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The Genome-Wide Interaction Network of Nutrient Stress Genes in Escherichia coli

View Article: PubMed Central - PubMed

ABSTRACT

Conventional efforts to describe essential genes in bacteria have typically emphasized nutrient-rich growth conditions. Of note, however, are the set of genes that become essential when bacteria are grown under nutrient stress. For example, more than 100 genes become indispensable when the model bacterium Escherichia coli is grown on nutrient-limited media, and many of these nutrient stress genes have also been shown to be important for the growth of various bacterial pathogens in vivo. To better understand the genetic network that underpins nutrient stress in E. coli, we performed a genome-scale cross of strains harboring deletions in some 82 nutrient stress genes with the entire E. coli gene deletion collection (Keio) to create 315,400 double deletion mutants. An analysis of the growth of the resulting strains on rich microbiological media revealed an average of 23 synthetic sick or lethal genetic interactions for each nutrient stress gene, suggesting that the network defining nutrient stress is surprisingly complex. A vast majority of these interactions involved genes of unknown function or genes of unrelated pathways. The most profound synthetic lethal interactions were between nutrient acquisition and biosynthesis. Further, the interaction map reveals remarkable metabolic robustness in E. coli through pathway redundancies. In all, the genetic interaction network provides a powerful tool to mine and identify missing links in nutrient synthesis and to further characterize genes of unknown function in E. coli. Moreover, understanding of bacterial growth under nutrient stress could aid in the development of novel antibiotic discovery platforms.

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High-throughput array to detect synthetic sick and lethal interactions. Shown here is an example of data from the mating of the argA deletion mutant with strains of the E. coli (Keio) deletion collection. (A) Example of a selection plate that contains 1,536 double deletion mutants in quadruplicate to give a total of 6,144 colonies per plate. (B) Replica plot of the integrated densities of two biological duplicates of the cross of the argA deletion mutant with the Keio collection. (C) Multiplicative approach to detect synthetic sick or lethal interactions. The growth of the single deletion and double deletion mutants are relative to that of wild-type (WT) E. coli strain BW25113. The dotted line delineates the expected growth defect from the accumulation of the single deletions as described in detail in Materials and Methods. (D) Index plot showing the synthetic interaction value of every double deletion mutant. (E) Correction of the dip using a rolling median as described in Materials and Methods.
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fig1: High-throughput array to detect synthetic sick and lethal interactions. Shown here is an example of data from the mating of the argA deletion mutant with strains of the E. coli (Keio) deletion collection. (A) Example of a selection plate that contains 1,536 double deletion mutants in quadruplicate to give a total of 6,144 colonies per plate. (B) Replica plot of the integrated densities of two biological duplicates of the cross of the argA deletion mutant with the Keio collection. (C) Multiplicative approach to detect synthetic sick or lethal interactions. The growth of the single deletion and double deletion mutants are relative to that of wild-type (WT) E. coli strain BW25113. The dotted line delineates the expected growth defect from the accumulation of the single deletions as described in detail in Materials and Methods. (D) Index plot showing the synthetic interaction value of every double deletion mutant. (E) Correction of the dip using a rolling median as described in Materials and Methods.

Mentions: In E. coli, 119 genes become essential when cells are grown in nutrient-limited media. In order to better understand gene essentiality during nutrient stress, we crossed bacteria with single gene deletions of these 119 genes with mutants in the genome-scale single deletion set (Keio) using synthetic genetic array methodology (25, 26). The approach relies on the high-throughput engineering of double deletion mutants by bacterial conjugation, where a query gene deletion is combined with every single gene deletion mutant in the Keio collection (see Fig. S1 in the supplemental material). We conducted the conjugation on plates containing 1,536 colonies and transferred each colony in quadruplicate onto the selection plates to obtain 6,144 colonies per plate (Fig. 1A and Fig. S1). Finally, we monitored the growth of every double deletion mutant over 18 h using the method of French et al. (27).


