Proceedings: Repair of injury in Salmonella anatum cells after freezing and thawing in milk

Fast freezing and slow thawing of Salmonella anatum cells in nonfat milk solids resulted in about 20% death and 50% injury of the cells surviving the treatment. Death was defined as the inability to form colonies on a nonselective plating medium [xylose-lysine-peptone agar (XLP)] after freezing and thawing. Injury was defined as the inability to form colonies on a selective plating medium (XLP with 0.2% sodium desoxycholate added). The injured cells repaired rapidly and within 2 hr at 25 °C, in the presence of 0.1% milk solids; all the injured cells regained the ability to form colonies on the selective medium. The treated cells showed a 1-hr extended lag phase of growth as compared to the unfrozen cells. Milk solids concentration in the freezing and repair menstrua influenced injury, repair of injury, and death. The repair process was affected by the pH and temperature of environment in which the injured cells were incubated. Maximum repair occurred at pH values between 6.0 and 7.4 and temperatures from 25 to 42 °C. The data suggested repair did not require the synthesis of protein, ribonucleic acid, or cell-wall mucopeptide but did require energy synthesis.     

Characterization of the Repair of Injury Induced by Freezing Salmonella anatum

Fast freezing and slow thawing of Salmonella anatum cells suspended in water resulted in injury of more than 90% of the cells that survived the treatment. The injured cells failed to form colonies on the selective medium (xyloselysine-peptone-agar with 0.2% sodium deoxycholate) but did form colonies on a nonselective (xylose-lysine-peptone-agar) plating medium. In Tryptic soy plus 0.3% yeast extract broth or minimal broth, most of the injured cells repaired within 1 to 2 hr at 25 C. Tryptic soy plus yeast extract broth supported repair to a greater extent than minimal broth. Phosphate or citrate at concentrations found in minimal broth supported repair of some cells. MgSO(4), when present with inorganic phosphate or citrate or both, increased the extent of repair. The repair process in the presence of phosphate was not prevented by actinomycin D, chloramphenicol, and D-cycloserine, but was prevented by cyanide and 2,4-dinitrophenol (only at pH 6). This suggested that the repair process might involve energy metabolism in the form of adenosine triphosphate. The freeze-injured cells were highly sensitive to lysozyme, whereas unfrozen fresh cells were not. In the presence of phosphate or minimal broth this sensitivity was greatly reduced. This suggested that, at least in some of the cells, the injury involved the lipopolysaccharide of the cell wall and adenosine triphosphate synthesis was required for repair.     

Effect of Rehydration on Recovery, Repair, and Growth of Injured Freeze-Dried Salmonella anatum

From 70 to 90% of the Salmonella anatum cells that survived freeze-drying in nonfat milk solids were injured. After rehydration, these injured survivors failed to grow on a selective plating medium containing deoxycholate but could form colonies on a nonselective medium. In a suitable environment after rehydration, injury disappeared in most of these cells. The rate of this repair at 25 C was very rapid initially and, in a medium containing milk solids, was completed within 1 hr after rehydration. The repaired cells initiated growth about 1 hr later than normal cells and grew at a slower rate. In a medium containing milk solids, initial recovery, extent of repair of injury, initiation of growth, and rate of growth were not influenced by supplementation with extra nutrients in other rehydration media. Rehydration controlled by modifying the concentrations of lactose, sucrose, or milk solids in the rehydration medium influenced the recovery of cells and the time that growth was initiated. Glycerol failed to increase recovery. Higher numbers of cells were recovered by rehydrating at 15 to 25 C, but an earlier initiation of growth and more rapid growth were observed at 35 C.     

Influence of Milk Components on the Injury, Repair of Injury, and Death of Salmonella anatum Cells Subjected to Freezing and Thawing

Fast freezing and slow thawing Salmonella anatum cells in various milk components inactivated from 20 to 98% of the cells and damaged 40 to 90% of the cells surviving the treatments. Injured cells failed to form colonies on a selective medium (xylose-lysine-peptone agar with 0.2% sodium deoxycholate) but did form colonies on a nonselective plating medium (xylose-lysine-peptone agar). The major milk components-lactose, milk salts, casein, and whey proteins-influenced the extent of injury, repair of injury, and death. The percentages of cells injured and inactivated were decreased by the presence of any milk components except whey proteins. Also, repair of injury was promoted by the presence of each milk component except whey proteins, which in contrast inhibited repair. Phosphate was the most influential milk salts component that protected the cells and promoted repair of injury. These individual milk components may have decreased the extent of freezing-induced death and cellular damage by stabilizing the S. anatum cell envelope.     

