Sporulation of the "thermophilic anaerobes".

A reasonable degree of synchrony in the sporulation of Clostridium thermosaccharolyticum 3814 was obtained by using three 10% transfers of 8-hr cultures in a medium containing 0.5% L-arabinose, 0.5% peptone, 0.5% yeast extract, and Gc minerals. Sporulation was stimulated by L-arabinose and L-xylose, but was repressed by glucose, mannose, fructose, and D-pentoses. Sporulating cells were long and thin, whereas repressed cells were shorter and thicker. The optimal pH for sporulation was in the range of pH 5.0 to 5.5. As sporulation continued, the accumulated acetate decreased. Label studies indicated that a significant amount of acetate-2-C(14) was incorporated into the spore lipid. The calcium, phosphorus, and dipicolinic acid (DPA) concentrations on a dry weight basis were 2.55, 2.60, and 7.25%, respectively. The molar ratio of Ca-DPA was 1.47.

Regeneration of ribosomes and ribosomal ribonucleic acid during repair of thermal injury to Staphylococcus.

Heating Staphylococcus aureus MF31 at 55 C for 15 min renders the organisms unable to reproduce on agar containing 7.5% NaCl (1). The heated organisms exhibited an extended lag period during which the organisms regained their ability to grow on the 7.5% NaCl-agar. Inhibitor and antibiotic data indicated that protein synthesis is not involved in this recovery process but nucleic acid synthesis is suggested (3). The data presented here further substantiate the noninvolvement of protein synthesis during recovery and further demonstrate the site of the thermally induced nucleic acid lesion. Methylated albumin kieselguhr column analysis showed the lesion site to be the ribosomal ribonucleic acid (rRNA). The rRNA is resynthesized during the extended lag period. Sucrose gradient analysis demonstrated that a ribosomal peak was undetectable subsequent to the thermal treatment, but this peak was regenerated during the recovery period.

Morphology of the spore of some strains of Clostridium botulinum type E.

Hodgkiss, W. (Torry Research Station, Aberdeen, Scotland), and Z. John Ordal. Morphology of the spore of some strains of Clostridium botulinum type E. J. Bacteriol. 91:2031-2036. 1966.-The spores of four strains of C. botulinum type E show an unusual and elaborate morphology. Numerous tubular appendages radiate from the surface of the spore. The spore and its appendages are enclosed in a delicate exosporium. The electron microscopic morphology of the spores, as seen in metal-shadowed and negatively stained preparations, and by the carbon-replica technique, is described.

Thermal injury and recovery of Salmonella typhimurium and its effect on enumeration procedures.

Exposure of Salmonella typhimurium 7136 to sublethal heating produced a temporary change in the tolerance of the organism to a particular stress medium. After sublethal heat treatment at 48 C for 30 min, greater than 90% of the viable population was unable to reproduce on Levine Eosin Methylene Blue Agar containing 2% NaCl. This sensitivity was dependent on the pH of the heating menstruum. In addition, the heated cells displayed a sensitivity to Brilliant Green Agar, Levine Eosin Methylene Blue Agar, Salmonella-Shigella Agar, and Desoxycholate Citrate Agar. Unheated cells displayed a sensitivity to Brilliant Green Agar, Salmonella-Shigella Agar, and Desoxycholate Citrate Agar. When the injured cells were placed in a suitable medium (Trypticase Soy Broth), they recovered and grew at a rate equal to that of normal cells. Recovery was also possible in Nutrient Broth, Lactose Broth, and Lauryl Tryptose Broth. Although recovery of the injured cell occurred in Tetrathionate Broth and Selenite F Broth, they were less than ideal growth media for the organism.

Thermal stress of Pseudomonas fluorescens in complex media.

Pseudomonas fluorescens (P7) cells were stressed by incubation at 43 degrees C for 2 h. The stress induced a 9-h lag in replication after the return of the temperature of the culture to 25 degrees C. Stressed cells demonstrated a sensitivity to diluents and plating media during the recovery period. Data from utilization of selective inhibitors suggested that ribonucleic acid and protein, but not deoxyribonucleic acid, syntheses were required for recovery by the cells. The cells lost uracil- and leucine-labeled material as a result of the stress, further suggesting that ribonucleic acid and protein damage had occurred. Membrane damage was indicated by sensitivity to sodium dodecyl sulfate near the end of the lag period. Membrane damage was also suggested by the failure of cells to incorporate labeled material from the recovery medium. The lesions induced in this foodlike system are compared with those previously reported for a minimal media model system (Gray et al., Appl. Microbiol. 26:78-85, 1973; Gray et al., Appl. Environ. Microbiol. 33:1074-1078, 1977).

Cell-bound lipase and esterase of Brevibacterium linens.

