Dorsey Caleb W, Tomaras Andrew P, Actis Luis A
Department of Microbiology, Miami University, Oxford, Ohio 45056, USA.
Appl Environ Microbiol. 2002 Dec;68(12):6353-60. doi: 10.1128/AEM.68.12.6353-6360.2002.
Acinetobacter baumannii is a metabolically versatile pathogen that causes severe infections in compromised patients. However, little is known about the genes and factors involved in its basic physiology and virulence properties. Insertion mutagenesis was used to initiate the identification and characterization of some of these factors and genes in the prototype strain 19606. The utilization of the pLOFKm suicide delivery vector, which harbors a suicide mini-Tn10 derivative, proved to be unsuccessful for this purpose. The EZ::TN <R6Kgammaori/KAN-2> Tnp transposome system available from Epicentre was then used in conjunction with electroporation to generate isogenic insertional derivatives of A. baumannii 19606. Replica plating showed that 2% of the colonies that grew after electroporation on agar plates without antibiotics also grew in the presence of 40 micro g of kanamycin per ml. DNA hybridization proved that all of the kanamycin-resistant derivatives contained the EZ::TN <R6Kgammaori/KAN-2> insertion element, which was mapped to different genomic locations. Replica plating on Simmons citrate agar and microtiter plate-plastic tube assays identified growth- and biofilm-defective derivatives, respectively. The location of the insertion in several of these derivatives was determined by self-ligation of NdeI- or EcoRI-digested genomic DNA and electroporation of Escherichia coli TransforMax EC100D (pir(+)). Sequence analysis of the recovered plasmids showed that some of the A. baumannii 19606 growth-defective derivatives contain insertions within genes encoding activities required for the generation of energy and cell wall components and for the biosynthesis of amino acids and purines. A gene encoding a protein similar to the GacS sensor kinase was interrupted in four derivatives, while another had an insertion in a gene coding for a hypothetical sensor kinase. A. baumannii 19606 derivatives with defective attachment or biofilm phenotypes had insertions within genes that appear to be part of a chaperone-usher transport system described for other bacteria. DNA hybridization experiments showed that the presence of strain 19606 genes encoding regulatory and attachment or biofilm functions is widespread among other A. baumannii clinical isolates.
鲍曼不动杆菌是一种代谢功能多样的病原体，可在免疫功能低下的患者中引起严重感染。然而，对于其基本生理学和毒力特性所涉及的基因和因素，人们了解甚少。插入诱变被用于在原型菌株19606中初步鉴定和表征其中一些因素和基因。事实证明，携带自杀性mini-Tn10衍生物的pLOFKm自杀传递载体在此目的上并不成功。随后，使用Epicentre公司提供的EZ::TN <R6Kgammaori/KAN-2> Tnp转座体系统结合电穿孔来产生鲍曼不动杆菌19606的同基因插入衍生物。影印平板法显示，在不含抗生素的琼脂平板上经电穿孔后生长的菌落中有2%在每毫升含40微克卡那霉素的情况下也能生长。DNA杂交证明，所有抗卡那霉素的衍生物都含有EZ::TN <R6Kgammaori/KAN-2>插入元件，该元件被定位到不同的基因组位置。在西蒙斯柠檬酸盐琼脂上进行影印平板法以及在微量滴定板 - 塑料管试验中分别鉴定出了生长缺陷型和生物膜缺陷型衍生物。通过对经NdeI或EcoRI消化的基因组DNA进行自我连接以及对大肠杆菌TransforMax EC100D（pir(+)）进行电穿孔，确定了其中几种衍生物中插入的位置。对回收质粒的序列分析表明，一些鲍曼不动杆菌19606生长缺陷型衍生物在编码能量和细胞壁成分生成以及氨基酸和嘌呤生物合成所需活性的基因内含有插入片段。在四种衍生物中，一个编码类似于GacS传感激酶的蛋白质的基因被中断，而另一个在编码假定传感激酶的基因中有插入片段。具有附着缺陷或生物膜表型缺陷的鲍曼不动杆菌19606衍生物在似乎是其他细菌所描述的伴侣 - 引导转运系统一部分的基因内含有插入片段。DNA杂交实验表明，编码调控以及附着或生物膜功能的19606菌株基因在其他鲍曼不动杆菌临床分离株中广泛存在。
