Effect of the internal water activity of bacterial spores on their heat resistance in water

The water contents and effective a_w of the core, cortex, and coat of spores in water, as well as the masses of the core, cortex, and coat plus exosporium in the dry state, are calculated from volumes, dry densities, and water absorption isotherms of the sporal components. From data presented here for spores ofBacillus subtilis var.niger andBacillus cereus T, and from previously published data forBacillus stearothermophilus, the logarithm of the heat resistance of the spores in water is linearly related to the effective a_w of their core and cortex.     

Peptidoglycan synthesis in bacilli: I. Effect of temperature on the in vitro system from Bacillus megaterium and Bacillus stearothermophilus

A study has been made of the cycle of reactions involved in peptidoglycan synthesis in particulate preparations from Bacillus megaterium and Bacillus stearothermophilus and some of the properties of the system are described. The particulate enzyme preparation from B. megaterium is inactivated rapidly at 37° and to a lesser extent at 30°; the most sensitive reaction appears to be the dephosphorylation of C-55 isoprenoid alcohol pyrophosphate. It has not been established whether a thermosensitive enzyme is involved or whether disorganization of a lipid-protein complex takes place. The cycle of reactions in the particles from B. stearothermophilus is more stable to high temperatures; only 25% of the activity in the polymerisation reaction is lost in 2 h at 55°. The activity of a second enzyme, d,d-alanine carboxypeptidase, is unaffected after 3 h at 65°. The stability of this membrane-bound multi-enzyme system from B. stearothermophilus in vitro provides further evidence that the organism can survive at high temperatures without inactivation of its enzyme proteins and the consequent need to replace them.     

Effect of water activity on the mechanical glass transition and dynamical transition of bacteria

The purpose of this study was to clarify the glass-transition behavior of bacteria (Cronobacter sakazakii) as a function of water activity (a_w). From the water sorption isotherm (298 K) for C. sakazakii, monolayer water content and monolayer a_w were determined to be 0.0724 g/g-dry matter and 0.252, respectively. Mechanical relaxation was investigated at 298 K. In a higher a_w range of over 0.529, the degree of mechanical relaxation increased with an increase in a_w. From the effect of a_w on the degree of mechanical relaxation, the mechanical a_wc (a_w at which mechanical glass transition occurs at 298 K) was determined to be 0.667. Mean-square displacement of atoms in the bacteria was investigated by incoherent elastic neutron scattering. The mean-square displacement increased gradually with an increase in temperature depending on the a_w of samples. From the linear fitting, two or three dynamical transition temperatures (low, middle, and high T_ds) were determined at each a_w. The low-T_d values (142–158 K) were almost independent from a_w. There was a minor effect of a_w on the middle T_d (214–234 K) except for the anhydrous sample (261 K). The high T_d (252–322 K) largely increased with the decrease in a_w. From the a_w dependence of the high T_d, the dynamical a_wc was determined to be 0.675, which was almost equivalent to the mechanical a_wc. The high T_d was assumed to be the glass-transition temperature (T_g), and anhydrous T_g was estimated to be 409 K. In addition, molecular relaxation time (τ) of the bacteria was calculated as a function of a_w. From the result, it is suggested that the progress of metabolism in the bacterial system requires a lower τ than approximately 6 × 10^?5 s.     

A study of mixed continuous cultures of sulfate-reducing and methane-producing bacteria

Ecological relationships between sulfate-reducing and methane-producing bacteria in mud of Lake Vechten have been studied by continuous culture studies using the chemostat technique. The maximum specific growth rate (μ _max) and saturation constant (K _s) were, respectively, 0.36 hr⁻¹ and 0.047 mM for lactate-limited growth ofDesulfovibrio desulfuricans and 0,011 hr⁻¹ and 0.17 mM for acetate-limited growth ofMethanobacterium sp. Calculated values for the true molar growth yieldsY _G) and maintenance coefficients (m) were 30.6 g bacterial mass/mole of lactate and 0.53 g substrate/g dry wt hr forD. desulfuricans and 37.8 g bacterial mass/mole of acetate and 0.54 g substrate/g dry wt hr forMethanobacterium. No growth ofMethanobacterium was observed at apS²⁻ value (the hydrogen sulfide potential) of more than 11 and there was no effect on the growth atpS²⁻ values above 13. In mixed continuous culture experiments the concentration of acetate decreased in the secondstage growth vessel, whereas that of methane increased stoichiometrically. If the substrate concentration in the reservoirs (S _r) was increased from 0.1 to 0.5 mg/ml, the population ofDesulfovibrio increased and that ofMethanobacterium was washed out of the culture vessel, since the concentration of hydrogen sulfide reached apS²⁻ value of 10.5. From the mixed continuous culture experiments a commensalism between the two species can be described, i.e., the acetate-fermentingMethanobacterium benefits from the acetate released byDesulfovibrio which is, in turn, not affected in the presence of the former.     

