The kinetics of germination in bacterial spores

The entire absorbance vs. time curve of a sample of germinating bacterial spores can be accurately described by a model which considers that the spores rate of entry into the phase initiation of germination is determined by transitions between three spore states. The first of these transitions is easily identified with the triggering event, while the existence of the intermediate state, and its identification with the triggered spore, can be established from theoretical as well as experimental considerations. The observed sample lag time is seen to arise from the position of the measured event in the single spore in the sequence of indices of germination. Consideration that the single spore may effect the measured change in a complex way over a finite interval of time leads to a mathematical formulation of our model which can describe the germination process whatever the endpoint chosen for its observation.     

Yeast flocculation: factors affecting the measurement of flocculence

The physical meaning of the residual absorbance of a yeast suspension after flocculation and settling has been investigated. Starting with a dispersed suspension, agitation accelerates flocculation by increasing the probability of collision between particles. As flocculation advances, agitation also breaks the flocs. A stationary state is reached when flocculence (tendency to flocculate) is counterpoised by agitation. If the intensity of agitation is maintained constant, the free cell concentration reflects the flocculence, provided the stationary state is reached. The residual absorbance, determined after settling of the flocs, is a measure of the free cell concentration and represents an adequate parameter to characterize yeast flocculence.     

Commitment of bacterial spores to germinate. A measure of the trigger reaction.

The rate of commitment of bacterial spores to germinate after short exposure to L-alanine increases exponentially from the time of addition of L-alanine. This absence of a lag facilitates kinetic analysis and allows the dependence of commitment on temperature and pH to be determined. The pH profile of commitment has been compared with that obtained from measurements of absorbance decreases during germination, and the two profiles exhibit differing pK values. It is suggested that because the decrease in A600 of spore suspensions is a late event in germination, it is an unsuitable parameter for studying germination-triggering reactions. Commitment has been shown to be temperature-dependent, with an optimum at approx. 37 degrees C and an activation energy (mu) of 1.08 X 10(5) J/mol. The data obtained from the present studies have been used to develop a model for the triggering of germination.     

Inhibition of germination ofBacillus cereus T spores by phenylglyoxal

Phenylgloxal at a concentration of 0.6 mM inhibited germination of Bacillus cereus T spores as characterized by a decrease in absorbance, dipicolinic acid and loss in heat resistance in a chemically defined growth and sporulation medium. In a germination medium containing L-alanine and adenosine, phenylglyoxal inhibited decrease in absorbance and affected partial loss of viability. It is postulated that phenylglyoxal interacts with free amino groups of various enzymes or amino compounds present in the spore structure thereby causing the inhibition of germination.     

Studies of glutamate dehydrogenase. Regulation of glutamate dehydrogenase from Candida utilis by a pH and temperature-dependent conformational transition.

Glutamate dehydrogenase from Candida utilis undergoes a reversible conformational transition between an active and an inactive state at low pH AND low temperature. This conformational transition can also be followed by fluorescence measurements. The temperature-dependent equilibrium between the active and the inactive state is characterized by a transition temperature of 10.7 degrees C and a delta H value of 148 kcal/mol (620 kJ/mol). The temperature dependence of the enzymic activity above 15 degrees C yields an activation energy of 15 kcal/mol (63 kJ/mol), a larger value than that for the beef liver enzyme (9 kcal/mol; 38 kJ/mol). In contrast to the yeast enzyme the Arrhenius plot is linear and, therefore, the beef liver enzyme is not transformed into an inactive conformation at low temperatures. Sedimentation analysis shows that the inactivation of the Candida utilis enzyme is not caused by change in the quaternary structure. The pH dependence of the conformational transition at low pH measured by fluorescence change is characterized by a pK value of 7.01 for the enzyme in the absence and of 6.89 for the enzyme in the presence of 2-oxoglutarate with a Hill coefficient of 3.4 in both cases. Similar results are found when the pH dependence of the enzymic activity is analyzed. With the beef liver enzyme the same pK value is obtained but with a Hill coefficient of 1 indicating cooperativity only in the case of the Candida utilis enzyme. The best fit of the pH dependence of the rate constants of the fluorescence changes was obtained with pK values of 7.45 and 6.45 for the active and the inactive state respectively. In this model the lowest time constant which is obtained at the pH of the equilibrium was found to be 0.05 s-1. Preincubation experiments with the substrate 2-oxoglutarate but not with the coenzyme shift the equilibrium to the active conformation. The coenzyme obviously reduces the rate constant of the conformational transition. The sedimentation coefficient (SO20, w) and the molecular weight were found to be 11.0 S and 276 000, respectively. The enzyme molecule is built up by six polypeptide chains each having a molecular weight of 47 000.     

