Isoleucine synthesis by Clostridium sporogenes from propionate or alpha-methylbutyrate.

Preliminary studies demonstrated that Clostridium sporogenes synthesized isoleucine by a pathway not involving threonine or threonine dehydratase. Radiotracer experiments with cells grown in a defined carbohydrate-free medium showed that radioactivity from [U-14C]serine, [3-14C]pyruvate, [14C]NaHCO3 and [1-], [2-] and [3-14C]propionate was incorporated into isoleucine. Conversely, there was no detectable incorporation of 14C into isoleucine during growth with [U-14C]glutamate, [U-14C]threonine, [U-14C]valine, [U-14C]leucine or [U-14C]methionine. Crude extracts of the bacteria grown in a minimal medium contained levels of alpha-acetohydroxyacid synthase activities comparable to those in Escherichia coli K12 grown in minimal medium. Stepwise degradation of isoleucine obtained from C. sporogenes grown in the presence of specifically-labelled precursors indicated that C. sporogenes can make isoleucine via the reductive carboxylation of propionate to yield alpha-oxobutyrate, which is metabolized to isoleucine in the classical fashion. Isoleucine was also formed by C. sporogenes via the reductive carboxylation of alpha-methylbutyrate to alpha-oxo-beta-methylvalerate.

Interconversion of valine and leucine by Clostridium sporogenes.

Clostridium sporogenes has been found to require L-leucine and L-valine for growth in a minimal medium, although valine can be replaced by isobutyrate and leucine by isovalerate. Cells grown in minimal media incorporated significant 14C from [14C]valine into leucine and from [14C]leucine into valine. Growth with [4,5-3H]leucine also resulted in the incorporation of 3H into valine. These results indicate that these bacteria can interconvert leucine and valine.

Purification and partial characterization of proline dehydrogenase from Clostridium sporogenes.

A proline dehydrogenase which catalyzes the nicotinamide adenine dinucleotide (NAD) dependent oxidation of proline and the NADH-dependent reduction of delta 1-pyrroline 5-carboxylic acid (PCA) was purified from extracts of Clostridium sporogenes. Following purification, only one protein band was found on analytical polyacrylamide disc gels and on sodium dodecyl sulfate (SDS) - polyacrylamide disc gels. Sucrose density gradient centrifugation and SDS-gel electrophoresis indicated that the enzyme has a molecular weight of approximately 217 000 and consists of two subunits of equal size. During purification of proline dehydrogenase on hydroxylapatite the ratio of dehydrogenase activity to reductase activity decreases significantly, and a similar change in ratio was brought about by storage of partially purified enzyme preparations in low ionic strength buffers. Subsequent purification did not change the ratio. The dehydrogenase activity of proline dehydrogenase was inhibited by L-glutamate (Ki = 0.32 mM at pH 7.4 and Ki = 0.65 mM at ph 10.2). However, the reductase activity of the purified enzyme was not affected by 100 mM L-glutamate.

Molds in brined cucumbers: cause of softening during air-purging of fermentations.

Softening of cucumbers in fermentations purged at high air-flow rates was caused by molds growing in the brined cucumbers, not in the brine. This conclusion is based on the following results: (i) no microorganisms were isolated in significant numbers from brines that caused softening of pasteurized brined cucumbers, (ii) no pectinolytic enzyme activities were produced in cucumber brines in the absence of cucumbers, (iii) the pickles in some air-purged fermentations became very soft without the appearance of any pectinolytic enzyme activity in the brine, (iv) mold hyphae were consistently observed in tissues of soft pickles, (v) molds consistently developed in cultures of slices of surface sterilized cucumbers taken from fermentations in which soft pickles were subsequently found, and (vi) molds belonging to the genera Alternaria, Fusarium, and Mucor isolated from slices all softened pasteurized brined cucumbers.

Identity of proline dehydrogenase and delta1-pyrroline-5-carboxylic acid reductase in Clostridium sporogenes.