Properties of pullulan-based blend films as affected by alginate content and relative humidity

Pullulan-sodium alginate blend films were prepared and characterized as a function of water activity (a_w). At low a_w, the incorporation of alginate into pullulan film increased the tensile strength and elastic modulus, but decreased the elongation at break of the composite films; the opposite trends were observed at elevated a_w. Above 0.43 a_w, water exerted a typical plasticization effect upon the biopolymer blends. As a_w increased from 0.23 to 0.43, an anti-plasticization effect was observed as tensile strength and elastic modulus increased. The glass transition temperature of all samples decreased substantially as aw increased from 0.23 to 0.84 due to the plasticization effect of water. Within this a_w range, one transition temperature was observed for all film specimens. The stretching vibration band of O-H was investigated using attenuated total reflection Fourier transform infrared spectroscopy to identify the various species of water interacting with the polysaccharide films.     

Influence of oxygen transfer rate on the accumulation of poly(3-hydroxybutyrate) by Bacillus megaterium

Poly(3-hydroxybutyrate)—P(3HB)—is a natural biodegradable polyester synthesized by several bacteria, produced from renewable resources. The effects of oxygen transfer rate on the intracellular accumulation of P(3HB) was evaluated, aiming at increasing P(3HB) synthesized by Bacillus megaterium DSM 32^T in bioreactor batch cultures. Bench-scale bioreactor cultivations were performed under different volumetric oxygen mass transfer coefficients, k_La, setting stirrer speed on specified values. The results of this work show that oxygen transfer is a key factor on P(3HB) accumulation by B. megaterium, increasing the P(3HB) intracellular mass fraction from 39% to 62% of CDW at k_La condition of 0.006 s^?1.     

Phosphorylation of skeletal muscle myosin light chain kinase by the catalytic subunit of cAMP-dependent protein kinase

Phosphatidylethanolamine (PE) was isolated from membranes of Bacillus megaterium. The organism was grown at 20°C and 55°C. The phase equilibria in PE/water systems were studied by ²H and ³¹P nuclear magnetic resonance, and by polarized light microscopy. PE isolated from B. megaterium grown at 20°C forms a lamellar liquid crystalline phase at the growth temperature, and at low water contents a cubic liquid crystalline phase at 58°C. The ratio iso/ante-iso acyl chains was 0.3 in this lipid. PE isolated from this organism grown at 55°C forms only a lamellar liquid crystalline phase up to at least 65°C. In this lipid the ratio iso/ante-iso acyl chains was 3.2.     

Evidence of an association between poly(3-hydroxybutyrate) accumulation and phosphotransbutyrylase expression in<Emphasis Type="Italic"> Bacillus megaterium</Emphasis>

Molecular analysis of a genomic region of Bacillus megaterium, a polyhydroxybutyrate (PHB)-producing microorganism, revealed the presence of a gene coding for the enzyme phosphotransbutyrylase (Ptb). Enzyme activity was measured throughout the different growth phases of B. megaterium and was found to correlate with PHB accumulation during the late-exponential growth phase. Ptb expression was repressed by glucose and activated by the branched amino acids isoleucine and valine. Overexpression of Act_Bm, a ⁵⁴ regulator from B. megaterium whose gene is located upstream from ptb, caused an increase in Ptb activity and PHB accumulation in B. megaterium.     