Determination of dipicolinic acid in bacterial spores by derivative spectroscopy

Dipicolinic acid was extracted from approximately 0.1 mg spores or 0.5 ml of sporulating culture with 20 mM HCl for 10 min at 100 degrees C. The suspension was diluted with 5 mM Ca2+, 100 mM Tris, pH 7.6, centrifuged, and the first derivative of the uv absorbance spectrum recorded from 275 nm to 285 nm. DPA concentration was determined from the difference between the maximum at 276.6 nm and the minimum at 280 nm. The use of the difference between two first derivative values removed possible interference from sloping baselines. Turbidity, nucleic acids, and bacteriological media did not interfere. Analysis time for four extracts was 4 min using a spectrophotometer reading at 0.1-nm intervals. Dipicolinate at 0.1 mM gave 0.184 absorbance/nm at 25 degrees C. The coefficient of variation was 1.5%, and the detection limit 1 microM.     

Liquid crystal-type assembly of native cellulose-glucuronoxylans extracted from plant cell wall.

In numerous plant cell walls, the cellulose microfibrils are arranged in a helicoidal pattern which has been considered as an analog to a cholesteric order. Here, we report on the spontaneous helicoidal organization which occurs in acellular conditions from aqueous suspensions of cellulose. The cellulosic mucilage of mature seeds of quince (Cydonia oblonga L) was studied both in situ (pre-release mucilage) and after water extraction and in in vitro re-assembly (prolonged high speed ultracentrifugation, further progressive dehydration and embedding in LR White methacrylate or hydrosoluble melamine resin). The cellulosic component was characterized by the use of cellobiohydrolase (CBH1) bound to colloidal gold, and the glucuronic acid residues of the xylan matrix were characterized by the use of cationised gold. Inside the seeds, the pre-release mucilage is mostly helicoidal, with the occurrence of more or less ordered domains, which indicate a fluid organization relevant to an actual liquid crystal state. Cytochemical tests revealed the tight association between cellulose and glucuronoxylans, the latter constituting a charged coat around each microfibril. Following the hydration of the seed, a cellulosic suspension was extracted in which microfibrils were totally dispersed. The progressive dehydration of the suspension gave rise to concentrated viscous drops. Ultrastructural observations revealed the occurrence of multidomain organization, from non-ordered to cholesteric-like regions, revealing that the mucilage is at the same time crystalline and liquid. This constitutes the first demonstration that liquid crystal type assemblies can arise from crystalline and biological cellulose in aqueous suspension. It strengthens the hypothesis that a transient liquid crystal state must occur during the cellulose ordering. The possible morphogenetic role of the glucuronoxylans in the cholesteric organization of the cellulose is discussed.     

Streptomyces viridochromogenes spore germination initiated by calcium ions.

Initiation of germination of heat-activated Streptomyces viridochromogenes spore occurs in media containing only calcium ions and organic buffer. The calcium-induced initiation of germination was accompanied by a decrease in absorbance of the spore suspension, an increased rate of endogenous metabolism, the loss of spore carbon, and the loss of heat resistance. Calcium amounts to 0.28% of the dry weight of freshly harvested spores. The amount of calcium remained the same after incubation of spores in water after heat activation. The spore content of calcium doubled after incubation in 0.5 mM CaCl2 for 5 min at 4 degrees C and during calcium-induced germination. Nearly all of the calcim appears to be bound to sites external to the spore membrane, since the chelating agents (ethylenedinitrilo) tetraacetic acid and arsenazo III removed virtually all of the calcium ions. The calcium ions must be present during the entire initiation of germination period. Germination ceases after an (ethylenedinitrilo) tetraacetic acid wash and begins again immediately after addition of calcium ions.     

