| Staphylococcus aureus is one of the most important pathogenic bacteria of contaminated frozen food,which is easy to cause great economic losses and adverse social effects.The objects of this research were Staphylococcus aureus and the same strain with but the luxS gene was deleted,The repair and growth of ?luxS frozen sublethal cells and stable prophase cells under different acidic conditions and their growth and the fate in acidic juice food were studied,At the same time,the ultrastructural changes were observed by scanning and transmission electron microscopy,and the difference of genome-wide transcriptional expression profile of cryopreserved sublethal cells during repair and initiation was analyzed by transcriptome sequencing(RNA-seq),It provides a theoretical basis for further exploring the mechanism of repair and initiation of sublethal cells induced by freezing of Staphylococcus aureus,The main findings were as follows:After frozen,the sublethal wild type of Staphylococcus aureus and its mutant?luxS cells survived in the form of small colony cells,that is,SCVs cells;The resistance to 10%NaCl concentration was significantly decreased,and ?luxS cells had the weakest resistance to NaCl;At 37? and the PBS condition of pH7.0,the repair could be completed well,but in pH 5.0 and pH3.5 the repair couldn't be completed,the resistance of cells to acid decreased more significantly after frozen treatment,especially in pH3.5 the mutant decreased by 3.39 log and the control decreased by 1.89 log;In the same concentration of acid(0.04m)and different concentrations of organic acid solution,it was inhibited,especially in the frozen cells of mutant ?luxS,the decrease of malic acid was particularly significant;Freezing treatment not only enhanced the tolerance of cells to ascorbic acid and lactic acid,but also enhanced the viability of mutant ?luxS sublethal cells in grape juice,orange juice and apple juice,especially in orange juice refrigerated at 4?.In this study,transmission electron microscopy(TEM)was used to observe the ultrastructural and morphological changes of Staphylococcus aureus wild bacteria and mutant ?luxS treated with 40mg/mL ascorbic acid(-18? for 50 days),It was found that the appearance of the cells treated with ascorbic acid changed obviously,frozen cells showed more significant changes,the cells overflowed with the contents of the hole,and the outline of the cell was blurred.Scanning electron microscopy(SEM)and transmission electron microscopy(TEM)were used to observe the morphological changes of Staphylococcus aureus after 2 hours of repair,The results showed that after TSBYE repair for 2 hours,the cell morphology of Staphylococcus aureus would gradually recover and had little difference from the cell morphology at the later stage of the index,but the cell surface became more sturdy and compact.Transcriptional sequencing(RNA-seq)was used to compare and analyze the differences of genome-wide transcriptional expression profiles of frozen sublethal cells during the initiation of ascorbic acid stress tolerance repair.In this study,ascorbic acid significantly decreased the survival of wild type and mutant ?luxS cells of Staphylococcus aureus with sublethal injury induced by freezing,accompanied by abnormal morphological changes.Transcriptome analysis showed that 431 genes were differentially expressed after the cells were treated with ascorbic acid,Including Staphylococcus aureus wild type and its mutant index late stage cell(1zc-2zc),Staphylococcus aureus mutant index later stage and its frozen treatment cell(2zc-21d),wild type and its mutant frozen cell(1ld-2ld)and wild type index later and its frozen cells(1zc-lld),There were 67,341,5 and 217 differential genes,respectively;The GO enrichment analysis of differentially expressed genes showed that ascorbic acid was the main factor affecting the morphological changes,cell metabolism,amino acid metabolism and biological process changes of bacterial cells;Through the analysis of KEGG metabolic pathway,there were 6,1,0 and 12 significant metabolic pathways in 1zc-2zc,2zc-21d,1ld-21d and 1zc-1ld,respectively.The synthesis and metabolism of amino acids play an important role in the metabolic pathway of differentially expressed genes during the initiation of frozen under ascorbic acid stress.Therefore,the change of resistance may be closely related to the regulation of luxS gene.In conclusion,through transcriptome analysis,the number of differentially expressed genes between ascorbic acid-induced luxS gene deletion mutants and wild strain cells was even smaller after freezing treatment.These results further suggest that luxS gene plays an important regulatory role in acid stress tolerance of Staphylococcus aureus. |
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