The activities of glycerol ester hydrolase, lipase (EC 3.1.1.3) and carboxylesterase, and esterase (EC 3.1.1.1) were determined for whole cell preparations of Brevibacterium linens by using the pH-stat assay. The culture growth liquors were inactive against the three substrates, tributyrin emulsion, triacetin, and methyl butyrate. Cells washed in water had less activity than cells washed in 5% NaCl; the ratio of activities was close to 1:2 for all strains using tributyrin emulsion as the substrate. For the esterase substrates, this relationship varied widely and was strain dependent. The ability to hydrolyze the two esterase substrates varied independently of the level of lipase activity.

Sporulation of Clostridium thermosaccharolyticum.

Good sporulation was induced in batch culture using carbon sources that have in common the effect of reducing growth rate.

Effect of sublethal heat on the metabolic activity of Staphylococcus aureus.

Cells of Staphylococcus aureus MF-31 which have been heat-injured at 52 C have an altered metabolic activity. Analyses of whole-cell preparations by means of the Thunberg technique and Warburg manometry showed decreased dehydrogenase activity and oxygen uptake on a variety of substrates. In cell-free extracts prepared from injured cells, it was demonstrated that the specific activity of fructose diphosphate aldolase, lactate dehydrogenase, and butanediol dehydrogenase was less than that of extracts prepared from normal unheated cells. Recovery of the heat-injured cells in a suitable medium supported a return of the dehydrogenase activity and oxygen uptake, but the activity of the enzymes in cell-free extracts prepared from such partially recovered cells did not fully return to the level of normal (unheated) preparations. Addition of chloramphenicol or actinomycin D to the recovery medium, singly or in combination, retarded the return of the normal metabolic activity. Radiorespirometric experiments indicated that the percentage participation of the Embden-Meyerhoff Parnas and hexose monophosphate pathways remained the same for normal and heat-injured cells. The sublethal heat treatment decreased the catabolic capabilities of S. aureus and the production of selected end products associated with the metabolism of glucose.

Sporulation of Clostridium thermosaccharolyticum under conditions of restricted growth.

Glucose served as an exogenous energy source for sporulation when a limited amount was fed continuously. Excess glucose repressed sporulation.

Comparative metabolism of vegetative and sporulating cultures of Clostridium thermosaccharolyticum.

Cultures of Clostridium thermosaccharolyticum, under conditions of restricted growth achieved by slow feeding of glucose, showed a high degree of sporulation. Analysis of the end products showed an accumulation of ethyl alcohol in addition to butyrate and acetate, whereas, in the nonsporulating cultures, acetate and butyrate were the principal products. Incorporation of uniformly labeled (14)C-glucose by sporulating cells was three to four times higher than by nonsporulating cells. The efficiency of acetate assimilation into the lipid fraction of sporulating cells was at least two times higher than that of glucose. When starch was used as the carbon source, the growth rate was reduced; sporulation occurred, and the end products and carbon distribution were similar. Alcohol dehydrogenase, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase were preferentially formed by sporulating cells. In vegetative cells, the formation of these enzymes was repressed if the glucose concentration in the medium was increased. The change in enzyme activity appeared to be related to a morphological change in the cells and indicated an altered metabolic pattern for sporulating cells.

Precursor ribosomal ribonucleic acid and ribosome accumulation in vivo during the recovery of Salmonella typhimurium from thermal injury.

When cells of S. typhimurium were heated at 48 C for 30 min in phosphate buffer (pH 6.0), they became sensitive to Levine Eosin Methylene Blue Agar containing 2% NaCl (EMB-NaCl). The inoculation of injured cells into fresh growth medium supported the return of their normal tolerance to EMB-NaCl within 6 hr. The fractionation of ribosomal ribonucleic acid (rRNA) from unheated and heat-injured cells by polyacrylamide gel electrophoresis demonstrated that after injury the 16S RNA species was totally degraded and the 23S RNA was partially degraded. Sucrose gradient analysis demonstrated that after injury the 30S ribosomal subunit was totally destroyed and the sedimentation coefficient of the 50S particle was decreased to 47S. During the recovery of cells from thermal injury, four species of rRNA accumulated which were demonstrated to have the following sedimentation coefficients: 16, 17, 23, and 24S. Under identical recovery conditions, 22, 26, and 28S precursors of the 30S ribosomal subunit and 31 and 48S precursors of the 50S ribosomal subunit accumulated along with both the 30 and 50S mature particles. The addition of chloramphenicol to the recovery medium inhibited both the maturation of 17S RNA and the production of mature 30S ribosomal subunits, but permitted the accumulation of a single 22S precursor particle. Chloramphenicol did not affect either the maturation of 24S RNA or the mechanism of formation of 50S ribosomal subunits during recovery. Very little old ribosomal protein was associated with the new rRNA synthesized during recovery. New ribosomal proteins were synthesized during recovery and they were found associated with the new rRNA in ribosomal particles. The rate-limiting step in the recovery of S. typhimurium from thermal injury was in the maturation of the newly synthesized rRNA.