Swelling and contraction of the mitochondrial matrix. I. A structural interpretation of the relationship between light scattering and matrix volume

The amount of light scattered by a mitochondrial suspension depends on matrix volume (Tedeschi, H., and Harris, D.L. (1955) Arch. Biochem. Biophys. 58, 52-67), a correlation which has been extensively exploited for qualitative studies of solute transport across the inner membrane. To obtain reliable, quantitative estimates of solute transport, it is first necessary to characterize the factors determining mitochondrial light scattering. We show that the dependence of absorbance on mitochondrial concentration can be linearized, resulting in an intrinsic light scattering parameter which is independent of the concentration and source of mitochondria. We show that the absorbance osmotic curve is segmentally linear, exhibiting discontinuities which disappear irreversibly following preswelling. In contrast, direct measurements reveal matrix volume to be reversibly and linearly dependent on inverse osmolality. This divergence is a consequence of the fact that the optical technique samples total particle volume, including contributions from folded membranes and trapped medium. These contributions are minimized by structural components, such as intermembrane connections and the outer membrane, which contribute to efficient packaging of the mitochondrion. When these structures are broken, the mitochondrion cannot return to its native state. We observe that the swelling-induced, irreversible transition from efficient packaging to a random packing state begins at a matrix volume of 1.9 microliter/mg and is complete at 3.1 microliter/mg. These findings complicate the interpretation of light scattering results but do not appear to present an insurmountable obstacle to the quantitative application of this technique to transport kinetics.     

Fluorescence of Dyes in Solutions with High Absorbance. Inner Filter Effect Correction

Fluorescence is a proven tool in all fields of knowledge, including biology and medicine. A significant obstacle in its use is the nonlinearity of the dependence of the fluorescence intensity on fluorophore concentration that is caused by the so-called primary inner filter effect. The existing methods for correcting the fluorescence intensity are hard to implement in practice; thus, it is generally considered best to use dilute solutions. We showed that correction must be performed always. Furthermore, high-concentration solutions (high absorbance) are inherent condition in studying of the photophysical properties of fluorescent dyes and the functionally significant interactions of biological macromolecules. We proposed an easy to use method to correct the experimentally recorded total fluorescence intensity and showed that informative component of fluorescence intensity numerically equals to the product of the absorbance and the fluorescence quantum yield of the object. It is shown that if dye molecules do not interact with each other and there is no reabsorption (as for NATA) and spectrofluorimeter provides the proportionality of the detected fluorescence intensity to the part of the absorbed light (that is possible for spectrofluorimeter with horizontal slits) then the dependence of experimentally detected total fluorescence intensity of the dye on its absorbance coincides with the calculated dependence and the correction factor for eliminating the primary inner filter effect can be calculated on the basis of solution absorbance. It was experimentally shown for NATA fluorescence in the wide range of absorbance (at least up to 60). For ATTO-425, which fluorescence and absorption spectra overlap, the elimination of the primary and secondary filter effects and additional spectral analysis allow to conclude that the most probable reason of the deviation of experimentally detected fluorescence intensity dependence on solution absorbance from the calculated dependence is the dye molecules self-quenching, which accompanies resonance radiationless excitation energy transfer.     

Spectroscopic and quantitative analysis of the oxygenated and peroxy states of the purified cytochrome d complex of Escherichia coli

Oxygenated and peroxy states of the cytochrome d complex of Escherichia coli have been proposed as intermediates in the reaction mechanism of this ubiquinol oxidase. In this report, several stable states of the purified enzyme were examined spectroscopically at room temperature. As purified, the cytochrome d complex exists in an oxygenated state characterized by an absorbance band at 650 nm. Removal of oxygen results in loss of absorbance at this wavelength, which is restored upon the return of oxygen. The presence of one oxygen molecule in the oxygenated state was quantified by measuring oxygen released when excess hydrogen peroxide was added to the oxygenated state by passage of argon generates a "partially reduced" state with an absorbance peak at 628 nm, apparently due to reduced cytochrome d. Addition of equimolar hydrogen peroxide to the fully oxidized state produces the peroxy state. This peroxy state is also formed upon addition of excess hydrogen peroxide to the oxygenated state via a stable intermediate termed "peroxy intermediate." It is likely that 1) the oxygenated state consists of one molecule of oxygen bound to reduced heme d, and 2) there are at least two stable states that have bound peroxide at room temperature, the peroxy state and a newly discovered peroxy intermediate.     

