Tinker J K, Clegg S
Department of Microbiology, College of Medicine, University of Iowa, Iowa City, IA 52242, USA.
Mol Microbiol. 2001 May;40(3):757-68. doi: 10.1046/j.1365-2958.2001.02430.x.
Expression of type 1 fimbriae in Salmonella enterica serovar Typhimurium undergoes phase variation or alteration between a fimbriate and a non-fimbriate phenotype. This variation is known to be dependent upon environmental conditions in vitro and is thought to be a complex process involving regulation by a number of proteins. The regulatory genes located within the fim cluster include fimZ, fimY and fimW. A fourth gene of the cluster, fimU, encodes a tRNA molecule specific for rare arginine codons. We have shown previously that fimU affects the expression of S. typhimurium type 1 fimbriae, and that fimU is functionally related to the Escherichia coli gene argU. A high frequency of rare arginine codons was found within the three fim regulatory genes, and five of these codons were clustered within fimY alone. To investigate the affects of fimU on FimY production, a FimY fusion with the E. coli maltose-binding protein was constructed and expressed in an E. coli argU background. Western blots of extracts from the argU mutant and parental strain indicated that production of FimY was significantly reduced in the absence of a functional tRNAArg(UCU). FimY production in this mutant could be restored to high levels when fimU was introduced on a plasmid, and also when three rare arginine codons, located within the first 14 positions within fimY, were exchanged for major arginine codons. A Tn10 insertion from a Salmonella enteritidis fimU mutant was transduced into S. typhimurium, and this strain was analysed for the expression of type 1 fimbriae. The resulting S. typhimurium fimU mutant was found to be non-fimbriate under all conditions tested and could be complemented by the introduction of fimU alone on a plasmid. In addition, this mutant could be complemented by transformation with fimY altered in the first three rare arginine codons. Reverse transcriptase-polymerase chain reaction confirmed that the fimY transcript was present at similar levels in the fimU mutant and parental strain. These results indicated that the observed inhibition of protein expression was not occurring at the transcriptional level. Analysis of expression of the malEfimY fusion in the S. typhimurium fimU mutant and parental strain confirmed the data observed in E. coli. In contrast, a FimW fusion was found to be produced at similar levels in both the fimU mutant and the parental strain. Together, these data indicate that the absence of a functional fimU results in the inhibition of efficient FimY translation, and thus type 1 fimbrial production in S. typhimurium.
鼠伤寒沙门氏菌1型菌毛的表达会发生相变，即在菌毛表型和非菌毛表型之间转变。已知这种变异取决于体外环境条件，并且被认为是一个涉及多种蛋白质调控的复杂过程。位于fim基因簇内的调控基因包括fimZ、fimY和fimW。该基因簇的第四个基因fimU编码一种特异于稀有精氨酸密码子的tRNA分子。我们之前已经表明fimU影响鼠伤寒沙门氏菌1型菌毛的表达，并且fimU在功能上与大肠杆菌基因argU相关。在三个fim调控基因中发现了高频的稀有精氨酸密码子，其中五个密码子仅聚集在fimY内。为了研究fimU对FimY产生的影响，构建了一个与大肠杆菌麦芽糖结合蛋白融合的FimY，并在大肠杆菌argU背景中表达。来自argU突变体和亲本菌株的提取物的蛋白质免疫印迹表明，在缺乏功能性tRNAArg(UCU)的情况下，FimY的产生显著减少。当在质粒上引入fimU时，以及当fimY内前14个位置的三个稀有精氨酸密码子被替换为主要精氨酸密码子时，该突变体中FimY的产生可以恢复到高水平。将肠炎沙门氏菌fimU突变体的Tn10插入片段转导到鼠伤寒沙门氏菌中，并分析该菌株1型菌毛的表达。结果发现，所得的鼠伤寒沙门氏菌fimU突变体在所有测试条件下均无菌毛，并且通过在质粒上单独引入fimU可以互补。此外，该突变体可以通过用前三个稀有精氨酸密码子改变的fimY进行转化来互补。逆转录聚合酶链反应证实fimY转录本在fimU突变体和亲本菌株中的水平相似。这些结果表明，观察到的蛋白质表达抑制不是发生在转录水平。对鼠伤寒沙门氏菌fimU突变体和亲本菌株中malEfimY融合蛋白表达的分析证实了在大肠杆菌中观察到的数据。相反，发现FimW融合蛋白在fimU突变体和亲本菌株中的产生水平相似。总之，这些数据表明缺乏功能性fimU会导致FimY有效翻译的抑制，从而导致鼠伤寒沙门氏菌中1型菌毛的产生受到抑制。
