{"title":"Use of Polymerase Chain Reaction for the Determination of About 2.5 kb fpvA and fpvB Gene Sequences in Pseudomonas aeruginosa Strains","authors":"J. Osayande","doi":"10.5772/23983","DOIUrl":null,"url":null,"abstract":"Pseudomonas aeruginosa produces three different pyoverdines, types I-III (Cornelis et al., 1989), which are able to chelate iron and form ferripyoverdine complexes that are recognized and transported by different ferripyoverdine receptors present on the outer membrane. The ferripyoverdine receptor gene, fpvA of P. aeruginosa (PAO1) has been characterized previously (Poole et al., 1993). In addition, the other iron-repressible outer membrane receptor proteins for types II and III ferripyoverdine complexes were recently identified and characterized by cloning (De Chial et al., 2003). Following the observation that an fpvA mutant could demonstrate low ferripyoverdine uptake compared with wild type (Poole et al., 1991; Gensberg et al., 1992), an alternative ferripyoverdine receptor gene fpvB was identified and a fragment (562 bp) was amplified by polymerase chain reaction (Ghysels et al. 2004). In addition, the growth of several P. aeruginosa pyoverdine-negative mutants, found to inhabit the lungs of cystic fibrosis patients, were stimulated by existing pyoverdine types, providing additional confirmation for the existence of an alternative route for ferripyoverdine uptake (De Vos et al., 2001; Ghysels et al., 2004). PCR was developed in 1983 by Kary Mullis (Karry Mullis Nobel Lecture, December 8, 1993) and involves the selective amplification of specific regions of DNA for extensive use in molecular biology (Sambrook and Russell, 2001). Using primers designed in this study, the complete sequence of the ferripyoverdine receptor genes (fpvA and fpvB) from several P. aeruginosa clinical and environmental isolates were amplified and sequenced, allowing the identification of variant forms of these receptor genes.","PeriodicalId":22514,"journal":{"name":"The Internet journal of microbiology","volume":null,"pages":null},"PeriodicalIF":0.0000,"publicationDate":"2011-10-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"4","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"The Internet journal of microbiology","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.5772/23983","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
引用次数: 4
Abstract
Pseudomonas aeruginosa produces three different pyoverdines, types I-III (Cornelis et al., 1989), which are able to chelate iron and form ferripyoverdine complexes that are recognized and transported by different ferripyoverdine receptors present on the outer membrane. The ferripyoverdine receptor gene, fpvA of P. aeruginosa (PAO1) has been characterized previously (Poole et al., 1993). In addition, the other iron-repressible outer membrane receptor proteins for types II and III ferripyoverdine complexes were recently identified and characterized by cloning (De Chial et al., 2003). Following the observation that an fpvA mutant could demonstrate low ferripyoverdine uptake compared with wild type (Poole et al., 1991; Gensberg et al., 1992), an alternative ferripyoverdine receptor gene fpvB was identified and a fragment (562 bp) was amplified by polymerase chain reaction (Ghysels et al. 2004). In addition, the growth of several P. aeruginosa pyoverdine-negative mutants, found to inhabit the lungs of cystic fibrosis patients, were stimulated by existing pyoverdine types, providing additional confirmation for the existence of an alternative route for ferripyoverdine uptake (De Vos et al., 2001; Ghysels et al., 2004). PCR was developed in 1983 by Kary Mullis (Karry Mullis Nobel Lecture, December 8, 1993) and involves the selective amplification of specific regions of DNA for extensive use in molecular biology (Sambrook and Russell, 2001). Using primers designed in this study, the complete sequence of the ferripyoverdine receptor genes (fpvA and fpvB) from several P. aeruginosa clinical and environmental isolates were amplified and sequenced, allowing the identification of variant forms of these receptor genes.
铜绿假单胞菌产生三种不同的吡啶,I-III型(Cornelis et al., 1989),它们能够螯合铁并形成铁吡啶复合物,这些复合物被存在于外膜上的不同铁吡啶受体识别和运输。铜绿假单胞菌(P. aeruginosa, PAO1)的铁嘌呤受体基因fpvA先前已被鉴定(Poole et al., 1993)。此外,II型和III型铁吡啶复合物的其他铁抑制外膜受体蛋白最近被鉴定并通过克隆进行了表征(De cil et al., 2003)。在观察到与野生型相比,fpvA突变体可以表现出较低的铁嘌呤摄取(Poole等,1991;Gensberg等人,1992),鉴定了另一种铁嘌呤受体基因fpvB,并通过聚合酶链反应扩增了一个片段(562 bp) (Ghysels等人,2004)。此外,囊性纤维化患者肺中发现的几种P. aeruginosa pyoverdine阴性突变体的生长受到现有pyoverdine类型的刺激,进一步证实了铁嘧啶(ferripyoverdine)摄取的替代途径的存在(De Vos et al., 2001;Ghysels et al., 2004)。PCR是由Kary Mullis于1983年发明的(1993年12月8日,Karry Mullis诺贝尔奖演讲),涉及DNA特定区域的选择性扩增,广泛应用于分子生物学(Sambrook和Russell, 2001)。利用本研究设计的引物,对来自铜绿假单胞菌临床和环境分离株的铁吡啶受体基因(fpvA和fpvB)的完整序列进行扩增和测序,从而鉴定出这些受体基因的变异形式。