Phase transition kinetics of phosphatidic acid bilayers A stopped-flow study of the electrostatically induced transition

The kinetics of the electrostatically induced phase transition of dimyristoyl phosphatidic acid bilayers was followed using the stopped-flow technique. The phase transition was triggered by a fast change in the pH or the magnesium ion concentration and followed by recording the time dependence of the absorbance. When the phase transition was induced by a pH jump the time course of the absorbance could be described by two exponentials, their time constants displaying the for cooperative processes characteristic maximum at the transition midpoint. The time constants are in the 10 and 100 ms range for the H⁺ triggered transition from the fluid to the ordered state. A third slower process shows no appreciable temperature dependence and is probably caused by vesicle aggregation. For the OH^--induced transition fron the ordered to the fluid state the time constants are in the 100 and 1000 ms range. The fluid-ordered transition could also be triggered by addition of magnesium ions. Of the several observed processes only the fastest in the 10–100 ms time range could definitely be assigned to the fluid-ordered transition while the others are due to aggregation phenomena. The experimental data were compared with results obtained from pressure jump experiments and could be interpreted on the basis of theories for non-equilibrium relaxation.     

Physicochemical characterization of bovine retinal arrestin

The native conformation of bovine retinal arrestin has been characterized by a variety of spectroscopic methods. The purified protein gives rise to a near uv absorption band centered at 279 nm which results from the absorbance of its 14 tyrosine and one tryptophan residue. The extinction coefficient for this absorption band was determined to be 38.64 mM-1, cm-1 using the tyrosinate-tyrosine difference spectrum method; this extinction coefficient is ca. 17% lower than the previously reported value, and provides estimates of protein concentration which are in good agreement with estimates from the Bradford colorimetric assay. When native arrestin is purified to homogeneity, it displays a fluorescence spectrum which is dominated by tyrosine emission with no discernible contribution from tryptophan. Observation of the tyrosine-like fluorescence is dependent on the purity and structural integrity of the protein. Denaturation of arrestin by guanidine hydrochloride results in a diminution of tyrosine fluorescence and the concomitant appearance of a second fluorescence maximum at ca. 340 nm, presumably due to the single tryptophan residue. Thermal denaturation of arrestin leads to a conformation characterized by a broad fluorescence band centered at ca. 325 nm. Study of the arrestin fluorescence spectrum as a function of temperature indicates that the thermal denaturation is well modeled as a two-state transition with a transition midpoint of 60 degrees C. Temperature-dependent far uv circular dichroism studies indicate that changes in secondary structure occur coincident with the change in fluorescence. Studies of the temperature dependence of arrestin binding to light-adapted phosphorylated rhodopsin shows a strong correlation between the fluorescence spectral features of arrestin and its ability to bind rhodopsin. These data suggest that the relative intensities of tyrosine and tryptophan fluorescence are sensitive to the structural integrity of the native (i.e., rhodopsin binding) state of arrestin, and can thus serve as useful markers of conformational transitions of this protein. The lack of tryptophan fluorescence for native arrestin suggests an unusual environment for this residue. Possible mechanisms for this tryptophan fluorescence quenching are discussed.     

Kinetics of the helix-coil transition in DNA

The kinetics of the helix-coil transition have been investigated for T2 and T7 phage DNA in a formamide-water-salt mixed solvent using a slow temperature perturbation technique (applicable to kinetic processes with rate constants ? 3 min^?1). In this solvent degradation of the DNA is effectively suppressed. Complex kinetic curves are observed by absorbance and viscosity measurements for the response to denaturing perturbations in the transition region. Analysis of the decay curves indicates that the denaturation reaction in this time range can be treated as a first-order reaction with a variable first-order rate parameter, k, the derivative of the logarithm of the absorbance or viscosity change with respect to time. In the approach to denaturation equilibrium in the transition region, the rate parameter is determined only by the instantaneous extent of denaturation of the molecules. Near equilibrium, the rate parameter assumes a constant value characteristic of the equilibrium state. In this region, where the denaturation reaction proceeds as a simple first-order process, both the decay of absorbance (reflected local conformational change) and the decay of solution viscosity (reflecting macromolecular conformational change) are characterized by the same constant value of k. In 83% formamide, 0.3M Na⁺, the rate parameter k for T2 DNA decreases from an extrapolated value of 2.0 min^?1 at 0% denaturation to 0.11 min^?1 at 90% denaturation. Rate parameters determined for T7 DNA at the same counterion concentration and fraction of denaturation are approximately five times as large as those cited for T2 DNA, indicating an inverse proportionality of rate constant to molecular length. On the other hand, simple first-order kinetic responses with constant k are obtained for renaturing perturbations within the transition, indicating that the mechanism of rewinding differs, in most cases, from that of unwinding. Only in the limit of very small perturbations about a given equilibrium position are the rate constants k obtained from denaturing and renaturing perturbations equal. For perturbations of finite size, it appears possible that an intramolecular initiation or nucleation event may precede rewinding and limit the rate of this reaction. The rate parameters again are approximately inversely proportional to molecular weight. The one exception to the first-power dependence on molecular weight appears when temperature jumps are made upward into the post-transition region. Here the molecular-weight dependence is second power, but complications arising from the different strand-separation properties of T2 and T7 DNA's make interpretation difficult. The previously used model of friction-limited unwinding appears to fit all the observations except for the molecular-weight dependence.     

