Resistance to Actinomycin D of Escherichia Coli After Frozen Storage

Bretz, Harold W.; Kocka, Frank E.

doi:10.1139/m67-121

Cited by 18 publications

(9 citation statements)

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“…However, this surface alteration was repaired quickly, and the repaired cells again became impermeable to these molecules. Similar permeability losses due to different sublethal stresses including freeze-drying have been reported by many workers (4,13,15,20,26,29). Chloramphenicol and tetracycline inhibit protein synthesis (25).…”

Section: Incubation Was At 25 Csupporting

confidence: 70%

Repair of Injury in Freeze-Dried Salmonella anatum1

Ray

Jezeski

Busta

1971

Applied Microbiology

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Repair of injury induced by freeze-drying Salmonella anatum in nonfat milk solids occurred rapidly after rehydration. Injury in surviving cells was defined as the inability to form colonies on a plating medium containing deoxycholate. Death was defined as inability to form colonies in the same medium without this selective agent. The rate of repair of injury was reduced by lowering the temperature from 35 C to 10 C and was extremely low at 1 C. Repair was independent of influence of pH between 6.0 and 7.0. Repair did not require synthesis of protein, ribonucleic acid, or cell wall mucopeptide, but did require energy in the form of adenosine triphosphate (ATP) synthesized through oxidative phosphorylation. The requirement for ATP was based on dinitrophenol or cyanide interference with repair. Dinitrophenol activity was pH-dependent; no repair occurred at pH 6.0 and some repair was observed at pH 6.5 and above. Injured cells were extremely sensitive to low concentrations of ethylenedinitrilotetraacetate. This indicated that freezedrying injury of S. anatum may involve the lipopolysaccharide portion of the cell wall and that repair of this damage requires ATP synthesis.

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Section: Incubation Was At 25 Csupporting

confidence: 70%

Repair of Injury in Freeze-Dried Salmonella anatum1

Ray

Jezeski

Busta

1971

Applied Microbiology

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“…Chemicals. The following compounds were used in this study: penicillin G sodium (1,650 units/mg), rifampin, and 2, 4-dinitrophenol (Mann Research Lab, New York, N.Y.); D-cycloserine, streptomycin sulfate, actinomycin D, lysozyme from egg white (1,800 units/mg), ATP disodium salt (Schwarz Mann, Orangeburg, N.Y.); chloramphenicol, kanamycin, 5-fluorouracil, hydroxyurea, gramicidin, oligomycin, sodium lauryl sulfate (Sigma Co., St. Louis, Mo. ); tetracycline-hydrochloride and puromycin (Nutritional Biochemicals Corp., Cleveland, Ohio); sodium cyanide, sodium azide, sodium deoxycholate (Fisher Scientific Co., Fairlawn, N.J.).…”

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confidence: 99%

Metabolic Process During the Repair of Freeze-Injury in Escherichia coli1

Ray¹,

Speck²

1972

Applied Microbiology

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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% K2HPO4 at pH 7.0 in the presence of specific metabolic inhibitors. The repair in K 2HPOa 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 K2HPO,, 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. Injury induced by freezing and other sublethal stresses in gram-negative bacteria can be repaired in the presence of many simple and complex compounds. The cellular events which occur during the repair process involve the synthesis of: ribonucleic acid (RNA) and proteins in freeze-dried Escherichia coli (17); adenosine triphosphate (ATP) in freeze-dried Salmonella anatum (13); lipids and proteins in frozen E. coli (D. A. Gabis, Ph.D. thesis, North Carolina State University, Raleigh, 1970); ATP in frozen S. anatum (12); RNA, proteins, and ATP in heat-shocked S. typhimurium (19); ATP in ethylenediaminetetraacetate (EDTA)and N-hydroxyurethan-treated E. coli (6, 9). The repair process, in most cases, has been studied in media which also facilitate growth. In a previous paper (14) we reported that frozen E. coli cells repaired their injury in the presence of compounds which by themselves are unable to support growth, such as in K2HPO4 and sodium pyruvate. Similar observations have also been reported for freezeinjured S. anatum (12). In this report we describe the possible nature of the metabolic process that occurs during the repair of freezeinjury in E. coli cells in the presence of K2HPO4. ' Paper no. 3784 of the Journal Series of the North Carolina State Agricultural Experiment Station, Raleigh, N.C.

