Effect of divalent metal ions on collagenase from Clostridium histolyticum.

The collagenase from Clostridium histolyticum (EC 3.4.24.3) degrades type IV collagen with Km 32 nM, indicating a high affinity for this substrate. Ferrous and ferric ions can inhibit Clostridium collagenase. Inhibition by Fe++ was of the mixed, non-competitive type, with Ki 90 microM. The inhibitory effect of Fe++ may be due to Zn++ displacement from the intrinsic functional center of this metalloprotease, since in the presence of excess amounts of Zn++ enzyme activity is retained. This inhibitory effect of Fe++ may be common for all types of collagenases, since this ion can also inhibit type IV collagenase purified from Walker 256 carcinoma, with IC50 80 microM. Cu++ can only partially inhibit Clostridium collagenase, while other divalent metal ions such as Cd++, Co++, Hg++, Mg++, Ni++ or Zn++ are devoid of any inhibitory effect on the enzyme.     

Characterization of Clostridium perfringens spores that lack SpoVA proteins and dipicolinic acid

Spores of Clostridium perfringens possess high heat resistance, and when these spores germinate and return to active growth, they can cause gastrointestinal disease. Work with Bacillus subtilis has shown that the spore's dipicolinic acid (DPA) level can markedly influence both spore germination and resistance and that the proteins encoded by the spoVA operon are essential for DPA uptake by the developing spore during sporulation. We now find that proteins encoded by the spoVA operon are also essential for the uptake of Ca(2+) and DPA into the developing spore during C. perfringens sporulation. Spores of a spoVA mutant had little, if any, Ca(2+) and DPA, and their core water content was approximately twofold higher than that of wild-type spores. These DPA-less spores did not germinate spontaneously, as DPA-less B. subtilis spores do. Indeed, wild-type and spoVA C. perfringens spores germinated similarly with a mixture of l-asparagine and KCl (AK), KCl alone, or a 1:1 chelate of Ca(2+) and DPA (Ca-DPA). However, the viability of C. perfringens spoVA spores was 20-fold lower than the viability of wild-type spores. Decoated wild-type and spoVA spores exhibited little, if any, germination with AK, KCl, or exogenous Ca-DPA, and their colony-forming efficiency was 10(3)- to 10(4)-fold lower than that of intact spores. However, lysozyme treatment rescued these decoated spores. Although the levels of DNA-protective alpha/beta-type, small, acid-soluble spore proteins in spoVA spores were similar to those in wild-type spores, spoVA spores exhibited markedly lower resistance to moist heat, formaldehyde, HCl, hydrogen peroxide, nitrous acid, and UV radiation than wild-type spores did. In sum, these results suggest the following. (i) SpoVA proteins are essential for Ca-DPA uptake by developing spores during C. perfringens sporulation. (ii) SpoVA proteins and Ca-DPA release are not required for C. perfringens spore germination. (iii) A low spore core water content is essential for full resistance of C. perfringens spores to moist heat, UV radiation, and chemicals.     

The effect of citric acid on growth of proteolytic strains of Clostridium botulinum

In strictly anaerobic conditions in a culture medium adjusted to pH 5.2 with HCl and incubated at 30 degrees C, inocula containing less than 10 vegetative bacteria of Clostridium botulinum ZK3 (type A) multiplied to give greater than 10(8) bacteria per ml in 3 d. Growth from an inoculum of between 10 and 100 spores occurred after a delay of 10-20 weeks. Citric acid concentrations of 10-50 mmol/l at pH 5.2 inhibited growth from both vegetative bacteria and spore inocula, a concentration of 50 mmol/l increasing the number of vegetative bacteria or of spores required to produce growth by a factor of approximately 10(6). The citric acid also reduced the concentration of free Ca2+ in the medium. The inhibitory effect of citric acid on vegetative bacteria at pH 5.2 could be prevented by the addition of Ca2+ or Mg2+ and greatly reduced by Fe2+ and Mn2+. The addition of Ca2+, but not of the remaining divalent metal ions, restored the concentration of free Ca2+ in the medium to that in the citrate-free medium. The inhibitory effect of citric acid on growth from a spore inoculum was only partially prevented by Ca2+. Citric acid (50 mmol/l) did not inhibit growth of strain ZK3 at pH 6 despite the greater chelating activity of citrate at pH 6 than at pH 5.2. The effect of citric acid and Ca2+ at pH 5.2 on vegetative bacteria of strains VL1 (type A) and 2346 and B6 (proteolytic type B) was similar to that on strain ZK3.     

