CHARACTERIZATION OF DEFINED OMPR MUTANTS OF SALMONELLA-TYPHI - OMPR IS INVOLVED IN THE REGULATION OF VI POLYSACCHARIDE EXPRESSION
CHARACTERIZATION OF DEFINED OMPR MUTANTS OF SALMONELLA-TYPHI - OMPR IS INVOLVED IN THE REGULATION OF VI POLYSACCHARIDE EXPRESSION
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DOI：
10.1128/iai.62.9.3984-3993.1994
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发表时间：
1994-09-01
影响因子：
3.1
通讯作者：
CHATFIELD, S
中科院分区：
文献类型：
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作者：
PICKARD, D;LI, JL;CHATFIELD, S
由ompR和envZ基因组成的ompB操纵子从伤寒沙门氏菌Ty2粘粒文库中克隆出来，并通过DNA序列分析进行了鉴定。伤寒沙门氏菌的ompR和envZ基因分别包含编码240个和451个氨基酸的蛋白质的开放阅读框。与鼠伤寒沙门氏菌OmpB蛋白质序列比较显示有99.5%的同源性。DNA序列数据被用于确定合适的限制性内切酶位点，以便在ompR基因的开放阅读框内产生一个517bp的确定缺失。这个缺失通过同源重组被引入到两个伤寒沙门氏菌菌株的染色体中，这两个菌株在aroC和aroD基因中已经有确定的缺失。通过Southern杂交以及对缺失区域周围的DNA片段进行PCR测序，证实了ompR内缺失的存在。如对外膜提取物的检测所示，伤寒沙门氏菌ompR突变体的OmpC和OmpF孔蛋白表达显著降低。还发现携带确定的ompR缺失的伤寒沙门氏菌菌株不再与Vi抗血清发生凝集。然而，当一个有功能的ompB操纵子通过多拷贝质粒或作为单拷贝染色体替换被重新引入到伤寒沙门氏菌ompR突变体中时，Vi(+)表型得以恢复。还发现Vi合成水平对生长培养基中不同浓度的氯化钠敏感，尽管不同的伤寒沙门氏菌分离株之间的敏感程度有所不同。因此得出结论，ompR - envZ双组分调控系统在伤寒沙门氏菌Vi多糖合成的调控中起重要作用，并且这种调控的环境信号之一可能是渗透压。
The ompB operon, comprising the ompR and envZ genes, was cloned from a Salmonella typhi Ty2 cosmid bank and characterized by DNA sequence analysis. The S. typhi ompR and envZ genes contained open reading frames encoding proteins of 240 and 451 amino acids, respectively. Comparison with the Salmonella typhimurium OmpB protein sequences revealed 99.5% homology. The DNA sequence data were used to identify appropriate restriction sites for generating a defined deletion of 517 bp within the open reading frame of the ompR gene. This deletion was introduced by homologous recombination into the chromosomes of two S. typhi strains which already harbored defined deletions in both the aroC and aroD genes. The presence of the deletions within ompR was confirmed by Southern hybridization and sequencing of the DNA fragments surrounding the deleted regions by PCR. The S. typhi ompR mutants displayed a marked decrease in OmpC and OmpF porin expression as demonstrated by examination of outer membrane preparations. It was also found that S. typhi strains harboring the defined ompR deletions no longer agglutinated with Vi antiserum. However, when a functional ompB operon was introduced back into the S. typhi ompR mutants, either on a multicopy plasmid or as a single-copy chromosomal replacement, the Vi(+) phenotype was restored. The levels of Vi synthesis were also found to be sensitive to different concentrations of sodium chloride present in the growth medium, although the levels of sensitivity varied between different isolates of S. typhi. It is therefore concluded that the ompR-envZ two component regulatory system plays an important role in the regulation of Vi polysaccharide synthesis in S. typhi and that one of the environmental signals for this regulation may be osmolarity.