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“…The damage to the stressed cells' permeability barriers is conclusively evidenced by their sensitivity to 40 jig/ml lysozyme as similarly demonstrated by Kohn (1960) in E . coli cells subjected to freeze-thawing, 50 pg/ml actinomycin D (Bretz & Kocka 1967) and 100 pg/ml ribonuclease (Postgate & Hunter 1963;Ray & Speck 1973). The observed lysozyme sensitivity is therefore indicative of damage to the lipopolysaccharide barrier that is generally considered to offer protection in Gram-negative cells to the otherwise sensitive inner located peptidoglycancontaining layer on which the lysozyme acts (Ray & Speck 1973;Souzu 1980).…”

Section: Discussionmentioning

confidence: 99%

“…The observed lysozyme sensitivity is therefore indicative of damage to the lipopolysaccharide barrier that is generally considered to offer protection in Gram-negative cells to the otherwise sensitive inner located peptidoglycancontaining layer on which the lysozyme acts (Ray & Speck 1973;Souzu 1980). Furthermore, actinomycin D, an RNAinhibiting antibiotic (Russell 1984), is considered incapable of penetrating the Gram-negative cells unless there has been structural damage to the permeability barriers (Bretz & Kocka 1967). Such damage would have allowed cellular penetration by actinomycin D leading to inactivation of RNA systems and would thus result in the observed loss of colony forming ability that is reflected in the viability reduction obtained for stressed cells.…”

Section: Discussionmentioning

confidence: 99%

Mechanisms of triple stress‐mediated damage in stationary phase cells of Salmonella typhimurium exposed to succinate‐acidified hypochlorite system at 5° C

Obafemi

Davies

1986

Journal of Applied Bacteriology

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Exposure for 20 min of stationary phase cells of Salmonella typhimurium to a combined triple stress system (TSS) treatment comprising hypochlorite derived 5 ppm free available chlorine in solution acidified with 1% succinate (pH 2.5) and at a chill shock temperature of 5 degrees C resulted in symptoms of injury. Cells became sensitive to 40 micrograms/ml lysozyme, 50 micrograms/ml actinomycin D and 100 micrograms/ml ribonuclease B, to which control cells were resistant. Metabolic injury was indicated by reduction in colony forming ability of stressed cells on minimal salts glucose agar M9 medium. There was no detectable leakage loss of 260-280 nm-absorbing materials. This was also confirmed by assay of the cellular RNA material components. Loss of alkaline phosphatase activity was observed in the stressed cells. The intensity of induced cellular damage as measured by lysozyme sensitivity was greatest in the cells exposed to the complete TSS, followed by those stressed in 1% succinate at 5 degrees C, then 5 ppm chlorine at 5 degrees C and the singular chill shock stress at 5 degrees C, respectively. The magnitudes of cellular damage, however, were suggestive of synergistic interactions among the component stress factors of the TSS. The findings obtained indicated impairment of the structural integrity and functional capabilities of the permeability barriers and the inactivation of certain periplasmic enzymes. The resultant cumulative cellular damage from the TSS exposure may therefore enhance greater sensitivity of treated cells to subsequent stress factors.

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Resistance to Actinomycin D of Escherichia Coli After Frozen Storage

Cited by 18 publications

References 0 publications

Repair of Injury in Freeze-Dried Salmonella anatum1

Repair of Injury in Freeze-Dried Salmonella anatum1

Metabolic Process During the Repair of Freeze-Injury in Escherichia coli1

Mechanisms of triple stress‐mediated damage in stationary phase cells of Salmonella typhimurium exposed to succinate‐acidified hypochlorite system at 5° C

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