Zinc stimulates sporulation inClostridium botulinum 113B

The presence of 0.5–1.0 mM zinc (Zn) in a complex sporulation medium stimulated spore formation in certain strains ofClostridium botulinum. Zinc increased both the titer of free refractile spores (spores per liter) and the percentage conversion of vegetative cells to spores. Certain other transition metals including iron (Fe) and manganese (Mn) also improved sporulation, but not so effectively as zinc. Sporulation was drastically decreased by the addition to the medium of 0.5–1.0 mM copper (Cu). Copper was shown to compete with the acquisition of zinc by the sporulating cells. Spores were separated from their progenitor vegetative cells to 98% homogeneity by incorporation of a density-separation step in the extensive washing procedure. Analysis of the metal contents of the purified spores showed that zinc levels in spores were reduced considerably in culture media containing excess copper. The results imply that either the availability of zinc or the limitation of copper stimulates sporulation inC. botulinum. In addition toC. botulinum 113B, zinc also increased sporulation in several type A, B, and E strains and one proteolytic type F strain ofC. botulinum.     

Surface density of calcium ions and calcium spikes in the barnacle muscle fiber membrane

The effects of various divalent cations in the external solution upon the Ca spike of the barnacle muscle fiber membrane were studied using intracellular recording and polarizing techniques. Analysis of the maximum rate of rise of the spike potential indicates that different species of divalent cations bind the same membrane sites competitively with different dissociation constants. The overshoot of the spike potential is determined by the density of Ca (Sr) ions in the membrane sites while the threshold membrane potential for spike initiation depends on the total density of divalent cations. The order of binding among different divalent and trivalent cations is the following: La+++, UO2++ > Zn++, Co++, Fe++ > Mn++ > Ni++ > Ca++ > Mg++, Sr++.     

Time of Enterotoxin Formation and Release During Sporulation of Clostridium perfringens Type A

Clostridium perfringens enterotoxin was detected intracellularly about 3 hr after the inoculation of vegetative cells into sporulation medium. The subsequent increase in intracellular enterotoxin concentration roughly paralleled but followed by 2.5 to 5 hr the increase in number of heat-resistant spores. The increase in biologically active toxin coincided with the increase in enterotoxin antigen. Enterotoxin was released from the sporangium with its lysis, concomitantly with the mature spore release.     

Rat ovarian and adrenal prolactin receptors. Sizes and effects of divalent metal ions

Receptor fractions were prepared from follicle-rich ovaries (for FSH), luteal cell-rich ovaries (for LH and PRL), and adrenals (for PRL) of rats. Divalent metal ions, Mg++, Ca++, and Mn++ showed inhibitory effects on the binding of LH and FSH to their receptors. The binding of the former was more sensitive to these ions than the latter. On the other hand they showed bell-shaped promotive effects on PRL-ovarian receptor binding, the maximal effects being observed at 10-20 mM. Besides these ions, Ba++ also had a promotive effect, while other divalent metal ions such as Zn++, Cd++, Ni++, and Co++ showed inhibitory effects on PRL-ovarian receptor binding at 5 mM. Mg++ and Ca++ also promoted PRL-adrenal receptor binding, while Mn++ promoted the binding at 10 mM but inhibited it at higher concentrations. Association constant (Ka) and binding capacity (Bmax) of PRL receptors of the ovary and the adrenal were significantly different (ovary: Ka = 0.69 X 10(10) M-1, Bmax = 62 fmol/mg protein, adrenal: Ka = 0.21 X 10(10) M-1, Bmax = 99 fmol/mg protein). Ka of the ovarian PRL receptor was not influenced by these divalent ions, while that of the adrenal receptor was doubled by Ca and Mn ions, Bmax of the latter was also increased. A cooperative effect of Mg and Ca ions was observed on Ka and Bmax of the adrenal receptor. The sizes of the PRL binding sites of these organs revealed by affinity labelling were 17K and 40K in the ovary, and 40K and 110K in the adrenal. These results indicate the different properties of receptors in these different target organs.     