Survival of Bacteria and Fungi in Relation to Water Activity and the Solvent Properties of Water in Biopolymer Gels

Survival of bacteria (Rhizobium, Agrobacterium, and Arthrobacter spp.), fungal spores (Penicillium sp.), and yeasts (Saccharomyces sp.) was studied in relation to water activity (a_w) and the presence of nutritive solutes. The cells were entrapped in polysaccharide gels, as is done to immobilize cells or enzymes, and then dehydrated. The number of living cells (10¹⁰ g of dry polymer⁻¹) remained constant for periods of storage of >3 years at 28°C when the inocula were kept at an a_w of <0.069. At a_w values between 0.069 and 0.83 the number of survivors diminished more and more rapidly as the a_w was raised. For a given a_w and organism, there were large differences in survival rate as a function of the nutritive solutes used to culture the microorganisms. Low-molecular-weight compounds (with three or five carbon atoms) had a deleterious effect on survival, whereas compounds of higher molecular weight (C₆ to C₁₂) had a protecting effect. Thus, the a_w alone was not a sufficient explanation for the deterioration of the inocula. Survival seemed to be more directly related to some properties of the water in the biopolymer. New concepts such as the discontinuity of properties of water and the point of mobilization of solutes, already proposed by Duckworth and Kelly (J. Food Technol. 8:105-113, 1973) and Seow (J. Sci. Food Agric., 26:535-536, 1975), have been taken into consideration to explain the interactions of water with the biopolymer and their specific effects on the microorganisms.     

Intracellular proteolytic activity during sporulation ofBacillus megaterium

Intracellular proteolytic activity increased during incubation of the sporogenic strain ofBacillus megaterium KM in a sporulation medium together with excretion of an extracellular metalloprotease. The exocellular protease activity in a constant volume of the medium reached a 100-fold value with respeot to the intracellular activity. Maximal values of the activity of both the extracellular and intracellular enzyme were reached after 3 – 5 h of incubation. After 7 h 20 – 50% cells formed refractile spores. The intracellular proteolytic system hydrolyzed denatured proteinsin vitro at a rate up to 150 μg mg_-1 h_-1 and native proteins at a rate up to 70 μg mg^-1 h^-1. Degradation of proteinsin vivo proceeded from the beginning of transfer to the sporulation medium at a constant rate of 40 μg mg^-1 h^-1 and the inactivation of beta-galactosidase at a rate of 70 μg mg^-1 h^-1. The intracellular proteolytic activity was inhibited to 65 – 88% by EDTA, to 23 – 76% by PMSF. Proteolysis of denatured proteins was inhibited both by EDTA and PMSF more pronouncedly than proteolysis of native proteins; 50 – 65% of the activity were localized in protoplasts. Another strain ofBacillus megaterium (J) characterized by a high (up to 90%) and synchronous sporulation activity was found to behave in a similar way, but the rate of protein turnover in this strain was almost twice as high. The asporogenic strain ofBacillus megaterium KM synthesized the exocellular protease in the sporulation medium, but its protein turnover was found to decrease substantially after 3 – 4 h. The intraeellular proteolytic system of the sporogenic strain J and the asporogenic strain KM were also inhibited by EDTA and PMSF.     

Geobacillus stearothermophilus V ubiE gene product is involved in the evolution of dimethyl telluride in Escherichia coli K-12 cultures amended with potassium tellurate but not with potassium tellurite

A 3.8-kb fragment of chromosomal DNA of Geobacillus stearothermophilus V cloned in pSP72 (p1VH) confers resistance to potassium tellurite (K₂TeO₃) and to potassium tellurate (K₂TeO₄) when the encoded genes are expressed in Escherichia coli K-12. The nt sequence of the cloned fragment predicts three ORFs of 780, 399, and 600 bp, whose encoded protein products exhibit about 80% similarity with the SUMT methyltransferase and the BtuR protein of Bacillus megaterium, and with the UbiE methyltransferase of Bacillus anthracis A2012, respectively. In addition, E. coli/p1VH cells evolved dimethyl telluride, which was released into the headspace gas above liquid cultures when amended with K₂TeO₃ or with K₂TeO₄. After 48 h of growth in the presence of these compounds, a protein of about 25 kDa was found at a significantly higher level when crude extracts were analyzed by SDS-PAGE. The N-terminal amino acid (aa) sequence of this protein, obtained by Edman degradation, matched the deduced aa sequence predicted by the G. stearothermophilus V ubiE gene. This gene was amplified by PCR, subcloned in pET21b, and transformed into E. coli JM109(DE3). Interestingly, DMTe evolution occurred when these modified cells were grown in K₂TeO₄ – but not in K₂TeO₃ – amended media. These results may be indicative that the two Te oxyanions could be detoxified in the cell by different metabolic pathways.     

