Two of the best characterized are CwlO and LytE: cells lacking both enzymes have a lethal block in cell elongation. Here we show that activity of CwlO is regulated by an ABC transporter, FtsEX, which is required for cell elongation, unlike cell division as in Escherichia coli. Actin‐like MreB proteins are thought to play a key role in orchestrating cell wall morphogenesis. B. subtilis has three MreB isologues with partially differentiated functions. We now show that the three MreB isologues have differential roles in regulation of the CwlO and LytE systems and that autolysins control different aspects of cell morphogenesis. The results add major autolytic activities to the growing list of functions controlled by MreB isologues in bacteria and provide new insights into the different specialized functions of essential cell wall autolysins.     

Comparative Sensitivity to UV-B Radiation of Two Bacillus thuringiensis Subspecies and Other Bacillus sp.

Susceptibility of Bacillus thuringiensis spores and toxins to the UV-B range (280–330 nm) of the solar spectrum reaching Earth's surface may be responsible for its inactivation and low persistence in nature. Spores of the mosquito larvicidal B. thuringiensis subsp. israelensis were significantly more resistant to UV-B than spores of the lepidopteran-active subsp. kurstaki. Spores of subsp. israelensis were as resistant to UV-B as spores of B. subtilis and more resistant than spores of the closely related B. cereus and another mosquito larvicidal species B. sphaericus. Sensitivity of B. thuringiensis subsp. israelensis spores to UV-B radiation depended upon their culture age; 24-h cultures, approaching maximal larvicidal activity, were still sensitive. Maximal resistance to UV-B was achieved only at 48 h. Received: 13 December 2000/Accepted: 19 January 2001     

Effect of radioprotective agents in sporulation medium on Bacillus subtilis spore resistance to hydrogen peroxide, wet heat and germicidal and environmentally relevant UV radiation

Aims: To determine the effects of cysteine, cystine, proline and thioproline as sporulation medium supplements on Bacillus subtilis spore resistance to hydrogen peroxide (H₂O₂), wet heat, and germicidal 254 nm and simulated environmental UV radiation. Methods and Results: Bacillus subtilis spores were prepared in a chemically defined liquid medium, with and without supplementation of cysteine, cystine, proline or thioproline. Spores produced with thioproline, cysteine or cystine were more resistant to environmentally relevant UV radiation at 280–400 and 320–400 nm, while proline supplementation had no effect. Spores prepared with cysteine, cystine or thioproline were also more resistant to H₂O₂ but not to wet heat or 254‐nm UV radiation. The increases in spore resistance attributed to the sporulation supplements were eliminated if spores were chemically decoated. Conclusions: Supplementation of sporulation medium with cysteine, cystine or thioproline increases spore resistance to solar UV radiation reaching the Earth’s surface and to H₂O₂. These effects were eliminated if the spores were decoated, indicating that alterations in coat proteins by different sporulation conditions can affect spore resistance to some agents. Significance and Impact of the Study: This study provides further evidence that the composition of the sporulation medium can have significant effects on B. subtilis spore resistance to UV radiation and H₂O₂. This knowledge provides further insight into factors influencing spore resistance and inactivation.     

An efficient procedure for the isolation of PCR-competent DNA from Bacillus endospores germinated in soil

DNA extraction techniques for endospore-forming bacteria in soil are often labour-intensive and unreliable. Our objective in this work was to investigate whether good quality DNA could be obtained from spores germinated in soil. To this end, endospores from Bacillus subtilis, B. megaterium and B. thuringiensis were inoculated into soil microcosms and germination was induced by addition of LB medium supplemented with l-alanine, glucose, fructose and KCl. Heat resistance count was reduced to 80% for B. subtilis and more than 90% for B. thuringiensis and B. megaterium after a few minutes. Isolation of DNA from soil with a procedure which did not work on spores was shown to be as efficient for in situ-germinated spores as for inoculated vegetative cells. Furthermore, we developed a simple procedure that allowed us to use the recovered DNA in PCR amplifications. The present methodology is simple and efficient; it avoids the use of special equipment and harsh spore rupturing methods and can be carried out with multiple samples.     

Peptidoglycan metabolism is controlled by the WalRK (YycFG) and PhoPR two‐component systems in phosphate‐limited Bacillus subtilis cells

In Bacillus subtilis, the WalRK (YycFG) two‐component system controls peptidoglycan metabolism in exponentially growing cells while PhoPR controls the response to phosphate limitation. Here we examine the roles of WalRK and PhoPR in peptidoglycan metabolism in phosphate‐limited cells. We show that B. subtilis cells remain viable in a phosphate‐limited state for an extended period and resume growth rapidly upon phosphate addition, even in the absence of a PhoPR‐mediated response. Peptidoglycan synthesis occurs in phosphate‐limited wild‐type cells at ～27% the rate of exponentially growing cells, and at ～18% the rate of exponentially growing cells in the absence of PhoPR. In phosphate‐limited cells, the WalRK regulon genes yocH, cwlO(yvcE), lytE and ydjM are expressed in a manner that is dependent on the WalR recognition sequence and deleting these genes individually reduces the rate of peptidoglycan synthesis. We show that ydjM expression can be activated by PhoP～P in vitro and that PhoP occupies its promoter in phosphate‐limited cells. However, iseA(yoeB) expression cannot be repressed by PhoP～P in vitro, but can be repressed by non‐phosphorylated WalR in vitro. Therefore, we conclude that peptidoglycan metabolism is controlled by both WalRK and PhoPR in phosphate‐limited B. subtilis cells.     

Study of Bacillus subtilis Endospores in Soil by Use of a Modified Endospore Stain

The Schaeffer-Fulton endospore stain was modified so that it would stain Bacillus subtilis endospores in soil smears. The modified stain differentiated among dormant spores, spores undergoing activation, and spores which had germinated but had not yet shown outgrowth. These differentiations were seen for spores in soil and for pure spore preparations in the laboratory. This stain was used to show reversible B. subtilis spore activation promoted by an Ensifer adhaerens-like indigenous bacterium in soil and by pure cultures of E. adhaerens added to spores in the laboratory. Under the specific conditions in the laboratory, spore germination did not proceed beyond the activation stage, and relatively little change occurred in the numbers of both E. adhaerens and B. subtilis. This was also true in soil, although some germination with destruction of spores and vegetative cells did occur if the soil had been nutritionally enriched by preincubation with incorporated ground alfalfa.     

The preparation,germination properties and stability of superdormant spores of Bacillus cereus

Aims: To determine yields, germination and stability of superdormant Bacillus cereus spores. Methods and Results: Superdormant B. cereus spores were isolated by germination with high concentrations of inosine or l ‐alanine in 2–5% yield and did not germinate with high concentrations of either of these germinants, but germinated like starting spores with Ca‐DPA, dodecylamine, l ‐alanine plus inosine or concentrated complete medium. Yields of superdormant spores from germinations with low inosine concentrations were higher, and these spores germinated poorly with low inosine, but relatively normally with high inosine. Yields of superdormant spores were also higher when nonheat‐activated spores were germinated. Superdormant spores stored at 4°C slowly recovered some germination capacity, but recovery was slowed significantly at ?20°C and ?80°C. Conclusions: Factors that influence levels of superdormant B. cereus spores and the properties of such spores are similar to those in B. megaterium and B. subtilis, suggesting there are common mechanisms involved in superdormancy of Bacillus spores. Significance: Superdormant spores are a major concern in the food industry, because the presence of such spores precludes decontamination strategies based on triggering spore germination followed by mild killing treatments. Studies of the properties of superdormant spores may suggest ways to eliminate them.