Inhibition of Clostridium perfringens sporulation by Bacteroides fragilis and short-chain fatty acids

The effect a common fecal organism, Bacteroides fragilis, has on the sporulation of Clostridium perfringens, an organism linked to some cases of antibiotic associated diarrhea, was examined. Established B. fragilis cultures significantly decreased the number of heat resistant spores formed by C. perfringens ATCC 12915 and increased the number of vegetative cells. To determine if short-chain fatty acids (SCFA), fermentation products of B. fragilis, inhibited sporulation, the SCFA were added to sporulation broth. Sporulation decreased in the presence of acetate, isobutyrate, isovalerate, and succinate. Vegetative cell number for C. perfringens decreased in the cultures with isobutyrate. Propionate did not affect sporulation or vegetative cell number. The data support the hypothesis that the decrease in short-chain fatty acid concentration following antibiotic therapy predisposes patients to diarrheas caused by C. perfringens.     

Influence of transition metals added during sporulation on heat resistance of Clostridium botulinum 113B spores

Sporulation of Clostridium botulinum 113B in a complex medium supplemented with certain transition metals (Fe, Mn, Cu, or Zn) at 0.01 to 1.0 mM gave spores that were increased two to sevenfold in their contents of the added metals. The contents of calcium, magnesium, and other metals in the purified spores were relatively unchanged. Inclusion of sodium citrate (3 g/liter) in the medium enhanced metal accumulation and gave consistency in the transition metal contents of independent spore crops. In citrate-supplemented media, C. botulinum formed spores with very high contents of Zn (approximately 1% of the dry weight). Spores containing an increased content of Fe (0.1 to 0.2%) were more susceptible to thermal killing than were native spores or spores containing increased Zn or Mn. The spores formed with added Fe or Cu also appeared less able to repair heat-induced injuries than the spores with added Mn or Zn. Fe-increased spores appeared to germinate and outgrow at a higher frequency than did native and Mn-increased spores. This study shows that C. botulinum spores can be sensitized to increased thermal destruction by incorporation of Fe in the spores.     

Influence of transition metals added during sporulation on heat resistance of Clostridium botulinum 113B spores.

Sporulation of Clostridium botulinum 113B in a complex medium supplemented with certain transition metals (Fe, Mn, Cu, or Zn) at 0.01 to 1.0 mM gave spores that were increased two to sevenfold in their contents of the added metals. The contents of calcium, magnesium, and other metals in the purified spores were relatively unchanged. Inclusion of sodium citrate (3 g/liter) in the medium enhanced metal accumulation and gave consistency in the transition metal contents of independent spore crops. In citrate-supplemented media, C. botulinum formed spores with very high contents of Zn (approximately 1% of the dry weight). Spores containing an increased content of Fe (0.1 to 0.2%) were more susceptible to thermal killing than were native spores or spores containing increased Zn or Mn. The spores formed with added Fe or Cu also appeared less able to repair heat-induced injuries than the spores with added Mn or Zn. Fe-increased spores appeared to germinate and outgrow at a higher frequency than did native and Mn-increased spores. This study shows that C. botulinum spores can be sensitized to increased thermal destruction by incorporation of Fe in the spores.     

