RamR突变协同靶位基因GyrA突变调控RamA表达水平的研究

Mutations in RamR together with mutations in GyrA played important roles in regulation of RamA expression

为了探讨RamR突变类型及对RamA表达调控的影响,靶位突变与外排泵过表达之间的关联,从临床分离的34株沙门菌中筛选不同RamR突变类型菌株,通过外排泵能量抑制(CCCP)试验和有机溶剂耐受试验(OST)验证外排泵的活性,构建不同RamR突变类型的质粒,互补到RamR野生型突变菌株中,采用RT-PCR方法检测各菌株RamA的表达水平。结果表明,15株ramR存在4种突变类型:M83T(n=6),A37S(n=3),T18P(n=3)和氨基酸的缺失(n=3)。CCCP抑制试验和OST试验证实,15株菌株中有10株存在明显的有活力的外排泵作用,4株存在活力微弱的外排泵。RamR三种氨基酸突变类型都上调了RamA的表达水平。靶位基因GyrA第83位的突变协同RamR的A37S或M83T突变均可引起RamA的表达显著增强,而T18P突变没有明显影响。相反,GyrA第87位的突变协同RamR的A37S或M83T突变则下调RamA的表达水平。本研究表明,所有的RamR的突变都可引起RamA表达水平增高,RamR与靶位基因突变协同调控RamA的表达水平,协同GyrA第83位突变可明显促进RamA的表达,而协同GyrA第87位突变则下调RamA的表达。

Salmonella strains with mutations in RamR were screened from 34 clinical isolates,and the efflux activity of the mutants was further detected by efflux pump inhibition assay with CCCP and organic solvent tolerance test（OST）. The rareR-complementing plasmids of mutants were constructed and trans- formed into wild type rarer strains harboring single mutation in position 83 or 87 in GyrA. Reverse tran- scription-PCR was used to assess the expression of ramA of various complementary strains. Total of 4 types of mutations within ramR among 15 of 34 Salmonella strains,including M83T（n= 6）, A37S（n= 3）, T18P（n=3） and nucleotide deletions（n=3）. Active or weak efflux pumps were detected by CCCP and OST assays. Both mutation types were associated with up regulation of expression of RamA. A single mutation at posi- tion 83 within GyrA combined with mutation A37S or M83T in RamR enhanced the expression of RamA. By con- trast,a single mutation of GyrA 87 together with RamR mutation decreased the expression of RamA.