The essential genome ofEscherichia coliK-12

Abstract

word count: 189 14 Text word count: 6,604 15 ABSTRACT 16Transposon-Directed Insertion-site Sequencing (TraDIS) is a high-throughput method 17 coupling transposon mutagenesis with short-fragment DNA sequencing. It is commonly used 18 to identify essential genes. Single gene deletion libraries are considered the gold standard for 19 identifying essential genes. Currently, the TraDIS method has not been benchmarked against 20 such libraries and therefore it remains unclear whether the two methodologies are 21 comparable. To address this, a high density transposon library was constructed in 22

Iron is a ligand of SecA-like metal-binding domains in vivo

“…Identification of genes that affect the susceptibility of E. coli to sodium azide. To gain insight into the effect of azide on cells, we identified transposon mutations that influence the susceptibility of E. coli to azide using TraDIS (22,23). To this end, we determined the concentration of azide that inhibited, but did not prevent, the growth of E. coli BW25113.…”

“…Growth curves in the presence of increasing concentrations of azide indicated that 250 and 500 μM sodium azide were the highest concentrations that did not completely inhibit growth in LB (supporting figure S2). We then grew a library of > 1 million independent BW25113 mini-Tn5 transposon mutants (23) in the presence of subinhibitory concentrations of azide and determined the change in the relative proportion of mutations in each insertion using Illumina sequencing. Growth of the library in the presence of 0.25 mM or 0.5 mM azide resulted in a strong depletion (> log2 1.5-fold) in insertions in approximately 180 of the 4346 genes included in the analysis.…”

“…Both proteins contain domains of unknown function (UPF0149 in YecA and UPF0225 in YchJ (45)) and are found in a broad range of bacterial species. Neither protein is universally conserved (31) or essential for viability (23,46). However, the conservation of the amino acids that mediate the interaction between the SecA MBD and SecB in the C-terminal MBDs of YecA and YchJ suggest that that these proteins could also potentially interact with the SecB or ribosomes.…”

“…TraDIS. TraDIS experiments were conducted as described previously (22,23). 50 ml of LB broth containing 0, 0.25 or 0.5 mM NaN3 were inoculated with 10 μl of a library of ~1 million E. coli BW25113 mini-Tn5 insertion mutants, and the cultures were grown to OD600 1.0.…”

“…In order to gain insight into the effect of azide on protein translocation in vivo, we determined the effect of azide on the relative growth rates of >1 million independent transposon mutants using transposon-directed insertion-site sequencing (TraDIS) (22,23). The results of this screen suggested that azide caused CTT-mediated autoinhibition of SecA, potentially by disrupting the MBD.…”