Identification of a Germination System Involved in the Heat Injury of Bacillus Subtilis Spores

Bacillus subtilis A spores were injured by exposure to heat treatments of 110 to 132 C. Injury was demonstrated by the inability to form colonies on fortified nutrient agar (FNA) unless the medium was supplemented with CaCl(2) and Na(2) dipicolinate (CNA). A preliminary heat treatment fully heat-activated the spores, was not lethal, and did not prevent injury by subsequent secondary heat treatment. Exposure of heat-activated spores to 122 C reduced germination in FNA. The primary germination agents in FNA were identified, and a defined germination medium of glucose, NaCl, l-alanine, and sodium phosphate (GNAP) was developed. Germination of heat-activated spores in GNAP was equivalent to germination in FNA. Injury measured by colony formation on FNA and CNA was correlated to injury measured by reduced germination in both FNA and GNAP. Inactivation of the FNA and GNAP germination systems by secondary treatment exhibited similar kinetics. Therefore, injury expressed as the inability to form colonies on FNA involved alteration of the GNAP germination system.     

Repair of Injury Induced by Freezing Escherichia coli as Influenced by Recovery Medium

Freezing an aqueous suspension of Escherichia coli NCSM at -78 C for 10 min, followed by thawing in water at 8 C for 30 min, resulted in the death of approximately 50% of the cells, as determined by their inability to form colonies on Trypticase soy agar containing 0.3% yeast extract (TSYA). Among the survivors, more than 90% of the cells were injured, as they failed to form colonies on TSYA containing 0.1% deoxycholate. Microscope counts and optical density determinations at 600 nm suggested that death from freezing was not due to lysis of the cells. Death and the injury were accompanied by the loss of 260- and 280-nm absorbing materials from the intracellular pool. Injury was reversible as the injured cells repaired in many suitable media. The rate of repair was rapid and maximum in a complex nutrient medium such as Trypticase soy broth supplemented with yeast extract. However, inorganic phosphate, with or without MgSO₄, was able to facilitate repair. Repair in phosphate was dependent on the pH, the temperature, and the concentration of phosphate.     

ADP effects on bleomycin-induced DNA repair synthesis and adenylate kinase activity in permeable mouse sarcoma cells

DNA repair in bleomycin-pretreated, permeable mouse sarcoma (SR-C3H/He) cells requires ATP for at least two steps, the repair DNA synthesis step and the repair patch ligation step. ADP can apparently replace ATP in both steps. Maximal, 1.5-2 fold stimulation of repair DNA synthesis was observed with 5-10 mM ADP as well as 2.5-5 mM ATP. Repair patch ligation in the presence of 2.5 mM ADP occurred at almost the same high efficiency as it did in the presence of ATP. The ADP effect on DNA repair patch ligation was attributed to ATP formed from ADP by adenylate kinase in permeable cells, however the ADP effect on repair DNA synthesis could not be attributed solely to the formation of ATP in the same manner.     

Influence of postirradiation incubation temperature on recovery of radiation-injured Clostridium botulinum 62A spores.

The number of colonies formed by unirradiated Clostridium botulinum 62A spores was independent of temperature, in the range from 20 to 45 degrees C (in 5 degrees C increments); no colonies developed at 50 degrees C. Spores irradiated at 1.2 or 1.4 Mrads produced more macrocolonies at 40 degrees C than at higher or lower temperatures. Apparently, radiation-injured spores were capable of repair of 40 degrees C than at the other temperatures studied. More than 99% of the radiation (1.2 Mrads) survivors were injured and were unable to form macrocolonies in the presence of 5% NaCl. The germinated radiation-injured spores were also sensitive to dilution, resulting in the loss of viability of 77 to 79% of the radiation survivors. At 30 and 40 degrees C, the irradiated spores did not differ significantly in the extent of germination (greater than 99% at both 30 and 40 degrees C), emergence (64% at 30 degrees C and 67% at 40 degrees C), and the maximum number of emerged cells that started to elongate (69% at 30 degrees C and 79% at 40 degrees C). However, elongation was remarkably more extensive at 40 degrees C than at 30 degrees C. Many elongated cells lysed within 48 h at 30 degrees C, indicating an impaired repair mechanism. If the radiation-injured spores were incubated at 40 degrees C in the recovery (repair) medium for 8 to 10 h, they germinated, emerged, and elongated extensively and were capable of repair. If, after 8 to 10 h at 40 degrees C, these cultures were shifted to 30 degrees C, the recovery at 30 increased by more than eightfold, resulting in similar colony counts at 30 and 40 degrees C. Thus, repair appeared to be associated with outgrowth. Repair did not occur in the presence of chloramphenicol at 40 degrees C, whereas penicillin had no effect, suggesting that the repair involved protein synthesis but did not require multiplication.     