Effect of sporulation and recovery medium on the heat resistance and amount of injury of spores from spoilage bacilli

AIMS: To assess the influence of sporulation media on heat resistance, and the use of stress recovery media to measure preservation injury of spores of five representative spoilage bacilli. METHODS AND RESULTS: Bacillus spores prepared on nutrient agar supplemented with Ca2+, Mg2+, Mn2+, Fe2+ and K+ were more heat-resistant than spores obtained from nutrient agar with Mn2+. This increased heat resistance correlated with a decrease in the protoplast water content as determined by buoyant density sedimentation. The degree of preservation injury severity could be assessed on media containing NaCl at moderate pH and organic acids at acid pH. Ca-DPA, K+ or proline were added to the recovery media to demonstrate that heat probably caused injury to both spore germination and the outgrowth system. SIGNIFICANCE AND IMPACT OF THE STUDY: The metal content of sporulation media can strongly effect the validity of preservation resistance studies. The distinctive recovery media developed here can be relevant for assessing and comparing new preservation technologies.     

Roles of DacB and spm proteins in clostridium perfringens spore resistance to moist heat, chemicals, and UV radiation

Clostridium perfringens food poisoning is caused mainly by enterotoxigenic type A isolates that typically possess high spore heat resistance. Previous studies have shown that alpha/beta-type small, acid-soluble proteins (SASP) play a major role in the resistance of Bacillus subtilis and C. perfringens spores to moist heat, UV radiation, and some chemicals. Additional major factors in B. subtilis spore resistance are the spore's core water content and cortex peptidoglycan (PG) structure, with the latter properties modulated by the spm and dacB gene products and the sporulation temperature. In the current work, we have shown that the spm and dacB genes are expressed only during C. perfringens sporulation and have examined the effects of spm and dacB mutations and sporulation temperature on spore core water content and spore resistance to moist heat, UV radiation, and a number of chemicals. The results of these analyses indicate that for C. perfringens SM101 (i) core water content and, probably, cortex PG structure have little if any role in spore resistance to UV and formaldehyde, presumably because these spores' DNA is saturated with alpha/beta-type SASP; (ii) spore resistance to moist heat and nitrous acid is determined to a large extent by core water content and, probably, cortex structure; (iii) core water content and cortex PG cross-linking play little or no role in spore resistance to hydrogen peroxide; (iv) spore core water content decreases with higher sporulation temperatures, resulting in spores that are more resistant to moist heat; and (v) factors in addition to SpmAB, DacB, and sporulation temperature play roles in determining spore core water content and thus, spore resistance to moist heat.     

Influence of carbohydrates on growth and sporulation of Clostridium perfringens in a defined medium with or without guanosine

Clostridium perfringens strains NCTC 8238, NCTC 8798, NCTC 8679, 8-6, FD-1, and PS52 formed high levels of heat-resistant spores in a defined medium (D) with various sugars as energy sources. Strain PS49 formed high levels of heat-resistant spores when grown with dextrin and methylxanthines. The experiments showed the possibility of carrying out experiments on the sporulation of certain C. perfringens strains in a completely defined medium, without using the ill-defined polysaccharide dextrin. The addition of guanosine and sucrose to D medium generally suppressed sporulation in most strains and made it possible to prepare overnight cultures consisting mainly of vegetative cells. These cultures could be used to inoculate D medium directly, eliminating both the need to wash cells and the lag which normally occurs when cells have been grown in a different medium. Except for strains PS52 and NCTC 8238, guanosine generally increased growth rates and reduced sporulation for all strains when grown on simple sugars. Methylxanthines decreased growth rates and increased sporulation of NCTC 8679 and PS49 when present in D medium with dextrin. In the absence of guanosine, strains NCTC 8798 and 8-6 grew much slower on glucose than on disaccharides. Strain PS52 grew on lactose only after a prolonged lag. For strains requiring dextrin for good sporulation, a commercial dextrin (Difco Laboratories) was found to be readily filter sterilized, making it possible to prepare large amounts of media for use in the production of spores (or enterotoxin).     

Influence of carbohydrates on growth and sporulation of Clostridium perfringens in a defined medium with or without guanosine.

