Effect of Sodium Nitrite, Sodium Chloride, and Sodium Nitrate on Germination and Outgrowth of Anaerobic Spores

The effects of meat-curing agents on germination and outgrowth of putrefactive anaerobe 3679h (PA 3679h) spores were studied in microcultures. Nitrite concentrations up to 0.06% at pH 6.0 or between 0.8 and 1% at pH 7.0 allowed emergence and elongation of vegetative cells but blocked cell division. The newly emerged cells then lysed. With more than 0.06% nitrite at pH 6.0 or more than 0.8 to 1% at pH 7.0, the spores lost refractility and swelled, but vegetative cells did not emerge. Even as much as 4% nitrite failed to prevent germination (complete loss of refractility) and swelling of the spores. Sodium chloride concentrations above 6% prevented complete germination (i.e., the spores retained a refractile core). In the presence of 3 to 6% sodium chloride, most of the spores germinated and produced vegetative cells, but cell division was often blocked. Sodium nitrate had no apparent effect on germination and outgrowth at concentrations up to 2%.     

Autolysis and ageing of Penicillium chrysogenum under carbon starvation: respiration and glucose oxidase production

During the exponential growth phase of Penicillium chrysogenum NCAIM 00237 the effective conversion of glucose and O2 to gluconate and H2O2 by glucose oxidase (GOX) was the most likely source of intracellular ROS measured. In glucose-supplemented autolysing cultures, the increased of intracellular ROS concentration was attributed to respiration in the absence of any significant GOX activity. The induction of GOX and catalase by glucose and H2O2 was clearly age-dependent in P. chrysogenum. In ageing cryptic growth phase cultures, superoxide dismutase and cyanide-resistant respiration were the major elements of antioxidative defence but these activities were insufficient to prevent the progressive accumulation of ROS and the concomitant decrease in cell vitality.     

Factors Affecting the Rate of Heat-induced Spore Germination in Dictyostelium discoideum

Washed spores of Dictyostelium discoideum, strains NC-4H, NC-4D, and V-12, germinated rapidly after being heat shocked at or near 45.0 C for 30 min. Cultures of the slime molds were grown in association with Escherichia coli B/r as the host bacterium; spores taken from plates of synthetic medium had a higher final germination value than spores from complex medium containing peptone and yeast extract. Young spores germinated more rapidly than older spores. Optimal germination occurred between pH 6.0 and 7.0, and, of the buffers tested, potassium phosphate allowed the most rapid germination. After heat shocking, spores were diluted into fresh oxygenated buffer to provide enough oxygen for completion of germination. Germination occurred most rapidly between incubation temperatures of 22 and 25 C.     

Characterization of spore germination of a thermoacidophilic spore-forming bacterium, Alicyclobacillus acidoterrestris

The germination behaviors of spores of Alicyclobacillus acidoterrestris, which has been considered to be a causative microorganism of flat sour type spoilage in acidic beverages, were investigated. The spores of A. acidoterrestris showed efficient germination and outgrowth after heat activation (80 degrees C, 20 min) in Potato dextrose medium (pH 4.0). Further, the spores treated with heat activation germinated in McIlvaine buffer (pH 4.0) in the presence of a germinative substance (L-alanine) and commercial fruit juices, although not in phosphate buffer (pH 7.0). Heat activation was necessary for germination. The spores of A. acidoterrestris, which easily survived the heat treatment in acidic conditions, lost their resistance to heat during germination. Our results suggest that the models obtained from spore germination of A. acidoterrestris might be beneficial to determine adequate thermal process in preventing the growth of potential spoilage bacteria in acidic beverages.     

The Germination of Vavraia culicis Spores1

Buffered solutions of KCl and NaCl were tested for their stimulatory effect on the germination of variously-aged spores of Vavraia culicis. Germination was optimal in 0.2 M KCl, pH 6.5 for one isolate, and, for another isolate, peaks of germination occurred at pH 7.0 and 9.5. Spores incubated for several hours in suboptimal solutions became unable to germinate under optimal conditions. After being returned to water, they regained their ability to germinate. Calcium chloride, magnesium chloride, and ammonium chloride inhibited germination. After ingestion by mosquito larvae, spores germinated near the posterior end of the midgut.     

