Bacillus Megaterium Spores Research Articles

PRODUCTION OF SELF-HEALING MORTAR USING BACTERIAL SPORES ENCAPSULATED WITH JUTE FIBRE

Cement-based composites have various advantages such as low cost, easy shaping, and high compressive strength. Therefore, they are widely used in the construction industry. However, their brittle structure makes them prone to cracking, which must be repaired promptly to avoid possible loss of strength and durability and ultimate structural failures. Microbially-induced calcite precipitation (MICP) offers an effective method for healing these cracks. Unlike other treatments, MICP method is a natural and environment-friendly option for self-healing of cracks. However, bacteria can be damaged by cement and mechanical impacts; therefore, encapsulation is necessary to protect them. Encapsulation in fibres is a simpler and more cost-effective method than the other techniques. This study tested the effectiveness of encapsulating Bacillus megaterium spores in jute fibres and added them to cement-based mortars. Different nutrient supplements were used, and physical analyses and compressive strength tests were conducted on 28-day cured cube specimens. Present findings revealed that Bacillus megaterium-type bacteria encapsulated in jute fibres improved the compressive strength recovery and healed the loading-induced cracks.

Levels and Characteristics of mRNAs in Spores of Firmicute Species.

Spores of firmicute species contain 100s of mRNAs, whose major function in Bacillus subtilis is to provide ribonucleotides for new RNA synthesis when spores germinate. To determine if this is a general phenomenon, RNA was isolated from spores of multiple firmicute species and relative mRNA levels determined by transcriptome sequencing (RNA-seq). Determination of RNA levels in single spores allowed calculation of RNA nucleotides/spore, and assuming mRNA is 3% of spore RNA indicated that only ∼6% of spore mRNAs were present at >1/spore. Bacillus subtilis, Bacillus atrophaeus, and Clostridioides difficile spores had 49, 42, and 51 mRNAs at >1/spore, and numbers of mRNAs at ≥1/spore were ∼10 to 50% higher in Geobacillus stearothermophilus and Bacillus thuringiensis Al Hakam spores and ∼4-fold higher in Bacillus megaterium spores. In all species, some to many abundant spore mRNAs (i) were transcribed by RNA polymerase with forespore-specific σ factors, (ii) encoded proteins that were homologs of those encoded by abundant B. subtilis spore mRNAs and are proteins in dormant spores, and (iii) were likely transcribed in the mother cell compartment of the sporulating cell. Analysis of the coverage of RNA-seq reads on mRNAs from all species suggested that abundant spore mRNAs were fragmented, as was confirmed by reverse transcriptase quantitative PCR (RT-qPCR) analysis of abundant B. subtilis and C. difficile spore mRNAs. These data add to evidence indicating that the function of at least the great majority of mRNAs in all firmicute spores is to be degraded to generate ribonucleotides for new RNA synthesis when spores germinate. IMPORTANCE Only ∼6% of mRNAs in spores of six firmicute species are at ≥1 molecule/spore, many abundant spore mRNAs encode proteins similar to B. subtilis spore proteins, and some abundant B. subtilis and C. difficile spore mRNAs were fragmented. Most of the abundant B. subtilis and other Bacillales spore mRNAs are transcribed under the control of the forespore-specific RNA polymerase σ factors, F or G, and these results may stimulate transcription analyses in developing spores of species other than B. subtilis. These findings, plus the absence of key nucleotide biosynthetic enzymes in spores, suggest that firmicute spores' abundant mRNAs are not translated when spores germinate but instead are degraded to generate ribonucleotides for new RNA synthesis by the germinated spore.

Impact of a probiotic-based cleaning product on the microbiological profile of broiler litters and chicken caeca microbiota.

This study investigated for the first time the decontamination efficacy of a probiotic-based cleaning product containing Bacillus subtilis, Bacillus pumilus, and Bacillus megaterium spores on fresh and reused broiler litters during 3 rearing cycles of 6 wk each. Moreover, the impact of reused litters treated with the cleaning product on the chicken caeca microbiota was assessed at the end of the rearing cycles in comparison to untreated litter. The Bacillus spores provided with the cleaning treatment were able to successfully colonize the reused poultry litters, decreasing the mean counts of total aerobic bacteria, Enterobacteriaceae, and coagulase positive Staphylococci. The decrease of Enterobacteriaceae, mainly represented by the genus Escherichia, was also observed in the caeca of broilers reared on reused litters treated with the cleaning product. Moreover, the treatment retained the caeca content of Ruminococcaceae and Faecalibacterium as well as the level of biodiversity among the bacteria genera colonizing the caeca of animals reared on reused litter. Overall, the results of this study highlight a positive effect of the probiotic-based cleaning strategy on the microbial decontamination of reused litters and on broiler caeca stability, thereby enhancing animal health and prevention of poultry diseases.

