Browsing by Author "Freed NE"
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- ItemA simple screen to identify promoters conferring high levels of phenotypic noise.(PUBLIC LIBRARY SCIENCE, 2008-12) Freed NE; Silander OK; Stecher B; Böhm A; Hardt W-D; Ackermann MGenetically identical populations of unicellular organisms often show marked variation in some phenotypic traits. To investigate the molecular causes and possible biological functions of this phenotypic noise, it would be useful to have a method to identify genes whose expression varies stochastically on a certain time scale. Here, we developed such a method and used it for identifying genes with high levels of phenotypic noise in Salmonella enterica ssp. I serovar Typhimurium (S. Typhimurium). We created a genomic plasmid library fused to a green fluorescent protein (GFP) reporter and subjected replicate populations harboring this library to fluctuating selection for GFP expression using fluorescent-activated cell sorting (FACS). After seven rounds of fluctuating selection, the populations were strongly enriched for promoters that showed a high amount of noise in gene expression. Our results indicate that the activity of some promoters of S. Typhimurium varies on such a short time scale that these promoters can absorb rapid fluctuations in the direction of selection, as imposed during our experiment. The genomic fragments that conferred the highest levels of phenotypic variation were promoters controlling the synthesis of flagella, which are associated with virulence and host-pathogen interactions. This confirms earlier reports that phenotypic noise may play a role in pathogenesis and indicates that these promoters have among the highest levels of noise in the S. Typhimurium genome. This approach can be applied to many other bacterial and eukaryotic systems as a simple method for identifying genes with noisy expression.
- ItemCombining Tn-seq with comparative genomics identifies proteins uniquely essential in Shigella flexneri(2015-09-03) Freed NE; Bumann D; Silander OProtein functions that are essential for the growth of bacterial pathogens provide promising targets for antibacterial treatment. This is especially true if those functions are uniquely essential for the pathogen, as this might allow the development of targeted antibiotics, i.e. those that disrupt essential functions only for the pathogenic bacteria. Here we present the results of a Tn-seq experiment designed to detect essential protein coding genes in Shigella flexneri 2a 2457T on a genome-wide scale. Our results suggest that 471 protein-coding genes in this organism are critical for cellular growth in rich media. Comparing this set of essential genes (the essential gene complement) with their orthologues in the closely related organism Escherichia coli K12 BW25113 revealed a significant number of genes that are essential in Shigella but not in E. coli, suggesting that the functional correspondence of these proteins had changed. Notably, we also identified a set of functionally related genes that are essential in Shigella but which have no orthologues in E. coli. We found an extreme bias in proteins that have evolved to provide essential functions, with many proteins essential in Shigella but not E. coli, but with none (or very few) being essential in E. coli but not Shigella. We also identify a set- of genes involved in nucleotide biosynthesis that are essential in Shigella, but which lack orthologues in E. coli. Consequently, the data presented here suggest that the essential gene complement can quickly become organism specific, especially for pathogenic organisms whose genomes might have reduced robustness in their metabolic capacity (e.g. functional redundancy), or a reduced numbers of protein coding genes. These results thus open the possibility of developing antibiotic treatments that target differentially essential genes, which may exist even between very closely related strains of bacteria.