Proteolysis in dairy lactic acid bacteria has been studied in great detail by genetic, biochemical and ultrastructural methods. From these studies the picture emerges that the proteolytic systems of lactococci and lactobacilli are remarkably similar in their components and mode of action. The proteolytic system consists of an extracellularly located serine-proteinase, transport systems specific for di-tripeptides and oligopeptides (> 3 residues), and a multitude of intracellular peptidases. This review describes the properties and regulation of individual components as well as studies that have led to identification of their cellular localization. Targeted mutational techniques developed in recent years have made it possible to investigate the role of individual and combinations of enzymes in vivo. Based on these results as well as in vitro studies of the enzymes and transporters, a model for the proteolytic pathway is proposed. The main features are: (i) proteinases have a broad specificity and are capable of releasing a large number of different oligopeptides, of which a large fraction falls in the range of 4 to 8 amino acid residues; (ii) oligopeptide transport is the main route for nitrogen entry into the cell; (iii) all peptidases are located intracellularly and concerted action of peptidases is required for complete degradation of accumulated peptides.

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The proteolytic systems of lactic acid bacteria

The conversion of peptides to free amino acids and their subsequent utilization is a central metabolic activity in prokaryotes. At least 16 peptidases from lactic acid bacteria (LAB) have been characterized biochemically and/or genetically. Among LAB, the peptidase systems of Lactobacillus helveticus and Lactococcus lactis have been examined in greatest detail. While there are homologous enzymes common to both systems, significant differences exist in the peptidase complement of these organisms. The characterization of single and multiple peptidase mutants indicate that these strains generally exhibit reduced specific growth rates in milk compared to the parental strains. LAB can also catabolize amino acids produced by peptide hydrolysis. While the catabolism of amino acids such as Arg, Thr, and His is well understood, few other amino acid catabolic pathways from lactic acid bacteria have been characterized in significant detail. Increasing research attention is being directed toward elucidating these pathways as well as characterizing their physiological and industrial significance.

Peptidases and amino acid catabolism in lactic acid bacteria

Microbial stress response in minimal processing

High hydrostatic pressure has the potential to produce high quality foods that are microbiologically safe and with an extended shelf-life. Micro-organisms vary in their response to high pressure. Bacterial spores are the most resistant group and they cannot be significantly inactivated by pressure alone. Combination treatments using high pressure and heat have been proposed as a method of producing shelf-stable low acid foods. Viruses are less resistant than bacterial spores and their infectivity can be abolished without destroying their ability to elicit antibodies, leading to the possibility of vaccine production. Yeasts, moulds and vegetative bacteria vary in their response to pressure, depending on factors such as species, strain, processing temperature and substrate, and these are reviewed in the paper. A knowledge of how these factors interact is necessary in order to select the optimum processing conditions for foods. A number of pressure-treated foods are already commercially available and these are discussed in the paper.

https://sfamjournals.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1365-2672.2005.02564.x

Microbiology of pressure‐treated foods

Monitoring of verotoxigenic Escherichia coli (VTEC) and identification of human pathogenic VTEC types 1 Scientific Opinion of the Panel on Biological Hazards

The relationship among growth temperature, membrane fatty acid composition, and pressure resistance was examined in Escherichia coli NCTC 8164. The pressure resistance of exponential-phase cells was maximal in cells grown at 10°C and decreased with increasing growth temperatures up to 45°C. By contrast, the pressure resistance of stationary-phase cells was lowest in cells grown at 10°C and increased with increasing growth temperature, reaching a maximum at 30 to 37°C before decreasing at 45°C. The proportion of unsaturated fatty acids in the membrane lipids decreased with increasing growth temperature in both exponential- and stationary-phase cells and correlated closely with the melting point of the phospholipids extracted from whole cells examined by differential scanning calorimetry. Therefore, in exponential-phase cells, pressure resistance increased with greater membrane fluidity, whereas in stationary-phase cells, there was apparently no simple relationship between membrane fluidity and pressure resistance. When exponential-phase or stationary-phase cells were pressure treated at different temperatures, resistance in both cell types increased with increasing temperatures of pressurization (between 10 and 30°C). Based on the above observations, we propose that membrane fluidity affects the pressure resistance of exponential- and stationary-phase cells in a similar way, but it is the dominant factor in exponential-phase cells whereas in stationary-phase cells, its effects are superimposed on a separate but larger effect of the physiological stationary-phase response that is itself temperature dependent.

https://aem.asm.org/content/aem/68/12/5965.full.pdf

Role of membrane fluidity in pressure resistance of Escherichia coli NCTC 8164.