Enhanced oxygen transfer rates in fermentation using soybean oil-in-water dispersions

Summary The effect of soybean oil on the volumetric oxygen transfer coefficient during the cultivation ofAerobacter aerogenes cells is presented. For our aeration-agitation conditions (0.278 vvm and 500 rpm), it has been demonstrated that the use 19% (v/v) of soybean oil enabled a 1.85-fold increase of thek _l a coefficient (calculated on a per liter aqueous phase basis). For smaller volumetric oil fractions,k _L a increased linearly with the oil loading. Because of the oxygen-vector properties of soybean oil, this oil is able to significantly increase thek _L a of a bioreactor.Nomenclature C^*, C saturation and actual dissolved oxygen concentrations respectively (g/m³) - K_La volumetric oxygen transfer coefficient (h^–1) - K_La_initial k _La measured before the oil addition (h^–1) - M_O2 molar mass of oxygen (dalton) - N oxygen transfer rate (g/m³. h) - P_O2. P_N2 partial pressures ofO ₂ andN ₂ in the gas (atm) - P_H2OT partial pressure of water in air at the temperatureT (atm) - P_T total pressure (atm) - Q₀ volumetric flow rate of outlet air before seeding (m³/h) - Sp spreading coefficient (dynes/cm) - T absolute temperature of outlet gas (K) - V_i volume of the liquidi in the fermentor (m³) - V_M molar volume at 273 K and 1 atm (m³/mole) - _ij interfacial tension betweeni andj componants (dynes/cm) - _v volumetric fraction of the oil (v/v) - G gas - O oil - W water - i inlet - o outlet     

Biolytic extraction of poly(3-hydroxybutyrate) from Bacillus megaterium Ti3 using the lytic enzyme of Streptomyces albus Tia1

The applicability of Streptomyces sp. cell lytic enzymes for devising a simple and competent biological polyhydroxyalkanoate (PHA) recovery approach from Bacillus megaterium cells was investigated. B. megaterium strain Ti3 produced 50% (w/w) PHA using glucose as carbon source. The intracellular PHA was recovered employing a non-PHA accumulating actinomycetes (Tia1) identified as Streptomyces albus, having potent lytic activity against living and heat inactivated B. megaterium. Interestingly, maximum biomass (2.53?±?0.6?g/L by 24?h) of the lytic actinomycete was obtained in PHA production medium itself thus circumventing the prior actinomycete acclimatization just by co-inoculation with B. megaterium as an inducer. Maximum lytic activity was observed at pH 6.0, 40?°C, 220?mg of biomass and 33.3?mL of concentrated culture filtrate in a 100?mL reaction mixture. Preliminary biochemical investigations confirmed the proteolytic and caseinolytic nature of the lytic enzyme. PHA yield of 0.55?g/g by co-inoculation extraction approach was comparable with the conventional sodium hypochlorite based extraction method. Interestingly, S. albus also demonstrated a broad spectrum lytic potential against varied Gram-negative and Gram-positive PHA producers highlighting the extensive applicability of this biolytic PHA recovery approach. The lytic enzyme retained almost 100% relative activity on storage at ?20?°C upto two months. ¹H Nuclear magnetic resonance analysis of the extracted polymer confirmed it as a homopolymer composed of 3-hydroxybutyrate monomeric units. This is the first report on Streptomyces sp. based biological and eco-friendly, intracellular PHA recovery from Bacillus spp.     

