Food-Related Gram-Positive Bacteria: Enterotoxin A Expression in Staphylococcus aureus and a New Regulation Mechanism in Lactococcus lactis

Sammanfattning: Staphylococcal enterotoxin A (SEA) is the most common enterotoxin found in outbreaks of staphylococcal food poisoning (SFP). Based on the amount of SEA produced, S. aureus strains were divided into two main groups, high- and low-SEA-producing strains. The differences in the amounts of SEA formed were found to be associated with the levels of expression of the sea gene and the bacteriophage version of sea, i.e. sea1 or sea2. Furthermore, differences in the nucleotide sequence of the Siphoviridae phage region showed two clonal lineages of the high-SEA-producing strains. One of these lines was associated with the capacity for a massive release of SEA through prophage induction, as demonstrating using mitomycin C (MC). This was also established by the detection of additional sea mRNA transcripts presumed to be initiated by a latent phage promoter located upstream of the endogenous sea promoter. The low-SEA-producing group and the high-SEA-producing subgroup lacking prophage-activated transcript of sea showed no increase in the production of SEA after the addition of MC. Thus, sea gene expression was found to be associated with the clonal lineage of the sea-encoding Siphoviridae phages. During the processing and preservation of food, organic acids such as acetic acid and lactic acid are widely used. The effects of acetic acid on the expression and formation of SEA in S. aureus were investigated in pH-controlled batch cultures carried out with two strains belonging to two clonal lineages of the high-SEA-producing strains. The sea expression profiles of both strains were comparable, with the relative expression peaking in the transition between exponential and stationary growth phase and falling during stationary phase at all pH values tested. The elevated sea expression in S. aureus was also found to be influenced by acetic acid, which induced the sea-encoding prophage and increased the intracellular copy number of the sea gene, linking SEA formation to the life cycle of the phage. The expression and formation of SEA was also studied in situ in pork sausage at low temperature (15°C). Three strains with different sea genetic backbones, the two clonal lineages of the high-SEA-producing strains (sea1) and the low-producing-strains (sea2), respectively, were used. Prolonged periods of expression and formation of SEA—of at least fourteen days—were found relative to the short growth-associated periods of SEA production observed in planktonic batch cultivations. The growth patterns were similar in all three strains; however, a clear difference in SEA levels between the two high-SEA-producing strains and the low-SEA-producing strain was seen. More importantly, the SEA formation of the high-SEA-producing strain harbouring the prophage-inducible sea1 gene was about three times higher than in the high-SEA-producing strain lacking phage-activated sea transcription. These results were in agreement with the previous prophage-activation results using MC. Without MC, this difference was not seen, indicating on-going prophage induction in situ. The complex food matrix of the frankfurter sausage and low temperature most likely influenced the formation of SEA in a way similar to that in MC treatment. In food, the high-SEA-producing group, and in particular the prophage-inducible sea1 group, may be more relevant in SFP than the low-SEA-producing group, which mainly harbours sea2. Environmental factors, including many preservatives and unfavourable temperature, can stimulate the expression and formation of SEA. Lactococcus lactis is very commonly used in the manufacture of many food products and as a biopreservative in food. The main function of L. lactis in food is to lower the pH by producing acids, e.g. formate, acetate, and lactate. A regulation mechanism that contributes to readjustment of the flux of ATP production in L. lactis was investigated. To varying degrees, ATP and ADP inhibit several dehydrogenases of the central carbon metabolism of Lactococcus lactis ATCC 19435 in vitro, i.e. glyceraldehyde-3-phosphate dehydrogenase (GAPDH), lactate dehydrogenase (LDH), and alcohol dehydrogenase (ADH). Inhibition types and parameters by single and multiple inhibitors were determined; a mathematical model using Hill-type kinetics was introduced and developed, and showed greater flexibility than the usual parabolic inhibition equation. Model discrimination suggested that the weak allosteric inhibition of GAPDH had no relevance when multiple inhibitors were present. Interestingly, with ADH and GAPDH, the combination of ATP and ADP showed lower dissociation constants than with either inhibitor alone. Moreover, the concerted inhibition of ADH and GAPDH, but not of LDH, showed synergy between the two nucleotides. Similar kinetics, but without synergies, were found for ADHs from horse liver and yeast, indicating that dehydrogenases can be modulated by these nucleotides in a non-linear manner in many organisms. The action of an elevated pool of ATP and ADP may effectively inactivate lactococcal ADH, but not GAPDH and LDH, providing leverage for the observed metabolic shift to homolactic acid formation in resting lactococcal cells supplied with maltose.

  Denna avhandling är EVENTUELLT nedladdningsbar som PDF. Kolla denna länk för att se om den går att ladda ner.