We characterized the sae operon, a global regulator for virulence gene expression in Staphylococcus aureus. A Tn917 sae mutant was obtained by screening a Tn917 library of the agr mutant ISP479Mu for clones with altered hemolytic activity. Sequence analysis of the sae operon revealed two additional open reading frames (ORFs) (ORF3 and ORF4) upstream of the two-component regulatory genes saeR and saeS. Four overlapping sae-specific transcripts (T1 to T4) were detected by Northern blot analysis, and the transcriptional initiation points were mapped by primer extension analysis. The T1, T2, and T3 mRNAs are probably terminated at the same stem-loop sequence downstream of saeS. The T1 message (3.1 kb) initiates upstream of ORF4, T2 (2.4 kb) initiates upstream of ORF3, and T3 (2.0 kb) initiates in front of saeR. T4 (0.7 kb) represents a monocistronic mRNA encompassing ORF4 only. sae-specific transcripts were detectable in all of the 40 different clinical S. aureus isolates investigated. Transcript levels were at maximum during the post-exponential growth phase. The sae mutant showed a significantly reduced rate of invasion of human endothelial cells, consistent with diminished transcription and expression of fnbA. The expression of type 5 capsular polysaccharide is activated in the sae mutant of strain Newman, as shown by immunofluorescence and promoter-reporter fusion experiments. In summary, the sae operon constitutes a four-component regulator system which acts on virulence gene expression in S. aureus.

译文

:我们表征了sae operon,它是金黄色葡萄球菌中毒力基因表达的全球调节物。通过筛选agr突变体ISP479Mu的Tn917文库中具有改变的溶血活性的克隆来获得Tn917 sae突变体。 sae操纵子的序列分析揭示了两个成分的调控基因saeR和saeS上游的两个额外的开放阅读框(ORF)(ORF3和ORF4)。通过Northern印迹分析检测到四个重叠的sae特异性转录物(T1至T4),并且通过引物延伸分析来绘制转录起始点。 T1,T2和T3 mRNA可能在saeS下游的相同茎环序列处终止。 T1消息(3.1 kb)在ORF4的上游启动,T2(2.4 kb)在ORF3的上游启动,而T3(2.0 kb)在saeR前面启动。 T4(0.7 kb)代表仅包含ORF4的单顺反子mRNA。在所研究的40种不同临床金黄色葡萄球菌分离株中,均可检测到sae特异的转录本。在指数后的生长阶段,转录物水平最高。 sae突变体显示出人类内皮细胞的侵袭率显着降低,这与fnbA转录和表达的减少相一致。如免疫荧光和启动子-报告子融合实验所示,在纽曼菌株的sae突变体中激活了5型荚膜多糖的表达。总之,sae操纵子构成了一个四组分调节系统,该系统作用于金黄色葡萄球菌中的毒力基因表达。

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