Metabolic Process During the Repair of Freeze-Injury in Escherichia coli

After Escherichia coli was injured by freezing, the repair process was studied during incubation of the cells for 2 hr at 25 C in 0.5% K(2)HPO(4) at pH 7.0 in the presence of specific metabolic inhibitors. The repair in K(2)HPO(4) was not affected by inhibitors of the synthesis of protein, nucleic acids, and mucopeptide. These inhibitors prevented growth of the repaired cells in a minimal broth at 35 C for 24 hr (except actinomycin D and hydroxyurea). Several uncouplers of adenosine triphosphate (ATP) synthesis reduced the repair process in K(2)HPO(4), but only cyanide and azide prevented growth in minimal medium. Data indicated that the cells synthesized energy in the form of ATP and probably utilized it for the repair process. Addition of ATP also facilitated the repair of injury. The freeze-injured cells showed extreme susceptibility to surface-active agents and lysozyme. The repaired cells, like the uninjured cells, became relatively resistant to these compounds.     

Characterization of Mild Thermal Stress in Pseudomonas fluorescens and Its Repair

The exposure of exponentially growing Pseudomonas fluorescens P7 cells to heating at 36 C for 2 h in a defined medium, followed by cooling to 25 C and further incubation at this, the optimal growth temperature, resulted in the apparent death of approximately 99% of the cells, as determined by their inability to form colonies on Trypticase soy agar. Continued incubation at 25 C resulted in an extremely rapid increase in the Trypticase soy agar count, demonstrating that the phenomenon observed was not death but rather injury. Presumptive evidence of heat-stimulated ribonucleic acid (RNA) degradation and membrane damage was provided by the observed loss of 260-nm absorbing materials. Confirmation of RNA degradation was obtained by colorimetric analysis. Ribosomal RNA from normal and injured cells, which was electrophoretically separated on polyacrylamide gels, revealed that the 23S and 16S species were only partially destroyed. Inhibitor studies demonstrated, however, that RNA synthesis was necessary for recovery. The unusual accumulation of 17S RNA during recovery pointed to the presence of a heat-induced lesion in the RNA maturation process. A thermally induced membrane lesion is also discussed.     

Habituation to acid in Escherichia coli: conditions for habituation and its effects on plasmid transfer.

Induction of acid resistance (habituation) in Escherichia coli at pH 5.0 took ca 5 min in broth at 37 degrees C and 30-60 min in minimal medium. Induction occurred at a range of pH values from 4.0 to 6.0; it was dependent on continuing protein and RNA synthesis but substantial acid resistance appeared in the presence of nalidixic acid. Acid resistance was long-lasting; organisms grown at pH 5.0 retained most of their resistance after 2 h growth at pH 7.0. Organisms grown at pH 5.0 showed increased synthesis of a number of cytoplasmic proteins compared with the level in cells grown at pH 7.0. DNA repair-deficient strains carrying recA, uvrA or polA1 mutations were more acid-sensitive than the repair-proficient parents but were able to habituate at pH 5.0. Organisms grown at pH 5.0 transferred the ColV plasmid much more effectively at acid pH than did those grown at pH 7.0 and habituated recipients appeared better able to repair incoming acid-damaged plasmid DNA than did those that were non-habituated. Induction of acid resistance at pH 5.0 may be significant for the survival of organisms exposed to periodic discharges of acid effluent in the aquatic environment and habituation may also allow plasmid transfer and repair of acid-damaged plasmid DNA during or after such exposure.     

