Biology subjects
Publications and source records attributed to Robertson, E. B..
Withdrawal statementThis manuscript has been withdrawn due to concerns about missing raw data. This work should not be cited as reference for the project. If you have any questions, please contact the corresponding author.
Biological collections, including arrayed libraries of single transposon or deletion mutants, greatly accelerate the pace of bacterial genetics research. Despite the importance of these resources, few protocols exist for the replication and distribution of these materials. Here, we describe a protocol for creating multiple replicates of an arrayed bacterial Tn library consisting of approximately 6,800 mutants in 73 x 96-well plates. Our protocol provides multiple checkpoints to guard against contamination and minimize genetic drift caused by freeze/thaw cycles. This approach can also be scaled for arrayed culture collections of various sizes. Overall, this protocol is a valuable resource for other researchers considering the construction and distribution of arrayed culture collection resources for the benefit of the greater scientific community. ImportanceArrayed mutant collections drive robust genetic screens, yet few protocols exist for replication of these resources and subsequent quality control. Increasing distribution of arrayed biological collections will increase accessibility to and use of these resources. Developing standardized techniques for replication of these resources is essential for ensuring their quality and usefulness to the scientific community.
Enterococcus faecalis is a common commensal bacterium in the gastrointestinal tract as well as a frequent nosocomial pathogen. The secreted metalloprotease gelatinase (GelE) is an important E. faecalis virulence factor that contributes to numerous cellular activities such as autolysis, biofilm formation, and biofilm-associated antibiotic resistance. Expression of gelE has been extensively studied and is regulated by the Fsr quorum-sensing system. Here, we identify two additional factors regulating gelatinase expression and activity in E. faecalis OG1RF. The Bph phosphatase is required for expression of gelE in an Fsr-dependent manner. Additionally, the membrane-anchored protein foldase PrsA is required for GelE activity, but not fsr or gelE gene expression. Disrupting prsA also leads to increased antibiotic sensitivity in biofilms independent of the loss of GelE activity. Together, our results expand the model for gelatinase production in E. faecalis, which has important implications for fundamental studies of GelE function in Enterococcus and also E. faecalis pathogenesis. ImportanceIn Enterococcus faecalis, gelatinase (GelE) is a virulence factor that is also important for biofilm formation and interactions with other microbes as well as the host immune system. The long-standing model for GelE production is that the Fsr quorum sensing system positively regulates expression of gelE. Here, we update that model by identifying two additional factors that contribute to gelatinase production. The biofilm-associated Bph phosphatase regulates the expression of gelE through Fsr, and the peptidyl-prolyl isomerase PrsA is required for production of active GelE through an Fsr-independent mechanism. This provides important insight into how regulatory networks outside of the fsr locus coordinate expression of gelatinase.