The Genome-Wide Interaction Network of Nutrient Stress Genes in Escherichia coli
High-throughput array to detect synthetic sick and lethal interactions. Shown here is an example of data from the mating of the argA deletion mutant with strains of the E. coli (Keio) deletion collection. (A) Example of a selection plate that contains 1,536 double deletion mutants in quadruplicate to give a total of 6,144 colonies per plate. (B) Replica plot of the integrated densities of two biological duplicates of the cross of the argA deletion mutant with the Keio collection. (C) Multiplicative approach to detect synthetic sick or lethal interactions. The growth of the single deletion and double deletion mutants are relative to that of wild-type (WT) E. coli strain BW25113. The dotted line delineates the expected growth defect from the accumulation of the single deletions as described in detail in Materials and Methods. (D) Index plot showing the synthetic interaction value of every double deletion mutant. (E) Correction of the dip using a rolling median as described in Materials and Methods.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5120140&req=5

fig1: High-throughput array to detect synthetic sick and lethal interactions. Shown here is an example of data from the mating of the argA deletion mutant with strains of the E. coli (Keio) deletion collection. (A) Example of a selection plate that contains 1,536 double deletion mutants in quadruplicate to give a total of 6,144 colonies per plate. (B) Replica plot of the integrated densities of two biological duplicates of the cross of the argA deletion mutant with the Keio collection. (C) Multiplicative approach to detect synthetic sick or lethal interactions. The growth of the single deletion and double deletion mutants are relative to that of wild-type (WT) E. coli strain BW25113. The dotted line delineates the expected growth defect from the accumulation of the single deletions as described in detail in Materials and Methods. (D) Index plot showing the synthetic interaction value of every double deletion mutant. (E) Correction of the dip using a rolling median as described in Materials and Methods.
Mentions: In E. coli, 119 genes become essential when cells are grown in nutrient-limited media. In order to better understand gene essentiality during nutrient stress, we crossed bacteria with single gene deletions of these 119 genes with mutants in the genome-scale single deletion set (Keio) using synthetic genetic array methodology (25, 26). The approach relies on the high-throughput engineering of double deletion mutants by bacterial conjugation, where a query gene deletion is combined with every single gene deletion mutant in the Keio collection (see Fig. S1 in the supplemental material). We conducted the conjugation on plates containing 1,536 colonies and transferred each colony in quadruplicate onto the selection plates to obtain 6,144 colonies per plate (Fig. 1A and Fig. S1). Finally, we monitored the growth of every double deletion mutant over 18 h using the method of French et al. (27).

View Article: PubMed Central - PubMed

ABSTRACT

Conventional efforts to describe essential genes in bacteria have typically emphasized nutrient-rich growth conditions. Of note, however, are the set of genes that become essential when bacteria are grown under nutrient stress. For example, more than 100 genes become indispensable when the model bacterium Escherichia coli is grown on nutrient-limited media, and many of these nutrient stress genes have also been shown to be important for the growth of various bacterial pathogens in vivo. To better understand the genetic network that underpins nutrient stress in E. coli, we performed a genome-scale cross of strains harboring deletions in some 82 nutrient stress genes with the entire E. coli gene deletion collection (Keio) to create 315,400 double deletion mutants. An analysis of the growth of the resulting strains on rich microbiological media revealed an average of 23 synthetic sick or lethal genetic interactions for each nutrient stress gene, suggesting that the network defining nutrient stress is surprisingly complex. A vast majority of these interactions involved genes of unknown function or genes of unrelated pathways. The most profound synthetic lethal interactions were between nutrient acquisition and biosynthesis. Further, the interaction map reveals remarkable metabolic robustness in E. coli through pathway redundancies. In all, the genetic interaction network provides a powerful tool to mine and identify missing links in nutrient synthesis and to further characterize genes of unknown function in E. coli. Moreover, understanding of bacterial growth under nutrient stress could aid in the development of novel antibiotic discovery platforms.

No MeSH data available.


Related in: MedlinePlus