Nitrite-induced germination of putefactive anaerobe 3679h spores

Sodium nitrite alone has been shown to stimulate germination of PA 3679h spores. The process was accelerated by using increased concentrations of sodium nitrite, a low pH, and a high temperature of incubation. At low concentrations of nitrite (0.01 to 0.2%), the delay of 36 to 48 hr occurred before germination commenced at 37 C. However, with 3.45% nitrite at 45 C and pH 6.0, most of the spores germinated within 1 hr. At pH 7.0, the germination rate decreased markedly, and at pH 8.0 it was nil. The greatest acceleration in germination rate occurred near 60 C. Hydroxylamine was completely inhibitory to nitrite-induced germination. Sodium nitrite, in turn, inhibited germination by l-alanine, the degree of inhibition being influenced by nitrite concentration and pH.     

The glucose oxidase of Penicillium variabile P16: gene cloning, sequencing and expression

AIMS: Isolation and characterization of the glucose oxidase (GOX)-encoding gene from a Penicillium variabile strain (P16) having a high level of GOX activity and comparison of its expression with that of another strain of P. variabile (NRRL 1048) characterized by low GOX activity. METHODS AND RESULTS: The gene, isolated by PCR consisted of 1818 bp encoding 605 amino acid residues. Gene expression was analysed by Northern blotting and compared with that of P. variabile NRRL 1048. The higher GOX activity of strain P16 appeared likely because of de novo mRNA synthesis. Southern blotting analyses of the genomic DNA showed that the hybridization pattern of the two strains differed for the size of hybridizing fragment detected by the probe and slightly for their signal intensity. CONCLUSIONS: The GOX-encoding gene of P. variabile P16 was isolated and characterized to identify the molecular bases of its high level of expression and in view of improving enzyme production by developing a process based on heterologous expression. SIGNIFICANCE AND IMPACT OF THE STUDY: GOX-encoding genes can be subjected to high difference in their expression levels. The P16 strain of P. variable producing large amount of GOX as well as its encoding gene might be exploited for industrial applications.     

Spore lytic enzyme released from Clostridium perfringens spores during germination.

The exudate of fully germinated spores of Clostridium perfringens was found to contain a large amount of a spore lytic enzyme which acted directly on alkali-treated spores of the organism to cause germination. Although no detectable amount of the enzyme was found in dormant spores during germination in a KCl medium, the enzyme was produced rapidly and released into the medium. The optimal conditions for enzyme activity were pH 6.0 and 45 degrees C. Maximum activity occurred in the presence of various univalent cations at a concentration of 50 mM. The enzyme was readily inactivated by several sulfhydryl reagents. A strong reducing condition was generated in the ionic germination of the spores, a minimum Eh level of -350 mV being reached 30 min after initiation of germination. Furthermore, adenosine triphosphate-dependent pyruvate:ferredoxin oxidoreductase (EC 1.2.7.1) was identified in both dorman and germinated spores. The relationship between the release of active enzyme and the generation of reducing conditions during germination is discussed.     

Endotrophic calcium, strontium, and barium spores of Bacillus megaterium and Bacillus cereus

Foerster, Harold F. (The University of Texas, Austin), and J. W. Foster. Endotrophic calcium, strontium, and barium spores of Bacillus megaterium and Bacillus cereus. J. Bacteriol. 91:1333-1345. 1966.-Spores were produced by washed vegetative cells suspended in deionized water supplemented with CaCl(2), SrCl(2), or BaCl(2). Normal, refractile spores were produced in each case; a portion of the barium spores lost refractility and darkened. Thin-section electron micrographs revealed no apparent anatomical differences among the three types of spores. Analyses revealed that the different spore types were enriched specifically in the metal to which they were exposed during sporogenesis. The calcium content of the strontium and the barium spores was very small. From binary equimolar mixtures of the metal salts, endotrophic spores accumulated both metals to nearly the same extent. Viability of the barium spores was considerably less than that of the other two types. Strontium and barium spores were heat-resistant; however, calcium was essential for maximal heat resistance. Significant differences existed in the rates of germination; calcium spores germinated fastest, strontium spores were slower, and barium spores were slowest. Calcium-barium and calcium-strontium spores germinated readily. Endotrophic calcium and strontium spores germinated without the prior heat activation essential for growth spores. Chemical germination of the different metal-type spores with n-dodecylamine took place at the same relative rates as physiological germination. Heat-induced release of dipicolinic acid occurred much faster with barium and strontium spores than with calcium spores. The washed "coat fraction" from disrupted spores contained little of the spore calcium but most of the spore barium. The metal in this fraction was released by dilute acid. The demineralized coats reabsorbed calcium and barium at neutral pH.     