Requirements of Salmonella typhimurium for recovery from thermal injury.

The heating of Salmonella typhimurium 7136 at 48 C for 30 min produces a population of cells that are incompetent at division on Levine Eosin Methylene Blue Agar containing 2.0% NaCl (EMB-NaCl). When these injured cells were placed in fresh citrate salts medium they recovered, and regained their tolerance to the EMB-NaCl medium and grew out. The addition of the selective inhibitors rifamycin, 5-fluorouracil, 2,4-dinitrophenol, chlorotetracycline, chloramphenicol, and 5-methyl-tryptophan to the recovery medium showed that the recovery process was dependent on ribosomal ribonucleic acid (RNA) synthesis, adenosine triphosphate synthesis, and the synthesis of new protein. These results were substantiated by incorporation experiments, which demonstrated that during recovery no deoxyribonucleic acid synthesis, and hence no cell division, occurred. Ribosomal RNA was synthesized during recovery, but its synthesis was not the rate-limiting step. A small but significant amount of protein was also formed during the latter part of the recovery period.

Interference-contrast and phase-contrast microscopy of sporulation in clostridium thermosaccharolyticum grown under strict anaerobiosis.

Cells of Clostridium thermosaccharolyticum grown under strict anaerobiosis (modified Hungate technique) were examined during growth and sporulation by employing Nomarski interference-contrast and Zernike phase-contrast optics to delineate the sequence of morphological changes leading to the formation of free, mature spores. A 0.5% l-arabinose, liquid, complex medium was used to obtain a yield of 30 to 40% free, refractile spores (ca. 10(8)/ml) by 48 hr of incubation. The mean doubling time for the glucose culture (vegetative cells) was found to be 80 min, and that for the l-arabinose culture (sporulating cells), 498 min. By 8 hr of incubation, beginning spore formation became evident in the arabinose culture by the development of a distinct arrowhead-shaped terminal swelling. By 32 hr of incubation or shortly thereafter, Nomarski optics showed the mature spore to be uniformly spherical, whereas the enlarged terminal swelling containing it was not. The use of phase-contrast and interference-contrast optics permitted the characterization of the distinctive morphological changes occurring during sporulation of C. thermosaccharolyticum.

Oxidative activation of Bacillus cereus spores.

A study was made of the activation of Bacillus cereus strain T spores by using the oxidizing agent sodium perborate. The degree of activation was measured with constant germination conditions by using L-alanine, inosine, adenosine, and L-alanine plus adenosine as germination stimulants. The germinal response following the various treatments was compared with the responses obtained with heat activation. It was concluded that the optimal time for activation with 30 mM sodium perborate at room temperature was about 4 hr. If the exposure time was greatly extended, the spores would germinate spontaneously. When the perborate treatment followed heat activation, the germinal response to L-alanine was stimulated, to inosine retarded and without apparent effect for adenosine or L-alanine plus adenosine. Results of experiments designed to demonstrate deactivation by slow oxidation showed that spores activated with sodium perborate were not deactivated by slow oxidation, whereas those activated by heat were. A deactivation study using mercaptoethanol as the deactivation agent showed that both methods of activation could be deactivated after a 24-hr exposure, but this deactivation was reversible by extending the exposure to mercaptoethanol. The results of heat-sensitivity studies revealed that about 70% of the sodium perborate-activated spores were heat sensitive after 60 min in a germination menstruum of L-alanine plus adenosine, whereas similarly treated heat-activated and nonactivated spores were about 99.99% heat sensitive, respectively.

Thermal injury and recovery of Bacillus subtilis.

Exposure of Bacillus subtilis NCTC 8236 to sublethal temperatures produced a change in the sensitivity of the organism to salt and polymyxin. After 30 min at 47 C, 90% of the population was unable to grow on a modified sulfite polymyxin sulfadiazine agar containing an added 1% NaCl, 1% glucose, and 1% asparagine. The data presented demonstrate that thermal injury results in degradation of both 16S and 23S ribonucleic acid (RNA) and in damage to the cell membrane, suggested by leakage into the heating mestruum of material absorbing at 260 nm. When the cells were placed in a recovery medium (Trypticase soy broth), complete recovery, indicated by a returned tolerance to salt and polymyxin, occurred within 2 hr. The presence of a protein inhibitor (chloramphenicol) and cell wall inhibitors (vancomycin and penicillin) during recovery had no effect, whereas the presence of an RNA inhibitor (actinomycin D) effectively inhibited recovery. Further data demonstrated that the injured cells were able to resynthesize both species of ribosomal RNA during recovery by using the fragments which resulted from the injury process. Also, precursor 16S and precursor 23S particles accumulated during recovery. The maturation of the precursor particles during recovery was not affected by the presence of chloramphenicol in the recovery medium.