A novel oligonucleotide cassette for the overproduction ofEscherichia coli aspartate transcarbamylase inBacillus stearothermophilus

Summary A novel double-stranded oligonucleotide cassette was devised in order to express heterologous genes inBacillus stearothermophilus. By linking the cassette to the upstream ofEscherichia coli aspartate transcarbamylase gene, this enzyme was overexpressed inB. stearothermophilus cells.     

Hydration Potential of Lysozyme: Protein Dehydration Using a Single Microparticle Technique

For biological molecules in aqueous solution, the hydration pressure as a function of distance from the molecular surface represents a very short-range repulsive pressure that limits atom-atom contact, opposing the attractive van der Waals pressure. Whereas the separation distance for molecules that easily arrange into ordered arrays (e.g., lipids, DNA, collagen fibers) can be determined from x-ray diffraction, many globular proteins are not as easily structured. Using a new micropipette technique, spherical, glassified protein microbeads can be made that allow determination of protein hydration as a function of the water activity (a_w) in a surrounding medium (decanol). By adjusting a_w of the dehydration medium, the final protein concentration of the solid microbead is controlled, and ranges from 700 to 1150 mg/mL. By controlling a_w (and thus the osmotic pressure) around lysozyme, the repulsive pressure was determined as a function of distance between each globular, ellipsoid protein. For separation distances, d, between 2.5 and 9 Å, the repulsive decay length was 1.7 Å and the pressure extrapolated to d = 0 was 2.2 × 10⁸ N/m², indicating that the hydration pressure for lysozyme is similar to other biological interfaces such as phospholipid bilayers.     

Sporicidal activity of tertiary butyl hydroperoxide

Tertiary butyl hydroperoxide (t-BOOH) was found to be sporicidal for Bacillus megaterium ATCC19213. Sporicidal action was very temperature dependent, and the potency of t-BOOH increased about tenfold for each increase in temperature of 15 °C over the range from 30° to 70 °C. At still higher temperatures, heat and molar levels of t-BOOH were mutually potentiating for killing. Vegetative cells and germinated spores were some thousand times less resistant to t-BOOH than dormant spores. The order of resistance for spores was: Bacillus stearothermophilus ATCC7953 > Bacillus subtilis var. niger = Bacillus megaterium ATCC33729 > Bacillus megaterium ATCC19213. Killing was not enhanced by decoating and occurred without germination or loss of refractility of the spores. Spore resistance to t-BOOH was lower at more acid pH values and was decreased also by demineralization. Spores could be protected by the chelator o-phenanthroline, especially in association with Fe²⁺. Overall, it seemed that killing was associated with nonmetabolic formation of alkyl peroxyl radicals, which are thought to be responsible for killing of vegetative cells by organic hydroperoxides.Abbreviation A-BOOH tertiary butyl hydroperoxide     

Studies on rDNA from the extreme thermophilic eubacterium Thermus thermophilus HB8: The 23 S rDNA region D

23 S ribosomal ribonucleic acid gene from the extreme thermophile eubacterium Thermus thermophilus HB8 has been cloned in pBR322, and the nucleotide sequence of region D has been determined, which encompasses 873 nucleotides at the 3′-end of the RNA. We compare the primary and secondary structure of this region with the respective part of the 23 S rRNA from Escherichia coli and Bacillus stearothermophilus. A high level of structural conservation can be observed, throughout the RNA domain, albeit the usage of G/C basepairs is substantial even in comparison with another thermophilic eubacterium B. stearothermophilus. It is surprising that, in contrast to the usage of ^3′U-G^5′, the occurrence of ^3′G-U^5′ is comparable in E. coli as well as in B. stearothermophilus and T. thermophilus. Furthermore, it is most remarkable that the use of ^3′A-U^5′ and ^3′U-A^5′ is, compared to E. coli, only slightly reduced in B. stearothermophilus, but drastically decreased in T. thermophilus.     