Proline dehydrogenase and delta1-pyrroline-5-carboxylic acid (PCA) reductase activities were copurified 60- and 130-fold, respectively, from extracts of Clostridium sporogenes. The primary change in the ratio of activites was the result of a loss of proline dehydrogenase activity during dialysis. Both activities were eluted in single peaks from diethylaminoethyl-cellulose, hydroxylapatite, and Sephadex G-200 columns. They had identical sedimentation coefficients (10.3S), as determined in linear sucrose gradients, and identical isoelectric points (4.95 to 5.12) based on isoelectric focusing. The proline dehydrogenase activity was dependent on nicotinamide adenine dinucleotide and L-proline, and the PCA reductase required L-PCA and reduced nicotinamide adenine dinucleotide. The optimum pH for the assay of proline dehydrogenase was approximately 10.2, whereas that for PCA reductase was 6.5 to 7.5. An increase in pH from 8.0 to 10.2 greatly decreased the apparent Michaelis constant observed for L-proline, and an increase from pH 8.3 to 8.6 resulted in a large shift in the reaction equilibrium toward PCA. Both the dehydrogenase and reductase activities were stabilized to heating at 65 degrees C for 5 min by solutes of high ionic strength and were inactivated in a similar fashion when dissolved in low-ionic-strength buffer. The specific activities for both were reduced by about 50% when glucose was added to the growth medium. The data support the conclusion that L-proline and L-PCA are interconverted by either a single enzyme or an enzyme complex in extracts of C. sporogenes cells.

Uptake of glucose and maltose by Bacillus popillae.

Results of experiments with glucose and its analog, methyl alpha-D-glucopyranoside, indicated that when glucose was present at low concentrations, it was transported into Bacillus popilliae NRRL B-2309MC cells as glucose 6-phosphate by a phosphoenolpyruvate:sugar phosphotransferase system. An additional mode(s) of entry may be operative at higher glucose concentrations. Maltose appeared to enter the cells by a nonphosphorylative process and was hydrolyzed intracellularly to glucose. No phosphoryl donor was necessary for this hydrolysis.

Selenium requirement for the growth of Clostridium sporogenes with glycine as the oxidant in stickland reaction systems.

Clostridium sporogenes was found to have an absolute requirement for selenium to utilize glycine but not proline as oxidant in Stickland-type fermentations. No glycine reductase activity was detectable in cells from media without added selenium. The data indicate that this organism could be used for microbiological assays for very low levels of selenium in certain forms.

Microbial inhibition by an isolate of pediococcus from cucumber brines.

We reported earlier that Pediococcus cerevisiae FBB-61 inhibited Lactobacillus plantarum FBB-67 in mixed species inoculation used for the fermentation of brined cucumbers. Herein, 16 isolates of the Pediococcus genus from various sources were tested for inhibitory activity against L. plantarum and other microorganisms by a seeded-agar screening technique. Only two of the 16 isolates gave consistent and distinctive zones of inhibition, and both were isolated from fermenting cucumber brines on separate occasions. These two isolates did not inhibit each other but did inhibit the other 14 Pediococcus isolates in addition to L. plantarum. They also inhibited several other gram-positive bacteria, but not four species each of gram-negative bacteria and yeasts tested. Inoculation of cucumber juice broth with P. cerevisiae FBB-61 and L. plantarum WSO resulted in a drastic reduction in the plate count of L. plantarum WSO during day 1, but counts increased rapidly thereafter. Consequently, acid production by L. plantarum WSO was delayed. Noninhibitory isolates of Pediococcus had no appreciable effect on growth and acid production by L. plantarum WSO.

Minimal growth requirements for Clostridium perfringens and isolation of auxotrophic mutants.

The minimal growth requirements for two strains of Clostridium perfringens were defined, and both synthetic and semisynthetic plating media were developed. Plate counts of the wild-type strains on both of these minimal media were equivalent to those on complex media. A number of auxotrophic mutants of each strain were isolated, and their phenotypes were defined.

Trehalose metabolism by Bacillus popilliae.