Summary: Differential scanning calorimetry of whole Escherichia coli cells allowed the detection in vivo of changes in ribosome conformation. This enabled for the first time an analysis of the effects of high hydrostatic pressures on ribosomes in living cells. A correlation was observed between loss of cell viability and decrease in ribosome-associated enthalpy in cells subjected to pressures of 50-250 MPa for 20 min. Cell death and ribosome damage were therefore closely related phenomena. In pressure-treated cells, the thermogram peak temperatures decreased, suggesting that the remaining ribosomes had adopted a less stable conformation. During subsequent incubation of the cultures at 37°C, peak temperatures and enthalpies gradually increased over a period of 5 h. This change in ribosome conformation had no apparent effect on cell survival, as viability continued to decrease. The addition of 5 mM MgCl2 before pressure treatment of cells prevented the reduction in stability of surviving ribosomes but had no effect on the initial loss of enthalpy or on cell viability.

The effects of hydrostatic pressure on ribosome conformation in Escherichia coli: an in vivo study using differential scanning calorimetry

An aminopeptidase A (EC 3.4.11.7) was purified from Lactococcus lactis subsp. lactis NCDO 712. Of the 18 aminoacyl-alanine dipeptides tested, the enzyme hydrolysed Asp-Ala, Glu-Ala and Ser-Ala. It was also active against tripeptide substrates but did not hydrolyse Ala-Asp or Ala-Glu. The kinetics of dipeptide hydrolysis were allosteric with positive cooperativity of substrate binding. The Hill constants were 1.52 for Asp-Ala, 1.51 for Glu-Ala and 1.61 for Ser-Ala. The M
r was found to be 245000 by gel-filtration chromatography. A single band corresponding to an M
r of 41 000 was detected by SDS-PAGE of the purified enzyme which indicated that the enzyme is hexameric. Activity against glutamate p-nitroanilide was optimum at 65 °C and pH 8. Activity was inhibited by 1 mM-EDTA, implying that the enzyme is metal-containing. Cu2+, Mn2+ and Zn2+ (all at 1 mM) inhibited activity and Co2+ was stimulatory.

Purification and characterization of aminopeptidase a from lactococcus lactis subsp. lactis ncdo 712

The distributions of times to first cell division were determined for populations of Escherichia coli stationary-phase cells inoculated onto agar media. This was accomplished by using automated analysis of digital images of individual cells growing on agar and calculation of the “box area ratio.” Using approximately 300 cells per experiment, the mean time to first division and standard deviation for cells grown in liquid medium at 37°C and inoculated on agar and incubated at 20°C were determined as 3.0 h and 0.7 h, respectively. Distributions were observed to tail toward the higher values, but no definitive model distribution was identified. Both preinoculation stress by heating cultures at 50°C and postinoculation stress by growth in the presence of higher concentrations of NaCl increased mean times to first division. Both stresses also resulted in an increase in the spread of the distributions that was proportional to the mean division time, the coefficient of variation being constant at approximately 0.2 in all cases. The “relative division time,” which is the time to first division for individual cells expressed in terms of the cell size doubling time, was used as measure of the “work to be done” to prepare for cell division. Relative division times were greater for heat-stressed cells than for those growing under osmotic stress.

Influence of environmental stress on distributions of times to first division in Escherichia coli populations, as determined by digital-image analysis of individual cells.

Ribosome modulation factor (RMF) was shown to have an influence on the survival of Escherichia coli under acid stress during stationary phase, since the viability of cultures of a mutant strain lacking functional RMF decreased more rapidly than that of the parent strain at pH 3. Loss of ribosomes was observed in both strains when exposed to low pH, although this occurred at a higher rate in the RMF-deficient mutant strain, which also suffered from higher levels of rRNA degradation. It was concluded that the action of RMF in limiting the damage to rRNA contributed to the protection of E. coli under acid stress. Expression of the rmf gene was lower during stationary phase after growth in acidified media compared to media containing no added acid, and the increased rmf expression associated with transition from exponential phase to stationary phase was much reduced in acidified media. It was demonstrated that RMF was not involved in the stationary-phase acid-tolerance response in E. coli by which growth under acidic conditions confers protection against subsequent acid shock. This response was sufficient to overcome the increased vulnerability of the RMF-deficient mutant strain to acid stress at pH values between 6.5 and 5.5.

Gordon W. Niven

Papers

Role of membrane fluidity in pressure resistance of Escherichia coli NCTC 8164.

The effects of hydrostatic pressure on ribosome conformation in Escherichia coli: an in vivo study using differential scanning calorimetry

Purification and characterization of aminopeptidase a from lactococcus lactis subsp. lactis ncdo 712

Influence of environmental stress on distributions of times to first division in Escherichia coli populations, as determined by digital-image analysis of individual cells.

The influence of ribosome modulation factor on the survival of stationary-phase Escherichia coli during acid stress.