Enumeration of Escherichia coli in Frozen Samples After Recovery from Injury

More than 90% of the surviving cells of Escherichia coli NCSM were injured after freezing in water at -78 C. Injury was determined by the ability of cells to form colonies on Trypticase soy agar with yeast extract but not on violet red-bile agar and deoxycholate-lactose agar. Exposure of the injured cells to Brilliant Green-bile broth and lauryl sulfate broth prevented subsequent colony formation on Trypticase soy agar with yeast extract. The freeze-injury could be repaired rapidly in a medium such as Trypticase soy broth with yeast extract (TSYB). The repaired cells formed colonies on violet red-bile agar and deoxycholate-lactose agar and were not inhibited by Brilliant Green-bile broth and lauryl sulfate broth. At least 90% of the cells repaired in TSYB within 30 min at 20 to 45 C and began multiplication within 2 h at 25 C. When the cells were frozen in different foods, 60 to 90% of the survivors were injured. Repair of the injured cells occurred in foods during 1 h at 25 C, but generally repair was greater and more reproducible when the foods were incubated in TSYB. The study indicated that the repair of freeze-injured coliform bacteria should be accomplished before such cells are exposed to selective media for their enumeration.     

The Recovery of Sublethally Injured Escherichia coli from Frozen Meat

Sublethal injury to Escherichia coli , measured as the inability of surviving cells to grow on media containing bile salts, was monitored during frozen storage on meat at —5, —10 and —20° C. More rapid increases in injury occurred at the higher subzero temperatures and log phase cells were more susceptible than those in the stationary phase of growth. Repair of injury in non-selective liquid media took between 2 and 6 h at 25° and was often accompanied by an increase in total viable count. Incubation for a fixed period in broth was, therefore, unsuitable for the quantitative recovery of freeze-injured Esch. coli. Resuscitation on membrane filters avoided confusing repair of injury with multiplication of uninjured or repaired cells. The mean recovery of injured cells following incubation on membranes for 4 h at 35°C on tryptone soya agar, was 94%.     

Activity of ATP synthetase complex after low temperature treatment or freeze-drying of mitochondria isolated from skeletal muscles

The influence of freezing, thawing, or freeze-drying on ATP synthetase complex of isolated skeletal muscle mitochondria was studied. Cooling to -60 or to -196 degrees C and rapid thawing did not change activity significantly. Slow warming stimulated the release of latent ATP-ase activity and decreased ATP synthesis. These changes were more pronounced after freeze-drying.     

Regulation of steady state level of phosphoenzyme and ATP synthesis in sarcoplasmic reticulum vesicles during reversal of the Ca2+ pump.

The role of the Ca2+ concentration gradient in ATP synthesis and membrane phosphorylation by Pi was investigated in sarcoplasmic reticulum vesicles isolated from rabbit skeletal muscle. The Pi concentration required to attain 50% of the maximal membrane phosphorylation varies significantly in the pH range of 5.5 to 4.5, the optimal being at pH 6.0. In the pH range of 6.0 to 7.0, this concentration of Pi was 4- to 10-fold higher in empty vesicles than in vesicles loaded with calcium phosphate, i.e. having transmembrane Ca2+ concentration gradient. ATP, ADP, and Ca2+ inhibit the membrane phosphorylation by Pi, the inhibition being greater at pH 7.0 than at pH 6.0. The pH profile for ATP synthesis shows a higher optimum than for membrane phosphorylation. The optimum pH for synthesis, but not for phosphorylation depends on whether the vesicles were previously loaded with calcium phosphate or with calcium oxalate. Addition of Ca2+ to the assay medium inhibits the extent of membrane phosphorylation and the rate of ATP synthesis to different extents. Evidence is presented that the rate of membrane phosphorylation by Pi is higher than the rate by which the phosphoprotein transfers its pohsphate to ADP for the ATP synthesis.     

Cold Shock Lethality and Injury in Clostridium perfringens

Several observations have been made in regard to cold shock lethality of Clostridium perfringens: (i) loss of viability was not consequence of exposure of the cells to air; (ii) stationary-phase cells were much more resistant to cold shock at 4 C than exponential-phase cells; (iii) at 4 C 96% of an initial population of exponential-phase cells was killed upon cold shock and 95% of the remaining population was killed within 90 min of continued exposure at 4 C; (iv) the minimal temperature differential for detectable cold shock lethality was between 17 and 23 C, and the maximum beyond which lethality was not appreciably increased was between 28 and 33 C. Up to 75% of viable cold-shocked cells were injured, as demonstrated by cold shocking late exponential-phase cells at 10 C and using differential plating procedure for recovery. Repair of injury was temperature dependent, and occurred in a complex medium and 0.1% peptone but not water. Nalidixic acid, chloramphenicol, and rifampin did not inhibit repair of injury.     

Substrate regulation of the sarcoplasmic reticulum ATPase. Transient kinetic studies.