Pathogenicity of Paecilomyces fumosoroseus isolates to diamondback moth, Plutella xylostella: correlation with spore size, germination speed, and attachment to cuticle

Infectivity to larvae of the diamondback moth, Plutella xylostella, was compared among eight Paecilomyces fumosoroseus isolates. Isolate infectivity was assessed for correlation with spore length and germination speed. Four isolates applied to P. xylostella cuticle were also compared for number of spores remaining on the cuticle after washing and for percentage germination after 36 h. Infection of larvae inoculated with the different isolates at an average dosage of 4000 conidia/cm2 ranged from 2 to 47%. The correlation of infectivity with spore length and germination speed in broth was highly significant. Fewer spores of the least infective isolate, ARSEF 1576, attached to larval cuticle compared to spores of the more infective isolates ARSEF 3682, 4461, and 4482 (P < 0.05). After 36 h on larval cuticle, the percentage of spores germinated for isolates 1576 and 3682 was 3 and 95%, respectively. Spores of isolate 1576 were smaller, germinated more slowly, and attached to cuticle in smaller numbers than spores of the more infective isolates. Further research will expand our understanding of the mechanisms of virulence among isolates of P. fumosoroseus.     

Inhibition of Bacillus licheniformis spore growth in milk by nisin, monolaurin, and pH combinations

The effects of nisin and monolaurin, alone and in combination, were investigated on Bacillus licheniformis spores in milk at 37 degrees C. In the absence of inhibitors, germinated spores developed into growing vegetative cells and started sporulation at the end of the exponential phase. In the presence of nisin (25 IU ml-1), spore outgrowth was inhibited (4 log10 reduction at 10 h). Regrowth appeared between 10 and 24 h and reached a high population level (1.25 x 10(8) cfu ml-1) after 7 d. Monolaurin (250 micrograms ml-1) had a bacteriostatic effect during the first 10 h but thereafter, regrowth occurred slowly with a population level after 7 d (4 x 10(5) cfu ml-1) lower than that of nisin. Different combined effects of nisin (between 0 and 42 IU ml-1), monolaurin (ranging from 0 to 300 micrograms ml-1), pH values (between 5.0 and 7.0) and spore loads (10(3), 10(4), 10(5) spores ml-1) were investigated using a Doehlert matrix in order to study the main effects of these factors and the different interactions. Results were analysed using the Response Surface Methodology (RSM) and indicated that nisin and monolaurin had no action on spores before germination; only pH values had a significant effect (P < or = 0.001), i.e. spore count decreased as the pH value increased in relation to germination. Sublethal concentrations of nisin (30 IU ml-1) and monolaurin (100 micrograms ml-1) in combination acted synergistically on outgrown spores and vegetative cells, showing total inhibition at pH 6.0, without regrowth, within 7 d at 37 degrees C.     

Plate assay for determining the time of production of protease, cellulase, and pectinases by germinating fungal spores

A new method for detecting enzymes produced by fungal spores during germination is described here. With this method, the production of enzymes such as protease, cellulase, or pectinase can be correlated with the extent of spore germination. Germination is studied in vitro on agar-based media containing protein, cellulose, or pectin. The spores are immobilized on a permeable membrane mounted on the substrate-containing medium. At various times after inoculation the membrane-bound spores are removed and the medium is stained. The extent of germination is assessed by microscopic examination of the spores and the presence of active hydrolytic enzymes is revealed by the staining. The staining methods are sensitive; detection limits are 1 X 10(-3) unit of cellulase; 2 X 10(-4) unit of protease; 3 X 10(-3) unit of pectin lyase; 3.5 units of polygalacturonase; 2 X 10(-3) unit of pectin methyl esterase. The method has been demonstrated by studying the production of enzymes by germinating conidia of Botrytis cinerea. Cellulase and protease were present before any spores germinated. Pectin lyase was first observed when at least 80% of the spores had germinated. Pectin methyl esterase and polygalacturonase were not produced by the spores.     

Regulation of alpha-aminoadipate reductase from Penicillium chrysogenum in relation to the flux from alpha-aminoadipate into penicillin biosynthesis.

