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Concurrent growth rate and transcript analyses reveal essential gene stringency in Escherichia coli.

Goh S, Boberek JM, Nakashima N, Stach J, Good L - PLoS ONE (2009)

Bottom Line: The relationship between mRNA decline and growth rate decline reflects the degree of essentiality, or stringency, of an essential gene, which is here defined by the minimum transcript level for a 50% reduction in growth rate (MTL(50)).When applied to four growth essential genes, both RNA silencing methods resulted in MTL(50) values that reveal acpP as the most stringently required of the four genes examined, with ftsZ the next most stringently required.This method may be used to validate existing essential genes and to quantify drug target requirement.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Molecular Biology, Karolinska Institute, Stockholm, Sweden.

ABSTRACT

Background: Genes essential for bacterial growth are of particular scientific interest. Many putative essential genes have been identified or predicted in several species, however, little is known about gene expression requirement stringency, which may be an important aspect of bacterial physiology and likely a determining factor in drug target development.

Methodology/principal findings: Working from the premise that essential genes differ in absolute requirement for growth, we describe silencing of putative essential genes in E. coli to obtain a titration of declining growth rates and transcript levels by using antisense peptide nucleic acids (PNA) and expressed antisense RNA. The relationship between mRNA decline and growth rate decline reflects the degree of essentiality, or stringency, of an essential gene, which is here defined by the minimum transcript level for a 50% reduction in growth rate (MTL(50)). When applied to four growth essential genes, both RNA silencing methods resulted in MTL(50) values that reveal acpP as the most stringently required of the four genes examined, with ftsZ the next most stringently required. The established antibacterial targets murA and fabI were less stringently required.

Conclusions: RNA silencing can reveal stringent requirements for gene expression with respect to growth. This method may be used to validate existing essential genes and to quantify drug target requirement.

Show MeSH
Dose dependent growth inhibition of antisense PNA-treated AS19 cells.Overnight AS19 cultures were sub-cultured in fresh media containing different PNA doses for each gene, and its growth monitored by turbidity. Six doses were chosen for each PNA, so as to obtain a titration in growth inhibition. Growth profiles terminated when all cultures were harvested at ΔOD550∼0.1 of the untreated culture, for transcript analyses.
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pone-0006061-g002: Dose dependent growth inhibition of antisense PNA-treated AS19 cells.Overnight AS19 cultures were sub-cultured in fresh media containing different PNA doses for each gene, and its growth monitored by turbidity. Six doses were chosen for each PNA, so as to obtain a titration in growth inhibition. Growth profiles terminated when all cultures were harvested at ΔOD550∼0.1 of the untreated culture, for transcript analyses.

Mentions: To test the hypothesis that essential genes are differentially required to maintain a similar level of growth, we titrated down the growth rate of E. coli using either antisense PNA or expressed antisense and determined mRNA levels of the targeted essential gene. To enable parallel analyses of multiple genes in this study, we used RT-PCR to quantify mRNA levels and assume it as a proxy to protein measure in bacteria [39]. For PNA-mediated antisense effects, we used the hyper-permeable E. coli strain AS19 to obtain efficient uptake [40]. Six doses of each antisense PNA were selected in order to achieve a titration in E. coli growth rate reduction. Hence, the dose range of each antisense PNA was unique, resulting in growth inhibition in a dose dependent manner (Figure 2). For expressed antisense, plasmids were transformed into TOP10 E. coli cells (Table 2) and antisense RNA expression was induced using IPTG at concentrations that provided a titration of decreasing growth rates (not shown).


Concurrent growth rate and transcript analyses reveal essential gene stringency in Escherichia coli.

Goh S, Boberek JM, Nakashima N, Stach J, Good L - PLoS ONE (2009)

Dose dependent growth inhibition of antisense PNA-treated AS19 cells.Overnight AS19 cultures were sub-cultured in fresh media containing different PNA doses for each gene, and its growth monitored by turbidity. Six doses were chosen for each PNA, so as to obtain a titration in growth inhibition. Growth profiles terminated when all cultures were harvested at ΔOD550∼0.1 of the untreated culture, for transcript analyses.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2698124&req=5

pone-0006061-g002: Dose dependent growth inhibition of antisense PNA-treated AS19 cells.Overnight AS19 cultures were sub-cultured in fresh media containing different PNA doses for each gene, and its growth monitored by turbidity. Six doses were chosen for each PNA, so as to obtain a titration in growth inhibition. Growth profiles terminated when all cultures were harvested at ΔOD550∼0.1 of the untreated culture, for transcript analyses.
Mentions: To test the hypothesis that essential genes are differentially required to maintain a similar level of growth, we titrated down the growth rate of E. coli using either antisense PNA or expressed antisense and determined mRNA levels of the targeted essential gene. To enable parallel analyses of multiple genes in this study, we used RT-PCR to quantify mRNA levels and assume it as a proxy to protein measure in bacteria [39]. For PNA-mediated antisense effects, we used the hyper-permeable E. coli strain AS19 to obtain efficient uptake [40]. Six doses of each antisense PNA were selected in order to achieve a titration in E. coli growth rate reduction. Hence, the dose range of each antisense PNA was unique, resulting in growth inhibition in a dose dependent manner (Figure 2). For expressed antisense, plasmids were transformed into TOP10 E. coli cells (Table 2) and antisense RNA expression was induced using IPTG at concentrations that provided a titration of decreasing growth rates (not shown).

Bottom Line: The relationship between mRNA decline and growth rate decline reflects the degree of essentiality, or stringency, of an essential gene, which is here defined by the minimum transcript level for a 50% reduction in growth rate (MTL(50)).When applied to four growth essential genes, both RNA silencing methods resulted in MTL(50) values that reveal acpP as the most stringently required of the four genes examined, with ftsZ the next most stringently required.This method may be used to validate existing essential genes and to quantify drug target requirement.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Molecular Biology, Karolinska Institute, Stockholm, Sweden.

ABSTRACT

Background: Genes essential for bacterial growth are of particular scientific interest. Many putative essential genes have been identified or predicted in several species, however, little is known about gene expression requirement stringency, which may be an important aspect of bacterial physiology and likely a determining factor in drug target development.

Methodology/principal findings: Working from the premise that essential genes differ in absolute requirement for growth, we describe silencing of putative essential genes in E. coli to obtain a titration of declining growth rates and transcript levels by using antisense peptide nucleic acids (PNA) and expressed antisense RNA. The relationship between mRNA decline and growth rate decline reflects the degree of essentiality, or stringency, of an essential gene, which is here defined by the minimum transcript level for a 50% reduction in growth rate (MTL(50)). When applied to four growth essential genes, both RNA silencing methods resulted in MTL(50) values that reveal acpP as the most stringently required of the four genes examined, with ftsZ the next most stringently required. The established antibacterial targets murA and fabI were less stringently required.

Conclusions: RNA silencing can reveal stringent requirements for gene expression with respect to growth. This method may be used to validate existing essential genes and to quantify drug target requirement.

Show MeSH