Effect of dioxane on the structure and hydration–dehydration of α-chymotrypsin as measured by FTIR spectroscopy

A new experimental approach based on FTIR spectroscopic measurements was proposed to study simultaneously the adsorption/desorption of water and organic solvent on solid enzyme and corresponding changes in the enzyme secondary structure in the water activity range from 0 to 1.0 at 25 °C. The effect of dioxane on the hydration/dehydration and structure of bovine pancreatic α-chymotrypsin (CT) was characterized by means of this approach. Dioxane sorption exhibits pronounced hysteresis. No sorbed dioxane was observed at low water activities (a_w < 0.5) during hydration. At a_w about 0.5, a sharp increase in the amount of sorbed dioxane was observed. Dioxane sorption isotherm obtained during dehydration resembles a smooth curve. In this case, CT binds about 150 mol dioxane/mol enzyme at the lowest water activities. Three different effects of dioxane on the water binding by the initially dried CT were observed. At a_w < 0.5, water adsorption is similar in the presence and absence of dioxane. It was concluded that the presence of dioxane has little effect on the interaction between enzyme and tightly bound water at low a_w. At a_w > 0.5, dioxane increases the amount of water bound by CT during hydration. This behavior was interpreted as a dioxane-assisted effect on water binding. Upon dehydration at low water activities, dioxane decreases the water content at a given a_w. This behavior suggests that the suppression in the uptake of water during dehydration may be due to a competition for water-binding sites on chymotrypsin by dioxane. Changes in the secondary structure of CT were determined from infrared spectra by analyzing the structure of amide I band. Dioxane induced a strong band at 1628 cm^?1 that was assigned to the intermolecular β-sheet aggregation. Changes in the intensity of the 1628 cm^?1 band agree well with changes in the dioxane sorption by CT. An explanation of the dioxane effect on the CT hydration and structure was provided on the basis of hypothesis on water-assisted disruption of polar contacts in the solid enzyme. The reported results demonstrate that the hydration and structure of α-chymotrypsin depend markedly on how enzyme has been hydrated — whether in the presence or in the absence of organic solvent. A qualitative model was proposed to classify the effect of hydration history on the enzyme activity-a_w profiles.     

Increased xerotolerance of Saccharomyces cerevisiae during an osmotic pressure ramp over several generations

Although mechanisms involved in response of Saccharomyces cerevisiae to osmotic challenge are well described for low and sudden stresses, little is known about how cells respond to a gradual increase of the osmotic pressure (reduced water activity; a_w) over several generations as it could encounter during drying in nature or in food processes. Using glycerol as a stressor, we propagated S. cerevisiae through a ramp of the osmotic pressure (up to high molar concentrations to achieve testing-to-destruction) at the rate of 1.5 MPa day^-1 from 1.38 to 58.5 MPa (0.990–0.635 a_w). Cultivability (measured at 1.38 MPa and at the harvest osmotic pressure) and glucose consumption compared with the corresponding sudden stress showed that yeasts were able to grow until about 10.5 MPa (0.926 a_w) and to survive until about 58.5 MPa, whereas glucose consumption occurred until 13.5 MPa (about 0.915 a_w). Nevertheless, the ramp conferred an advantage since yeasts harvested at 10.5 and 34.5 MPa (0.778 a_w) showed a greater cultivability than glycerol-shocked cells after a subsequent shock at 200 MPa (0.234 a_w) for 2 days. FTIR analysis revealed structural changes in wall and proteins in the range 1.38–10.5 MPa, which would be likely to be involved in the resistance at extreme osmotic pressure.     

Identification andin silico characterisation of putative conjugative transfer genes onGeobacillus stearothermophilus plasmids

We repor the first data demonstrating the presence of putative conjugative transfer genes on plasmids of the speciesGeobacillus stearothermophilus. Partial sequence analysis of the plasmid pGS18 fromG. stearothermophilus 18 was determined. It contained eleven complete open reading frames. Five of them encoded proteins which are homologous toBacillus megaterium pBM300 Mob/TraA,Lactococcus lactis pMRC01 TrsD and TrsE,Staphylococcus aureus pGO1 TrsG andS. aureus subsp.aureus pUSA03 TraL, the proteins that are associated with conjugative plasmid transfer. Southern hybridizations were performed on two other plasmids isolated fromG. stearothermophilus 3 andG. stearothermophilus 19 strains using the most homologous parts of those five genes as probes. Data from different hybridization patterns show a close homology of putative conjugative transfer genes between pGS18 and pGS3 hypothesizing a similar molecular organization of putative conjugative plasmid transfer region of both plasmids.