Structural and functional analyses of the N-terminal domain of the A subunit of a Bacillus megaterium spore germinant receptor

Germination of Bacillus spores is induced by the interaction of specific nutrient molecules with germinant receptors (GRs) localized in the spore's inner membrane. GRs typically consist of three subunits referred to as A, B, and C, although functions of individual subunits are not known. Here we present the crystal structure of the N-terminal domain (NTD) of the A subunit of the Bacillus megaterium GerK3 GR, revealing two distinct globular subdomains bisected by a cleft, a fold with strong homology to substrate-binding proteins in bacterial ABC transporters. Molecular docking, chemical shift perturbation measurement, and mutagenesis coupled with spore germination analyses support a proposed model that the interface between the two subdomains in the NTD of GR A subunits serves as the germinant binding site and plays a critical role in spore germination. Our findings provide a conceptual framework for understanding the germinant recruitment mechanism by which GRs trigger spore germination.

Paramagnetism in Bacillus spores: Opportunities for novel biotechnological applications.

Spores of Bacillus megaterium, Bacillus cereus, and Bacillus subtilis were found to exhibit intrinsic paramagnetic properties as a result of the accumulation of manganese ions. All three Bacillus species displayed strong yet distinctive magnetic properties arising from differences in manganese quantity and valency. Manganese ions were found to accumulate both within the spore core as well as being associated with the surface of the spore. Bacillus megaterium spores accumulated up to 1 wt.% manganese (II) within, with a further 0.6 wt.% adsorbed onto the surface. At room temperature, Bacillus spores possess average magnetic susceptibilities in the range of 10−6 to 10−5. Three spore‐related biotechnological applications—magnetic sensing, magnetic separation and metal ion adsorption—were assessed subsequently, with the latter two considered as having the most potential for development.

Changes in Bacillus Spore Small Molecules, rRNA, Germination, and Outgrowth after Extended Sublethal Exposure to Various Temperatures: Evidence that Protein Synthesis Is Not Essential for Spore Germination.

rRNAs of dormant spores of Bacillus subtilis were >95% degraded during extended incubation at 50°C, as reported previously (E. Segev, Y. Smith, and S. Ben-Yehuda, Cell 148:139-114, 2012, doi:http://dx.doi.org/10.1016/j.cell.2011.11.059), and this was also true of spores of Bacillus megaterium Incubation of spores of these two species for ∼20 h at 75 to 80°C also resulted in the degradation of all or the great majority of the 23S and 16S rRNAs, although this rRNA degradation was slower than nonenzymatic hydrolysis of purified rRNAs at these temperatures. This rRNA degradation at high temperature generated almost exclusively oligonucleotides with minimal levels of mononucleotides. RNase Y, suggested to be involved in rRNA hydrolysis during B. subtilis spore incubation at 50°C, did not play a role in B. subtilis spore rRNA breakdown at 80°C. Twenty hours of incubation of Bacillus spores at 70°C also decreased the already minimal levels of ATP in dormant spores 10- to 30-fold, to ≤0.01% of the total free adenine nucleotide levels. Spores depleted of rRNA were viable and germinated relatively normally, often even faster than starting spores. Their return to vegetative growth was also similar to that of untreated spores for B. megaterium spores and slower for heat-treated B. subtilis spores; accumulation of rRNA took place only after completion of spore germination. These findings thus strongly suggest that protein synthesis is not essential for Bacillus spore germination.IMPORTANCE A recent report (L. Sinai, A. Rosenberg, Y. Smith, E. Segev, and S. Ben-Yehuda, Mol Cell 57:3486-3495, 2015, doi:http://dx.doi.org/10.1016/j.molcel.2014.12.019) suggested that protein synthesis is essential for early steps in the germination of dormant spores of Bacillus subtilis If true, this would be a paradigm shift in our understanding of spore germination. We now show that essentially all of the rRNA can be eliminated from spores of Bacillus megaterium or B. subtilis, and these rRNA-depleted spores are viable and germinate as well as or better than spores with normal rRNA levels. Thus, protein synthesis is not required in the process of spore germination.

