Thermal denaturation of whole cells and cell components of Escherichia coli examined by differential scanning calorimetry.
Thermograms of whole cells of Escherichia coli obtained by differential scanning calorimetry contained ten main peaks (denoted f, l, m1, m2, m3, n, p, q, r and s) occurring at temperatures of approximately 25, 54, 61, 71, 76, 81, 95, 105, 118 and 124 degrees C, respectively. After cooling to 5 degrees C and reheating, peaks denoted fr, mr and pr were observed at 23, 73 and 94 degrees C, respectively. By examining thermograms of different cell fractions we have identified the following thermal denaturation events. During primary heating there is a broad endotherm (f) beginning below 20 degrees C and extending to just above 40 degrees C that is caused by melting of membrane lipids. Superimposed on this is an exothermic process associated with a change of state of the peptidoglycan. The first irreversible denaturation event occurs just above 47 degrees C, associated with the onset of denaturation of the 30S ribosomal subunit and soluble cytoplasmic proteins. Ribosome melting is a complex process occurring between 47 and 85 degrees C and is characterized by peaks m1, m2 and n. Peak m3 at 75-76 degrees C is of unknown identity but may possibly represent melting of tRNA. Peak p at 95 degrees C results from melting of a portion of the cellular DNA combined with denaturation of a cell wall component. Peak q at 105 degrees C is multicomponent and may be caused by melting of a different region of DNA together with denaturation of another cell wall component. The complex events denoted r and s at 118 and 125 degrees C, respectively, are associated with denaturation of a component of the cell envelope, and possibly also of DNA. Following cooling and reheating there is a broad endotherm with a maximum at 23 degrees C caused by remelting of membrane lipid and a very broad endotherm extending between 40 and 100 degrees C caused by the remelting of ribosomal RNA. Peak pr at 94 degrees C is caused by the melting of reannealed DNA. Additional features not appearing in whole cells were evident in some cell fractions. These observations should allow us to distinguish events that may lead to loss of viability from those that do not.
Mackey
B M
BM
AFRC Institute of Food Research, Bristol Laboratory, Langford, UK.
Miles
C A
CA
Parsons
S E
SE
Seymour
D A
DA
D001426:Bacterial Proteins / Q000737:chemistry; D002152:Calorimetry, Differential Scanning; D002458:Cell Fractionation; D002462:Cell Membrane / Q000737:chemistry; D004269:DNA, Bacterial / Q000737:chemistry; D004926:Escherichia coli / Q000737:chemistry*; D006358:Hot Temperature; D008563:Membrane Lipids / Q000737:chemistry; D009691:Nucleic Acid Denaturation; D011489:Protein Denaturation; D012329:RNA, Bacterial / Q000737:chemistry; D012270:Ribosomes / Q000737:chemistry
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D008563:Membrane Lipids; D012329:RNA, Bacterial