Physiological Changes And The Difference Between The Transcriptome Expression Of Sublethal Staphylococcus Aureus Under Frozen Stress

The experiment by Staphylococcus aureus (ATCC6538) as the research object, to-18℃condition for research and study of Staphylococcus aureus under different freezing timeand the sublethal rate and physiological index change rule, at the same time use RNA-seqsequencing technologies compared the sublethal Staphylococcus aureus under frozen stress andnormal strain of the transcription of all information, then preliminary study the sublethalStaphylococcus aureus molecular mechanisms under-18℃. The main research content was asfollows:The Staphylococcus aureus frozen under-18℃shows a decrease in survival rate within therange from0-180d at first, and then maintains the trend. The survival rate drops remarkablywithin0-90d, and shows no obvious decrease within the range of90-180d; and the sublethalityrate increases in the beginning and then keeps the trend, which reaches over99%when frozen to90d and shows no distinct change along with the progress of freezing duration. The resultsdemonstrate that the Staphylococcus aureus, under the normal condition at-18℃lost its vitalitymainly in the earlier stage of freezing, and the sublethal cells do not increase with the extension offreezing duration, proving that the tolerance toward freeze increases dramatically.Through tests of conductivity, protein leakage, contents of total sugar and the cell membranebreakage of bacterial cells during different frozen period, we find that the frozen Staphylococcusaureus under normal conditions would undergo apoptosis because of the leakage of insolublesubstances from the cell structure; and the Staphylococcus aureus sublethal because of the frozenthreat could maintain a complete cell structure and metabolic activities, thus surviving for a longtime under low temperatures.The comparison between sublethal samples and the normal samples in terms of transcriptioninformation through RNA-seq discovered66differential genes, of which45are up-regulatedwhile21are down-regulated. Among the differential genes tested by Real time-PCR, the energymetabolism-related genes, such as cytB shows a trend of down regulation, which might be causedby the minimum level of energy consumption that the sublethal cells keeps in order to maintainvital activities under the threat of low temperature for a long time. Some amino acidmetabolism-related genes, such as serine also show obvious down-regulation. For thestress-related genes, such as pressure stress, acid salt stress and light stress show the trend ofup-regulation. Among them, the cold-shock proteins （CspLA） function genes are alsoup-regulated remarkably as an important way to help maintain its functions under the adverseenvironment. The virulence factors of msrR and clfA are also up-regulated, showing that theStaphylococcus aureus, though under freezing threat for a long duration, remains pathogenic, thusposing threat against the safety of frozen food. On the other hand, norB, an efflux pump ofMSF-the major tolerance channel of Staphylococcus aureus, shows a trend of down regulation, alikely consequence of the weaker tolerance toward some drug of the sublethal Staphylococcusaureus under freezing threat. From the conclusions above, the paper gets a preliminaryunderstanding of the survival and physiological phenomenon of the sublethal Staphylococcus aureus under freezing threat. The sublethal cells might form a molecule-regulation networkagainst the freezing stress through the coordination of multiple genes. The results might be helpfulfor the risk evaluation of food safety and the improvement of food control techniques based uponthe molecule mechanism during food processing and delivery.