Detection of Bacillus anthracis spores in liquid using phage-based magnetoelastic micro-resonators

This paper presents the results of an investigation to detect Bacillus anthracis spores in liquids using microfabricated magnetoelastic particles (MEP). Free standing, magnetoelastic resonating particles, as small as 500 μm × 100 μm × 4 μm, have been made using microelectronics fabrication techniques. These magnetoelastic resonators feature wireless signal transmission and are suitable for use in air and liquids. Affinity-selected filamentous bacteriophage was employed as the bio-molecular recognition agent and was immobilized onto all surfaces of the resonators. This phage was selected from a landscape phage library specifically for binding B. anthracis spores, the causative agent for the disease anthrax. The biosensors were tested in B. anthracis spore solutions with concentrations from 103 to 108 cfu/ml. A detection limit of 103 cfu/ml, with a sensitivity of 6.5 kHz/decade was observed. Scanning electron microscopy (SEM) micrographs of the tested sensors were used for visual correlation of the sensors’ responses. To investigate specificity, the biosensors were exposed to mixed spore solutions containing B. anthracis Sterne, Bacillus cereus and Bacillus megaterium spore strains. The phage-coated biosensors were proven to have better binding preference to B. anthracis spores than other strains. The study on the binding kinetics have shown that the dissociation constant (Kd) and the binding valency for the sensor in B. anthracis spores solutions are 193 cfu/ml and 2.32.

Effects of Freezing on the Survival of Escherichia coli and Bacillus and Response to UV and Chlorine After Freezing

Escherichia coli (E. coli) and Bacillus megaterium bacteria were frozen at -15 degrees C using a freezer and a spray freezing method. The frozen Bacillus spores were also exposed to UV and free chlorine. An average of 4.7-log inactivation was obtained from the spray ice with 2-day storage time, while the freezer freezing only caused 0.84-log reduction with the same storage time. Significantly higher inactivation levels were observed for the E. coli cells with 2-day storage compared with those without storage. The spray freezing was found more effective in killing the E. coli cells, while more cells were sublethally injured by the freezer freezing. Freezing did not kill the Bacillus megaterium spores, but affected their response to UV and chlorine. Greater inactivation levels were observed at higher free chlorine doses or longer contact time, and the UV fluence-response curve showed initial rapid kill followed by tailing for the frozen spores.

Location of metal ions in Bacillus megaterium spores by high-resolution electron probe x-ray microanalysis

Location of metal ions in Bacillus megaterium spores by high-resolution electron probe x-ray microanalysis

Microfabricated, Wireless, Magnetoelastic Micro-Particles for the Detection of Bacillus Anthracis Spores

ABSTRACTMagnetoelastic resonance biosensors were fabricated by immobilizing a bio-molecular recognition element onto the surface of Fe79B21 magnetoelastic particles (MEP). These sensors can be measured wirelessly and remotely for both in-air and in-liquid bacteria detection. Filamentous bacterio-phage that was selected specifically for the detection of Bacillus anthracis spores was employed as the biomolecular recognition element and immobilized onto the MEPs' surfaces. Attachment of the spores to the sensor surface due to specific phage-spore binding results in a shift in the resonance frequency of the biosensor. Insitu measurement of the resonance frequency of biosensors of 5×100×500 microns were used to determine the sensor response as a function of spore concentrations of 103 to 108 cfu/ml. Specificity of the sensor was evaluated by conducting tests using a mixture of Bacillus anthracis Sterne strain, Bacillus cereus and Bacillus megaterium spores.

A Novel Semiquantitative Fluorescence-Based Multiplex Polymerase Chain Reaction Assay for Rapid Simultaneous Detection of Bacterial and Parasitic Pathogens from Blood

A multiplex polymerase chain reaction assay was developed for the rapid simultaneous detection of category A select bacterial agents (Bacillus anthracis and Yersinia pestis) and parasitic pathogens (Leishmania species) in blood using the Cepheid Smart Cycler platform. B. anthracis (Sterne) and Yersinia. pseudotuberculosis were used in the assay for optimization for B. anthracis and Y. pestis, respectively. The specificity of the target amplicons [protective antigen gene of B. anthracis and rRNA genes of other pathogens or human (internal control)] was evaluated by staining the amplicons with SYBR Green I and determining their individual melting temperatures (T(m)). As a novel approach for pathogen semiquantitation, the Tm peak height of the amplicon was correlated with a known standard curve of pathogen-spiked samples. This assay was able to detect DNA in blood spiked with less than 50 target cells/ml for all of the pathogens. The sensitivity of this assay in blood was 100% for the detection of Leishmania donovani from leishmaniasis patients and B. anthracis (Sterne) from symptomatic mice. The time necessary for performing this assay including sample preparation was less than 1.5 hours, making this a potentially useful method for rapidly diagnosing and monitoring the efficacy of drugs or vaccines in infected individuals.

