Litwin C M, Calderwood S B
Infectious Disease Unit, Massachusetts General Hospital, Boston 02114.
J Bacteriol. 1993 Feb;175(3):706-15. doi: 10.1128/jb.175.3.706-715.1993.
Vibrio vulnificus infections have been associated with iron overload and preexisting liver disease. Iron may play a major role in the pathogenesis of V. vulnificus infections. Many virulence genes, as well as genes involved in the transport of iron by bacteria, are regulated by iron, with increased expression under low-iron conditions. In Escherichia coli and Vibrio cholerae, transcriptional regulation by iron depends on the fur gene. We utilized Southern hybridization under low- and high-stringency conditions with both E. coli and V. cholerae fur gene probes to demonstrate that there are fur-homologous sequences in the DNAs of V. vulnificus, Vibrio fischeri, and Aeromonas sp. but not in the DNAs of the other bacterial species tested. We developed a restriction map and cloned the fur-homologous sequence from V. vulnificus. The hybridizing clone of V. vulnificus chromosomal DNA complemented a V. cholerae fur mutant. DNA sequence analysis confirmed the presence of a 149-amino-acid open reading frame that was 77% homologous to E. coli Fur and 93% homologous to V. cholerae Fur. Primer extension localized a single promoter for the V. vulnificus fur gene. Northern (RNA) blot analysis and beta-galactosidase assays of an operon fusion to lacZ suggested that there was not significant regulation of transcription of V. vulnificus fur by iron or the E. coli Fur protein. We used marker exchange to construct a V. vulnificus fur deletion mutant and confirmed its phenotype by observing overexpression of iron-regulated outer membrane proteins on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The fur deletion mutant of V. vulnificus will be helpful in future studies of the role of iron in V. vulnificus pathogenesis.
创伤弧菌感染与铁过载及既往存在的肝脏疾病有关。铁可能在创伤弧菌感染的发病机制中起主要作用。许多毒力基因以及参与细菌铁转运的基因都受铁的调控，在低铁条件下表达增加。在大肠杆菌和霍乱弧菌中，铁的转录调控依赖于fur基因。我们在低严谨度和高严谨度条件下，使用大肠杆菌和霍乱弧菌的fur基因探针进行Southern杂交，以证明创伤弧菌、费氏弧菌和气单胞菌属的DNA中存在fur同源序列，但在所测试的其他细菌物种的DNA中不存在。我们绘制了限制酶切图谱，并从创伤弧菌中克隆了fur同源序列。创伤弧菌染色体DNA的杂交克隆互补了霍乱弧菌的fur突变体。DNA序列分析证实存在一个149个氨基酸的开放阅读框，与大肠杆菌Fur的同源性为77%，与霍乱弧菌Fur的同源性为93%。引物延伸定位了创伤弧菌fur基因的一个单一启动子。对与lacZ的操纵子融合进行Northern（RNA）印迹分析和β-半乳糖苷酶测定表明，铁或大肠杆菌Fur蛋白对创伤弧菌fur转录没有显著调控。我们使用标记交换构建了创伤弧菌fur缺失突变体，并通过在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上观察铁调节外膜蛋白的过表达来确认其表型。创伤弧菌的fur缺失突变体将有助于未来研究铁在创伤弧菌发病机制中的作用。
