The use of lux enzymes to investigate the association between irreversible protein denaturation and pressure-mediated inactivation of Escherichia coli
Abstract
Pressure processing represents an effective technology for food preservation as it eliminates undesired microorganisms while retaining foods nutritional and organoleptic properties. The proposed mechanisms behind pressure-mediated bacterial inactivation include protein denaturation, membrane perturbation and oxidative stress. The overall objective of this research was to use bioluminescence as an approach to investigate the association between protein denaturation and the onset of loss of cell viability. A laboratory scale hydrostatic pressure system coupled with a photomultiplier tube was used for in situ photon detection from Escherichia coli cells carrying plasmids containing either luxCDABE or luxAB genes from Vibrio fischeri or Photorhabdus luminescens. The specific objectives of this study were: (i) evaluate pressure (0.1, 50, 100 and 150 MPa) and thermal (30, 35, 40 and 45°C) effects on Escherichia coli lux; (ii) determine growth media (LB and M9-glucose) effects on bacterial pressure resistance; (iii) determine RpoS sigma factor involvement in the regulation of pressure stress response in exponentially growing cells. Findings suggest that the denaturation and renaturation of the different lux enzyme systems proceed in a similar manner as in thermal denaturation. Higher pressure and temperature levels result in lower bioluminescence emission and irreversible enzyme denaturation. Interestingly, Escherichia coli in exponential growth phase showed 2 to 4 log reductions at 150 MPa in LB, whereas no inactivation was observed in M9-glucose media. The lack of inactivation in M9-glucose media was attributed to the consequences of nutrient starvation such as the activation of the RpoS sigma factor that induces the transcription of stress genes, providing a survival mechanism for the cells. However, when an Escherichia coli rpoS mutant strain was exposed to the same pressure conditions, bioluminescence and bacterial survival were not significantly different as compared to the wild type. Therefore, it is possible that an alternative sigma factor is responsible for the transcriptional regulation of genes needed for survival under pressure stress. In conclusion, enzyme denaturation represents the initial step towards pressure-mediated bacterial inactivation, confirmed in this study by the irreversible loss of bioluminescence and viability in Escherichia coli cells.
Degree
Ph.D.
Advisors
San Martin-Gonzalez, Purdue University.
Subject Area
Molecular biology|Food Science|Microbiology
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