Photoabsorption study of Bacillus megaterium, DNA and related biological materials in the phosphorus K-shell edge region.

We measured the X-ray transmission spectra of several biologically related samples in the phosphorus K-shell edge absorption region. These include red phosphorus, hydrated sodium phosphate (Na(3)PO(4).12 H(2)O), deoxyribonucleic acid (DNA), adenosine triphosphate (ATP), diolylphosphatidyl choline (DOPC), and Bacillus megaterium spores. Red phosphorus essentially displays an edge-jump. All other spectra are similar in form and energy position: Each is dominated by a narrower, more intense first peak and a broader but less intense second peak. The corresponding K-shell edge absorption thresholds are shifted toward higher energy relative to that for red phosphorus, as expected for increasing degrees of phosphorus oxidation. The B. megaterium spectrum has aspects common to both the phosphate and DNA spectra and is therefore interpreted as a composite of spectra arising from DNA, ribonucleic acid (RNA) and phosphates within the spore. The B. megaterium spore spectrum provides information for resonant radiation damage studies in the phosphorus K-shell edge absorption region by identifying candidate photoexcitations. In addition, the absorption spectra will be useful in X-ray microscopy and macromolecular crystallography studies at the phosphorus K-shell edge.

Target Selection in Designing Pasteurization Processes for Shelf-Stable High-Acid Fruit Products

This study is focused on the search for targets and criteria for the design of pasteurization processes for high-acid shelf-stable fruit products, such as juices, nectars, pastes, purées, concentrates, jams, jellies, etc. First, an overview of pasteurization is presented and then, frequently used targets for pasteurization processes are reviewed. Enzymes naturally present in fruits, in decreasing order of heat resistance, were pectinesterase, peroxidase, and polyphenoloxidase, and they may be used as pasteurization targets. The heat resistance of each enzyme is strongly dependent on its fruit origin. The most heat resistant micro-organisms capable of spoiling high acid fruit products include ascospores of Neosartorya fischeri, Byssochlamys nivea, Talaromyces flavus, Eupenicillium javanicum, and Byssochlamys fulva moulds, as well as bacterial spores of Clostridium butyricum, Bacillus coagulans, and Bacillus megaterium. These micro-organisms, spores, and enzymes were, in general, less heat resistant than the spores of a particular spoilage micro-organism named Alicyclobacillus acidoterrestris, which has been causing problems in the fruit industry. Therefore, the use of Alicyclobacillus acidoterrestris spores as a reference micro-organism in the design of pasteurization processes for high-acid shelf-stable fruit products is suggested.

Bacillus megaterium spore germination is influenced by inoculum size.

The effect of spore density on the germination (time-to-germination, percent germination) of Bacillus megaterium spores on tryptic soy agar was determined using direct microscopic observation. Inoculum size varied from approximately 10(3) to 10(8) cfu ml(-1) in a medium where pH=7 and the sodium chloride concentration was 0.5% w/v. Inoculum size was measured by global inoculum size (the concentration of spores on a microscope slide) and local inoculum size (the number of spores observed in a given microscope field of observation). Both global and local inoculum sizes had a significant effect on time-to-germination (TTG), but only the global inoculum size influenced the percentage germination of the observed spores. These results show that higher concentrations of Bacillus megaterium spores encourage more rapid germination and more spores to germinate, indicating that low spore populations do not behave similarly to high spore populations. A likely explanation for the inoculum size-dependency of germination would be chemical signalling or quorum sensing between Bacillus spores.