The activity and regulation of alpha-aminoadipate reductase in three Penicillium chrysogenum strains (Q176, D6/1014/A, and P2), producing different amounts of penicillin, were studied. The enzyme exhibited decreasing affinity for alpha-aminoadipate with increasing capacity of the respective strain to produce penicillin. The enzyme from all three strains was inhibited by L-lysine, and the enzyme from the lowest producer, Q176, was least sensitive. Between pH 7.5 and 6.5, inhibition of alpha-aminoadipate reductase by L-lysine was pH dependent, being more pronounced at lower pH. The highest producer strain, P2, displayed the lowest alpha-aminoadipate reductase activity at pH 7.0. In Q176, the addition of 0.5-1 mM of exogenous lysine stimulated penicillin formation, whereas the same concentration was ineffective or inhibitory with strains D6/1014/A and P2. The addition of higher (up to 5 mM) lysine concentrations inhibited penicillin production in all three strains. In mutants of P. chrysogenum D6/1014/A, selected for resistance to 20 mM alpha-aminoadipate, highest penicillin production was observed in those strains whose alpha-aminoadipate reductase was most strongly inhibited by L-lysine. The results support the conclusion that the in vivo activity of alpha-aminoadipate reductase from superior penicillin producer strains of P. chrysogenum is more strongly inhibited by lysine, and that this is related to their ability to accumulate increased amounts of alpha-aminoadipate, and hence penicillin.     

Relationship between Heat Activation and Percentage Colony Formation for Bacillus stearothermophilus Spores: Effects of Storage and pH of the Recovery Medium

Colony counts of unheated spores were higher in a medium at pH 5·9 than in media of higher pH. The pattern was reversed as the spores were heated. Slide germination studies showed that about 8% or less of unheated spores germinated in slide culture. Optimal heat activation resulted in about 50% germination. The colony counts of heat activated spores dropped significantly upon storage for 9 months, but those of unheated spores did not.     

Influence of NaCl, NaNO2 and oxygen on the germination and growth of Bacillus licheniformis, a spoilage organism of chub-packed luncheon meat

The thermal resistance of Bacillus licheniformis spores was increased from a D70-value of 590 min to one of 900 min by the addition of 4% NaCl to the heating medium [tryptone-yeast extract-glucose (TYG) broth, pH 6.8], but was decreased to 470 min in TYG broth acidified to pH 4.4. Sodium nitrite (0.02%) enhanced spore destruction at 80 degrees C but not at 70 degrees C; addition of 4% NaCl eliminated this effect. Less than half the number of spores surviving heat comparable to commercial cooking were heat-damaged to the extent of being unable to grow aerobically in the presence of 4% NaCl. No growth occurred during anaerobic incubation even when the media contained no added NaCl. Oxygen was not required to trigger spore germination, but trace amounts were needed for the successful outgrowth of germinated spores. Spore germination was accelerated and enhanced by the presence of at least 2% NaCl. Therefore under anaerobic conditions NaCl promotes microbiological stability because the germinated spores cannot develop further and become moribund. It is concluded that the plastic casing of luncheon-meat chubs is not sufficiently oxygen-impermeable to allow the product a long shelf-life other than at chill temperatures unless the chubs are stored in an oxygen-free atmosphere.     

Penicillium chrysogenum glucose oxidase -- a study on its antifungal effects

AIMS: Purification and characterization of the high molecular mass Candida albicans-killing protein secreted by Penicillium chrysogenum. METHODS AND RESULTS: The protein was purified by a combination of ultrafiltration, chromatofocusing and gel filtration. Enzymological characteristics [relative molecular mass (M(r)) = 155 000, subunit structure alpha(2) with M(r,alpha) = 76 000, isoelectric point (pI) = 5.4] were determined using SDS-PAGE and 2D-electrophoresis. N-terminal amino acid sequencing and homology search demonstrated that the antifungal protein was the glucose oxidase (GOX) of the fungus. The enzyme was cytotoxic for a series of bacteria, yeasts and filamentous fungi. Vitamin C (1.0 mg ml(-1)) prevented oxidative cell injuries triggered by 0.004 U GOX in Emericella nidulans cultures but bovine liver catalase was ineffective even at a GOX : catalase activity ratio of 0.004 : 200 U. A secondary inhibition of growth in E. nidulans cultures by the oxygen-depleting GOX-catalase system was likely to replace the primary inhibition exerted by H(2)O(2). CONCLUSIONS: Penicillium chrysogenum GOX possesses similar enzymological features to those described earlier for other Penicillium GOXs. Its cytotoxicity was dependent on the inherent antioxidant potential of the test micro-organisms. SIGNIFICANCE AND IMPACT OF THE STUDY: Penicillium chrysogenum GOX may find future applications in glucose biosensor production, the disinfection of medical implants or in the food industry as an antimicrobial and/or preservative agent.     