Germination and peptidoglycan solubilization in Bacillus megaterium spores.

During initiation of Bacillus megaterium QM B1551 spore germination, trichloroacetic acid-soluble, nondialyzable peptidoglycan fragments with an average molecular weight of 20,000 were excreted. This solubilization of peptidoglycan was measured in vitro as the amount of trichloroacetic acid-soluble hexosamine released from a suspension of broken spores. HgC12, a potent inhibitor of initiation, had no effect on the in vitro solubilization of peptidoglycan. In vivo, HgC12 had no effect on peptidoglycan release from spores that had lost heat resistance, but HgC12 did block complete absorbance loss. These results suggest that mercury inhibits some reactions that normally occur before loss in heat resistance but not the subsequent peptidoglycan release, and mercury inhibits other reactions involved with complete absorbance loss.     

Model phospholipid membranes affect the holomyoglobin structure: conformational changes at pH 6.2

The influence of model negatively charged membranes on the sperm whale holomyoglobin structure at pH 6.2 has been investigated by different techniques (far and near UV circular dichroism, tryptophan fluorescence, absorbance at Soret band, differential scanning microcalorimetry and fast performance liquid chromatography). It is shown that the holomyoglobin structure undergoes a conformational transition from the native to intermediate state analogous to its apo-form. This state is characterized by the absence of a rigid tertiary structure and the native heme environment. At the same time, the content of alpha-helical secondary structure remains almost native. To change the holomyoglobin structure similarly to that of its apo-form in the presence of membranes, a higher molar phospholipids/protein ratio is required. The properties of holomyoglobin in the presence of negatively charged membranes resemble those of the molten globule state of its apo-form protein in aqueous solution. A possible functional role of the discovered non-native myoglobin state is discussed.     

The lipoamide dehydrogenase from Mycobacterium tuberculosis permits the direct observation of flavin intermediates in catalysis