Regulatory effect of pyruvate on the glucose metabolism of Clostridium thermosaccharolyticum.

Whole cells and cell-free extracts of Clostridium thermosaccharolyticum 3814 grown in media containing 0.5% glucose or 0.6% pyruvate were evaluated for their metabolic activities toward these compounds. Glucose-grown cells rapidly fermented glucose with the production of gases (CO(2) and H(2)), acids, and alcohol, but they did not ferment pyruvate well. Pyruvate-grown cells, on the other hand, readily fermented pyruvate, while fermenting glucose at a rate of one-half that of pyruvate. An analysis of the enzyme levels in the two cell culture conditions revealed that pyruvate-grown cells had lower levels of most of the glycolytic enzymes and increased levels of the hexose monophosphate pathway enzymes. Incorporation studies with the use of labeled glucose demonstrated that cells do have a control mechanism(s) whereby they can discriminate between a carbon (glucose) and an energy (pyruvate) source, selectively utilizing glucose in the synthetic pathway while obtaining energy from the phosphoroclastic degradation of pyruvate.

Repair of thermal injury of Staphylococcus aureus.

Iandolo, John J. (University of Illinois, Urbana), and Z. John Ordal. Repair of thermal injury of Staphylococcus aureus. J. Bacteriol. 91:134-142. 1966.-Exposure of Staphylococcus aureus MF 31 to sublethal temperatures produced a temporary change in the salt tolerance and growth of the organism. After sublethal heat treatment at 55 C for 15 min, more than 99% of the viable population was unable to reproduce on media containing 7.5% NaCl. The data presented demonstrate that thermal injury, in part, occurred owing to changes in the cell membrane, which allowed soluble cellular components to leak into the heating menstruum. When the cells were placed in a limiting medium, complete recovery did not occur, regardless of the incubation time. The temperature and the pH which produced the optimal rate of recovery were similar to those described previously for the multiplication of uninjured cells. However, the rate of recovery as well as the unchanging total count during recovery indicated that cell multiplication was not a factor during the recovery process. The nutrient requirements for the complete recovery of injured cells consisted of a solution containing an energy source, such as glucose, a mixture of amino acids, and phosphate. The use of the metabolic inhibitors, penicillin, cycloserine, 2,4-dinitrophenol, and chloramphenicol, did not inhibit recovery. Actinomycin D, however, completely suppressed recovery. This result implied that ribonucleic acid synthesis was particularly involved; this inference was substantiated by radio tracer experiments. The rate at which label was incorporated in the nucleic acid fraction paralleled that of recovery and the return of salt tolerance.

Thermal injury and recovery of Streptococcus faecalis.

Exposure of Streptococcus faecalis R57 to sublethal heating produced a temporary change in the salt tolerance and growth of the organism. After sublethal heat treatment at 60 C for 15 min, greater than 99.0% of the viable population was unable to reproduce on media containing 6% NaCl. In addition, the heated cells displayed a sensitivity to incubation temperature, pH, and 0.01% methylene blue. When the injured cells were placed in a synthetic medium, recovery occurred at a much slower rate than in a complex medium. However, both media supported comparable growth of the uninjured organism. Various media used for the enrichment of streptococci also provided a suitable environment for the recovery of the injured cells. Generally, as more selective agents were present in the medium, the rates of recovery decreased. Metabolic inhibitor studies with chloramphenicol, penicillin, and actinomycin D substantiated the fact that the process involved was recovery and not growth, and that this recovery was linked to ribonucleic acid synthesis.

Glycerol ester hydrolase activity of lactic acid bacteria.

Seventeen strains of lactic acid bacteria were assayed for their glycerol ester hydrolase activity by using an improved agar-well technique, and eight strains by determining the activity in cell-free extracts using a pH-stat procedure. All cultures tested showed activity and hydrolyzed tributyrin more actively than they did tricaproin. The cell extract studies demonstrated that the cells contained intracellular esterases and lipases. The culture supernatant fluid was without activity. The lipase and the esterase differed in their relative activity to each other in the different extracts and in the ease by which they could be freed from the cellular debris. It is suggested that the lipase of these organisms is an endoenzyme and the esterase an ectoenzyme.

Continuous Propagation of Trichosporon cutaneum in Cheese Whey.

Trichosporon cutaneum, a nonfermenting yeast, was used to convert cheese whey lactose into microbial cell material. The doubling time for this organism in a laboratory-scale continuous propagator was 2 hr in a whey medium fortified with ammonium sulfate and corn steep liquor. Cellular growth and efficiency of conversion of lactose to cell material was higher than with Saccharomyces fragilis. When grown in whey, the nitrogen content of T. cutaneum was 3.5% and the distribution of amino acids per gram of cell protein was similar to that of commercial food yeasts.