Clostridium perfringens strains NCTC 8238, NCTC 8798, NCTC 8679, 8-6, FD-1, and PS52 formed high levels of heat-resistant spores in a defined medium (D) with various sugars as energy sources. Strain PS49 formed high levels of heat-resistant spores when grown with dextrin and methylxanthines. The experiments showed the possibility of carrying out experiments on the sporulation of certain C. perfringens strains in a completely defined medium, without using the ill-defined polysaccharide dextrin. The addition of guanosine and sucrose to D medium generally suppressed sporulation in most strains and made it possible to prepare overnight cultures consisting mainly of vegetative cells. These cultures could be used to inoculate D medium directly, eliminating both the need to wash cells and the lag which normally occurs when cells have been grown in a different medium. Except for strains PS52 and NCTC 8238, guanosine generally increased growth rates and reduced sporulation for all strains when grown on simple sugars. Methylxanthines decreased growth rates and increased sporulation of NCTC 8679 and PS49 when present in D medium with dextrin. In the absence of guanosine, strains NCTC 8798 and 8-6 grew much slower on glucose than on disaccharides. Strain PS52 grew on lactose only after a prolonged lag. For strains requiring dextrin for good sporulation, a commercial dextrin (Difco Laboratories) was found to be readily filter sterilized, making it possible to prepare large amounts of media for use in the production of spores (or enterotoxin).     

Cationic metal-specific structures adopted by the poly(dG) region and the direct repeats in the chicken adult beta A globin gene promoter.

Naturally occurring contiguous deoxyguanine residues and their surrounding sequences in the chicken adult beta A globin gene promoter were analyzed for their inherent potential to adopt non-B DNA structures in supercoiled plasmid DNA. In particular, cationic effects on structure were studied by treating the supercoiled plasmid DNA harboring the chicken adult beta A globin 5' flanking sequence with an unpaired DNA base-specific probe, chloroacetaldehyde in the presence of either Mg++, Cu++, Zn++, Ca++ or Co++ ions. The chloroacetaldehyde-reactive bases were mapped at a single base resolution by a chemical cleavage method that specifically cleaves DNA at the chloroacetaldehyde modified sites. These experiments revealed that while Mg++ and Ca++ ions induce a dG.dG.dC triple helix structure at the contiguous dG residues, Zn++, Cu++ and Co++ ions induce yet another structure at the direct repeats immediately 5' of the dG residues. When Mg++ and Zn++ ions are both present, Zn++ inhibits the dG.dG.dC triplex at the contiguous dG residues and induces a particular non-B DNA structure at the adjacent direct repeats. The specific induction of non-B DNA structures by metal ions at the two adjacent sequences within the promoter region may be of biological significance.     

Role of chelation and water binding of calcium in dormancy and heat resistance of bacterial endospores.

The possible relationship between the water binding by bacterial endospores and their dormancy and heat resistances has been examined in terms of the coordination characteristics of the spore-bound calcium. Stabilities of the calcium complexes of typical cytoplasmic and structural spore components were determined by potentiometric equilibrium pH measurements in model systems consisting of DPA, glycine, alanine, glutamic acid, alanyl-glutamic acid, triglycine, and tetraglycine. The Ca++-form and H+-form spores of Clostridium botulinum 33A were investigated in vivo with respect to their water sorption and heat-resistance characteristics. The results suggest that the complexing of calcium and Ca(II)-DPA may be biologically significant for spore resistance and dormancy at the following three levels: (1) complexing with spore cytoplasmic pool constituents consistent with the idea of a metal-chelate cross-linked cytoplasm or spore cement stabilizing the essential biological macromolecules, (2) complexing with structural components of the spore as indicated by the interaction with model peptides, and (3) coordination with water to produce an apparently dehydrated environment in the spore as evident from the much greater water-sorption capacity of the Ca++-form spores vs the much smaller water sorption of the H+-form spores. Interestingly enough, DPA itself, in the absence of metal ion, showed some interaction with di-, tri-, and tetrapeptides and a weak but detectable interaction with amino acids. Although the exact mode of the DPA-peptide interaction is not clear, it is attractive to speculate about its possible involvement in the control of spore dormancy and resistance.     