Demonstrating the integrity of a large scale microfiltration plant using a Bacillus spore challenge test

For protection against Cryptosporidium and Giardia New Zealand drinking water regulations established a goal of removal of all particles of 1μm and larger. The City of Tauranga, New Zealand, commissioned a 36 ML/d (9.5 mgd) microfiltration (MF) plant, the Joyce Road Water Processing Plant (JRWPP), to meet this stringent reduction goal. The plant consists of ten USF Memcor 90M10C units. This paper details a bacillus spore challenge test conducted on the JRWPP. The objectives were to (1) confirm that the plant could achieve the required 5 log reduction of particles, and (2) evaluate direct and indirect integrity testing methods as means to confirm log-5 particle reduction performance over the life of the plant. The test used Bacillus megaterium spores dosed into the feed water with the plant operated to maximise the challenge on the MF hollow fibre membranes. The results showed (1) the ability of an integral MF plant to reject Bacillus megaterium spores and achieve at least log 5.9 reduction in organisms; (2) Bacillus spores are a good medium for testing as they are robust, easy to handle, cultured in large quantities, and easily identified; (3) the spore challenge test cannot be economically used to monitor full-scale plant performance; (4) particle counting results correlated poorly with bacillus counts in feed or filtrate; and (5) the USF Memcor integrity test methods, which included a pressure hold test and a diffusive airflow test, underestimated spore rejection.

Netropsin suppresses the rise of activity of an intracellular proteolytic system in sporulatingBacillus megaterium

Intracellular catabolism of proteins labeled at the end of the exponential growth proceeded in two phases during sporulation. The first phase was induced by starvation and took place also in cells whose sporulation was inhibited by netropsin. The second phase of degradation, which was triggered at the onset of the irreversible sporulation phase, was inhibited by netropsin. Intracellular proteolytic activity determined in disintegrated cells, i.e., primarily the activity of the cytoplasmic Ca2+-dependent serine proteinase(s) at the first place, was increasing throughout the sporulation process and reached its maximum during the irreversible sporulation phase. Its increase was suppressed by netropsin. Fractionation of the cell sap by HPLC revealed a similar distribution of proteolytic activities in the extract from control and netropsin-inhibited cells. The antibiotic thus probably affected the activation, not the formation of the cytoplasmic serine proteinase(s). Netropsin also inhibited an increase of proteolytic activity in the membrane fraction, probably owing to the presence of two different proteolytic enzymes.

Variability of Steady-State Bacterial Fluorescence with Respect to Growth Conditions

The UV-excited (∼280 nm) fluorescence emission spectra from suspensions of several different species of bacteria are examined to determine to what extent differences in condition of growth change their spectra. This is an important factor if these spectra are considered for use for identification of species. It was found that the spectra were relatively invariant with respect to whether the bacteria were harvested during growth (log phase) or after the medium was depleted (stationary phase). For a given strain of Escherichia coli it was found that the emission spectrum did not change for growth in rich, compared to minimal, medium; however, there was some change for other species. For bacterial spores, the emission spectra were found to be similar for several species. An estimate was made for the quantum yield for Bacillus megaterium spores. The emission spectrum from spores and bacteria was found to change substantially on drying.

Netropsin inhibits the increase of intracellular Ca(2+)-dependent serine proteinase activity in sporulating Bacillus megaterium.

Netropsin suppressed the increase of intracellular proteolytic activity when added to B. megaterium incubated in a sporulation medium. The inhibited enzyme was a Ca(2+)-dependent serine proteinase. Sporulation and protein turnover in later sporulation phases were inhibited as well. Different concentrations of netropsin affected various aspects of protein catabolism differently.

Biochemical defects of outermost layer deficient mutants during sporulation of Bacillus megaterium.

To determine the regulation of morphogenesis of the outermost layer, the thick layer outside the inner coat, of the Bacillus megaterium spore, we isolated 15 outermost layer deficient mutants of B. megaterium using transposon Tn917. Three mutant strains lacked both synthesis of the 48-kDa outermost layer protein and induction of two initial enzymes for galactosamine-6-phosphate polymer synthesis, evidence that these biochemical events are regulated in the cascade system during morphogenesis of the outermost layer.

Perturbation of the heat resistance of bacterial spores by sporulation temperature and ethanol

Spores ofBacillus megaterium, B. subtilis, andB. stearothermophilus, harvested from cultures grown and sporulated at different temperatures or in the presence of ethanol, had different thermal resistance. There was a direct relationship between the sporulation temperature and the spore-killing temperature. The spores were more temperature-sensitive when formed in ethanol-supplemented media. Temperature and ethanol are known to perturb the degree of order within membranes and to alter membrane functions. Thus, alteration of spore membranes is an additional factor in the multifactorial nature of heat resistance. Another interpretation may be that heat shock proteins, known to be induced by heat, are formed during sporulation and may increase the thermostability of the spores.