Glutamic Acid Decarboxylase in Spores of Bacillus megaterium and Its Possible Involvement in Spore Germination

Spores of Bacillus megaterium were examined for glutamic acid decarboxylase (GAD). Although dormant spores showed no GAD activity, spores given sonic treatment and heat-activated spores had high activities when assayed for this enzyme. Several parameters of GAD in heat-activated spores were examined. The effects of KCN, NaN(3), 2,4-dinitrophenol, and KF on GAD activity were examined. Only KCN was an effective inhibitor of GAD activity in heated spores and was also shown to be the only effective inhibitor of GAD activity in vegetative bacteria. Similar patterns of inhibition were obtained with GAD activity and with spore germination, KCN being the only effective inhibitor of both, although at different concentrations. Spore GAD activity in heat-activated spores showed a loss with storage at 4 C; on the other hand, storage at 25 C was not accompanied by a loss, but, to the contrary, showed an increase in GAD activity of about 30%. A comparison of GAD activity at different times during germination, growth, and sporulation showed it to be highest in freshly germinated spores. Although vegetative cells contained GAD activity, the level in log-phase cells was approximately one-half the level obtained with freshly germinated spores. Heat-activated mutant spores with a requirement of gamma-aminobutyric acid for germination gave no GAD activity. GAD activity appeared in mutant spores after germination and increased to levels comparable to parent spores after 9 min of germination.     

Inhibition by potassium ion of the pregerminative response to L-alanine of unactivated spores of Bacillus cereus T.

The effect of potassium ion on L-alanine-inosine-induced germination of unactivated spores of Bacillus cereus T was studied. Unactivated spores germinated in 0.1 M sodium phosphate buffer (NaPB), but not 0.1 M potassium phosphate buffer (KPB), at pH 8.0 and at 30 C. Inhibition of germination was also observed on incubation of unactivated spores in NaPB containing potassium chloride. Previously it was demonstrated that germination of unactivated spores involves at least two steps, one induced by L-alanine, and the other by inosine. Potassium ion seems to inhibit the response of the spores to inosine, because: (1) Spores that had been preincubated with L-alanine in NaPB or KPB, germinated in NaPB but not KPB in the presence of inosine. (2) During germination in NaPB, incorporation of L-[14C]alanine showed bimodal kinetics with a rapid first phase and a second continuous phase, but in KPB the second phase of incorporation did not occur. The events occurring before germination of unactivated spores are discussed with reference to the initiation of germination.     

Changes in Terminal Respiratory Pathways of Bacillus subtilis During Germination, Outgrowth, and Vegetative Growth

The chemical and enzymatic properties of the cytochrome system in the particulate preparations obtained from dormant spores, germinated spores, young vegetative cells, and vegetative cells of Bacillus subtilis PCI219 were investigated. Difference spectra of particulate fractions from dormant spores of this strain suggested the presence of cytochromes a, a(3), b, c(+c(1)), and o. All of the cytochrome components were present in dormant spores and in germinated spores and vegetative cells at all stages which were investigated. Concentrations of cytochromes a, a(3), b, and c(+c(1)) increased during germination, outgrowth, and vegetative growth, but that of cytochrome o was highest in dormant spores. As the cytochrome components were reducible by reduced nicotinamide adenine dinucleotide (NADH), they were believed to be metabolically active. Difference spectra of whole-cell suspensions of dormant spores and vegetative cells were coincident with those of the particulate fractions. NADH oxidase and cytochrome c oxidase were present in dormant spores, germinated spores, and vegetative cells at all stages after germination, but succinate cytochrome c reductase was not present in dormant spores. Cytochrome c oxidase and succinate cytochrome c reductase activities increased with growth, but NADH oxidase activity was highest in germinated spores and lowest in vegetative cells. There was no striking difference between the effects of respiratory inhibitors on NADH oxidase in dormant spores and those on NADH oxidase in vegetative cells.