Identification of Genes Involved in Biofilm Formation in Acinetobacter baumannii
¹Matthew Saling, ²Chen Niu, and ^2,3Phil Rather
¹Washington State University, Pullman, WA;
²Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA;
³Bacterial Pathogenesis Section, Atlanta VA Medical Center, Atlanta, GA

Abstract

Using transposon mutagenesis in Acinetobacter baumannii, genes that function in biofilm formation were identified. This involved the creation of several A. baumannii libraries containing random insertions of the transposon EZ::TnKanR. This transposon contains resistance to kanamycin and only clones containing the EZ::TnKanR are able to grow on LB plates containing kanamycin. For measuring biofilm formation A. baumannii mutants were inoculated into two microtiter plates and incubated for four hours and twelve hours. After their respective incubation times, the OD600 of the growth was measured. Following growth measurements biofilm density was recorded by staining the biofilms with crystal violet and measuring the OD580 values. A. baumannii mutants showing at least a two-fold increase or decrease in biofilm formation, as compared to the Metro-2 wild-type, were subject to further genetic analysis. By using this method we were able to find 14 mutants out of a total of 504 screened, which had either increased or decreased biofilm production as compared to the wild-type. The DNA of these clones were cut using the restriction enzyme XbaI, and a Southern blot was performed to determine the location of the EZ::TnKanR transposon. The transposon contained a plasmid origin of replication, therefore chromosomal DNA was religated and electroporated into E. coli cells to isolate the transposon and flanking chromosomal DNA. Due to time constraints, only a limited number of E. coli transformations could be performed by project’s end. Further transformations of E. coli and sequencing of genes involved in biofilm formation will be carried out in the next few weeks.