Analysis of Pools of Targeted Salmonella Deletion Mutants Identifies Novel Genes Affecting Fitness during Competitive Infection in Mice

PLoS Pathogens

Helene L. Andrews-Polymenis¹  M. Megan Reynolds¹  Sang-Ho Choi²  Carlos A. Santiviago²  Steffen Porwollik²  Fred Long²  Michael McClelland² 

[1] Department of Microbial and Molecular Pathogenesis, College of Medicine, Texas A&M University System Health Science Center, College Station, Texas, United States of America;Sidney Kimmel Cancer Center, San Diego, California, United States of America

关键词: Gene pool;    Polymerase chain reaction;    Salmonella;    Salmonellosis;    Phenotypes;    Gene targeting;    Genetic screens;    RNA hybridization;   

DOI  :  10.1371/journal.ppat.1000477

学科分类：生物科学（综合）

来源: Public Library of Science

【 摘 要 】

Pools of mutants of minimal complexity but maximal coverage of genes of interest facilitate screening for genes under selection in a particular environment. We constructed individual deletion mutants in 1,023 Salmonella enterica serovar Typhimurium genes, including almost all genes found in Salmonella but not in related genera. All mutations were confirmed simultaneously using a novel amplification strategy to produce labeled RNA from a T7 RNA polymerase promoter, introduced during the construction of each mutant, followed by hybridization of this labeled RNA to a Typhimurium genome tiling array. To demonstrate the ability to identify fitness phenotypes using our pool of mutants, the pool was subjected to selection by intraperitoneal injection into BALB/c mice and subsequent recovery from spleens. Changes in the representation of each mutant were monitored using T7 transcripts hybridized to a novel inexpensive minimal microarray. Among the top 120 statistically significant spleen colonization phenotypes, more than 40 were mutations in genes with no previously known role in this model. Fifteen phenotypes were tested using individual mutants in competitive assays of intraperitoneal infection in mice and eleven were confirmed, including the first two examples of attenuation for sRNA mutants in Salmonella. We refer to the method as Array-based analysis of cistrons under selection (ABACUS).

【 授权许可】

CC BY