| Brucella spp.encounter formidable environmental stresses when interaction with the host cells.Successful adaptation depends on changes in gene expression,and these changes may happen in both transcription level and post-transcription level.Compared to widely studies in transcriptional regulation,only a limited number of studies have performed on post-transcriptional regulation（especially small RNAs）in Brucella spp..In this study,two sRNA prediction programs were used for the genome-wide prediction of sRNA candidates in the B.abortus 2308 genomes,and 129 sRNA candidates were identified.The prediction result was tested by RT-PCR,and 50 could be verified.The putative targets of these candidates were also predicted and the results indicated that sRNAs mainly participated in regulating expressions of genes involved in biofilm formation,amino acid metabolism,post transcriptional modification,signal transduction,iron metabolism and transcriptional regulation.To study the expression patterns of sRNAs,the expression levels of twenty-two sRNAs were detected at different growth stages and various stress conditions.The results demonstrated that the expression levels of twenty-one sRNAs changed at different growth stages and the expression levels of all sRNAs were also affected under various stress conditions.To study how the expression of sRNAs were regulated,the mutants of vjbR,rpoE1,rpoE2,and hfq were constructed,and the expression levels of sRNAs were also tested in these mutants and wide type strain.The results demonstrated that VjbR,RpoEl,RpoE2 and Hfq could affect the expression of 16,20,10 and 7 sRNAs respectively.According to the studies of sRNAs’ targets,We found an sRNA that could inhibit the expression of hemH and called it BsrH（Brucella sRNA regulating HemH）.The expression level of BsrH was tested in four different stress conditions.A significant up-regulation was detected during the growth in acidic and Brucella minimal media,as well as in the presence of hydroxyl peroxide,while iron deficiency caused an opposite effect.BsrH strongly affected the survival of the Brucella cells under iron-limitation conditions,though over-expression of BsrH did not affect Brucella virulence.Thus,we conclude that BsrH plays a regulatory role in bacterial iron metabolism.To identify virulence related sRNAs,50 sRNA over-expression strains were constructed and analyzed using the mouse macrophage-like cell line J774A.1.The results demonstrated that one sRNA（BASRC Ⅰ 74）was involved in Brucella virulence in macrophages infection model.To study why BASRC Ⅰ 74 over-expression strain was attenuated in vitro,the stress assays were performed and the target of BASRC Ⅰ 74 was also predicted.BASRC Ⅰ 74 strongly affected the survival of the Brucella cells in Brucella minimal media and iron limiting medium.Through a two-plasmid report system,a target genes BAB 11154 of BASRC Ⅰ 74 was found.BABI1154 encoded a cytosine-N4-specific DNA-methyltransferase,which protected the cell DNA from the restriction endonuclease in bacterial.Thus,we concluded that BASRC Ⅰ 74 played an important role in Brucella survival ability in macrophage infection model,which was possibly caused by its connection of stress response or impact on restriction-modification system.Altogether,this study paved the way for future work aimed at sRNAs related post-transcriptional regulation and pathogenic mechanism in Brucella spp.. |
