Withers T Ryan, Yin Yeshi, Yu Hongwei D
Department of Biochemistry and Microbiology, Joan C. Edwards School of Medicine, Marshall University.
Department of Biochemistry and Microbiology, Joan C. Edwards School of Medicine, Marshall University;
J Vis Exp. 2014 Mar 10(85):51346. doi: 10.3791/51346.
Pseudomonas aeruginosa is a Gram-negative, environmental bacterium with versatile metabolic capabilities. P. aeruginosa is an opportunistic bacterial pathogen which establishes chronic pulmonary infections in patients with cystic fibrosis (CF). The overproduction of a capsular polysaccharide called alginate, also known as mucoidy, promotes the formation of mucoid biofilms which are more resistant than planktonic cells to antibiotic chemotherapy and host defenses. Additionally, the conversion from the nonmucoid to mucoid phenotype is a clinical marker for the onset of chronic infection in CF. Alginate overproduction by P. aeruginosa is an endergonic process which heavily taxes cellular energy. Therefore, alginate production is highly regulated in P. aeruginosa. To better understand alginate regulation, we describe a protocol using the mini-himar1 transposon mutagenesis for the identification of novel alginate regulators in a prototypic strain PAO1. The procedure consists of two basic steps. First, we transferred the mini-himar1 transposon (pFAC) from host E. coli SM10/λpir into recipient P. aeruginosa PAO1 via biparental conjugation to create a high-density insertion mutant library, which were selected on Pseudomonas isolation agar plates supplemented with gentamycin. Secondly, we screened and isolated the mucoid colonies to map the insertion site through inverse PCR using DNA primers pointing outward from the gentamycin cassette and DNA sequencing. Using this protocol, we have identified two novel alginate regulators, mucE (PA4033) and kinB (PA5484), in strain PAO1 with a wild-type mucA encoding the anti-sigma factor MucA for the master alginate regulator AlgU (AlgT, σ(22)). This high-throughput mutagenesis protocol can be modified for the identification of other virulence-related genes causing change in colony morphology.
铜绿假单胞菌是一种革兰氏阴性环境细菌，具有多种代谢能力。铜绿假单胞菌是一种机会性细菌病原体，可在囊性纤维化（CF）患者中引发慢性肺部感染。一种名为藻酸盐的荚膜多糖过量产生，也称为黏液样变性，会促进黏液样生物膜的形成，这种生物膜比浮游细胞更能抵抗抗生素化疗和宿主防御。此外，从非黏液样表型向黏液样表型的转变是CF中慢性感染发作的临床标志物。铜绿假单胞菌过量产生藻酸盐是一个耗能过程，会严重消耗细胞能量。因此，铜绿假单胞菌中藻酸盐的产生受到高度调控。为了更好地理解藻酸盐调控，我们描述了一种使用mini-himar1转座子诱变的方案，用于在原型菌株PAO1中鉴定新的藻酸盐调节因子。该过程包括两个基本步骤。首先，我们通过双亲接合将mini-himar1转座子（pFAC）从宿主大肠杆菌SM10/λpir转移到受体铜绿假单胞菌PAO1中，以创建一个高密度插入突变体文库，该文库在补充有庆大霉素的假单胞菌分离琼脂平板上进行筛选。其次，我们筛选并分离出黏液样菌落，使用从庆大霉素盒向外指向的DNA引物通过反向PCR和DNA测序来定位插入位点。使用该方案，我们在菌株PAO1中鉴定出两个新的藻酸盐调节因子，mucE（PA4033）和kinB（PA5484），该菌株具有编码主要藻酸盐调节因子AlgU（AlgT，σ(22)）的抗σ因子MucA的野生型mucA。这种高通量诱变方案可进行修改，以鉴定导致菌落形态变化的其他毒力相关基因。
