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

Abstract

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.

Inactivation ofEscherichia coliby citral

“…The mechanism of microbial inactivation by citral appears to be a complex phenomenon involving the occurrence of different types of injury. Production of energy is very often required when cell membrane or other structures are damaged by PEF (García et al 2006), freezing (Ray and Speck 1972) or HHP (Chilton et al 2001); however, the energy requirement does not reveal any kind of specific cell damage. In contrast, because in E. coli, lipids are only located in their membranes, the requirement for lipid synthesis for the repair of injury, also provides evidence that cell envelopes are one of the vital structures affected by citral.…”

Biosynthetic requirements for the repair of sublethal membrane damage in Escherichia coli cells after pulsed electric fields

“…The energy requirement does not reveal any kind of specific cell damage. In fact, production of energy is very often required when cell membrane or other structures are damaged by other physical stresses such as a freezing (Ray and Speck 1972) or high hydrostatic pressure (Chilton et al 2001). In contrast, since in procaryote cells lipids are only located in their membranes, the requirement of the lipid synthesis for the repair of the PEF-damaged cells clearly reveals that the cell envelopes are one of the vital structures affected by PEF.…”

Biosynthetic requirements for the repair of sublethally injuredSaccharomyces cerevisiaecells after pulsed electric fields

“…The energy requirement would not reveal any kind of specific cell damage. In fact, energy production is required when cell membrane or other structures are damaged by other physical stresses, such as a freezing (Ray and Speck 1972) or high hydrostatic pressure (Chilton et al 2001). As membranes are mainly composed of lipids and proteins, but lipids did not seem involved in yeast sublethal membrane damages, several specific inhibitors of metabolic processes related to nucleic acid and protein synthesis in eukaryotic organisms were also included in the study.…”

“…Repair of membrane damage can be examined by incubating injured cells in broth and by monitoring their changes in selective plating media (Mackey 2000). The addition of inhibitors of specific metabolic processes to the repair liquid medium allows to detect the biosynthetic requirements for membrane repair (Ray and Speck 1972;Mackey 2000;Chilton et al 2001;García et al 2006). Ray and Speck (1972) and Chilton et al (2001) have shown the efficacy of the presence of chloramphenicol, penicillin G, rifampicin and sodium azide, as specific inhibitors of protein synthesis, peptidoglican synthesis, RNA synthesis and energy metabolism, respectively, in the prevention of the repair of freeze-injured and high-pressure-injured Escherichia coli cells.…”

“…The addition of inhibitors of specific metabolic processes to the repair liquid medium allows to detect the biosynthetic requirements for membrane repair (Ray and Speck 1972;Mackey 2000;Chilton et al 2001;García et al 2006). Ray and Speck (1972) and Chilton et al (2001) have shown the efficacy of the presence of chloramphenicol, penicillin G, rifampicin and sodium azide, as specific inhibitors of protein synthesis, peptidoglican synthesis, RNA synthesis and energy metabolism, respectively, in the prevention of the repair of freeze-injured and high-pressure-injured Escherichia coli cells. A recent report on the study of the biosynthetic requirements for the repair of PEF-injured E. coli cells (García et al 2006) has demonstrated that a large proportion of sublethally injured cells required energy and synthesis of lipids to be repaired.…”