High calcium content in Streptomyces spores and its release as an early event during spore germination.

The metal ion content of spores of five Streptomyces species was studied. A general feature of this study was the finding of a very high calcium content (1.1 to 2.1% of the dry weight). Accumulation of calcium occurred preferentially during the sporulation process. Spore calcium was located in the integument fraction, and more than 95% of the calcium was removed from intact spores by ethylene glycol-bis(beta-aminoethyl ether)-N,N-tetraacetic acid. Several divalent cations (Mg2+, Mn2+, Zn2+, and Fe2+) which induced darkening of spores and loss of heat resistance also caused the release of calcium from spores. In addition, darkening of spores was blocked by metabolic inhibitors, whereas calcium excretion was not affected. Two different categories of events in the initiation of germination may be differentiated; first, calcium release from spores which is not energy dependent and is a consequence of triggering of germination by some divalent cations, and second, some other events including loss of heat resistance, loss of spore refractility, and a decrease in absorbance, with at least one energy-dependent step.     

A new growth and in vitro sporulation medium for Clostridium perfringens

Growth and in vitro sporulation capabilities of three related Clostridium perfringens strains (NCTC 8798, 8-6 and R3) were followed in a new sporulation medium (NSM), with notable changes from a maintenance medium originally designed for strictly anaerobic bacteria. Compared with thioglycollate (FTG) medium, the new sporulation medium promoted growth of Cl. perfringens with a shorter lag phase and a 20% higher biomass production. The age of inoculum did not change Cl. perfringens growth kinetics. When compared with reference conditions, in vitro spore production kinetics were different in the new sporulation medium, but both conditions led routinely to 100% sporulation and spore counts of approximately 10(8) ml-1. The ease of preparation of the NSM, and the use of the same culture medium for good growth, high sporulation yields and spore production, represent an attractive alternative to the complex media routinely used for in vitro studies of Cl. perfringens physiology.     

Characterization of calcium-activated bifunctional peptidase of the psychrotrophic Bacillus cereus

The protease purified from Bacillus cereus JH108 has the function of leucine specific endopeptidase. When measured by hydrolysis of synthetic substrate (N-succinyl-Ala-Ala-Pro-Leu-p-nitroanilide), the enzyme activity exhibited optimal activity at pH 9.0, 60 degrees C. The endopeptidase activity was stimulated by Ca++, Co++, Mn++, Mg++, and Ni++, and was inhibited by metal chelating agents such as EDTA, 1,10-phenanthroline, and EGTA. Addition of serine protease inhibitor, PMSF, resulted in the elimination of the activity. The endopeptidase activity was fully recovered from the inhibition of EDTA by the addition of 1 mM Ca++, and was partially restored by Co++ and Mn++, indicating that the enzyme was stabilized and activated by divalent cations and has a serine residue at the active site. Addition of Ca++ increased the pH and heat stability of endopeptidase activity. These results show that endopeptidase requires calcium ions for activity and/or stability. A Lineweaver-Burk plot analysis indicated that the Km value of endopeptidase is 0.315 mM and Vmax is 0.222 mmol of N-succinyl-Ala-Ala-Pro-Leu-p-nitroanilide per min. Bestatin was shown to act as a competitive inhibitor to the endopeptidase activity.     

Sporulation and enterotoxin production by Clostridium perfringens type A at 37 and 43 degrees C.

Enterotoxin-positive strains of Clostridium perfringens were grown in Duncan-Strong sporulation medium in the presence of 0.4% (7.9 mM) raffinose at 37 and 43 degrees C. Enterotoxin- and heat-resistant spores were produced at similar concentrations but sooner at 43 degrees C than at 37 degrees C. There was a direct relationship between spore heat resistance and sporulation temperature (32, 37, and 43 degrees C).