Lipoamide dehydrogenase catalyses the NAD(+)-dependent oxidation of the dihydrolipoyl cofactors that are covalently attached to the acyltransferase components of the pyruvate dehydrogenase, alpha-ketoglutarate dehydrogenase, and glycine reductase multienzyme complexes. It contains a tightly, but noncovalently, bound FAD and a redox-active disulfide, which cycle between the oxidized and reduced forms during catalysis. The mechanism of reduction of the Mycobacterium tuberculosis lipoamide dehydrogenase by NADH and [4S-(2)H]-NADH was studied anaerobically at 4 degrees C and pH 7.5 by stopped-flow spectrophotometry. Three phases of enzyme reduction were observed. The first phase, characterized by a decrease in absorbance at 400-500 nm and an increase in absorbance at 550-700 nm, was fast (k(for) = 1260 s(-)(1), k(rev) = 590 s(-)(1)) and represents the formation of FADH(2).NAD(+), an intermediate that has never been observed before in any wild-type lipoamide dehydrogenase. A primary deuterium kinetic isotope effect [(D)(k(for) + k(rev)) approximately 4.2] was observed on this phase. The second phase, characterized by regain of the absorbance at 400-500 nm, loss of the 550-700 nm absorbance, and gain of 500-550 nm absorbance, was slower (k(obs) = 200 s(-)(1)). This phase represents the intramolecular transfer of electrons from FADH(2) to the redox-active disulfide to generate the anaerobically stable two-electron reduced enzyme, EH(2). The third phase, characterized by a decrease in absorbance at 400-550 nm, represents the formation of the four-electron reduced form of the enzyme, EH(4). The observed rate constant for this phase showed a decreasing NADH concentration dependence, and results from the slow (k(for) = 57 s(-)(1), k(rev) = 128 s(-)(1)) isomerization of EH(2) or slow release of NAD(+) before rapid NADH binding and reaction to form EH(4). The mechanism of oxidation of EH(2) by NAD(+) was also investigated under the same conditions. The 530 nm charge-transfer absorbance of EH(2) shifted to 600 nm upon NAD(+) binding in the dead time of mixing of the stopped-flow instrument and represents formation of the EH(2).NAD(+) complex. This was followed by two phases. The first phase (k(obs) = 750 s(-)(1)), characterized by a small decrease in absorbance at 435 and 458 nm, probably represents limited accumulation of FADH(2).NAD(+). The second phase was characterized by an increase in absorbance at 435 and 458 nm and a decrease in absorbance at 530 and 670 nm. The observed rate constant that describes this phase of approximately 115 s(-)(1) probably represents the overall rate of formation of E(ox) and NADH from EH(2) and NAD(+), and is largely determined by the slower rates of the coupled sequence of reactions preceding flavin oxidation.     

Spectrophotometric quantitation of anchorage-dependent cell numbers using extraction of naphthol blue-black-stained cellular protein

A convenient method is described by which the actual or relative number of cells in anchorage culture is determined. After removal of the growth medium, cells are subjected to a double-fixation procedure. The cellular protein content is subsequently quantitatively stained with naphthol blue-black. After a period of removal of unbound stain, dye-protein complexes are hydrolytically released and measured spectrophotometrically at 620 nm. A linear correlation exists (r = 0.994) between cell concentration, in the range 3 X 10(4) to 8 X 10(5) cells/ml of final assay volume, and absorbance up to reading values of 3.8. The technical reproducibility of the assay, as judged from assessments of cell numbers in suspension culture, displays a coefficient of variation of 5%. The method was developed for 9.6-cm2 culture dishes, but it should be possible to transform it for the use of microtiter plates.     

Protein flexibility and conformational state: a comparison of collective vibrational modes of wild-type and D96N bacteriorhodopsin

Far infrared (FIR) spectral measurements of wild-type (WT) and D96N mutant bacteriorhodopsin thin films have been carried out using terahertz time domain spectroscopy as a function of hydration, temperature, and conformational state. The results are compared to calculated spectra generated via normal mode analyses using CHARMM. We find that the FIR absorbance is slowly increasing with frequency and without strong narrow features over the range of 2-60 cm(-1) and up to a resolution of 0.17 cm(-1). The broad absorption shifts in frequency with decreasing temperature as expected with a strongly anharmonic potential and in agreement with neutron inelastic scattering results. Decreasing hydration shifts the absorption to higher frequencies, possibly resulting from decreased coupling mediated by the interior water molecules. Ground-state FIR absorbances have nearly identical frequency dependence, with the mutant having less optical density than the WT. In the M state, the FIR absorbance of the WT increases whereas there is no change for D96N. These results represent the first measurement of FIR absorbance change as a function of conformational state.     

An automated method for rapid determination of diffusion coefficients via measurements of boundary spreading

The use of a simple device by which a layer of solvent may be deposited onto a solution of an optically absorbing solute in a cylindrical quartz tube, without substantial mixing of solution and solvent, is described. The spreading of the boundary thus formed may be monitored as a function of time using an automated absorbance scanning device previously described [A. K. Attri and A. P. Minton (1983) Anal. Biochem. 133, 142-152]. A semiautomatic procedure for determining the diffusion coefficient from the time dependence of the shape of the boundary is described and is particularly well-suited for real-time data analysis with a laboratory microcomputer. The diffusion coefficients of several proteins have been measured using the technique reported, and the results are generally in good agreement with values reported in the literature. The feasibility of using this technique in combination with a previously described method for measuring the sedimentation coefficient [A. K. Attri and A. P. Minton (1984) Anal. Biochem. 136, 407-415] to rapidly determine the molecular weight of a protein is established.