Trehalose was found to be utilized more readily than glucose for the growth of Bacillus popilliae NRRL B-2309MC. The pathway of degradation of trehalose was elucidated and found to differ from that reported for other organisms. Trehalase and trehalose phosphorylase activities could not be detected. Rather, trehalose was found to undergo phosphoenolpyruvate (PEP)-dependent phosphorylation, and the resulting trehalose 6-phosphate was cleaved by a phosphotrehalase to equimolar amounts of glucose and glucose 6-phosphate. The phosphotrehalase was purified 34-fold and shown to have a pH optimum of 6.5 to 7.0 and a K(m) for trehalose 6-phosphate of 1.8 mM. A mutant missing the phosphotrehalase failed to grow on trehalose but grew normally on other sugars. The mutant accumulated [(14)C]trehalose as [(14)C]trehalose 6-phosphate. Phosphorylation of trehalose by dialyzed extracts was at least 25 times faster with PEP than with adenosine 5'-triphosphate, and the phosphorylation activity was associated primarily with the particulate fraction. These data and the results of studies of [(14)C]trehalose uptake suggest that trehalose is transported into the cell as trehalose 6-phosphate by a PEP:sugar phosphotransferase system. Cell extracts of other strains of B. popilliae were also found to produce [(14)C]sugar phosphate from [(14)C]trehalose and to have phosphotrehalase activity.

Influence of temperature and humidity on microbial, enzymatic, and physical changes of stored, pickling cucumbers.

Pickling cucumbers stored at five temperatures and four relative humidities were examined for growth of eight microbial groups, the activities of two enzyme systems (pectinolytic and cellulolytic), and weight loss. Twenty-four storage tests for 6 days each were conducted during the 2-year study. In general, microbial populations of the eight groups increased rapidly at the higher temperature (>21 C) and humidity (>70%) treatments. Moisture loss of the cucumbers was rapid with combinations of high temperatures and low humidities. The results suggest that the best environmental conditions for storage or transport of cucumbers would be a combination of low temperature (10 C) with high relative humidity (about 95%). These conditions minimized undesirable microbial, enzymatic, and physical changes of stored, pickling cucumbers.

Superoxide dismutase in Bacillus popilliae.

Vegetative cells of Bacillus popilliae were devoid of catalase but had high levels of superoxide dismutase. This provides further support of a theory that oxygen tolerance by an organism is more dependent on superoxide dismutase than on catalase.

Physiological studies of an oligosporogenous strain of Bacillus popilliae.

A relatively small but consistent increase in the frequency of spore formation by an oligosporogenous strain of Bacillus popilliae (NRRL B-2309M) was obtained by adding 0.1% sodium pyruvate to the sporulation medium. The frequency of spore formation was essentially the same when a low level of glucose, trehalose, or glucose-6-phosphate or a high level of alpha-methyl-d-mannoside was added as the carbon and energy source. Many other variations in the cultural medium and cultural conditions failed to enhance spore formation of 2309M, and no spores were found in four asporogenic strains under any of the conditions tried. There were no significant differences between the 2309M strain and three nonsporeforming cultures with respect to (i) the rate and extent of growth, (ii) the rates of glucose utilization, or (iii) volatile acid production and utilization. None of the cultures tested was found to produce detectable levels of extracellular protease or an antibiotic. The only consistent marker found associated with spore formation was the development of catalase activity, and this activity was stimulated by heating at 80 C for 10 min. This was not found unless morphological evidence of spore formation was observed. The germination of the spores formed by 2309M in vitro was stimulated by heat shock and by the addition of pyruvate to the germination medium.

Oxidation of acetate by various strains of Bacillus popilliae.

A number of strains of Bacillus popilliae were examined for their ability to oxidize acetate. Some of these would not sporulate in vitro, and some were oligosporogenous. The ability to oxidize acetate varied widely among the strains tested. A culture derived from spores of the parent strain produced in vivo and one of the asporogenous strains derived from it failed to produce any significant levels of (14)CO(2) from [(14)C]acetate. Oligosporogenous strains derived from the same parent culture all produced (14)CO(2) from both [1-(14)C] and [2-(14)C]acetate but at relatively low rates. The highest rates of acetate oxidation were observed with three strains which did not produce spores in vitro. When cultured under appropriate conditions, one of these strains displayed a secondary growth response concomitant with a decrease in the titratable acidity and an increase in the pH of the medium. The data indicate that B. popilliae has a complete citric acid cycle but that the activity of the cycle is strongly repressed in wild-type strains under the usual conditions used for in vitro cultivation.