The rate of phosphorylation of the Ca2+-dependent ATPase of sarcoplasmic reticulum vesicles by ITP and ATP was studied using a millisecond mixing and quenching device. The rate of phosphorylation was slower when the vesicles were preincubated in a Ca2+-free medium than when preincubated with Ca2+, regardless of the substrate used and of the pH of the medium. When the vesicles were preincubated with Ca2+ at pH 7.4 an overshoot of phosphorylation was observed in the presence of ITP. The overshoot was abolished when the pH of the medium was decreased to 6.0 or when the vesicles were preincubated in a Ca2+-free medium. Using vesicles preincubated with Ca2+ the apparent Km for ITP found was 2.5 mM at pH 6.0 and 1.0 mM at pH 7.4. The Vmax observed (77 mumol g-1 s-1) did not change with the pH of the medium. Both at pH 6.0 and 7.4 the apparent Km for ATP was 3 microM when preincubated in a Ca2+-free medium. At pH 6.0 the Vmax for ATP varied from 96 to 33 mumol g-1 s-1 depending on whether the vesicles were preincubated in the presence or absence of Ca2+. At pH 7.4 the Vmax for ATP was 90 mumol g-1 s-1 in both conditions. The rate of phosphorylation of the vesicles was dependent on the relative Ca2+ and Mg2+ concentrations of the reaction medium regardless of the substrate used.     

Spore membrane(s) as the site of damage within heated Clostridium perfringens spores.

Clostridium perfringens spores were injured by ultrahigh-temperature treatment at 105 C for 5 min. Injury was manifested as an increased sensitivity to polymyxin and neomycin. Since many of the survivors could not germinate normally the ultrahigh-temperature-treated spores were sensitized to and germinated by lysozyme. Polymyxin reportedly acts upon the cell membrane. Neomycin may inhibit protein synthesis and has surface-active properties. Injured spores were increasingly sensitive to known surface-active agents, sodium lauryl sulfate, sodium deoxycholate, and Roccal, a quaternary ammonium compound. Injured spores sensitive to polymyxin and neomycin also were osmotically fragile and died during outgrowth in a liquid medium unless the medium was supplemented with 20% sucrose, 10% dextran, or 10% polyvinylpyrrolidone. The results suggested that a spore structure destined to become cell membrane or cell wall was the site of injury. Repair of injury during outgrowth in the presence of protein, deoxyribonucleic acid, ribonucleic acid and cell wall synthesis inhibitors was consistent with this hypothesis.     

The regulation of ATPase-ATPase interactions in sarcoplasmic reticulum membrane. I. The effects of Ca2+, ATP, and inorganic phosphate

Two-dimensional crystalline arrays of Ca2+-ATPase molecules develop after treatment of sarcoplasmic reticulum vesicles with Na3VO4 in calcium-free medium (Dux, L., and Martonosi, A. (1983) J. Biol. Chem. 258, 2599-2603). The formation of Ca2+-ATPase crystals is inhibited by Ca2+ (2 microM), or ATP (5 mM), but not by ADP, 5'-adenylylimidodiphosphate, or adenylylmethylenediphosphonate. ATPase crystals did not form at 37 degrees C and exposure of preformed crystals to 37 degrees C for 1 h caused the disappearance of crystal lattice. Inorganic orthophosphate (1 mM at pH 6.0) promoted the formation of a distinct crystal form of Ca2+-ATPase, which was different from that produced by Na3VO4. These observations indicate that Ca2+, ATP, inorganic phosphate, pH, and temperature influence the interactions between ATPase molecules in the sarcoplasmic reticulum membrane.     

Simple and efficient synthesis of 5' pre-adenylated DNA using thermostable RNA ligase

We report a simple method of enzymatic synthesis of pre-adenylated DNA linkers/adapters for next-generation sequencing using thermostable RNA ligase from Methanobacterium thermoautotrophicum (MthRnl). Using RNA ligase for the reaction instead of the existing chemical or T4 DNA ligase-based methods allows quantitative conversion of 5'-phosphorylated single-stranded DNA (ssDNA) to the adenylated form. The MthRnl adenylation reaction is specific for ATP and either ssDNA or RNA. In the presence of Mg(+2), the reaction has a pH optimum of 6.0-6.5. Unlike reactions that use T4 DNA ligase, this protocol does not require synthesis of a template strand for adenylation. The high yield of the reaction simplifies isolation and purification of the adenylated product. Conducting the adenylation reaction at the elevated temperature (65°C) reduces structural constraints, while increased ATP concentrations allow quantitative adenylation of DNA with a 3'-unprotected end.